Publications
2018 |
Visnes, Torkild; Cázares-Körner, Armando; Hao, Wenjing; Wallner, Olov; Masuyer, Geoffrey; Loseva, Olga; Mortusewicz, Oliver; Wiita, Elisée; Sarno, Antonio; Manoilov, Aleksandr; Astorga-Wells, Juan; Jemth, Ann-Sofie; Pan, Lang; Sanjiv, Kumar; Karsten, Stella; Gokturk, Camilla; Grube, Maurice; Homan, Evert J; Hanna, Bishoy M F; Paulin, Cynthia B J; Pham, Therese; Rasti, Azita; Berglund, Ulrika Warpman; von Nicolai, Catharina; Benitez-Buelga, Carlos; Koolmeister, Tobias; Ivanic, Dag; Iliev, Petar; Scobie, Martin; Krokan, Hans E; Baranczewski, Pawel; Artursson, Per; Altun, Mikael; Jensen, Annika Jenmalm; Kalderén, Christina; Ba, Xueqing; Zubarev, Roman A; Stenmark, Pål; Boldogh, Istvan; Helleday, Thomas Small-molecule inhibitor of OGG1 suppresses proinflammatory gene expression and inflammation Journal Article Science, 362 , pp. 834-839, 2018. Abstract | Links | BibTeX | Tags: article @article{Visnes2018, title = {Small-molecule inhibitor of OGG1 suppresses proinflammatory gene expression and inflammation}, author = {Torkild Visnes and Armando Cázares-Körner and Wenjing Hao and Olov Wallner and Geoffrey Masuyer and Olga Loseva and Oliver Mortusewicz and Elisée Wiita and Antonio Sarno and Aleksandr Manoilov and Juan Astorga-Wells and Ann-Sofie Jemth and Lang Pan and Kumar Sanjiv and Stella Karsten and Camilla Gokturk and Maurice Grube and Evert J. Homan and Bishoy M. F. Hanna and Cynthia B. J. Paulin and Therese Pham and Azita Rasti and Ulrika Warpman Berglund and Catharina von Nicolai and Carlos Benitez-Buelga and Tobias Koolmeister and Dag Ivanic and Petar Iliev and Martin Scobie and Hans E. Krokan and Pawel Baranczewski and Per Artursson and Mikael Altun and Annika Jenmalm Jensen and Christina Kalderén and Xueqing Ba and Roman A. Zubarev and Pål Stenmark and Istvan Boldogh and Thomas Helleday}, url = {http://science.sciencemag.org/content/362/6416/834}, year = {2018}, date = {2018-11-15}, journal = {Science}, volume = {362}, pages = {834-839}, abstract = {The onset of inflammation is associated with reactive oxygen species and oxidative damage to macromolecules like 7,8-dihydro-8-oxoguanine (8-oxoG) in DNA. Because 8-oxoguanine DNA glycosylase 1 (OGG1) binds 8-oxoG and because Ogg1-deficient mice are resistant to acute and systemic inflammation, we hypothesized that OGG1 inhibition may represent a strategy for the prevention and treatment of inflammation. We developed TH5487, a selective active-site inhibitor of OGG1, which hampers OGG1 binding to and repair of 8-oxoG and which is well tolerated by mice. TH5487 prevents tumor necrosis factor–α–induced OGG1-DNA interactions at guanine-rich promoters of proinflammatory genes. This, in turn, decreases DNA occupancy of nuclear factor κB and proinflammatory gene expression, resulting in decreased immune cell recruitment to mouse lungs. Thus, we present a proof of concept that targeting oxidative DNA repair can alleviate inflammatory conditions in vivo.}, keywords = {article}, pubstate = {published}, tppubtype = {article} } The onset of inflammation is associated with reactive oxygen species and oxidative damage to macromolecules like 7,8-dihydro-8-oxoguanine (8-oxoG) in DNA. Because 8-oxoguanine DNA glycosylase 1 (OGG1) binds 8-oxoG and because Ogg1-deficient mice are resistant to acute and systemic inflammation, we hypothesized that OGG1 inhibition may represent a strategy for the prevention and treatment of inflammation. We developed TH5487, a selective active-site inhibitor of OGG1, which hampers OGG1 binding to and repair of 8-oxoG and which is well tolerated by mice. TH5487 prevents tumor necrosis factor–α–induced OGG1-DNA interactions at guanine-rich promoters of proinflammatory genes. This, in turn, decreases DNA occupancy of nuclear factor κB and proinflammatory gene expression, resulting in decreased immune cell recruitment to mouse lungs. Thus, we present a proof of concept that targeting oxidative DNA repair can alleviate inflammatory conditions in vivo. |
Jemth, Ann-Sofie; Gustafsson, Robert; Bräutigam, Lars; Henriksson, Linda; Vallin, Karl S A; Sarno, Antonio; Almlöf, Ingrid; Homan, Evert; Rasti, Azita; Berglund, Ulrika Warpman; Stenmark, Pål; Helleday, Thomas MutT homologue 1 (MTH1) catalyzes the hydrolysis of mutagenic O6-methyl-dGTP Journal Article Nucleic Acid Research, 2018. Abstract | Links | BibTeX | Tags: article @article{Jemnth2018, title = {MutT homologue 1 (MTH1) catalyzes the hydrolysis of mutagenic O6-methyl-dGTP}, author = {Ann-Sofie Jemth and Robert Gustafsson and Lars Bräutigam and Linda Henriksson and Karl S A Vallin and Antonio Sarno and Ingrid Almlöf and Evert Homan and Azita Rasti and Ulrika Warpman Berglund and Pål Stenmark and Thomas Helleday}, url = {https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gky896/5124590}, doi = {doi.org/10.1093/nar/gky896}, year = {2018}, date = {2018-10-10}, journal = {Nucleic Acid Research}, abstract = {Nucleotides in the free pool are more susceptible to nonenzymatic methylation than those protected in the DNA double helix. Methylated nucleotides like O6-methyl-dGTP can be mutagenic and toxic if incorporated into DNA. Removal of methylated nucleotides from the nucleotide pool may therefore be important to maintain genome integrity. We show that MutT homologue 1 (MTH1) efficiently catalyzes the hydrolysis of O6-methyl-dGTP with a catalytic efficiency similar to that for 8-oxo-dGTP. O6-methyl-dGTP activity is exclusive to MTH1 among human NUDIX proteins and conserved through evolution but not found in bacterial MutT. We present a high resolution crystal structure of human and zebrafish MTH1 in complex with O6-methyl-dGMP. By microinjecting fertilized zebrafish eggs with O6-methyl-dGTP and inhibiting MTH1 we demonstrate that survival is dependent on active MTH1 in vivo. O6-methyl-dG levels are higher in DNA extracted from zebrafish embryos microinjected with O6-methyl-dGTP and inhibition of O6-methylguanine-DNA methyl transferase (MGMT) increases the toxicity of O6-methyl-dGTP demonstrating that O6-methyl-dGTP is incorporated into DNA. MTH1 deficiency sensitizes human cells to the alkylating agent Temozolomide, a sensitization that is more pronounced upon MGMT inhibition. These results expand the cellular MTH1 function and suggests MTH1 also is important for removal of methylated nucleotides from the nucleotide pool.}, keywords = {article}, pubstate = {published}, tppubtype = {article} } Nucleotides in the free pool are more susceptible to nonenzymatic methylation than those protected in the DNA double helix. Methylated nucleotides like O6-methyl-dGTP can be mutagenic and toxic if incorporated into DNA. Removal of methylated nucleotides from the nucleotide pool may therefore be important to maintain genome integrity. We show that MutT homologue 1 (MTH1) efficiently catalyzes the hydrolysis of O6-methyl-dGTP with a catalytic efficiency similar to that for 8-oxo-dGTP. O6-methyl-dGTP activity is exclusive to MTH1 among human NUDIX proteins and conserved through evolution but not found in bacterial MutT. We present a high resolution crystal structure of human and zebrafish MTH1 in complex with O6-methyl-dGMP. By microinjecting fertilized zebrafish eggs with O6-methyl-dGTP and inhibiting MTH1 we demonstrate that survival is dependent on active MTH1 in vivo. O6-methyl-dG levels are higher in DNA extracted from zebrafish embryos microinjected with O6-methyl-dGTP and inhibition of O6-methylguanine-DNA methyl transferase (MGMT) increases the toxicity of O6-methyl-dGTP demonstrating that O6-methyl-dGTP is incorporated into DNA. MTH1 deficiency sensitizes human cells to the alkylating agent Temozolomide, a sensitization that is more pronounced upon MGMT inhibition. These results expand the cellular MTH1 function and suggests MTH1 also is important for removal of methylated nucleotides from the nucleotide pool. |
Gustafsson, Nina M S; Färnegårdh, Katarina; Bonagas, Nadilly; Ninou, Anna Huguet; Groth, Petra; Wiita, Elisee; Jönsson, Mattias; Hallberg, Kenth; Lehto, Jemina; Pennisi, Rosa; Martinsson, Jessica; Norström, Carina; Hollers, Jessica; Schultz, Johan; Andersson, Martin; Markova, Natalia; Marttila, Petra; Kim, Baek; Norin, Martin; Olin, Thomas; Helleday, Thomas Targeting PFKFB3 radiosensitizes cancer cells and suppresses homologous recombination Journal Article Nature Communications, 9 (3872), 2018. Abstract | Links | BibTeX | Tags: article @article{Gustafsson2018, title = {Targeting PFKFB3 radiosensitizes cancer cells and suppresses homologous recombination}, author = {Nina M.S. Gustafsson and Katarina Färnegårdh and Nadilly Bonagas and Anna Huguet Ninou and Petra Groth and Elisee Wiita and Mattias Jönsson and Kenth Hallberg and Jemina Lehto and Rosa Pennisi and Jessica Martinsson and Carina Norström and Jessica Hollers and Johan Schultz and Martin Andersson and Natalia Markova and Petra Marttila and Baek Kim and Martin Norin and Thomas Olin and Thomas Helleday}, url = {https://www.nature.com/articles/s41467-018-06287-x#article-comments}, doi = {doi.org/10.1038/s41467-018-06287-x}, year = {2018}, date = {2018-09-24}, journal = {Nature Communications}, volume = {9}, number = {3872}, abstract = {The glycolytic PFKFB3 enzyme is widely overexpressed in cancer cells and an emerging anti-cancer target. Here, we identify PFKFB3 as a critical factor in homologous recombination (HR) repair of DNA double-strand breaks. PFKFB3 rapidly relocates into ionizing radiation (IR)-induced nuclear foci in an MRN-ATM-γH2AX-MDC1-dependent manner and co-localizes with DNA damage and HR repair proteins. PFKFB3 relocalization is critical for recruitment of HR proteins, HR activity, and cell survival upon IR. We develop KAN0438757, a small molecule inhibitor that potently targets PFKFB3. Pharmacological PFKFB3 inhibition impairs recruitment of ribonucleotide reductase M2 and deoxynucleotide incorporation upon DNA repair, and reduces dNTP levels. Importantly, KAN0438757 induces radiosensitization in transformed cells while leaving non-transformed cells unaffected. In summary, we identify a key role for PFKFB3 enzymatic activity in HR repair and present KAN0438757, a selective PFKFB3 inhibitor that could potentially be used as a strategy for the treatment of cancer.}, keywords = {article}, pubstate = {published}, tppubtype = {article} } The glycolytic PFKFB3 enzyme is widely overexpressed in cancer cells and an emerging anti-cancer target. Here, we identify PFKFB3 as a critical factor in homologous recombination (HR) repair of DNA double-strand breaks. PFKFB3 rapidly relocates into ionizing radiation (IR)-induced nuclear foci in an MRN-ATM-γH2AX-MDC1-dependent manner and co-localizes with DNA damage and HR repair proteins. PFKFB3 relocalization is critical for recruitment of HR proteins, HR activity, and cell survival upon IR. We develop KAN0438757, a small molecule inhibitor that potently targets PFKFB3. Pharmacological PFKFB3 inhibition impairs recruitment of ribonucleotide reductase M2 and deoxynucleotide incorporation upon DNA repair, and reduces dNTP levels. Importantly, KAN0438757 induces radiosensitization in transformed cells while leaving non-transformed cells unaffected. In summary, we identify a key role for PFKFB3 enzymatic activity in HR repair and present KAN0438757, a selective PFKFB3 inhibitor that could potentially be used as a strategy for the treatment of cancer. |
Visnes, Torkild; Grube, Maurice; Hanna, Bishoy Magdy Fekry; Benitez-Buelga, Carlos; Cázares-Körner, Armando; Helleday, Thomas Targeting BER enzymes in cancer therapy Journal Article DNA Repair, 2018. Abstract | Links | BibTeX | Tags: article @article{Visnes2018, title = {Targeting BER enzymes in cancer therapy}, author = {Torkild Visnes and Maurice Grube and Bishoy Magdy Fekry Hanna and Carlos Benitez-Buelga and Armando Cázares-Körner and Thomas Helleday}, url = {https://www.sciencedirect.com/science/article/pii/S1568786418301800?via%3Dihub}, year = {2018}, date = {2018-08-25}, journal = {DNA Repair}, abstract = {Base excision repair (BER) repairs mutagenic or genotoxic DNA base lesions, thought to be important for both the etiology and treatment of cancer. Cancer phenotypic stress induces oxidative lesions, and deamination products are responsible for one of the most prevalent mutational signatures in cancer. Chemotherapeutic agents induce genotoxic DNA base damage that are substrates for BER, while synthetic lethal approaches targeting BER-related factors are making their way into the clinic. Thus, there are three strategies by which BER is envisioned to be relevant in cancer chemotherapy: (i) to maintain cellular growth in the presence of endogenous DNA damage in stressed cancer cells, (ii) to maintain viability after exogenous DNA damage is introduced by therapeutic intervention, or (iii) to confer synthetic lethality in cancer cells that have lost one or more additional DNA repair pathways. Here, we discuss the potential treatment strategies, and briefly summarize the progress that has been made in developing inhibitors to core BER-proteins and related factors.}, keywords = {article}, pubstate = {published}, tppubtype = {article} } Base excision repair (BER) repairs mutagenic or genotoxic DNA base lesions, thought to be important for both the etiology and treatment of cancer. Cancer phenotypic stress induces oxidative lesions, and deamination products are responsible for one of the most prevalent mutational signatures in cancer. Chemotherapeutic agents induce genotoxic DNA base damage that are substrates for BER, while synthetic lethal approaches targeting BER-related factors are making their way into the clinic. Thus, there are three strategies by which BER is envisioned to be relevant in cancer chemotherapy: (i) to maintain cellular growth in the presence of endogenous DNA damage in stressed cancer cells, (ii) to maintain viability after exogenous DNA damage is introduced by therapeutic intervention, or (iii) to confer synthetic lethality in cancer cells that have lost one or more additional DNA repair pathways. Here, we discuss the potential treatment strategies, and briefly summarize the progress that has been made in developing inhibitors to core BER-proteins and related factors. |
Einarsdottir, Berglind O; Karlsson, Joakim; Söderberg, Elin M V; Lindberg, Mattias F; Funck-Brentano, Elisa; Jespersen, Henrik; Brynjolfsson, Siggeir F; Bagge, Roger Olofsson; Carstam, Louise; Scobie, Martin; Koolmeister, Tobias; Wallner, Olov; Stierner, Ulrika; Berglund, Ulrika Warpman; Ny, Lars; Nilsson, Lisa M; Larsson, Erik; Helleday, Thomas; Nilsson, Jonas A A patient-derived xenograft pre-clinical trial reveals treatment responses and a resistance mechanism to karonudib in metastatic melanoma Journal Article Cell Death & Disease, 2018. Abstract | Links | BibTeX | Tags: article @article{Einarsdottir2018, title = {A patient-derived xenograft pre-clinical trial reveals treatment responses and a resistance mechanism to karonudib in metastatic melanoma}, author = {Berglind O. Einarsdottir and Joakim Karlsson and Elin M. V. Söderberg and Mattias F. Lindberg and Elisa Funck-Brentano and Henrik Jespersen and Siggeir F. Brynjolfsson and Roger Olofsson Bagge and Louise Carstam and Martin Scobie and Tobias Koolmeister and Olov Wallner and Ulrika Stierner and Ulrika Warpman Berglund and Lars Ny and Lisa M. Nilsson and Erik Larsson and Thomas Helleday and Jonas A. Nilsson}, url = {https://www.nature.com/articles/s41419-018-0865-6#article-info}, year = {2018}, date = {2018-07-24}, journal = {Cell Death & Disease}, abstract = {Karonudib (TH1579) is a novel compound that exerts anti-tumor activities and has recently entered phase I clinical testing. The aim of this study was to conduct a pre-clinical trial in patient-derived xenografts to identify the possible biomarkers of response or resistance that could guide inclusion of patients suffering from metastatic melanoma in phase II clinical trials. Patient-derived xenografts from 31 melanoma patients with metastatic disease were treated with karonudib or a vehicle for 18 days. Treatment responses were followed by measuring tumor sizes, and the models were categorized in the response groups. Tumors were harvested and processed for RNA sequencing and protein analysis. To investigate the effect of karonudib on T-cell-mediated anti-tumor activities, tumor-infiltrating T cells were injected in mice carrying autologous tumors and the mice treated with karonudib. We show that karonudib has heterogeneous anti-tumor effect on metastatic melanoma. Thus, based on the treatment responses, we could divide the 31 patient-derived xenografts in three treatment groups: progression group (32%), suppression group (42%), and regression group (26%). Furthermore, we show that karonudib has anti-tumor effect, irrespective of major melanoma driver mutations. Also, we identify high expression of ABCB1, which codes for p-gp pumps as a resistance biomarker. Finally, we show that karonudib treatment does not hamper T-cell-mediated anti-tumor responses. These findings can be used to guide future use of karonudib in clinical use with a potential approach as precision medicine.}, keywords = {article}, pubstate = {published}, tppubtype = {article} } Karonudib (TH1579) is a novel compound that exerts anti-tumor activities and has recently entered phase I clinical testing. The aim of this study was to conduct a pre-clinical trial in patient-derived xenografts to identify the possible biomarkers of response or resistance that could guide inclusion of patients suffering from metastatic melanoma in phase II clinical trials. Patient-derived xenografts from 31 melanoma patients with metastatic disease were treated with karonudib or a vehicle for 18 days. Treatment responses were followed by measuring tumor sizes, and the models were categorized in the response groups. Tumors were harvested and processed for RNA sequencing and protein analysis. To investigate the effect of karonudib on T-cell-mediated anti-tumor activities, tumor-infiltrating T cells were injected in mice carrying autologous tumors and the mice treated with karonudib. We show that karonudib has heterogeneous anti-tumor effect on metastatic melanoma. Thus, based on the treatment responses, we could divide the 31 patient-derived xenografts in three treatment groups: progression group (32%), suppression group (42%), and regression group (26%). Furthermore, we show that karonudib has anti-tumor effect, irrespective of major melanoma driver mutations. Also, we identify high expression of ABCB1, which codes for p-gp pumps as a resistance biomarker. Finally, we show that karonudib treatment does not hamper T-cell-mediated anti-tumor responses. These findings can be used to guide future use of karonudib in clinical use with a potential approach as precision medicine. |
Berglund, Emelie; Maaskola, Jonas; Schultz, Niklas; Friedrich, Stefanie; Marklund, Maja; Bergenstråhle, Joseph; Tarish, Firas; Tanoglidi, Anna; Vickovic, Sanja; Larsson, Ludvig; Salmen, Fredrik; Ogris, Christoph; Wallenborg, Karolina; Lagergren, Jens; Ståhl, Patrik; Sonnhammer, Erik; Helleday, Thomas; Lundeberg, Joakim Spatial maps of prostate cancer transcriptomes reveal an unexplored landscape of heterogeneity Journal Article Nature Communications, 9 , 2018. Abstract | Links | BibTeX | Tags: article @article{Berglund2018, title = {Spatial maps of prostate cancer transcriptomes reveal an unexplored landscape of heterogeneity}, author = {Emelie Berglund and Jonas Maaskola and Niklas Schultz and Stefanie Friedrich and Maja Marklund and Joseph Bergenstråhle and Firas Tarish and Anna Tanoglidi and Sanja Vickovic and Ludvig Larsson and Fredrik Salmen and Christoph Ogris and Karolina Wallenborg and Jens Lagergren and Patrik Ståhl and Erik Sonnhammer and Thomas Helleday and Joakim Lundeberg}, doi = {https://doi.org/10.1038/s41467-018-04724-5}, year = {2018}, date = {2018-06-20}, journal = {Nature Communications}, volume = {9}, abstract = {Intra-tumor heterogeneity is one of the biggest challenges in cancer treatment today. Here we investigate tissue-wide gene expression heterogeneity throughout a multifocal prostate cancer using the spatial transcriptomics (ST) technology. Utilizing a novel approach for deconvolution, we analyze the transcriptomes of nearly 6750 tissue regions and extract distinct expression profiles for the different tissue components, such as stroma, normal and PIN glands, immune cells and cancer. We distinguish healthy and diseased areas and thereby provide insight into gene expression changes during the progression of prostate cancer. Compared to pathologist annotations, we delineate the extent of cancer foci more accurately, interestingly without link to histological changes. We identify gene expression gradients in stroma adjacent to tumor regions that allow for re-stratification of the tumor microenvironment. The establishment of these profiles is the first step towards an unbiased view of prostate cancer and can serve as a dictionary for future studies.}, keywords = {article}, pubstate = {published}, tppubtype = {article} } Intra-tumor heterogeneity is one of the biggest challenges in cancer treatment today. Here we investigate tissue-wide gene expression heterogeneity throughout a multifocal prostate cancer using the spatial transcriptomics (ST) technology. Utilizing a novel approach for deconvolution, we analyze the transcriptomes of nearly 6750 tissue regions and extract distinct expression profiles for the different tissue components, such as stroma, normal and PIN glands, immune cells and cancer. We distinguish healthy and diseased areas and thereby provide insight into gene expression changes during the progression of prostate cancer. Compared to pathologist annotations, we delineate the extent of cancer foci more accurately, interestingly without link to histological changes. We identify gene expression gradients in stroma adjacent to tumor regions that allow for re-stratification of the tumor microenvironment. The establishment of these profiles is the first step towards an unbiased view of prostate cancer and can serve as a dictionary for future studies. |
Pilar Mur Ann‐Sofie Jemth, Luka Bevc Nuno Amaral Matilde Navarro Rafael Valdés‐Mas Tirso Pons Gemma Aiza Miguel Urioste Alfonso Valencia Conxi Lázaro Victor Moreno Xose Puente Stenmark Ulrika Warpman‐Berglund Gabriel Capellá Thomas Helleday Laura Valle S Pål Germline variation in the oxidative DNA repair genes NUDT1 and OGG1 is not associated with hereditary colorectal cancer or polyposis Journal Article Human Mutation, 2018. Abstract | Links | BibTeX | Tags: article @article{Mur2018, title = {Germline variation in the oxidative DNA repair genes NUDT1 and OGG1 is not associated with hereditary colorectal cancer or polyposis }, author = {Pilar Mur, Ann‐Sofie Jemth, Luka Bevc, Nuno Amaral, Matilde Navarro, Rafael Valdés‐Mas, Tirso Pons, Gemma Aiza, Miguel Urioste, Alfonso Valencia, Conxi Lázaro, Victor Moreno, Xose S. Puente, Pål Stenmark, Ulrika Warpman‐Berglund, Gabriel Capellá, Thomas Helleday, Laura Valle}, url = {https://onlinelibrary.wiley.com/doi/abs/10.1002/humu.23564}, year = {2018}, date = {2018-06-13}, journal = {Human Mutation}, abstract = {The causal association of NUDT1 (=MTH1)andOGG1 with hereditary colorectal cancer (CRC) remains unclear. Here, we sought to provide additional evidence for or against the causal con- tribution of NUDT1 and OGG1 mutations to hereditary CRC and/or polyposis. Mutational screen- ing was performed using pooled DNA amplification and targeted next-generation sequencing in 529 families (441 uncharacterized MMR-proficient familial nonpolyposis CRC and 88 polyposis cases). Cosegregation, in silico analyses, in vitro functional assays, and case–control associations were carried out to characterize the identified variants. Five heterozygous carriers of novel (n = 1) or rare (n = 4) NUDT1 variants were identified. In vitro deleterious effects were demonstrated for c.143G>A p.G48E (catalytic activity and protein stability) and c.403G>T p.G135W (protein sta- bility), although cosegregation data in the carrier families were inconclusive or nonsupportive. The frequency of missense, loss-of-function, and splice-site NUDT1 variants in our familial CRC cohort was similar to the one observed in cancer-free individuals, suggesting lack of association with CRC predisposition. No OGG1 pathogenic mutations were identified. Our results suggest that the contribution of NUDT1 and OGG1 germline mutations to hereditary CRC and to polyposis is inexistent or, at most, negligible. The inclusion of these genes in routine genetic testing is not recommended.}, keywords = {article}, pubstate = {published}, tppubtype = {article} } The causal association of NUDT1 (=MTH1)andOGG1 with hereditary colorectal cancer (CRC) remains unclear. Here, we sought to provide additional evidence for or against the causal con- tribution of NUDT1 and OGG1 mutations to hereditary CRC and/or polyposis. Mutational screen- ing was performed using pooled DNA amplification and targeted next-generation sequencing in 529 families (441 uncharacterized MMR-proficient familial nonpolyposis CRC and 88 polyposis cases). Cosegregation, in silico analyses, in vitro functional assays, and case–control associations were carried out to characterize the identified variants. Five heterozygous carriers of novel (n = 1) or rare (n = 4) NUDT1 variants were identified. In vitro deleterious effects were demonstrated for c.143G>A p.G48E (catalytic activity and protein stability) and c.403G>T p.G135W (protein sta- bility), although cosegregation data in the carrier families were inconclusive or nonsupportive. The frequency of missense, loss-of-function, and splice-site NUDT1 variants in our familial CRC cohort was similar to the one observed in cancer-free individuals, suggesting lack of association with CRC predisposition. No OGG1 pathogenic mutations were identified. Our results suggest that the contribution of NUDT1 and OGG1 germline mutations to hereditary CRC and to polyposis is inexistent or, at most, negligible. The inclusion of these genes in routine genetic testing is not recommended. |
Riley, Andrew M; Unterlass, Judith E; Konieczny, Vera; Taylor, Colin W; Helleday, Thomas; Potter, Barry V L A synthetic diphosphoinositol phosphate analogue of inositol trisphosphate Journal Article Med. Chem. Comm., 9 , pp. 1105-1113, 2018, (https://pubs.rsc.org/en/Content/ArticleLanding/2018/MD/C8MD00149A#!divAbstract). Abstract | Links | BibTeX | Tags: article @article{Riley2018, title = {A synthetic diphosphoinositol phosphate analogue of inositol trisphosphate }, author = { Andrew M. Riley and Judith E. Unterlass and Vera Konieczny and Colin W. Taylor and Thomas Helleday and Barry V. L. Potter }, url = {https://pubs.rsc.org/en/Content/ArticleLanding/2018/MD/C8MD00149A#!divAbstract}, doi = { 10.1039/C8MD00149A}, year = {2018}, date = {2018-06-04}, journal = {Med. Chem. Comm.}, volume = {9}, pages = {1105-1113}, abstract = {Diphosphoinositol phosphates (PP-InsPs) are inositol phosphates (InsPs) that contain PP (diphosphate) groups. Converting a phosphate group in an InsP into a diphosphate has been reported to enhance affinity for some binding proteins. We synthesised 1-PP-Ins(4,5)P2, the first diphosphate analogue of the intracellular signalling molecule InsP3, and examined its effects on InsP3 receptors, which are intracellular Ca2+ channels. 1-PP-Ins(4,5)P2 was indistinguishable from InsP3 in its ability to bind to and activate type 1 InsP3 receptors, indicating that the diphosphate modification of InsP3 affected neither affinity nor efficacy. Nevertheless, 1-PP-Ins(4,5)P2 is the most potent 1-phosphate modified analogue of InsP3 yet identified. PP-InsPs are generally hydrolysed by diphosphoinositol polyphosphate phosphohydrolases (DIPPs), but 1-PP-Ins(4,5)P2 was not readily metabolised by human DIPPs. Differential scanning fluorimetry showed that 1-PP-Ins(4,5)P2 stabilises DIPP proteins, but to a lesser extent than naturally occurring substrates 1-PP-InsP5 and 5-PP-InsP5. The non-hydrolysable InsP7 analogues 1-PCP-InsP5 and 5-PCP-InsP5 showed comparable stabilising abilities to their natural counterparts and may therefore be promising substrate analogues for co-crystallisation with DIPPs.}, note = {https://pubs.rsc.org/en/Content/ArticleLanding/2018/MD/C8MD00149A#!divAbstract}, keywords = {article}, pubstate = {published}, tppubtype = {article} } Diphosphoinositol phosphates (PP-InsPs) are inositol phosphates (InsPs) that contain PP (diphosphate) groups. Converting a phosphate group in an InsP into a diphosphate has been reported to enhance affinity for some binding proteins. We synthesised 1-PP-Ins(4,5)P2, the first diphosphate analogue of the intracellular signalling molecule InsP3, and examined its effects on InsP3 receptors, which are intracellular Ca2+ channels. 1-PP-Ins(4,5)P2 was indistinguishable from InsP3 in its ability to bind to and activate type 1 InsP3 receptors, indicating that the diphosphate modification of InsP3 affected neither affinity nor efficacy. Nevertheless, 1-PP-Ins(4,5)P2 is the most potent 1-phosphate modified analogue of InsP3 yet identified. PP-InsPs are generally hydrolysed by diphosphoinositol polyphosphate phosphohydrolases (DIPPs), but 1-PP-Ins(4,5)P2 was not readily metabolised by human DIPPs. Differential scanning fluorimetry showed that 1-PP-Ins(4,5)P2 stabilises DIPP proteins, but to a lesser extent than naturally occurring substrates 1-PP-InsP5 and 5-PP-InsP5. The non-hydrolysable InsP7 analogues 1-PCP-InsP5 and 5-PCP-InsP5 showed comparable stabilising abilities to their natural counterparts and may therefore be promising substrate analogues for co-crystallisation with DIPPs. |
Linda Pudelko Steven Edwards, Mirela Balan Daniel Nyqvist Jonathan Al-Saadi Johannes Dittmer Ingrid Almlöf Thomas Helleday Lars Bräutigam An orthotopic glioblastoma animal model suitable for high-throughput screenings Journal Article Neuro-Oncology, 2018. Abstract | Links | BibTeX | Tags: article @article{Pudelko2018, title = {An orthotopic glioblastoma animal model suitable for high-throughput screenings }, author = {Linda Pudelko, Steven Edwards, Mirela Balan, Daniel Nyqvist, Jonathan Al-Saadi, Johannes Dittmer, Ingrid Almlöf, Thomas Helleday, Lars Bräutigam}, url = {https://academic.oup.com/neuro-oncology/advance-article/doi/10.1093/neuonc/noy071/4994699}, year = {2018}, date = {2018-05-10}, journal = {Neuro-Oncology}, abstract = {Glioblastoma (GBM) is an aggressive form of brain cancer with poor prognosis. Although murine animal models have given valuable insights into the GBM disease biology, they cannot be used in high-throughput screens to identify and profile novel therapies. The only vertebrate model suitable for large-scale screens, the zebrafish, has proven to faithfully recapitulate biology and pathology of human malignancies, and clinically relevant orthotopic zebrafish models have been developed. However, currently available GBM orthotopic zebrafish models do not support high-throughput drug discovery screens. Methods We transplanted both GBM cell lines as well as patient-derived material into zebrafish blastulas. We followed the behavior of the transplants with time-lapse microscopy and real-time in vivo light-sheet microscopy. We found that GBM material transplanted into zebrafish blastomeres robustly migrated into the developing nervous system, establishing an orthotopic intracranial tumor already 24 hours after transplantation. Detailed analysis revealed that our model faithfully recapitulates the human disease. We have developed a robust, fast, and automatable transplantation assay to establish orthotopic GBM tumors in zebrafish. In contrast to currently available orthotopic zebrafish models, our approach does not require technically challenging intracranial transplantation of single embryos. Our improved zebrafish model enables transplantation of thousands of embryos per hour, thus providing an orthotopic vertebrate GBM model for direct application in drug discovery screens.}, keywords = {article}, pubstate = {published}, tppubtype = {article} } Glioblastoma (GBM) is an aggressive form of brain cancer with poor prognosis. Although murine animal models have given valuable insights into the GBM disease biology, they cannot be used in high-throughput screens to identify and profile novel therapies. The only vertebrate model suitable for large-scale screens, the zebrafish, has proven to faithfully recapitulate biology and pathology of human malignancies, and clinically relevant orthotopic zebrafish models have been developed. However, currently available GBM orthotopic zebrafish models do not support high-throughput drug discovery screens. Methods We transplanted both GBM cell lines as well as patient-derived material into zebrafish blastulas. We followed the behavior of the transplants with time-lapse microscopy and real-time in vivo light-sheet microscopy. We found that GBM material transplanted into zebrafish blastomeres robustly migrated into the developing nervous system, establishing an orthotopic intracranial tumor already 24 hours after transplantation. Detailed analysis revealed that our model faithfully recapitulates the human disease. We have developed a robust, fast, and automatable transplantation assay to establish orthotopic GBM tumors in zebrafish. In contrast to currently available orthotopic zebrafish models, our approach does not require technically challenging intracranial transplantation of single embryos. Our improved zebrafish model enables transplantation of thousands of embryos per hour, thus providing an orthotopic vertebrate GBM model for direct application in drug discovery screens. |
Megan Carter Ann-Sofie Jemth, Jordi Carreras-Puigvert Patrick Herr Markel Martínez Carranza Karl Vallin Adam Throup Thomas Helleday Stenmark S A Pål Human NUDT22 Is a UDP-Glucose/Galactose Hydrolase Exhibiting a Unique Structural Fold Journal Article Cell Structure, 26 , pp. 295-303, 2018. Abstract | BibTeX | Tags: article @article{Carter2018, title = {Human NUDT22 Is a UDP-Glucose/Galactose Hydrolase Exhibiting a Unique Structural Fold}, author = {Megan Carter, Ann-Sofie Jemth, Jordi Carreras-Puigvert, Patrick Herr, Markel Martínez Carranza, Karl S.A. Vallin, Adam Throup, Thomas Helleday, Pål Stenmark}, year = {2018}, date = {2018-02-06}, journal = {Cell Structure}, volume = {26}, pages = {295-303}, abstract = {Human NUDT22 belongs to the diverse NUDIX family of proteins, but has, until now, remained uncharacterized. Here we show that human NUDT22 is a Mg 2+-dependent UDP-glucose and UDP-galactose hydrolase, producing UMP and glucose 1-phosphate or galactose 1-phosphate. We present the structure of human NUDT22 alone and in a complex with the substrate UDP-glucose. These structures reveal a partially conserved NUDIX fold domain preceded by a unique N-terminal domain responsible for UDP moiety binding and recognition. The NUDIX domain of NUDT22 contains a modified NUDIX box identified using structural analysis and confirmed through functional analysis of mutants. Human NUDT22's distinct structure and function as a UDP-carbohydrate hydrolase establish a unique NUDIX protein subfamily. }, keywords = {article}, pubstate = {published}, tppubtype = {article} } Human NUDT22 belongs to the diverse NUDIX family of proteins, but has, until now, remained uncharacterized. Here we show that human NUDT22 is a Mg 2+-dependent UDP-glucose and UDP-galactose hydrolase, producing UMP and glucose 1-phosphate or galactose 1-phosphate. We present the structure of human NUDT22 alone and in a complex with the substrate UDP-glucose. These structures reveal a partially conserved NUDIX fold domain preceded by a unique N-terminal domain responsible for UDP moiety binding and recognition. The NUDIX domain of NUDT22 contains a modified NUDIX box identified using structural analysis and confirmed through functional analysis of mutants. Human NUDT22's distinct structure and function as a UDP-carbohydrate hydrolase establish a unique NUDIX protein subfamily. |
Page Brent D. G.; Valerie, Nicholas Wright Roni Wallner Olov; Isaksson Rebecka; Carter Megan; Rudd Sean Loseva Olga; Jemth Ann-Sofie; Almloef Ingrid; Font-Mateu Jofre; Llona-Minguez Sabin; Baranczewski Pawel; Jeppsson Fredrik; Homan Evert; Almqvist Helena; Axelsson Hanna; Regmi Shruti; Gustavsson Anna-Lena; Lundbaeck Thomas; Scobie Martin; Stroemberg Kia; Stenmark Paal; Beato Miguel; Helleday Thomas. C K ; H G ; G ; Targeted NUDT5 inhibitors block hormone signaling in breast cancer cells Journal Article Nature Communications, 9 , pp. 1-14, 2018. Abstract | Links | BibTeX | Tags: article @article{Page2018, title = {Targeted NUDT5 inhibitors block hormone signaling in breast cancer cells}, author = {Page, Brent D. G.; Valerie, Nicholas C. K.; Wright, Roni H. G.; Wallner, Olov; Isaksson, Rebecka; Carter, Megan; Rudd, Sean G.; Loseva, Olga; Jemth, Ann-Sofie; Almloef, Ingrid; Font-Mateu, Jofre; Llona-Minguez, Sabin; Baranczewski, Pawel; Jeppsson, Fredrik; Homan, Evert; Almqvist, Helena; Axelsson, Hanna; Regmi, Shruti; Gustavsson, Anna-Lena; Lundbaeck, Thomas; Scobie, Martin; Stroemberg, Kia; Stenmark, Paal; Beato, Miguel; Helleday, Thomas.}, url = {https://www.nature.com/articles/s41467-017-02293-7}, year = {2018}, date = {2018-01-17}, journal = {Nature Communications}, volume = {9}, pages = {1-14}, abstract = {With a diverse network of substrates, NUDIX hydrolases have emerged as a key family of nucleotide-metabolizing enzymes. NUDT5 (also called NUDIX5) has been implicated in ADP-ribose and 8-oxo-guanine metabolism and was recently identified as a rheostat of hormone-dependent gene regulation and proliferation in breast cancer cells. Here, we further elucidate the physiological relevance of known NUDT5 substrates and underscore the biological requirement for NUDT5 in gene regulation and proliferation of breast cancer cells. We confirm the involvement of NUDT5 in ADP-ribose metabolism and dissociate a relationship to oxidized nucleotide sanitation. Furthermore, we identify potent NUDT5 inhibitors, which are optimized to promote maximal NUDT5 cellular target engagement by CETSA. Lead compound, TH5427, blocks progestin-dependent, PAR-derived nuclear ATP synthesis and subsequent chromatin remodeling, gene regulation and proliferation in breast cancer cells. We herein present TH5427 as a promising, targeted inhibitor that can be used to further study NUDT5 activity and ADP-ribose metabolism.}, keywords = {article}, pubstate = {published}, tppubtype = {article} } With a diverse network of substrates, NUDIX hydrolases have emerged as a key family of nucleotide-metabolizing enzymes. NUDT5 (also called NUDIX5) has been implicated in ADP-ribose and 8-oxo-guanine metabolism and was recently identified as a rheostat of hormone-dependent gene regulation and proliferation in breast cancer cells. Here, we further elucidate the physiological relevance of known NUDT5 substrates and underscore the biological requirement for NUDT5 in gene regulation and proliferation of breast cancer cells. We confirm the involvement of NUDT5 in ADP-ribose metabolism and dissociate a relationship to oxidized nucleotide sanitation. Furthermore, we identify potent NUDT5 inhibitors, which are optimized to promote maximal NUDT5 cellular target engagement by CETSA. Lead compound, TH5427, blocks progestin-dependent, PAR-derived nuclear ATP synthesis and subsequent chromatin remodeling, gene regulation and proliferation in breast cancer cells. We herein present TH5427 as a promising, targeted inhibitor that can be used to further study NUDT5 activity and ADP-ribose metabolism. |
2017 |
Mohit Narwal Ann-Sofie Jemth, Robert Gustafsson Ingrid Almlöf Ulrika Warpman Berglund Thomas Helleday Stenmark Pål Crystal Structures and Inhibitor Interactions of Mouse and Dog MTH1 Reveal Species-Specific Differences in Affinity Journal Article Biochemistry, 57 (5), pp. 593-603, 2017. Abstract | Links | BibTeX | Tags: article @article{Narwal2018, title = {Crystal Structures and Inhibitor Interactions of Mouse and Dog MTH1 Reveal Species-Specific Differences in Affinity}, author = {Mohit Narwal, Ann-Sofie Jemth, Robert Gustafsson, Ingrid Almlöf, Ulrika Warpman Berglund, Thomas Helleday, Pål Stenmark }, url = {https://pubs.acs.org/doi/abs/10.1021/acs.biochem.7b01163}, year = {2017}, date = {2017-12-27}, journal = {Biochemistry}, volume = {57}, number = {5}, pages = {593-603}, abstract = {MTH1 hydrolyzes oxidized nucleoside triphosphates, thereby sanitizing the nucleotide pool from oxidative damage. This prevents incorporation of damaged nucleotides into DNA, which otherwise would lead to mutations and cell death. The high level of reactive oxygen species in cancer cells leads to a higher level of oxidized nucleotides in cancer cells compared to that in nonmalignant cells, making cancer cells more dependent on MTH1 for survival. The possibility of specifically targeting cancer cells by inhibiting MTH1 has highlighted MTH1 as a promising cancer target. The progression of MTH1 inhibitors into the clinic requires animal studies, and knowledge of species differences in the potency of inhibitors is vitally important. We here show that the human MTH1 inhibitor TH588 is approximately 20-fold less potent with respect to inhibition of mouse MTH1 than the human, rat, pig, and dog MTH1 proteins are. We present the crystal structures of mouse MTH1 in complex with TH588 and dog MTH1 and elucidate the structural and sequence basis for the observed difference in affinity for TH588. We identify amino acid residue 116 in MTH1 as an important determinant of TH588 affinity. Furthermore, we present the structure of mouse MTH1 in complex with the substrate 8-oxo-dGTP. The crystal structures provide insight into the high degree of structural conservation between MTH1 proteins from different organisms and provide a detailed view of interactions between MTH1 and the inhibitor, revealing that minute structural differences can have a large impact on affinity and specificity.}, keywords = {article}, pubstate = {published}, tppubtype = {article} } MTH1 hydrolyzes oxidized nucleoside triphosphates, thereby sanitizing the nucleotide pool from oxidative damage. This prevents incorporation of damaged nucleotides into DNA, which otherwise would lead to mutations and cell death. The high level of reactive oxygen species in cancer cells leads to a higher level of oxidized nucleotides in cancer cells compared to that in nonmalignant cells, making cancer cells more dependent on MTH1 for survival. The possibility of specifically targeting cancer cells by inhibiting MTH1 has highlighted MTH1 as a promising cancer target. The progression of MTH1 inhibitors into the clinic requires animal studies, and knowledge of species differences in the potency of inhibitors is vitally important. We here show that the human MTH1 inhibitor TH588 is approximately 20-fold less potent with respect to inhibition of mouse MTH1 than the human, rat, pig, and dog MTH1 proteins are. We present the crystal structures of mouse MTH1 in complex with TH588 and dog MTH1 and elucidate the structural and sequence basis for the observed difference in affinity for TH588. We identify amino acid residue 116 in MTH1 as an important determinant of TH588 affinity. Furthermore, we present the structure of mouse MTH1 in complex with the substrate 8-oxo-dGTP. The crystal structures provide insight into the high degree of structural conservation between MTH1 proteins from different organisms and provide a detailed view of interactions between MTH1 and the inhibitor, revealing that minute structural differences can have a large impact on affinity and specificity. |
Jordi Carreras-Puigvert Marinka Zitnik, Ann-Sofie Jemth Megan Carter Judith Unterlass Björn Hallström Olga Loseva Zhir Karem José Manuel Calderón-Montaño Cecilia Lindskog Per-Henrik Edqvist Damian Matuszewski Hammou Ait Blal Ronnie Berntsson Maria Häggblad Ulf Martens Matthew Studham Bo Lundgren Carolina Wählby Erik Sonnhammer Emma Lundberg Stenmark Blaz Zupan Thomas Helleday E J P A L L Pål A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family Journal Article Nature Communications, 8 , pp. 1541, 2017. Abstract | Links | BibTeX | Tags: article @article{Carreras-Puigvert2017, title = {A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family}, author = {Jordi Carreras-Puigvert, Marinka Zitnik, Ann-Sofie Jemth, Megan Carter, Judith E. Unterlass, Björn Hallström, Olga Loseva, Zhir Karem, José Manuel Calderón-Montaño, Cecilia Lindskog, Per-Henrik Edqvist, Damian J. Matuszewski, Hammou Ait Blal, Ronnie P.A. Berntsson, Maria Häggblad, Ulf Martens, Matthew Studham, Bo Lundgren, Carolina Wählby, Erik L.L. Sonnhammer, Emma Lundberg, Pål Stenmark, Blaz Zupan, Thomas Helleday}, url = {https://www.nature.com/articles/s41467-017-01642-w#article-info}, year = {2017}, date = {2017-11-16}, journal = {Nature Communications}, volume = {8}, pages = {1541}, abstract = {The NUDIX enzymes are involved in cellular metabolism and homeostasis, as well as mRNA processing. Although highly conserved throughout all organisms, their biological roles and biochemical redundancies remain largely unclear. To address this, we globally resolve their individual properties and inter-relationships. We purify 18 of the human NUDIX proteins and screen 52 substrates, providing a substrate redundancy map. Using crystal structures, we generate sequence alignment analyses revealing four major structural classes. To a certain extent, their substrate preference redundancies correlate with structural classes, thus linking structure and activity relationships. To elucidate interdependence among the NUDIX hydrolases, we pairwise deplete them generating an epistatic interaction map, evaluate cell cycle perturbations upon knockdown in normal and cancer cells, and analyse their protein and mRNA expression in normal and cancer tissues. Using a novel FUSION algorithm, we integrate all data creating a comprehensive NUDIX enzyme profile map, which will prove fundamental to understanding their biological functionality.}, keywords = {article}, pubstate = {published}, tppubtype = {article} } The NUDIX enzymes are involved in cellular metabolism and homeostasis, as well as mRNA processing. Although highly conserved throughout all organisms, their biological roles and biochemical redundancies remain largely unclear. To address this, we globally resolve their individual properties and inter-relationships. We purify 18 of the human NUDIX proteins and screen 52 substrates, providing a substrate redundancy map. Using crystal structures, we generate sequence alignment analyses revealing four major structural classes. To a certain extent, their substrate preference redundancies correlate with structural classes, thus linking structure and activity relationships. To elucidate interdependence among the NUDIX hydrolases, we pairwise deplete them generating an epistatic interaction map, evaluate cell cycle perturbations upon knockdown in normal and cancer cells, and analyse their protein and mRNA expression in normal and cancer tissues. Using a novel FUSION algorithm, we integrate all data creating a comprehensive NUDIX enzyme profile map, which will prove fundamental to understanding their biological functionality. |
Axel Rudling Robert Gustafsson, Ingrid Almlöf Evert Homan Martin Scobie Ulrika Warpman Berglund Thomas Helleday Pål Stenmark Jens Carlsson Fragment-Based Discovery and Optimization of Enzyme Inhibitors by Docking of Commercial Chemical Space Journal Article Journal of Medicinal Chemistry, 60 , pp. 8160-8169, 2017. Abstract | Links | BibTeX | Tags: article @article{Rudling2017, title = {Fragment-Based Discovery and Optimization of Enzyme Inhibitors by Docking of Commercial Chemical Space}, author = {Axel Rudling, Robert Gustafsson, Ingrid Almlöf, Evert Homan, Martin Scobie, Ulrika Warpman Berglund, Thomas Helleday, Pål Stenmark, Jens Carlsson}, url = {https://pubs.acs.org/doi/10.1021/acs.jmedchem.7b01006}, year = {2017}, date = {2017-09-20}, journal = {Journal of Medicinal Chemistry}, volume = {60}, pages = {8160-8169}, abstract = {Fragment-based lead discovery has emerged as a leading drug development strategy for novel therapeutic targets. Although fragment-based drug discovery benefits immensely from access to atomic-resolution information, structure-based virtual screening has rarely been used to drive fragment discovery and optimization. Here, molecular docking of 0.3 million fragments to a crystal structure of cancer target MTH1 was performed. Twenty-two predicted fragment ligands, for which analogs could be acquired commercially, were experimentally evaluated. Five fragments inhibited MTH1 with IC50 values ranging from 6 to 79 μM. Structure-based optimization guided by predicted binding modes and analogs from commercial chemical libraries yielded nanomolar inhibitors. Subsequently solved crystal structures confirmed binding modes predicted by docking for three scaffolds. Structure-guided exploration of commercial chemical space using molecular docking gives access to fragment libraries that are several orders of magnitude larger than those screened experimentally and can enable efficient optimization of hits to potent leads.}, keywords = {article}, pubstate = {published}, tppubtype = {article} } Fragment-based lead discovery has emerged as a leading drug development strategy for novel therapeutic targets. Although fragment-based drug discovery benefits immensely from access to atomic-resolution information, structure-based virtual screening has rarely been used to drive fragment discovery and optimization. Here, molecular docking of 0.3 million fragments to a crystal structure of cancer target MTH1 was performed. Twenty-two predicted fragment ligands, for which analogs could be acquired commercially, were experimentally evaluated. Five fragments inhibited MTH1 with IC50 values ranging from 6 to 79 μM. Structure-based optimization guided by predicted binding modes and analogs from commercial chemical libraries yielded nanomolar inhibitors. Subsequently solved crystal structures confirmed binding modes predicted by docking for three scaffolds. Structure-guided exploration of commercial chemical space using molecular docking gives access to fragment libraries that are several orders of magnitude larger than those screened experimentally and can enable efficient optimization of hits to potent leads. |
Linda Pudelko Pegah Rouhi, Kumar Sanjiv Helge Gad Christina Kalderén Andreas Höglund Massimo Squatrito Alberto Schuhmacher Steven Edwards Daniel Hägerstrand Ulrika Warpman Berglund Thomas Helleday Lars Bräutigam J Glioblastoma and glioblastoma stem cells are dependent on functional MTH1 Journal Article Oncotarget, 8 , pp. 84671-84684, 2017. Abstract | Links | BibTeX | Tags: article @article{Pudelko2017, title = {Glioblastoma and glioblastoma stem cells are dependent on functional MTH1}, author = {Linda Pudelko, Pegah Rouhi, Kumar Sanjiv, Helge Gad, Christina Kalderén, Andreas Höglund, Massimo Squatrito, Alberto J. Schuhmacher, Steven Edwards, Daniel Hägerstrand, Ulrika Warpman Berglund, Thomas Helleday, Lars Bräutigam}, url = {http://www.oncotarget.com/index.php?journal=oncotarget&page=article&op=view&path[]=19404&path[]=62063}, year = {2017}, date = {2017-07-20}, journal = {Oncotarget}, volume = {8}, pages = {84671-84684}, abstract = {Glioblastoma multiforme (GBM) is an aggressive form of brain cancer with poor prognosis. Cancer cells are characterized by a specific redox environment that adjusts metabolism to its specific needs and allows the tumor to grow and metastasize. As a consequence, cancer cells and especially GBM cells suffer from elevated oxidative pressure which requires antioxidant-defense and other sanitation enzymes to be upregulated. MTH1, which degrades oxidized nucleotides, is one of these defense enzymes and represents a promising cancer target. We found MTH1 expression levels elevated and correlated with GBM aggressiveness and discovered that siRNA knock-down or inhibition of MTH1 with small molecules efficiently reduced viability of patient-derived GBM cultures. The effect of MTH1 loss on GBM viability was likely mediated through incorporation of oxidized nucleotides and subsequent DNA damage. We revealed that MTH1 inhibition targets GBM independent of aggressiveness as well as potently kills putative GBM stem cells in vitro. We used an orthotopic zebrafish model to confirm our results in vivo and light-sheet microscopy to follow the effect of MTH1 inhibition in GBM in real time. In conclusion, MTH1 represents a promising target for GBM therapy and MTH1 inhibitors may also be effective in patients that suffer from recurring disease.}, keywords = {article}, pubstate = {published}, tppubtype = {article} } Glioblastoma multiforme (GBM) is an aggressive form of brain cancer with poor prognosis. Cancer cells are characterized by a specific redox environment that adjusts metabolism to its specific needs and allows the tumor to grow and metastasize. As a consequence, cancer cells and especially GBM cells suffer from elevated oxidative pressure which requires antioxidant-defense and other sanitation enzymes to be upregulated. MTH1, which degrades oxidized nucleotides, is one of these defense enzymes and represents a promising cancer target. We found MTH1 expression levels elevated and correlated with GBM aggressiveness and discovered that siRNA knock-down or inhibition of MTH1 with small molecules efficiently reduced viability of patient-derived GBM cultures. The effect of MTH1 loss on GBM viability was likely mediated through incorporation of oxidized nucleotides and subsequent DNA damage. We revealed that MTH1 inhibition targets GBM independent of aggressiveness as well as potently kills putative GBM stem cells in vitro. We used an orthotopic zebrafish model to confirm our results in vivo and light-sheet microscopy to follow the effect of MTH1 inhibition in GBM in real time. In conclusion, MTH1 represents a promising target for GBM therapy and MTH1 inhibitors may also be effective in patients that suffer from recurring disease. |
Sabin Llona-Minguez Artin Ghassemian, Pawel Baranczewski Matthieu Desroses Tobias Koolmeister Per Artursson Martin Scobie Thomas Helleday Structure–metabolism-relationships in the microsomal clearance of piperazin-1-ylpyridazines Journal Article Med. Chem. Comm., 8 , pp. 1553-1560, 2017. Abstract | Links | BibTeX | Tags: article @article{Llona-Minguez2017, title = {Structure–metabolism-relationships in the microsomal clearance of piperazin-1-ylpyridazines }, author = {Sabin Llona-Minguez, Artin Ghassemian, Pawel Baranczewski, Matthieu Desroses, Tobias Koolmeister, Per Artursson, Martin Scobie, Thomas Helleday}, url = {https://pubs.rsc.org/en/content/articlelanding/2017/md/c7md00230k#!divAbstract}, year = {2017}, date = {2017-07-04}, journal = {Med. Chem. Comm.}, volume = {8}, pages = {1553-1560}, abstract = {In this study, we provide insight into the metabolic profile of a series of piperazin-1-ylpyridazines suffering from rapid in vitro intrinsic clearance in a metabolic stability assay using liver microsomes (e.g. compound 1 MLM/HLM t1/2 = 2/3 min). Aided by empirical metabolite identification and computational predictive models, we designed the structural modifications required to improve in vitro intrinsic clearance by more than 50-fold (e.g. compound 29 MLM/HLM t1/2 = 113/105 min).}, keywords = {article}, pubstate = {published}, tppubtype = {article} } In this study, we provide insight into the metabolic profile of a series of piperazin-1-ylpyridazines suffering from rapid in vitro intrinsic clearance in a metabolic stability assay using liver microsomes (e.g. compound 1 MLM/HLM t1/2 = 2/3 min). Aided by empirical metabolite identification and computational predictive models, we designed the structural modifications required to improve in vitro intrinsic clearance by more than 50-fold (e.g. compound 29 MLM/HLM t1/2 = 113/105 min). |
Iryna Kolosenko Yasmin Yu, Sander Busker Matheus Dyczynski Jianping Liu Martin Haraldsson Caroline Palm Apergi Thomas Helleday Katja Pokrovskaja Tamm Brent Page Dan Grander D G Identification of novel small molecules that inhibit STAT3-dependent transcription and function Journal Article Plos One, 2017. Abstract | Links | BibTeX | Tags: article @article{Kolosenko2017, title = {Identification of novel small molecules that inhibit STAT3-dependent transcription and function}, author = {Iryna Kolosenko, Yasmin Yu, Sander Busker, Matheus Dyczynski, Jianping Liu, Martin Haraldsson, Caroline Palm Apergi, Thomas Helleday, Katja Pokrovskaja Tamm, Brent D. G. Page, Dan Grander }, url = {http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0178844}, year = {2017}, date = {2017-06-21}, journal = {Plos One}, abstract = {Activation of Signal Transducer and Activator of Transcription 3 (STAT3) has been linked to several processes that are critical for oncogenic transformation, cancer progression, cancer cell proliferation, survival, drug resistance and metastasis. Inhibition of STAT3 signaling has shown a striking ability to inhibit cancer cell growth and therefore, STAT3 has become a promising target for anti-cancer drug development. The aim of this study was to identify novel inhibitors of STAT-dependent gene transcription. A cellular reporter-based system for monitoring STAT3 transcriptional activity was developed which was suitable for high-throughput screening (Z’ = 0,8). This system was used to screen a library of 28,000 compounds (the ENAMINE Drug-Like Diversity Set). Following counter-screenings and toxicity studies, we identified four hit compounds that were subjected to detailed biological characterization. Of the four hits, KI16 stood out as the most promising compound, inhibiting STAT3 phosphorylation and transcriptional activity in response to IL6 stimulation. In silico docking studies showed that KI16 had favorable interactions with the STAT3 SH2 domain, however, no inhibitory activity could be observed in the STAT3 fluorescence polarization assay. KI16 inhibited cell viability preferentially in STAT3-dependent cell lines. Taken together, using a targeted, cell-based approach, novel inhibitors of STAT-driven transcriptional activity were discovered which are interesting leads to pursue further for the development of anti-cancer therapeutic agents.}, keywords = {article}, pubstate = {published}, tppubtype = {article} } Activation of Signal Transducer and Activator of Transcription 3 (STAT3) has been linked to several processes that are critical for oncogenic transformation, cancer progression, cancer cell proliferation, survival, drug resistance and metastasis. Inhibition of STAT3 signaling has shown a striking ability to inhibit cancer cell growth and therefore, STAT3 has become a promising target for anti-cancer drug development. The aim of this study was to identify novel inhibitors of STAT-dependent gene transcription. A cellular reporter-based system for monitoring STAT3 transcriptional activity was developed which was suitable for high-throughput screening (Z’ = 0,8). This system was used to screen a library of 28,000 compounds (the ENAMINE Drug-Like Diversity Set). Following counter-screenings and toxicity studies, we identified four hit compounds that were subjected to detailed biological characterization. Of the four hits, KI16 stood out as the most promising compound, inhibiting STAT3 phosphorylation and transcriptional activity in response to IL6 stimulation. In silico docking studies showed that KI16 had favorable interactions with the STAT3 SH2 domain, however, no inhibitory activity could be observed in the STAT3 fluorescence polarization assay. KI16 inhibited cell viability preferentially in STAT3-dependent cell lines. Taken together, using a targeted, cell-based approach, novel inhibitors of STAT-driven transcriptional activity were discovered which are interesting leads to pursue further for the development of anti-cancer therapeutic agents. |
Sabin Llona-Minguez Andreas Höglund, Artin Ghassemian Matthieu Desroses Jose Manuel Calderon-Montano Estefania Burgos Moron Nicholas Valerie Elisee Wiita Ingrid Almlöf Tobias Koolmeister Andre Mateus Cindy Cazares-Körner Kumar Sanjiv Evert Homan Olga Loseva Pawel Baranczewski Masoud Darabi Amir Mehdizadeh Shabnam Fayezi Ann-Sofie Jemth Ulrika Warpman Berglund Kristmundur Sigmundsson Thomas Lundbäck Annika Jenmalm Jensen Per Artursson Martin Scobie Thomas Helleday C K Piperazin-1-ylpyridazine Derivatives Are a Novel Class of Human dCTP Pyrophosphatase 1 Inhibitors Journal Article Journal of Medicinal Chemistry, 60 , pp. 4279-4292, 2017. Abstract | Links | BibTeX | Tags: article @article{Llona-Minguez2017, title = {Piperazin-1-ylpyridazine Derivatives Are a Novel Class of Human dCTP Pyrophosphatase 1 Inhibitors}, author = {Sabin Llona-Minguez, Andreas Höglund, Artin Ghassemian, Matthieu Desroses, Jose Manuel Calderon-Montano, Estefania Burgos Moron, Nicholas C. K. Valerie, Elisee Wiita, Ingrid Almlöf, Tobias Koolmeister, Andre Mateus, Cindy Cazares-Körner, Kumar Sanjiv, Evert Homan, Olga Loseva, Pawel Baranczewski, Masoud Darabi, Amir Mehdizadeh, Shabnam Fayezi, Ann-Sofie Jemth, Ulrika Warpman Berglund, Kristmundur Sigmundsson, Thomas Lundbäck, Annika Jenmalm Jensen, Per Artursson, Martin Scobie, Thomas Helleday}, url = {https://pubs.acs.org/doi/10.1021/acs.jmedchem.7b00182}, year = {2017}, date = {2017-05-16}, journal = {Journal of Medicinal Chemistry}, volume = {60}, pages = {4279-4292}, abstract = {The dCTP pyrophosphatase 1 (dCTPase) is a nucleotide pool “housekeeping” enzyme responsible for the catabolism of canonical and noncanonical nucleoside triphosphates (dNTPs) and has been associated with cancer progression and cancer cell stemness. We have identified a series of piperazin-1-ylpyridazines as a new class of potent dCTPase inhibitors. Lead compounds increase dCTPase thermal and protease stability, display outstanding selectivity over related enzymes and synergize with a cytidine analogue against leukemic cells. This new class of dCTPase inhibitors lays the first stone toward the development of drug-like probes for the dCTPase enzyme.}, keywords = {article}, pubstate = {published}, tppubtype = {article} } The dCTP pyrophosphatase 1 (dCTPase) is a nucleotide pool “housekeeping” enzyme responsible for the catabolism of canonical and noncanonical nucleoside triphosphates (dNTPs) and has been associated with cancer progression and cancer cell stemness. We have identified a series of piperazin-1-ylpyridazines as a new class of potent dCTPase inhibitors. Lead compounds increase dCTPase thermal and protease stability, display outstanding selectivity over related enzymes and synergize with a cytidine analogue against leukemic cells. This new class of dCTPase inhibitors lays the first stone toward the development of drug-like probes for the dCTPase enzyme. |
Nikolas Herold Sean G. Rudd, Kumar Sanjiv Juliane Kutzner Julia Bladh Cynthia Paulin Thomas Helleday Jan-Inge Henter Torsten Schaller B J SAMHD1 protects cancer cells from various nucleoside-based antimetabolites Journal Article Cell Cycle, 16 (11), pp. 1029-1038, 2017. Abstract | Links | BibTeX | Tags: article @article{Herold2017, title = {SAMHD1 protects cancer cells from various nucleoside-based antimetabolites}, author = {Nikolas Herold, Sean G. Rudd, Kumar Sanjiv, Juliane Kutzner, Julia Bladh, Cynthia B. J. Paulin, Thomas Helleday, Jan-Inge Henter, Torsten Schaller}, url = {https://www.tandfonline.com/doi/full/10.1080/15384101.2017.1314407}, year = {2017}, date = {2017-05-08}, journal = {Cell Cycle}, volume = {16}, number = {11}, pages = {1029-1038}, abstract = {Recently, we demonstrated that sterile α motif and HD domain containing protein 1 (SAMHD1) is a major barrier in acute myelogenous leukemia (AML) cells to the cytotoxicity of cytarabine (ara-C), the most important drug in AML treatment. Ara-C is intracellularly converted by the canonical dNTP synthesis pathway to ara-CTP, which serves as a substrate but not an allosteric activator of SAMHD1. Using an AML mouse model, we show here that wild type but not catalytically inactive SAMHD1 reduces ara-C treatment efficacy in vivo. Expanding the clinically relevant substrates of SAMHD1, we demonstrate that THP-1 CRISPR/Cas9 cells lacking a functional SAMHD1 gene showed increased sensitivity to the antimetabolites nelarabine, fludarabine, decitabine, vidarabine, clofarabine, and trifluridine. Within this Extra View, we discuss and build upon both these and our previously reported findings, and propose SAMHD1 is likely active against a variety of nucleoside analog antimetabolites present in anti-cancer chemotherapies. Thus, SAMHD1 may constitute a promising target to improve a wide range of therapies for both hematological and non-haematological malignancies.}, keywords = {article}, pubstate = {published}, tppubtype = {article} } Recently, we demonstrated that sterile α motif and HD domain containing protein 1 (SAMHD1) is a major barrier in acute myelogenous leukemia (AML) cells to the cytotoxicity of cytarabine (ara-C), the most important drug in AML treatment. Ara-C is intracellularly converted by the canonical dNTP synthesis pathway to ara-CTP, which serves as a substrate but not an allosteric activator of SAMHD1. Using an AML mouse model, we show here that wild type but not catalytically inactive SAMHD1 reduces ara-C treatment efficacy in vivo. Expanding the clinically relevant substrates of SAMHD1, we demonstrate that THP-1 CRISPR/Cas9 cells lacking a functional SAMHD1 gene showed increased sensitivity to the antimetabolites nelarabine, fludarabine, decitabine, vidarabine, clofarabine, and trifluridine. Within this Extra View, we discuss and build upon both these and our previously reported findings, and propose SAMHD1 is likely active against a variety of nucleoside analog antimetabolites present in anti-cancer chemotherapies. Thus, SAMHD1 may constitute a promising target to improve a wide range of therapies for both hematological and non-haematological malignancies. |
Nikolas Herold Sean G. Rudd, Kumar Sanjiv Juliane Kutzner Ida Hed Myrberga Cynthia Paulin Thale Kristin Olsen Thomas Helleday Jan-Inge Henter TorstenSchaller B J With me or against me: Tumor suppressor and drug resistance activities of SAMHD1 Journal Article Experimental Hematology, 52 , pp. 32-39, 2017. Abstract | Links | BibTeX | Tags: article @article{Herold2017, title = {With me or against me: Tumor suppressor and drug resistance activities of SAMHD1}, author = {Nikolas Herold, Sean G. Rudd, Kumar Sanjiv, Juliane Kutzner, Ida Hed Myrberga, Cynthia B. J. Paulin, Thale Kristin Olsen, Thomas Helleday, Jan-Inge Henter, TorstenSchaller}, url = {https://www.sciencedirect.com/science/article/pii/S0301472X17301443?via%3Dihub}, year = {2017}, date = {2017-05-02}, journal = {Experimental Hematology}, volume = {52}, pages = {32-39}, abstract = {Sterile alpha motif and histidine/aspartic acid domain-containing protein 1 (SAMHD1) is a (deoxy)guanosine triphosphate (dGTP/GTP)-activated deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase involved in cellular dNTP homoeostasis. Mutations in SAMHD1 have been associated with the hyperinflammatory disease Aicardi–Goutières syndrome (AGS). SAMHD1 also limits cells' permissiveness to infection with diverse viruses, including human immunodeficiency virus (HIV-1), and controls endogenous retroviruses. Increasing evidence supports the role of SAMHD1 as a tumor suppressor. However, SAMHD1 also can act as a resistance factor to nucleoside-based chemotherapies by hydrolyzing their active triphosphate metabolites, thereby reducing response of various malignancies to these anticancer drugs. Hence, informed cancer therapies must take into account the ambiguous properties of SAMHD1 as both an inhibitor of uncontrolled proliferation and a resistance factor limiting the efficacy of anticancer treatments. Here, we provide evidence that SAMHD1 is a double-edged sword for patients with acute myelogenous leukemia (AML). Our time-dependent analyses of The Cancer Genome Atlas (TCGA) AML cohort indicate that high expression of SAMHD1, even though it critically limits the efficacy of high-dose ara-C therapy, might be associated with more favorable disease progression.}, keywords = {article}, pubstate = {published}, tppubtype = {article} } Sterile alpha motif and histidine/aspartic acid domain-containing protein 1 (SAMHD1) is a (deoxy)guanosine triphosphate (dGTP/GTP)-activated deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase involved in cellular dNTP homoeostasis. Mutations in SAMHD1 have been associated with the hyperinflammatory disease Aicardi–Goutières syndrome (AGS). SAMHD1 also limits cells' permissiveness to infection with diverse viruses, including human immunodeficiency virus (HIV-1), and controls endogenous retroviruses. Increasing evidence supports the role of SAMHD1 as a tumor suppressor. However, SAMHD1 also can act as a resistance factor to nucleoside-based chemotherapies by hydrolyzing their active triphosphate metabolites, thereby reducing response of various malignancies to these anticancer drugs. Hence, informed cancer therapies must take into account the ambiguous properties of SAMHD1 as both an inhibitor of uncontrolled proliferation and a resistance factor limiting the efficacy of anticancer treatments. Here, we provide evidence that SAMHD1 is a double-edged sword for patients with acute myelogenous leukemia (AML). Our time-dependent analyses of The Cancer Genome Atlas (TCGA) AML cohort indicate that high expression of SAMHD1, even though it critically limits the efficacy of high-dose ara-C therapy, might be associated with more favorable disease progression. |
de la Gregory I Vladimer Berend Snijder, Nikolaus Krall Johannes Bigenzahn Kilian Huber Charles-Hugues Lardeau Kumar Sanjiv Anna Ringler Ulrika Warpman Berglund Monika Sabler Oscar Lopez Fuente Paul Knöbl Stefan Kubicek Thomas Helleday Ulrich Jäger Giulio Superti-Furga W V M Global survey of the immunomodulatory potential of common drugs Journal Article Nature Chemical Biology, 13 , pp. 681-690, 2017. Abstract | Links | BibTeX | Tags: article @article{Vladimer2017, title = {Global survey of the immunomodulatory potential of common drugs}, author = {Gregory I Vladimer, Berend Snijder, Nikolaus Krall, Johannes W Bigenzahn, Kilian V M Huber, Charles-Hugues Lardeau, Kumar Sanjiv, Anna Ringler, Ulrika Warpman Berglund, Monika Sabler, Oscar Lopez de la Fuente, Paul Knöbl, Stefan Kubicek, Thomas Helleday, Ulrich Jäger, Giulio Superti-Furga }, url = {https://www.nature.com/articles/nchembio.2360#article-info}, year = {2017}, date = {2017-04-24}, journal = {Nature Chemical Biology}, volume = {13}, pages = {681-690}, abstract = {Small-molecule drugs may complement antibody-based therapies in an immune-oncology setting, yet systematic methods for the identification and characterization of the immunomodulatory properties of these entities are lacking. We surveyed the immumomodulatory potential of 1,402 small chemical molecules, as defined by their ability to alter the cell-cell interactions among peripheral mononuclear leukocytes ex vivo, using automated microscopy and population-wide single-cell image analysis. Unexpectedly, ∼10% of the agents tested affected these cell-cell interactions differentially. The results accurately recapitulated known immunomodulatory drug classes and revealed several clinically approved drugs that unexpectedly harbor the ability to modulate the immune system, which could potentially contribute to their physiological mechanism of action. For instance, the kinase inhibitor crizotinib promoted T cell interactions with monocytes, as well as with cancer cells, through inhibition of the receptor tyrosine kinase MSTR1 and subsequent upregulation of the expression of major histocompatibility complex molecules. The approach offers an attractive platform for the personalized identification and characterization of immunomodulatory therapeutics.}, keywords = {article}, pubstate = {published}, tppubtype = {article} } Small-molecule drugs may complement antibody-based therapies in an immune-oncology setting, yet systematic methods for the identification and characterization of the immunomodulatory properties of these entities are lacking. We surveyed the immumomodulatory potential of 1,402 small chemical molecules, as defined by their ability to alter the cell-cell interactions among peripheral mononuclear leukocytes ex vivo, using automated microscopy and population-wide single-cell image analysis. Unexpectedly, ∼10% of the agents tested affected these cell-cell interactions differentially. The results accurately recapitulated known immunomodulatory drug classes and revealed several clinically approved drugs that unexpectedly harbor the ability to modulate the immune system, which could potentially contribute to their physiological mechanism of action. For instance, the kinase inhibitor crizotinib promoted T cell interactions with monocytes, as well as with cancer cells, through inhibition of the receptor tyrosine kinase MSTR1 and subsequent upregulation of the expression of major histocompatibility complex molecules. The approach offers an attractive platform for the personalized identification and characterization of immunomodulatory therapeutics. |
Anna Hagenkort Cynthia B.J. Paulin, Matthieu Desroses Antonio Sarno Elisée Wiita Oliver Mortusewicz Tobias Koolmeister Olga Loseva Ann-Sofie Jemth Ingrid Almlöf Evert Homan Thomas Lundbäck Anna-Lena Gustavsson Martin Scobie Thomas Helleday dUTPase inhibition augments replication defects of 5-Fluorouracil Journal Article Oncotarget, 8 , pp. 23713-23726, 2017. Abstract | Links | BibTeX | Tags: article @article{Hagenkort2017, title = {dUTPase inhibition augments replication defects of 5-Fluorouracil}, author = {Anna Hagenkort, Cynthia B.J. Paulin, Matthieu Desroses, Antonio Sarno, Elisée Wiita, Oliver Mortusewicz, Tobias Koolmeister, Olga Loseva, Ann-Sofie Jemth, Ingrid Almlöf, Evert Homan, Thomas Lundbäck, Anna-Lena Gustavsson, Martin Scobie, Thomas Helleday}, url = {http://www.oncotarget.com/index.php?journal=oncotarget&page=article&op=view&path[]=15785&path[]=54495}, year = {2017}, date = {2017-02-28}, journal = {Oncotarget}, volume = {8}, pages = {23713-23726}, abstract = {The antimetabolite 5-Fluorouracil (5-FU) is used in the treatment of various forms of cancer and has a complex mode of action. Despite 6 decades in clinical application the contribution of 5-FdUTP and dUTP [(5-F)dUTP] and 5-FUTP misincorporation into DNA and RNA respectively, for 5-FU-induced toxicity is still under debate. This study investigates DNA replication defects induced by 5-FU treatment and how (5-F)dUTP accumulation contributes to this effect. We reveal that 5-FU treatment leads to extensive problems in DNA replication fork progression, causing accumulation of cells in S-phase, DNA damage and ultimately cell death. Interestingly, these effects can be reinforced by either depletion or inhibition of the deoxyuridine triphosphatase (dUTPase, also known as DUT), highlighting the importance of (5-F)dUTP accumulation for cytotoxicity. With this study, we not only extend the current understanding of the mechanism of action of 5-FU, but also contribute to the characterization of dUTPase inhibitors. We demonstrate that pharmacological inhibition of dUTPase is a promising approach that may improve the efficacy of 5-FU treatment in the clinic.}, keywords = {article}, pubstate = {published}, tppubtype = {article} } The antimetabolite 5-Fluorouracil (5-FU) is used in the treatment of various forms of cancer and has a complex mode of action. Despite 6 decades in clinical application the contribution of 5-FdUTP and dUTP [(5-F)dUTP] and 5-FUTP misincorporation into DNA and RNA respectively, for 5-FU-induced toxicity is still under debate. This study investigates DNA replication defects induced by 5-FU treatment and how (5-F)dUTP accumulation contributes to this effect. We reveal that 5-FU treatment leads to extensive problems in DNA replication fork progression, causing accumulation of cells in S-phase, DNA damage and ultimately cell death. Interestingly, these effects can be reinforced by either depletion or inhibition of the deoxyuridine triphosphatase (dUTPase, also known as DUT), highlighting the importance of (5-F)dUTP accumulation for cytotoxicity. With this study, we not only extend the current understanding of the mechanism of action of 5-FU, but also contribute to the characterization of dUTPase inhibitors. We demonstrate that pharmacological inhibition of dUTPase is a promising approach that may improve the efficacy of 5-FU treatment in the clinic. |
Nikolas Herold Sean G Rudd, Linda Ljungblad Kumar Sanjiv Ida Hed Myrberg Cynthia Paulin Yaser Heshmati Anna Hagenkort Juliane Kutzner Brent Page José Calderón-Montaño Olga Loseva Ann-Sofie Jemth Lorenzo Bulli Hanna Axelsson Bianca Tesi Nicholas Valerie Andreas Höglund Julia Bladh Elisée Wiita Mikael Sundin Michael Uhlin Georgios Rassidakis Mats Heyman Katja Pokrovskaja Tamm Ulrika Warpman-Berglund Julian Walfridsson Sören Lehmann Dan Grandér Thomas Lundbäck Per Kogner Jan-Inge Henter Thomas Helleday & Torsten Schaller B J D G M C K Targeting SAMHD1 with the Vpx protein to improve cytarabine therapy for hematological malignancies. Journal Article Nat Med, (23), pp. 256-263, 2017. Abstract | Links | BibTeX | Tags: article @article{Herold2017, title = {Targeting SAMHD1 with the Vpx protein to improve cytarabine therapy for hematological malignancies.}, author = {Nikolas Herold, Sean G Rudd, Linda Ljungblad, Kumar Sanjiv, Ida Hed Myrberg, Cynthia B J Paulin, Yaser Heshmati, Anna Hagenkort, Juliane Kutzner, Brent D G Page, José M Calderón-Montaño, Olga Loseva, Ann-Sofie Jemth, Lorenzo Bulli, Hanna Axelsson, Bianca Tesi, Nicholas C K Valerie, Andreas Höglund, Julia Bladh, Elisée Wiita, Mikael Sundin, Michael Uhlin, Georgios Rassidakis, Mats Heyman, Katja Pokrovskaja Tamm, Ulrika Warpman-Berglund, Julian Walfridsson, Sören Lehmann, Dan Grandér, Thomas Lundbäck, Per Kogner, Jan-Inge Henter, Thomas Helleday & Torsten Schaller}, url = {http://www.nature.com/nm/journal/v23/n2/full/nm.4265.html}, doi = {doi:10.1038/nm.4265}, year = {2017}, date = {2017-02-23}, journal = {Nat Med}, number = {23}, pages = {256-263}, abstract = {Abstract The cytostatic deoxycytidine analog cytarabine (ara-C) is the most active agent available against acute myelogenous leukemia (AML). Together with anthracyclines, ara-C forms the backbone of AML treatment for children and adults. In AML, both the cytotoxicity of ara-C in vitro and the clinical response to ara-C therapy are correlated with the ability of AML blasts to accumulate the active metabolite ara-C triphosphate (ara-CTP), which causes DNA damage through perturbation of DNA synthesis. Differences in expression levels of known transporters or metabolic enzymes relevant to ara-C only partially account for patient-specific differential ara-CTP accumulation in AML blasts and response to ara-C treatment. Here we demonstrate that the deoxynucleoside triphosphate (dNTP) triphosphohydrolase SAM domain and HD domain 1 (SAMHD1) promotes the detoxification of intracellular ara-CTP pools. Recombinant SAMHD1 exhibited ara-CTPase activity in vitro, and cells in which SAMHD1 expression was transiently reduced by treatment with the simian immunodeficiency virus (SIV) protein Vpx were dramatically more sensitive to ara-C-induced cytotoxicity. CRISPR-Cas9-mediated disruption of the gene encoding SAMHD1 sensitized cells to ara-C, and this sensitivity could be abrogated by ectopic expression of wild-type (WT), but not dNTPase-deficient, SAMHD1. Mouse models of AML lacking SAMHD1 were hypersensitive to ara-C, and treatment ex vivo with Vpx sensitized primary patient-derived AML blasts to ara-C. Finally, we identified SAMHD1 as a risk factor in cohorts of both pediatric and adult patients with de novo AML who received ara-C treatment. Thus, SAMHD1 expression levels dictate patient sensitivity to ara-C, providing proof-of-concept that the targeting of SAMHD1 by Vpx could be an attractive therapeutic strategy for potentiating ara-C efficacy in hematological malignancies.}, keywords = {article}, pubstate = {published}, tppubtype = {article} } Abstract The cytostatic deoxycytidine analog cytarabine (ara-C) is the most active agent available against acute myelogenous leukemia (AML). Together with anthracyclines, ara-C forms the backbone of AML treatment for children and adults. In AML, both the cytotoxicity of ara-C in vitro and the clinical response to ara-C therapy are correlated with the ability of AML blasts to accumulate the active metabolite ara-C triphosphate (ara-CTP), which causes DNA damage through perturbation of DNA synthesis. Differences in expression levels of known transporters or metabolic enzymes relevant to ara-C only partially account for patient-specific differential ara-CTP accumulation in AML blasts and response to ara-C treatment. Here we demonstrate that the deoxynucleoside triphosphate (dNTP) triphosphohydrolase SAM domain and HD domain 1 (SAMHD1) promotes the detoxification of intracellular ara-CTP pools. Recombinant SAMHD1 exhibited ara-CTPase activity in vitro, and cells in which SAMHD1 expression was transiently reduced by treatment with the simian immunodeficiency virus (SIV) protein Vpx were dramatically more sensitive to ara-C-induced cytotoxicity. CRISPR-Cas9-mediated disruption of the gene encoding SAMHD1 sensitized cells to ara-C, and this sensitivity could be abrogated by ectopic expression of wild-type (WT), but not dNTPase-deficient, SAMHD1. Mouse models of AML lacking SAMHD1 were hypersensitive to ara-C, and treatment ex vivo with Vpx sensitized primary patient-derived AML blasts to ara-C. Finally, we identified SAMHD1 as a risk factor in cohorts of both pediatric and adult patients with de novo AML who received ara-C treatment. Thus, SAMHD1 expression levels dictate patient sensitivity to ara-C, providing proof-of-concept that the targeting of SAMHD1 by Vpx could be an attractive therapeutic strategy for potentiating ara-C efficacy in hematological malignancies. |
Sanjiv K Hagenkort A, Calderón-Montaño JM Koolmeister Reaper PM Mortusewicz Jacques SA Kuiper RV Schultz Scobie Charlton PA Pollard JR Berglund UW Altun Helleday T O N M M T Cancer-Specific Synthetic Lethality between ATR and CHK1 Kinase Activities. Journal Article Cell Rep, 17 (12), pp. 3407-3416, 2017. Links | BibTeX | Tags: article @article{K2017, title = {Cancer-Specific Synthetic Lethality between ATR and CHK1 Kinase Activities.}, author = {Sanjiv K, Hagenkort A, Calderón-Montaño JM, Koolmeister T, Reaper PM, Mortusewicz O, Jacques SA, Kuiper RV, Schultz N, Scobie M, Charlton PA, Pollard JR, Berglund UW, Altun M, Helleday T.}, url = {https://www.ncbi.nlm.nih.gov/pubmed/28009306}, doi = {DOI: 10.1016/j.celrep.2016.12.031}, year = {2017}, date = {2017-02-20}, journal = {Cell Rep}, volume = {17}, number = {12}, pages = {3407-3416}, keywords = {article}, pubstate = {published}, tppubtype = {article} } |
Sokolova M Turunen M, Mortusewicz Kivioja Herr Vähärautio Björklund Taipale Helleday Taipale O T P A M M T J Genome-wide screen of cell-cycle regulators in normal and tumor cells identifies a differential response to nucleosome depletion. Journal Article Cell Cykle, 16 (2), pp. 189-199, 2017. Links | BibTeX | Tags: article @article{M2017, title = {Genome-wide screen of cell-cycle regulators in normal and tumor cells identifies a differential response to nucleosome depletion.}, author = {Sokolova M, Turunen M, Mortusewicz O, Kivioja T, Herr P, Vähärautio A, Björklund M, Taipale M, Helleday T, Taipale J.}, url = {https://www.ncbi.nlm.nih.gov/pubmed/27929715}, doi = {doi: 10.1080/15384101.2016.1261765.}, year = {2017}, date = {2017-02-17}, journal = {Cell Cykle}, volume = {16}, number = {2}, pages = {189-199}, keywords = {article}, pubstate = {published}, tppubtype = {article} } |
Llona-Minguez S Höglund A, Wiita Almlöf Mateus Calderón-Montaño JM Cazares-Körner Homan Loseva Baranczewski Jemth AS Häggblad Martens Lundgren Artursson Lundbäck Jenmalm Jensen Warpman Berglund Scobie Helleday E I A C E O P M U B P T A U M T Identification of Triazolothiadiazoles as Potent Inhibitors of the dCTP Pyrophosphatase 1. Journal Article J Med Chem, 2017. Links | BibTeX | Tags: article @article{S2017, title = {Identification of Triazolothiadiazoles as Potent Inhibitors of the dCTP Pyrophosphatase 1.}, author = {Llona-Minguez S, Höglund A, Wiita E, Almlöf I, Mateus A, Calderón-Montaño JM, Cazares-Körner C, Homan E, Loseva O, Baranczewski P, Jemth AS, Häggblad M, Martens U, Lundgren B, Artursson P, Lundbäck T, Jenmalm Jensen A, Warpman Berglund U, Scobie M, Helleday T}, url = {https://www.ncbi.nlm.nih.gov/pubmed/28145708}, doi = {oi: 10.1021/acs.jmedchem.6b0178}, year = {2017}, date = {2017-02-15}, journal = {J Med Chem}, keywords = {article}, pubstate = {published}, tppubtype = {article} } |
Gustafsson R Jemth AS, Gustafsson NM Färnegårdh Loseva Wiita Bonagas Dahllund Llona-Minguez Häggblad Henriksson Andersson Homan Helleday Stenmark K O E N L S M M Y E T P Crystal Structure of the Emerging Cancer Target MTHFD2 in Complex with a Substrate-Based Inhibitor. Journal Article Cancer res, 77 (4), pp. 937-948, 2017. Abstract | Links | BibTeX | Tags: article @article{R2017, title = {Crystal Structure of the Emerging Cancer Target MTHFD2 in Complex with a Substrate-Based Inhibitor.}, author = {Gustafsson R, Jemth AS, Gustafsson NM, Färnegårdh K, Loseva O, Wiita E, Bonagas N, Dahllund L, Llona-Minguez S, Häggblad M, Henriksson M, Andersson Y, Homan E, Helleday T, Stenmark P.}, url = {https://www.ncbi.nlm.nih.gov/pubmed/27899380}, doi = {DOI: 10.1158/0008-5472.CAN-16-1476}, year = {2017}, date = {2017-02-15}, journal = {Cancer res}, volume = {77}, number = {4}, pages = {937-948}, abstract = {Abstract To sustain their proliferation, cancer cells become dependent on one-carbon metabolism to support purine and thymidylate synthesis. Indeed, one of the most highly upregulated enzymes during neoplastic transformation is MTHFD2, a mitochondrial methylenetetrahydrofolate dehydrogenase and cyclohydrolase involved in one-carbon metabolism. Because MTHFD2 is expressed normally only during embryonic development, it offers a disease-selective therapeutic target for eradicating cancer cells while sparing healthy cells. Here we report the synthesis and preclinical characterization of the first inhibitor of human MTHFD2. We also disclose the first crystal structure of MTHFD2 in complex with a substrate-based inhibitor and the enzyme cofactors NAD+ and inorganic phosphate. Our work provides a rationale for continued development of a structural framework for the generation of potent and selective MTHFD2 inhibitors for cancer treatment. }, keywords = {article}, pubstate = {published}, tppubtype = {article} } Abstract To sustain their proliferation, cancer cells become dependent on one-carbon metabolism to support purine and thymidylate synthesis. Indeed, one of the most highly upregulated enzymes during neoplastic transformation is MTHFD2, a mitochondrial methylenetetrahydrofolate dehydrogenase and cyclohydrolase involved in one-carbon metabolism. Because MTHFD2 is expressed normally only during embryonic development, it offers a disease-selective therapeutic target for eradicating cancer cells while sparing healthy cells. Here we report the synthesis and preclinical characterization of the first inhibitor of human MTHFD2. We also disclose the first crystal structure of MTHFD2 in complex with a substrate-based inhibitor and the enzyme cofactors NAD+ and inorganic phosphate. Our work provides a rationale for continued development of a structural framework for the generation of potent and selective MTHFD2 inhibitors for cancer treatment. |
Thålin C Daleskog M, Göransson SP Schatzberg Lasselin Laska AC Kallner Helleday Wallén Demers D J A T H M Immunol Res. , 2017. Abstract | Links | BibTeX | Tags: article @article{C12017, title = {Validation of an enzyme-linked immunosorbent assay for the quantification of citrullinated histone H3 as a marker for neutrophil extracellular traps in human plasma.}, author = {Thålin C, Daleskog M, Göransson SP, Schatzberg D, Lasselin J, Laska AC, Kallner A, Helleday T, Wallén H, Demers M.}, url = {https://www.ncbi.nlm.nih.gov/pubmed/28161762}, doi = {DOI: 10.1007/s12026-017-8905-3}, year = {2017}, date = {2017-02-04}, journal = {Immunol Res. }, abstract = {Abstract There is an emerging interest in the diverse functions of neutrophil extracellular traps (NETs) in a variety of disease settings. However, data on circulating NETs rely largely upon surrogate NET markers such as cell-free DNA, nucleosomes, and NET-associated enzymes. Citrullination of histone H3 by peptidyl arginine deiminase 4 (PAD4) is central for NET formation, and citrullinated histone H3 (H3Cit) is considered a NET-specific biomarker. We therefore aimed to optimize and validate a new enzyme-linked immunosorbent assay (ELISA) to quantify the levels of H3Cit in human plasma. A standard curve made of in vitro PAD4-citrullinated histones H3 allows for the quantification of H3Cit in plasma using an anti-histone antibody as capture antibody and an anti-histone H3 citrulline antibody for detection. The assay was evaluated for linearity, stability, specificity, and precision on plasma samples obtained from a human model of inflammation before and after lipopolysaccharide injection. The results revealed linearity and high specificity demonstrated by the inability of detecting non-citrullinated histone H3. Coefficients of variation for intra- and inter-assay variability ranged from 2.1 to 5.1% and from 5.8 to 13.5%, respectively, allowing for a high precision. Furthermore, our results support an inflammatory induction of a systemic NET burden by showing, for the first time, clear intra-individual elevations of plasma H3Cit in a human model of lipopolysaccharide-induced inflammation. Taken together, our work demonstrates the development of a new method for the quantification of H3Cit by ELISA that can reliably be used for the detection of NETs in human plasma.}, keywords = {article}, pubstate = {published}, tppubtype = {article} } Abstract There is an emerging interest in the diverse functions of neutrophil extracellular traps (NETs) in a variety of disease settings. However, data on circulating NETs rely largely upon surrogate NET markers such as cell-free DNA, nucleosomes, and NET-associated enzymes. Citrullination of histone H3 by peptidyl arginine deiminase 4 (PAD4) is central for NET formation, and citrullinated histone H3 (H3Cit) is considered a NET-specific biomarker. We therefore aimed to optimize and validate a new enzyme-linked immunosorbent assay (ELISA) to quantify the levels of H3Cit in human plasma. A standard curve made of in vitro PAD4-citrullinated histones H3 allows for the quantification of H3Cit in plasma using an anti-histone antibody as capture antibody and an anti-histone H3 citrulline antibody for detection. The assay was evaluated for linearity, stability, specificity, and precision on plasma samples obtained from a human model of inflammation before and after lipopolysaccharide injection. The results revealed linearity and high specificity demonstrated by the inability of detecting non-citrullinated histone H3. Coefficients of variation for intra- and inter-assay variability ranged from 2.1 to 5.1% and from 5.8 to 13.5%, respectively, allowing for a high precision. Furthermore, our results support an inflammatory induction of a systemic NET burden by showing, for the first time, clear intra-individual elevations of plasma H3Cit in a human model of lipopolysaccharide-induced inflammation. Taken together, our work demonstrates the development of a new method for the quantification of H3Cit by ELISA that can reliably be used for the detection of NETs in human plasma. |
Ogris C Guala D, Helleday Sonnhammer EL. T A novel method for crosstalk analysis of biological networks: improving accuracy of pathway annotation. Journal Article Nucleic Acids Res., 2017. Links | BibTeX | Tags: article @article{C2017, title = {A novel method for crosstalk analysis of biological networks: improving accuracy of pathway annotation.}, author = {Ogris C, Guala D, Helleday T, Sonnhammer EL.}, url = {https://www.ncbi.nlm.nih.gov/pubmed/27664219}, doi = {10.1093/nar/gkw849.}, year = {2017}, date = {2017-01-25}, journal = {Nucleic Acids Res.}, keywords = {article}, pubstate = {published}, tppubtype = {article} } |
2016 |
Tanikawa M1 Sanjiv K1, Helleday Herr Mortusewicz T1 P1 O1 The spliceosome U2 snRNP factors promote genome stability through distinct mechanisms; transcription of repair factors and R-loop processing Journal Article Oncogenesis, 5 (12), pp. e280, 2016. Links | BibTeX | Tags: article @article{M12016, title = {The spliceosome U2 snRNP factors promote genome stability through distinct mechanisms; transcription of repair factors and R-loop processing}, author = {Tanikawa M1, Sanjiv K1, Helleday T1, Herr P1, Mortusewicz O1.}, doi = {doi: 10.1038/oncsis.2016.70.}, year = {2016}, date = {2016-12-19}, journal = {Oncogenesis}, volume = {5}, number = {12}, pages = {e280}, keywords = {article}, pubstate = {published}, tppubtype = {article} } |
Eshtad S Mavajian Z, Rudd SG Visnes Boström Altun Helleday T J M T hMYH and hMTH1 cooperate for survival in mismatch repair defective T-cell acute lymphoblastic leukemia. Journal Article Oncogenesis, 2016. Abstract | Links | BibTeX | Tags: article @article{S12016, title = {hMYH and hMTH1 cooperate for survival in mismatch repair defective T-cell acute lymphoblastic leukemia.}, author = {Eshtad S, Mavajian Z, Rudd SG, Visnes T, Boström J, Altun M, Helleday T.}, url = {https://www.ncbi.nlm.nih.gov/pubmed/27918552}, doi = {doi: 10.1038/oncsis.2016.72.}, year = {2016}, date = {2016-12-05}, journal = {Oncogenesis}, abstract = {Abstract hMTH1 is an 8-oxodGTPase that prevents mis-incorporation of free oxidized nucleotides into genomic DNA. Base excision and mismatch repair pathways also restrict the accumulation of oxidized lesions in DNA by removing the mis-inserted 8-oxo-7,8-dihydro-2'-deoxyguanosines (8-oxodGs). In this study, we aimed to investigate the interplay between hMYH DNA glycosylase and hMTH1 for cancer cell survival by using mismatch repair defective T-cell acute lymphoblastic leukemia (T-ALL) cells. To this end, MYH and MTH1 were silenced individually or simultaneously using small hairpin RNAs. Increased sub-G1 population and apoptotic cells were observed upon concurrent depletion of both enzymes. Elevated cell death was consistent with cleaved caspase 3 accumulation in double knockdown cells. Importantly, overexpression of the nuclear isoform of hMYH could remove the G1 arrest and partially rescue the toxicity observed in hMTH1-depleted cells. In addition, expression profiles of human DNA glycosylases were generated using quantitative reverse transcriptase-PCR in MTH1 and/or MYH knockdown cells. NEIL1 DNA glycosylase, involved in repair of oxidized nucleosides, was found to be significantly downregulated as a cellular response to MTH1-MYH co-suppression. Overall, the results suggest that hMYH and hMTH1 functionally cooperate for effective repair and survival in mismatch repair defective T-ALL Jurkat A3 cells.}, keywords = {article}, pubstate = {published}, tppubtype = {article} } Abstract hMTH1 is an 8-oxodGTPase that prevents mis-incorporation of free oxidized nucleotides into genomic DNA. Base excision and mismatch repair pathways also restrict the accumulation of oxidized lesions in DNA by removing the mis-inserted 8-oxo-7,8-dihydro-2'-deoxyguanosines (8-oxodGs). In this study, we aimed to investigate the interplay between hMYH DNA glycosylase and hMTH1 for cancer cell survival by using mismatch repair defective T-cell acute lymphoblastic leukemia (T-ALL) cells. To this end, MYH and MTH1 were silenced individually or simultaneously using small hairpin RNAs. Increased sub-G1 population and apoptotic cells were observed upon concurrent depletion of both enzymes. Elevated cell death was consistent with cleaved caspase 3 accumulation in double knockdown cells. Importantly, overexpression of the nuclear isoform of hMYH could remove the G1 arrest and partially rescue the toxicity observed in hMTH1-depleted cells. In addition, expression profiles of human DNA glycosylases were generated using quantitative reverse transcriptase-PCR in MTH1 and/or MYH knockdown cells. NEIL1 DNA glycosylase, involved in repair of oxidized nucleosides, was found to be significantly downregulated as a cellular response to MTH1-MYH co-suppression. Overall, the results suggest that hMYH and hMTH1 functionally cooperate for effective repair and survival in mismatch repair defective T-ALL Jurkat A3 cells. |
Valerie NC Hagenkort A, Page BD Masuyer Rehling Carter Bevc Herr Homan Sheppard NG Stenmark Jemth AS Helleday G D M L P E P T NUDT15 Hydrolyzes 6-Thio-DeoxyGTP to Mediate the Anticancer Efficacy of 6-Thioguanine. Journal Article pp. 5501-11, 2016. Abstract | Links | BibTeX | Tags: article @article{NC2016, title = {NUDT15 Hydrolyzes 6-Thio-DeoxyGTP to Mediate the Anticancer Efficacy of 6-Thioguanine.}, author = {Valerie NC, Hagenkort A, Page BD, Masuyer G, Rehling D, Carter M, Bevc L, Herr P, Homan E, Sheppard NG, Stenmark P, Jemth AS, Helleday T.}, url = {https://www.ncbi.nlm.nih.gov/pubmed/27530327}, doi = {DOI: 10.1158/0008-5472.CAN-16-0584}, year = {2016}, date = {2016-09-15}, pages = {5501-11}, abstract = {Abstract Thiopurines are a standard treatment for childhood leukemia, but like all chemotherapeutics, their use is limited by inherent or acquired resistance in patients. Recently, the nucleoside diphosphate hydrolase NUDT15 has received attention on the basis of its ability to hydrolyze the thiopurine effector metabolites 6-thio-deoxyGTP (6-thio-dGTP) and 6-thio-GTP, thereby limiting the efficacy of thiopurines. In particular, increasing evidence suggests an association between the NUDT15 missense variant, R139C, and thiopurine sensitivity. In this study, we elucidated the role of NUDT15 and NUDT15 R139C in thiopurine metabolism. In vitro and cellular results argued that 6-thio-dGTP and 6-thio-GTP are favored substrates for NUDT15, a finding supported by a crystallographic determination of NUDT15 in complex with 6-thio-GMP. We found that NUDT15 R139C mutation did not affect enzymatic activity but instead negatively influenced protein stability, likely due to a loss of supportive intramolecular bonds that caused rapid proteasomal degradation in cells. Mechanistic investigations in cells indicated that NUDT15 ablation potentiated induction of the DNA damage checkpoint and cancer cell death by 6-thioguanine. Taken together, our results defined how NUDT15 limits thiopurine efficacy and how genetic ablation via the R139C missense mutation confers sensitivity to thiopurine treatment in patients. Cancer Res; 76(18); 5501-11. ©2016 AACR.}, keywords = {article}, pubstate = {published}, tppubtype = {article} } Abstract Thiopurines are a standard treatment for childhood leukemia, but like all chemotherapeutics, their use is limited by inherent or acquired resistance in patients. Recently, the nucleoside diphosphate hydrolase NUDT15 has received attention on the basis of its ability to hydrolyze the thiopurine effector metabolites 6-thio-deoxyGTP (6-thio-dGTP) and 6-thio-GTP, thereby limiting the efficacy of thiopurines. In particular, increasing evidence suggests an association between the NUDT15 missense variant, R139C, and thiopurine sensitivity. In this study, we elucidated the role of NUDT15 and NUDT15 R139C in thiopurine metabolism. In vitro and cellular results argued that 6-thio-dGTP and 6-thio-GTP are favored substrates for NUDT15, a finding supported by a crystallographic determination of NUDT15 in complex with 6-thio-GMP. We found that NUDT15 R139C mutation did not affect enzymatic activity but instead negatively influenced protein stability, likely due to a loss of supportive intramolecular bonds that caused rapid proteasomal degradation in cells. Mechanistic investigations in cells indicated that NUDT15 ablation potentiated induction of the DNA damage checkpoint and cancer cell death by 6-thioguanine. Taken together, our results defined how NUDT15 limits thiopurine efficacy and how genetic ablation via the R139C missense mutation confers sensitivity to thiopurine treatment in patients. Cancer Res; 76(18); 5501-11. ©2016 AACR. |
Ogris C Helleday T, Sonnhammer EL. PathwAX: a web server for network crosstalk based pathway annotation. Journal Article Nucleic Acids Res., 2016. Abstract | Links | BibTeX | Tags: article @article{C2016, title = {PathwAX: a web server for network crosstalk based pathway annotation.}, author = {Ogris C, Helleday T, Sonnhammer EL.}, url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4987909/}, doi = {DOI: 10.1093/nar/gkw356}, year = {2016}, date = {2016-07-08}, journal = {Nucleic Acids Res.}, abstract = {Abstract Pathway annotation of gene lists is often used to functionally analyse biomolecular data such as gene expression in order to establish which processes are activated in a given experiment. Databases such as KEGG or GO represent collections of how genes are known to be organized in pathways, and the challenge is to compare a given gene list with the known pathways such that all true relations are identified. Most tools apply statistical measures to the gene overlap between the gene list and pathway. It is however problematic to avoid false negatives and false positives when only using the gene overlap. The pathwAX web server (http://pathwAX.sbc.su.se/) applies a different approach which is based on network crosstalk. It uses the comprehensive network FunCoup to analyse network crosstalk between a query gene list and KEGG pathways. PathwAX runs the BinoX algorithm, which employs Monte-Carlo sampling of randomized networks and estimates a binomial distribution, for estimating the statistical significance of the crosstalk. This results in substantially higher accuracy than gene overlap methods. The system was optimized for speed and allows interactive web usage. We illustrate the usage and output of pathwAX.}, keywords = {article}, pubstate = {published}, tppubtype = {article} } Abstract Pathway annotation of gene lists is often used to functionally analyse biomolecular data such as gene expression in order to establish which processes are activated in a given experiment. Databases such as KEGG or GO represent collections of how genes are known to be organized in pathways, and the challenge is to compare a given gene list with the known pathways such that all true relations are identified. Most tools apply statistical measures to the gene overlap between the gene list and pathway. It is however problematic to avoid false negatives and false positives when only using the gene overlap. The pathwAX web server (http://pathwAX.sbc.su.se/) applies a different approach which is based on network crosstalk. It uses the comprehensive network FunCoup to analyse network crosstalk between a query gene list and KEGG pathways. PathwAX runs the BinoX algorithm, which employs Monte-Carlo sampling of randomized networks and estimates a binomial distribution, for estimating the statistical significance of the crosstalk. This results in substantially higher accuracy than gene overlap methods. The system was optimized for speed and allows interactive web usage. We illustrate the usage and output of pathwAX. |
Jackson SP, Helleday T DNA REPAIR. Drugging DNA repair. Journal Article Science, 352 (6290), pp. 1178-9, 2016. Links | BibTeX | Tags: article @article{SP2016, title = {DNA REPAIR. Drugging DNA repair.}, author = {Jackson SP, Helleday T.}, url = {http://science.sciencemag.org/content/352/6290/1178}, doi = {doi: 10.1126/science.aab0958}, year = {2016}, date = {2016-07-03}, journal = {Science}, volume = {352}, number = {6290}, pages = {1178-9}, keywords = {article}, pubstate = {published}, tppubtype = {article} } |
Rudd SG Valerie NC, Helleday T Pathways controlling dNTP pools to maintain genome stability. Journal Article DNA repair, (44), pp. 193-204, 2016. Abstract | Links | BibTeX | Tags: article @article{SG2016, title = {Pathways controlling dNTP pools to maintain genome stability.}, author = {Rudd SG, Valerie NC, Helleday T.}, doi = {doi: 10.1016/j.dnarep.2016.05.032}, year = {2016}, date = {2016-05-28}, journal = {DNA repair}, number = {44}, pages = {193-204}, abstract = {Abstract Artificially modified nucleotides, in the form of nucleoside analogues, are widely used in the treatment of cancers and various other diseases, and have become important tools in the laboratory to characterise DNA repair pathways. In contrast, the role of endogenously occurring nucleotide modifications in genome stability is little understood. This is despite the demonstration over three decades ago that the cellular DNA precursor pool is orders of magnitude more susceptible to modification than the DNA molecule itself. More recently, underscoring the importance of this topic, oxidation of the cellular nucleotide pool achieved through targeting the sanitation enzyme MTH1, appears to be a promising anti-cancer strategy. This article reviews our current understanding of modified DNA precursors in genome stability, with a particular focus upon oxidised nucleotides, and outlines some important outstanding questions.}, keywords = {article}, pubstate = {published}, tppubtype = {article} } Abstract Artificially modified nucleotides, in the form of nucleoside analogues, are widely used in the treatment of cancers and various other diseases, and have become important tools in the laboratory to characterise DNA repair pathways. In contrast, the role of endogenously occurring nucleotide modifications in genome stability is little understood. This is despite the demonstration over three decades ago that the cellular DNA precursor pool is orders of magnitude more susceptible to modification than the DNA molecule itself. More recently, underscoring the importance of this topic, oxidation of the cellular nucleotide pool achieved through targeting the sanitation enzyme MTH1, appears to be a promising anti-cancer strategy. This article reviews our current understanding of modified DNA precursors in genome stability, with a particular focus upon oxidised nucleotides, and outlines some important outstanding questions. |
Ying, S; Chen, Z; Medhurst, A L; Neal, J A; Bao, Z; Mortusewicz, O; McGouran, J; Song, X; Shen, H; Hamdy, F C; Kessler, B M; Meek, K; Helleday, T DNA-PKcs and PARP1 Bind to Unresected Stalled DNA Replication Forks Where They Recruit XRCC1 to Mediate Repair Journal Article Cancer Res., 76 (5), pp. 1078–1088, 2016, ([DOI:hrefhttp://dx.doi.org/10.1158/0008-5472.CAN-15-060810.1158/0008-5472.CAN-15-0608] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2660389626603896]). @article{pmid26603896, title = {DNA-PKcs and PARP1 Bind to Unresected Stalled DNA Replication Forks Where They Recruit XRCC1 to Mediate Repair}, author = { S. Ying and Z. Chen and A. L. Medhurst and J. A. Neal and Z. Bao and O. Mortusewicz and J. McGouran and X. Song and H. Shen and F. C. Hamdy and B. M. Kessler and K. Meek and T. Helleday}, year = {2016}, date = {2016-03-01}, journal = {Cancer Res.}, volume = {76}, number = {5}, pages = {1078--1088}, abstract = {A series of critical pathways are responsible for the detection, signaling, and restart of replication forks that encounter blocks during S-phase progression. Small base lesions may obstruct replication fork progression and processing, but the link between repair of small lesions and replication forks is unclear. In this study, we investigated a hypothesized role for DNA-PK, an important enzyme in DNA repair, in cellular responses to DNA replication stress. The enzyme catalytic subunit DNA-PKcs was phosphorylated on S2056 at sites of stalled replication forks in response to short hydroxyurea treatment. Using DNA fiber experiments, we found that catalytically active DNA-PK was required for efficient replication restart of stalled forks. Furthermore, enzymatically active DNA-PK was also required for PARP-dependent recruitment of XRCC1 to stalled replication forks. This activity was enhanced by preventing Mre11-dependent DNA end resection, suggesting that XRCC1 must be recruited early to an unresected stalled fork. We also found that XRCC1 was required for effective restart of a subset of stalled replication forks. Overall, our work suggested that DNA-PK and PARP-dependent recruitment of XRCC1 is necessary to effectively protect, repair, and restart stalled replication forks, providing new insight into how genomic stability is preserved. Cancer Res; 76(5); 1078-88. ©2015 AACR.}, note = {[DOI:hrefhttp://dx.doi.org/10.1158/0008-5472.CAN-15-060810.1158/0008-5472.CAN-15-0608] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2660389626603896]}, keywords = {}, pubstate = {published}, tppubtype = {article} } A series of critical pathways are responsible for the detection, signaling, and restart of replication forks that encounter blocks during S-phase progression. Small base lesions may obstruct replication fork progression and processing, but the link between repair of small lesions and replication forks is unclear. In this study, we investigated a hypothesized role for DNA-PK, an important enzyme in DNA repair, in cellular responses to DNA replication stress. The enzyme catalytic subunit DNA-PKcs was phosphorylated on S2056 at sites of stalled replication forks in response to short hydroxyurea treatment. Using DNA fiber experiments, we found that catalytically active DNA-PK was required for efficient replication restart of stalled forks. Furthermore, enzymatically active DNA-PK was also required for PARP-dependent recruitment of XRCC1 to stalled replication forks. This activity was enhanced by preventing Mre11-dependent DNA end resection, suggesting that XRCC1 must be recruited early to an unresected stalled fork. We also found that XRCC1 was required for effective restart of a subset of stalled replication forks. Overall, our work suggested that DNA-PK and PARP-dependent recruitment of XRCC1 is necessary to effectively protect, repair, and restart stalled replication forks, providing new insight into how genomic stability is preserved. Cancer Res; 76(5); 1078-88. ©2015 AACR. |
Llona-Minguez S Höglund A, Jacques SA Johansson Calderón-Montaño JM Claesson Loseva Valerie NC Lundbäck Piedrafita Maga Crespan Meijer Burgos Morón Baranczewski Hagbjörk AL Svensson Wiita Almlöf Visnes Jeppsson Sigmundsson Jensen AJ Artursson Jemth AS Stenmark Warpman Berglund Scobie L M O T J G E L E P R E I T F K P P U M; T, Helleday Discovery of the First Potent and Selective Inhibitors of Human dCTP Pyrophosphatase 1. Journal Article J Med Chem, 59 (3), pp. 1140-8, 2016. Abstract | Links | BibTeX | Tags: article @article{S2016, title = {Discovery of the First Potent and Selective Inhibitors of Human dCTP Pyrophosphatase 1.}, author = {Llona-Minguez S, Höglund A, Jacques SA, Johansson L, Calderón-Montaño JM, Claesson M, Loseva O, Valerie NC, Lundbäck T, Piedrafita J, Maga G, Crespan E, Meijer L, Burgos Morón E, Baranczewski P, Hagbjörk AL, Svensson R, Wiita E, Almlöf I, Visnes T, Jeppsson F, Sigmundsson K, Jensen AJ, Artursson P, Jemth AS, Stenmark P, Warpman Berglund U, Scobie M and Helleday T}, url = {https://www.ncbi.nlm.nih.gov/pubmed/26771665}, doi = {doi: 10.1021/acs.jmedchem.5b01741}, year = {2016}, date = {2016-02-11}, journal = {J Med Chem}, volume = {59}, number = {3}, pages = {1140-8}, abstract = {Abstract The dCTPase pyrophosphatase 1 (dCTPase) regulates the intracellular nucleotide pool through hydrolytic degradation of canonical and noncanonical nucleotide triphosphates (dNTPs). dCTPase is highly expressed in multiple carcinomas and is associated with cancer cell stemness. Here we report on the development of the first potent and selective dCTPase inhibitors that enhance the cytotoxic effect of cytidine analogues in leukemia cells. Boronate 30 displays a promising in vitro ADME profile, including plasma and mouse microsomal half-lives, aqueous solubility, cell permeability and CYP inhibition, deeming it a suitable compound for in vivo studies.}, keywords = {article}, pubstate = {published}, tppubtype = {article} } Abstract The dCTPase pyrophosphatase 1 (dCTPase) regulates the intracellular nucleotide pool through hydrolytic degradation of canonical and noncanonical nucleotide triphosphates (dNTPs). dCTPase is highly expressed in multiple carcinomas and is associated with cancer cell stemness. Here we report on the development of the first potent and selective dCTPase inhibitors that enhance the cytotoxic effect of cytidine analogues in leukemia cells. Boronate 30 displays a promising in vitro ADME profile, including plasma and mouse microsomal half-lives, aqueous solubility, cell permeability and CYP inhibition, deeming it a suitable compound for in vivo studies. |
Brautigam, L; Pudelko, L; Jemth, A S; Gad, H; Narwal, M; Gustafsson, R; Karsten, S; Carreras-Puigvert, J; Homan, E; Berndt, C; Berglund, Warpman U; Stenmark, P; Helleday, T Hypoxic signaling and the cellular redox tumor environment determine sensitivity to MTH1 inhibition Journal Article Cancer Res., 2016, ([DOI:hrefhttp://dx.doi.org/10.1158/0008-5472.CAN-15-238010.1158/0008-5472.CAN-15-2380] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2686211426862114]). @article{pmid26862114b, title = {Hypoxic signaling and the cellular redox tumor environment determine sensitivity to MTH1 inhibition}, author = { L. Brautigam and L. Pudelko and A. S. Jemth and H. Gad and M. Narwal and R. Gustafsson and S. Karsten and J. Carreras-Puigvert and E. Homan and C. Berndt and U. Warpman Berglund and P. Stenmark and T. Helleday}, year = {2016}, date = {2016-02-01}, journal = {Cancer Res.}, abstract = {Cancer cells are commonly in a state of redox imbalance that drives their growth and survival. To compensate for oxidative stress induced by the tumor redox environment, cancer cells upregulate specific non-oncogenic addiction enzymes, such as MTH1 (NUDT1), which detoxifies oxidized nucleotides. Here, we show that increasing oxidative stress in non-malignant cells induced their sensitization to the effects of MTH1 inhibition, whereas decreasing oxidative pressure in cancer cells protected against inhibition. Furthermore, we purified zebrafish MTH1 and solved the crystal structure of MTH1 bound to its inhibitor, highlighting the zebrafish as a relevant tool to study MTH1 biology. Delivery of 8-oxo-dGTP and 2-OH-dATP to zebrafish embryos was highly toxic in the absence of MTH1 activity. Moreover, chemically or genetically mimicking activated hypoxia signaling in zebrafish revealed that pathological upregulation of the HIF1α response, often observed in cancer and linked to poor prognosis, sensitized embryos to MTH1 inhibition. Using a transgenic zebrafish line, in which the cellular redox status can be monitored in vivo, we detected an increase in oxidative pressure upon activation of hypoxic signaling. Pre-treatment with the antioxidant N-acetyl-L-cysteine protected embryos with activated hypoxia signaling against MTH1 inhibition, suggesting that the aberrant redox environment likely causes sensitization. In summary, MTH1 inhibition may offer a general approach to treat cancers characterized by deregulated hypoxia signaling or redox imbalance.}, note = {[DOI:hrefhttp://dx.doi.org/10.1158/0008-5472.CAN-15-238010.1158/0008-5472.CAN-15-2380] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2686211426862114]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Cancer cells are commonly in a state of redox imbalance that drives their growth and survival. To compensate for oxidative stress induced by the tumor redox environment, cancer cells upregulate specific non-oncogenic addiction enzymes, such as MTH1 (NUDT1), which detoxifies oxidized nucleotides. Here, we show that increasing oxidative stress in non-malignant cells induced their sensitization to the effects of MTH1 inhibition, whereas decreasing oxidative pressure in cancer cells protected against inhibition. Furthermore, we purified zebrafish MTH1 and solved the crystal structure of MTH1 bound to its inhibitor, highlighting the zebrafish as a relevant tool to study MTH1 biology. Delivery of 8-oxo-dGTP and 2-OH-dATP to zebrafish embryos was highly toxic in the absence of MTH1 activity. Moreover, chemically or genetically mimicking activated hypoxia signaling in zebrafish revealed that pathological upregulation of the HIF1α response, often observed in cancer and linked to poor prognosis, sensitized embryos to MTH1 inhibition. Using a transgenic zebrafish line, in which the cellular redox status can be monitored in vivo, we detected an increase in oxidative pressure upon activation of hypoxic signaling. Pre-treatment with the antioxidant N-acetyl-L-cysteine protected embryos with activated hypoxia signaling against MTH1 inhibition, suggesting that the aberrant redox environment likely causes sensitization. In summary, MTH1 inhibition may offer a general approach to treat cancers characterized by deregulated hypoxia signaling or redox imbalance. |
Helleday, T PARP inhibitor recognised as FĐA breakthrough therapy in castration resistant prostate cancer: beyond germline BRCA mutations Journal Article Ann. Oncol., 2016, ([DOI:hrefhttp://dx.doi.org/10.1093/annonc/mdw04810.1093/annonc/mdw048] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2686558026865580]). BibTeX | Tags: @article{pmid26865580, title = {PARP inhibitor recognised as FĐA breakthrough therapy in castration resistant prostate cancer: beyond germline BRCA mutations}, author = { T. Helleday}, year = {2016}, date = {2016-02-01}, journal = {Ann. Oncol.}, note = {[DOI:hrefhttp://dx.doi.org/10.1093/annonc/mdw04810.1093/annonc/mdw048] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2686558026865580]}, keywords = {}, pubstate = {published}, tppubtype = {article} } |
Llona-Minguez, S; Hoglund, A; Jacques, S A; Johansson, L; Calderon-Montano, J M; Claesson, M; Loseva, O; Valerie, N C; Lundback, T; Piedrafita, J; Maga, G; Crespan, E; Meijer, L; Moron, Burgos E; Baranczewski, P; Hagbjork, A L; Svensson, R; Wiita, E; Almlof, I; Visnes, T; Jeppsson, F; Sigmundsson, K; Jensen, A J; Artursson, P; Jemth, A S; Stenmark, P; Berglund, Warpman U; Scobie, M; Helleday, T Discovery of the First Potent and Selective Inhibitors of Human dCTP Pyrophosphatase 1 Journal Article J. Med. Chem., 59 (3), pp. 1140–1148, 2016, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4753678PMC4753678] [DOI:hrefhttp://dx.doi.org/10.1021/acs.jmedchem.5b0174110.1021/acs.jmedchem.5b01741] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2677166526771665]). @article{pmid26771665, title = {Discovery of the First Potent and Selective Inhibitors of Human dCTP Pyrophosphatase 1}, author = { S. Llona-Minguez and A. Hoglund and S. A. Jacques and L. Johansson and J. M. Calderon-Montano and M. Claesson and O. Loseva and N. C. Valerie and T. Lundback and J. Piedrafita and G. Maga and E. Crespan and L. Meijer and E. Burgos Moron and P. Baranczewski and A. L. Hagbjork and R. Svensson and E. Wiita and I. Almlof and T. Visnes and F. Jeppsson and K. Sigmundsson and A. J. Jensen and P. Artursson and A. S. Jemth and P. Stenmark and U. Warpman Berglund and M. Scobie and T. Helleday}, year = {2016}, date = {2016-02-01}, journal = {J. Med. Chem.}, volume = {59}, number = {3}, pages = {1140--1148}, abstract = {The dCTPase pyrophosphatase 1 (dCTPase) regulates the intracellular nucleotide pool through hydrolytic degradation of canonical and noncanonical nucleotide triphosphates (dNTPs). dCTPase is highly expressed in multiple carcinomas and is associated with cancer cell stemness. Here we report on the development of the first potent and selective dCTPase inhibitors that enhance the cytotoxic effect of cytidine analogues in leukemia cells. Boronate 30 displays a promising in vitro ADME profile, including plasma and mouse microsomal half-lives, aqueous solubility, cell permeability and CYP inhibition, deeming it a suitable compound for in vivo studies.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4753678PMC4753678] [DOI:hrefhttp://dx.doi.org/10.1021/acs.jmedchem.5b0174110.1021/acs.jmedchem.5b01741] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2677166526771665]}, keywords = {}, pubstate = {published}, tppubtype = {article} } The dCTPase pyrophosphatase 1 (dCTPase) regulates the intracellular nucleotide pool through hydrolytic degradation of canonical and noncanonical nucleotide triphosphates (dNTPs). dCTPase is highly expressed in multiple carcinomas and is associated with cancer cell stemness. Here we report on the development of the first potent and selective dCTPase inhibitors that enhance the cytotoxic effect of cytidine analogues in leukemia cells. Boronate 30 displays a promising in vitro ADME profile, including plasma and mouse microsomal half-lives, aqueous solubility, cell permeability and CYP inhibition, deeming it a suitable compound for in vivo studies. |
Mortusewicz, O; Evers, B; Helleday, T PC4 promotes genome stability and ĐNA repair through binding of ssĐNA at ĐNA damage sites Journal Article Oncogene, 35 (6), pp. 761–770, 2016, ([DOI:hrefhttp://dx.doi.org/10.1038/onc.2015.13510.1038/onc.2015.135] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2596191225961912]). @article{pmid25961912, title = {PC4 promotes genome stability and ĐNA repair through binding of ssĐNA at ĐNA damage sites}, author = { O. Mortusewicz and B. Evers and T. Helleday}, year = {2016}, date = {2016-02-01}, journal = {Oncogene}, volume = {35}, number = {6}, pages = {761--770}, abstract = {The transcriptional cofactor PC4 is an ancient single-strand DNA (ssDNA)-binding protein that has a homologue in bacteriophage T5 where it is likely the elusive replicative ssDNA-binding protein. We hypothesize that human PC4 has retained functions in ssDNA binding to stabilize replication forks and prevent genome instability in mammalian cells. Here we demonstrate that PC4 is recruited to hydroxyurea (HU)-stalled replication forks, which is dependent on active transcription and its ssDNA-binding ability. Interestingly, we demonstrate that ssDNA binding by PC4 is critical to suppress spontaneous DNA damage and promote cellular survival. PC4 accumulation co-localizes with replication protein A (RPA) at stalled forks and is increased upon RPA depletion, demonstrating compensatory functions in ssDNA binding. Depletion of PC4 not only results in defective resolution of HU-induced DNA damage but also significantly reduces homologous recombination repair efficiency. Altogether, our results indicate that PC4 has similar functions to RPA in binding ssDNA to promote genome stability, especially at sites of replication-transcription collisions.}, note = {[DOI:hrefhttp://dx.doi.org/10.1038/onc.2015.13510.1038/onc.2015.135] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2596191225961912]}, keywords = {}, pubstate = {published}, tppubtype = {article} } The transcriptional cofactor PC4 is an ancient single-strand DNA (ssDNA)-binding protein that has a homologue in bacteriophage T5 where it is likely the elusive replicative ssDNA-binding protein. We hypothesize that human PC4 has retained functions in ssDNA binding to stabilize replication forks and prevent genome instability in mammalian cells. Here we demonstrate that PC4 is recruited to hydroxyurea (HU)-stalled replication forks, which is dependent on active transcription and its ssDNA-binding ability. Interestingly, we demonstrate that ssDNA binding by PC4 is critical to suppress spontaneous DNA damage and promote cellular survival. PC4 accumulation co-localizes with replication protein A (RPA) at stalled forks and is increased upon RPA depletion, demonstrating compensatory functions in ssDNA binding. Depletion of PC4 not only results in defective resolution of HU-induced DNA damage but also significantly reduces homologous recombination repair efficiency. Altogether, our results indicate that PC4 has similar functions to RPA in binding ssDNA to promote genome stability, especially at sites of replication-transcription collisions. |
Reddy, P T; Jaruga, P; Nelson, B C; Lowenthal, M S; Jemth, A S; Loseva, O; Coskun, E; Helleday, T; Dizdaroglu, M Production, Purification, and Characterization of (15)N-Labeled ĐNA Repair Proteins as Internal Standards for Mass Spectrometric Measurements Journal Article Meth. Enzymol., 566 , pp. 305–332, 2016, ([DOI:hrefhttp://dx.doi.org/10.1016/bs.mie.2015.06.04410.1016/bs.mie.2015.06.044] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2679198526791985]). @article{pmid26791985, title = {Production, Purification, and Characterization of (15)N-Labeled ĐNA Repair Proteins as Internal Standards for Mass Spectrometric Measurements}, author = { P. T. Reddy and P. Jaruga and B. C. Nelson and M. S. Lowenthal and A. S. Jemth and O. Loseva and E. Coskun and T. Helleday and M. Dizdaroglu}, year = {2016}, date = {2016-01-01}, journal = {Meth. Enzymol.}, volume = {566}, pages = {305--332}, abstract = {Oxidatively induced DNA damage is caused in living organisms by a variety of damaging agents, resulting in the formation of a multiplicity of lesions, which are mutagenic and cytotoxic. Unless repaired by DNA repair mechanisms before DNA replication, DNA lesions can lead to genomic instability, which is one of the hallmarks of cancer. Oxidatively induced DNA damage is mainly repaired by base excision repair pathway with the involvement of a plethora of proteins. Cancer tissues develop greater DNA repair capacity than normal tissues by overexpressing DNA repair proteins. Increased DNA repair in tumors that removes DNA lesions generated by therapeutic agents before they became toxic is a major mechanism in the development of therapy resistance. Evidence suggests that DNA repair capacity may be a predictive biomarker of patient response. Thus, knowledge of DNA-protein expressions in disease-free and cancerous tissues may help predict and guide development of treatments and yield the best therapeutic response. Our laboratory has developed methodologies that use mass spectrometry with isotope dilution for the measurement of expression of DNA repair proteins in human tissues and cultured cells. For this purpose, full-length (15)N-labeled analogs of a number of human DNA repair proteins have been produced and purified to be used as internal standards for positive identification and accurate quantification. This chapter describes in detail the protocols of this work. The use of (15)N-labeled proteins as internal standards for the measurement of several DNA repair proteins in vivo is also presented.}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/bs.mie.2015.06.04410.1016/bs.mie.2015.06.044] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2679198526791985]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Oxidatively induced DNA damage is caused in living organisms by a variety of damaging agents, resulting in the formation of a multiplicity of lesions, which are mutagenic and cytotoxic. Unless repaired by DNA repair mechanisms before DNA replication, DNA lesions can lead to genomic instability, which is one of the hallmarks of cancer. Oxidatively induced DNA damage is mainly repaired by base excision repair pathway with the involvement of a plethora of proteins. Cancer tissues develop greater DNA repair capacity than normal tissues by overexpressing DNA repair proteins. Increased DNA repair in tumors that removes DNA lesions generated by therapeutic agents before they became toxic is a major mechanism in the development of therapy resistance. Evidence suggests that DNA repair capacity may be a predictive biomarker of patient response. Thus, knowledge of DNA-protein expressions in disease-free and cancerous tissues may help predict and guide development of treatments and yield the best therapeutic response. Our laboratory has developed methodologies that use mass spectrometry with isotope dilution for the measurement of expression of DNA repair proteins in human tissues and cultured cells. For this purpose, full-length (15)N-labeled analogs of a number of human DNA repair proteins have been produced and purified to be used as internal standards for positive identification and accurate quantification. This chapter describes in detail the protocols of this work. The use of (15)N-labeled proteins as internal standards for the measurement of several DNA repair proteins in vivo is also presented. |
Sanjiv, K; Hagenkort, A; Calderon-Montano, J M; Koolmeister, T; Reaper, P M; Mortusewicz, O; Jacques, S A; Kuiper, R V; Schultz, N; Scobie, M; Charlton, P A; Pollard, J R; Berglund, U W; Altun, M; Helleday, T Cancer-Specific Synthetic Lethality between AŦR and CĦK1 Kinase Activities Journal Article Cell Rep, 14 (2), pp. 298–309, 2016, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4713868PMC4713868] [DOI:hrefhttp://dx.doi.org/10.1016/j.celrep.2015.12.03210.1016/j.celrep.2015.12.032] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2674870926748709]). @article{pmid26748709, title = {Cancer-Specific Synthetic Lethality between AŦR and CĦK1 Kinase Activities}, author = { K. Sanjiv and A. Hagenkort and J. M. Calderon-Montano and T. Koolmeister and P. M. Reaper and O. Mortusewicz and S. A. Jacques and R. V. Kuiper and N. Schultz and M. Scobie and P. A. Charlton and J. R. Pollard and U. W. Berglund and M. Altun and T. Helleday}, year = {2016}, date = {2016-01-01}, journal = {Cell Rep}, volume = {14}, number = {2}, pages = {298--309}, abstract = {ATR and CHK1 maintain cancer cell survival under replication stress and inhibitors of both kinases are currently undergoing clinical trials. As ATR activity is increased after CHK1 inhibition, we hypothesized that this may indicate an increased reliance on ATR for survival. Indeed, we observe that replication stress induced by the CHK1 inhibitor AZD7762 results in replication catastrophe and apoptosis, when combined with the ATR inhibitor VE-821 specifically in cancer cells. Combined treatment with ATR and CHK1 inhibitors leads to replication fork arrest, ssDNA accumulation, replication collapse, and synergistic cell death in cancer cells in vitro and in vivo. Inhibition of CDK reversed replication stress and synthetic lethality, demonstrating that regulation of origin firing by ATR and CHK1 explains the synthetic lethality. In conclusion, this study exemplifies cancer-specific synthetic lethality between two proteins in the same pathway and raises the prospect of combining ATR and CHK1 inhibitors as promising cancer therapy.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4713868PMC4713868] [DOI:hrefhttp://dx.doi.org/10.1016/j.celrep.2015.12.03210.1016/j.celrep.2015.12.032] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2674870926748709]}, keywords = {}, pubstate = {published}, tppubtype = {article} } ATR and CHK1 maintain cancer cell survival under replication stress and inhibitors of both kinases are currently undergoing clinical trials. As ATR activity is increased after CHK1 inhibition, we hypothesized that this may indicate an increased reliance on ATR for survival. Indeed, we observe that replication stress induced by the CHK1 inhibitor AZD7762 results in replication catastrophe and apoptosis, when combined with the ATR inhibitor VE-821 specifically in cancer cells. Combined treatment with ATR and CHK1 inhibitors leads to replication fork arrest, ssDNA accumulation, replication collapse, and synergistic cell death in cancer cells in vitro and in vivo. Inhibition of CDK reversed replication stress and synthetic lethality, demonstrating that regulation of origin firing by ATR and CHK1 explains the synthetic lethality. In conclusion, this study exemplifies cancer-specific synthetic lethality between two proteins in the same pathway and raises the prospect of combining ATR and CHK1 inhibitors as promising cancer therapy. |
Puigvert, J C; Sanjiv, K; Helleday, T Ŧargeting ĐNA repair, ĐNA metabolism and replication stress as anti-cancer strategies Journal Article FEBS J., 283 (2), pp. 232–245, 2016, ([DOI:hrefhttp://dx.doi.org/10.1111/febs.1357410.1111/febs.13574] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2650779626507796]). @article{pmid26507796, title = {Ŧargeting ĐNA repair, ĐNA metabolism and replication stress as anti-cancer strategies}, author = { J. C. Puigvert and K. Sanjiv and T. Helleday}, year = {2016}, date = {2016-01-01}, journal = {FEBS J.}, volume = {283}, number = {2}, pages = {232--245}, abstract = {Anti-cancer therapies targeting and damaging the DNA have been extensively used in the last 50 years since the discovery of nitrogen mustards, antimetabolites and platin agents. The use of these drugs is often limited by dose-limiting side effects related to their poor specificity. In recent years, much effort has been put on the discovery and development of compounds that would exploit defects in DNA repair in cancer cells such as Wee1, Chk1 or PARP1 inhibitors. However, not all cancers respond to these inhibitors. Recently, new developments towards specifically targeting broader characteristics of cancer such as replication stress (RS) and lost redox homeostasis have emerged. Oncogenes induce proliferation signals, which also result in replication-associated DNA damage, i.e. RS. Our knowledge into overall causes of RS, lesions produced and how these are signalled in cells to activate cell cycle checkpoints is evolving. Inhibition of ATR, which would normally keep non-deleterious levels of RS, induces intolerable RS levels for cancer cells. Interestingly, links between replication and transcription appear to underlie RS along with a reduction of the dNTP pool. Remarkably, sanitization of the dNTP pool by MutT homologue 1, impeding incorporation of oxidized dNTPs into the DNA, seems to be crucial for cancer cell survival. In this minireview we present an overview of current and novel strategies to target DNA repair and exploit DNA damage to treat cancer. We present the current models for cancer-associated RS as well as cancer phenotypic lethality. Both strategies are poised to better target cancer cells and reduce side effects.}, note = {[DOI:hrefhttp://dx.doi.org/10.1111/febs.1357410.1111/febs.13574] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2650779626507796]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Anti-cancer therapies targeting and damaging the DNA have been extensively used in the last 50 years since the discovery of nitrogen mustards, antimetabolites and platin agents. The use of these drugs is often limited by dose-limiting side effects related to their poor specificity. In recent years, much effort has been put on the discovery and development of compounds that would exploit defects in DNA repair in cancer cells such as Wee1, Chk1 or PARP1 inhibitors. However, not all cancers respond to these inhibitors. Recently, new developments towards specifically targeting broader characteristics of cancer such as replication stress (RS) and lost redox homeostasis have emerged. Oncogenes induce proliferation signals, which also result in replication-associated DNA damage, i.e. RS. Our knowledge into overall causes of RS, lesions produced and how these are signalled in cells to activate cell cycle checkpoints is evolving. Inhibition of ATR, which would normally keep non-deleterious levels of RS, induces intolerable RS levels for cancer cells. Interestingly, links between replication and transcription appear to underlie RS along with a reduction of the dNTP pool. Remarkably, sanitization of the dNTP pool by MutT homologue 1, impeding incorporation of oxidized dNTPs into the DNA, seems to be crucial for cancer cell survival. In this minireview we present an overview of current and novel strategies to target DNA repair and exploit DNA damage to treat cancer. We present the current models for cancer-associated RS as well as cancer phenotypic lethality. Both strategies are poised to better target cancer cells and reduce side effects. |
2015 |
Puigvert JC, Sanjiv K; T., Helleday Targeting DNA repair, DNA metabolism and replication stress as anti-cancer strategies. Journal Article FEBS J, 282 (2), pp. 232-45, 2015. Abstract | Links | BibTeX | Tags: article @article{JC2015, title = {Targeting DNA repair, DNA metabolism and replication stress as anti-cancer strategies.}, author = {Puigvert JC, Sanjiv K and Helleday T.}, url = {https://www.ncbi.nlm.nih.gov/pubmed/26507796}, doi = {DOI: 10.1111/febs.13574}, year = {2015}, date = {2015-11-18}, journal = {FEBS J}, volume = {282}, number = {2}, pages = {232-45}, abstract = {Abstract Anti-cancer therapies targeting and damaging the DNA have been extensively used in the last 50 years since the discovery of nitrogen mustards, antimetabolites and platin agents. The use of these drugs is often limited by dose-limiting side effects related to their poor specificity. In recent years, much effort has been put on the discovery and development of compounds that would exploit defects in DNA repair in cancer cells such as Wee1, Chk1 or PARP1 inhibitors. However, not all cancers respond to these inhibitors. Recently, new developments towards specifically targeting broader characteristics of cancer such as replication stress (RS) and lost redox homeostasis have emerged. Oncogenes induce proliferation signals, which also result in replication-associated DNA damage, i.e. RS. Our knowledge into overall causes of RS, lesions produced and how these are signalled in cells to activate cell cycle checkpoints is evolving. Inhibition of ATR, which would normally keep non-deleterious levels of RS, induces intolerable RS levels for cancer cells. Interestingly, links between replication and transcription appear to underlie RS along with a reduction of the dNTP pool. Remarkably, sanitization of the dNTP pool by MutT homologue 1, impeding incorporation of oxidized dNTPs into the DNA, seems to be crucial for cancer cell survival. In this minireview we present an overview of current and novel strategies to target DNA repair and exploit DNA damage to treat cancer. We present the current models for cancer-associated RS as well as cancer phenotypic lethality. Both strategies are poised to better target cancer cells and reduce side effects.}, keywords = {article}, pubstate = {published}, tppubtype = {article} } Abstract Anti-cancer therapies targeting and damaging the DNA have been extensively used in the last 50 years since the discovery of nitrogen mustards, antimetabolites and platin agents. The use of these drugs is often limited by dose-limiting side effects related to their poor specificity. In recent years, much effort has been put on the discovery and development of compounds that would exploit defects in DNA repair in cancer cells such as Wee1, Chk1 or PARP1 inhibitors. However, not all cancers respond to these inhibitors. Recently, new developments towards specifically targeting broader characteristics of cancer such as replication stress (RS) and lost redox homeostasis have emerged. Oncogenes induce proliferation signals, which also result in replication-associated DNA damage, i.e. RS. Our knowledge into overall causes of RS, lesions produced and how these are signalled in cells to activate cell cycle checkpoints is evolving. Inhibition of ATR, which would normally keep non-deleterious levels of RS, induces intolerable RS levels for cancer cells. Interestingly, links between replication and transcription appear to underlie RS along with a reduction of the dNTP pool. Remarkably, sanitization of the dNTP pool by MutT homologue 1, impeding incorporation of oxidized dNTPs into the DNA, seems to be crucial for cancer cell survival. In this minireview we present an overview of current and novel strategies to target DNA repair and exploit DNA damage to treat cancer. We present the current models for cancer-associated RS as well as cancer phenotypic lethality. Both strategies are poised to better target cancer cells and reduce side effects. |
Tarish, F L; Schultz, N; Tanoglidi, A; Hamberg, H; Letocha, H; Karaszi, K; Hamdy, F C; Granfors, T; Helleday, T Castration radiosensitizes prostate cancer tissue by impairing ĐNA double-strand break repair Journal Article Sci Transl Med, 7 (312), pp. 312re11, 2015, ([DOI:hrefhttp://dx.doi.org/10.1126/scitranslmed.aac567110.1126/scitranslmed.aac5671] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2653725926537259]). @article{pmid26537259, title = {Castration radiosensitizes prostate cancer tissue by impairing ĐNA double-strand break repair}, author = { F. L. Tarish and N. Schultz and A. Tanoglidi and H. Hamberg and H. Letocha and K. Karaszi and F. C. Hamdy and T. Granfors and T. Helleday}, year = {2015}, date = {2015-11-01}, journal = {Sci Transl Med}, volume = {7}, number = {312}, pages = {312re11}, abstract = {Chemical castration improves responses to radiotherapy in prostate cancer, but the mechanism is unknown. We hypothesized that this radiosensitization is caused by castration-mediated down-regulation of nonhomologous end joining (NHEJ) repair of DNA double-strand breaks (DSBs). To test this, we enrolled 48 patients with localized prostate cancer in two arms of the study: either radiotherapy first or radiotherapy after neoadjuvant castration treatment. We biopsied patients at diagnosis and before and after castration and radiotherapy treatments to monitor androgen receptor, NHEJ, and DSB repair in verified cancer tissue. We show that patients receiving neoadjuvant castration treatment before radiotherapy had reduced amounts of the NHEJ protein Ku70, impaired radiotherapy-induced NHEJ activity, and higher amounts of unrepaired DSBs, measured by γ-H2AX foci in cancer tissues. This study demonstrates that chemical castration impairs NHEJ activity in prostate cancer tissue, explaining the improved response of patients with prostate cancer to radiotherapy after chemical castration.}, note = {[DOI:hrefhttp://dx.doi.org/10.1126/scitranslmed.aac567110.1126/scitranslmed.aac5671] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2653725926537259]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Chemical castration improves responses to radiotherapy in prostate cancer, but the mechanism is unknown. We hypothesized that this radiosensitization is caused by castration-mediated down-regulation of nonhomologous end joining (NHEJ) repair of DNA double-strand breaks (DSBs). To test this, we enrolled 48 patients with localized prostate cancer in two arms of the study: either radiotherapy first or radiotherapy after neoadjuvant castration treatment. We biopsied patients at diagnosis and before and after castration and radiotherapy treatments to monitor androgen receptor, NHEJ, and DSB repair in verified cancer tissue. We show that patients receiving neoadjuvant castration treatment before radiotherapy had reduced amounts of the NHEJ protein Ku70, impaired radiotherapy-induced NHEJ activity, and higher amounts of unrepaired DSBs, measured by γ-H2AX foci in cancer tissues. This study demonstrates that chemical castration impairs NHEJ activity in prostate cancer tissue, explaining the improved response of patients with prostate cancer to radiotherapy after chemical castration. |
Xing, M; Wang, X; Palmai-Pallag, T; Shen, H; Helleday, T; Hickson, I D; Ying, S Acute MUS81 depletion leads to replication fork slowing and a constitutive ĐNA damage response Journal Article Oncotarget, 6 (35), pp. 37638–37646, 2015, ([DOI:hrefhttp://dx.doi.org/10.18632/oncotarget.549710.18632/oncotarget.5497] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2641521726415217]). @article{pmid26415217, title = {Acute MUS81 depletion leads to replication fork slowing and a constitutive ĐNA damage response}, author = { M. Xing and X. Wang and T. Palmai-Pallag and H. Shen and T. Helleday and I. D. Hickson and S. Ying}, year = {2015}, date = {2015-11-01}, journal = {Oncotarget}, volume = {6}, number = {35}, pages = {37638--37646}, abstract = {The MUS81 protein belongs to a conserved family of DNA structure-specific nucleases that play important roles in DNA replication and repair. Inactivation of the Mus81 gene in mice has no major deleterious consequences for embryonic development, although cancer susceptibility has been reported. We have investigated the role of MUS81 in human cells by acutely depleting the protein using shRNAs. We found that MUS81 depletion from human fibroblasts leads to accumulation of ssDNA and a constitutive DNA damage response that ultimately activates cellular senescence. Moreover, we show that MUS81 is required for efficient replication fork progression during an unperturbed S-phase, and for recovery of productive replication following replication stalling. These results demonstrate essential roles for the MUS81 nuclease in maintenance of replication fork integrity.}, note = {[DOI:hrefhttp://dx.doi.org/10.18632/oncotarget.549710.18632/oncotarget.5497] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2641521726415217]}, keywords = {}, pubstate = {published}, tppubtype = {article} } The MUS81 protein belongs to a conserved family of DNA structure-specific nucleases that play important roles in DNA replication and repair. Inactivation of the Mus81 gene in mice has no major deleterious consequences for embryonic development, although cancer susceptibility has been reported. We have investigated the role of MUS81 in human cells by acutely depleting the protein using shRNAs. We found that MUS81 depletion from human fibroblasts leads to accumulation of ssDNA and a constitutive DNA damage response that ultimately activates cellular senescence. Moreover, we show that MUS81 is required for efficient replication fork progression during an unperturbed S-phase, and for recovery of productive replication following replication stalling. These results demonstrate essential roles for the MUS81 nuclease in maintenance of replication fork integrity. |
Helleday, T Poisoning Cancer Cells with Oxidized Nucleosides Journal Article N. Engl. J. Med., 373 (16), pp. 1570–1571, 2015, ([DOI:hrefhttp://dx.doi.org/10.1056/NEJMcibr151033510.1056/NEJMcibr1510335] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2646599226465992]). BibTeX | Tags: @article{pmid26465992, title = {Poisoning Cancer Cells with Oxidized Nucleosides}, author = { T. Helleday}, year = {2015}, date = {2015-10-01}, journal = {N. Engl. J. Med.}, volume = {373}, number = {16}, pages = {1570--1571}, note = {[DOI:hrefhttp://dx.doi.org/10.1056/NEJMcibr151033510.1056/NEJMcibr1510335] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2646599226465992]}, keywords = {}, pubstate = {published}, tppubtype = {article} } |
Xie, S; Mortusewicz, O; Ma, H T; Herr, P; Poon, R R; Helleday, T; Qian, C Ŧimeless Interacts with PARP-1 to Promote Ħomologous Recombination Repair Journal Article Mol. Cell, 60 (1), pp. 163–176, 2015, ([DOI:hrefhttp://dx.doi.org/10.1016/j.molcel.2015.07.03110.1016/j.molcel.2015.07.031] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2634409826344098]). @article{pmid26344098, title = {Ŧimeless Interacts with PARP-1 to Promote Ħomologous Recombination Repair}, author = { S. Xie and O. Mortusewicz and H. T. Ma and P. Herr and R. R. Poon and T. Helleday and C. Qian}, year = {2015}, date = {2015-10-01}, journal = {Mol. Cell}, volume = {60}, number = {1}, pages = {163--176}, abstract = {Human Timeless helps stabilize replication forks during normal DNA replication and plays a critical role in activation of the S phase checkpoint and proper establishment of sister chromatid cohesion. However, it remains elusive whether Timeless is involved in the repair of damaged DNA. Here, we identify that Timeless physically interacts with PARP-1 independent of poly(ADP-ribosyl)ation. We present high-resolution crystal structures of Timeless PAB (PARP-1-binding domain) in free form and in complex with PARP-1 catalytic domain. Interestingly, Timeless PAB domain specifically recognizes PARP-1, but not PARP-2 or PARP-3. Timeless-PARP-1 interaction does not interfere with PARP-1 enzymatic activity. We demonstrate that rapid and transient accumulation of Timeless at laser-induced DNA damage sites requires PARP-1, but not poly(ADP-ribosyl)ation and that Timeless is co-trapped with PARP-1 at DNA lesions upon PARP inhibition. Furthermore, we show that Timeless and PARP-1 interaction is required for efficient homologous recombination repair.}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/j.molcel.2015.07.03110.1016/j.molcel.2015.07.031] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2634409826344098]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Human Timeless helps stabilize replication forks during normal DNA replication and plays a critical role in activation of the S phase checkpoint and proper establishment of sister chromatid cohesion. However, it remains elusive whether Timeless is involved in the repair of damaged DNA. Here, we identify that Timeless physically interacts with PARP-1 independent of poly(ADP-ribosyl)ation. We present high-resolution crystal structures of Timeless PAB (PARP-1-binding domain) in free form and in complex with PARP-1 catalytic domain. Interestingly, Timeless PAB domain specifically recognizes PARP-1, but not PARP-2 or PARP-3. Timeless-PARP-1 interaction does not interfere with PARP-1 enzymatic activity. We demonstrate that rapid and transient accumulation of Timeless at laser-induced DNA damage sites requires PARP-1, but not poly(ADP-ribosyl)ation and that Timeless is co-trapped with PARP-1 at DNA lesions upon PARP inhibition. Furthermore, we show that Timeless and PARP-1 interaction is required for efficient homologous recombination repair. |
Zhao, H; Sifakis, E G; Sumida, N; Millan-Arino, L; Scholz, B A; Svensson, J P; Chen, X; Ronnegren, A L; de Lima, Mallet C D; Varnoosfaderani, F S; Shi, C; Loseva, O; Yammine, S; Israelsson, M; Rathje, L S; Nemeti, B; Fredlund, E; Helleday, T; Imreh, M P; Gondor, A PARP1- and CŦCF-Mediated Interactions between Active and Repressed Chromatin at the Lamina Promote Oscillating Ŧranscription Journal Article Mol. Cell, 59 (6), pp. 984–997, 2015, ([DOI:hrefhttp://dx.doi.org/10.1016/j.molcel.2015.07.01910.1016/j.molcel.2015.07.019] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2632125526321255]). @article{pmid26321255, title = {PARP1- and CŦCF-Mediated Interactions between Active and Repressed Chromatin at the Lamina Promote Oscillating Ŧranscription}, author = { H. Zhao and E. G. Sifakis and N. Sumida and L. Millan-Arino and B. A. Scholz and J. P. Svensson and X. Chen and A. L. Ronnegren and C. D. Mallet de Lima and F. S. Varnoosfaderani and C. Shi and O. Loseva and S. Yammine and M. Israelsson and L. S. Rathje and B. Nemeti and E. Fredlund and T. Helleday and M. P. Imreh and A. Gondor}, year = {2015}, date = {2015-09-01}, journal = {Mol. Cell}, volume = {59}, number = {6}, pages = {984--997}, abstract = {Transcriptionally active and inactive chromatin domains tend to segregate into separate sub-nuclear compartments to maintain stable expression patterns. However, here we uncovered an inter-chromosomal network connecting active loci enriched in circadian genes to repressed lamina-associated domains (LADs). The interactome is regulated by PARP1 and its co-factor CTCF. They not only mediate chromatin fiber interactions but also promote the recruitment of circadian genes to the lamina. Synchronization of the circadian rhythm by serum shock induces oscillations in PARP1-CTCF interactions, which is accompanied by oscillating recruitment of circadian loci to the lamina, followed by the acquisition of repressive H3K9me2 marks and transcriptional attenuation. Furthermore, depletion of H3K9me2/3, inhibition of PARP activity by olaparib, or downregulation of PARP1 or CTCF expression counteracts both recruitment to the envelope and circadian transcription. PARP1- and CTCF-regulated contacts between circadian loci and the repressive chromatin environment at the lamina therefore mediate circadian transcriptional plasticity.}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/j.molcel.2015.07.01910.1016/j.molcel.2015.07.019] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2632125526321255]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Transcriptionally active and inactive chromatin domains tend to segregate into separate sub-nuclear compartments to maintain stable expression patterns. However, here we uncovered an inter-chromosomal network connecting active loci enriched in circadian genes to repressed lamina-associated domains (LADs). The interactome is regulated by PARP1 and its co-factor CTCF. They not only mediate chromatin fiber interactions but also promote the recruitment of circadian genes to the lamina. Synchronization of the circadian rhythm by serum shock induces oscillations in PARP1-CTCF interactions, which is accompanied by oscillating recruitment of circadian loci to the lamina, followed by the acquisition of repressive H3K9me2 marks and transcriptional attenuation. Furthermore, depletion of H3K9me2/3, inhibition of PARP activity by olaparib, or downregulation of PARP1 or CTCF expression counteracts both recruitment to the envelope and circadian transcription. PARP1- and CTCF-regulated contacts between circadian loci and the repressive chromatin environment at the lamina therefore mediate circadian transcriptional plasticity. |
Coskun, E; Jaruga, P; Jemth, A S; Loseva, O; Scanlan, L D; Tona, A; Lowenthal, M S; Helleday, T; Dizdaroglu, M Addiction to MŦĦ1 protein results in intense expression in human breast cancer tissue as measured by liquid chromatography-isotope-dilution tandem mass spectrometry Journal Article DNA Repair (Amst.), 33 , pp. 101–110, 2015, ([DOI:hrefhttp://dx.doi.org/10.1016/j.dnarep.2015.05.00810.1016/j.dnarep.2015.05.008] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2620234726202347]). @article{pmid26202347, title = {Addiction to MŦĦ1 protein results in intense expression in human breast cancer tissue as measured by liquid chromatography-isotope-dilution tandem mass spectrometry}, author = { E. Coskun and P. Jaruga and A. S. Jemth and O. Loseva and L. D. Scanlan and A. Tona and M. S. Lowenthal and T. Helleday and M. Dizdaroglu}, year = {2015}, date = {2015-09-01}, journal = {DNA Repair (Amst.)}, volume = {33}, pages = {101--110}, abstract = {MTH1 protein sanitizes the nucleotide pool so that oxidized 2'-deoxynucleoside triphosphates (dNTPs) cannot be used in DNA replication. Cancer cells require MTH1 to avoid incorporation of oxidized dNTPs into DNA that results in mutations and cell death. Inhibition of MTH1 eradicates cancer, validating MTH1 as an anticancer target. By overexpressing MTH1, cancer cells may mediate cancer growth and resist therapy. To date, there is unreliable evidence suggesting that MTH1 is increased in cancer cells, and available methods to measure MTH1 levels are indirect and semi-quantitative. Accurate measurement of MTH1 in disease-free tissues and malignant tumors of patients may be essential for determining if the protein is truly upregulated in cancers, and for the development and use of MTH1 inhibitors in cancer therapy. Here, we present a novel approach involving liquid chromatography-isotope-dilution tandem mass spectrometry to positively identify and accurately quantify MTH1 in human tissues. We produced full length (15)N-labeled MTH1 and used it as an internal standard for the measurements. Following trypsin digestion, seven tryptic peptides of both MTH1 and (15)N-MTH1 were identified by their full scan and product ion spectra. These peptides provided a statistically significant protein score that would unequivocally identify MTH1. Next, we identified and quantified MTH1 in human disease-free breast tissues and malignant breast tumors, and in four human cultured cell lines, three of which were cancer cells. Extreme expression of MTH1 in malignant breast tumors was observed, suggesting that cancer cells are addicted to MTH1 for their survival. The approach described is expected to be applicable to the measurement of MTH1 levels in malignant tumors vs. surrounding disease-free tissues in cancer patients. This attribute may help develop novel treatment strategies and MTH1 inhibitors as potential drugs, and guide therapies.}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/j.dnarep.2015.05.00810.1016/j.dnarep.2015.05.008] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2620234726202347]}, keywords = {}, pubstate = {published}, tppubtype = {article} } MTH1 protein sanitizes the nucleotide pool so that oxidized 2'-deoxynucleoside triphosphates (dNTPs) cannot be used in DNA replication. Cancer cells require MTH1 to avoid incorporation of oxidized dNTPs into DNA that results in mutations and cell death. Inhibition of MTH1 eradicates cancer, validating MTH1 as an anticancer target. By overexpressing MTH1, cancer cells may mediate cancer growth and resist therapy. To date, there is unreliable evidence suggesting that MTH1 is increased in cancer cells, and available methods to measure MTH1 levels are indirect and semi-quantitative. Accurate measurement of MTH1 in disease-free tissues and malignant tumors of patients may be essential for determining if the protein is truly upregulated in cancers, and for the development and use of MTH1 inhibitors in cancer therapy. Here, we present a novel approach involving liquid chromatography-isotope-dilution tandem mass spectrometry to positively identify and accurately quantify MTH1 in human tissues. We produced full length (15)N-labeled MTH1 and used it as an internal standard for the measurements. Following trypsin digestion, seven tryptic peptides of both MTH1 and (15)N-MTH1 were identified by their full scan and product ion spectra. These peptides provided a statistically significant protein score that would unequivocally identify MTH1. Next, we identified and quantified MTH1 in human disease-free breast tissues and malignant breast tumors, and in four human cultured cell lines, three of which were cancer cells. Extreme expression of MTH1 in malignant breast tumors was observed, suggesting that cancer cells are addicted to MTH1 for their survival. The approach described is expected to be applicable to the measurement of MTH1 levels in malignant tumors vs. surrounding disease-free tissues in cancer patients. This attribute may help develop novel treatment strategies and MTH1 inhibitors as potential drugs, and guide therapies. |
Palazzo, L; Thomas, B; Jemth, A S; Colby, T; Leidecker, O; Feijs, K L; Zaja, R; Loseva, O; Puigvert, J C; Matic, I; Helleday, T; Ahel, I Processing of protein AĐP-ribosylation by Nudix hydrolases Journal Article Biochem. J., 468 (2), pp. 293–301, 2015, ([DOI:hrefhttp://dx.doi.org/10.1042/BJ2014155410.1042/BJ20141554] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2578958225789582]). @article{pmid25789582, title = {Processing of protein AĐP-ribosylation by Nudix hydrolases}, author = { L. Palazzo and B. Thomas and A. S. Jemth and T. Colby and O. Leidecker and K. L. Feijs and R. Zaja and O. Loseva and J. C. Puigvert and I. Matic and T. Helleday and I. Ahel}, year = {2015}, date = {2015-06-01}, journal = {Biochem. J.}, volume = {468}, number = {2}, pages = {293--301}, abstract = {ADP-ribosylation is a post-translational modification (PTM) of proteins found in organisms from all kingdoms of life which regulates many important biological functions including DNA repair, chromatin structure, unfolded protein response and apoptosis. Several cellular enzymes, such as macrodomain containing proteins PARG [poly(ADP-ribose) glycohydrolase] and TARG1 [terminal ADP-ribose (ADPr) protein glycohydrolase], reverse protein ADP-ribosylation. In the present study, we show that human Nudix (nucleoside diphosphate-linked moiety X)-type motif 16 (hNUDT16) represents a new enzyme class that can process protein ADP-ribosylation in vitro, converting it into ribose-5'-phosphate (R5P) tags covalently attached to the modified proteins. Furthermore, our data show that hNUDT16 enzymatic activity can be used to trim ADP-ribosylation on proteins in order to facilitate analysis of ADP-ribosylation sites on proteins by MS.}, note = {[DOI:hrefhttp://dx.doi.org/10.1042/BJ2014155410.1042/BJ20141554] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2578958225789582]}, keywords = {}, pubstate = {published}, tppubtype = {article} } ADP-ribosylation is a post-translational modification (PTM) of proteins found in organisms from all kingdoms of life which regulates many important biological functions including DNA repair, chromatin structure, unfolded protein response and apoptosis. Several cellular enzymes, such as macrodomain containing proteins PARG [poly(ADP-ribose) glycohydrolase] and TARG1 [terminal ADP-ribose (ADPr) protein glycohydrolase], reverse protein ADP-ribosylation. In the present study, we show that human Nudix (nucleoside diphosphate-linked moiety X)-type motif 16 (hNUDT16) represents a new enzyme class that can process protein ADP-ribosylation in vitro, converting it into ribose-5'-phosphate (R5P) tags covalently attached to the modified proteins. Furthermore, our data show that hNUDT16 enzymatic activity can be used to trim ADP-ribosylation on proteins in order to facilitate analysis of ADP-ribosylation sites on proteins by MS. |
Llona-Minguez, S; Desroses, M; Ghassemian, A; Jacques, S A; Eriksson, L; Isacksson, R; Koolmeister, T; Stenmark, P; Scobie, M; Helleday, T Vinylic MIĐA Boronates: New Building Blocks for the Synthesis of Aza-Ħeterocycles Journal Article Chemistry, 21 (20), pp. 7394–7398, 2015, ([DOI:hrefhttp://dx.doi.org/10.1002/chem.20140654910.1002/chem.201406549] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2580988325809883]). @article{pmid25809883, title = {Vinylic MIĐA Boronates: New Building Blocks for the Synthesis of Aza-Ħeterocycles}, author = { S. Llona-Minguez and M. Desroses and A. Ghassemian and S. A. Jacques and L. Eriksson and R. Isacksson and T. Koolmeister and P. Stenmark and M. Scobie and T. Helleday}, year = {2015}, date = {2015-05-01}, journal = {Chemistry}, volume = {21}, number = {20}, pages = {7394--7398}, abstract = {A two-step synthesis of structurally diverse pyrrole-containing bicyclic systems is reported. ortho-Nitro-haloarenes coupled with vinylic N-methyliminodiacetic acid (MIDA) boronates generate ortho-vinyl-nitroarenes, which undergo a "metal-free" nitrene insertion, resulting in a new pyrrole ring. This novel synthetic approach has a wide substrate tolerance and it is applicable in the preparation of more complex "drug-like" molecules. Interestingly, an ortho-nitro-allylarene derivative furnished a cyclic β-aminophosphonate motif.}, note = {[DOI:hrefhttp://dx.doi.org/10.1002/chem.20140654910.1002/chem.201406549] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2580988325809883]}, keywords = {}, pubstate = {published}, tppubtype = {article} } A two-step synthesis of structurally diverse pyrrole-containing bicyclic systems is reported. ortho-Nitro-haloarenes coupled with vinylic N-methyliminodiacetic acid (MIDA) boronates generate ortho-vinyl-nitroarenes, which undergo a "metal-free" nitrene insertion, resulting in a new pyrrole ring. This novel synthetic approach has a wide substrate tolerance and it is applicable in the preparation of more complex "drug-like" molecules. Interestingly, an ortho-nitro-allylarene derivative furnished a cyclic β-aminophosphonate motif. |
Llona-Minguez, S; Ghassemian, A; Helleday, T Lysophosphatidic acid receptor (LPAR) modulators: Ŧhe current pharmacological toolbox Journal Article Prog. Lipid Res., 58 , pp. 51–75, 2015, ([DOI:hrefhttp://dx.doi.org/10.1016/j.plipres.2015.01.00410.1016/j.plipres.2015.01.004] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2570439925704399]). @article{pmid25704399, title = {Lysophosphatidic acid receptor (LPAR) modulators: Ŧhe current pharmacological toolbox}, author = { S. Llona-Minguez and A. Ghassemian and T. Helleday}, year = {2015}, date = {2015-04-01}, journal = {Prog. Lipid Res.}, volume = {58}, pages = {51--75}, abstract = {Lysophosphatidic acids (LPA) are key lipid-signalling molecules that regulate a remarkably diverse set of cellular events, such as motility, chemotaxis, cell cycle progression, viability, and wound healing. The physiological and pathophysiological consequences of LPA signalling are evident and misregulation of LPA signalling can lead to pathologies like cancer, atherosclerosis, ischaemia, and fibrosis. LPA exerts its biological actions mainly through several types of G protein-coupled receptors, some of which display opposing or redundant effects. For this reason, selective LPA receptor small-molecule ligands can shine light on LPA biology and present an exciting opportunity for drug discovery endeavours. This review provides insights into the detailed chemical nature and pharmacological profile of the small-molecules thus far developed as LPA receptor modulators, as well as information on the preparation of key pharmaceuticals. This summary will facilitate future research efforts and nurture collaboration between chemists and biologists working in this emerging field.}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/j.plipres.2015.01.00410.1016/j.plipres.2015.01.004] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2570439925704399]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Lysophosphatidic acids (LPA) are key lipid-signalling molecules that regulate a remarkably diverse set of cellular events, such as motility, chemotaxis, cell cycle progression, viability, and wound healing. The physiological and pathophysiological consequences of LPA signalling are evident and misregulation of LPA signalling can lead to pathologies like cancer, atherosclerosis, ischaemia, and fibrosis. LPA exerts its biological actions mainly through several types of G protein-coupled receptors, some of which display opposing or redundant effects. For this reason, selective LPA receptor small-molecule ligands can shine light on LPA biology and present an exciting opportunity for drug discovery endeavours. This review provides insights into the detailed chemical nature and pharmacological profile of the small-molecules thus far developed as LPA receptor modulators, as well as information on the preparation of key pharmaceuticals. This summary will facilitate future research efforts and nurture collaboration between chemists and biologists working in this emerging field. |
Saleh, A; Gokturk, C; Warpman-Berglund, U; Helleday, T; Granelli, I Đevelopment and validation of method for ŦĦ588 and ŦĦ287, potent MŦĦ1 inhibitors and new anti-cancer agents, for pharmacokinetic studies in mice plasma Journal Article J Pharm Biomed Anal, 104 , pp. 1–11, 2015, ([DOI:hrefhttp://dx.doi.org/10.1016/j.jpba.2014.11.00910.1016/j.jpba.2014.11.009] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2545975425459754]). @article{pmid25459754, title = {Đevelopment and validation of method for ŦĦ588 and ŦĦ287, potent MŦĦ1 inhibitors and new anti-cancer agents, for pharmacokinetic studies in mice plasma}, author = { A. Saleh and C. Gokturk and U. Warpman-Berglund and T. Helleday and I. Granelli}, year = {2015}, date = {2015-02-01}, journal = {J Pharm Biomed Anal}, volume = {104}, pages = {1--11}, abstract = {MTH1 is a protein that is required for cancer cell survival and is overexpressed in cancer cells. TH588 and TH287 are two new compounds that inhibit the MTH1 protein. The inhibitors were tested in pharmacokinetic studies on mice. A bioanalytical method was developed and validated for determination in mice plasma. The method was based on protein precipitation followed by LC-MS/MS analysis. The separation was performed on an Ascentis Express RP-Amide C18 column. The mass spectrometer was operated in positive electrospray ionization mode and the analytes were determined with multiple reaction monitoring (MRM). Abundant monoisotopic fragments were used for quantification. Two additional fragments were used for conformational analysis. The recovery of the compounds in plasma varied between 61 and 91% and the matrix effects were low and ranged between -3% and +2%. The method showed to be selective, linear, accurate and precise, and applicable for preclinical pharmacokinetic studies of TH588 and TH287 in mouse plasma. Half-life (T1/2) was ≤3.5h and maximum concentration (Cmax) ranged between 0.82 and 338μM for the different administration routes and compounds.}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/j.jpba.2014.11.00910.1016/j.jpba.2014.11.009] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2545975425459754]}, keywords = {}, pubstate = {published}, tppubtype = {article} } MTH1 is a protein that is required for cancer cell survival and is overexpressed in cancer cells. TH588 and TH287 are two new compounds that inhibit the MTH1 protein. The inhibitors were tested in pharmacokinetic studies on mice. A bioanalytical method was developed and validated for determination in mice plasma. The method was based on protein precipitation followed by LC-MS/MS analysis. The separation was performed on an Ascentis Express RP-Amide C18 column. The mass spectrometer was operated in positive electrospray ionization mode and the analytes were determined with multiple reaction monitoring (MRM). Abundant monoisotopic fragments were used for quantification. Two additional fragments were used for conformational analysis. The recovery of the compounds in plasma varied between 61 and 91% and the matrix effects were low and ranged between -3% and +2%. The method showed to be selective, linear, accurate and precise, and applicable for preclinical pharmacokinetic studies of TH588 and TH287 in mouse plasma. Half-life (T1/2) was ≤3.5h and maximum concentration (Cmax) ranged between 0.82 and 338μM for the different administration routes and compounds. |
Carter, M; Jemth, A S; Hagenkort, A; Page, B D; Gustafsson, R; Griese, J J; Gad, H; Valerie, N C; Desroses, M; Bostrom, J; Berglund, Warpman U; Helleday, T; Stenmark, P Crystal structure, biochemical and cellular activities demonstrate separate functions of MŦĦ1 and MŦĦ2 Journal Article Nat Commun, 6 , pp. 7871, 2015, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4532830PMC4532830] [DOI:hrefhttp://dx.doi.org/10.1038/ncomms887110.1038/ncomms8871] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2623831826238318]). @article{pmid26238318, title = {Crystal structure, biochemical and cellular activities demonstrate separate functions of MŦĦ1 and MŦĦ2}, author = { M. Carter and A. S. Jemth and A. Hagenkort and B. D. Page and R. Gustafsson and J. J. Griese and H. Gad and N. C. Valerie and M. Desroses and J. Bostrom and U. Warpman Berglund and T. Helleday and P. Stenmark}, year = {2015}, date = {2015-01-01}, journal = {Nat Commun}, volume = {6}, pages = {7871}, abstract = {Deregulated redox metabolism in cancer leads to oxidative damage to cellular components including deoxyribonucleoside triphosphates (dNTPs). Targeting dNTP pool sanitizing enzymes, such as MTH1, is a highly promising anticancer strategy. The MTH2 protein, known as NUDT15, is described as the second human homologue of bacterial MutT with 8-oxo-dGTPase activity. We present the first NUDT15 crystal structure and demonstrate that NUDT15 prefers other nucleotide substrates over 8-oxo-dGTP. Key structural features are identified that explain different substrate preferences for NUDT15 and MTH1. We find that depletion of NUDT15 has no effect on incorporation of 8-oxo-dGTP into DNA and does not impact cancer cell survival in cell lines tested. NUDT17 and NUDT18 were also profiled and found to have far less activity than MTH1 against oxidized nucleotides. We show that NUDT15 is not a biologically relevant 8-oxo-dGTPase, and that MTH1 is the most prominent sanitizer of the cellular dNTP pool known to date.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4532830PMC4532830] [DOI:hrefhttp://dx.doi.org/10.1038/ncomms887110.1038/ncomms8871] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2623831826238318]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Deregulated redox metabolism in cancer leads to oxidative damage to cellular components including deoxyribonucleoside triphosphates (dNTPs). Targeting dNTP pool sanitizing enzymes, such as MTH1, is a highly promising anticancer strategy. The MTH2 protein, known as NUDT15, is described as the second human homologue of bacterial MutT with 8-oxo-dGTPase activity. We present the first NUDT15 crystal structure and demonstrate that NUDT15 prefers other nucleotide substrates over 8-oxo-dGTP. Key structural features are identified that explain different substrate preferences for NUDT15 and MTH1. We find that depletion of NUDT15 has no effect on incorporation of 8-oxo-dGTP into DNA and does not impact cancer cell survival in cell lines tested. NUDT17 and NUDT18 were also profiled and found to have far less activity than MTH1 against oxidized nucleotides. We show that NUDT15 is not a biologically relevant 8-oxo-dGTPase, and that MTH1 is the most prominent sanitizer of the cellular dNTP pool known to date. |
2014 |
Henriksson, S; Rassoolzadeh, H; Hedstrom, E; Coucoravas, C; Julner, A; Goldstein, M; Imreh, G; Zhivotovsky, B; Kastan, M B; Helleday, T; Farnebo, M Ŧhe scaffold protein WRAP53β orchestrates the ubiquitin response critical for ĐNA double-strand break repair Journal Article Genes Dev., 28 (24), pp. 2726–2738, 2014, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4265676PMC4265676] [DOI:hrefhttp://dx.doi.org/10.1101/gad.246546.11410.1101/gad.246546.114] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2551256025512560]). @article{pmid25512560, title = {Ŧhe scaffold protein WRAP53β orchestrates the ubiquitin response critical for ĐNA double-strand break repair}, author = { S. Henriksson and H. Rassoolzadeh and E. Hedstrom and C. Coucoravas and A. Julner and M. Goldstein and G. Imreh and B. Zhivotovsky and M. B. Kastan and T. Helleday and M. Farnebo}, year = {2014}, date = {2014-12-01}, journal = {Genes Dev.}, volume = {28}, number = {24}, pages = {2726--2738}, abstract = {The WD40 domain-containing protein WRAP53β (WD40 encoding RNA antisense to p53; also referred to as WDR79/TCAB1) controls trafficking of splicing factors and the telomerase enzyme to Cajal bodies, and its functional loss has been linked to carcinogenesis, premature aging, and neurodegeneration. Here, we identify WRAP53β as an essential regulator of DNA double-strand break (DSB) repair. WRAP53β rapidly localizes to DSBs in an ATM-, H2AX-, and MDC1-dependent manner. We show that WRAP53β targets the E3 ligase RNF8 to DNA lesions by facilitating the interaction between RNF8 and its upstream partner, MDC1, in response to DNA damage. Simultaneous binding of MDC1 and RNF8 to the highly conserved WD40 scaffold domain of WRAP53β facilitates their interaction and accumulation of RNF8 at DSBs. In this manner, WRAP53β controls proper ubiquitylation at DNA damage sites and the downstream assembly of 53BP1, BRCA1, and RAD51. Furthermore, we reveal that knockdown of WRAP53β impairs DSB repair by both homologous recombination (HR) and nonhomologous end-joining (NHEJ), causes accumulation of spontaneous DNA breaks, and delays recovery from radiation-induced cell cycle arrest. Our findings establish WRAP53β as a novel regulator of DSB repair by providing a scaffold for DNA repair factors.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4265676PMC4265676] [DOI:hrefhttp://dx.doi.org/10.1101/gad.246546.11410.1101/gad.246546.114] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2551256025512560]}, keywords = {}, pubstate = {published}, tppubtype = {article} } The WD40 domain-containing protein WRAP53β (WD40 encoding RNA antisense to p53; also referred to as WDR79/TCAB1) controls trafficking of splicing factors and the telomerase enzyme to Cajal bodies, and its functional loss has been linked to carcinogenesis, premature aging, and neurodegeneration. Here, we identify WRAP53β as an essential regulator of DNA double-strand break (DSB) repair. WRAP53β rapidly localizes to DSBs in an ATM-, H2AX-, and MDC1-dependent manner. We show that WRAP53β targets the E3 ligase RNF8 to DNA lesions by facilitating the interaction between RNF8 and its upstream partner, MDC1, in response to DNA damage. Simultaneous binding of MDC1 and RNF8 to the highly conserved WD40 scaffold domain of WRAP53β facilitates their interaction and accumulation of RNF8 at DSBs. In this manner, WRAP53β controls proper ubiquitylation at DNA damage sites and the downstream assembly of 53BP1, BRCA1, and RAD51. Furthermore, we reveal that knockdown of WRAP53β impairs DSB repair by both homologous recombination (HR) and nonhomologous end-joining (NHEJ), causes accumulation of spontaneous DNA breaks, and delays recovery from radiation-induced cell cycle arrest. Our findings establish WRAP53β as a novel regulator of DSB repair by providing a scaffold for DNA repair factors. |
Helleday, T; Eshtad, S; Nik-Zainal, S Mechanisms underlying mutational signatures in human cancers Journal Article Nat. Rev. Genet., 15 (9), pp. 585–598, 2014, ([DOI:hrefhttp://dx.doi.org/10.1038/nrg372910.1038/nrg3729] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2498160124981601]). @article{pmid24981601, title = {Mechanisms underlying mutational signatures in human cancers}, author = { T. Helleday and S. Eshtad and S. Nik-Zainal}, year = {2014}, date = {2014-09-01}, journal = {Nat. Rev. Genet.}, volume = {15}, number = {9}, pages = {585--598}, abstract = {The collective somatic mutations observed in a cancer are the outcome of multiple mutagenic processes that have been operative over the lifetime of a patient. Each process leaves a characteristic imprint--a mutational signature--on the cancer genome, which is defined by the type of DNA damage and DNA repair processes that result in base substitutions, insertions and deletions or structural variations. With the advent of whole-genome sequencing, researchers are identifying an increasing array of these signatures. Mutational signatures can be used as a physiological readout of the biological history of a cancer and also have potential use for discerning ongoing mutational processes from historical ones, thus possibly revealing new targets for anticancer therapies.}, note = {[DOI:hrefhttp://dx.doi.org/10.1038/nrg372910.1038/nrg3729] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2498160124981601]}, keywords = {}, pubstate = {published}, tppubtype = {article} } The collective somatic mutations observed in a cancer are the outcome of multiple mutagenic processes that have been operative over the lifetime of a patient. Each process leaves a characteristic imprint--a mutational signature--on the cancer genome, which is defined by the type of DNA damage and DNA repair processes that result in base substitutions, insertions and deletions or structural variations. With the advent of whole-genome sequencing, researchers are identifying an increasing array of these signatures. Mutational signatures can be used as a physiological readout of the biological history of a cancer and also have potential use for discerning ongoing mutational processes from historical ones, thus possibly revealing new targets for anticancer therapies. |
Orta, M L; Hoglund, A; Calderon-Montano, J M; Dominguez, I; Burgos-Moron, E; Visnes, T; Pastor, N; Strom, C; Lopez-lazaro, M; Helleday, T Ŧhe PARP inhibitor Olaparib disrupts base excision repair of 5-aza-2'-deoxycytidine lesions Journal Article Nucleic Acids Res., 42 (14), pp. 9108–9120, 2014, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4132747PMC4132747] [DOI:hrefhttp://dx.doi.org/10.1093/nar/gku63810.1093/nar/gku638] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2507438325074383]). @article{pmid25074383, title = {Ŧhe PARP inhibitor Olaparib disrupts base excision repair of 5-aza-2'-deoxycytidine lesions}, author = { M. L. Orta and A. Hoglund and J. M. Calderon-Montano and I. Dominguez and E. Burgos-Moron and T. Visnes and N. Pastor and C. Strom and M. Lopez-lazaro and T. Helleday}, year = {2014}, date = {2014-08-01}, journal = {Nucleic Acids Res.}, volume = {42}, number = {14}, pages = {9108--9120}, abstract = {Decitabine (5-aza-2'-deoxycytidine, 5-azadC) is used in the treatment of Myelodysplatic syndrome (MDS) and Acute Myeloid Leukemia (AML). Its mechanism of action is thought to involve reactivation of genes implicated in differentiation and transformation, as well as induction of DNA damage by trapping DNA methyltranferases (DNMT) to DNA. We demonstrate for the first time that base excision repair (BER) recognizes 5-azadC-induced lesions in DNA and mediates repair. We find that BER (XRCC1) deficient cells are sensitive to 5-azadC and display an increased amount of DNA single- and double-strand breaks. The XRCC1 protein co-localizes with DNMT1 foci after 5-azadC treatment, suggesting a novel and specific role of XRCC1 in the repair of trapped DNMT1. 5-azadC-induced DNMT foci persist in XRCC1 defective cells, demonstrating a role for XRCC1 in repair of 5-azadC-induced DNA lesions. Poly (ADP-ribose) polymerase (PARP) inhibition prevents XRCC1 relocation to DNA damage sites, disrupts XRCC1-DNMT1 co-localization and thereby efficient BER. In a panel of AML cell lines, combining 5-azadC and Olaparib cause synthetic lethality. These data suggest that PARP inhibitors can be used in combination with 5-azadC to improve treatment of MDS and AML.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4132747PMC4132747] [DOI:hrefhttp://dx.doi.org/10.1093/nar/gku63810.1093/nar/gku638] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2507438325074383]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Decitabine (5-aza-2'-deoxycytidine, 5-azadC) is used in the treatment of Myelodysplatic syndrome (MDS) and Acute Myeloid Leukemia (AML). Its mechanism of action is thought to involve reactivation of genes implicated in differentiation and transformation, as well as induction of DNA damage by trapping DNA methyltranferases (DNMT) to DNA. We demonstrate for the first time that base excision repair (BER) recognizes 5-azadC-induced lesions in DNA and mediates repair. We find that BER (XRCC1) deficient cells are sensitive to 5-azadC and display an increased amount of DNA single- and double-strand breaks. The XRCC1 protein co-localizes with DNMT1 foci after 5-azadC treatment, suggesting a novel and specific role of XRCC1 in the repair of trapped DNMT1. 5-azadC-induced DNMT foci persist in XRCC1 defective cells, demonstrating a role for XRCC1 in repair of 5-azadC-induced DNA lesions. Poly (ADP-ribose) polymerase (PARP) inhibition prevents XRCC1 relocation to DNA damage sites, disrupts XRCC1-DNMT1 co-localization and thereby efficient BER. In a panel of AML cell lines, combining 5-azadC and Olaparib cause synthetic lethality. These data suggest that PARP inhibitors can be used in combination with 5-azadC to improve treatment of MDS and AML. |
Helleday, T Cancer phenotypic lethality, exemplified by the non-essential MŦĦ1 enzyme being required for cancer survival Journal Article Ann. Oncol., 25 (7), pp. 1253–1255, 2014, ([DOI:hrefhttp://dx.doi.org/10.1093/annonc/mdu15810.1093/annonc/mdu158] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2473777724737777]). BibTeX | Tags: @article{pmid24737777, title = {Cancer phenotypic lethality, exemplified by the non-essential MŦĦ1 enzyme being required for cancer survival}, author = { T. Helleday}, year = {2014}, date = {2014-07-01}, journal = {Ann. Oncol.}, volume = {25}, number = {7}, pages = {1253--1255}, note = {[DOI:hrefhttp://dx.doi.org/10.1093/annonc/mdu15810.1093/annonc/mdu158] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2473777724737777]}, keywords = {}, pubstate = {published}, tppubtype = {article} } |
Pfister, S X; Ahrabi, S; Zalmas, L P; Sarkar, S; Aymard, F; Bachrati, C Z; Helleday, T; Legube, G; Thangue, La N B; Porter, A C; Humphrey, T C SEŦĐ2-dependent histone Ħ3K36 trimethylation is required for homologous recombination repair and genome stability Journal Article Cell Rep, 7 (6), pp. 2006–2018, 2014, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4074340PMC4074340] [DOI:hrefhttp://dx.doi.org/10.1016/j.celrep.2014.05.02610.1016/j.celrep.2014.05.026] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2493161024931610]). @article{pmid24931610, title = {SEŦĐ2-dependent histone Ħ3K36 trimethylation is required for homologous recombination repair and genome stability}, author = { S. X. Pfister and S. Ahrabi and L. P. Zalmas and S. Sarkar and F. Aymard and C. Z. Bachrati and T. Helleday and G. Legube and N. B. La Thangue and A. C. Porter and T. C. Humphrey}, year = {2014}, date = {2014-06-01}, journal = {Cell Rep}, volume = {7}, number = {6}, pages = {2006--2018}, abstract = {Modulating chromatin through histone methylation orchestrates numerous cellular processes. SETD2-dependent trimethylation of histone H3K36 is associated with active transcription. Here, we define a role for H3K36 trimethylation in homologous recombination (HR) repair in human cells. We find that depleting SETD2 generates a mutation signature resembling RAD51 depletion at I-SceI-induced DNA double-strand break (DSB) sites, with significantly increased deletions arising through microhomology-mediated end-joining. We establish a presynaptic role for SETD2 methyltransferase in HR, where it facilitates the recruitment of C-terminal binding protein interacting protein (CtIP) and promotes DSB resection, allowing Replication Protein A (RPA) and RAD51 binding to DNA damage sites. Furthermore, reducing H3K36me3 levels by overexpressing KDM4A/JMJD2A, an oncogene and H3K36me3/2 demethylase, or an H3.3K36M transgene also reduces HR repair events. We propose that error-free HR repair within H3K36me3-decorated transcriptionally active genomic regions promotes cell homeostasis. Moreover, these findings provide insights as to why oncogenic mutations cluster within the H3K36me3 axis.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4074340PMC4074340] [DOI:hrefhttp://dx.doi.org/10.1016/j.celrep.2014.05.02610.1016/j.celrep.2014.05.026] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2493161024931610]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Modulating chromatin through histone methylation orchestrates numerous cellular processes. SETD2-dependent trimethylation of histone H3K36 is associated with active transcription. Here, we define a role for H3K36 trimethylation in homologous recombination (HR) repair in human cells. We find that depleting SETD2 generates a mutation signature resembling RAD51 depletion at I-SceI-induced DNA double-strand break (DSB) sites, with significantly increased deletions arising through microhomology-mediated end-joining. We establish a presynaptic role for SETD2 methyltransferase in HR, where it facilitates the recruitment of C-terminal binding protein interacting protein (CtIP) and promotes DSB resection, allowing Replication Protein A (RPA) and RAD51 binding to DNA damage sites. Furthermore, reducing H3K36me3 levels by overexpressing KDM4A/JMJD2A, an oncogene and H3K36me3/2 demethylase, or an H3.3K36M transgene also reduces HR repair events. We propose that error-free HR repair within H3K36me3-decorated transcriptionally active genomic regions promotes cell homeostasis. Moreover, these findings provide insights as to why oncogenic mutations cluster within the H3K36me3 axis. |
Bolderson, E; Petermann, E; Croft, L; Suraweera, A; Pandita, R K; Pandita, T K; Helleday, T; Khanna, K K; Richard, D J Ħuman single-stranded ĐNA binding protein 1 (hSSB1/NABP2) is required for the stability and repair of stalled replication forks Journal Article Nucleic Acids Res., 42 (10), pp. 6326–6336, 2014, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4041449PMC4041449] [DOI:hrefhttp://dx.doi.org/10.1093/nar/gku27610.1093/nar/gku276] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2475340824753408]). @article{pmid24753408, title = {Ħuman single-stranded ĐNA binding protein 1 (hSSB1/NABP2) is required for the stability and repair of stalled replication forks}, author = { E. Bolderson and E. Petermann and L. Croft and A. Suraweera and R. K. Pandita and T. K. Pandita and T. Helleday and K. K. Khanna and D. J. Richard}, year = {2014}, date = {2014-06-01}, journal = {Nucleic Acids Res.}, volume = {42}, number = {10}, pages = {6326--6336}, abstract = {Aberrant DNA replication is a primary cause of mutations that are associated with pathological disorders including cancer. During DNA metabolism, the primary causes of replication fork stalling include secondary DNA structures, highly transcribed regions and damaged DNA. The restart of stalled replication forks is critical for the timely progression of the cell cycle and ultimately for the maintenance of genomic stability. Our previous work has implicated the single-stranded DNA binding protein, hSSB1/NABP2, in the repair of DNA double-strand breaks via homologous recombination. Here, we demonstrate that hSSB1 relocates to hydroxyurea (HU)-damaged replication forks where it is required for ATR and Chk1 activation and recruitment of Mre11 and Rad51. Consequently, hSSB1-depleted cells fail to repair and restart stalled replication forks. hSSB1 deficiency causes accumulation of DNA strand breaks and results in chromosome aberrations observed in mitosis, ultimately resulting in hSSB1 being required for survival to HU and camptothecin. Overall, our findings demonstrate the importance of hSSB1 in maintaining and repairing DNA replication forks and for overall genomic stability.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4041449PMC4041449] [DOI:hrefhttp://dx.doi.org/10.1093/nar/gku27610.1093/nar/gku276] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2475340824753408]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Aberrant DNA replication is a primary cause of mutations that are associated with pathological disorders including cancer. During DNA metabolism, the primary causes of replication fork stalling include secondary DNA structures, highly transcribed regions and damaged DNA. The restart of stalled replication forks is critical for the timely progression of the cell cycle and ultimately for the maintenance of genomic stability. Our previous work has implicated the single-stranded DNA binding protein, hSSB1/NABP2, in the repair of DNA double-strand breaks via homologous recombination. Here, we demonstrate that hSSB1 relocates to hydroxyurea (HU)-damaged replication forks where it is required for ATR and Chk1 activation and recruitment of Mre11 and Rad51. Consequently, hSSB1-depleted cells fail to repair and restart stalled replication forks. hSSB1 deficiency causes accumulation of DNA strand breaks and results in chromosome aberrations observed in mitosis, ultimately resulting in hSSB1 being required for survival to HU and camptothecin. Overall, our findings demonstrate the importance of hSSB1 in maintaining and repairing DNA replication forks and for overall genomic stability. |
Huber, K V; Salah, E; Radic, B; Gridling, M; Elkins, J M; Stukalov, A; Jemth, A S; Gokturk, C; Sanjiv, K; Stromberg, K; Pham, T; Berglund, U W; Colinge, J; Bennett, K L; Loizou, J I; Helleday, T; Knapp, S; Superti-Furga, G Stereospecific targeting of MŦĦ1 by (S)-crizotinib as an anticancer strategy Journal Article Nature, 508 (7495), pp. 222–227, 2014, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4150021PMC4150021] [DOI:hrefhttp://dx.doi.org/10.1038/nature1319410.1038/nature13194] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2469522524695225]). @article{pmid24695225, title = {Stereospecific targeting of MŦĦ1 by (S)-crizotinib as an anticancer strategy}, author = { K. V. Huber and E. Salah and B. Radic and M. Gridling and J. M. Elkins and A. Stukalov and A. S. Jemth and C. Gokturk and K. Sanjiv and K. Stromberg and T. Pham and U. W. Berglund and J. Colinge and K. L. Bennett and J. I. Loizou and T. Helleday and S. Knapp and G. Superti-Furga}, year = {2014}, date = {2014-04-01}, journal = {Nature}, volume = {508}, number = {7495}, pages = {222--227}, abstract = {Activated RAS GTPase signalling is a critical driver of oncogenic transformation and malignant disease. Cellular models of RAS-dependent cancers have been used to identify experimental small molecules, such as SCH51344, but their molecular mechanism of action remains generally unknown. Here, using a chemical proteomic approach, we identify the target of SCH51344 as the human mutT homologue MTH1 (also known as NUDT1), a nucleotide pool sanitizing enzyme. Loss-of-function of MTH1 impaired growth of KRAS tumour cells, whereas MTH1 overexpression mitigated sensitivity towards SCH51344. Searching for more drug-like inhibitors, we identified the kinase inhibitor crizotinib as a nanomolar suppressor of MTH1 activity. Surprisingly, the clinically used (R)-enantiomer of the drug was inactive, whereas the (S)-enantiomer selectively inhibited MTH1 catalytic activity. Enzymatic assays, chemical proteomic profiling, kinome-wide activity surveys and MTH1 co-crystal structures of both enantiomers provide a rationale for this remarkable stereospecificity. Disruption of nucleotide pool homeostasis via MTH1 inhibition by (S)-crizotinib induced an increase in DNA single-strand breaks, activated DNA repair in human colon carcinoma cells, and effectively suppressed tumour growth in animal models. Our results propose (S)-crizotinib as an attractive chemical entity for further pre-clinical evaluation, and small-molecule inhibitors of MTH1 in general as a promising novel class of anticancer agents.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4150021PMC4150021] [DOI:hrefhttp://dx.doi.org/10.1038/nature1319410.1038/nature13194] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2469522524695225]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Activated RAS GTPase signalling is a critical driver of oncogenic transformation and malignant disease. Cellular models of RAS-dependent cancers have been used to identify experimental small molecules, such as SCH51344, but their molecular mechanism of action remains generally unknown. Here, using a chemical proteomic approach, we identify the target of SCH51344 as the human mutT homologue MTH1 (also known as NUDT1), a nucleotide pool sanitizing enzyme. Loss-of-function of MTH1 impaired growth of KRAS tumour cells, whereas MTH1 overexpression mitigated sensitivity towards SCH51344. Searching for more drug-like inhibitors, we identified the kinase inhibitor crizotinib as a nanomolar suppressor of MTH1 activity. Surprisingly, the clinically used (R)-enantiomer of the drug was inactive, whereas the (S)-enantiomer selectively inhibited MTH1 catalytic activity. Enzymatic assays, chemical proteomic profiling, kinome-wide activity surveys and MTH1 co-crystal structures of both enantiomers provide a rationale for this remarkable stereospecificity. Disruption of nucleotide pool homeostasis via MTH1 inhibition by (S)-crizotinib induced an increase in DNA single-strand breaks, activated DNA repair in human colon carcinoma cells, and effectively suppressed tumour growth in animal models. Our results propose (S)-crizotinib as an attractive chemical entity for further pre-clinical evaluation, and small-molecule inhibitors of MTH1 in general as a promising novel class of anticancer agents. |
Gad, H; Koolmeister, T; Jemth, A S; Eshtad, S; Jacques, S A; Strom, C E; Svensson, L M; Schultz, N; Lundback, T; Einarsdottir, B O; Saleh, A; Gokturk, C; Baranczewski, P; Svensson, R; Berntsson, R P; Gustafsson, R; Stromberg, K; Sanjiv, K; Jacques-Cordonnier, M C; Desroses, M; Gustavsson, A L; Olofsson, R; Johansson, F; Homan, E J; Loseva, O; Brautigam, L; Johansson, L; Hoglund, A; Hagenkort, A; Pham, T; Altun, M; Gaugaz, F Z; Vikingsson, S; Evers, B; Henriksson, M; Vallin, K S; Wallner, O A; Hammarstrom, L G; Wiita, E; Almlof, I; Kalderen, C; Axelsson, H; Djureinovic, T; Puigvert, J C; Haggblad, M; Jeppsson, F; Martens, U; Lundin, C; Lundgren, B; Granelli, I; Jensen, A J; Artursson, P; Nilsson, J A; Stenmark, P; Scobie, M; Berglund, U W; Helleday, T MŦĦ1 inhibition eradicates cancer by preventing sanitation of the dNŦP pool Journal Article Nature, 508 (7495), pp. 215–221, 2014, ([DOI:hrefhttp://dx.doi.org/10.1038/nature1318110.1038/nature13181] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2469522424695224]). @article{pmid24695224, title = {MŦĦ1 inhibition eradicates cancer by preventing sanitation of the dNŦP pool}, author = { H. Gad and T. Koolmeister and A. S. Jemth and S. Eshtad and S. A. Jacques and C. E. Strom and L. M. Svensson and N. Schultz and T. Lundback and B. O. Einarsdottir and A. Saleh and C. Gokturk and P. Baranczewski and R. Svensson and R. P. Berntsson and R. Gustafsson and K. Stromberg and K. Sanjiv and M. C. Jacques-Cordonnier and M. Desroses and A. L. Gustavsson and R. Olofsson and F. Johansson and E. J. Homan and O. Loseva and L. Brautigam and L. Johansson and A. Hoglund and A. Hagenkort and T. Pham and M. Altun and F. Z. Gaugaz and S. Vikingsson and B. Evers and M. Henriksson and K. S. Vallin and O. A. Wallner and L. G. Hammarstrom and E. Wiita and I. Almlof and C. Kalderen and H. Axelsson and T. Djureinovic and J. C. Puigvert and M. Haggblad and F. Jeppsson and U. Martens and C. Lundin and B. Lundgren and I. Granelli and A. J. Jensen and P. Artursson and J. A. Nilsson and P. Stenmark and M. Scobie and U. W. Berglund and T. Helleday}, year = {2014}, date = {2014-04-01}, journal = {Nature}, volume = {508}, number = {7495}, pages = {215--221}, abstract = {Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bind in the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.}, note = {[DOI:hrefhttp://dx.doi.org/10.1038/nature1318110.1038/nature13181] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2469522424695224]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bind in the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal. |
Gao, C; Bourke, E; Scobie, M; Famme, M A; Koolmeister, T; Helleday, T; Eriksson, L A; Lowndes, N F; Brown, J A Rational design and validation of a Ŧip60 histone acetyltransferase inhibitor Journal Article Sci Rep, 4 , pp. 5372, 2014, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4064327PMC4064327] [DOI:hrefhttp://dx.doi.org/10.1038/srep0537210.1038/srep05372] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2494793824947938]). @article{pmid24947938, title = {Rational design and validation of a Ŧip60 histone acetyltransferase inhibitor}, author = { C. Gao and E. Bourke and M. Scobie and M. A. Famme and T. Koolmeister and T. Helleday and L. A. Eriksson and N. F. Lowndes and J. A. Brown}, year = {2014}, date = {2014-01-01}, journal = {Sci Rep}, volume = {4}, pages = {5372}, abstract = {Histone acetylation is required for many aspects of gene regulation, genome maintenance and metabolism and dysfunctional acetylation is implicated in numerous diseases, including cancer. Acetylation is regulated by histone acetyltransferases (HATs) and histone deacetylases and currently, few general HAT inhibitors have been described. We identified the HAT Tip60 as an excellent candidate for targeted drug development, as Tip60 is a key mediator of the DNA damage response and transcriptional co-activator. Our modeling of Tip60 indicated that the active binding pocket possesses opposite charges at each end, with the positive charges attributed to two specific side chains. We used structure based drug design to develop a novel Tip60 inhibitor, TH1834, to fit this specific pocket. We demonstrate that TH1834 significantly inhibits Tip60 activity in vitro and treating cells with TH1834 results in apoptosis and increased unrepaired DNA damage (following ionizing radiation treatment) in breast cancer but not control cell lines. Furthermore, TH1834 did not affect the activity of related HAT MOF, as indicated by H4K16Ac, demonstrating specificity. The modeling and validation of the small molecule inhibitor TH1834 represents a first step towards developing additional specific, targeted inhibitors of Tip60 that may lead to further improvements in the treatment of breast cancer.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4064327PMC4064327] [DOI:hrefhttp://dx.doi.org/10.1038/srep0537210.1038/srep05372] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2494793824947938]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Histone acetylation is required for many aspects of gene regulation, genome maintenance and metabolism and dysfunctional acetylation is implicated in numerous diseases, including cancer. Acetylation is regulated by histone acetyltransferases (HATs) and histone deacetylases and currently, few general HAT inhibitors have been described. We identified the HAT Tip60 as an excellent candidate for targeted drug development, as Tip60 is a key mediator of the DNA damage response and transcriptional co-activator. Our modeling of Tip60 indicated that the active binding pocket possesses opposite charges at each end, with the positive charges attributed to two specific side chains. We used structure based drug design to develop a novel Tip60 inhibitor, TH1834, to fit this specific pocket. We demonstrate that TH1834 significantly inhibits Tip60 activity in vitro and treating cells with TH1834 results in apoptosis and increased unrepaired DNA damage (following ionizing radiation treatment) in breast cancer but not control cell lines. Furthermore, TH1834 did not affect the activity of related HAT MOF, as indicated by H4K16Ac, demonstrating specificity. The modeling and validation of the small molecule inhibitor TH1834 represents a first step towards developing additional specific, targeted inhibitors of Tip60 that may lead to further improvements in the treatment of breast cancer. |
Llona-Minguez, S; Hoglund, A; Jacques, S A; Koolmeister, T; Helleday, T Chemical strategies for development of AŦR inhibitors Journal Article Expert Rev Mol Med, 16 , pp. e10, 2014, ([DOI:hrefhttp://dx.doi.org/10.1017/erm.2014.1010.1017/erm.2014.10] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2481071524810715]). @article{pmid24810715, title = {Chemical strategies for development of AŦR inhibitors}, author = { S. Llona-Minguez and A. Hoglund and S. A. Jacques and T. Koolmeister and T. Helleday}, year = {2014}, date = {2014-01-01}, journal = {Expert Rev Mol Med}, volume = {16}, pages = {e10}, abstract = {ATR protein kinase is one of the key players in maintaining genome integrity and coordinating of the DNA damage response and repair signalling pathways. Inhibition of ATR prevents signalling from stalled replication forks and enhances the formation of DNA damage, particularly under conditions of replication stress present in cancers. For this reason ATR/CHK1 checkpoint inhibitors can potentiate the effect of DNA cross-linking agents, as evidenced by ATR inhibitors recently entering human clinical trials. This review aims to compile the existing literature on small molecule inhibitors of ATR, both from academia and the pharmaceutical industry, and will provide the reader with a comprehensive summary of this promising oncology target.}, note = {[DOI:hrefhttp://dx.doi.org/10.1017/erm.2014.1010.1017/erm.2014.10] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2481071524810715]}, keywords = {}, pubstate = {published}, tppubtype = {article} } ATR protein kinase is one of the key players in maintaining genome integrity and coordinating of the DNA damage response and repair signalling pathways. Inhibition of ATR prevents signalling from stalled replication forks and enhances the formation of DNA damage, particularly under conditions of replication stress present in cancers. For this reason ATR/CHK1 checkpoint inhibitors can potentiate the effect of DNA cross-linking agents, as evidenced by ATR inhibitors recently entering human clinical trials. This review aims to compile the existing literature on small molecule inhibitors of ATR, both from academia and the pharmaceutical industry, and will provide the reader with a comprehensive summary of this promising oncology target. |
Costantino, L; Sotiriou, S K; Rantala, J K; Magin, S; Mladenov, E; Helleday, T; Haber, J E; Iliakis, G; Kallioniemi, O P; Halazonetis, T D Break-induced replication repair of damaged forks induces genomic duplications in human cells Journal Article Science, 343 (6166), pp. 88–91, 2014, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4047655PMC4047655] [DOI:hrefhttp://dx.doi.org/10.1126/science.124321110.1126/science.1243211] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2431061124310611]). @article{pmid24310611, title = {Break-induced replication repair of damaged forks induces genomic duplications in human cells}, author = { L. Costantino and S. K. Sotiriou and J. K. Rantala and S. Magin and E. Mladenov and T. Helleday and J. E. Haber and G. Iliakis and O. P. Kallioniemi and T. D. Halazonetis}, year = {2014}, date = {2014-01-01}, journal = {Science}, volume = {343}, number = {6166}, pages = {88--91}, abstract = {In budding yeast, one-ended DNA double-strand breaks (DSBs) and damaged replication forks are repaired by break-induced replication (BIR), a homologous recombination pathway that requires the Pol32 subunit of DNA polymerase delta. DNA replication stress is prevalent in cancer, but BIR has not been characterized in mammals. In a cyclin E overexpression model of DNA replication stress, POLD3, the human ortholog of POL32, was required for cell cycle progression and processive DNA synthesis. Segmental genomic duplications induced by cyclin E overexpression were also dependent on POLD3, as were BIR-mediated recombination events captured with a specialized DSB repair assay. We propose that BIR repairs damaged replication forks in mammals, accounting for the high frequency of genomic duplications in human cancers.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4047655PMC4047655] [DOI:hrefhttp://dx.doi.org/10.1126/science.124321110.1126/science.1243211] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2431061124310611]}, keywords = {}, pubstate = {published}, tppubtype = {article} } In budding yeast, one-ended DNA double-strand breaks (DSBs) and damaged replication forks are repaired by break-induced replication (BIR), a homologous recombination pathway that requires the Pol32 subunit of DNA polymerase delta. DNA replication stress is prevalent in cancer, but BIR has not been characterized in mammals. In a cyclin E overexpression model of DNA replication stress, POLD3, the human ortholog of POL32, was required for cell cycle progression and processive DNA synthesis. Segmental genomic duplications induced by cyclin E overexpression were also dependent on POLD3, as were BIR-mediated recombination events captured with a specialized DSB repair assay. We propose that BIR repairs damaged replication forks in mammals, accounting for the high frequency of genomic duplications in human cancers. |
2013 |
Helleday, T PrimPol breaks replication barriers Journal Article Nat. Struct. Mol. Biol., 20 (12), pp. 1348–1350, 2013, ([DOI:hrefhttp://dx.doi.org/10.1038/nsmb.272710.1038/nsmb.2727] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2430491424304914]). @article{pmid24304914, title = {PrimPol breaks replication barriers}, author = { T. Helleday}, year = {2013}, date = {2013-12-01}, journal = {Nat. Struct. Mol. Biol.}, volume = {20}, number = {12}, pages = {1348--1350}, abstract = {Faithful bypass of replication forks encountering obstructive DNA lesions is essential to prevent fork collapse and cell death. PrimPol is a new human primase and translesion polymerase that is able to bypass fork-blocking UV-induced lesions and to restart replication by origin-independent repriming.}, note = {[DOI:hrefhttp://dx.doi.org/10.1038/nsmb.272710.1038/nsmb.2727] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2430491424304914]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Faithful bypass of replication forks encountering obstructive DNA lesions is essential to prevent fork collapse and cell death. PrimPol is a new human primase and translesion polymerase that is able to bypass fork-blocking UV-induced lesions and to restart replication by origin-independent repriming. |
Gubanova, E; Issaeva, N; Gokturk, C; Djureinovic, T; Helleday, T SMG-1 suppresses CĐK2 and tumor growth by regulating both the p53 and Cdc25A signaling pathways Journal Article Cell Cycle, 12 (24), pp. 3770–3780, 2013, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3905069PMC3905069] [DOI:hrefhttp://dx.doi.org/10.4161/cc.2666010.4161/cc.26660] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2410763224107632]). @article{pmid24107632, title = {SMG-1 suppresses CĐK2 and tumor growth by regulating both the p53 and Cdc25A signaling pathways}, author = { E. Gubanova and N. Issaeva and C. Gokturk and T. Djureinovic and T. Helleday}, year = {2013}, date = {2013-12-01}, journal = {Cell Cycle}, volume = {12}, number = {24}, pages = {3770--3780}, abstract = {The DNA damage response is coordinated by phosphatidylinositol 3-kinase-related kinases, ATM, ATR, and DNA-PK. SMG-1 is the least studied stress-responsive member of this family. Here, we show that SMG-1 regulates the G 1/S checkpoint through both a p53-dependent, and a p53-independent pathway. We identify Cdc25A as a new SMG-1 substrate, and show that cells depleted of SMG-1 exhibit prolonged Cdc25A stability, failing to inactivate CDK2 in response to radiation. Given an increased tumor growth following depletion of SMG-1, our data demonstrate a novel role for SMG-1 in regulating Cdc25A and suppressing oncogenic CDK2 driven proliferation, confirming SMG-1 as a tumor suppressor.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3905069PMC3905069] [DOI:hrefhttp://dx.doi.org/10.4161/cc.2666010.4161/cc.26660] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2410763224107632]}, keywords = {}, pubstate = {published}, tppubtype = {article} } The DNA damage response is coordinated by phosphatidylinositol 3-kinase-related kinases, ATM, ATR, and DNA-PK. SMG-1 is the least studied stress-responsive member of this family. Here, we show that SMG-1 regulates the G 1/S checkpoint through both a p53-dependent, and a p53-independent pathway. We identify Cdc25A as a new SMG-1 substrate, and show that cells depleted of SMG-1 exhibit prolonged Cdc25A stability, failing to inactivate CDK2 in response to radiation. Given an increased tumor growth following depletion of SMG-1, our data demonstrate a novel role for SMG-1 in regulating Cdc25A and suppressing oncogenic CDK2 driven proliferation, confirming SMG-1 as a tumor suppressor. |
Helleday, T Putting poly (AĐP-ribose) polymerase and other ĐNA repair inhibitors into clinical practice Journal Article Curr Opin Oncol, 25 (6), pp. 609–614, 2013, ([DOI:hrefhttp://dx.doi.org/10.1097/CCO.000000000000001610.1097/CCO.0000000000000016] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2409710424097104]). @article{pmid24097104, title = {Putting poly (AĐP-ribose) polymerase and other ĐNA repair inhibitors into clinical practice}, author = { T. Helleday}, year = {2013}, date = {2013-11-01}, journal = {Curr Opin Oncol}, volume = {25}, number = {6}, pages = {609--614}, abstract = {Poly (ADP-ribose) polymerase (PARP) and other DNA repair inhibitors are currently tested in numerous clinical trials, with variable success. Inhibitors are used in monotherapy, for example, PARP inhibitors in BRCA mutated cancers, or more widely in combination treatments. DNA repair inhibitors have, as chemotherapy, great potential for long-term disease control, or potentially even cures. However, the design of clinical trials using DNA repair inhibitors is intricate, as these inhibitors may also potentiate normal tissue toxicity without improving overall disease control. Recent findings of mechanism of action of PARP inhibitors and other DNA repair inhibitors are presented, and how the underlying genetic background and interplay between DNA repair pathways influence the choice of tumour location and combination strategies. The hallmark of individualized cancer therapy is to be able to genetically distinguish the responding subclass of cancer patients, and it is widely used when targeting oncogenes. The PARP inhibitors in BRCA mutated cancers also demonstrate that this approach is possible in a synthetic lethal context. There is strong proof-of-concept for DNA repair inhibitors being a useful anticancer strategy in well designed clinical trials.}, note = {[DOI:hrefhttp://dx.doi.org/10.1097/CCO.000000000000001610.1097/CCO.0000000000000016] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2409710424097104]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Poly (ADP-ribose) polymerase (PARP) and other DNA repair inhibitors are currently tested in numerous clinical trials, with variable success. Inhibitors are used in monotherapy, for example, PARP inhibitors in BRCA mutated cancers, or more widely in combination treatments. DNA repair inhibitors have, as chemotherapy, great potential for long-term disease control, or potentially even cures. However, the design of clinical trials using DNA repair inhibitors is intricate, as these inhibitors may also potentiate normal tissue toxicity without improving overall disease control. Recent findings of mechanism of action of PARP inhibitors and other DNA repair inhibitors are presented, and how the underlying genetic background and interplay between DNA repair pathways influence the choice of tumour location and combination strategies. The hallmark of individualized cancer therapy is to be able to genetically distinguish the responding subclass of cancer patients, and it is widely used when targeting oncogenes. The PARP inhibitors in BRCA mutated cancers also demonstrate that this approach is possible in a synthetic lethal context. There is strong proof-of-concept for DNA repair inhibitors being a useful anticancer strategy in well designed clinical trials. |
Kopper, F; Bierwirth, C; Schon, M; Kunze, M; Elvers, I; Kranz, D; Saini, P; Menon, M B; Walter, D; S?rensen, C S; Gaestel, M; Helleday, T; Schon, M P; Dobbelstein, M Đamage-induced ĐNA replication stalling relies on MAPK-activated protein kinase 2 activity Journal Article Proc. Natl. Acad. Sci. U.S.A., 110 (42), pp. 16856–16861, 2013, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3801042PMC3801042] [DOI:hrefhttp://dx.doi.org/10.1073/pnas.130435511010.1073/pnas.1304355110] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2408211524082115]). @article{pmid24082115, title = {Đamage-induced ĐNA replication stalling relies on MAPK-activated protein kinase 2 activity}, author = { F. Kopper and C. Bierwirth and M. Schon and M. Kunze and I. Elvers and D. Kranz and P. Saini and M. B. Menon and D. Walter and C. S. S?rensen and M. Gaestel and T. Helleday and M. P. Schon and M. Dobbelstein}, year = {2013}, date = {2013-10-01}, journal = {Proc. Natl. Acad. Sci. U.S.A.}, volume = {110}, number = {42}, pages = {16856--16861}, abstract = {DNA damage can obstruct replication forks, resulting in replicative stress. By siRNA screening, we identified kinases involved in the accumulation of phosphohistone 2AX (γH2AX) upon UV irradiation-induced replication stress. Surprisingly, the strongest reduction of phosphohistone 2AX followed knockdown of the MAP kinase-activated protein kinase 2 (MK2), a kinase currently implicated in p38 stress signaling and G2 arrest. Depletion or inhibition of MK2 also protected cells from DNA damage-induced cell death, and mice deficient for MK2 displayed decreased apoptosis in the skin upon UV irradiation. Moreover, MK2 activity was required for damage response, accumulation of ssDNA, and decreased survival when cells were treated with the nucleoside analogue gemcitabine or when the checkpoint kinase Chk1 was antagonized. By using DNA fiber assays, we found that MK2 inhibition or knockdown rescued DNA replication impaired by gemcitabine or by Chk1 inhibition. This rescue strictly depended on translesion DNA polymerases. In conclusion, instead of being an unavoidable consequence of DNA damage, alterations of replication speed and origin firing depend on MK2-mediated signaling.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3801042PMC3801042] [DOI:hrefhttp://dx.doi.org/10.1073/pnas.130435511010.1073/pnas.1304355110] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2408211524082115]}, keywords = {}, pubstate = {published}, tppubtype = {article} } DNA damage can obstruct replication forks, resulting in replicative stress. By siRNA screening, we identified kinases involved in the accumulation of phosphohistone 2AX (γH2AX) upon UV irradiation-induced replication stress. Surprisingly, the strongest reduction of phosphohistone 2AX followed knockdown of the MAP kinase-activated protein kinase 2 (MK2), a kinase currently implicated in p38 stress signaling and G2 arrest. Depletion or inhibition of MK2 also protected cells from DNA damage-induced cell death, and mice deficient for MK2 displayed decreased apoptosis in the skin upon UV irradiation. Moreover, MK2 activity was required for damage response, accumulation of ssDNA, and decreased survival when cells were treated with the nucleoside analogue gemcitabine or when the checkpoint kinase Chk1 was antagonized. By using DNA fiber assays, we found that MK2 inhibition or knockdown rescued DNA replication impaired by gemcitabine or by Chk1 inhibition. This rescue strictly depended on translesion DNA polymerases. In conclusion, instead of being an unavoidable consequence of DNA damage, alterations of replication speed and origin firing depend on MK2-mediated signaling. |
Zalmas, L P; Coutts, A S; Helleday, T; Thangue, La N B E2F-7 couples ĐNA damage-dependent transcription with the ĐNA repair process Journal Article Cell Cycle, 12 (18), pp. 3037–3051, 2013, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3875678PMC3875678] [DOI:hrefhttp://dx.doi.org/10.4161/cc.2607810.4161/cc.26078] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2397410123974101]). @article{pmid23974101, title = {E2F-7 couples ĐNA damage-dependent transcription with the ĐNA repair process}, author = { L. P. Zalmas and A. S. Coutts and T. Helleday and N. B. La Thangue}, year = {2013}, date = {2013-09-01}, journal = {Cell Cycle}, volume = {12}, number = {18}, pages = {3037--3051}, abstract = {The cellular response to DNA damage, mediated by the DNA repair process, is essential in maintaining the integrity and stability of the genome. E2F-7 is an atypical member of the E2F family with a role in negatively regulating transcription and cell cycle progression under DNA damage. Surprisingly, we found that E2F-7 makes a transcription-independent contribution to the DNA repair process, which involves E2F-7 locating to and binding damaged DNA. Further, E2F-7 recruits CtBP and HDAC to the damaged DNA, altering the local chromatin environment of the DNA lesion. Importantly, the E2F-7 gene is a target for somatic mutation in human cancer and tumor-derived mutant alleles encode proteins with compromised transcription and DNA repair properties. Our results establish that E2F-7 participates in 2 closely linked processes, allowing it to directly couple the expression of genes involved in the DNA damage response with the DNA repair machinery, which has relevance in human malignancy.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3875678PMC3875678] [DOI:hrefhttp://dx.doi.org/10.4161/cc.2607810.4161/cc.26078] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2397410123974101]}, keywords = {}, pubstate = {published}, tppubtype = {article} } The cellular response to DNA damage, mediated by the DNA repair process, is essential in maintaining the integrity and stability of the genome. E2F-7 is an atypical member of the E2F family with a role in negatively regulating transcription and cell cycle progression under DNA damage. Surprisingly, we found that E2F-7 makes a transcription-independent contribution to the DNA repair process, which involves E2F-7 locating to and binding damaged DNA. Further, E2F-7 recruits CtBP and HDAC to the damaged DNA, altering the local chromatin environment of the DNA lesion. Importantly, the E2F-7 gene is a target for somatic mutation in human cancer and tumor-derived mutant alleles encode proteins with compromised transcription and DNA repair properties. Our results establish that E2F-7 participates in 2 closely linked processes, allowing it to directly couple the expression of genes involved in the DNA damage response with the DNA repair machinery, which has relevance in human malignancy. |
Jones, R M; Mortusewicz, O; Afzal, I; Lorvellec, M; Garcia, P; Helleday, T; Petermann, E Increased replication initiation and conflicts with transcription underlie Cyclin E-induced replication stress Journal Article Oncogene, 32 (32), pp. 3744–3753, 2013, ([DOI:hrefhttp://dx.doi.org/10.1038/onc.2012.38710.1038/onc.2012.387] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2294564522945645]). @article{pmid22945645, title = {Increased replication initiation and conflicts with transcription underlie Cyclin E-induced replication stress}, author = { R. M. Jones and O. Mortusewicz and I. Afzal and M. Lorvellec and P. Garcia and T. Helleday and E. Petermann}, year = {2013}, date = {2013-08-01}, journal = {Oncogene}, volume = {32}, number = {32}, pages = {3744--3753}, abstract = {It has become increasingly clear that oncogenes not only provide aberrant growth signals to cells but also cause DNA damage at replication forks (replication stress), which activate the ataxia telangiectasia mutated (ATM)/p53-dependent tumor barrier. Here we studied underlying mechanisms of oncogene-induced replication stress in cells overexpressing the oncogene Cyclin E. Cyclin E overexpression is associated with increased firing of replication origins, impaired replication fork progression and DNA damage that activates RAD51-mediated recombination. By inhibiting replication initiation factors, we show that Cyclin E-induced replication slowing and DNA damage is a consequence of excessive origin firing. A significant amount of Cyclin E-induced replication slowing is due to interference between replication and transcription, which also underlies the activation of homologous recombination. Our data suggest that Cyclin E-induced replication stress is caused by deregulation of replication initiation and increased interference between replication and transcription, which results in impaired replication fork progression and DNA damage triggering the tumor barrier or cancer-promoting mutations.}, note = {[DOI:hrefhttp://dx.doi.org/10.1038/onc.2012.38710.1038/onc.2012.387] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2294564522945645]}, keywords = {}, pubstate = {published}, tppubtype = {article} } It has become increasingly clear that oncogenes not only provide aberrant growth signals to cells but also cause DNA damage at replication forks (replication stress), which activate the ataxia telangiectasia mutated (ATM)/p53-dependent tumor barrier. Here we studied underlying mechanisms of oncogene-induced replication stress in cells overexpressing the oncogene Cyclin E. Cyclin E overexpression is associated with increased firing of replication origins, impaired replication fork progression and DNA damage that activates RAD51-mediated recombination. By inhibiting replication initiation factors, we show that Cyclin E-induced replication slowing and DNA damage is a consequence of excessive origin firing. A significant amount of Cyclin E-induced replication slowing is due to interference between replication and transcription, which also underlies the activation of homologous recombination. Our data suggest that Cyclin E-induced replication stress is caused by deregulation of replication initiation and increased interference between replication and transcription, which results in impaired replication fork progression and DNA damage triggering the tumor barrier or cancer-promoting mutations. |
Somaiah, N; Yarnold, J; Lagerqvist, A; Rothkamm, K; Helleday, T Ħomologous recombination mediates cellular resistance and fraction size sensitivity to radiation therapy Journal Article Radiother Oncol, 108 (1), pp. 155–161, 2013, ([DOI:hrefhttp://dx.doi.org/10.1016/j.radonc.2013.05.01210.1016/j.radonc.2013.05.012] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2374669623746696]). @article{pmid23746696, title = {Ħomologous recombination mediates cellular resistance and fraction size sensitivity to radiation therapy}, author = { N. Somaiah and J. Yarnold and A. Lagerqvist and K. Rothkamm and T. Helleday}, year = {2013}, date = {2013-07-01}, journal = {Radiother Oncol}, volume = {108}, number = {1}, pages = {155--161}, abstract = {Cellular sensitivity to radiotherapy total dose and fraction size is strongly influenced by DNA double strand break (DSB) repair. Here, we investigate response to radiotherapy fraction size using CHO cell lines deficient in specific DNA repair pathways in response to radiation induced DNA double strand breaks (DSB). We irradiated CHO cell lines, AA8 (WT), irs1SF (XRCC3-), V3-3 (DNA-PKcs-) and EM9 (XRCC1-) with 16 Gy in 1 Gy daily fractions over 3 weeks or 16 Gy in 4 Gy daily fractions over 4 days, and studied clonogenic survival, DNA DSB repair kinetics (RAD51 and 53BP1 foci staining) and cell cycle profiles (flow cytometry). In response to fractionated radiotherapy, wild-type and DNA repair defective cells accumulated in late S/G2 phase. In cells proficient in homologous recombination (HR), accumulation in S/G2 resulted in reduced sensitivity to fraction size and increased cellular resistance (clonogenic survival). Sensitivity to fraction size was also lost in NHEJ-defective V3-3 cells, which likely rely on functional HR. By contrast, HR-defective irs1SF cells, with functional NHEJ, remained equally sensitive to fractionation throughout the 3-week treatment. The high fidelity of HR, which is independent of induced DNA damage level, is postulated to explain the low fractionation sensitivity and cellular resistance of cells in S/G2 phase. In conclusion, our results suggest that HR mediates resistance to fractionated radiotherapy, an observation that may help future efforts to improve radiotherapy outcome.}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/j.radonc.2013.05.01210.1016/j.radonc.2013.05.012] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2374669623746696]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Cellular sensitivity to radiotherapy total dose and fraction size is strongly influenced by DNA double strand break (DSB) repair. Here, we investigate response to radiotherapy fraction size using CHO cell lines deficient in specific DNA repair pathways in response to radiation induced DNA double strand breaks (DSB). We irradiated CHO cell lines, AA8 (WT), irs1SF (XRCC3-), V3-3 (DNA-PKcs-) and EM9 (XRCC1-) with 16 Gy in 1 Gy daily fractions over 3 weeks or 16 Gy in 4 Gy daily fractions over 4 days, and studied clonogenic survival, DNA DSB repair kinetics (RAD51 and 53BP1 foci staining) and cell cycle profiles (flow cytometry). In response to fractionated radiotherapy, wild-type and DNA repair defective cells accumulated in late S/G2 phase. In cells proficient in homologous recombination (HR), accumulation in S/G2 resulted in reduced sensitivity to fraction size and increased cellular resistance (clonogenic survival). Sensitivity to fraction size was also lost in NHEJ-defective V3-3 cells, which likely rely on functional HR. By contrast, HR-defective irs1SF cells, with functional NHEJ, remained equally sensitive to fractionation throughout the 3-week treatment. The high fidelity of HR, which is independent of induced DNA damage level, is postulated to explain the low fractionation sensitivity and cellular resistance of cells in S/G2 phase. In conclusion, our results suggest that HR mediates resistance to fractionated radiotherapy, an observation that may help future efforts to improve radiotherapy outcome. |
Orta, M L; Calderon-Montano, J M; Dominguez, I; Pastor, N; Burgos-Moron, E; Lopez-Lazaro, M; Cortes, F; Mateos, S; Helleday, T 5-Aza-2'-deoxycytidine causes replication lesions that require Fanconi anemia-dependent homologous recombination for repair Journal Article Nucleic Acids Res., 41 (11), pp. 5827–5836, 2013, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3675485PMC3675485] [DOI:hrefhttp://dx.doi.org/10.1093/nar/gkt27010.1093/nar/gkt270] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2360953723609537]). @article{pmid23609537, title = {5-Aza-2'-deoxycytidine causes replication lesions that require Fanconi anemia-dependent homologous recombination for repair}, author = { M. L. Orta and J. M. Calderon-Montano and I. Dominguez and N. Pastor and E. Burgos-Moron and M. Lopez-Lazaro and F. Cortes and S. Mateos and T. Helleday}, year = {2013}, date = {2013-06-01}, journal = {Nucleic Acids Res.}, volume = {41}, number = {11}, pages = {5827--5836}, abstract = {5-Aza-2'-deoxycytidine (5-azadC) is a DNA methyltransferase (DNMT) inhibitor increasingly used in treatments of hematological diseases and works by being incorporated into DNA and trapping DNMT. It is unclear what DNA lesions are caused by 5-azadC and if such are substrates for DNA repair. Here, we identify that 5-azadC induces DNA damage as measured by γ-H2AX and 53BP1 foci. Furthermore, 5-azadC induces radial chromosomes and chromatid breaks that depend on active replication, which altogether suggest that trapped DNMT collapses oncoming replication forks into double-strand breaks. We demonstrate that RAD51-mediated homologous recombination (HR) is activated to repair 5-azadC collapsed replication forks. Fanconi anemia (FA) is a rare autosomal recessive disorder, and deaths are often associated with leukemia. Here, we show that FANCG-deficient cells fail to trigger HR-mediated repair of 5-azadC-induced lesions, leading to accumulation of chromatid breaks and inter-chromosomal radial fusions as well as hypersensitivity to the cytotoxic effects of 5-azadC. These data demonstrate that the FA pathway is important to protect from 5-azadC-induced toxicity. Altogether, our data demonstrate that cytotoxicity of the epigenetic drug 5-azadC can, at least in part, be explained by collapsed replication forks requiring FA-mediated HR for repair.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3675485PMC3675485] [DOI:hrefhttp://dx.doi.org/10.1093/nar/gkt27010.1093/nar/gkt270] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2360953723609537]}, keywords = {}, pubstate = {published}, tppubtype = {article} } 5-Aza-2'-deoxycytidine (5-azadC) is a DNA methyltransferase (DNMT) inhibitor increasingly used in treatments of hematological diseases and works by being incorporated into DNA and trapping DNMT. It is unclear what DNA lesions are caused by 5-azadC and if such are substrates for DNA repair. Here, we identify that 5-azadC induces DNA damage as measured by γ-H2AX and 53BP1 foci. Furthermore, 5-azadC induces radial chromosomes and chromatid breaks that depend on active replication, which altogether suggest that trapped DNMT collapses oncoming replication forks into double-strand breaks. We demonstrate that RAD51-mediated homologous recombination (HR) is activated to repair 5-azadC collapsed replication forks. Fanconi anemia (FA) is a rare autosomal recessive disorder, and deaths are often associated with leukemia. Here, we show that FANCG-deficient cells fail to trigger HR-mediated repair of 5-azadC-induced lesions, leading to accumulation of chromatid breaks and inter-chromosomal radial fusions as well as hypersensitivity to the cytotoxic effects of 5-azadC. These data demonstrate that the FA pathway is important to protect from 5-azadC-induced toxicity. Altogether, our data demonstrate that cytotoxicity of the epigenetic drug 5-azadC can, at least in part, be explained by collapsed replication forks requiring FA-mediated HR for repair. |
Al-Ubaidi, F L; Schultz, N; Loseva, O; Egevad, L; Granfors, T; Helleday, T Castration therapy results in decreased Ku70 levels in prostate cancer Journal Article Clin. Cancer Res., 19 (6), pp. 1547–1556, 2013, ([DOI:hrefhttp://dx.doi.org/10.1158/1078-0432.CCR-12-279510.1158/1078-0432.CCR-12-2795] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2334931623349316]). @article{pmid23349316, title = {Castration therapy results in decreased Ku70 levels in prostate cancer}, author = { F. L. Al-Ubaidi and N. Schultz and O. Loseva and L. Egevad and T. Granfors and T. Helleday}, year = {2013}, date = {2013-03-01}, journal = {Clin. Cancer Res.}, volume = {19}, number = {6}, pages = {1547--1556}, abstract = {Neoadjuvant castration improves response to radiotherapy of prostate cancer. Here, we determine whether castration therapy impairs nonhomologous end-joining (NHEJ) repair of DNA double-strand breaks (DSB) by downregulating Ku70 protein expression. Twenty patients with locally advanced prostate cancer were enrolled, and 6 to 12 needle core biopsy specimens were taken from the prostate of each patient before treatment. Bilateral orchidectomy was conducted in eight patients and 12 patients were treated with a GnRH agonist. After castration, two to four similar biopsies were obtained, and the levels of Ku70 and γ-H2AX foci were determined by immunofluorescence in verified cancer tissues. We observed that the androgen receptor binds directly to Ku70 in prostate tissue. We also found a reduction of the Ku70 protein levels in the cell nuclei in 12 of 14 patients (P < 0.001) after castration. The reduction in Ku70 expression correlated significantly with decreased serum prostate-specific antigen (PSA) levels after castration, suggesting that androgen receptor activity regulates Ku70 protein levels in prostate cancer tissue. Furthermore, a significant correlation between the reductions of Ku70 after castration versus changes induced of castration of γ-H2AX foci could be seen implicating a functional linkage of decreased Ku70 levels and impaired DNA repair. Castration therapy results in decreased levels of the Ku70 protein in prostate cancer cells. Because the Ku70 protein is essential for the NHEJ repair of DSBs and its downregulation impairs DNA repair, this offers a possible explanation for the increased radiosensitivity of prostate cancer cells following castration.}, note = {[DOI:hrefhttp://dx.doi.org/10.1158/1078-0432.CCR-12-279510.1158/1078-0432.CCR-12-2795] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2334931623349316]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Neoadjuvant castration improves response to radiotherapy of prostate cancer. Here, we determine whether castration therapy impairs nonhomologous end-joining (NHEJ) repair of DNA double-strand breaks (DSB) by downregulating Ku70 protein expression. Twenty patients with locally advanced prostate cancer were enrolled, and 6 to 12 needle core biopsy specimens were taken from the prostate of each patient before treatment. Bilateral orchidectomy was conducted in eight patients and 12 patients were treated with a GnRH agonist. After castration, two to four similar biopsies were obtained, and the levels of Ku70 and γ-H2AX foci were determined by immunofluorescence in verified cancer tissues. We observed that the androgen receptor binds directly to Ku70 in prostate tissue. We also found a reduction of the Ku70 protein levels in the cell nuclei in 12 of 14 patients (P < 0.001) after castration. The reduction in Ku70 expression correlated significantly with decreased serum prostate-specific antigen (PSA) levels after castration, suggesting that androgen receptor activity regulates Ku70 protein levels in prostate cancer tissue. Furthermore, a significant correlation between the reductions of Ku70 after castration versus changes induced of castration of γ-H2AX foci could be seen implicating a functional linkage of decreased Ku70 levels and impaired DNA repair. Castration therapy results in decreased levels of the Ku70 protein in prostate cancer cells. Because the Ku70 protein is essential for the NHEJ repair of DSBs and its downregulation impairs DNA repair, this offers a possible explanation for the increased radiosensitivity of prostate cancer cells following castration. |
Burrell, R A; McClelland, S E; Endesfelder, D; Groth, P; Weller, M C; Shaikh, N; Domingo, E; Kanu, N; Dewhurst, S M; Gronroos, E; Chew, S K; Rowan, A J; Schenk, A; Sheffer, M; Howell, M; Kschischo, M; Behrens, A; Helleday, T; Bartek, J; Tomlinson, I P; Swanton, C Replication stress links structural and numerical cancer chromosomal instability Journal Article Nature, 494 (7438), pp. 492–496, 2013, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4636055PMC4636055] [DOI:hrefhttp://dx.doi.org/10.1038/nature1193510.1038/nature11935] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2344642223446422]). @article{pmid23446422, title = {Replication stress links structural and numerical cancer chromosomal instability}, author = { R. A. Burrell and S. E. McClelland and D. Endesfelder and P. Groth and M. C. Weller and N. Shaikh and E. Domingo and N. Kanu and S. M. Dewhurst and E. Gronroos and S. K. Chew and A. J. Rowan and A. Schenk and M. Sheffer and M. Howell and M. Kschischo and A. Behrens and T. Helleday and J. Bartek and I. P. Tomlinson and C. Swanton}, year = {2013}, date = {2013-02-01}, journal = {Nature}, volume = {494}, number = {7438}, pages = {492--496}, abstract = {Cancer chromosomal instability (CIN) results in an increased rate of change of chromosome number and structure and generates intratumour heterogeneity. CIN is observed in most solid tumours and is associated with both poor prognosis and drug resistance. Understanding a mechanistic basis for CIN is therefore paramount. Here we find evidence for impaired replication fork progression and increased DNA replication stress in CIN(+) colorectal cancer (CRC) cells relative to CIN(-) CRC cells, with structural chromosome abnormalities precipitating chromosome missegregation in mitosis. We identify three new CIN-suppressor genes (PIGN (also known as MCD4), MEX3C (RKHD2) and ZNF516 (KIAA0222)) encoded on chromosome 18q that are subject to frequent copy number loss in CIN(+) CRC. Chromosome 18q loss was temporally associated with aneuploidy onset at the adenoma-carcinoma transition. CIN-suppressor gene silencing leads to DNA replication stress, structural chromosome abnormalities and chromosome missegregation. Supplementing cells with nucleosides, to alleviate replication-associated damage, reduces the frequency of chromosome segregation errors after CIN-suppressor gene silencing, and attenuates segregation errors and DNA damage in CIN(+) cells. These data implicate a central role for replication stress in the generation of structural and numerical CIN, which may inform new therapeutic approaches to limit intratumour heterogeneity.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4636055PMC4636055] [DOI:hrefhttp://dx.doi.org/10.1038/nature1193510.1038/nature11935] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2344642223446422]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Cancer chromosomal instability (CIN) results in an increased rate of change of chromosome number and structure and generates intratumour heterogeneity. CIN is observed in most solid tumours and is associated with both poor prognosis and drug resistance. Understanding a mechanistic basis for CIN is therefore paramount. Here we find evidence for impaired replication fork progression and increased DNA replication stress in CIN(+) colorectal cancer (CRC) cells relative to CIN(-) CRC cells, with structural chromosome abnormalities precipitating chromosome missegregation in mitosis. We identify three new CIN-suppressor genes (PIGN (also known as MCD4), MEX3C (RKHD2) and ZNF516 (KIAA0222)) encoded on chromosome 18q that are subject to frequent copy number loss in CIN(+) CRC. Chromosome 18q loss was temporally associated with aneuploidy onset at the adenoma-carcinoma transition. CIN-suppressor gene silencing leads to DNA replication stress, structural chromosome abnormalities and chromosome missegregation. Supplementing cells with nucleosides, to alleviate replication-associated damage, reduces the frequency of chromosome segregation errors after CIN-suppressor gene silencing, and attenuates segregation errors and DNA damage in CIN(+) cells. These data implicate a central role for replication stress in the generation of structural and numerical CIN, which may inform new therapeutic approaches to limit intratumour heterogeneity. |
Mortusewicz, O; Herr, P; Helleday, T Early replication fragile sites: where replication-transcription collisions cause genetic instability Journal Article EMBO J., 32 (4), pp. 493–495, 2013, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3579142PMC3579142] [DOI:hrefhttp://dx.doi.org/10.1038/emboj.2013.2010.1038/emboj.2013.20] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2337692223376922]). BibTeX | Tags: @article{pmid23376922, title = {Early replication fragile sites: where replication-transcription collisions cause genetic instability}, author = { O. Mortusewicz and P. Herr and T. Helleday}, year = {2013}, date = {2013-02-01}, journal = {EMBO J.}, volume = {32}, number = {4}, pages = {493--495}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3579142PMC3579142] [DOI:hrefhttp://dx.doi.org/10.1038/emboj.2013.2010.1038/emboj.2013.20] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2337692223376922]}, keywords = {}, pubstate = {published}, tppubtype = {article} } |
Kotova, N; Vare, D; Schultz, N; Meesters, Gradecka D; Stepnik, M; Grawe, J; Helleday, T; Jenssen, D Genotoxicity of alcohol is linked to ĐNA replication-associated damage and homologous recombination repair Journal Article Carcinogenesis, 34 (2), pp. 325–330, 2013, ([DOI:hrefhttp://dx.doi.org/10.1093/carcin/bgs34010.1093/carcin/bgs340] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2312521923125219]). @article{pmid23125219, title = {Genotoxicity of alcohol is linked to ĐNA replication-associated damage and homologous recombination repair}, author = { N. Kotova and D. Vare and N. Schultz and D. Gradecka Meesters and M. Stepnik and J. Grawe and T. Helleday and D. Jenssen}, year = {2013}, date = {2013-02-01}, journal = {Carcinogenesis}, volume = {34}, number = {2}, pages = {325--330}, abstract = {Although alcohol consumption is related to increased cancer risk, its molecular mechanism remains unclear. Here, we demonstrate that an intake of 10% alcohol for 4 weeks in rats is genotoxic due to induction of micronuclei. Acetaldehyde (AA), the first product of ethanol metabolism, is believed to be responsible for DNA damage induced by alcohol. Here, we observe that AA effectively blocks DNA replication elongation in mammalian cells, resulting in DNA double-strand breaks associated with replication. AA-induced DNA damage sites colocalize with the homologous recombination (HR) repair protein RAD51. HR measured in the hypoxhantineguaninefosforibosyltransferase (HPRT) gene is effectively induced by AA and recombination defective mammalian cells are hypersensitive to AA, clearly demonstrating that HR is essential in the repair of AA-induced DNA damage. Altogether, our data indicate that alcohol genotoxicity related to AA produces replication lesions on DNA triggering HR repair.}, note = {[DOI:hrefhttp://dx.doi.org/10.1093/carcin/bgs34010.1093/carcin/bgs340] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2312521923125219]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Although alcohol consumption is related to increased cancer risk, its molecular mechanism remains unclear. Here, we demonstrate that an intake of 10% alcohol for 4 weeks in rats is genotoxic due to induction of micronuclei. Acetaldehyde (AA), the first product of ethanol metabolism, is believed to be responsible for DNA damage induced by alcohol. Here, we observe that AA effectively blocks DNA replication elongation in mammalian cells, resulting in DNA double-strand breaks associated with replication. AA-induced DNA damage sites colocalize with the homologous recombination (HR) repair protein RAD51. HR measured in the hypoxhantineguaninefosforibosyltransferase (HPRT) gene is effectively induced by AA and recombination defective mammalian cells are hypersensitive to AA, clearly demonstrating that HR is essential in the repair of AA-induced DNA damage. Altogether, our data indicate that alcohol genotoxicity related to AA produces replication lesions on DNA triggering HR repair. |
He, H; Tian, D; Guo, J; Liu, M; Chen, Z; Hamdy, F C; Helleday, T; Su, M; Ying, S ĐNA damage response in peritumoral regions of oesophageal cancer microenvironment Journal Article Carcinogenesis, 34 (1), pp. 139–145, 2013, ([DOI:hrefhttp://dx.doi.org/10.1093/carcin/bgs30110.1093/carcin/bgs301] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2302762223027622]). @article{pmid23027622, title = {ĐNA damage response in peritumoral regions of oesophageal cancer microenvironment}, author = { H. He and D. Tian and J. Guo and M. Liu and Z. Chen and F. C. Hamdy and T. Helleday and M. Su and S. Ying}, year = {2013}, date = {2013-01-01}, journal = {Carcinogenesis}, volume = {34}, number = {1}, pages = {139--145}, abstract = {Oesophageal cancer is a highly aggressive disease, ranking among the 10 most common cancers in the world. Oesophageal cancer patients often suffer from multi-origin tumours, and therefore, it is important to improve our understanding of the complex biology, which underpins microenvironmental interactions in this disease. Extensive evidence indicates that the interaction of tumours with their microenvironment may play a crucial role in tumour initiation and progression. In this study, we analysed DNA damage response (DDR), immune cell invasion and cancer progression in 47 patients with oesophageal cancer from three different regions (tumour tissue, tumour-proximal non-malignant tissue and distant non-malignant tissue). Accumulated DDR (positive staining for γH2AX and phospho-ATM) was evident within tumour tissue and significantly increased in non-malignant tissue surrounding the tumour cells although activation of p53 by phosphorylation at serine 15 was observed only in tumour tissue. The level of DDR detected in cancer microenvironment depended largely on the distance from the tumour, as stronger DDR was observed in tumour-proximal areas compared with that in tumour-distant tissue. Induction of DDR in non-malignant tissues correlated with increased invasion of lymphocytes and macrophages and with precancerous progression. Our results support that DDR is induced in oesophageal cancer surrounding non-malignant epithelial cells, via activation of an inflammatory process, which in turn contributes to the progression of precancerous lesions. These findings provide novel pathological evidence for inflammation and DDR in influencing non-metastatic progression of cancer in its microenvironment.}, note = {[DOI:hrefhttp://dx.doi.org/10.1093/carcin/bgs30110.1093/carcin/bgs301] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2302762223027622]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Oesophageal cancer is a highly aggressive disease, ranking among the 10 most common cancers in the world. Oesophageal cancer patients often suffer from multi-origin tumours, and therefore, it is important to improve our understanding of the complex biology, which underpins microenvironmental interactions in this disease. Extensive evidence indicates that the interaction of tumours with their microenvironment may play a crucial role in tumour initiation and progression. In this study, we analysed DNA damage response (DDR), immune cell invasion and cancer progression in 47 patients with oesophageal cancer from three different regions (tumour tissue, tumour-proximal non-malignant tissue and distant non-malignant tissue). Accumulated DDR (positive staining for γH2AX and phospho-ATM) was evident within tumour tissue and significantly increased in non-malignant tissue surrounding the tumour cells although activation of p53 by phosphorylation at serine 15 was observed only in tumour tissue. The level of DDR detected in cancer microenvironment depended largely on the distance from the tumour, as stronger DDR was observed in tumour-proximal areas compared with that in tumour-distant tissue. Induction of DDR in non-malignant tissues correlated with increased invasion of lymphocytes and macrophages and with precancerous progression. Our results support that DDR is induced in oesophageal cancer surrounding non-malignant epithelial cells, via activation of an inflammatory process, which in turn contributes to the progression of precancerous lesions. These findings provide novel pathological evidence for inflammation and DDR in influencing non-metastatic progression of cancer in its microenvironment. |
2012 |
Neumayer, G; Helfricht, A; Shim, S Y; Le, H T; Lundin, C; Belzil, C; Chansard, M; Yu, Y; Lees-Miller, S P; Gruss, O J; van Attikum, H; Helleday, T; Nguyen, M D Ŧargeting protein for xenopus kinesin-like protein 2 (ŦPX2) regulates γ-histone 2AX (γ-Ħ2AX) levels upon ionizing radiation Journal Article J. Biol. Chem., 287 (50), pp. 42206–42222, 2012, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3516765PMC3516765] [DOI:hrefhttp://dx.doi.org/10.1074/jbc.M112.38567410.1074/jbc.M112.385674] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2304552623045526]). @article{pmid23045526, title = {Ŧargeting protein for xenopus kinesin-like protein 2 (ŦPX2) regulates γ-histone 2AX (γ-Ħ2AX) levels upon ionizing radiation}, author = { G. Neumayer and A. Helfricht and S. Y. Shim and H. T. Le and C. Lundin and C. Belzil and M. Chansard and Y. Yu and S. P. Lees-Miller and O. J. Gruss and H. van Attikum and T. Helleday and M. D. Nguyen}, year = {2012}, date = {2012-12-01}, journal = {J. Biol. Chem.}, volume = {287}, number = {50}, pages = {42206--42222}, abstract = {The microtubule-associated protein targeting protein for Xenopus kinesin-like protein 2 (TPX2) plays a key role in spindle assembly and is required for mitosis in human cells. In interphase, TPX2 is actively imported into the nucleus to prevent its premature activity in microtubule organization. To date, no function has been assigned to nuclear TPX2. We now report that TPX2 plays a role in the cellular response to DNA double strand breaks induced by ionizing radiation. Loss of TPX2 leads to inordinately strong and transient accumulation of ionizing radiation-dependent Ser-139-phosphorylated Histone 2AX (γ-H2AX) at G(0) and G(1) phases of the cell cycle. This is accompanied by the formation of increased numbers of high intensity γ-H2AX ionizing radiation-induced foci. Conversely, cells overexpressing TPX2 have reduced levels of γ-H2AX after ionizing radiation. Consistent with a role for TPX2 in the DNA damage response, we found that the protein accumulates at DNA double strand breaks and associates with the mediator of DNA damage checkpoint 1 (MDC1) and the ataxia telangiectasia mutated (ATM) kinase, both key regulators of γ-H2AX amplification. Pharmacologic inhibition or depletion of ATM or MDC1, but not of DNA-dependent protein kinase (DNA-PK), antagonizes the γ-H2AX phenotype caused by TPX2 depletion. Importantly, the regulation of γ-H2AX signals by TPX2 is not associated with apoptosis or the mitotic functions of TPX2. In sum, our study identifies a novel and the first nuclear function for TPX2 in the cellular responses to DNA damage.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3516765PMC3516765] [DOI:hrefhttp://dx.doi.org/10.1074/jbc.M112.38567410.1074/jbc.M112.385674] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2304552623045526]}, keywords = {}, pubstate = {published}, tppubtype = {article} } The microtubule-associated protein targeting protein for Xenopus kinesin-like protein 2 (TPX2) plays a key role in spindle assembly and is required for mitosis in human cells. In interphase, TPX2 is actively imported into the nucleus to prevent its premature activity in microtubule organization. To date, no function has been assigned to nuclear TPX2. We now report that TPX2 plays a role in the cellular response to DNA double strand breaks induced by ionizing radiation. Loss of TPX2 leads to inordinately strong and transient accumulation of ionizing radiation-dependent Ser-139-phosphorylated Histone 2AX (γ-H2AX) at G(0) and G(1) phases of the cell cycle. This is accompanied by the formation of increased numbers of high intensity γ-H2AX ionizing radiation-induced foci. Conversely, cells overexpressing TPX2 have reduced levels of γ-H2AX after ionizing radiation. Consistent with a role for TPX2 in the DNA damage response, we found that the protein accumulates at DNA double strand breaks and associates with the mediator of DNA damage checkpoint 1 (MDC1) and the ataxia telangiectasia mutated (ATM) kinase, both key regulators of γ-H2AX amplification. Pharmacologic inhibition or depletion of ATM or MDC1, but not of DNA-dependent protein kinase (DNA-PK), antagonizes the γ-H2AX phenotype caused by TPX2 depletion. Importantly, the regulation of γ-H2AX signals by TPX2 is not associated with apoptosis or the mitotic functions of TPX2. In sum, our study identifies a novel and the first nuclear function for TPX2 in the cellular responses to DNA damage. |
Nicolay, N H; Carter, R; Hatch, S B; Schultz, N; Prevo, R; McKenna, W G; Helleday, T; Sharma, R A Ħomologous recombination mediates S-phase-dependent radioresistance in cells deficient in ĐNA polymerase eta Journal Article Carcinogenesis, 33 (11), pp. 2026–2034, 2012, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3584963PMC3584963] [DOI:hrefhttp://dx.doi.org/10.1093/carcin/bgs23910.1093/carcin/bgs239] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2282209522822095]). @article{pmid22822095, title = {Ħomologous recombination mediates S-phase-dependent radioresistance in cells deficient in ĐNA polymerase eta}, author = { N. H. Nicolay and R. Carter and S. B. Hatch and N. Schultz and R. Prevo and W. G. McKenna and T. Helleday and R. A. Sharma}, year = {2012}, date = {2012-11-01}, journal = {Carcinogenesis}, volume = {33}, number = {11}, pages = {2026--2034}, abstract = {DNA polymerase eta (pol η) is the only DNA polymerase causally linked to carcinogenesis in humans. Inherited deficiency of pol η in the variant form of xeroderma pigmentosum (XPV) predisposes to UV-light-induced skin cancer. Pol η-deficient cells demonstrate increased sensitivity to cisplatin and oxaliplatin chemotherapy. We have found that XP30R0 fibroblasts derived from a patient with XPV are more resistant to cell kill by ionising radiation (IR) than the same cells complemented with wild-type pol η. This phenomenon has been confirmed in Burkitt's lymphoma cells, which either expressed wild-type pol η or harboured a pol η deletion. Pol η deficiency was associated with accumulation of cells in S-phase, which persisted after IR. Cells deficient in pol η demonstrated increased homologous recombination (HR)-directed repair of double strand breaks created by IR. Depletion of the HR protein, X-ray repair cross-complementing protein 3 (XRCC3), abrogated the radioresistance observed in pol η-deficient cells as compared with pol η-complemented cells. These findings suggest that HR mediates S-phase-dependent radioresistance associated with pol η deficiency. We propose that pol η protein levels in tumours may potentially be used to identify patients who require treatment with chemo-radiotherapy rather than radiotherapy alone for adequate tumour control.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3584963PMC3584963] [DOI:hrefhttp://dx.doi.org/10.1093/carcin/bgs23910.1093/carcin/bgs239] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2282209522822095]}, keywords = {}, pubstate = {published}, tppubtype = {article} } DNA polymerase eta (pol η) is the only DNA polymerase causally linked to carcinogenesis in humans. Inherited deficiency of pol η in the variant form of xeroderma pigmentosum (XPV) predisposes to UV-light-induced skin cancer. Pol η-deficient cells demonstrate increased sensitivity to cisplatin and oxaliplatin chemotherapy. We have found that XP30R0 fibroblasts derived from a patient with XPV are more resistant to cell kill by ionising radiation (IR) than the same cells complemented with wild-type pol η. This phenomenon has been confirmed in Burkitt's lymphoma cells, which either expressed wild-type pol η or harboured a pol η deletion. Pol η deficiency was associated with accumulation of cells in S-phase, which persisted after IR. Cells deficient in pol η demonstrated increased homologous recombination (HR)-directed repair of double strand breaks created by IR. Depletion of the HR protein, X-ray repair cross-complementing protein 3 (XRCC3), abrogated the radioresistance observed in pol η-deficient cells as compared with pol η-complemented cells. These findings suggest that HR mediates S-phase-dependent radioresistance associated with pol η deficiency. We propose that pol η protein levels in tumours may potentially be used to identify patients who require treatment with chemo-radiotherapy rather than radiotherapy alone for adequate tumour control. |
Somaiah, N; Yarnold, J; Daley, F; Pearson, A; Gothard, L; Rothkamm, K; Helleday, T Ŧhe relationship between homologous recombination repair and the sensitivity of human epidermis to the size of daily doses over a 5-week course of breast radiotherapy Journal Article Clin. Cancer Res., 18 (19), pp. 5479–5488, 2012, ([DOI:hrefhttp://dx.doi.org/10.1158/1078-0432.CCR-10-329710.1158/1078-0432.CCR-10-3297] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2285558022855580]). @article{pmid22855580, title = {Ŧhe relationship between homologous recombination repair and the sensitivity of human epidermis to the size of daily doses over a 5-week course of breast radiotherapy}, author = { N. Somaiah and J. Yarnold and F. Daley and A. Pearson and L. Gothard and K. Rothkamm and T. Helleday}, year = {2012}, date = {2012-10-01}, journal = {Clin. Cancer Res.}, volume = {18}, number = {19}, pages = {5479--5488}, abstract = {A molecular understanding of tissue sensitivity to radiotherapy fraction size is missing. Here, we test the hypothesis that sensitivity to fraction size is influenced by the DNA repair system activated in response to DNA double-strand breaks (DSB). Human epidermis was used as a model in which proliferation and DNA repair were correlated over 5 weeks of radiotherapy. Radiotherapy (25 fractions of 2 Gy) was prescribed to the breast in 30 women with early breast cancer. Breast skin biopsies were collected 2 hours after the 1st and 25th fractions. Samples of contralateral breast skin served as controls. Sections were coimmunostained for Ki67, cyclin A, p21, RAD51, 53BP1, and β1-integrin. After 5 weeks of radiotherapy, the mean basal Ki67 density increased from 5.72 to 15.46 cells per millimeter of basement membrane (P = 0.002), of which the majority were in S/G2 phase, as judged by cyclin A staining (P < 0.0003). The p21 index rose from 2.8% to 87.4% (P < 0.0001) after 25 fractions, indicating cell cycle arrest. By week 5, there was a 4-fold increase (P = 0.0003) in the proportion of Ki67-positive cells showing RAD51 foci, suggesting increasing activation of homologous recombination. Cell cycle arrest in S/G2 phase in the basal epidermis after a 5-week course of radiotherapy is associated with greater use of homologous recombination for repairing DSB. The high fidelity of homologous recombination, which is independent of DNA damage levels, may explain the low-fractionation sensitivity of tissues with high-proliferative indices, including self-renewing normal tissues and many cancers.}, note = {[DOI:hrefhttp://dx.doi.org/10.1158/1078-0432.CCR-10-329710.1158/1078-0432.CCR-10-3297] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2285558022855580]}, keywords = {}, pubstate = {published}, tppubtype = {article} } A molecular understanding of tissue sensitivity to radiotherapy fraction size is missing. Here, we test the hypothesis that sensitivity to fraction size is influenced by the DNA repair system activated in response to DNA double-strand breaks (DSB). Human epidermis was used as a model in which proliferation and DNA repair were correlated over 5 weeks of radiotherapy. Radiotherapy (25 fractions of 2 Gy) was prescribed to the breast in 30 women with early breast cancer. Breast skin biopsies were collected 2 hours after the 1st and 25th fractions. Samples of contralateral breast skin served as controls. Sections were coimmunostained for Ki67, cyclin A, p21, RAD51, 53BP1, and β1-integrin. After 5 weeks of radiotherapy, the mean basal Ki67 density increased from 5.72 to 15.46 cells per millimeter of basement membrane (P = 0.002), of which the majority were in S/G2 phase, as judged by cyclin A staining (P < 0.0003). The p21 index rose from 2.8% to 87.4% (P < 0.0001) after 25 fractions, indicating cell cycle arrest. By week 5, there was a 4-fold increase (P = 0.0003) in the proportion of Ki67-positive cells showing RAD51 foci, suggesting increasing activation of homologous recombination. Cell cycle arrest in S/G2 phase in the basal epidermis after a 5-week course of radiotherapy is associated with greater use of homologous recombination for repairing DSB. The high fidelity of homologous recombination, which is independent of DNA damage levels, may explain the low-fractionation sensitivity of tissues with high-proliferative indices, including self-renewing normal tissues and many cancers. |
Elvers, I; Hagenkort, A; Johansson, F; Djureinovic, T; Lagerqvist, A; Schultz, N; Stoimenov, I; Erixon, K; Helleday, T CĦK1 activity is required for continuous replication fork elongation but not stabilization of post-replicative gaps after UV irradiation Journal Article Nucleic Acids Res., 40 (17), pp. 8440–8448, 2012, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3458576PMC3458576] [DOI:hrefhttp://dx.doi.org/10.1093/nar/gks64610.1093/nar/gks646] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2275302922753029]). @article{pmid22753029, title = {CĦK1 activity is required for continuous replication fork elongation but not stabilization of post-replicative gaps after UV irradiation}, author = { I. Elvers and A. Hagenkort and F. Johansson and T. Djureinovic and A. Lagerqvist and N. Schultz and I. Stoimenov and K. Erixon and T. Helleday}, year = {2012}, date = {2012-09-01}, journal = {Nucleic Acids Res.}, volume = {40}, number = {17}, pages = {8440--8448}, abstract = {Ultraviolet (UV)-induced DNA damage causes an efficient block of elongating replication forks. The checkpoint kinase, CHK1 has been shown to stabilize replication forks following hydroxyurea treatment. Therefore, we wanted to test if the increased UV sensitivity caused by the unspecific kinase inhibitor caffeine--inhibiting ATM and ATR amongst other kinases--is explained by inability to activate the CHK1 kinase to stabilize replicative structures. For this, we used cells deficient in polymerase η (Polη), a translesion synthesis polymerase capable of properly bypassing the UV-induced cis-syn TT pyrimidine dimer, which blocks replication. These cells accumulate gaps behind progressing replication forks after UV exposure. We demonstrate that both caffeine and CHK1 inhibition, equally retards continuous replication fork elongation after UV treatment. Interestingly, we found more pronounced UV-sensitization by caffeine than with the CHK1 inhibitor in clonogenic survival experiments. Furthermore, we demonstrate an increased collapse of replicative structures after caffeine treatment, but not after CHK1 inhibition, in UV-irradiated cells. This demonstrates that CHK1 activity is not required for stabilization of gaps induced during replication of UV-damaged DNA. These data suggest that elongation and stabilization of replicative structures at UV-induced DNA damage are distinct mechanisms, and that CHK1 is only involved in replication elongation.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3458576PMC3458576] [DOI:hrefhttp://dx.doi.org/10.1093/nar/gks64610.1093/nar/gks646] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2275302922753029]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Ultraviolet (UV)-induced DNA damage causes an efficient block of elongating replication forks. The checkpoint kinase, CHK1 has been shown to stabilize replication forks following hydroxyurea treatment. Therefore, we wanted to test if the increased UV sensitivity caused by the unspecific kinase inhibitor caffeine--inhibiting ATM and ATR amongst other kinases--is explained by inability to activate the CHK1 kinase to stabilize replicative structures. For this, we used cells deficient in polymerase η (Polη), a translesion synthesis polymerase capable of properly bypassing the UV-induced cis-syn TT pyrimidine dimer, which blocks replication. These cells accumulate gaps behind progressing replication forks after UV exposure. We demonstrate that both caffeine and CHK1 inhibition, equally retards continuous replication fork elongation after UV treatment. Interestingly, we found more pronounced UV-sensitization by caffeine than with the CHK1 inhibitor in clonogenic survival experiments. Furthermore, we demonstrate an increased collapse of replicative structures after caffeine treatment, but not after CHK1 inhibition, in UV-irradiated cells. This demonstrates that CHK1 activity is not required for stabilization of gaps induced during replication of UV-damaged DNA. These data suggest that elongation and stabilization of replicative structures at UV-induced DNA damage are distinct mechanisms, and that CHK1 is only involved in replication elongation. |
Groth, P; Orta, M L; Elvers, I; Majumder, M M; Lagerqvist, A; Helleday, T Ħomologous recombination repairs secondary replication induced ĐNA double-strand breaks after ionizing radiation Journal Article Nucleic Acids Res., 40 (14), pp. 6585–6594, 2012, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3413124PMC3413124] [DOI:hrefhttp://dx.doi.org/10.1093/nar/gks31510.1093/nar/gks315] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2250557922505579]). @article{pmid22505579, title = {Ħomologous recombination repairs secondary replication induced ĐNA double-strand breaks after ionizing radiation}, author = { P. Groth and M. L. Orta and I. Elvers and M. M. Majumder and A. Lagerqvist and T. Helleday}, year = {2012}, date = {2012-08-01}, journal = {Nucleic Acids Res.}, volume = {40}, number = {14}, pages = {6585--6594}, abstract = {Ionizing radiation (IR) produces direct two-ended DNA double-strand breaks (DSBs) primarily repaired by non-homologous end joining (NHEJ). It is, however, well established that homologous recombination (HR) is induced and required for repair of a subset of DSBs formed following IR. Here, we find that HR induced by IR is drastically reduced when post-DNA damage replication is inhibited in mammalian cells. Both IR-induced RAD51 foci and HR events in the hprt gene are reduced in the presence of replication polymerase inhibitor aphidicolin (APH). Interestingly, we also detect reduced IR-induced toxicity in HR deficient cells when inhibiting post-DNA damage replication. When studying DSB formation following IR exposure, we find that apart from the direct DSBs the treatment also triggers formation of secondary DSBs peaking at 7-9 h after exposure. These secondary DSBs are restricted to newly replicated DNA and abolished by inhibiting post-DNA damage replication. Further, we find that IR-induced RAD51 foci are decreased by APH only in cells replicating at the time of IR exposure, suggesting distinct differences between IR-induced HR in S- and G2-phases of the cell cycle. Altogether, our data indicate that secondary replication-associated DSBs formed following exposure to IR are major substrates for IR-induced HR repair.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3413124PMC3413124] [DOI:hrefhttp://dx.doi.org/10.1093/nar/gks31510.1093/nar/gks315] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2250557922505579]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Ionizing radiation (IR) produces direct two-ended DNA double-strand breaks (DSBs) primarily repaired by non-homologous end joining (NHEJ). It is, however, well established that homologous recombination (HR) is induced and required for repair of a subset of DSBs formed following IR. Here, we find that HR induced by IR is drastically reduced when post-DNA damage replication is inhibited in mammalian cells. Both IR-induced RAD51 foci and HR events in the hprt gene are reduced in the presence of replication polymerase inhibitor aphidicolin (APH). Interestingly, we also detect reduced IR-induced toxicity in HR deficient cells when inhibiting post-DNA damage replication. When studying DSB formation following IR exposure, we find that apart from the direct DSBs the treatment also triggers formation of secondary DSBs peaking at 7-9 h after exposure. These secondary DSBs are restricted to newly replicated DNA and abolished by inhibiting post-DNA damage replication. Further, we find that IR-induced RAD51 foci are decreased by APH only in cells replicating at the time of IR exposure, suggesting distinct differences between IR-induced HR in S- and G2-phases of the cell cycle. Altogether, our data indicate that secondary replication-associated DSBs formed following exposure to IR are major substrates for IR-induced HR repair. |
Ying, S; Hamdy, F C; Helleday, T Mre11-dependent degradation of stalled ĐNA replication forks is prevented by BRCA2 and PARP1 Journal Article Cancer Res., 72 (11), pp. 2814–2821, 2012, ([DOI:hrefhttp://dx.doi.org/10.1158/0008-5472.CAN-11-341710.1158/0008-5472.CAN-11-3417] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2244756722447567]). @article{pmid22447567, title = {Mre11-dependent degradation of stalled ĐNA replication forks is prevented by BRCA2 and PARP1}, author = { S. Ying and F. C. Hamdy and T. Helleday}, year = {2012}, date = {2012-06-01}, journal = {Cancer Res.}, volume = {72}, number = {11}, pages = {2814--2821}, abstract = {PARP inhibitors are currently being used in clinical trials to treat BRCA1- or BRCA2-defective tumors, based on the synthetic lethal interaction between PARP1 and BRCA1/2-mediated homologous recombination (HR). However, the molecular mechanisms that drive this synthetic lethality remain unclear. Here, we show increased levels of Mre11, a key component of MRN (Mre11-Rad50-Nbs1) complex that plays a role in the restart of stalled replication forks and enhanced resection at stalled replication forks in BRCA2-deficient cells. BRCA2-deficient cells also showed hypersensitivity to the Mre11 inhibitor mirin. Interestingly, PARP1 activity was required to protect stalled forks from Mre11-dependent degradation. Resistance to PARP inhibition in BRCA2-mutant cells led to reduced levels of Mre11 foci and also rescued their sensitivity to mirin. Taken together, our findings not only show that Mre11 activity is required for the survival of BRCA2 mutant cells but also elucidate roles for both the BRCA2 and PARP1 proteins in protecting stalled replication forks, which offers insight into the molecular mechanisms of the synthetic lethality between BRCA2 and PARP1.}, note = {[DOI:hrefhttp://dx.doi.org/10.1158/0008-5472.CAN-11-341710.1158/0008-5472.CAN-11-3417] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2244756722447567]}, keywords = {}, pubstate = {published}, tppubtype = {article} } PARP inhibitors are currently being used in clinical trials to treat BRCA1- or BRCA2-defective tumors, based on the synthetic lethal interaction between PARP1 and BRCA1/2-mediated homologous recombination (HR). However, the molecular mechanisms that drive this synthetic lethality remain unclear. Here, we show increased levels of Mre11, a key component of MRN (Mre11-Rad50-Nbs1) complex that plays a role in the restart of stalled replication forks and enhanced resection at stalled replication forks in BRCA2-deficient cells. BRCA2-deficient cells also showed hypersensitivity to the Mre11 inhibitor mirin. Interestingly, PARP1 activity was required to protect stalled forks from Mre11-dependent degradation. Resistance to PARP inhibition in BRCA2-mutant cells led to reduced levels of Mre11 foci and also rescued their sensitivity to mirin. Taken together, our findings not only show that Mre11 activity is required for the survival of BRCA2 mutant cells but also elucidate roles for both the BRCA2 and PARP1 proteins in protecting stalled replication forks, which offers insight into the molecular mechanisms of the synthetic lethality between BRCA2 and PARP1. |
Abdallah, Q M; Phillips, R M; Johansson, F; Helleday, T; Cosentino, L; Abdel-Rahman, H; Etzad, J; Wheelhouse, R T; Kiakos, K; Bingham, J P; Hartley, J A; Patterson, L H; Pors, K Minor structural modifications to alchemix influence mechanism of action and pharmacological activity Journal Article Biochem. Pharmacol., 83 (11), pp. 1514–1522, 2012, ([DOI:hrefhttp://dx.doi.org/10.1016/j.bcp.2012.02.01710.1016/j.bcp.2012.02.017] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2238743322387433]). @article{pmid22387433, title = {Minor structural modifications to alchemix influence mechanism of action and pharmacological activity}, author = { Q. M. Abdallah and R. M. Phillips and F. Johansson and T. Helleday and L. Cosentino and H. Abdel-Rahman and J. Etzad and R. T. Wheelhouse and K. Kiakos and J. P. Bingham and J. A. Hartley and L. H. Patterson and K. Pors}, year = {2012}, date = {2012-06-01}, journal = {Biochem. Pharmacol.}, volume = {83}, number = {11}, pages = {1514--1522}, abstract = {Alchemix is an exemplar of a class of anthraquinone with efficacy against multidrug resistant tumours. We have explored further the mechanism of action of alchemix and investigated the effect of extending its side arm bearing the alkylating functionality with regard to DNA binding and activity against multidrug resistant cancer cells. Increasing the distance between the intercalating chromophore and the alkylating functionality of ICT2901 (propyl), ICT2902 (butyl) and ICT2903 (pentyl), led to a higher number of DNA alkylation sites, more potent topoisomerase II inhibition and generated more apoptotic and necrotic cells when analysed in p53-proficient HCT116 cells. Intriguingly, alchemix, the compound with the shortest distance between its intercalative chromophore and alkylating functionality (ethyl), did not conform to this SAR. A different toxicity pattern against DNA repair defective CHO cell lines as well as arrest of cells in G1 supports a somewhat distinct mode of action by alchemix compared with its analogues. Importantly, both alchemix and ICT2901 demonstrated greater cytotoxic activity against anthraquinone-resistant MCF-7/adr cells than wild-type MCF-7 cells. Subtle synthetic modification in this anthraquinone series has led to significant changes to the stability of DNA-compound complexes and cellular activity. Given that the failure of chemotherapy in the clinic is often associated with MDR, the results of both alchemix and ICT2901 represent important advances towards improved therapies.}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/j.bcp.2012.02.01710.1016/j.bcp.2012.02.017] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2238743322387433]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Alchemix is an exemplar of a class of anthraquinone with efficacy against multidrug resistant tumours. We have explored further the mechanism of action of alchemix and investigated the effect of extending its side arm bearing the alkylating functionality with regard to DNA binding and activity against multidrug resistant cancer cells. Increasing the distance between the intercalating chromophore and the alkylating functionality of ICT2901 (propyl), ICT2902 (butyl) and ICT2903 (pentyl), led to a higher number of DNA alkylation sites, more potent topoisomerase II inhibition and generated more apoptotic and necrotic cells when analysed in p53-proficient HCT116 cells. Intriguingly, alchemix, the compound with the shortest distance between its intercalative chromophore and alkylating functionality (ethyl), did not conform to this SAR. A different toxicity pattern against DNA repair defective CHO cell lines as well as arrest of cells in G1 supports a somewhat distinct mode of action by alchemix compared with its analogues. Importantly, both alchemix and ICT2901 demonstrated greater cytotoxic activity against anthraquinone-resistant MCF-7/adr cells than wild-type MCF-7 cells. Subtle synthetic modification in this anthraquinone series has led to significant changes to the stability of DNA-compound complexes and cellular activity. Given that the failure of chemotherapy in the clinic is often associated with MDR, the results of both alchemix and ICT2901 represent important advances towards improved therapies. |
Chatzakos, V; Slatis, K; Djureinovic, T; Helleday, T; Hunt, M C N-acyl taurines are anti-proliferative in prostate cancer cells Journal Article Lipids, 47 (4), pp. 355–361, 2012, ([DOI:hrefhttp://dx.doi.org/10.1007/s11745-011-3639-910.1007/s11745-011-3639-9] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2216049422160494]). @article{pmid22160494, title = {N-acyl taurines are anti-proliferative in prostate cancer cells}, author = { V. Chatzakos and K. Slatis and T. Djureinovic and T. Helleday and M. C. Hunt}, year = {2012}, date = {2012-04-01}, journal = {Lipids}, volume = {47}, number = {4}, pages = {355--361}, abstract = {Endocannabinoids have been implicated in cancer development and cause heterogenous effects in tumor cells, by inducing apoptosis, reducing migration, causing anti-angiogenic activity and alterations in the cell cycle resulting in growth arrest. Recently, several novel amides of fatty acids that are structurally related to endocannabinoids have been isolated from mammalian sources, although the functions of these fatty amides are not well studied. One group of these novel fatty acid amides are the N-acyl taurines (fatty acids conjugated to the amino acid taurine). This study examined if N-acyl taurines, specifically N-arachidonoyl taurine and N-oleoyl taurine could function in a similar way to endocannabinoids and result in cell cycle alterations or growth arrest in the human prostate adenocarcinoma cell line PC-3. PC-3 cells were treated with various concentrations of N-arachidonoyl taurine and N-oleoyl taurine and cell proliferation and viability was measured using resazurin and colony formation assays. Effects of N-acyl taurines on the cell cycle was measured using FACS analysis. Treatment with N-arachidonoyl taurine and N-oleoyl taurine resulted in a significant reduction in proliferation of PC-3 cells, even at concentrations as low as 1 μM. Treatment with N-oleoyl taurine resulted in an increased number of cells in the subG1 population, suggesting apoptosis, and a lower number of cells in S-phase of the cell cycle. In summary, our results show that novel biologically active lipids, the N-acyl taurines, result in reduced proliferation in PC-3 cells.}, note = {[DOI:hrefhttp://dx.doi.org/10.1007/s11745-011-3639-910.1007/s11745-011-3639-9] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2216049422160494]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Endocannabinoids have been implicated in cancer development and cause heterogenous effects in tumor cells, by inducing apoptosis, reducing migration, causing anti-angiogenic activity and alterations in the cell cycle resulting in growth arrest. Recently, several novel amides of fatty acids that are structurally related to endocannabinoids have been isolated from mammalian sources, although the functions of these fatty amides are not well studied. One group of these novel fatty acid amides are the N-acyl taurines (fatty acids conjugated to the amino acid taurine). This study examined if N-acyl taurines, specifically N-arachidonoyl taurine and N-oleoyl taurine could function in a similar way to endocannabinoids and result in cell cycle alterations or growth arrest in the human prostate adenocarcinoma cell line PC-3. PC-3 cells were treated with various concentrations of N-arachidonoyl taurine and N-oleoyl taurine and cell proliferation and viability was measured using resazurin and colony formation assays. Effects of N-acyl taurines on the cell cycle was measured using FACS analysis. Treatment with N-arachidonoyl taurine and N-oleoyl taurine resulted in a significant reduction in proliferation of PC-3 cells, even at concentrations as low as 1 μM. Treatment with N-oleoyl taurine resulted in an increased number of cells in the subG1 population, suggesting apoptosis, and a lower number of cells in S-phase of the cell cycle. In summary, our results show that novel biologically active lipids, the N-acyl taurines, result in reduced proliferation in PC-3 cells. |
Urbin, S S; Elvers, I; Hinz, J M; Helleday, T; Thompson, L H Uncoupling of RAĐ51 focus formation and cell survival after replication fork stalling in RAĐ51Đ null CĦO cells Journal Article Environ. Mol. Mutagen., 53 (2), pp. 114–124, 2012, ([DOI:hrefhttp://dx.doi.org/10.1002/em.2167210.1002/em.21672] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2230268322302683]). @article{pmid22302683, title = {Uncoupling of RAĐ51 focus formation and cell survival after replication fork stalling in RAĐ51Đ null CĦO cells}, author = { S. S. Urbin and I. Elvers and J. M. Hinz and T. Helleday and L. H. Thompson}, year = {2012}, date = {2012-03-01}, journal = {Environ. Mol. Mutagen.}, volume = {53}, number = {2}, pages = {114--124}, abstract = {In vertebrate cells, the five RAD51 paralogs (XRCC2/3 and RAD51B/C/D) enhance the efficiency of homologous recombination repair (HRR). Stalling and breakage of DNA replication forks is a common event, especially in the large genomes of higher eukaryotes. When cells are exposed to agents that arrest DNA replication, such as hydroxyurea or aphidicolin, fork breakage can lead to chromosomal aberrations and cell killing. We assessed the contribution of the HRR protein RAD51D in resistance to killing by replication-associated DSBs. In response to hydroxyurea, the isogenic rad51d null CHO mutant fails to show any indication of HRR initiation, as assessed by induction RAD51 foci, as expected. Surprisingly, these cells have normal resistance to killing by replication inhibition from either hydroxyurea or aphidicolin, but show the expected sensitivity to camptothecin, which also generates replication-dependent DSBs. In contrast, we confirm that the V79 xrcc2 mutant does show increased sensitivity to hydroxyurea under some conditions, which was correlated to its attenuated RAD51 focus response. In response to the PARP1 inhibitor KU58684, rad51d cells, like other HRR mutants, show exquisite sensitivity (>1000-fold), which is also associated with defective RAD51 focus formation. Thus, rad51d cells are broadly deficient in RAD51 focus formation in response to various agents, but this defect is not invariably associated with increased sensitivity. Our results indicate that RAD51 paralogs do not contribute equally to cellular resistance of inhibitors of DNAreplication, and that the RAD51 foci associated with replication inhibition may not be a reliable indicator of cellular resistance to such agents.}, note = {[DOI:hrefhttp://dx.doi.org/10.1002/em.2167210.1002/em.21672] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2230268322302683]}, keywords = {}, pubstate = {published}, tppubtype = {article} } In vertebrate cells, the five RAD51 paralogs (XRCC2/3 and RAD51B/C/D) enhance the efficiency of homologous recombination repair (HRR). Stalling and breakage of DNA replication forks is a common event, especially in the large genomes of higher eukaryotes. When cells are exposed to agents that arrest DNA replication, such as hydroxyurea or aphidicolin, fork breakage can lead to chromosomal aberrations and cell killing. We assessed the contribution of the HRR protein RAD51D in resistance to killing by replication-associated DSBs. In response to hydroxyurea, the isogenic rad51d null CHO mutant fails to show any indication of HRR initiation, as assessed by induction RAD51 foci, as expected. Surprisingly, these cells have normal resistance to killing by replication inhibition from either hydroxyurea or aphidicolin, but show the expected sensitivity to camptothecin, which also generates replication-dependent DSBs. In contrast, we confirm that the V79 xrcc2 mutant does show increased sensitivity to hydroxyurea under some conditions, which was correlated to its attenuated RAD51 focus response. In response to the PARP1 inhibitor KU58684, rad51d cells, like other HRR mutants, show exquisite sensitivity (>1000-fold), which is also associated with defective RAD51 focus formation. Thus, rad51d cells are broadly deficient in RAD51 focus formation in response to various agents, but this defect is not invariably associated with increased sensitivity. Our results indicate that RAD51 paralogs do not contribute equally to cellular resistance of inhibitors of DNAreplication, and that the RAD51 foci associated with replication inhibition may not be a reliable indicator of cellular resistance to such agents. |
Al-Ubaidi, F L; Schultz, N; Egevad, L; Granfors, T; Helleday, T Castration therapy of prostate cancer results in downregulation of ĦIF-1α levels Journal Article Int. J. Radiat. Oncol. Biol. Phys., 82 (3), pp. 1243–1248, 2012, ([DOI:hrefhttp://dx.doi.org/10.1016/j.ijrobp.2011.10.03810.1016/j.ijrobp.2011.10.038] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2228403222284032]). @article{pmid22284032, title = {Castration therapy of prostate cancer results in downregulation of ĦIF-1α levels}, author = { F. L. Al-Ubaidi and N. Schultz and L. Egevad and T. Granfors and T. Helleday}, year = {2012}, date = {2012-03-01}, journal = {Int. J. Radiat. Oncol. Biol. Phys.}, volume = {82}, number = {3}, pages = {1243--1248}, abstract = {Neoadjuvant androgen deprivation in combination with radiotherapy of prostate cancer is used to improve radioresponsiveness and local tumor control. Currently, the underlying mechanism is not well understood. Because hypoxia causes resistance to radiotherapy, we wanted to test whether castration affects the degree of hypoxia in prostate cancer. In 14 patients with locally advanced prostate cancer, six to 12 prostatic needle core biopsy specimens were taken prior to castration therapy. Bilateral orchidectomy was performed in 7 patients, and 7 were treated with a GnRH-agonist (leuprorelin). After castrationm two to four prostatic core biopsy specimens were taken, and the level of hypoxia-inducible factor-1α (HIF-1α) in cancer was determined by immunofluorescence. Among biopsy specimens taken before castration, strong HIF-1α expression (mean intensity above 30) was shown in 5 patients, weak expression (mean intensity 10-30) in 3 patients, and background levels of HIF-1α (mean intensity 0-10) in 6 patients. Downregulation of HIF-1α expression after castration was observed in all 5 patients with strong HIF-1α precastration expression. HIF-1α expression was also reduced in 2 of 3 patients with weak HIF-1α precastration expression. Our data suggest that neoadjuvant castration decreases tumor cell hypoxia in prostate cancer, which may explain increased radiosensitivity after castration.}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/j.ijrobp.2011.10.03810.1016/j.ijrobp.2011.10.038] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2228403222284032]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Neoadjuvant androgen deprivation in combination with radiotherapy of prostate cancer is used to improve radioresponsiveness and local tumor control. Currently, the underlying mechanism is not well understood. Because hypoxia causes resistance to radiotherapy, we wanted to test whether castration affects the degree of hypoxia in prostate cancer. In 14 patients with locally advanced prostate cancer, six to 12 prostatic needle core biopsy specimens were taken prior to castration therapy. Bilateral orchidectomy was performed in 7 patients, and 7 were treated with a GnRH-agonist (leuprorelin). After castrationm two to four prostatic core biopsy specimens were taken, and the level of hypoxia-inducible factor-1α (HIF-1α) in cancer was determined by immunofluorescence. Among biopsy specimens taken before castration, strong HIF-1α expression (mean intensity above 30) was shown in 5 patients, weak expression (mean intensity 10-30) in 3 patients, and background levels of HIF-1α (mean intensity 0-10) in 6 patients. Downregulation of HIF-1α expression after castration was observed in all 5 patients with strong HIF-1α precastration expression. HIF-1α expression was also reduced in 2 of 3 patients with weak HIF-1α precastration expression. Our data suggest that neoadjuvant castration decreases tumor cell hypoxia in prostate cancer, which may explain increased radiosensitivity after castration. |
Gubanova, E; Brown, B; Ivanov, S V; Helleday, T; Mills, G B; Yarbrough, W G; Issaeva, N Đownregulation of SMG-1 in ĦPV-positive head and neck squamous cell carcinoma due to promoter hypermethylation correlates with improved survival Journal Article Clin. Cancer Res., 18 (5), pp. 1257–1267, 2012, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4010255PMC4010255] [DOI:hrefhttp://dx.doi.org/10.1158/1078-0432.CCR-11-205810.1158/1078-0432.CCR-11-2058] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2224749522247495]). @article{pmid22247495, title = {Đownregulation of SMG-1 in ĦPV-positive head and neck squamous cell carcinoma due to promoter hypermethylation correlates with improved survival}, author = { E. Gubanova and B. Brown and S. V. Ivanov and T. Helleday and G. B. Mills and W. G. Yarbrough and N. Issaeva}, year = {2012}, date = {2012-03-01}, journal = {Clin. Cancer Res.}, volume = {18}, number = {5}, pages = {1257--1267}, abstract = {Human papillomavirus (HPV) is linked with a subset of head and neck squamous cell carcinomas (HNSCC). HPV-positive HNSCCs show a better prognosis than HPV-negative HNSCCs, which may be explained by sensitivity of the HPV-positive HNSCCs to ionizing radiation (IR). Although the molecular mechanism behind sensitivity to IR in HPV-positive HNSCCs is unresolved, DNA damage response (DDR) might be a significant determinant of IR sensitivity. An important player in the DDR, SMG-1 (suppressor with morphogenetic effect on genitalia), is a potential tumor suppressor and may therefore be deregulated in cancer. No studies have yet been conducted linking defects in SMG-1 expression with cancer. We investigated whether deregulation of SMG-1 could be responsible for defects in the DDR in oropharyngeal HNSCC. Expression and promoter methylation status of SMG-1 were investigated in HNSCCs. To identify a functional link between HPV infection and SMG-1, we transfected the HPV-negative cells with an E6/E7 expression construct. SMG-1 short hairpin RNAs were expressed in HPV-negative cells to estimate survival upon IR. Forced E6/E7 expression in HPV-negative cells resulted in SMG-1 promoter hypermethylation and decreased SMG-1 expression. Due to promoter hypermethylation, HPV-positive HNSCC cells and tumors express SMG-1 at lower levels than HPV-negative SCCs. Depletion of SMG-1 in HPV-negative HNSCC cells resulted in increased radiation sensitivity, whereas SMG-1 overexpression protected HPV-positive tumor cells from irradiation. Levels of SMG-1 expression negatively correlated with HPV status in cancer cell lines and tumors. Diminished SMG-1 expression may contribute to the enhanced response to therapy exhibited by HPV-positive HNSCCs.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4010255PMC4010255] [DOI:hrefhttp://dx.doi.org/10.1158/1078-0432.CCR-11-205810.1158/1078-0432.CCR-11-2058] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2224749522247495]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Human papillomavirus (HPV) is linked with a subset of head and neck squamous cell carcinomas (HNSCC). HPV-positive HNSCCs show a better prognosis than HPV-negative HNSCCs, which may be explained by sensitivity of the HPV-positive HNSCCs to ionizing radiation (IR). Although the molecular mechanism behind sensitivity to IR in HPV-positive HNSCCs is unresolved, DNA damage response (DDR) might be a significant determinant of IR sensitivity. An important player in the DDR, SMG-1 (suppressor with morphogenetic effect on genitalia), is a potential tumor suppressor and may therefore be deregulated in cancer. No studies have yet been conducted linking defects in SMG-1 expression with cancer. We investigated whether deregulation of SMG-1 could be responsible for defects in the DDR in oropharyngeal HNSCC. Expression and promoter methylation status of SMG-1 were investigated in HNSCCs. To identify a functional link between HPV infection and SMG-1, we transfected the HPV-negative cells with an E6/E7 expression construct. SMG-1 short hairpin RNAs were expressed in HPV-negative cells to estimate survival upon IR. Forced E6/E7 expression in HPV-negative cells resulted in SMG-1 promoter hypermethylation and decreased SMG-1 expression. Due to promoter hypermethylation, HPV-positive HNSCC cells and tumors express SMG-1 at lower levels than HPV-negative SCCs. Depletion of SMG-1 in HPV-negative HNSCC cells resulted in increased radiation sensitivity, whereas SMG-1 overexpression protected HPV-positive tumor cells from irradiation. Levels of SMG-1 expression negatively correlated with HPV status in cancer cell lines and tumors. Diminished SMG-1 expression may contribute to the enhanced response to therapy exhibited by HPV-positive HNSCCs. |
Fraser, M; Zhao, H; Luoto, K R; Lundin, C; Coackley, C; Chan, N; Joshua, A M; Bismar, T A; Evans, A; Helleday, T; Bristow, R G PŦEN deletion in prostate cancer cells does not associate with loss of RAĐ51 function: implications for radiotherapy and chemotherapy Journal Article Clin. Cancer Res., 18 (4), pp. 1015–1027, 2012, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3378487PMC3378487] [DOI:hrefhttp://dx.doi.org/10.1158/1078-0432.CCR-11-218910.1158/1078-0432.CCR-11-2189] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2211413822114138]). @article{pmid22114138, title = {PŦEN deletion in prostate cancer cells does not associate with loss of RAĐ51 function: implications for radiotherapy and chemotherapy}, author = { M. Fraser and H. Zhao and K. R. Luoto and C. Lundin and C. Coackley and N. Chan and A. M. Joshua and T. A. Bismar and A. Evans and T. Helleday and R. G. Bristow}, year = {2012}, date = {2012-02-01}, journal = {Clin. Cancer Res.}, volume = {18}, number = {4}, pages = {1015--1027}, abstract = {PTEN deletions in prostate cancer are associated with tumor aggression and poor outcome. Recent studies have implicated PTEN as a determinant of homologous recombination (HR) through defective RAD51 function. Similar to BRCA1/2-defective tumor cells, PTEN-null prostate and other cancer cells have been reported to be sensitive to PARP inhibitors (PARPi). To date, no direct comparison between PTEN and RAD51 expression in primary prostate tumors has been reported. Prostate cancer cell lines and xenografts with known PTEN status (22RV1-PTEN(+/+), DU145-PTEN(+/-), PC3-PTEN(-/-)) and H1299 and HCT116 cancer cells were used to evaluate how PTEN loss affects RAD51 expression and PARPi sensitivity. Primary prostate cancers with known PTEN status were analyzed for RAD51 expression. PTEN status is not associated with reduced RAD51 mRNA or protein expression in primary prostate cancers. Decreased PTEN expression did not reduce RAD51 expression or clonogenic survival following PARPi among prostate cancer cells that vary in TP53 and PTEN. PARPi sensitivity instead associated with a defect in MRE11 expression. PTEN-deficient cells had only mild PARPi sensitivity and no loss of HR or RAD51 recruitment. Clonogenic cell survival following a series of DNA damaging agents was variable: PTEN-deficient cells were sensitive to ionizing radiation, mitomycin-C, UV, H(2)O(2), and methyl methanesulfonate but not to cisplatin, camptothecin, or paclitaxel. These data suggest that the relationship between PTEN status and survival following DNA damage is indirect and complex. It is unlikely that PTEN status will be a direct biomarker for HR status or PARPi response in prostate cancer clinical trials.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3378487PMC3378487] [DOI:hrefhttp://dx.doi.org/10.1158/1078-0432.CCR-11-218910.1158/1078-0432.CCR-11-2189] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2211413822114138]}, keywords = {}, pubstate = {published}, tppubtype = {article} } PTEN deletions in prostate cancer are associated with tumor aggression and poor outcome. Recent studies have implicated PTEN as a determinant of homologous recombination (HR) through defective RAD51 function. Similar to BRCA1/2-defective tumor cells, PTEN-null prostate and other cancer cells have been reported to be sensitive to PARP inhibitors (PARPi). To date, no direct comparison between PTEN and RAD51 expression in primary prostate tumors has been reported. Prostate cancer cell lines and xenografts with known PTEN status (22RV1-PTEN(+/+), DU145-PTEN(+/-), PC3-PTEN(-/-)) and H1299 and HCT116 cancer cells were used to evaluate how PTEN loss affects RAD51 expression and PARPi sensitivity. Primary prostate cancers with known PTEN status were analyzed for RAD51 expression. PTEN status is not associated with reduced RAD51 mRNA or protein expression in primary prostate cancers. Decreased PTEN expression did not reduce RAD51 expression or clonogenic survival following PARPi among prostate cancer cells that vary in TP53 and PTEN. PARPi sensitivity instead associated with a defect in MRE11 expression. PTEN-deficient cells had only mild PARPi sensitivity and no loss of HR or RAD51 recruitment. Clonogenic cell survival following a series of DNA damaging agents was variable: PTEN-deficient cells were sensitive to ionizing radiation, mitomycin-C, UV, H(2)O(2), and methyl methanesulfonate but not to cisplatin, camptothecin, or paclitaxel. These data suggest that the relationship between PTEN status and survival following DNA damage is indirect and complex. It is unlikely that PTEN status will be a direct biomarker for HR status or PARPi response in prostate cancer clinical trials. |
Strom, C E; Helleday, T Strategies for the Use of Poly(adenosine diphosphate ribose) Polymerase (PARP) Inhibitors in Cancer Ŧherapy Journal Article Biomolecules, 2 (4), pp. 635–649, 2012, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4030856PMC4030856] [DOI:hrefhttp://dx.doi.org/10.3390/biom204063510.3390/biom2040635] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2497015324970153]). @article{pmid24970153, title = {Strategies for the Use of Poly(adenosine diphosphate ribose) Polymerase (PARP) Inhibitors in Cancer Ŧherapy}, author = { C. E. Strom and T. Helleday}, year = {2012}, date = {2012-01-01}, journal = {Biomolecules}, volume = {2}, number = {4}, pages = {635--649}, abstract = {Treatments with Poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors have offered patients carrying cancers with mutated BRCA1 or BRCA2 genes a new and in many cases effective option for disease control. There is potentially a large patient population that may also benefit from PARP inhibitor treatment, either in monotherapy or in combination with chemotherapy. Here, we describe the multifaceted role of PARP inhibitors and discuss which treatment options could potentially be useful to gain disease control without potentiating side effects.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC4030856PMC4030856] [DOI:hrefhttp://dx.doi.org/10.3390/biom204063510.3390/biom2040635] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2497015324970153]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Treatments with Poly(adenosine diphosphate ribose) polymerase (PARP) inhibitors have offered patients carrying cancers with mutated BRCA1 or BRCA2 genes a new and in many cases effective option for disease control. There is potentially a large patient population that may also benefit from PARP inhibitor treatment, either in monotherapy or in combination with chemotherapy. Here, we describe the multifaceted role of PARP inhibitors and discuss which treatment options could potentially be useful to gain disease control without potentiating side effects. |
Nicolay, N H; Helleday, T; Sharma, R A Biological relevance of ĐNA polymerase β and translesion synthesis polymerases to cancer and its treatment Journal Article Curr Mol Pharmacol, 5 (1), pp. 54–67, 2012, ([PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2212246422122464]). @article{pmid22122464, title = {Biological relevance of ĐNA polymerase β and translesion synthesis polymerases to cancer and its treatment}, author = { N. H. Nicolay and T. Helleday and R. A. Sharma}, year = {2012}, date = {2012-01-01}, journal = {Curr Mol Pharmacol}, volume = {5}, number = {1}, pages = {54--67}, abstract = {The cellular genome is constantly subject to DNA damage caused by endogenous factors or exogenously by damaging agents such as ionizing radiation or various anticancer agents. The base excision repair (BER) enzyme, DNA polymerase β, and the polymerases involved in translesion synthesis (TLS) have been shown to contribute to cellular tolerance and repair of DNA lesions by anticancer treatments, particularly the platinum cytotoxic drugs. Moreover, there is robust preclinical evidence linking alterations in DNA pol β and TLS polymerase levels to cancer. DNA polymerases may therefore be potential targets to increase the sensitivity of cancer cells to chemotherapy drugs. In this article, the physical and chemical properties of DNA polymerase β and the translesion synthesis polymerases are reviewed with a view to identifying how they may act as targets for anticancer treatment. The potential clinical role of new DNA polymerase inhibitors is discussed and how they may be combined with conventional cytotoxic agents.}, note = {[PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2212246422122464]}, keywords = {}, pubstate = {published}, tppubtype = {article} } The cellular genome is constantly subject to DNA damage caused by endogenous factors or exogenously by damaging agents such as ionizing radiation or various anticancer agents. The base excision repair (BER) enzyme, DNA polymerase β, and the polymerases involved in translesion synthesis (TLS) have been shown to contribute to cellular tolerance and repair of DNA lesions by anticancer treatments, particularly the platinum cytotoxic drugs. Moreover, there is robust preclinical evidence linking alterations in DNA pol β and TLS polymerase levels to cancer. DNA polymerases may therefore be potential targets to increase the sensitivity of cancer cells to chemotherapy drugs. In this article, the physical and chemical properties of DNA polymerase β and the translesion synthesis polymerases are reviewed with a view to identifying how they may act as targets for anticancer treatment. The potential clinical role of new DNA polymerase inhibitors is discussed and how they may be combined with conventional cytotoxic agents. |
Wilsker, D; Chung, J H; Pradilla, I; Petermann, E; Helleday, T; Bunz, F Ŧargeted mutations in the AŦR pathway define agent-specific requirements for cancer cell growth and survival Journal Article Mol. Cancer Ther., 11 (1), pp. 98–107, 2012, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3256256PMC3256256] [DOI:hrefhttp://dx.doi.org/10.1158/1535-7163.MCT-11-067510.1158/1535-7163.MCT-11-0675] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2208416922084169]). @article{pmid22084169, title = {Ŧargeted mutations in the AŦR pathway define agent-specific requirements for cancer cell growth and survival}, author = { D. Wilsker and J. H. Chung and I. Pradilla and E. Petermann and T. Helleday and F. Bunz}, year = {2012}, date = {2012-01-01}, journal = {Mol. Cancer Ther.}, volume = {11}, number = {1}, pages = {98--107}, abstract = {Many anticancer agents induce DNA strand breaks or cause the accumulation of DNA replication intermediates. The protein encoded by ataxia-telangiectasia mutated and Rad 3-related (ATR) generates signals in response to these altered DNA structures and activates cellular survival responses. Accordingly, ATR has drawn increased attention as a potential target for novel therapeutic strategies designed to potentiate the effects of existing drugs. In this study, we use a unique panel of genetically modified human cancer cells to unambiguously test the roles of upstream and downstream components of the ATR pathway in the responses to common therapeutic agents. Upstream, the S-phase-specific cyclin-dependent kinase (Cdk) 2 was required for robust activation of ATR in response to diverse chemotherapeutic agents. While Cdk2-mediated ATR activation promoted cell survival after treatment with many drugs, signaling from ATR directly to the checkpoint kinase Chk1 was required for survival responses to only a subset of the drugs tested. These results show that specifically inhibiting the Cdk2/ATR/Chk1 pathway via distinct regulators can differentially sensitize cancer cells to a wide range of therapeutic agents.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3256256PMC3256256] [DOI:hrefhttp://dx.doi.org/10.1158/1535-7163.MCT-11-067510.1158/1535-7163.MCT-11-0675] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2208416922084169]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Many anticancer agents induce DNA strand breaks or cause the accumulation of DNA replication intermediates. The protein encoded by ataxia-telangiectasia mutated and Rad 3-related (ATR) generates signals in response to these altered DNA structures and activates cellular survival responses. Accordingly, ATR has drawn increased attention as a potential target for novel therapeutic strategies designed to potentiate the effects of existing drugs. In this study, we use a unique panel of genetically modified human cancer cells to unambiguously test the roles of upstream and downstream components of the ATR pathway in the responses to common therapeutic agents. Upstream, the S-phase-specific cyclin-dependent kinase (Cdk) 2 was required for robust activation of ATR in response to diverse chemotherapeutic agents. While Cdk2-mediated ATR activation promoted cell survival after treatment with many drugs, signaling from ATR directly to the checkpoint kinase Chk1 was required for survival responses to only a subset of the drugs tested. These results show that specifically inhibiting the Cdk2/ATR/Chk1 pathway via distinct regulators can differentially sensitize cancer cells to a wide range of therapeutic agents. |
2011 |
Helleday, T ĐNA repair as treatment target Journal Article Eur. J. Cancer, 47 Suppl 3 , pp. S333–335, 2011, ([DOI:hrefhttp://dx.doi.org/10.1016/S0959-8049(11)70192-710.1016/S0959-8049(11)70192-7] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2194400421944004]). BibTeX | Tags: @article{pmid21944004, title = {ĐNA repair as treatment target}, author = { T. Helleday}, year = {2011}, date = {2011-09-01}, journal = {Eur. J. Cancer}, volume = {47 Suppl 3}, pages = {S333--335}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/S0959-8049(11)70192-710.1016/S0959-8049(11)70192-7] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2194400421944004]}, keywords = {}, pubstate = {published}, tppubtype = {article} } |
Glazer, P M; Le, Q T; Bristow, R; Helleday, T; Pelroy, R; Bernhard, E J New translational possibilities for microenvironmental modulation of radiosensitivity Journal Article Radiat. Res., 176 (3), pp. 412–414, 2011, ([PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2186743121867431]). BibTeX | Tags: @article{pmid21867431, title = {New translational possibilities for microenvironmental modulation of radiosensitivity}, author = { P. M. Glazer and Q. T. Le and R. Bristow and T. Helleday and R. Pelroy and E. J. Bernhard}, year = {2011}, date = {2011-09-01}, journal = {Radiat. Res.}, volume = {176}, number = {3}, pages = {412--414}, note = {[PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2186743121867431]}, keywords = {}, pubstate = {published}, tppubtype = {article} } |
Glazer, P M; Grandis, J; Powell, S N; Brown, J M; Helleday, T; Bristow, R; Powis, G; Hill, R P; Le, Q T; Pelroy, R; Mohla, S; Bernhard, E J; DeGregori, J; Ivan, M; Vaezi, A; Boothman, D A; Hammond, E; Larner, J; Wheeler, D; Koumenis, C; Chan, D; Chen, D; Czerniecki, B J; Dewhirst, M; Furdui, C; Lyden, D; McBride, W H; Pajonk, F; Powis, G; Rich, J; Haimovitz-Friedman, A Radiation Resistance in Cancer Ŧherapy: meeting summary and research opportunities. Report of an NCI Workshop held September 1-3, 2010 Journal Article Radiat. Res., 176 (3), pp. 0016–21, 2011, ([PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2186742821867428]). BibTeX | Tags: @article{pmid21867428, title = {Radiation Resistance in Cancer Ŧherapy: meeting summary and research opportunities. Report of an NCI Workshop held September 1-3, 2010}, author = { P. M. Glazer and J. Grandis and S. N. Powell and J. M. Brown and T. Helleday and R. Bristow and G. Powis and R. P. Hill and Q. T. Le and R. Pelroy and S. Mohla and E. J. Bernhard and J. DeGregori and M. Ivan and A. Vaezi and D. A. Boothman and E. Hammond and J. Larner and D. Wheeler and C. Koumenis and D. Chan and D. Chen and B. J. Czerniecki and M. Dewhirst and C. Furdui and D. Lyden and W. H. McBride and F. Pajonk and G. Powis and J. Rich and A. Haimovitz-Friedman}, year = {2011}, date = {2011-09-01}, journal = {Radiat. Res.}, volume = {176}, number = {3}, pages = {0016--21}, note = {[PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2186742821867428]}, keywords = {}, pubstate = {published}, tppubtype = {article} } |
Strom, C E; Mortusewicz, O; Finch, D; Parsons, J L; Lagerqvist, A; Johansson, F; Schultz, N; Erixon, K; Dianov, G L; Helleday, T CK2 phosphorylation of XRCC1 facilitates dissociation from ĐNA and single-strand break formation during base excision repair Journal Article DNA Repair (Amst.), 10 (9), pp. 961–969, 2011, ([DOI:hrefhttp://dx.doi.org/10.1016/j.dnarep.2011.07.00410.1016/j.dnarep.2011.07.004] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2184077521840775]). @article{pmid21840775, title = {CK2 phosphorylation of XRCC1 facilitates dissociation from ĐNA and single-strand break formation during base excision repair}, author = { C. E. Strom and O. Mortusewicz and D. Finch and J. L. Parsons and A. Lagerqvist and F. Johansson and N. Schultz and K. Erixon and G. L. Dianov and T. Helleday}, year = {2011}, date = {2011-09-01}, journal = {DNA Repair (Amst.)}, volume = {10}, number = {9}, pages = {961--969}, abstract = {CK2 phosphorylates the scaffold protein XRCC1, which is required for efficient DNA single-strand break (SSB) repair. Here, we express an XRCC1 protein (XRCC1(ckm)) that cannot be phosphorylated by CK2 in XRCC1 mutated EM9 cells and show that the role of this post-translational modification gives distinct phenotypes in SSB repair and base excision repair (BER). Interestingly, we find that fewer SSBs are formed during BER after treatment with the alkylating agent dimethyl sulfate (DMS) in EM9 cells expressing XRCC1(ckm) (CKM cells) or following inhibition with the CK2 inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT). We also show that XRCC1(ckm) protein has a higher affinity for DNA than wild type XRCC1 protein and resides in an immobile fraction on DNA, in particular after damage. We propose a model whereby the increased affinity for DNA sequesters XRCC1(ckm) and the repair enzymes associated with it, at the repair site, which retards kinetics of BER. In conclusion, our results indicate that phosphorylation of XRCC1 by CK2 facilitates the BER incision step, likely by promoting dissociation from DNA.}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/j.dnarep.2011.07.00410.1016/j.dnarep.2011.07.004] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2184077521840775]}, keywords = {}, pubstate = {published}, tppubtype = {article} } CK2 phosphorylates the scaffold protein XRCC1, which is required for efficient DNA single-strand break (SSB) repair. Here, we express an XRCC1 protein (XRCC1(ckm)) that cannot be phosphorylated by CK2 in XRCC1 mutated EM9 cells and show that the role of this post-translational modification gives distinct phenotypes in SSB repair and base excision repair (BER). Interestingly, we find that fewer SSBs are formed during BER after treatment with the alkylating agent dimethyl sulfate (DMS) in EM9 cells expressing XRCC1(ckm) (CKM cells) or following inhibition with the CK2 inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT). We also show that XRCC1(ckm) protein has a higher affinity for DNA than wild type XRCC1 protein and resides in an immobile fraction on DNA, in particular after damage. We propose a model whereby the increased affinity for DNA sequesters XRCC1(ckm) and the repair enzymes associated with it, at the repair site, which retards kinetics of BER. In conclusion, our results indicate that phosphorylation of XRCC1 by CK2 facilitates the BER incision step, likely by promoting dissociation from DNA. |
Elvers, I; Johansson, F; Groth, P; Erixon, K; Helleday, T UV stalled replication forks restart by re-priming in human fibroblasts Journal Article Nucleic Acids Res., 39 (16), pp. 7049–7057, 2011, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3167624PMC3167624] [DOI:hrefhttp://dx.doi.org/10.1093/nar/gkr42010.1093/nar/gkr420] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2164634021646340]). @article{pmid21646340, title = {UV stalled replication forks restart by re-priming in human fibroblasts}, author = { I. Elvers and F. Johansson and P. Groth and K. Erixon and T. Helleday}, year = {2011}, date = {2011-09-01}, journal = {Nucleic Acids Res.}, volume = {39}, number = {16}, pages = {7049--7057}, abstract = {Restarting stalled replication forks is vital to avoid fatal replication errors. Previously, it was demonstrated that hydroxyurea-stalled replication forks rescue replication either by an active restart mechanism or by new origin firing. To our surprise, using the DNA fibre assay, we only detect a slightly reduced fork speed on a UV-damaged template during the first hour after UV exposure, and no evidence for persistent replication fork arrest. Interestingly, no evidence for persistent UV-induced fork stalling was observed even in translesion synthesis defective, Polη(mut) cells. In contrast, using an assay to measure DNA molecule elongation at the fork, we observe that continuous DNA elongation is severely blocked by UV irradiation, particularly in UV-damaged Polη(mut) cells. In conclusion, our data suggest that UV-blocked replication forks restart effectively through re-priming past the lesion, leaving only a small gap opposite the lesion. This allows continuation of replication on damaged DNA. If left unfilled, the gaps may collapse into DNA double-strand breaks that are repaired by a recombination pathway, similar to the fate of replication forks collapsed after hydroxyurea treatment.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3167624PMC3167624] [DOI:hrefhttp://dx.doi.org/10.1093/nar/gkr42010.1093/nar/gkr420] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2164634021646340]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Restarting stalled replication forks is vital to avoid fatal replication errors. Previously, it was demonstrated that hydroxyurea-stalled replication forks rescue replication either by an active restart mechanism or by new origin firing. To our surprise, using the DNA fibre assay, we only detect a slightly reduced fork speed on a UV-damaged template during the first hour after UV exposure, and no evidence for persistent replication fork arrest. Interestingly, no evidence for persistent UV-induced fork stalling was observed even in translesion synthesis defective, Polη(mut) cells. In contrast, using an assay to measure DNA molecule elongation at the fork, we observe that continuous DNA elongation is severely blocked by UV irradiation, particularly in UV-damaged Polη(mut) cells. In conclusion, our data suggest that UV-blocked replication forks restart effectively through re-priming past the lesion, leaving only a small gap opposite the lesion. This allows continuation of replication on damaged DNA. If left unfilled, the gaps may collapse into DNA double-strand breaks that are repaired by a recombination pathway, similar to the fate of replication forks collapsed after hydroxyurea treatment. |
Helleday, T Ŧhe underlying mechanism for the PARP and BRCA synthetic lethality: clearing up the misunderstandings Journal Article Mol Oncol, 5 (4), pp. 387–393, 2011, ([DOI:hrefhttp://dx.doi.org/10.1016/j.molonc.2011.07.00110.1016/j.molonc.2011.07.001] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2182147521821475]). @article{pmid21821475, title = {Ŧhe underlying mechanism for the PARP and BRCA synthetic lethality: clearing up the misunderstandings}, author = { T. Helleday}, year = {2011}, date = {2011-08-01}, journal = {Mol Oncol}, volume = {5}, number = {4}, pages = {387--393}, abstract = {Poly (ADP-ribose) polymerase (PARP) inhibitors effectively kill tumours defective in the BRCA1 or BRCA2 genes through the concept of synthetic lethality. It is suggested that PARP inhibitors cause an increase in DNA single-strand breaks (SSBs), which are converted during replication to irreparable toxic DNA double-strand breaks (DSBs) in BRCA1/2 defective cells. There are a number of recent reports challenging this model. Here, alternative models that are not mutually exclusive are presented to explain the synthetic lethality between BRCA1/2 and PARP inhibitors. One such model proposes that PARP inhibition causes PARP-1 to be trapped onto DNA repair intermediates, especially during base excision repair. This may in turn cause obstruction to replication forks, which require BRCA-dependent homologous recombination to be resolved. In another model, PARP is directly involved in catalysing replication repair in a distinct pathway from homologous recombination. Experimental evidence supporting these novel models to explain the PARP-BRCA synthetic lethality are discussed.}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/j.molonc.2011.07.00110.1016/j.molonc.2011.07.001] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2182147521821475]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Poly (ADP-ribose) polymerase (PARP) inhibitors effectively kill tumours defective in the BRCA1 or BRCA2 genes through the concept of synthetic lethality. It is suggested that PARP inhibitors cause an increase in DNA single-strand breaks (SSBs), which are converted during replication to irreparable toxic DNA double-strand breaks (DSBs) in BRCA1/2 defective cells. There are a number of recent reports challenging this model. Here, alternative models that are not mutually exclusive are presented to explain the synthetic lethality between BRCA1/2 and PARP inhibitors. One such model proposes that PARP inhibition causes PARP-1 to be trapped onto DNA repair intermediates, especially during base excision repair. This may in turn cause obstruction to replication forks, which require BRCA-dependent homologous recombination to be resolved. In another model, PARP is directly involved in catalysing replication repair in a distinct pathway from homologous recombination. Experimental evidence supporting these novel models to explain the PARP-BRCA synthetic lethality are discussed. |
Svensson, L M; Jemth, A S; Desroses, M; Loseva, O; Helleday, T; Hogbom, M; Stenmark, P Crystal structure of human MŦĦ1 and the 8-oxo-dGMP product complex Journal Article FEBS Lett., 585 (16), pp. 2617–2621, 2011, ([DOI:hrefhttp://dx.doi.org/10.1016/j.febslet.2011.07.01710.1016/j.febslet.2011.07.017] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2178777221787772]). @article{pmid21787772, title = {Crystal structure of human MŦĦ1 and the 8-oxo-dGMP product complex}, author = { L. M. Svensson and A. S. Jemth and M. Desroses and O. Loseva and T. Helleday and M. Hogbom and P. Stenmark}, year = {2011}, date = {2011-08-01}, journal = {FEBS Lett.}, volume = {585}, number = {16}, pages = {2617--2621}, abstract = {MTH1 hydrolyzes oxidized nucleotide triphosphates, thereby preventing them from being incorporated into DNA. We here present the structures of human MTH1 (1.9Å) and its complex with the product 8-oxo-dGMP (1.8Å). Unexpectedly MTH1 binds the nucleotide in the anti conformation with no direct interaction between the 8-oxo group and the protein. We suggest that the specificity depends on the stabilization of an enol tautomer of the 8-oxo form of dGTP. The binding of the product induces no major structural changes. The structures reveal the mode of nucleotide binding in MTH1 and provide the structural basis for inhibitor design.}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/j.febslet.2011.07.01710.1016/j.febslet.2011.07.017] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2178777221787772]}, keywords = {}, pubstate = {published}, tppubtype = {article} } MTH1 hydrolyzes oxidized nucleotide triphosphates, thereby preventing them from being incorporated into DNA. We here present the structures of human MTH1 (1.9Å) and its complex with the product 8-oxo-dGMP (1.8Å). Unexpectedly MTH1 binds the nucleotide in the anti conformation with no direct interaction between the 8-oxo group and the protein. We suggest that the specificity depends on the stabilization of an enol tautomer of the 8-oxo form of dGTP. The binding of the product induces no major structural changes. The structures reveal the mode of nucleotide binding in MTH1 and provide the structural basis for inhibitor design. |
Glazer, P M; Grandis, J; Powell, S N; Brown, J M; Helleday, T; Bristow, R; Powis, G; Hill, R P; Le, Q T; Pelroy, R; Mohla, S; Bernhard, E J Radiation Resistance in Cancer Ŧherapy: Meeting Summary and Research Opportunities: Report of an NCI Workshop held September 1-3, 2010 Journal Article Radiat. Res., 2011, ([PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2174025121740251]). BibTeX | Tags: @article{pmid21740251, title = {Radiation Resistance in Cancer Ŧherapy: Meeting Summary and Research Opportunities: Report of an NCI Workshop held September 1-3, 2010}, author = { P. M. Glazer and J. Grandis and S. N. Powell and J. M. Brown and T. Helleday and R. Bristow and G. Powis and R. P. Hill and Q. T. Le and R. Pelroy and S. Mohla and E. J. Bernhard}, year = {2011}, date = {2011-07-01}, journal = {Radiat. Res.}, note = {[PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2174025121740251]}, keywords = {}, pubstate = {published}, tppubtype = {article} } |
Segal-Raz, H; Mass, G; Baranes-Bachar, K; Lerenthal, Y; Wang, S Y; Chung, Y M; Ziv-Lehrman, S; Strom, C E; Helleday, T; Hu, M C; Chen, D J; Shiloh, Y AŦM-mediated phosphorylation of polynucleotide kinase/phosphatase is required for effective ĐNA double-strand break repair Journal Article EMBO Rep., 12 (7), pp. 713–719, 2011, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3128972PMC3128972] [DOI:hrefhttp://dx.doi.org/10.1038/embor.2011.9610.1038/embor.2011.96] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2163729821637298]). @article{pmid21637298, title = {AŦM-mediated phosphorylation of polynucleotide kinase/phosphatase is required for effective ĐNA double-strand break repair}, author = { H. Segal-Raz and G. Mass and K. Baranes-Bachar and Y. Lerenthal and S. Y. Wang and Y. M. Chung and S. Ziv-Lehrman and C. E. Strom and T. Helleday and M. C. Hu and D. J. Chen and Y. Shiloh}, year = {2011}, date = {2011-07-01}, journal = {EMBO Rep.}, volume = {12}, number = {7}, pages = {713--719}, abstract = {The cellular response to double-strand breaks (DSBs) in DNA is a complex signalling network, mobilized by the nuclear protein kinase ataxia-telangiectasia mutated (ATM), which phosphorylates many factors in the various branches of this network. A main question is how ATM regulates DSB repair. Here, we identify the DNA repair enzyme polynucleotide kinase/phosphatase (PNKP) as an ATM target. PNKP phosphorylates 5'-OH and dephosphorylates 3'-phosphate DNA ends that are formed at DSB termini caused by DNA-damaging agents, thereby regenerating legitimate ends for further processing. We establish that the ATM phosphorylation targets on human PNKP-Ser 114 and Ser 126-are crucial for cellular survival following DSB induction and for effective DSB repair, being essential for damage-induced enhancement of the activity of PNKP and its proper accumulation at the sites of DNA damage. These findings show a direct functional link between ATM and the DSB-repair machinery.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3128972PMC3128972] [DOI:hrefhttp://dx.doi.org/10.1038/embor.2011.9610.1038/embor.2011.96] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2163729821637298]}, keywords = {}, pubstate = {published}, tppubtype = {article} } The cellular response to double-strand breaks (DSBs) in DNA is a complex signalling network, mobilized by the nuclear protein kinase ataxia-telangiectasia mutated (ATM), which phosphorylates many factors in the various branches of this network. A main question is how ATM regulates DSB repair. Here, we identify the DNA repair enzyme polynucleotide kinase/phosphatase (PNKP) as an ATM target. PNKP phosphorylates 5'-OH and dephosphorylates 3'-phosphate DNA ends that are formed at DSB termini caused by DNA-damaging agents, thereby regenerating legitimate ends for further processing. We establish that the ATM phosphorylation targets on human PNKP-Ser 114 and Ser 126-are crucial for cellular survival following DSB induction and for effective DSB repair, being essential for damage-induced enhancement of the activity of PNKP and its proper accumulation at the sites of DNA damage. These findings show a direct functional link between ATM and the DSB-repair machinery. |
Al-Assar, O; Mantoni, T; Lunardi, S; Kingham, G; Helleday, T; Brunner, T B Breast cancer stem-like cells show dominant homologous recombination due to a larger S-G2 fraction Journal Article Cancer Biol. Ther., 11 (12), pp. 1028–1035, 2011, ([PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2155878921558789]). @article{pmid21558789, title = {Breast cancer stem-like cells show dominant homologous recombination due to a larger S-G2 fraction}, author = { O. Al-Assar and T. Mantoni and S. Lunardi and G. Kingham and T. Helleday and T. B. Brunner}, year = {2011}, date = {2011-06-01}, journal = {Cancer Biol. Ther.}, volume = {11}, number = {12}, pages = {1028--1035}, abstract = {The concept of cancer stem cells is generally accepted in different malignancies. We have previously shown that the MDA-MB231 breast cancer cells were more radiation resistant when sorted for the two stem cell markers CD24 and ESA. In this study, we examined a possible mechanism that might underlie this phenotype by looking at cell cycle profile and the effect this has on DNA repair pathways. The cell cycle profile showed that there were more CD24(-) ESA(+) sorted MDA-MB231 cells in the S- and G(2)-phases compared with the unsorted cells, 60 and 38% respectively. Cyclin D and E protein levels supported the cell cycle profile and highlighted the possible involvement of homologous recombination (HR) repair in the radioresistant phenotype. To further support this, CD24(-) ESA(+) sorted MDA-MB231 cells demonstrated statistically significant more RAD51 and less γ-H2AX foci 2 h post 4Gy ionising radiation, compared with the unsorted population. Inhibition of the HR pathway effectively sterilised the CD24(- ) ESA(+) sorted MDA-MB231 cells but had no effect on the unsorted cells or MDA468 control breast cancer cell line. Although the changes we saw were specific to MDA-MB231, these results merit further investigation and can be crucial in identifying a mechanism responsible for cancer stem cells treatment resistance in primary tumors.}, note = {[PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2155878921558789]}, keywords = {}, pubstate = {published}, tppubtype = {article} } The concept of cancer stem cells is generally accepted in different malignancies. We have previously shown that the MDA-MB231 breast cancer cells were more radiation resistant when sorted for the two stem cell markers CD24 and ESA. In this study, we examined a possible mechanism that might underlie this phenotype by looking at cell cycle profile and the effect this has on DNA repair pathways. The cell cycle profile showed that there were more CD24(-) ESA(+) sorted MDA-MB231 cells in the S- and G(2)-phases compared with the unsorted cells, 60 and 38% respectively. Cyclin D and E protein levels supported the cell cycle profile and highlighted the possible involvement of homologous recombination (HR) repair in the radioresistant phenotype. To further support this, CD24(-) ESA(+) sorted MDA-MB231 cells demonstrated statistically significant more RAD51 and less γ-H2AX foci 2 h post 4Gy ionising radiation, compared with the unsorted population. Inhibition of the HR pathway effectively sterilised the CD24(- ) ESA(+) sorted MDA-MB231 cells but had no effect on the unsorted cells or MDA468 control breast cancer cell line. Although the changes we saw were specific to MDA-MB231, these results merit further investigation and can be crucial in identifying a mechanism responsible for cancer stem cells treatment resistance in primary tumors. |
Strom, C E; Johansson, F; Uhlen, M; Szigyarto, C A; Erixon, K; Helleday, T Poly (AĐP-ribose) polymerase (PARP) is not involved in base excision repair but PARP inhibition traps a single-strand intermediate Journal Article Nucleic Acids Res., 39 (8), pp. 3166–3175, 2011, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3082910PMC3082910] [DOI:hrefhttp://dx.doi.org/10.1093/nar/gkq124110.1093/nar/gkq1241] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2118346621183466]). @article{pmid21183466, title = {Poly (AĐP-ribose) polymerase (PARP) is not involved in base excision repair but PARP inhibition traps a single-strand intermediate}, author = { C. E. Strom and F. Johansson and M. Uhlen and C. A. Szigyarto and K. Erixon and T. Helleday}, year = {2011}, date = {2011-04-01}, journal = {Nucleic Acids Res.}, volume = {39}, number = {8}, pages = {3166--3175}, abstract = {Base excision repair (BER) represents the most important repair pathway of endogenous DNA lesions. Initially, a base damage is recognized, excised and a DNA single-strand break (SSB) intermediate forms. The SSB is then ligated, a process that employs proteins also involved in SSB repair, e.g. XRCC1, Ligase III and possibly PARP1. Here, we confirm the role of XRCC1 and PARP in direct SSB repair. Interestingly, we uncover a synthetic lethality between XRCC1 deficiency and PARP inhibition. We also treated cells with alkylating agent dimethyl sulfate (DMS) and monitored the SSB intermediates formed during BER. DMS-induced SSBs were quickly repaired in wild-type cells; while a rapid accumulation of SSBs was observed in cells where post-incision repair was blocked by a PARP inhibitor or by XRCC1 deficiency (EM9 cells). Interestingly, DMS-induced SSBs did not accumulate in PARP1 siRNA depleted cells, demonstrating that PARP1 is not required for efficient completion of BER. Based on these results we suggest no immediate role for PARP1 in BER, but that PARP inhibitors trap PARP on the SSB intermediate formed during BER. Unexpectedly, addition of PARP inhibitor 2 h after DMS treatment still increased SSB levels indicating ongoing repair even at this late time point.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3082910PMC3082910] [DOI:hrefhttp://dx.doi.org/10.1093/nar/gkq124110.1093/nar/gkq1241] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2118346621183466]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Base excision repair (BER) represents the most important repair pathway of endogenous DNA lesions. Initially, a base damage is recognized, excised and a DNA single-strand break (SSB) intermediate forms. The SSB is then ligated, a process that employs proteins also involved in SSB repair, e.g. XRCC1, Ligase III and possibly PARP1. Here, we confirm the role of XRCC1 and PARP in direct SSB repair. Interestingly, we uncover a synthetic lethality between XRCC1 deficiency and PARP inhibition. We also treated cells with alkylating agent dimethyl sulfate (DMS) and monitored the SSB intermediates formed during BER. DMS-induced SSBs were quickly repaired in wild-type cells; while a rapid accumulation of SSBs was observed in cells where post-incision repair was blocked by a PARP inhibitor or by XRCC1 deficiency (EM9 cells). Interestingly, DMS-induced SSBs did not accumulate in PARP1 siRNA depleted cells, demonstrating that PARP1 is not required for efficient completion of BER. Based on these results we suggest no immediate role for PARP1 in BER, but that PARP inhibitors trap PARP on the SSB intermediate formed during BER. Unexpectedly, addition of PARP inhibitor 2 h after DMS treatment still increased SSB levels indicating ongoing repair even at this late time point. |
Bauerschmidt, C; Woodcock, M; Stevens, D L; Hill, M A; Rothkamm, K; Helleday, T Cohesin phosphorylation and mobility of SMC1 at ionizing radiation-induced ĐNA double-strand breaks in human cells Journal Article Exp. Cell Res., 317 (3), pp. 330–337, 2011, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3087432PMC3087432] [DOI:hrefhttp://dx.doi.org/10.1016/j.yexcr.2010.10.02110.1016/j.yexcr.2010.10.021] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2105655621056556]). @article{pmid21056556, title = {Cohesin phosphorylation and mobility of SMC1 at ionizing radiation-induced ĐNA double-strand breaks in human cells}, author = { C. Bauerschmidt and M. Woodcock and D. L. Stevens and M. A. Hill and K. Rothkamm and T. Helleday}, year = {2011}, date = {2011-02-01}, journal = {Exp. Cell Res.}, volume = {317}, number = {3}, pages = {330--337}, abstract = {Cohesin, a hetero-tetrameric complex of SMC1, SMC3, Rad21 and Scc3, associates with chromatin after mitosis and holds sister chromatids together following DNA replication. Following DNA damage, cohesin accumulates at and promotes the repair of DNA double-strand breaks. In addition, phosphorylation of the SMC1/3 subunits contributes to DNA damage-induced cell cycle checkpoint regulation. The aim of this study was to determine the regulation and consequences of SMC1/3 phosphorylation as part of the cohesin complex. We show here that the ATM-dependent phosphorylation of SMC1 and SMC3 is mediated by H2AX, 53BP1 and MDC1. Depletion of RAD21 abolishes these phosphorylations, indicating that only the fully assembled complex is phosphorylated. Comparison of wild type SMC1 and SMC1S966A in fluorescence recovery after photo-bleaching experiments shows that phosphorylation of SMC1 is required for an increased mobility after DNA damage in G2-phase cells, suggesting that ATM-dependent phosphorylation facilitates mobilization of the cohesin complex after DNA damage.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3087432PMC3087432] [DOI:hrefhttp://dx.doi.org/10.1016/j.yexcr.2010.10.02110.1016/j.yexcr.2010.10.021] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2105655621056556]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Cohesin, a hetero-tetrameric complex of SMC1, SMC3, Rad21 and Scc3, associates with chromatin after mitosis and holds sister chromatids together following DNA replication. Following DNA damage, cohesin accumulates at and promotes the repair of DNA double-strand breaks. In addition, phosphorylation of the SMC1/3 subunits contributes to DNA damage-induced cell cycle checkpoint regulation. The aim of this study was to determine the regulation and consequences of SMC1/3 phosphorylation as part of the cohesin complex. We show here that the ATM-dependent phosphorylation of SMC1 and SMC3 is mediated by H2AX, 53BP1 and MDC1. Depletion of RAD21 abolishes these phosphorylations, indicating that only the fully assembled complex is phosphorylated. Comparison of wild type SMC1 and SMC1S966A in fluorescence recovery after photo-bleaching experiments shows that phosphorylation of SMC1 is required for an increased mobility after DNA damage in G2-phase cells, suggesting that ATM-dependent phosphorylation facilitates mobilization of the cohesin complex after DNA damage. |
Stoimenov, I; Schultz, N; Gottipati, P; Helleday, T Ŧranscription inhibition by ĐRB potentiates recombinational repair of UV lesions in mammalian cells Journal Article PLoS ONE, 6 (5), pp. e19492, 2011, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3088672PMC3088672] [DOI:hrefhttp://dx.doi.org/10.1371/journal.pone.001949210.1371/journal.pone.0019492] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2157316021573160]). @article{pmid21573160, title = {Ŧranscription inhibition by ĐRB potentiates recombinational repair of UV lesions in mammalian cells}, author = { I. Stoimenov and N. Schultz and P. Gottipati and T. Helleday}, year = {2011}, date = {2011-01-01}, journal = {PLoS ONE}, volume = {6}, number = {5}, pages = {e19492}, abstract = {Homologous recombination (HR) is intricately associated with replication, transcription and DNA repair in all organisms studied. However, the interplay between all these processes occurring simultaneously on the same DNA molecule is still poorly understood. Here, we study the interplay between transcription and HR during ultraviolet light (UV)-induced DNA damage in mammalian cells. Our results show that inhibition of transcription with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) increases the number of UV-induced DNA lesions (γH2AX, 53BP1 foci formation), which correlates with a decrease in the survival of wild type or nucleotide excision repair defective cells. Furthermore, we observe an increase in RAD51 foci formation, suggesting HR is triggered in response to an increase in UV-induced DSBs, while inhibiting transcription. Unexpectedly, we observe that DRB fails to sensitise HR defective cells to UV treatment. Thus, increased RAD51 foci formation correlates with increased cell death, suggesting the existence of a futile HR repair of UV-induced DSBs which is linked to transcription inhibition.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3088672PMC3088672] [DOI:hrefhttp://dx.doi.org/10.1371/journal.pone.001949210.1371/journal.pone.0019492] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2157316021573160]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Homologous recombination (HR) is intricately associated with replication, transcription and DNA repair in all organisms studied. However, the interplay between all these processes occurring simultaneously on the same DNA molecule is still poorly understood. Here, we study the interplay between transcription and HR during ultraviolet light (UV)-induced DNA damage in mammalian cells. Our results show that inhibition of transcription with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) increases the number of UV-induced DNA lesions (γH2AX, 53BP1 foci formation), which correlates with a decrease in the survival of wild type or nucleotide excision repair defective cells. Furthermore, we observe an increase in RAD51 foci formation, suggesting HR is triggered in response to an increase in UV-induced DSBs, while inhibiting transcription. Unexpectedly, we observe that DRB fails to sensitise HR defective cells to UV treatment. Thus, increased RAD51 foci formation correlates with increased cell death, suggesting the existence of a futile HR repair of UV-induced DSBs which is linked to transcription inhibition. |
Kondo, N; Takahashi, A; Mori, E; Noda, T; Zdzienicka, M Z; Thompson, L H; Helleday, T; Suzuki, M; Kinashi, Y; Masunaga, S; Ono, K; Hasegawa, M; Ohnishi, T FANCĐ1/BRCA2 plays predominant role in the repair of ĐNA damage induced by ACNU or ŦMZ Journal Article PLoS ONE, 6 (5), pp. e19659, 2011, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3090409PMC3090409] [DOI:hrefhttp://dx.doi.org/10.1371/journal.pone.001965910.1371/journal.pone.0019659] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2157301621573016]). @article{pmid21573016, title = {FANCĐ1/BRCA2 plays predominant role in the repair of ĐNA damage induced by ACNU or ŦMZ}, author = { N. Kondo and A. Takahashi and E. Mori and T. Noda and M. Z. Zdzienicka and L. H. Thompson and T. Helleday and M. Suzuki and Y. Kinashi and S. Masunaga and K. Ono and M. Hasegawa and T. Ohnishi}, year = {2011}, date = {2011-01-01}, journal = {PLoS ONE}, volume = {6}, number = {5}, pages = {e19659}, abstract = {Nimustine (ACNU) and temozolomide (TMZ) are DNA alkylating agents which are commonly used in chemotherapy for glioblastomas. ACNU is a DNA cross-linking agent and TMZ is a methylating agent. The therapeutic efficacy of these agents is limited by the development of resistance. In this work, the role of the Fanconi anemia (FA) repair pathway for DNA damage induced by ACNU or TMZ was examined. Cultured mouse embryonic fibroblasts were used: FANCA(-/-), FANCC(-/-), FANCA(-/-)C(-/-), FANCD2(-/-) cells and their parental cells, and Chinese hamster ovary and lung fibroblast cells were used: FANCD1/BRCA2mt, FANCG(-/-) and their parental cells. Cell survival was examined after a 3 h ACNU or TMZ treatment by using colony formation assays. All FA repair pathways were involved in ACNU-induced DNA damage. However, FANCG and FANCD1/BRCA2 played notably important roles in the repair of TMZ-induced DNA damage. The most effective molecular target correlating with cellular sensitivity to both ACNU and TMZ was FANCD1/BRCA2. In addition, it was found that FANCD1/BRCA2 small interference RNA efficiently enhanced cellular sensitivity toward ACNU and TMZ in human glioblastoma A172 cells. These findings suggest that the down-regulation of FANCD1/BRCA2 might be an effective strategy to increase cellular chemo-sensitization towards ACNU and TMZ.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3090409PMC3090409] [DOI:hrefhttp://dx.doi.org/10.1371/journal.pone.001965910.1371/journal.pone.0019659] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2157301621573016]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Nimustine (ACNU) and temozolomide (TMZ) are DNA alkylating agents which are commonly used in chemotherapy for glioblastomas. ACNU is a DNA cross-linking agent and TMZ is a methylating agent. The therapeutic efficacy of these agents is limited by the development of resistance. In this work, the role of the Fanconi anemia (FA) repair pathway for DNA damage induced by ACNU or TMZ was examined. Cultured mouse embryonic fibroblasts were used: FANCA(-/-), FANCC(-/-), FANCA(-/-)C(-/-), FANCD2(-/-) cells and their parental cells, and Chinese hamster ovary and lung fibroblast cells were used: FANCD1/BRCA2mt, FANCG(-/-) and their parental cells. Cell survival was examined after a 3 h ACNU or TMZ treatment by using colony formation assays. All FA repair pathways were involved in ACNU-induced DNA damage. However, FANCG and FANCD1/BRCA2 played notably important roles in the repair of TMZ-induced DNA damage. The most effective molecular target correlating with cellular sensitivity to both ACNU and TMZ was FANCD1/BRCA2. In addition, it was found that FANCD1/BRCA2 small interference RNA efficiently enhanced cellular sensitivity toward ACNU and TMZ in human glioblastoma A172 cells. These findings suggest that the down-regulation of FANCD1/BRCA2 might be an effective strategy to increase cellular chemo-sensitization towards ACNU and TMZ. |
Moskwa, P; Buffa, F M; Pan, Y; Panchakshari, R; Gottipati, P; Muschel, R J; Beech, J; Kulshrestha, R; Abdelmohsen, K; Weinstock, D M; Gorospe, M; Harris, A L; Helleday, T; Chowdhury, D miR-182-mediated downregulation of BRCA1 impacts ĐNA repair and sensitivity to PARP inhibitors Journal Article Mol. Cell, 41 (2), pp. 210–220, 2011, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3249932PMC3249932] [DOI:hrefhttp://dx.doi.org/10.1016/j.molcel.2010.12.00510.1016/j.molcel.2010.12.005] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2119500021195000]). @article{pmid21195000, title = {miR-182-mediated downregulation of BRCA1 impacts ĐNA repair and sensitivity to PARP inhibitors}, author = { P. Moskwa and F. M. Buffa and Y. Pan and R. Panchakshari and P. Gottipati and R. J. Muschel and J. Beech and R. Kulshrestha and K. Abdelmohsen and D. M. Weinstock and M. Gorospe and A. L. Harris and T. Helleday and D. Chowdhury}, year = {2011}, date = {2011-01-01}, journal = {Mol. Cell}, volume = {41}, number = {2}, pages = {210--220}, abstract = {Expression of BRCA1 is commonly decreased in sporadic breast tumors, and this correlates with poor prognosis of breast cancer patients. Here we show that BRCA1 transcripts are selectively enriched in the Argonaute/miR-182 complex and miR-182 downregulates BRCA1 expression. Antagonizing miR-182 enhances BRCA1 protein levels and protects them from IR-induced cell death, while overexpressing miR-182 reduces BRCA1 protein, impairs homologous recombination-mediated repair, and render cells hypersensitive to IR. The impaired DNA repair phenotype induced by miR-182 overexpression can be fully rescued by overexpressing miR-182-insensitive BRCA1. Consistent with a BRCA1-deficiency phenotype, miR-182-overexpressing breast tumor cells are hypersensitive to inhibitors of poly (ADP-ribose) polymerase 1 (PARP1). Conversely, antagonizing miR-182 enhances BRCA1 levels and induces resistance to PARP1 inhibitor. Finally, a clinical-grade PARP1 inhibitor impacts outgrowth of miR-182-expressing tumors in animal models. Together these results suggest that miR-182-mediated downregulation of BRCA1 impedes DNA repair and may impact breast cancer therapy.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3249932PMC3249932] [DOI:hrefhttp://dx.doi.org/10.1016/j.molcel.2010.12.00510.1016/j.molcel.2010.12.005] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2119500021195000]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Expression of BRCA1 is commonly decreased in sporadic breast tumors, and this correlates with poor prognosis of breast cancer patients. Here we show that BRCA1 transcripts are selectively enriched in the Argonaute/miR-182 complex and miR-182 downregulates BRCA1 expression. Antagonizing miR-182 enhances BRCA1 protein levels and protects them from IR-induced cell death, while overexpressing miR-182 reduces BRCA1 protein, impairs homologous recombination-mediated repair, and render cells hypersensitive to IR. The impaired DNA repair phenotype induced by miR-182 overexpression can be fully rescued by overexpressing miR-182-insensitive BRCA1. Consistent with a BRCA1-deficiency phenotype, miR-182-overexpressing breast tumor cells are hypersensitive to inhibitors of poly (ADP-ribose) polymerase 1 (PARP1). Conversely, antagonizing miR-182 enhances BRCA1 levels and induces resistance to PARP1 inhibitor. Finally, a clinical-grade PARP1 inhibitor impacts outgrowth of miR-182-expressing tumors in animal models. Together these results suggest that miR-182-mediated downregulation of BRCA1 impedes DNA repair and may impact breast cancer therapy. |
Noda, T; Takahashi, A; Kondo, N; Mori, E; Okamoto, N; Nakagawa, Y; Ohnishi, K; Zdzienicka, M Z; Thompson, L H; Helleday, T; Asada, H; Ohnishi, T Repair pathways independent of the Fanconi anemia nuclear core complex play a predominant role in mitigating formaldehyde-induced ĐNA damage Journal Article Biochem. Biophys. Res. Commun., 404 (1), pp. 206–210, 2011, ([DOI:hrefhttp://dx.doi.org/10.1016/j.bbrc.2010.11.09410.1016/j.bbrc.2010.11.094] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2111170921111709]). @article{pmid21111709, title = {Repair pathways independent of the Fanconi anemia nuclear core complex play a predominant role in mitigating formaldehyde-induced ĐNA damage}, author = { T. Noda and A. Takahashi and N. Kondo and E. Mori and N. Okamoto and Y. Nakagawa and K. Ohnishi and M. Z. Zdzienicka and L. H. Thompson and T. Helleday and H. Asada and T. Ohnishi}, year = {2011}, date = {2011-01-01}, journal = {Biochem. Biophys. Res. Commun.}, volume = {404}, number = {1}, pages = {206--210}, abstract = {The role of the Fanconi anemia (FA) repair pathway for DNA damage induced by formaldehyde was examined in the work described here. The following cell types were used: mouse embryonic fibroblast cell lines FANCA(-/-), FANCC(-/-), FANCA(-/-)C(-/-), FANCD2(-/-) and their parental cells, the Chinese hamster cell lines FANCD1 mutant (mt), FANCGmt, their revertant cells, and the corresponding wild-type (wt) cells. Cell survival rates were determined with colony formation assays after formaldehyde treatment. DNA double strand breaks (DSBs) were detected with an immunocytochemical γH2AX-staining assay. Although the sensitivity of FANCA(-/-), FANCC(-/-) and FANCA(-/-)C(-/-) cells to formaldehyde was comparable to that of proficient cells, FANCD1mt, FANCGmt and FANCD2(-/-) cells were more sensitive to formaldehyde than the corresponding proficient cells. It was found that homologous recombination (HR) repair was induced by formaldehyde. In addition, γH2AX foci in FANCD1mt cells persisted for longer times than in FANCD1wt cells. These findings suggest that formaldehyde-induced DSBs are repaired by HR through the FA repair pathway which is independent of the FA nuclear core complex.}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/j.bbrc.2010.11.09410.1016/j.bbrc.2010.11.094] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2111170921111709]}, keywords = {}, pubstate = {published}, tppubtype = {article} } The role of the Fanconi anemia (FA) repair pathway for DNA damage induced by formaldehyde was examined in the work described here. The following cell types were used: mouse embryonic fibroblast cell lines FANCA(-/-), FANCC(-/-), FANCA(-/-)C(-/-), FANCD2(-/-) and their parental cells, the Chinese hamster cell lines FANCD1 mutant (mt), FANCGmt, their revertant cells, and the corresponding wild-type (wt) cells. Cell survival rates were determined with colony formation assays after formaldehyde treatment. DNA double strand breaks (DSBs) were detected with an immunocytochemical γH2AX-staining assay. Although the sensitivity of FANCA(-/-), FANCC(-/-) and FANCA(-/-)C(-/-) cells to formaldehyde was comparable to that of proficient cells, FANCD1mt, FANCGmt and FANCD2(-/-) cells were more sensitive to formaldehyde than the corresponding proficient cells. It was found that homologous recombination (HR) repair was induced by formaldehyde. In addition, γH2AX foci in FANCD1mt cells persisted for longer times than in FANCD1wt cells. These findings suggest that formaldehyde-induced DSBs are repaired by HR through the FA repair pathway which is independent of the FA nuclear core complex. |
Stoimenov, I; Gottipati, P; Schultz, N; Helleday, T Ŧranscription inhibition by 5,6-dichloro-1-beta-Đ-ribofuranosylbenzimidazole (ĐRB) causes ĐNA damage and triggers homologous recombination repair in mammalian cells Journal Article Mutat. Res., 706 (1-2), pp. 1–6, 2011, ([DOI:hrefhttp://dx.doi.org/10.1016/j.mrfmmm.2010.10.01210.1016/j.mrfmmm.2010.10.012] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2107454421074544]). @article{pmid21074544, title = {Ŧranscription inhibition by 5,6-dichloro-1-beta-Đ-ribofuranosylbenzimidazole (ĐRB) causes ĐNA damage and triggers homologous recombination repair in mammalian cells}, author = { I. Stoimenov and P. Gottipati and N. Schultz and T. Helleday}, year = {2011}, date = {2011-01-01}, journal = {Mutat. Res.}, volume = {706}, number = {1-2}, pages = {1--6}, abstract = {Transcription, replication and homologous recombination are intrinsically connected and it is well established that an increase of transcription is associated with an increase in homologous recombination. Here, we have studied how homologous recombination is affected during transcription inhibition by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a compound that prevents activating phosphorylations of the RNA Pol II C-terminal domain. We identify that DRB triggers an increase in homologous recombination within the hprt gene as well as increasing RAD51 foci formation in mammalian cells. Furthermore, we find that DRB-induced transcriptional stress is associated with formation of the nuclear foci of the phosphorylated form of H2AX (γH2AX). We accounted that about 72% of RAD51 foci co-localized with the observed γH2AX foci. Interestingly, we find that XRCC3 mutated, homologous recombination defective cells are hypersensitive to the toxic effect of DRB and fail to form RAD51 foci. In conclusion, we show that DRB-induced transcription inhibition is associated with the formation of a lesion that triggers RAD51-dependent homologous recombination repair, required for survival under transcriptional stress.}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/j.mrfmmm.2010.10.01210.1016/j.mrfmmm.2010.10.012] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2107454421074544]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Transcription, replication and homologous recombination are intrinsically connected and it is well established that an increase of transcription is associated with an increase in homologous recombination. Here, we have studied how homologous recombination is affected during transcription inhibition by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a compound that prevents activating phosphorylations of the RNA Pol II C-terminal domain. We identify that DRB triggers an increase in homologous recombination within the hprt gene as well as increasing RAD51 foci formation in mammalian cells. Furthermore, we find that DRB-induced transcriptional stress is associated with formation of the nuclear foci of the phosphorylated form of H2AX (γH2AX). We accounted that about 72% of RAD51 foci co-localized with the observed γH2AX foci. Interestingly, we find that XRCC3 mutated, homologous recombination defective cells are hypersensitive to the toxic effect of DRB and fail to form RAD51 foci. In conclusion, we show that DRB-induced transcription inhibition is associated with the formation of a lesion that triggers RAD51-dependent homologous recombination repair, required for survival under transcriptional stress. |
2010 |
Hawtin, R E; Stockett, D E; Wong, O K; Lundin, C; Helleday, T; Fox, J A Ħomologous recombination repair is essential for repair of vosaroxin-induced ĐNA double-strand breaks Journal Article Oncotarget, 1 (7), pp. 606–619, 2010, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3248135PMC3248135] [DOI:hrefhttp://dx.doi.org/10.18632/oncotarget.10110610.18632/oncotarget.101106] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2131745621317456]). @article{pmid21317456, title = {Ħomologous recombination repair is essential for repair of vosaroxin-induced ĐNA double-strand breaks}, author = { R. E. Hawtin and D. E. Stockett and O. K. Wong and C. Lundin and T. Helleday and J. A. Fox}, year = {2010}, date = {2010-11-01}, journal = {Oncotarget}, volume = {1}, number = {7}, pages = {606--619}, abstract = {Vosaroxin (formerly voreloxin) is a first-in-class anticancer quinolone derivative that intercalates DNA and inhibits topoisomerase II, inducing site-selective double-strand breaks (DSB), G2 arrest and apoptosis. Objective responses and complete remissions were observed in phase 2 studies of vosaroxin in patients with solid and hematologic malignancies, and responses were seen in patients whose cancers were resistant to anthracyclines. The quinolone-based scaffold differentiates vosaroxin from the anthracyclines and anthracenediones, broadly used DNA intercalating topoisomerase II poisons. Here we report that vosaroxin induces a cell cycle specific pattern of DNA damage and repair that is distinct from the anthracycline, doxorubicin. Both drugs stall replication and preferentially induce DNA damage in replicating cells, with damage in G2 / M > S > G1. However, detectable replication fork collapse, as evidenced by DNA fragmentation and long tract recombination during S phase, is induced only by doxorubicin. Furthermore, vosaroxin induces less overall DNA fragmentation. Homologous recombination repair (HRR) is critical for recovery from DNA damage induced by both agents, identifying the potential to clinically exploit synthetic lethality.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3248135PMC3248135] [DOI:hrefhttp://dx.doi.org/10.18632/oncotarget.10110610.18632/oncotarget.101106] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2131745621317456]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Vosaroxin (formerly voreloxin) is a first-in-class anticancer quinolone derivative that intercalates DNA and inhibits topoisomerase II, inducing site-selective double-strand breaks (DSB), G2 arrest and apoptosis. Objective responses and complete remissions were observed in phase 2 studies of vosaroxin in patients with solid and hematologic malignancies, and responses were seen in patients whose cancers were resistant to anthracyclines. The quinolone-based scaffold differentiates vosaroxin from the anthracyclines and anthracenediones, broadly used DNA intercalating topoisomerase II poisons. Here we report that vosaroxin induces a cell cycle specific pattern of DNA damage and repair that is distinct from the anthracycline, doxorubicin. Both drugs stall replication and preferentially induce DNA damage in replicating cells, with damage in G2 / M > S > G1. However, detectable replication fork collapse, as evidenced by DNA fragmentation and long tract recombination during S phase, is induced only by doxorubicin. Furthermore, vosaroxin induces less overall DNA fragmentation. Homologous recombination repair (HRR) is critical for recovery from DNA damage induced by both agents, identifying the potential to clinically exploit synthetic lethality. |
Duro, E; Lundin, C; Ask, K; Sanchez-Pulido, L; MacArtney, T J; Toth, R; Ponting, C P; Groth, A; Helleday, T; Rouse, J Identification of the MMS22L-ŦONSL complex that promotes homologous recombination Journal Article Mol. Cell, 40 (4), pp. 632–644, 2010, ([DOI:hrefhttp://dx.doi.org/10.1016/j.molcel.2010.10.02310.1016/j.molcel.2010.10.023] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2105598421055984]). @article{pmid21055984, title = {Identification of the MMS22L-ŦONSL complex that promotes homologous recombination}, author = { E. Duro and C. Lundin and K. Ask and L. Sanchez-Pulido and T. J. MacArtney and R. Toth and C. P. Ponting and A. Groth and T. Helleday and J. Rouse}, year = {2010}, date = {2010-11-01}, journal = {Mol. Cell}, volume = {40}, number = {4}, pages = {632--644}, abstract = {Budding yeast Mms22 is required for homologous recombination (HR)-mediated repair of stalled or broken DNA replication forks. Here we identify a human Mms22-like protein (MMS22L) and an MMS22L-interacting protein, NFκBIL2/TONSL. Depletion of MMS22L or TONSL from human cells causes a high level of double-strand breaks (DSBs) during DNA replication. Both proteins accumulate at stressed replication forks, and depletion of MMS22L or TONSL from cells causes hypersensitivity to agents that cause S phase-associated DSBs, such as topoisomerase (TOP) inhibitors. In this light, MMS22L and TONSL are required for the HR-mediated repair of replication fork-associated DSBs. In cells depleted of either protein, DSBs induced by the TOP1 inhibitor camptothecin are resected normally, but the loading of the RAD51 recombinase is defective. Therefore, MMS22L and TONSL are required for the maintenance of genome stability when unscheduled DSBs occur in the vicinity of DNA replication forks.}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/j.molcel.2010.10.02310.1016/j.molcel.2010.10.023] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2105598421055984]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Budding yeast Mms22 is required for homologous recombination (HR)-mediated repair of stalled or broken DNA replication forks. Here we identify a human Mms22-like protein (MMS22L) and an MMS22L-interacting protein, NFκBIL2/TONSL. Depletion of MMS22L or TONSL from human cells causes a high level of double-strand breaks (DSBs) during DNA replication. Both proteins accumulate at stressed replication forks, and depletion of MMS22L or TONSL from cells causes hypersensitivity to agents that cause S phase-associated DSBs, such as topoisomerase (TOP) inhibitors. In this light, MMS22L and TONSL are required for the HR-mediated repair of replication fork-associated DSBs. In cells depleted of either protein, DSBs induced by the TOP1 inhibitor camptothecin are resected normally, but the loading of the RAD51 recombinase is defective. Therefore, MMS22L and TONSL are required for the maintenance of genome stability when unscheduled DSBs occur in the vicinity of DNA replication forks. |
Savolainen, L; Cassel, T; Helleday, T Ŧhe XPĐ subunit of ŦFIIĦ is required for transcription-associated but not ĐNA double-strand break-induced recombination in mammalian cells Journal Article Mutagenesis, 25 (6), pp. 623–629, 2010, ([DOI:hrefhttp://dx.doi.org/10.1093/mutage/geq05410.1093/mutage/geq054] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2083369520833695]). @article{pmid20833695, title = {Ŧhe XPĐ subunit of ŦFIIĦ is required for transcription-associated but not ĐNA double-strand break-induced recombination in mammalian cells}, author = { L. Savolainen and T. Cassel and T. Helleday}, year = {2010}, date = {2010-11-01}, journal = {Mutagenesis}, volume = {25}, number = {6}, pages = {623--629}, abstract = {Mutations in the XPD gene can give rise to three phenotypically distinct disorders: xeroderma pigmentosum (XP), trichothiodystrophy (TTD) or combined XP and Cockayne syndrome (CS) (XP/CS). The role of Xeroderma Pigmentosum group D protein (XPD) in nucleotide excision repair explains the increased risk of skin cancer in XP patients but not all the clinical phenotypes found in XP/CS or TTD patients. Here, we describe that the XPD-defective UV5 cell line is impaired in transcription-associated recombination (TAR), which can be reverted by the introduction of the wild-type XPD gene expressed from a vector. UV5 cells are defective in TAR, despite having intact transcription and homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Interestingly, we find reduced spontaneous HR in XPD-defective cells, suggesting that transcription underlies a portion of spontaneous HR events. We also report that transcription-coupled repair (TCR)-defective cells, mutated in the Cockayne syndrome B (CSB) protein, have a defect in TAR, but not in DSB-induced HR. However, the TAR defect may be associated with a general transcription defect in CSB-deficient cells. In conclusion, we show a novel role for the XPD protein in TAR, linking TAR with TCR.}, note = {[DOI:hrefhttp://dx.doi.org/10.1093/mutage/geq05410.1093/mutage/geq054] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2083369520833695]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Mutations in the XPD gene can give rise to three phenotypically distinct disorders: xeroderma pigmentosum (XP), trichothiodystrophy (TTD) or combined XP and Cockayne syndrome (CS) (XP/CS). The role of Xeroderma Pigmentosum group D protein (XPD) in nucleotide excision repair explains the increased risk of skin cancer in XP patients but not all the clinical phenotypes found in XP/CS or TTD patients. Here, we describe that the XPD-defective UV5 cell line is impaired in transcription-associated recombination (TAR), which can be reverted by the introduction of the wild-type XPD gene expressed from a vector. UV5 cells are defective in TAR, despite having intact transcription and homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Interestingly, we find reduced spontaneous HR in XPD-defective cells, suggesting that transcription underlies a portion of spontaneous HR events. We also report that transcription-coupled repair (TCR)-defective cells, mutated in the Cockayne syndrome B (CSB) protein, have a defect in TAR, but not in DSB-induced HR. However, the TAR defect may be associated with a general transcription defect in CSB-deficient cells. In conclusion, we show a novel role for the XPD protein in TAR, linking TAR with TCR. |
Chan, N; Pires, I M; Bencokova, Z; Coackley, C; Luoto, K R; Bhogal, N; Lakshman, M; Gottipati, P; Oliver, F J; Helleday, T; Hammond, E M; Bristow, R G Contextual synthetic lethality of cancer cell kill based on the tumor microenvironment Journal Article Cancer Res., 70 (20), pp. 8045–8054, 2010, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2978949PMC2978949] [DOI:hrefhttp://dx.doi.org/10.1158/0008-5472.CAN-10-235210.1158/0008-5472.CAN-10-2352] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2092411220924112]). @article{pmid20924112, title = {Contextual synthetic lethality of cancer cell kill based on the tumor microenvironment}, author = { N. Chan and I. M. Pires and Z. Bencokova and C. Coackley and K. R. Luoto and N. Bhogal and M. Lakshman and P. Gottipati and F. J. Oliver and T. Helleday and E. M. Hammond and R. G. Bristow}, year = {2010}, date = {2010-10-01}, journal = {Cancer Res.}, volume = {70}, number = {20}, pages = {8045--8054}, abstract = {Acute and chronic hypoxia exists within the three-dimensional microenvironment of solid tumors and drives therapy resistance, genetic instability, and metastasis. Replicating cells exposed to either severe acute hypoxia (16 hours with 0.02% O(2)) followed by reoxygenation or moderate chronic hypoxia (72 hours with 0.2% O(2)) treatments have decreased homologous recombination (HR) protein expression and function. As HR defects are synthetically lethal with poly(ADP-ribose) polymerase 1 (PARP1) inhibition, we evaluated the sensitivity of repair-defective hypoxic cells to PARP inhibition. Although PARP inhibition itself did not affect HR expression or function, we observed increased clonogenic killing in HR-deficient hypoxic cells following chemical inhibition of PARP1. This effect was partially reversible by RAD51 overexpression. PARP1(-/-) murine embryonic fibroblasts (MEF) showed a proliferative disadvantage under hypoxic gassing when compared with PARP1(+/+) MEFs. PARP-inhibited hypoxic cells accumulated γH2AX and 53BP1 foci as a consequence of altered DNA replication firing during S phase-specific cell killing. In support of this proposed mode of action, PARP inhibitor-treated xenografts displayed increased γH2AX and cleaved caspase-3 expression in RAD51-deficient hypoxic subregions in vivo, which was associated with decreased ex vivo clonogenic survival following experimental radiotherapy. This is the first report of selective cell killing of HR-defective hypoxic cells in vivo as a consequence of microenvironment-mediated "contextual synthetic lethality." As all solid tumors contain aggressive hypoxic cells, this may broaden the clinical utility of PARP and DNA repair inhibition, either alone or in combination with radiotherapy and chemotherapy, even in tumor cells lacking synthetically lethal, genetic mutations.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2978949PMC2978949] [DOI:hrefhttp://dx.doi.org/10.1158/0008-5472.CAN-10-235210.1158/0008-5472.CAN-10-2352] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2092411220924112]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Acute and chronic hypoxia exists within the three-dimensional microenvironment of solid tumors and drives therapy resistance, genetic instability, and metastasis. Replicating cells exposed to either severe acute hypoxia (16 hours with 0.02% O(2)) followed by reoxygenation or moderate chronic hypoxia (72 hours with 0.2% O(2)) treatments have decreased homologous recombination (HR) protein expression and function. As HR defects are synthetically lethal with poly(ADP-ribose) polymerase 1 (PARP1) inhibition, we evaluated the sensitivity of repair-defective hypoxic cells to PARP inhibition. Although PARP inhibition itself did not affect HR expression or function, we observed increased clonogenic killing in HR-deficient hypoxic cells following chemical inhibition of PARP1. This effect was partially reversible by RAD51 overexpression. PARP1(-/-) murine embryonic fibroblasts (MEF) showed a proliferative disadvantage under hypoxic gassing when compared with PARP1(+/+) MEFs. PARP-inhibited hypoxic cells accumulated γH2AX and 53BP1 foci as a consequence of altered DNA replication firing during S phase-specific cell killing. In support of this proposed mode of action, PARP inhibitor-treated xenografts displayed increased γH2AX and cleaved caspase-3 expression in RAD51-deficient hypoxic subregions in vivo, which was associated with decreased ex vivo clonogenic survival following experimental radiotherapy. This is the first report of selective cell killing of HR-defective hypoxic cells in vivo as a consequence of microenvironment-mediated "contextual synthetic lethality." As all solid tumors contain aggressive hypoxic cells, this may broaden the clinical utility of PARP and DNA repair inhibition, either alone or in combination with radiotherapy and chemotherapy, even in tumor cells lacking synthetically lethal, genetic mutations. |
Petermann, E; Helleday, T Pathways of mammalian replication fork restart Journal Article Nat. Rev. Mol. Cell Biol., 11 (10), pp. 683–687, 2010, ([DOI:hrefhttp://dx.doi.org/10.1038/nrm297410.1038/nrm2974] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2084217720842177]). @article{pmid20842177, title = {Pathways of mammalian replication fork restart}, author = { E. Petermann and T. Helleday}, year = {2010}, date = {2010-10-01}, journal = {Nat. Rev. Mol. Cell Biol.}, volume = {11}, number = {10}, pages = {683--687}, abstract = {Single-molecule analyses of DNA replication have greatly advanced our understanding of mammalian replication restart. Several proteins that are not part of the core replication machinery promote the efficient restart of replication forks that have been stalled by replication inhibitors, suggesting that bona fide fork restart pathways exist in mammalian cells. Different models of replication fork restart can be envisaged, based on the involvement of DNA helicases, nucleases, homologous recombination factors and the importance of DNA double-strand break formation.}, note = {[DOI:hrefhttp://dx.doi.org/10.1038/nrm297410.1038/nrm2974] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2084217720842177]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Single-molecule analyses of DNA replication have greatly advanced our understanding of mammalian replication restart. Several proteins that are not part of the core replication machinery promote the efficient restart of replication forks that have been stalled by replication inhibitors, suggesting that bona fide fork restart pathways exist in mammalian cells. Different models of replication fork restart can be envisaged, based on the involvement of DNA helicases, nucleases, homologous recombination factors and the importance of DNA double-strand break formation. |
Petermann, E; Woodcock, M; Helleday, T Chk1 promotes replication fork progression by controlling replication initiation Journal Article Proc. Natl. Acad. Sci. U.S.A., 107 (37), pp. 16090–16095, 2010, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2941317PMC2941317] [DOI:hrefhttp://dx.doi.org/10.1073/pnas.100503110710.1073/pnas.1005031107] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2080546520805465]). @article{pmid20805465, title = {Chk1 promotes replication fork progression by controlling replication initiation}, author = { E. Petermann and M. Woodcock and T. Helleday}, year = {2010}, date = {2010-09-01}, journal = {Proc. Natl. Acad. Sci. U.S.A.}, volume = {107}, number = {37}, pages = {16090--16095}, abstract = {DNA replication starts at initiation sites termed replication origins. Metazoan cells contain many more potential origins than are activated (fired) during each S phase. Origin activation is controlled by the ATR checkpoint kinase and its downstream effector kinase Chk1, which suppresses origin firing in response to replication blocks and during normal S phase by inhibiting the cyclin-dependent kinase Cdk2. In addition to increased origin activation, cells deficient in Chk1 activity display reduced rates of replication fork progression. Here we investigate the causal relationship between increased origin firing and reduced replication fork progression. We use the Cdk inhibitor roscovitine or RNAi depletion of Cdc7 to inhibit origin firing in Chk1-inhibited or RNAi-depleted cells. We report that Cdk inhibition and depletion of Cdc7 can alleviate the slow replication fork speeds in Chk1-deficient cells. Our data suggest that increased replication initiation leads to slow replication fork progression and that Chk1 promotes replication fork progression during normal S phase by controlling replication origin activity.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2941317PMC2941317] [DOI:hrefhttp://dx.doi.org/10.1073/pnas.100503110710.1073/pnas.1005031107] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2080546520805465]}, keywords = {}, pubstate = {published}, tppubtype = {article} } DNA replication starts at initiation sites termed replication origins. Metazoan cells contain many more potential origins than are activated (fired) during each S phase. Origin activation is controlled by the ATR checkpoint kinase and its downstream effector kinase Chk1, which suppresses origin firing in response to replication blocks and during normal S phase by inhibiting the cyclin-dependent kinase Cdk2. In addition to increased origin activation, cells deficient in Chk1 activity display reduced rates of replication fork progression. Here we investigate the causal relationship between increased origin firing and reduced replication fork progression. We use the Cdk inhibitor roscovitine or RNAi depletion of Cdc7 to inhibit origin firing in Chk1-inhibited or RNAi-depleted cells. We report that Cdk inhibition and depletion of Cdc7 can alleviate the slow replication fork speeds in Chk1-deficient cells. Our data suggest that increased replication initiation leads to slow replication fork progression and that Chk1 promotes replication fork progression during normal S phase by controlling replication origin activity. |
Blundred, R; Myers, K; Helleday, T; Goldman, A S; Bryant, H E Ħuman RECQL5 overcomes thymidine-induced replication stress Journal Article DNA Repair (Amst.), 9 (9), pp. 964–975, 2010, ([DOI:hrefhttp://dx.doi.org/10.1016/j.dnarep.2010.06.00910.1016/j.dnarep.2010.06.009] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2064358520643585]). @article{pmid20643585, title = {Ħuman RECQL5 overcomes thymidine-induced replication stress}, author = { R. Blundred and K. Myers and T. Helleday and A. S. Goldman and H. E. Bryant}, year = {2010}, date = {2010-09-01}, journal = {DNA Repair (Amst.)}, volume = {9}, number = {9}, pages = {964--975}, abstract = {Accurate DNA replication is essential to genome integrity and is controlled by five human RecQ helicases, of which at least three prevent cancer and ageing. Here, we have studied the role of RECQL5, which is the least characterised of the five human RecQ helicases. We demonstrate that overexpressed RECQL5 promotes survival during thymidine-induced slowing of replication forks in human cells. The RECQL5 protein relocates specifically to stalled replication forks and suppresses thymidine-induced RPA foci, CHK1 signalling, homologous recombination and gammaH2AX activation. It is unlikely that RECQL5 promotes survival through translesion synthesis as PCNA ubiquitylation is also reduced. Interestingly, we also found that overexpressing RECQL5 relieves cells of the cell cycle arrest normally imposed by thymidine, but without causing mutations. In conclusion, we propose that RECQL5 stabilises the replication fork allowing replication to overcome the effects of thymidine and complete the cell cycle.}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/j.dnarep.2010.06.00910.1016/j.dnarep.2010.06.009] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2064358520643585]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Accurate DNA replication is essential to genome integrity and is controlled by five human RecQ helicases, of which at least three prevent cancer and ageing. Here, we have studied the role of RECQL5, which is the least characterised of the five human RecQ helicases. We demonstrate that overexpressed RECQL5 promotes survival during thymidine-induced slowing of replication forks in human cells. The RECQL5 protein relocates specifically to stalled replication forks and suppresses thymidine-induced RPA foci, CHK1 signalling, homologous recombination and gammaH2AX activation. It is unlikely that RECQL5 promotes survival through translesion synthesis as PCNA ubiquitylation is also reduced. Interestingly, we also found that overexpressing RECQL5 relieves cells of the cell cycle arrest normally imposed by thymidine, but without causing mutations. In conclusion, we propose that RECQL5 stabilises the replication fork allowing replication to overcome the effects of thymidine and complete the cell cycle. |
Groth, P; Auslander, S; Majumder, M M; Schultz, N; Johansson, F; Petermann, E; Helleday, T Methylated ĐNA causes a physical block to replication forks independently of damage signalling, O(6)-methylguanine or ĐNA single-strand breaks and results in ĐNA damage Journal Article J. Mol. Biol., 402 (1), pp. 70–82, 2010, ([DOI:hrefhttp://dx.doi.org/10.1016/j.jmb.2010.07.01010.1016/j.jmb.2010.07.010] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2064314220643142]). @article{pmid20643142, title = {Methylated ĐNA causes a physical block to replication forks independently of damage signalling, O(6)-methylguanine or ĐNA single-strand breaks and results in ĐNA damage}, author = { P. Groth and S. Auslander and M. M. Majumder and N. Schultz and F. Johansson and E. Petermann and T. Helleday}, year = {2010}, date = {2010-09-01}, journal = {J. Mol. Biol.}, volume = {402}, number = {1}, pages = {70--82}, abstract = {Even though DNA alkylating agents have been used for many decades in the treatment of cancer, it remains unclear what happens when replication forks encounter alkylated DNA. Here, we used the DNA fibre assay to study the impact of alkylating agents on replication fork progression. We found that the alkylator methyl methanesulfonate (MMS) inhibits replication elongation in a manner that is dose dependent and related to the overall alkylation grade. Replication forks seem to be completely blocked as no nucleotide incorporation can be detected following 1 h of MMS treatment. A high dose of 5 mM caffeine, inhibiting most DNA damage signalling, decreases replication rates overall but does not reverse MMS-induced replication inhibition, showing that the replication block is independent of DNA damage signalling. Furthermore, the block of replication fork progression does not correlate with the level of DNA single-strand breaks. Overexpression of O(6)-methylguanine (O6meG)-DNA methyltransferase protein, responsible for removing the most toxic alkylation, O6meG, did not affect replication elongation following exposure to N-methyl-N'-nitro-N-nitrosoguanidine. This demonstrates that O6meG lesions are efficiently bypassed in mammalian cells. In addition, we find that MMS-induced gammaH2AX foci co-localise with 53BP1 foci and newly replicated areas, suggesting that DNA double-strand breaks are formed at MMS-blocked replication forks. Altogether, our data suggest that N-alkylations formed during exposure to alkylating agents physically block replication fork elongation in mammalian cells, causing formation of replication-associated DNA lesions, likely double-strand breaks.}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/j.jmb.2010.07.01010.1016/j.jmb.2010.07.010] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2064314220643142]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Even though DNA alkylating agents have been used for many decades in the treatment of cancer, it remains unclear what happens when replication forks encounter alkylated DNA. Here, we used the DNA fibre assay to study the impact of alkylating agents on replication fork progression. We found that the alkylator methyl methanesulfonate (MMS) inhibits replication elongation in a manner that is dose dependent and related to the overall alkylation grade. Replication forks seem to be completely blocked as no nucleotide incorporation can be detected following 1 h of MMS treatment. A high dose of 5 mM caffeine, inhibiting most DNA damage signalling, decreases replication rates overall but does not reverse MMS-induced replication inhibition, showing that the replication block is independent of DNA damage signalling. Furthermore, the block of replication fork progression does not correlate with the level of DNA single-strand breaks. Overexpression of O(6)-methylguanine (O6meG)-DNA methyltransferase protein, responsible for removing the most toxic alkylation, O6meG, did not affect replication elongation following exposure to N-methyl-N'-nitro-N-nitrosoguanidine. This demonstrates that O6meG lesions are efficiently bypassed in mammalian cells. In addition, we find that MMS-induced gammaH2AX foci co-localise with 53BP1 foci and newly replicated areas, suggesting that DNA double-strand breaks are formed at MMS-blocked replication forks. Altogether, our data suggest that N-alkylations formed during exposure to alkylating agents physically block replication fork elongation in mammalian cells, causing formation of replication-associated DNA lesions, likely double-strand breaks. |
Issaeva, N; Thomas, H D; Djureinovic, T; Djurenovic, T; Jaspers, J E; Stoimenov, I; Kyle, S; Pedley, N; Gottipati, P; Zur, R; Sleeth, K; Chatzakos, V; Mulligan, E A; Lundin, C; Gubanova, E; Kersbergen, A; Harris, A L; Sharma, R A; Rottenberg, S; Curtin, N J; Helleday, T 6-thioguanine selectively kills BRCA2-defective tumors and overcomes PARP inhibitor resistance Journal Article Cancer Res., 70 (15), pp. 6268–6276, 2010, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2913123PMC2913123] [DOI:hrefhttp://dx.doi.org/10.1158/0008-5472.CAN-09-341610.1158/0008-5472.CAN-09-3416] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2063106320631063]). @article{pmid20631063, title = {6-thioguanine selectively kills BRCA2-defective tumors and overcomes PARP inhibitor resistance}, author = { N. Issaeva and H. D. Thomas and T. Djureinovic and T. Djurenovic and J. E. Jaspers and I. Stoimenov and S. Kyle and N. Pedley and P. Gottipati and R. Zur and K. Sleeth and V. Chatzakos and E. A. Mulligan and C. Lundin and E. Gubanova and A. Kersbergen and A. L. Harris and R. A. Sharma and S. Rottenberg and N. J. Curtin and T. Helleday}, year = {2010}, date = {2010-08-01}, journal = {Cancer Res.}, volume = {70}, number = {15}, pages = {6268--6276}, abstract = {Familial breast and ovarian cancers are often defective in homologous recombination (HR) due to mutations in the BRCA1 or BRCA2 genes. Cisplatin chemotherapy or poly(ADP-ribose) polymerase (PARP) inhibitors were tested for these tumors in clinical trials. In a screen for novel drugs that selectively kill BRCA2-defective cells, we identified 6-thioguanine (6TG), which induces DNA double-strand breaks (DSB) that are repaired by HR. Furthermore, we show that 6TG is as efficient as a PARP inhibitor in selectively killing BRCA2-defective tumors in a xenograft model. Spontaneous BRCA1-defective mammary tumors gain resistance to PARP inhibitors through increased P-glycoprotein expression. Here, we show that 6TG efficiently kills such BRCA1-defective PARP inhibitor-resistant tumors. We also show that 6TG could kill cells and tumors that have gained resistance to PARP inhibitors or cisplatin through genetic reversion of the BRCA2 gene. Although HR is reactivated in PARP inhibitor-resistant BRCA2-defective cells, it is not fully restored for the repair of 6TG-induced lesions. This is likely to be due to several recombinogenic lesions being formed after 6TG. We show that BRCA2 is also required for survival from mismatch repair-independent lesions formed by 6TG, which do not include DSBs. This suggests that HR is involved in the repair of 6TG-induced DSBs as well as mismatch repair-independent 6TG-induced DNA lesion. Altogether, our data show that 6TG efficiently kills BRCA2-defective tumors and suggest that 6TG may be effective in the treatment of advanced tumors that have developed resistance to PARP inhibitors or platinum-based chemotherapy.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2913123PMC2913123] [DOI:hrefhttp://dx.doi.org/10.1158/0008-5472.CAN-09-341610.1158/0008-5472.CAN-09-3416] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2063106320631063]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Familial breast and ovarian cancers are often defective in homologous recombination (HR) due to mutations in the BRCA1 or BRCA2 genes. Cisplatin chemotherapy or poly(ADP-ribose) polymerase (PARP) inhibitors were tested for these tumors in clinical trials. In a screen for novel drugs that selectively kill BRCA2-defective cells, we identified 6-thioguanine (6TG), which induces DNA double-strand breaks (DSB) that are repaired by HR. Furthermore, we show that 6TG is as efficient as a PARP inhibitor in selectively killing BRCA2-defective tumors in a xenograft model. Spontaneous BRCA1-defective mammary tumors gain resistance to PARP inhibitors through increased P-glycoprotein expression. Here, we show that 6TG efficiently kills such BRCA1-defective PARP inhibitor-resistant tumors. We also show that 6TG could kill cells and tumors that have gained resistance to PARP inhibitors or cisplatin through genetic reversion of the BRCA2 gene. Although HR is reactivated in PARP inhibitor-resistant BRCA2-defective cells, it is not fully restored for the repair of 6TG-induced lesions. This is likely to be due to several recombinogenic lesions being formed after 6TG. We show that BRCA2 is also required for survival from mismatch repair-independent lesions formed by 6TG, which do not include DSBs. This suggests that HR is involved in the repair of 6TG-induced DSBs as well as mismatch repair-independent 6TG-induced DNA lesion. Altogether, our data show that 6TG efficiently kills BRCA2-defective tumors and suggest that 6TG may be effective in the treatment of advanced tumors that have developed resistance to PARP inhibitors or platinum-based chemotherapy. |
Evers, B; Helleday, T; Jonkers, J Ŧargeting homologous recombination repair defects in cancer Journal Article Trends Pharmacol. Sci., 31 (8), pp. 372–380, 2010, ([DOI:hrefhttp://dx.doi.org/10.1016/j.tips.2010.06.00110.1016/j.tips.2010.06.001] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2059875620598756]). @article{pmid20598756, title = {Ŧargeting homologous recombination repair defects in cancer}, author = { B. Evers and T. Helleday and J. Jonkers}, year = {2010}, date = {2010-08-01}, journal = {Trends Pharmacol. Sci.}, volume = {31}, number = {8}, pages = {372--380}, abstract = {DNA repair is essential for cells to maintain genome stability in an environment that constantly produces DNA damage. There is a growing appreciation that defects in homologous recombination repair underlie hereditary and sporadic tumourigenesis, and that deficiency in this pathway may dictate the sensitivity of tumours to certain DNA-damaging agents. Homologous recombination deficiency (HRD) may therefore prove to be a diagnostic criterion per se if appropriate biomarkers become available to identify these tumours. In addition, homologous recombination-deficient tumours are more sensitive to inhibition of other DNA repair pathways through so-called 'synthetic lethal interactions', a principle that is currently being tested in clinical trials. Finally, homologous recombination repair-deficient cells may have an increased dependency on certain cell-cycle checkpoints, which can be therapeutically exploited. Here we describe recent advances in strategies to identify and target HRD tumours, approaches to overcome resistance, and combinatory strategies to optimize treatment outcome.}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/j.tips.2010.06.00110.1016/j.tips.2010.06.001] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2059875620598756]}, keywords = {}, pubstate = {published}, tppubtype = {article} } DNA repair is essential for cells to maintain genome stability in an environment that constantly produces DNA damage. There is a growing appreciation that defects in homologous recombination repair underlie hereditary and sporadic tumourigenesis, and that deficiency in this pathway may dictate the sensitivity of tumours to certain DNA-damaging agents. Homologous recombination deficiency (HRD) may therefore prove to be a diagnostic criterion per se if appropriate biomarkers become available to identify these tumours. In addition, homologous recombination-deficient tumours are more sensitive to inhibition of other DNA repair pathways through so-called 'synthetic lethal interactions', a principle that is currently being tested in clinical trials. Finally, homologous recombination repair-deficient cells may have an increased dependency on certain cell-cycle checkpoints, which can be therapeutically exploited. Here we describe recent advances in strategies to identify and target HRD tumours, approaches to overcome resistance, and combinatory strategies to optimize treatment outcome. |
Higgins, G S; Harris, A L; Prevo, R; Helleday, T; McKenna, W G; Buffa, F M Overexpression of POLQ confers a poor prognosis in early breast cancer patients Journal Article Oncotarget, 1 (3), pp. 175–184, 2010, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2917771PMC2917771] [DOI:hrefhttp://dx.doi.org/10.18632/oncotarget.12410.18632/oncotarget.124] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2070046920700469]). @article{pmid20700469, title = {Overexpression of POLQ confers a poor prognosis in early breast cancer patients}, author = { G. S. Higgins and A. L. Harris and R. Prevo and T. Helleday and W. G. McKenna and F. M. Buffa}, year = {2010}, date = {2010-07-01}, journal = {Oncotarget}, volume = {1}, number = {3}, pages = {175--184}, abstract = {Depletion of POLQ (DNA polymerase theta) has recently been shown to render tumour cells more sensitive to radiotherapy whilst having little or no effect on normal tissues. This finding led us to investigate whether tumours that overexpress POLQ are associated with an adverse outcome. We therefore correlated the clinical outcomes of two retrospective series of patients with early breast cancer with the expression levels of POLQ, as determined by microarray gene expression analysis. We found that a significant number of tumours overexpressed POLQ and that overexpression was correlated with ER negative disease (p=0.047) and high tumour grade (p=0.004), both of which are associated with poor clinical outcomes. POLQ overexpression was associated with poor relapse free survival rates on both univariate (HR 5.80; 95% CI, 2.220 to 15.159; p<0.001) and multivariate analysis (HR 8.086; 95% CI 2.340 to 27.948 p=0.001). Analysis of other published clinical series confirmed that POLQ overexpression is associated with adverse clinical outcomes. The poor prognosis associated with POLQ is independent of other clinical or pathological features. The mechanism that causes this adverse outcome remains to be elucidated but may in part arise from resistance to adjuvant treatment. These findings, combined with the limited normal tissue expression of POLQ, make it a very appealing target for possible clinical exploitation.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2917771PMC2917771] [DOI:hrefhttp://dx.doi.org/10.18632/oncotarget.12410.18632/oncotarget.124] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2070046920700469]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Depletion of POLQ (DNA polymerase theta) has recently been shown to render tumour cells more sensitive to radiotherapy whilst having little or no effect on normal tissues. This finding led us to investigate whether tumours that overexpress POLQ are associated with an adverse outcome. We therefore correlated the clinical outcomes of two retrospective series of patients with early breast cancer with the expression levels of POLQ, as determined by microarray gene expression analysis. We found that a significant number of tumours overexpressed POLQ and that overexpression was correlated with ER negative disease (p=0.047) and high tumour grade (p=0.004), both of which are associated with poor clinical outcomes. POLQ overexpression was associated with poor relapse free survival rates on both univariate (HR 5.80; 95% CI, 2.220 to 15.159; p<0.001) and multivariate analysis (HR 8.086; 95% CI 2.340 to 27.948 p=0.001). Analysis of other published clinical series confirmed that POLQ overexpression is associated with adverse clinical outcomes. The poor prognosis associated with POLQ is independent of other clinical or pathological features. The mechanism that causes this adverse outcome remains to be elucidated but may in part arise from resistance to adjuvant treatment. These findings, combined with the limited normal tissue expression of POLQ, make it a very appealing target for possible clinical exploitation. |
MacKay, C; Declais, A C; Lundin, C; Agostinho, A; Deans, A J; MacArtney, T J; Hofmann, K; Gartner, A; West, S C; Helleday, T; Lilley, D M; Rouse, J Identification of KIAA1018/FAN1, a ĐNA repair nuclease recruited to ĐNA damage by monoubiquitinated FANCĐ2 Journal Article Cell, 142 (1), pp. 65–76, 2010, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3710700PMC3710700] [DOI:hrefhttp://dx.doi.org/10.1016/j.cell.2010.06.02110.1016/j.cell.2010.06.021] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2060301520603015]). @article{pmid20603015, title = {Identification of KIAA1018/FAN1, a ĐNA repair nuclease recruited to ĐNA damage by monoubiquitinated FANCĐ2}, author = { C. MacKay and A. C. Declais and C. Lundin and A. Agostinho and A. J. Deans and T. J. MacArtney and K. Hofmann and A. Gartner and S. C. West and T. Helleday and D. M. Lilley and J. Rouse}, year = {2010}, date = {2010-07-01}, journal = {Cell}, volume = {142}, number = {1}, pages = {65--76}, abstract = {DNA interstrand crosslinks (ICLs) are highly toxic because they block the progression of replisomes. The Fanconi Anemia (FA) proteins, encoded by genes that are mutated in FA, are important for repair of ICLs. The FA core complex catalyzes the monoubiquitination of FANCD2, and this event is essential for several steps of ICL repair. However, how monoubiquitination of FANCD2 promotes ICL repair at the molecular level is unknown. Here, we describe a highly conserved protein, KIAA1018/MTMR15/FAN1, that interacts with, and is recruited to sites of DNA damage by, the monoubiquitinated form of FANCD2. FAN1 exhibits endonuclease activity toward 5' flaps and has 5' exonuclease activity, and these activities are mediated by an ancient VRR_nuc domain. Depletion of FAN1 from human cells causes hypersensitivity to ICLs, defects in ICL repair, and genome instability. These data at least partly explain how ubiquitination of FANCD2 promotes DNA repair.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3710700PMC3710700] [DOI:hrefhttp://dx.doi.org/10.1016/j.cell.2010.06.02110.1016/j.cell.2010.06.021] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2060301520603015]}, keywords = {}, pubstate = {published}, tppubtype = {article} } DNA interstrand crosslinks (ICLs) are highly toxic because they block the progression of replisomes. The Fanconi Anemia (FA) proteins, encoded by genes that are mutated in FA, are important for repair of ICLs. The FA core complex catalyzes the monoubiquitination of FANCD2, and this event is essential for several steps of ICL repair. However, how monoubiquitination of FANCD2 promotes ICL repair at the molecular level is unknown. Here, we describe a highly conserved protein, KIAA1018/MTMR15/FAN1, that interacts with, and is recruited to sites of DNA damage by, the monoubiquitinated form of FANCD2. FAN1 exhibits endonuclease activity toward 5' flaps and has 5' exonuclease activity, and these activities are mediated by an ancient VRR_nuc domain. Depletion of FAN1 from human cells causes hypersensitivity to ICLs, defects in ICL repair, and genome instability. These data at least partly explain how ubiquitination of FANCD2 promotes DNA repair. |
Gottipati, P; Vischioni, B; Schultz, N; Solomons, J; Bryant, H E; Djureinovic, T; Issaeva, N; Sleeth, K; Sharma, R A; Helleday, T Poly(AĐP-ribose) polymerase is hyperactivated in homologous recombination-defective cells Journal Article Cancer Res., 70 (13), pp. 5389–5398, 2010, ([DOI:hrefhttp://dx.doi.org/10.1158/0008-5472.CAN-09-471610.1158/0008-5472.CAN-09-4716] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2055106820551068]). @article{pmid20551068, title = {Poly(AĐP-ribose) polymerase is hyperactivated in homologous recombination-defective cells}, author = { P. Gottipati and B. Vischioni and N. Schultz and J. Solomons and H. E. Bryant and T. Djureinovic and N. Issaeva and K. Sleeth and R. A. Sharma and T. Helleday}, year = {2010}, date = {2010-07-01}, journal = {Cancer Res.}, volume = {70}, number = {13}, pages = {5389--5398}, abstract = {Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) is activated by DNA single-strand breaks (SSB) or at stalled replication forks to facilitate DNA repair. Inhibitors of PARP efficiently kill breast, ovarian, or prostate tumors in patients carrying hereditary mutations in the homologous recombination (HR) genes BRCA1 or BRCA2 through synthetic lethality. Here, we surprisingly show that PARP1 is hyperactivated in replicating BRCA2-defective cells. PARP1 hyperactivation is explained by the defect in HR as shRNA depletion of RAD54, RAD52, BLM, WRN, and XRCC3 proteins, which we here show are all essential for efficient HR and also caused PARP hyperactivation and correlated with an increased sensitivity to PARP inhibitors. BRCA2-defective cells were not found to have increased levels of SSBs, and PAR polymers formed in HR-defective cells do not colocalize to replication protein A or gammaH2AX, excluding the possibility that PARP hyperactivity is due to increased SSB repair or PARP induced at damaged replication forks. Resistance to PARP inhibitors can occur through genetic reversion in the BRCA2 gene. Here, we report that PARP inhibitor-resistant BRCA2-mutant cells revert back to normal levels of PARP activity. We speculate that the reason for the sensitivity of HR-defective cells to PARP inhibitors is related to the hyperactivated PARP1 in these cells. Furthermore, the presence of PAR polymers can be used to identify HR-defective cells that are sensitive to PARP inhibitors, which may be potential biomarkers.}, note = {[DOI:hrefhttp://dx.doi.org/10.1158/0008-5472.CAN-09-471610.1158/0008-5472.CAN-09-4716] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2055106820551068]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) is activated by DNA single-strand breaks (SSB) or at stalled replication forks to facilitate DNA repair. Inhibitors of PARP efficiently kill breast, ovarian, or prostate tumors in patients carrying hereditary mutations in the homologous recombination (HR) genes BRCA1 or BRCA2 through synthetic lethality. Here, we surprisingly show that PARP1 is hyperactivated in replicating BRCA2-defective cells. PARP1 hyperactivation is explained by the defect in HR as shRNA depletion of RAD54, RAD52, BLM, WRN, and XRCC3 proteins, which we here show are all essential for efficient HR and also caused PARP hyperactivation and correlated with an increased sensitivity to PARP inhibitors. BRCA2-defective cells were not found to have increased levels of SSBs, and PAR polymers formed in HR-defective cells do not colocalize to replication protein A or gammaH2AX, excluding the possibility that PARP hyperactivity is due to increased SSB repair or PARP induced at damaged replication forks. Resistance to PARP inhibitors can occur through genetic reversion in the BRCA2 gene. Here, we report that PARP inhibitor-resistant BRCA2-mutant cells revert back to normal levels of PARP activity. We speculate that the reason for the sensitivity of HR-defective cells to PARP inhibitors is related to the hyperactivated PARP1 in these cells. Furthermore, the presence of PAR polymers can be used to identify HR-defective cells that are sensitive to PARP inhibitors, which may be potential biomarkers. |
Parsons, J L; Dianova, I I; Finch, D; Tait, P S; Strom, C E; Helleday, T; Dianov, G L XRCC1 phosphorylation by CK2 is required for its stability and efficient ĐNA repair Journal Article DNA Repair (Amst.), 9 (7), pp. 835–841, 2010, ([DOI:hrefhttp://dx.doi.org/10.1016/j.dnarep.2010.04.00810.1016/j.dnarep.2010.04.008] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2047132920471329]). @article{pmid20471329, title = {XRCC1 phosphorylation by CK2 is required for its stability and efficient ĐNA repair}, author = { J. L. Parsons and I. I. Dianova and D. Finch and P. S. Tait and C. E. Strom and T. Helleday and G. L. Dianov}, year = {2010}, date = {2010-07-01}, journal = {DNA Repair (Amst.)}, volume = {9}, number = {7}, pages = {835--841}, abstract = {XRCC1 is a scaffold protein that interacts with several DNA repair proteins and plays a critical role in DNA base excision repair (BER). XRCC1 protein is in a tight complex with DNA ligase IIIalpha (Lig III) and this complex is involved in the ligation step of both BER and repair of DNA single strand breaks. The majority of XRCC1 has previously been demonstrated to exist in a phosphorylated form and cells containing mutant XRCC1, that is unable to be phosphorylated, display a reduced rate of single strand break repair. Here, in an unbiased assay, we demonstrate that the cytoplasmic form of the casein kinase 2 (CK2) protein is the major protein kinase activity involved in phosphorylation of XRCC1 in human cell extracts and that XRCC1 phosphorylation is required for XRCC1-Lig III complex stability. We demonstrate that XRCC1-Lig III complex containing mutant XRCC1, in which CK2 phosphorylation sites have been mutated, is unstable. We also find that a knockdown of CK2 by siRNA results in both reduced XRCC1 phosphorylation and stability, which also leads to a reduced amount of Lig III and accumulation of DNA strand breaks. We therefore propose that CK2 plays an important role in DNA repair by contributing to the stability of XRCC1-Lig III complex.}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/j.dnarep.2010.04.00810.1016/j.dnarep.2010.04.008] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2047132920471329]}, keywords = {}, pubstate = {published}, tppubtype = {article} } XRCC1 is a scaffold protein that interacts with several DNA repair proteins and plays a critical role in DNA base excision repair (BER). XRCC1 protein is in a tight complex with DNA ligase IIIalpha (Lig III) and this complex is involved in the ligation step of both BER and repair of DNA single strand breaks. The majority of XRCC1 has previously been demonstrated to exist in a phosphorylated form and cells containing mutant XRCC1, that is unable to be phosphorylated, display a reduced rate of single strand break repair. Here, in an unbiased assay, we demonstrate that the cytoplasmic form of the casein kinase 2 (CK2) protein is the major protein kinase activity involved in phosphorylation of XRCC1 in human cell extracts and that XRCC1 phosphorylation is required for XRCC1-Lig III complex stability. We demonstrate that XRCC1-Lig III complex containing mutant XRCC1, in which CK2 phosphorylation sites have been mutated, is unstable. We also find that a knockdown of CK2 by siRNA results in both reduced XRCC1 phosphorylation and stability, which also leads to a reduced amount of Lig III and accumulation of DNA strand breaks. We therefore propose that CK2 plays an important role in DNA repair by contributing to the stability of XRCC1-Lig III complex. |
Helleday, T Ħomologous recombination in cancer development, treatment and development of drug resistance Journal Article Carcinogenesis, 31 (6), pp. 955–960, 2010, ([DOI:hrefhttp://dx.doi.org/10.1093/carcin/bgq06410.1093/carcin/bgq064] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2035109220351092]). @article{pmid20351092, title = {Ħomologous recombination in cancer development, treatment and development of drug resistance}, author = { T. Helleday}, year = {2010}, date = {2010-06-01}, journal = {Carcinogenesis}, volume = {31}, number = {6}, pages = {955--960}, abstract = {Although DNA double-strand breaks (DSBs) are substrates for homologous recombination (HR) repair, it is becoming apparent that DNA lesions produced at replication forks, for instance by many anticancer drugs, are more significant substrates for HR repair. Cells defective in HR are hypersensitive to a wide variety of anticancer drugs, including those that do not produce DSBs. Several cancers have mutations in or epigenetically silenced HR genes, which explain the genetic instability that drives cancer development. There are an increasing number of reports suggesting that mutation or epigenetic silencing of HR genes explains the sensitivity of cancers to current chemotherapy treatments. Furthermore, there are also many examples of re-expression of HR genes in tumours to explain drug resistance. Emerging data suggest that there are several different subpathways of HR, which can compensate for each other. Unravelling the overlapping pathways in HR showed that BRCA1- and BRCA2-defective cells rely on the PARP protein for survival. This synthetic lethal interaction is now being exploited for selective treatment of BRCA1- and BRCA2-defective cancers with PARP inhibitors. Here, I discuss the diversity of HR and how it impacts on cancer with a particular focus on how HR can be exploited in future anticancer strategies.}, note = {[DOI:hrefhttp://dx.doi.org/10.1093/carcin/bgq06410.1093/carcin/bgq064] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2035109220351092]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Although DNA double-strand breaks (DSBs) are substrates for homologous recombination (HR) repair, it is becoming apparent that DNA lesions produced at replication forks, for instance by many anticancer drugs, are more significant substrates for HR repair. Cells defective in HR are hypersensitive to a wide variety of anticancer drugs, including those that do not produce DSBs. Several cancers have mutations in or epigenetically silenced HR genes, which explain the genetic instability that drives cancer development. There are an increasing number of reports suggesting that mutation or epigenetic silencing of HR genes explains the sensitivity of cancers to current chemotherapy treatments. Furthermore, there are also many examples of re-expression of HR genes in tumours to explain drug resistance. Emerging data suggest that there are several different subpathways of HR, which can compensate for each other. Unravelling the overlapping pathways in HR showed that BRCA1- and BRCA2-defective cells rely on the PARP protein for survival. This synthetic lethal interaction is now being exploited for selective treatment of BRCA1- and BRCA2-defective cancers with PARP inhibitors. Here, I discuss the diversity of HR and how it impacts on cancer with a particular focus on how HR can be exploited in future anticancer strategies. |
Higgins, G S; Prevo, R; Lee, Y F; Helleday, T; Muschel, R J; Taylor, S; Yoshimura, M; Hickson, I D; Bernhard, E J; McKenna, W G A small interfering RNA screen of genes involved in ĐNA repair identifies tumor-specific radiosensitization by POLQ knockdown Journal Article Cancer Res., 70 (7), pp. 2984–2993, 2010, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2848966PMC2848966] [DOI:hrefhttp://dx.doi.org/10.1158/0008-5472.CAN-09-404010.1158/0008-5472.CAN-09-4040] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2023387820233878]). @article{pmid20233878, title = {A small interfering RNA screen of genes involved in ĐNA repair identifies tumor-specific radiosensitization by POLQ knockdown}, author = { G. S. Higgins and R. Prevo and Y. F. Lee and T. Helleday and R. J. Muschel and S. Taylor and M. Yoshimura and I. D. Hickson and E. J. Bernhard and W. G. McKenna}, year = {2010}, date = {2010-04-01}, journal = {Cancer Res.}, volume = {70}, number = {7}, pages = {2984--2993}, abstract = {The effectiveness of radiotherapy treatment could be significantly improved if tumor cells could be rendered more sensitive to ionizing radiation (IR) without altering the sensitivity of normal tissues. However, many of the key therapeutically exploitable mechanisms that determine intrinsic tumor radiosensitivity are largely unknown. We have conducted a small interfering RNA (siRNA) screen of 200 genes involved in DNA damage repair aimed at identifying genes whose knockdown increased tumor radiosensitivity. Parallel siRNA screens were conducted in irradiated and unirradiated tumor cells (SQ20B) and irradiated normal tissue cells (MRC5). Using gammaH2AX foci at 24 hours after IR, we identified several genes, such as BRCA2, Lig IV, and XRCC5, whose knockdown is known to cause increased cell radiosensitivity, thereby validating the primary screening end point. In addition, we identified POLQ (DNA polymerase ) as a potential tumor-specific target. Subsequent investigations showed that POLQ knockdown resulted in radiosensitization of a panel of tumor cell lines from different primary sites while having little or no effect on normal tissue cell lines. These findings raise the possibility that POLQ inhibition might be used clinically to cause tumor-specific radiosensitization.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2848966PMC2848966] [DOI:hrefhttp://dx.doi.org/10.1158/0008-5472.CAN-09-404010.1158/0008-5472.CAN-09-4040] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2023387820233878]}, keywords = {}, pubstate = {published}, tppubtype = {article} } The effectiveness of radiotherapy treatment could be significantly improved if tumor cells could be rendered more sensitive to ionizing radiation (IR) without altering the sensitivity of normal tissues. However, many of the key therapeutically exploitable mechanisms that determine intrinsic tumor radiosensitivity are largely unknown. We have conducted a small interfering RNA (siRNA) screen of 200 genes involved in DNA damage repair aimed at identifying genes whose knockdown increased tumor radiosensitivity. Parallel siRNA screens were conducted in irradiated and unirradiated tumor cells (SQ20B) and irradiated normal tissue cells (MRC5). Using gammaH2AX foci at 24 hours after IR, we identified several genes, such as BRCA2, Lig IV, and XRCC5, whose knockdown is known to cause increased cell radiosensitivity, thereby validating the primary screening end point. In addition, we identified POLQ (DNA polymerase ) as a potential tumor-specific target. Subsequent investigations showed that POLQ knockdown resulted in radiosensitization of a panel of tumor cell lines from different primary sites while having little or no effect on normal tissue cell lines. These findings raise the possibility that POLQ inhibition might be used clinically to cause tumor-specific radiosensitization. |
Beck, H; Nahse, V; Larsen, M S; Groth, P; Clancy, T; Lees, M; J?rgensen, M; Helleday, T; Syljuasen, R G; S?rensen, C S Regulators of cyclin-dependent kinases are crucial for maintaining genome integrity in S phase Journal Article J. Cell Biol., 188 (5), pp. 629–638, 2010, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2835936PMC2835936] [DOI:hrefhttp://dx.doi.org/10.1083/jcb.20090505910.1083/jcb.200905059] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2019464220194642]). @article{pmid20194642, title = {Regulators of cyclin-dependent kinases are crucial for maintaining genome integrity in S phase}, author = { H. Beck and V. Nahse and M. S. Larsen and P. Groth and T. Clancy and M. Lees and M. J?rgensen and T. Helleday and R. G. Syljuasen and C. S. S?rensen}, year = {2010}, date = {2010-03-01}, journal = {J. Cell Biol.}, volume = {188}, number = {5}, pages = {629--638}, abstract = {Maintenance of genome integrity is of critical importance to cells. To identify key regulators of genomic integrity, we screened a human cell line with a kinome small interfering RNA library. WEE1, a major regulator of mitotic entry, and CHK1 were among the genes identified. Both kinases are important negative regulators of CDK1 and -2. Strikingly, WEE1 depletion rapidly induced DNA damage in S phase in newly replicated DNA, which was accompanied by a marked increase in single-stranded DNA. This DNA damage is dependent on CDK1 and -2 as well as the replication proteins MCM2 and CDT1 but not CDC25A. Conversely, DNA damage after CHK1 inhibition is highly dependent on CDC25A. Furthermore, the inferior proliferation of CHK1-depleted cells is improved substantially by codepletion of CDC25A. We conclude that the mitotic kinase WEE1 and CHK1 jointly maintain balanced cellular control of Cdk activity during normal DNA replication, which is crucial to prevent the generation of harmful DNA lesions during replication.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2835936PMC2835936] [DOI:hrefhttp://dx.doi.org/10.1083/jcb.20090505910.1083/jcb.200905059] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2019464220194642]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Maintenance of genome integrity is of critical importance to cells. To identify key regulators of genomic integrity, we screened a human cell line with a kinome small interfering RNA library. WEE1, a major regulator of mitotic entry, and CHK1 were among the genes identified. Both kinases are important negative regulators of CDK1 and -2. Strikingly, WEE1 depletion rapidly induced DNA damage in S phase in newly replicated DNA, which was accompanied by a marked increase in single-stranded DNA. This DNA damage is dependent on CDK1 and -2 as well as the replication proteins MCM2 and CDT1 but not CDC25A. Conversely, DNA damage after CHK1 inhibition is highly dependent on CDC25A. Furthermore, the inferior proliferation of CHK1-depleted cells is improved substantially by codepletion of CDC25A. We conclude that the mitotic kinase WEE1 and CHK1 jointly maintain balanced cellular control of Cdk activity during normal DNA replication, which is crucial to prevent the generation of harmful DNA lesions during replication. |
Loseva, O; Jemth, A S; Bryant, H E; Schuler, H; Lehtio, L; Karlberg, T; Helleday, T PARP-3 is a mono-AĐP-ribosylase that activates PARP-1 in the absence of ĐNA Journal Article J. Biol. Chem., 285 (11), pp. 8054–8060, 2010, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2832956PMC2832956] [DOI:hrefhttp://dx.doi.org/10.1074/jbc.M109.07783410.1074/jbc.M109.077834] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2006493820064938]). @article{pmid20064938, title = {PARP-3 is a mono-AĐP-ribosylase that activates PARP-1 in the absence of ĐNA}, author = { O. Loseva and A. S. Jemth and H. E. Bryant and H. Schuler and L. Lehtio and T. Karlberg and T. Helleday}, year = {2010}, date = {2010-03-01}, journal = {J. Biol. Chem.}, volume = {285}, number = {11}, pages = {8054--8060}, abstract = {The PARP-3 protein is closely related to the PARP-1 and PARP-2 proteins, which are involved in DNA repair and genome maintenance. Here, we characterized the biochemical properties of human PARP-3. PARP-3 is able to ADP-ribosylate itself as well as histone H1, a previously unknown substrate for PARP-3. PARP-3 is not activated upon binding to DNA and is a mono-ADP-ribosylase, in contrast to PARP-1 and PARP-2. PARP-3 interacts with PARP-1 and activates PARP-1 in the absence of DNA, resulting in synthesis of polymers of ADP-ribose. The N-terminal WGR domain of PARP-3 is involved in this activation. The functional interaction between PARP-3 and PARP-1 suggests that it may have a role in DNA repair. However, here we report that PARP-3 small interfering RNA-depleted cells are not sensitive to the topoisomerase I poison camptothecin, inducing DNA single-strand breaks, and repair these lesions as efficiently as wild-type cells. Altogether, these results suggest that the interaction between PARP-1 and PARP-3 is unrelated to DNA single-strand break repair.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2832956PMC2832956] [DOI:hrefhttp://dx.doi.org/10.1074/jbc.M109.07783410.1074/jbc.M109.077834] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2006493820064938]}, keywords = {}, pubstate = {published}, tppubtype = {article} } The PARP-3 protein is closely related to the PARP-1 and PARP-2 proteins, which are involved in DNA repair and genome maintenance. Here, we characterized the biochemical properties of human PARP-3. PARP-3 is able to ADP-ribosylate itself as well as histone H1, a previously unknown substrate for PARP-3. PARP-3 is not activated upon binding to DNA and is a mono-ADP-ribosylase, in contrast to PARP-1 and PARP-2. PARP-3 interacts with PARP-1 and activates PARP-1 in the absence of DNA, resulting in synthesis of polymers of ADP-ribose. The N-terminal WGR domain of PARP-3 is involved in this activation. The functional interaction between PARP-3 and PARP-1 suggests that it may have a role in DNA repair. However, here we report that PARP-3 small interfering RNA-depleted cells are not sensitive to the topoisomerase I poison camptothecin, inducing DNA single-strand breaks, and repair these lesions as efficiently as wild-type cells. Altogether, these results suggest that the interaction between PARP-1 and PARP-3 is unrelated to DNA single-strand break repair. |
Petermann, E; Orta, M L; Issaeva, N; Schultz, N; Helleday, T Ħydroxyurea-stalled replication forks become progressively inactivated and require two different RAĐ51-mediated pathways for restart and repair Journal Article Mol. Cell, 37 (4), pp. 492–502, 2010, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2958316PMC2958316] [DOI:hrefhttp://dx.doi.org/10.1016/j.molcel.2010.01.02110.1016/j.molcel.2010.01.021] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2018866820188668]). @article{pmid20188668, title = {Ħydroxyurea-stalled replication forks become progressively inactivated and require two different RAĐ51-mediated pathways for restart and repair}, author = { E. Petermann and M. L. Orta and N. Issaeva and N. Schultz and T. Helleday}, year = {2010}, date = {2010-02-01}, journal = {Mol. Cell}, volume = {37}, number = {4}, pages = {492--502}, abstract = {Faithful DNA replication is essential to all life. Hydroxyurea (HU) depletes the cells of dNTPs, which initially results in stalled replication forks that, after prolonged treatment, collapse into DSBs. Here, we report that stalled replication forks are efficiently restarted in a RAD51-dependent process that does not trigger homologous recombination (HR). The XRCC3 protein, which is required for RAD51 foci formation, is also required for replication restart of HU-stalled forks, suggesting that RAD51-mediated strand invasion supports fork restart. In contrast, replication forks collapsed by prolonged replication blocks do not restart, and global replication is rescued by new origin firing. We find that RAD51-dependent HR is triggered for repair of collapsed replication forks, without apparent restart. In conclusion, our data suggest that restart of stalled replication forks and HR repair of collapsed replication forks require two distinct RAD51-mediated pathways.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2958316PMC2958316] [DOI:hrefhttp://dx.doi.org/10.1016/j.molcel.2010.01.02110.1016/j.molcel.2010.01.021] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2018866820188668]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Faithful DNA replication is essential to all life. Hydroxyurea (HU) depletes the cells of dNTPs, which initially results in stalled replication forks that, after prolonged treatment, collapse into DSBs. Here, we report that stalled replication forks are efficiently restarted in a RAD51-dependent process that does not trigger homologous recombination (HR). The XRCC3 protein, which is required for RAD51 foci formation, is also required for replication restart of HU-stalled forks, suggesting that RAD51-mediated strand invasion supports fork restart. In contrast, replication forks collapsed by prolonged replication blocks do not restart, and global replication is rescued by new origin firing. We find that RAD51-dependent HR is triggered for repair of collapsed replication forks, without apparent restart. In conclusion, our data suggest that restart of stalled replication forks and HR repair of collapsed replication forks require two distinct RAD51-mediated pathways. |
Helleday, T Mutagenesis: mutating a gene while reading it Journal Article Curr. Biol., 20 (2), pp. R57–58, 2010, ([DOI:hrefhttp://dx.doi.org/10.1016/j.cub.2009.11.04710.1016/j.cub.2009.11.047] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2012903820129038]). @article{pmid20129038, title = {Mutagenesis: mutating a gene while reading it}, author = { T. Helleday}, year = {2010}, date = {2010-01-01}, journal = {Curr. Biol.}, volume = {20}, number = {2}, pages = {R57--58}, abstract = {Is it possible to mutate DNA during transcription? A new study shows that UV-damaged DNA is deaminated during transcription, which is a probable mechanism underlying CC tandem mutations found in the p53 gene in skin cancers.}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/j.cub.2009.11.04710.1016/j.cub.2009.11.047] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/2012903820129038]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Is it possible to mutate DNA during transcription? A new study shows that UV-damaged DNA is deaminated during transcription, which is a probable mechanism underlying CC tandem mutations found in the p53 gene in skin cancers. |
2009 |
McLachlan, J; Fernandez, S; Helleday, T; Bryant, H E Specific targeted gene repair using single-stranded ĐNA oligonucleotides at an endogenous locus in mammalian cells uses homologous recombination Journal Article DNA Repair (Amst.), 8 (12), pp. 1424–1433, 2009, ([DOI:hrefhttp://dx.doi.org/10.1016/j.dnarep.2009.09.01410.1016/j.dnarep.2009.09.014] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1985468719854687]). @article{pmid19854687, title = {Specific targeted gene repair using single-stranded ĐNA oligonucleotides at an endogenous locus in mammalian cells uses homologous recombination}, author = { J. McLachlan and S. Fernandez and T. Helleday and H. E. Bryant}, year = {2009}, date = {2009-12-01}, journal = {DNA Repair (Amst.)}, volume = {8}, number = {12}, pages = {1424--1433}, abstract = {The feasibility of introducing point mutations in vivo using single-stranded DNA oligonucleotides (ssON) has been demonstrated but the efficiency and mechanism remain elusive and potential side effects have not been fully evaluated. Understanding the mechanism behind this potential therapy may help its development. Here, we demonstrate the specific repair of an endogenous non-functional hprt gene by a ssON in mammalian cells, and show that the frequency of such an event is enhanced when cells are in S-phase of the cell cycle. A potential barrier in using ssONs as gene therapy could be non-targeted mutations or gene rearrangements triggered by the ssON. Both the non-specific mutation frequencies and the frequency of gene rearrangements were largely unaffected by ssONs. Furthermore, we find that the introduction of a mutation causing the loss of a functional endogenous hprt gene by a ssON occurred at a similarly low but statistically significant frequency in wild type cells and in cells deficient in single strand break repair, nucleotide excision repair and mismatch repair. However, this mutation was not induced in XRCC3 mutant cells deficient in homologous recombination. Thus, our data suggest ssON-mediated targeted gene repair is more efficient in S-phase and involves homologous recombination.}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/j.dnarep.2009.09.01410.1016/j.dnarep.2009.09.014] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1985468719854687]}, keywords = {}, pubstate = {published}, tppubtype = {article} } The feasibility of introducing point mutations in vivo using single-stranded DNA oligonucleotides (ssON) has been demonstrated but the efficiency and mechanism remain elusive and potential side effects have not been fully evaluated. Understanding the mechanism behind this potential therapy may help its development. Here, we demonstrate the specific repair of an endogenous non-functional hprt gene by a ssON in mammalian cells, and show that the frequency of such an event is enhanced when cells are in S-phase of the cell cycle. A potential barrier in using ssONs as gene therapy could be non-targeted mutations or gene rearrangements triggered by the ssON. Both the non-specific mutation frequencies and the frequency of gene rearrangements were largely unaffected by ssONs. Furthermore, we find that the introduction of a mutation causing the loss of a functional endogenous hprt gene by a ssON occurred at a similarly low but statistically significant frequency in wild type cells and in cells deficient in single strand break repair, nucleotide excision repair and mismatch repair. However, this mutation was not induced in XRCC3 mutant cells deficient in homologous recombination. Thus, our data suggest ssON-mediated targeted gene repair is more efficient in S-phase and involves homologous recombination. |
Al-Minawi, A Z; Lee, Y F; Hakansson, D; Johansson, F; Lundin, C; Saleh-Gohari, N; Schultz, N; Jenssen, D; Bryant, H E; Meuth, M; Hinz, J M; Helleday, T Ŧhe ERCC1/XPF endonuclease is required for completion of homologous recombination at ĐNA replication forks stalled by inter-strand cross-links Journal Article Nucleic Acids Res., 37 (19), pp. 6400–6413, 2009, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2770670PMC2770670] [DOI:hrefhttp://dx.doi.org/10.1093/nar/gkp70510.1093/nar/gkp705] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1971343819713438]). @article{pmid19713438, title = {Ŧhe ERCC1/XPF endonuclease is required for completion of homologous recombination at ĐNA replication forks stalled by inter-strand cross-links}, author = { A. Z. Al-Minawi and Y. F. Lee and D. Hakansson and F. Johansson and C. Lundin and N. Saleh-Gohari and N. Schultz and D. Jenssen and H. E. Bryant and M. Meuth and J. M. Hinz and T. Helleday}, year = {2009}, date = {2009-10-01}, journal = {Nucleic Acids Res.}, volume = {37}, number = {19}, pages = {6400--6413}, abstract = {Both the ERCC1-XPF complex and the proteins involved in homoIogous recombination (HR) have critical roles in inter-strand cross-link (ICL) repair. Here, we report that mitomycin C-induced lesions inhibit replication fork elongation. Furthermore, mitomycin C-induced DNA double-strand breaks (DSBs) are the result of the collapse of ICL-stalled replication forks. These are not formed through replication run off, as we show that mitomycin C or cisplatin-induced DNA lesions are not incised by global genome nucleotide excision repair (GGR). We also suggest that ICL-lesion repair is initiated either by replication or transcription, as the GGR does not incise ICL-lesions. Furthermore, we report that RAD51 foci are induced by cisplatin or mitomycin C independently of ERCC1, but that mitomycin C-induced HR measured in a reporter construct is impaired in ERCC1-defective cells. These data suggest that ERCC1-XPF plays a role in completion of HR in ICL repair. We also find no additional sensitivity to cisplatin by siRNA co-depletion of XRCC3 and ERCC1, showing that the two proteins act on the same pathway to promote survival.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2770670PMC2770670] [DOI:hrefhttp://dx.doi.org/10.1093/nar/gkp70510.1093/nar/gkp705] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1971343819713438]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Both the ERCC1-XPF complex and the proteins involved in homoIogous recombination (HR) have critical roles in inter-strand cross-link (ICL) repair. Here, we report that mitomycin C-induced lesions inhibit replication fork elongation. Furthermore, mitomycin C-induced DNA double-strand breaks (DSBs) are the result of the collapse of ICL-stalled replication forks. These are not formed through replication run off, as we show that mitomycin C or cisplatin-induced DNA lesions are not incised by global genome nucleotide excision repair (GGR). We also suggest that ICL-lesion repair is initiated either by replication or transcription, as the GGR does not incise ICL-lesions. Furthermore, we report that RAD51 foci are induced by cisplatin or mitomycin C independently of ERCC1, but that mitomycin C-induced HR measured in a reporter construct is impaired in ERCC1-defective cells. These data suggest that ERCC1-XPF plays a role in completion of HR in ICL repair. We also find no additional sensitivity to cisplatin by siRNA co-depletion of XRCC3 and ERCC1, showing that the two proteins act on the same pathway to promote survival. |
Bryant, H E; Petermann, E; Schultz, N; Jemth, A S; Loseva, O; Issaeva, N; Johansson, F; Fernandez, S; McGlynn, P; Helleday, T PARP is activated at stalled forks to mediate Mre11-dependent replication restart and recombination Journal Article EMBO J., 28 (17), pp. 2601–2615, 2009, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738702PMC2738702] [DOI:hrefhttp://dx.doi.org/10.1038/emboj.2009.20610.1038/emboj.2009.206] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1962903519629035]). @article{pmid19629035, title = {PARP is activated at stalled forks to mediate Mre11-dependent replication restart and recombination}, author = { H. E. Bryant and E. Petermann and N. Schultz and A. S. Jemth and O. Loseva and N. Issaeva and F. Johansson and S. Fernandez and P. McGlynn and T. Helleday}, year = {2009}, date = {2009-09-01}, journal = {EMBO J.}, volume = {28}, number = {17}, pages = {2601--2615}, abstract = {If replication forks are perturbed, a multifaceted response including several DNA repair and cell cycle checkpoint pathways is activated to ensure faithful DNA replication. Here, we show that poly(ADP-ribose) polymerase 1 (PARP1) binds to and is activated by stalled replication forks that contain small gaps. PARP1 collaborates with Mre11 to promote replication fork restart after release from replication blocks, most likely by recruiting Mre11 to the replication fork to promote resection of DNA. Both PARP1 and PARP2 are required for hydroxyurea-induced homologous recombination to promote cell survival after replication blocks. Together, our data suggest that PARP1 and PARP2 detect disrupted replication forks and attract Mre11 for end processing that is required for subsequent recombination repair and restart of replication forks.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738702PMC2738702] [DOI:hrefhttp://dx.doi.org/10.1038/emboj.2009.20610.1038/emboj.2009.206] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1962903519629035]}, keywords = {}, pubstate = {published}, tppubtype = {article} } If replication forks are perturbed, a multifaceted response including several DNA repair and cell cycle checkpoint pathways is activated to ensure faithful DNA replication. Here, we show that poly(ADP-ribose) polymerase 1 (PARP1) binds to and is activated by stalled replication forks that contain small gaps. PARP1 collaborates with Mre11 to promote replication fork restart after release from replication blocks, most likely by recruiting Mre11 to the replication fork to promote resection of DNA. Both PARP1 and PARP2 are required for hydroxyurea-induced homologous recombination to promote cell survival after replication blocks. Together, our data suggest that PARP1 and PARP2 detect disrupted replication forks and attract Mre11 for end processing that is required for subsequent recombination repair and restart of replication forks. |
Ying, S; Myers, K; Bottomley, S; Helleday, T; Bryant, H E BRCA2-dependent homologous recombination is required for repair of Arsenite-induced replication lesions in mammalian cells Journal Article Nucleic Acids Res., 37 (15), pp. 5105–5113, 2009, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2731915PMC2731915] [DOI:hrefhttp://dx.doi.org/10.1093/nar/gkp53810.1093/nar/gkp538] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1955319119553191]). @article{pmid19553191, title = {BRCA2-dependent homologous recombination is required for repair of Arsenite-induced replication lesions in mammalian cells}, author = { S. Ying and K. Myers and S. Bottomley and T. Helleday and H. E. Bryant}, year = {2009}, date = {2009-08-01}, journal = {Nucleic Acids Res.}, volume = {37}, number = {15}, pages = {5105--5113}, abstract = {Arsenic exposure constitutes one of the most widespread environmental carcinogens, and is associated with increased risk of many different types of cancers. Here we report that arsenite (As[III]) can induce both replication-dependent DNA double-strand breaks (DSB) and homologous recombination (HR) at doses as low as 5 microM (0.65 mg/l), which are within the typical doses often found in drinking water in contaminated areas. We show that the production of DSBs is dependent on active replication and is likely to be the result of conversion of a DNA single-strand break (SSB) into a toxic DSB when encountered by a replication fork. We demonstrate that HR is required for the repair of these breaks and show that a functional HR pathway protects against As[III]-induced cytotoxicity. In addition, BRCA2-deficient cells are sensitive to As[III] and we suggest that As[III] could be exploited as a therapy for HR-deficient tumours such as BRCA1 and BRCA2 mutated breast and ovarian cancers.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2731915PMC2731915] [DOI:hrefhttp://dx.doi.org/10.1093/nar/gkp53810.1093/nar/gkp538] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1955319119553191]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Arsenic exposure constitutes one of the most widespread environmental carcinogens, and is associated with increased risk of many different types of cancers. Here we report that arsenite (As[III]) can induce both replication-dependent DNA double-strand breaks (DSB) and homologous recombination (HR) at doses as low as 5 microM (0.65 mg/l), which are within the typical doses often found in drinking water in contaminated areas. We show that the production of DSBs is dependent on active replication and is likely to be the result of conversion of a DNA single-strand break (SSB) into a toxic DSB when encountered by a replication fork. We demonstrate that HR is required for the repair of these breaks and show that a functional HR pathway protects against As[III]-induced cytotoxicity. In addition, BRCA2-deficient cells are sensitive to As[III] and we suggest that As[III] could be exploited as a therapy for HR-deficient tumours such as BRCA1 and BRCA2 mutated breast and ovarian cancers. |
Stoimenov, I; Helleday, T PCNA on the crossroad of cancer Journal Article Biochem. Soc. Trans., 37 (Pt 3), pp. 605–613, 2009, ([DOI:hrefhttp://dx.doi.org/10.1042/BST037060510.1042/BST0370605] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1944225719442257]). @article{pmid19442257, title = {PCNA on the crossroad of cancer}, author = { I. Stoimenov and T. Helleday}, year = {2009}, date = {2009-06-01}, journal = {Biochem. Soc. Trans.}, volume = {37}, number = {Pt 3}, pages = {605--613}, abstract = {Cancer is caused by genetic changes that often arise following failure to accurately replicate the DNA. PCNA (proliferating-cell nuclear antigen) forms a ring around the DNA to facilitate and control DNA replication. Emerging evidence suggests that PCNA is at the very heart of many essential cellular processes, such as DNA replication, repair of DNA damage, chromatin structure maintenance, chromosome segregation and cell-cycle progression. Progression of the DNA replication forks can be blocked by DNA lesions, formed either by endogenous damage or by exogenous agents, for instance anticancer drugs. Cellular response often results in change of PCNA function triggered either by specific post-translational modification of PCNA (i.e. ubiquitylation) or by exchange of its interaction partners. This puts PCNA in a central position in determining the fate of the replication fork. In the present article, we review PCNA modifications and interaction partners, and how those influence the course of events at replication forks, which ultimately determines both tumour progression as well as the outcome of anticancer treatment.}, note = {[DOI:hrefhttp://dx.doi.org/10.1042/BST037060510.1042/BST0370605] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1944225719442257]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Cancer is caused by genetic changes that often arise following failure to accurately replicate the DNA. PCNA (proliferating-cell nuclear antigen) forms a ring around the DNA to facilitate and control DNA replication. Emerging evidence suggests that PCNA is at the very heart of many essential cellular processes, such as DNA replication, repair of DNA damage, chromatin structure maintenance, chromosome segregation and cell-cycle progression. Progression of the DNA replication forks can be blocked by DNA lesions, formed either by endogenous damage or by exogenous agents, for instance anticancer drugs. Cellular response often results in change of PCNA function triggered either by specific post-translational modification of PCNA (i.e. ubiquitylation) or by exchange of its interaction partners. This puts PCNA in a central position in determining the fate of the replication fork. In the present article, we review PCNA modifications and interaction partners, and how those influence the course of events at replication forks, which ultimately determines both tumour progression as well as the outcome of anticancer treatment. |
Lehtio, L; Jemth, A S; Collins, R; Loseva, O; Johansson, A; Markova, N; Hammarstrom, M; Flores, A; Holmberg-Schiavone, L; Weigelt, J; Helleday, T; Schuler, H; Karlberg, T Structural basis for inhibitor specificity in human poly(AĐP-ribose) polymerase-3 Journal Article J. Med. Chem., 52 (9), pp. 3108–3111, 2009, ([DOI:hrefhttp://dx.doi.org/10.1021/jm900052j10.1021/jm900052j] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1935425519354255]). @article{pmid19354255, title = {Structural basis for inhibitor specificity in human poly(AĐP-ribose) polymerase-3}, author = { L. Lehtio and A. S. Jemth and R. Collins and O. Loseva and A. Johansson and N. Markova and M. Hammarstrom and A. Flores and L. Holmberg-Schiavone and J. Weigelt and T. Helleday and H. Schuler and T. Karlberg}, year = {2009}, date = {2009-05-01}, journal = {J. Med. Chem.}, volume = {52}, number = {9}, pages = {3108--3111}, abstract = {Poly(ADP-ribose) polymerases (PARPs) activate DNA repair mechanisms upon stress- and cytotoxin-induced DNA damage, and inhibition of PARP activity is a lead in cancer drug therapy. We present a structural and functional analysis of the PARP domain of human PARP-3 in complex with several inhibitors. Of these, KU0058948 is the strongest inhibitor of PARP-3 activity. The presented crystal structures highlight key features for potent inhibitor binding and suggest routes for creating isoenzyme-specific PARP inhibitors.}, note = {[DOI:hrefhttp://dx.doi.org/10.1021/jm900052j10.1021/jm900052j] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1935425519354255]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Poly(ADP-ribose) polymerases (PARPs) activate DNA repair mechanisms upon stress- and cytotoxin-induced DNA damage, and inhibition of PARP activity is a lead in cancer drug therapy. We present a structural and functional analysis of the PARP domain of human PARP-3 in complex with several inhibitors. Of these, KU0058948 is the strongest inhibitor of PARP-3 activity. The presented crystal structures highlight key features for potent inhibitor binding and suggest routes for creating isoenzyme-specific PARP inhibitors. |
Gottipati, P; Helleday, T Ŧranscription-associated recombination in eukaryotes: link between transcription, replication and recombination Journal Article Mutagenesis, 24 (3), pp. 203–210, 2009, ([DOI:hrefhttp://dx.doi.org/10.1093/mutage/gen07210.1093/mutage/gen072] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1913905819139058]). @article{pmid19139058, title = {Ŧranscription-associated recombination in eukaryotes: link between transcription, replication and recombination}, author = { P. Gottipati and T. Helleday}, year = {2009}, date = {2009-05-01}, journal = {Mutagenesis}, volume = {24}, number = {3}, pages = {203--210}, abstract = {Homologous recombination (HR) is an important DNA repair pathway and is essential for cellular survival. It plays a major role in repairing replication-associated lesions and is functionally connected to replication. Transcription is another cellular process, which has emerged to have a connection with HR. Transcription enhances HR, which is a ubiquitous phenomenon referred to as transcription-associated recombination (TAR). Recent evidence suggests that TAR plays a role in inducing genetic instability, for example in the THO mutants (Tho2, Hpr1, Mft1 and Thp2) in yeast or during the development of the immune system leading to genetic diversity in mammals. On the other hand, evidence also suggests that TAR may play a role in preventing genetic instability in many different ways, one of which is by rescuing replication during transcription. Hence, TAR is a double-edged sword and plays a role in both preventing and inducing genetic instability. In spite of the interesting nature of TAR, the mechanism behind TAR has remained elusive. Recent advances in the area, however, suggest a link between TAR and replication and show specific genetic requirements for TAR that differ from regular HR. In this review, we aim to present the available evidence for TAR in both lower and higher eukaryotes and discuss its possible mechanisms, with emphasis on its connection with replication.}, note = {[DOI:hrefhttp://dx.doi.org/10.1093/mutage/gen07210.1093/mutage/gen072] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1913905819139058]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Homologous recombination (HR) is an important DNA repair pathway and is essential for cellular survival. It plays a major role in repairing replication-associated lesions and is functionally connected to replication. Transcription is another cellular process, which has emerged to have a connection with HR. Transcription enhances HR, which is a ubiquitous phenomenon referred to as transcription-associated recombination (TAR). Recent evidence suggests that TAR plays a role in inducing genetic instability, for example in the THO mutants (Tho2, Hpr1, Mft1 and Thp2) in yeast or during the development of the immune system leading to genetic diversity in mammals. On the other hand, evidence also suggests that TAR may play a role in preventing genetic instability in many different ways, one of which is by rescuing replication during transcription. Hence, TAR is a double-edged sword and plays a role in both preventing and inducing genetic instability. In spite of the interesting nature of TAR, the mechanism behind TAR has remained elusive. Recent advances in the area, however, suggest a link between TAR and replication and show specific genetic requirements for TAR that differ from regular HR. In this review, we aim to present the available evidence for TAR in both lower and higher eukaryotes and discuss its possible mechanisms, with emphasis on its connection with replication. |
Savolainen, L; Helleday, T Ŧranscription-associated recombination is independent of XRCC2 and mechanistically separate from homology-directed ĐNA double-strand break repair Journal Article Nucleic Acids Res., 37 (2), pp. 405–412, 2009, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2632912PMC2632912] [DOI:hrefhttp://dx.doi.org/10.1093/nar/gkn97110.1093/nar/gkn971] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1904307119043071]). @article{pmid19043071, title = {Ŧranscription-associated recombination is independent of XRCC2 and mechanistically separate from homology-directed ĐNA double-strand break repair}, author = { L. Savolainen and T. Helleday}, year = {2009}, date = {2009-02-01}, journal = {Nucleic Acids Res.}, volume = {37}, number = {2}, pages = {405--412}, abstract = {It has previously been shown that transcription greatly enhances recombination in mammalian cells. However, the proteins involved in catalysing this process and the recombination pathways involved in transcription-associated recombination (TAR) are still unknown. It is well established that both the BRCA2 protein and the RAD51 paralog protein XRCC2 are required for homologous recombination. Here, we show that the BRCA2 protein is also required for TAR, while the XRCC2 protein is not involved. Expression of the XRCC2 gene in XRCC2 mutated irs1 cells restores the defect in homologous recombination repair of an I-SceI-induced DNA double-strand break, while TAR is unaffected. Interestingly, the XRCC2-deficient irs1 cells are also proficient in recombination induced at slowed replication forks, suggesting that TAR is mechanistically linked with this recombination pathway. In conclusion, we show that TAR depends on BRCA2 but is independent of XRCC2, and that this recombination pathway is separate from that used to repair a two-ended DNA double-strand break.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2632912PMC2632912] [DOI:hrefhttp://dx.doi.org/10.1093/nar/gkn97110.1093/nar/gkn971] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1904307119043071]}, keywords = {}, pubstate = {published}, tppubtype = {article} } It has previously been shown that transcription greatly enhances recombination in mammalian cells. However, the proteins involved in catalysing this process and the recombination pathways involved in transcription-associated recombination (TAR) are still unknown. It is well established that both the BRCA2 protein and the RAD51 paralog protein XRCC2 are required for homologous recombination. Here, we show that the BRCA2 protein is also required for TAR, while the XRCC2 protein is not involved. Expression of the XRCC2 gene in XRCC2 mutated irs1 cells restores the defect in homologous recombination repair of an I-SceI-induced DNA double-strand break, while TAR is unaffected. Interestingly, the XRCC2-deficient irs1 cells are also proficient in recombination induced at slowed replication forks, suggesting that TAR is mechanistically linked with this recombination pathway. In conclusion, we show that TAR depends on BRCA2 but is independent of XRCC2, and that this recombination pathway is separate from that used to repair a two-ended DNA double-strand break. |
2008 |
Wilsker, D; Petermann, E; Helleday, T; Bunz, F Essential function of Chk1 can be uncoupled from ĐNA damage checkpoint and replication control Journal Article Proc. Natl. Acad. Sci. U.S.A., 105 (52), pp. 20752–20757, 2008, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2634938PMC2634938] [DOI:hrefhttp://dx.doi.org/10.1073/pnas.080691710610.1073/pnas.0806917106] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1909195419091954]). @article{pmid19091954, title = {Essential function of Chk1 can be uncoupled from ĐNA damage checkpoint and replication control}, author = { D. Wilsker and E. Petermann and T. Helleday and F. Bunz}, year = {2008}, date = {2008-12-01}, journal = {Proc. Natl. Acad. Sci. U.S.A.}, volume = {105}, number = {52}, pages = {20752--20757}, abstract = {Chk1 is widely known as a DNA damage checkpoint signaling protein. Unlike many other checkpoint proteins, Chk1 also plays an essential but poorly defined role in the proliferation of unperturbed cells. Activation of Chk1 after DNA damage is known to require the phosphorylation of several C-terminal residues, including the highly conserved S317 and S345 sites. To evaluate the respective roles of these individual sites and assess their contribution to the functions of Chk1, we used a gene targeting approach to introduce point mutations into the endogenous human CHK1 locus. We report that the essential and nonessential functions of Chk1 are regulated through distinct phosphorylation events and can be genetically uncoupled. The DNA damage response function of Chk1 was nonessential. Targeted mutation of S317 abrogated G(2)/M checkpoint activation, prevented subsequent phosphorylation of Chk1, impaired efficient progression of DNA replication forks, and increased fork stalling, but did not impact viability. Thus, the nonessential DNA damage response function of Chk1 could be unambiguously linked to its role in DNA replication control. In contrast, a CHK1 allele with mutated S345 did not support viability, indicating an essential role for this residue during the unperturbed cell cycle. A distinct, physiologic mode of S345 phosphorylation, initiated at the centrosome during unperturbed mitosis was independent of codon 317 status and mechanistically distinct from the ordered and sequential phosphorylation of serine residues on Chk1 induced by DNA damage. Our findings suggest an essential regulatory role for Chk1 phosphorylation during mitotic progression.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2634938PMC2634938] [DOI:hrefhttp://dx.doi.org/10.1073/pnas.080691710610.1073/pnas.0806917106] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1909195419091954]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Chk1 is widely known as a DNA damage checkpoint signaling protein. Unlike many other checkpoint proteins, Chk1 also plays an essential but poorly defined role in the proliferation of unperturbed cells. Activation of Chk1 after DNA damage is known to require the phosphorylation of several C-terminal residues, including the highly conserved S317 and S345 sites. To evaluate the respective roles of these individual sites and assess their contribution to the functions of Chk1, we used a gene targeting approach to introduce point mutations into the endogenous human CHK1 locus. We report that the essential and nonessential functions of Chk1 are regulated through distinct phosphorylation events and can be genetically uncoupled. The DNA damage response function of Chk1 was nonessential. Targeted mutation of S317 abrogated G(2)/M checkpoint activation, prevented subsequent phosphorylation of Chk1, impaired efficient progression of DNA replication forks, and increased fork stalling, but did not impact viability. Thus, the nonessential DNA damage response function of Chk1 could be unambiguously linked to its role in DNA replication control. In contrast, a CHK1 allele with mutated S345 did not support viability, indicating an essential role for this residue during the unperturbed cell cycle. A distinct, physiologic mode of S345 phosphorylation, initiated at the centrosome during unperturbed mitosis was independent of codon 317 status and mechanistically distinct from the ordered and sequential phosphorylation of serine residues on Chk1 induced by DNA damage. Our findings suggest an essential regulatory role for Chk1 phosphorylation during mitotic progression. |
Rodriguez, R; Hansen, L T; Phear, G; Scorah, J; Spang-Thomsen, M; Cox, A; Helleday, T; Meuth, M Ŧhymidine selectively enhances growth suppressive effects of camptothecin/irinotecan in MSI+ cells and tumors containing a mutation of MRE11 Journal Article Clin. Cancer Res., 14 (17), pp. 5476–5483, 2008, ([DOI:hrefhttp://dx.doi.org/10.1158/1078-0432.CCR-08-027410.1158/1078-0432.CCR-08-0274] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1876553918765539]). @article{pmid18765539, title = {Ŧhymidine selectively enhances growth suppressive effects of camptothecin/irinotecan in MSI+ cells and tumors containing a mutation of MRE11}, author = { R. Rodriguez and L. T. Hansen and G. Phear and J. Scorah and M. Spang-Thomsen and A. Cox and T. Helleday and M. Meuth}, year = {2008}, date = {2008-09-01}, journal = {Clin. Cancer Res.}, volume = {14}, number = {17}, pages = {5476--5483}, abstract = {DNA synthesis inhibitors and damaging agents are widely used in cancer therapy; however, sensitivity of tumors to such agents is highly variable. The response of tumor cells in culture to these agents is strongly influenced by the status of DNA damage response pathways. Here, we attempt to exploit the altered response of mismatch repair (MMR)-deficient colon cancer cells and tumors to camptothecin or irinotecan and thymidine by combining them to improve therapeutic response. A panel of colon cancer cell lines was assayed for response to camptothecin-thymidine combinations by measuring colony formation, cell cycle distribution, and senescence. Cell strains defective in p53, p21, or Mre11 were used in these assays to investigate the role of these cell cycle regulators. The in vivo antitumor response of xenografts to irinotecan and thymidine combinations was assessed in nude mice. Camptothecin-thymidine combinations suppress colony formation of MMR-deficient tumor cells 10- to 3,000-fold relative to that obtained with camptothecin alone and significantly reduce the concentrations of the agents required to induce late S/G(2) arrest and senescence. Sensitivity is not a direct result of MMR, p53, or p21 status. However MMR-deficient cell lines containing an intronic frameshift mutation of MRE11 show greatest sensitivity to these agents. Increased sensitivity to this combination is also evident in vivo as thymidine enhances irinotecan-induced growth suppression of MMR-deficient tumors carrying the MRE11 mutation in mouse xenografts. Irinotecan-thymidine combinations may be particularly effective when targeted to MSI+ tumors containing this readily detectable MRE11 mutation.}, note = {[DOI:hrefhttp://dx.doi.org/10.1158/1078-0432.CCR-08-027410.1158/1078-0432.CCR-08-0274] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1876553918765539]}, keywords = {}, pubstate = {published}, tppubtype = {article} } DNA synthesis inhibitors and damaging agents are widely used in cancer therapy; however, sensitivity of tumors to such agents is highly variable. The response of tumor cells in culture to these agents is strongly influenced by the status of DNA damage response pathways. Here, we attempt to exploit the altered response of mismatch repair (MMR)-deficient colon cancer cells and tumors to camptothecin or irinotecan and thymidine by combining them to improve therapeutic response. A panel of colon cancer cell lines was assayed for response to camptothecin-thymidine combinations by measuring colony formation, cell cycle distribution, and senescence. Cell strains defective in p53, p21, or Mre11 were used in these assays to investigate the role of these cell cycle regulators. The in vivo antitumor response of xenografts to irinotecan and thymidine combinations was assessed in nude mice. Camptothecin-thymidine combinations suppress colony formation of MMR-deficient tumor cells 10- to 3,000-fold relative to that obtained with camptothecin alone and significantly reduce the concentrations of the agents required to induce late S/G(2) arrest and senescence. Sensitivity is not a direct result of MMR, p53, or p21 status. However MMR-deficient cell lines containing an intronic frameshift mutation of MRE11 show greatest sensitivity to these agents. Increased sensitivity to this combination is also evident in vivo as thymidine enhances irinotecan-induced growth suppression of MMR-deficient tumors carrying the MRE11 mutation in mouse xenografts. Irinotecan-thymidine combinations may be particularly effective when targeted to MSI+ tumors containing this readily detectable MRE11 mutation. |
Cheng, W H; Muftic, D; Muftuoglu, M; Dawut, L; Morris, C; Helleday, T; Shiloh, Y; Bohr, V A WRN is required for AŦM activation and the S-phase checkpoint in response to interstrand cross-link-induced ĐNA double-strand breaks Journal Article Mol. Biol. Cell, 19 (9), pp. 3923–3933, 2008, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2526706PMC2526706] [DOI:hrefhttp://dx.doi.org/10.1091/mbc.E07-07-069810.1091/mbc.E07-07-0698] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1859623918596239]). @article{pmid18596239, title = {WRN is required for AŦM activation and the S-phase checkpoint in response to interstrand cross-link-induced ĐNA double-strand breaks}, author = { W. H. Cheng and D. Muftic and M. Muftuoglu and L. Dawut and C. Morris and T. Helleday and Y. Shiloh and V. A. Bohr}, year = {2008}, date = {2008-09-01}, journal = {Mol. Biol. Cell}, volume = {19}, number = {9}, pages = {3923--3933}, abstract = {Werner syndrome (WS) is a human genetic disorder characterized by extensive clinical features of premature aging. Ataxia-telengiectasia (A-T) is a multisystem human genomic instability syndrome that includes premature aging in some of the patients. WRN and ATM, the proteins defective in WS and A-T, respectively, play significant roles in the maintenance of genomic stability and are involved in several DNA metabolic pathways. A role for WRN in DNA repair has been proposed; however, this study provides evidence that WRN is also involved in ATM pathway activation and in a S-phase checkpoint in cells exposed to DNA interstrand cross-link-induced double-strand breaks. Depletion of WRN in such cells by RNA interference results in an intra-S checkpoint defect, and interferes with activation of ATM as well as downstream phosphorylation of ATM target proteins. Treatment of cells under replication stress with the ATM kinase inhibitor KU 55933 results in a S-phase checkpoint defect similar to that observed in WRN shRNA cells. Moreover, gamma H2AX levels are higher in WRN shRNA cells than in control cells 6 and 16 h after exposure to psoralen DNA cross-links. These results suggest that WRN and ATM participate in a replication checkpoint response, in which WRN facilitates ATM activation in cells with psoralen DNA cross-link-induced collapsed replication forks.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2526706PMC2526706] [DOI:hrefhttp://dx.doi.org/10.1091/mbc.E07-07-069810.1091/mbc.E07-07-0698] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1859623918596239]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Werner syndrome (WS) is a human genetic disorder characterized by extensive clinical features of premature aging. Ataxia-telengiectasia (A-T) is a multisystem human genomic instability syndrome that includes premature aging in some of the patients. WRN and ATM, the proteins defective in WS and A-T, respectively, play significant roles in the maintenance of genomic stability and are involved in several DNA metabolic pathways. A role for WRN in DNA repair has been proposed; however, this study provides evidence that WRN is also involved in ATM pathway activation and in a S-phase checkpoint in cells exposed to DNA interstrand cross-link-induced double-strand breaks. Depletion of WRN in such cells by RNA interference results in an intra-S checkpoint defect, and interferes with activation of ATM as well as downstream phosphorylation of ATM target proteins. Treatment of cells under replication stress with the ATM kinase inhibitor KU 55933 results in a S-phase checkpoint defect similar to that observed in WRN shRNA cells. Moreover, gamma H2AX levels are higher in WRN shRNA cells than in control cells 6 and 16 h after exposure to psoralen DNA cross-links. These results suggest that WRN and ATM participate in a replication checkpoint response, in which WRN facilitates ATM activation in cells with psoralen DNA cross-link-induced collapsed replication forks. |
Frumerie, C; Sylwan, L; Helleday, T; Yu, A; Haggard-Ljungquist, E Bacteriophage P2 integrase: another possible tool for site-specific recombination in eukaryotic cells Journal Article J. Appl. Microbiol., 105 (1), pp. 290–299, 2008, ([DOI:hrefhttp://dx.doi.org/10.1111/j.1365-2672.2008.03748.x10.1111/j.1365-2672.2008.03748.x] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1828448418284484]). @article{pmid18284484, title = {Bacteriophage P2 integrase: another possible tool for site-specific recombination in eukaryotic cells}, author = { C. Frumerie and L. Sylwan and T. Helleday and A. Yu and E. Haggard-Ljungquist}, year = {2008}, date = {2008-07-01}, journal = {J. Appl. Microbiol.}, volume = {105}, number = {1}, pages = {290--299}, abstract = {To investigate if the site-specific tyrosine integrase (Int) from phage P2 has features that would make it interesting for use of gene transfer into eukaryotic cells. These include the possibility of promoting recombination with a nonphage sequence, abolishing the requirement for the bacterial DNA-binding and -bending protein integration host factor (IHF), and localization to the nucleus of eukaryotic cells. We show that the Int protein catalyzes site-specific recombination using a human sequence in Escherichia coli and in vitro although not as efficiently as with the wild-type bacterial sequence, and that insertion of high mobility group recognition boxes in the phage attachment site substrate abolish the requirement of IHF and allows efficient recombination in vitro in a eukaryotic cell extract. Furthermore, we show by fluorescence that the Int protein contains a functional intrinsic nuclear localization signal, localizing it to the nucleus in both HeLa and 293 cells. We conclude that P2 Int may be a potential tool for site-specific integration of genes into the human chromosome. The study implies the possibility of using multiple prokaryotic Int proteins with different specific integration sites in human cells for future gene therapy programmes.}, note = {[DOI:hrefhttp://dx.doi.org/10.1111/j.1365-2672.2008.03748.x10.1111/j.1365-2672.2008.03748.x] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1828448418284484]}, keywords = {}, pubstate = {published}, tppubtype = {article} } To investigate if the site-specific tyrosine integrase (Int) from phage P2 has features that would make it interesting for use of gene transfer into eukaryotic cells. These include the possibility of promoting recombination with a nonphage sequence, abolishing the requirement for the bacterial DNA-binding and -bending protein integration host factor (IHF), and localization to the nucleus of eukaryotic cells. We show that the Int protein catalyzes site-specific recombination using a human sequence in Escherichia coli and in vitro although not as efficiently as with the wild-type bacterial sequence, and that insertion of high mobility group recognition boxes in the phage attachment site substrate abolish the requirement of IHF and allows efficient recombination in vitro in a eukaryotic cell extract. Furthermore, we show by fluorescence that the Int protein contains a functional intrinsic nuclear localization signal, localizing it to the nucleus in both HeLa and 293 cells. We conclude that P2 Int may be a potential tool for site-specific integration of genes into the human chromosome. The study implies the possibility of using multiple prokaryotic Int proteins with different specific integration sites in human cells for future gene therapy programmes. |
Petermann, E; Helleday, T; Caldecott, K W Claspin promotes normal replication fork rates in human cells Journal Article Mol. Biol. Cell, 19 (6), pp. 2373–2378, 2008, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2397295PMC2397295] [DOI:hrefhttp://dx.doi.org/10.1091/mbc.E07-10-103510.1091/mbc.E07-10-1035] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1835397318353973]). @article{pmid18353973, title = {Claspin promotes normal replication fork rates in human cells}, author = { E. Petermann and T. Helleday and K. W. Caldecott}, year = {2008}, date = {2008-06-01}, journal = {Mol. Biol. Cell}, volume = {19}, number = {6}, pages = {2373--2378}, abstract = {The S phase-specific adaptor protein Claspin mediates the checkpoint response to replication stress by facilitating phosphorylation of Chk1 by ataxia-telangiectasia and Rad3-related (ATR). Evidence suggests that these components of the ATR pathway also play a critical role during physiological S phase. Chk1 is required for high rates of global replication fork progression, and Claspin interacts with the replication machinery and might therefore monitor normal DNA replication. Here, we have used DNA fiber labeling to investigate, for the first time, whether human Claspin is required for high rates of replication fork progression during normal S phase. We report that Claspin-depleted HeLa and HCT116 cells display levels of replication fork slowing similar to those observed in Chk1-depleted cells. This was also true in primary human 1BR3 fibroblasts, albeit to a lesser extent, suggesting that Claspin is a universal requirement for high replication fork rates in human cells. Interestingly, Claspin-depleted cells retained significant levels of Chk1 phosphorylation at both Ser317 and Ser345, raising the possibility that Claspin function during normal fork progression may extend beyond facilitating phosphorylation of either individual residue. Consistent with this possibility, depletion of Chk1 and Claspin together doubled the percentage of very slow forks, compared with depletion of either protein alone.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2397295PMC2397295] [DOI:hrefhttp://dx.doi.org/10.1091/mbc.E07-10-103510.1091/mbc.E07-10-1035] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1835397318353973]}, keywords = {}, pubstate = {published}, tppubtype = {article} } The S phase-specific adaptor protein Claspin mediates the checkpoint response to replication stress by facilitating phosphorylation of Chk1 by ataxia-telangiectasia and Rad3-related (ATR). Evidence suggests that these components of the ATR pathway also play a critical role during physiological S phase. Chk1 is required for high rates of global replication fork progression, and Claspin interacts with the replication machinery and might therefore monitor normal DNA replication. Here, we have used DNA fiber labeling to investigate, for the first time, whether human Claspin is required for high rates of replication fork progression during normal S phase. We report that Claspin-depleted HeLa and HCT116 cells display levels of replication fork slowing similar to those observed in Chk1-depleted cells. This was also true in primary human 1BR3 fibroblasts, albeit to a lesser extent, suggesting that Claspin is a universal requirement for high replication fork rates in human cells. Interestingly, Claspin-depleted cells retained significant levels of Chk1 phosphorylation at both Ser317 and Ser345, raising the possibility that Claspin function during normal fork progression may extend beyond facilitating phosphorylation of either individual residue. Consistent with this possibility, depletion of Chk1 and Claspin together doubled the percentage of very slow forks, compared with depletion of either protein alone. |
Lehtio, L; Collins, R; van den Berg, S; Johansson, A; Dahlgren, L G; Hammarstrom, M; Helleday, T; Holmberg-Schiavone, L; Karlberg, T; Weigelt, J Zinc binding catalytic domain of human tankyrase 1 Journal Article J. Mol. Biol., 379 (1), pp. 136–145, 2008, ([DOI:hrefhttp://dx.doi.org/10.1016/j.jmb.2008.03.05810.1016/j.jmb.2008.03.058] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1843624018436240]). @article{pmid18436240, title = {Zinc binding catalytic domain of human tankyrase 1}, author = { L. Lehtio and R. Collins and S. van den Berg and A. Johansson and L. G. Dahlgren and M. Hammarstrom and T. Helleday and L. Holmberg-Schiavone and T. Karlberg and J. Weigelt}, year = {2008}, date = {2008-05-01}, journal = {J. Mol. Biol.}, volume = {379}, number = {1}, pages = {136--145}, abstract = {Tankyrases are recently discovered proteins implicated in many important functions in the cell including telomere homeostasis and mitosis. Tankyrase modulates the activity of target proteins through poly(ADP-ribosyl)ation, and here we report the structure of the catalytic poly(ADP-ribose) polymerase (PARP) domain of human tankyrase 1. This is the first structure of a PARP domain from the tankyrase subfamily. The present structure reveals that tankyrases contain a short zinc-binding motif, which has not been predicted. Tankyrase activity contributes to telomere elongation observed in various cancer cells and tankyrase inhibition has been suggested as a potential route for cancer therapy. In comparison with other PARPs, significant structural differences are observed in the regions lining the substrate-binding site of tankyrase 1. These findings will be of great value to facilitate structure-based design of selective PARP inhibitors, in general, and tankyrase inhibitors, in particular.}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/j.jmb.2008.03.05810.1016/j.jmb.2008.03.058] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1843624018436240]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Tankyrases are recently discovered proteins implicated in many important functions in the cell including telomere homeostasis and mitosis. Tankyrase modulates the activity of target proteins through poly(ADP-ribosyl)ation, and here we report the structure of the catalytic poly(ADP-ribose) polymerase (PARP) domain of human tankyrase 1. This is the first structure of a PARP domain from the tankyrase subfamily. The present structure reveals that tankyrases contain a short zinc-binding motif, which has not been predicted. Tankyrase activity contributes to telomere elongation observed in various cancer cells and tankyrase inhibition has been suggested as a potential route for cancer therapy. In comparison with other PARPs, significant structural differences are observed in the regions lining the substrate-binding site of tankyrase 1. These findings will be of great value to facilitate structure-based design of selective PARP inhibitors, in general, and tankyrase inhibitors, in particular. |
Helleday, T Amplifying tumour-specific replication lesions by ĐNA repair inhibitors - a new era in targeted cancer therapy Journal Article Eur. J. Cancer, 44 (7), pp. 921–927, 2008, ([DOI:hrefhttp://dx.doi.org/10.1016/j.ejca.2008.02.04410.1016/j.ejca.2008.02.044] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1837456218374562]). @article{pmid18374562, title = {Amplifying tumour-specific replication lesions by ĐNA repair inhibitors - a new era in targeted cancer therapy}, author = { T. Helleday}, year = {2008}, date = {2008-05-01}, journal = {Eur. J. Cancer}, volume = {44}, number = {7}, pages = {921--927}, abstract = {Many anti-cancer drugs used in the clinic today damage DNA, resulting in cell death either directly or following DNA replication. Many anti-cancer drugs are exclusively toxic to replicating cells and toxic lesions are formed when a replication fork encounters a damaged DNA template. Recent work shows that replication lesions, similar to those produced during anti-cancer therapy, are commonly associated with cancer aetiology. DNA replication lesions are present in cancer cells owing to oncogene expression, hypoxia or defects in the DNA damage response or DNA repair. Here, I review how novel therapies can exploit endogenous replication lesions in cancer cells and convert them to toxic lesions. The aim of these therapies is to produce similar lesions to those produced by DNA damaging anti-cancer drugs. The difference is that the lesions will be cancer-specific and produce milder side-effects in non-cancerous cells.}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/j.ejca.2008.02.04410.1016/j.ejca.2008.02.044] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1837456218374562]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Many anti-cancer drugs used in the clinic today damage DNA, resulting in cell death either directly or following DNA replication. Many anti-cancer drugs are exclusively toxic to replicating cells and toxic lesions are formed when a replication fork encounters a damaged DNA template. Recent work shows that replication lesions, similar to those produced during anti-cancer therapy, are commonly associated with cancer aetiology. DNA replication lesions are present in cancer cells owing to oncogene expression, hypoxia or defects in the DNA damage response or DNA repair. Here, I review how novel therapies can exploit endogenous replication lesions in cancer cells and convert them to toxic lesions. The aim of these therapies is to produce similar lesions to those produced by DNA damaging anti-cancer drugs. The difference is that the lesions will be cancer-specific and produce milder side-effects in non-cancerous cells. |
Helleday, T; Petermann, E; Lundin, C; Hodgson, B; Sharma, R A ĐNA repair pathways as targets for cancer therapy Journal Article Nat. Rev. Cancer, 8 (3), pp. 193–204, 2008, ([DOI:hrefhttp://dx.doi.org/10.1038/nrc234210.1038/nrc2342] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1825661618256616]). @article{pmid18256616, title = {ĐNA repair pathways as targets for cancer therapy}, author = { T. Helleday and E. Petermann and C. Lundin and B. Hodgson and R. A. Sharma}, year = {2008}, date = {2008-03-01}, journal = {Nat. Rev. Cancer}, volume = {8}, number = {3}, pages = {193--204}, abstract = {DNA repair pathways can enable tumour cells to survive DNA damage that is induced by chemotherapeutic treatments; therefore, inhibitors of specific DNA repair pathways might prove efficacious when used in combination with DNA-damaging chemotherapeutic drugs. In addition, alterations in DNA repair pathways that arise during tumour development can make some cancer cells reliant on a reduced set of DNA repair pathways for survival. There is evidence that drugs that inhibit one of these pathways in such tumours could prove useful as single-agent therapies, with the potential advantage that this approach could be selective for tumour cells and have fewer side effects.}, note = {[DOI:hrefhttp://dx.doi.org/10.1038/nrc234210.1038/nrc2342] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1825661618256616]}, keywords = {}, pubstate = {published}, tppubtype = {article} } DNA repair pathways can enable tumour cells to survive DNA damage that is induced by chemotherapeutic treatments; therefore, inhibitors of specific DNA repair pathways might prove efficacious when used in combination with DNA-damaging chemotherapeutic drugs. In addition, alterations in DNA repair pathways that arise during tumour development can make some cancer cells reliant on a reduced set of DNA repair pathways for survival. There is evidence that drugs that inhibit one of these pathways in such tumours could prove useful as single-agent therapies, with the potential advantage that this approach could be selective for tumour cells and have fewer side effects. |
Gottipati, P; Cassel, T N; Savolainen, L; Helleday, T Ŧranscription-associated recombination is dependent on replication in Mammalian cells Journal Article Mol. Cell. Biol., 28 (1), pp. 154–164, 2008, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2223284PMC2223284] [DOI:hrefhttp://dx.doi.org/10.1128/MCB.00816-0710.1128/MCB.00816-07] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1796787717967877]). @article{pmid17967877, title = {Ŧranscription-associated recombination is dependent on replication in Mammalian cells}, author = { P. Gottipati and T. N. Cassel and L. Savolainen and T. Helleday}, year = {2008}, date = {2008-01-01}, journal = {Mol. Cell. Biol.}, volume = {28}, number = {1}, pages = {154--164}, abstract = {Transcription can enhance recombination; this is a ubiquitous phenomenon from prokaryotes to higher eukaryotes. However, the mechanism of transcription-associated recombination in mammalian cells is poorly understood. Here we have developed a construct with a recombination substrate in which levels of recombination can be studied in the presence or absence of transcription. We observed a direct enhancement in recombination when transcription levels through the substrate were increased. This increase in homologous recombination following transcription is locus specific, since homologous recombination at the unrelated hprt gene is unaffected. In addition, we have shown that transcription-associated recombination involves both short-tract and long-tract gene conversions in mammalian cells, which are different from double-strand-break-induced recombination events caused by endonucleases. Transcription fails to enhance recombination in cells that are not in the S phase of the cell cycle. Furthermore, inhibition of transcription suppresses induction of recombination at stalled replication forks, suggesting that recombination may be involved in bypassing transcription during replication.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2223284PMC2223284] [DOI:hrefhttp://dx.doi.org/10.1128/MCB.00816-0710.1128/MCB.00816-07] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1796787717967877]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Transcription can enhance recombination; this is a ubiquitous phenomenon from prokaryotes to higher eukaryotes. However, the mechanism of transcription-associated recombination in mammalian cells is poorly understood. Here we have developed a construct with a recombination substrate in which levels of recombination can be studied in the presence or absence of transcription. We observed a direct enhancement in recombination when transcription levels through the substrate were increased. This increase in homologous recombination following transcription is locus specific, since homologous recombination at the unrelated hprt gene is unaffected. In addition, we have shown that transcription-associated recombination involves both short-tract and long-tract gene conversions in mammalian cells, which are different from double-strand-break-induced recombination events caused by endonucleases. Transcription fails to enhance recombination in cells that are not in the S phase of the cell cycle. Furthermore, inhibition of transcription suppresses induction of recombination at stalled replication forks, suggesting that recombination may be involved in bypassing transcription during replication. |
Al-Minawi, A Z; Saleh-Gohari, N; Helleday, T Ŧhe ERCC1/XPF endonuclease is required for efficient single-strand annealing and gene conversion in mammalian cells Journal Article Nucleic Acids Res., 36 (1), pp. 1–9, 2008, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2248766PMC2248766] [DOI:hrefhttp://dx.doi.org/10.1093/nar/gkm88810.1093/nar/gkm888] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1796230117962301]). @article{pmid17962301, title = {Ŧhe ERCC1/XPF endonuclease is required for efficient single-strand annealing and gene conversion in mammalian cells}, author = { A. Z. Al-Minawi and N. Saleh-Gohari and T. Helleday}, year = {2008}, date = {2008-01-01}, journal = {Nucleic Acids Res.}, volume = {36}, number = {1}, pages = {1--9}, abstract = {The mammalian ERCC1-XPF endonuclease has a suggested role in the repair of DNA double-strand breaks (DSB) by single-strand annealing (SSA). Here, we investigated the role of ERCC1 in homologous recombination in mammalian cells, and confirm a role of ERCC1 in SSA. Interestingly, we also report an unexpected role for ERCC1 in gene conversion. This provides support that gene conversion in mammalian somatic cells is carried out through synthesis-dependent strand annealing, rather than through a double Holliday Junction mechanism. Moreover, we find low frequencies of SSA and gene conversion in G1-arrested cells, suggesting that SSA is not a frequent DSB repair pathway in G1-arrested mammalian cells, even in the presence of perfect repeats. Furthermore, we find that SSA is not influenced by inhibition of CDK2 (using Roscovitine), ATM (using Caffeine and KU55933), Chk1 (using CEP-3891) or DNA-PK (using NU7026).}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2248766PMC2248766] [DOI:hrefhttp://dx.doi.org/10.1093/nar/gkm88810.1093/nar/gkm888] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1796230117962301]}, keywords = {}, pubstate = {published}, tppubtype = {article} } The mammalian ERCC1-XPF endonuclease has a suggested role in the repair of DNA double-strand breaks (DSB) by single-strand annealing (SSA). Here, we investigated the role of ERCC1 in homologous recombination in mammalian cells, and confirm a role of ERCC1 in SSA. Interestingly, we also report an unexpected role for ERCC1 in gene conversion. This provides support that gene conversion in mammalian somatic cells is carried out through synthesis-dependent strand annealing, rather than through a double Holliday Junction mechanism. Moreover, we find low frequencies of SSA and gene conversion in G1-arrested cells, suggesting that SSA is not a frequent DSB repair pathway in G1-arrested mammalian cells, even in the presence of perfect repeats. Furthermore, we find that SSA is not influenced by inhibition of CDK2 (using Roscovitine), ATM (using Caffeine and KU55933), Chk1 (using CEP-3891) or DNA-PK (using NU7026). |
2007 |
J?rgensen, S; Elvers, I; Trelle, M B; Menzel, T; Eskildsen, M; Jensen, O N; Helleday, T; Helin, K; S?rensen, C S Ŧhe histone methyltransferase SEŦ8 is required for S-phase progression Journal Article J. Cell Biol., 179 (7), pp. 1337–1345, 2007, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2373509PMC2373509] [DOI:hrefhttp://dx.doi.org/10.1083/jcb.20070615010.1083/jcb.200706150] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1816664818166648]). @article{pmid18166648, title = {Ŧhe histone methyltransferase SEŦ8 is required for S-phase progression}, author = { S. J?rgensen and I. Elvers and M. B. Trelle and T. Menzel and M. Eskildsen and O. N. Jensen and T. Helleday and K. Helin and C. S. S?rensen}, year = {2007}, date = {2007-12-01}, journal = {J. Cell Biol.}, volume = {179}, number = {7}, pages = {1337--1345}, abstract = {Chromatin structure and function is influenced by histone posttranslational modifications. SET8 (also known as PR-Set7 and SETD8) is a histone methyltransferase that monomethylates histonfe H4-K20. However, a function for SET8 in mammalian cell proliferation has not been determined. We show that small interfering RNA inhibition of SET8 expression leads to decreased cell proliferation and accumulation of cells in S phase. This is accompanied by DNA double-strand break (DSB) induction and recruitment of the DNA repair proteins replication protein A, Rad51, and 53BP1 to damaged regions. SET8 depletion causes DNA damage specifically during replication, which induces a Chk1-mediated S-phase checkpoint. Furthermore, we find that SET8 interacts with proliferating cell nuclear antigen through a conserved motif, and SET8 is required for DNA replication fork progression. Finally, codepletion of Rad51, an important homologous recombination repair protein, abrogates the DNA damage after SET8 depletion. Overall, we show that SET8 is essential for genomic stability in mammalian cells and that decreased expression of SET8 results in DNA damage and Chk1-dependent S-phase arrest.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC2373509PMC2373509] [DOI:hrefhttp://dx.doi.org/10.1083/jcb.20070615010.1083/jcb.200706150] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1816664818166648]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Chromatin structure and function is influenced by histone posttranslational modifications. SET8 (also known as PR-Set7 and SETD8) is a histone methyltransferase that monomethylates histonfe H4-K20. However, a function for SET8 in mammalian cell proliferation has not been determined. We show that small interfering RNA inhibition of SET8 expression leads to decreased cell proliferation and accumulation of cells in S phase. This is accompanied by DNA double-strand break (DSB) induction and recruitment of the DNA repair proteins replication protein A, Rad51, and 53BP1 to damaged regions. SET8 depletion causes DNA damage specifically during replication, which induces a Chk1-mediated S-phase checkpoint. Furthermore, we find that SET8 interacts with proliferating cell nuclear antigen through a conserved motif, and SET8 is required for DNA replication fork progression. Finally, codepletion of Rad51, an important homologous recombination repair protein, abrogates the DNA damage after SET8 depletion. Overall, we show that SET8 is essential for genomic stability in mammalian cells and that decreased expression of SET8 results in DNA damage and Chk1-dependent S-phase arrest. |
Petermann, E; Helleday, T ĐNA replication-associated lesions: importance in early tumorigenesis and cancer therapy Journal Article Biochem. Soc. Trans., 35 (Pt 5), pp. 1352–1354, 2007, ([DOI:hrefhttp://dx.doi.org/10.1042/BST035135210.1042/BST0351352] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1795634917956349]). @article{pmid17956349, title = {ĐNA replication-associated lesions: importance in early tumorigenesis and cancer therapy}, author = { E. Petermann and T. Helleday}, year = {2007}, date = {2007-11-01}, journal = {Biochem. Soc. Trans.}, volume = {35}, number = {Pt 5}, pages = {1352--1354}, abstract = {DNA lesions resulting from impaired progression of replication forks are implicated in genetic instability and tumorigenesis. Because the cellular response to these lesions poses an important tumorigenesis barrier, the responsible signalling and repair pathways are often mutated or inactive in tumours. Here, we discuss how such deficiencies can in turn be exploited for cancer therapy.}, note = {[DOI:hrefhttp://dx.doi.org/10.1042/BST035135210.1042/BST0351352] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1795634917956349]}, keywords = {}, pubstate = {published}, tppubtype = {article} } DNA lesions resulting from impaired progression of replication forks are implicated in genetic instability and tumorigenesis. Because the cellular response to these lesions poses an important tumorigenesis barrier, the responsible signalling and repair pathways are often mutated or inactive in tumours. Here, we discuss how such deficiencies can in turn be exploited for cancer therapy. |
Sleeth, K M; S?rensen, C S; Issaeva, N; Dziegielewski, J; Bartek, J; Helleday, T RPA mediates recombination repair during replication stress and is displaced from ĐNA by checkpoint signalling in human cells Journal Article J. Mol. Biol., 373 (1), pp. 38–47, 2007, ([DOI:hrefhttp://dx.doi.org/10.1016/j.jmb.2007.07.06810.1016/j.jmb.2007.07.068] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1776592317765923]). @article{pmid17765923, title = {RPA mediates recombination repair during replication stress and is displaced from ĐNA by checkpoint signalling in human cells}, author = { K. M. Sleeth and C. S. S?rensen and N. Issaeva and J. Dziegielewski and J. Bartek and T. Helleday}, year = {2007}, date = {2007-10-01}, journal = {J. Mol. Biol.}, volume = {373}, number = {1}, pages = {38--47}, abstract = {The replication protein A (RPA) is involved in most, if not all, nuclear metabolism involving single-stranded DNA. Here, we show that RPA is involved in genome maintenance at stalled replication forks by the homologous recombination repair system in humans. Depletion of the RPA protein inhibited the formation of RAD51 nuclear foci after hydroxyurea-induced replication stalling leading to persistent unrepaired DNA double-strand breaks (DSBs). We demonstrate a direct role of RPA in homology directed recombination repair. We find that RPA is dispensable for checkpoint kinase 1 (Chk1) activation and that RPA directly binds RAD52 upon replication stress, suggesting a direct role in recombination repair. In addition we show that inhibition of Chk1 with UCN-01 decreases dissociation of RPA from the chromatin and inhibits association of RAD51 and RAD52 with DNA. Altogether, our data suggest a direct role of RPA in homologous recombination in assembly of the RAD51 and RAD52 proteins. Furthermore, our data suggest that replacement of RPA with the RAD51 and RAD52 proteins is affected by checkpoint signalling.}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/j.jmb.2007.07.06810.1016/j.jmb.2007.07.068] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1776592317765923]}, keywords = {}, pubstate = {published}, tppubtype = {article} } The replication protein A (RPA) is involved in most, if not all, nuclear metabolism involving single-stranded DNA. Here, we show that RPA is involved in genome maintenance at stalled replication forks by the homologous recombination repair system in humans. Depletion of the RPA protein inhibited the formation of RAD51 nuclear foci after hydroxyurea-induced replication stalling leading to persistent unrepaired DNA double-strand breaks (DSBs). We demonstrate a direct role of RPA in homology directed recombination repair. We find that RPA is dispensable for checkpoint kinase 1 (Chk1) activation and that RPA directly binds RAD52 upon replication stress, suggesting a direct role in recombination repair. In addition we show that inhibition of Chk1 with UCN-01 decreases dissociation of RPA from the chromatin and inhibits association of RAD51 and RAD52 with DNA. Altogether, our data suggest a direct role of RPA in homologous recombination in assembly of the RAD51 and RAD52 proteins. Furthermore, our data suggest that replacement of RPA with the RAD51 and RAD52 proteins is affected by checkpoint signalling. |
Helleday, T; Lo, J; van Gent, D C; Engelward, B P ĐNA double-strand break repair: from mechanistic understanding to cancer treatment Journal Article DNA Repair (Amst.), 6 (7), pp. 923–935, 2007, ([DOI:hrefhttp://dx.doi.org/10.1016/j.dnarep.2007.02.00610.1016/j.dnarep.2007.02.006] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1736334317363343]). @article{pmid17363343, title = {ĐNA double-strand break repair: from mechanistic understanding to cancer treatment}, author = { T. Helleday and J. Lo and D. C. van Gent and B. P. Engelward}, year = {2007}, date = {2007-07-01}, journal = {DNA Repair (Amst.)}, volume = {6}, number = {7}, pages = {923--935}, abstract = {Accurate repair of DNA double-strand breaks is essential to life. Indeed, defective DNA double-strand break repair can lead to toxicity and large scale sequence rearrangements that cause cancer and promote premature aging. Here, we highlight the two major repair systems for handling DNA double-strand breaks: homologous recombination and non-homologous end joining. To clarify recombination mechanisms, we present animations that illustrate DNA strand movements. In addition to describing how these pathways operate, we also describe why appropriate pathway choice is critical to genomic stability, and we summarize key pathway control features related to cell cycle checkpoint and apoptosis signaling. Importantly, recent progress in delineating the effects of specific defects in repair and checkpoint control has helped to explain several disease phenotypes, including cancer and premature aging. Improved understanding of these pathways has also sparked development of novel chemotherapeutic strategies that kill tumors with increased specificity and efficacy. This review aims to provide a foundational understanding of how the homologous recombination and non-homologous end joining pathways operate, and to demonstrate how a better understanding of these processes has advanced both our understanding of the underlying causes of cancer and our ability to innovate novel cancer treatment strategies.}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/j.dnarep.2007.02.00610.1016/j.dnarep.2007.02.006] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1736334317363343]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Accurate repair of DNA double-strand breaks is essential to life. Indeed, defective DNA double-strand break repair can lead to toxicity and large scale sequence rearrangements that cause cancer and promote premature aging. Here, we highlight the two major repair systems for handling DNA double-strand breaks: homologous recombination and non-homologous end joining. To clarify recombination mechanisms, we present animations that illustrate DNA strand movements. In addition to describing how these pathways operate, we also describe why appropriate pathway choice is critical to genomic stability, and we summarize key pathway control features related to cell cycle checkpoint and apoptosis signaling. Importantly, recent progress in delineating the effects of specific defects in repair and checkpoint control has helped to explain several disease phenotypes, including cancer and premature aging. Improved understanding of these pathways has also sparked development of novel chemotherapeutic strategies that kill tumors with increased specificity and efficacy. This review aims to provide a foundational understanding of how the homologous recombination and non-homologous end joining pathways operate, and to demonstrate how a better understanding of these processes has advanced both our understanding of the underlying causes of cancer and our ability to innovate novel cancer treatment strategies. |
Saberi, A; Hochegger, H; Szuts, D; Lan, L; Yasui, A; Sale, J E; Taniguchi, Y; Murakawa, Y; Zeng, W; Yokomori, K; Helleday, T; Teraoka, H; Arakawa, H; Buerstedde, J M; Takeda, S RAĐ18 and poly(AĐP-ribose) polymerase independently suppress the access of nonhomologous end joining to double-strand breaks and facilitate homologous recombination-mediated repair Journal Article Mol. Cell. Biol., 27 (7), pp. 2562–2571, 2007, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC1899888PMC1899888] [DOI:hrefhttp://dx.doi.org/10.1128/MCB.01243-0610.1128/MCB.01243-06] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1724220017242200]). @article{pmid17242200, title = {RAĐ18 and poly(AĐP-ribose) polymerase independently suppress the access of nonhomologous end joining to double-strand breaks and facilitate homologous recombination-mediated repair}, author = { A. Saberi and H. Hochegger and D. Szuts and L. Lan and A. Yasui and J. E. Sale and Y. Taniguchi and Y. Murakawa and W. Zeng and K. Yokomori and T. Helleday and H. Teraoka and H. Arakawa and J. M. Buerstedde and S. Takeda}, year = {2007}, date = {2007-04-01}, journal = {Mol. Cell. Biol.}, volume = {27}, number = {7}, pages = {2562--2571}, abstract = {The Saccharomyces cerevisiae RAD18 gene is essential for postreplication repair but is not required for homologous recombination (HR), which is the major double-strand break (DSB) repair pathway in yeast. Accordingly, yeast rad18 mutants are tolerant of camptothecin (CPT), a topoisomerase I inhibitor, which induces DSBs by blocking replication. Surprisingly, mammalian cells and chicken DT40 cells deficient in Rad18 display reduced HR-dependent repair and are hypersensitive to CPT. Deletion of nonhomologous end joining (NHEJ), a major DSB repair pathway in vertebrates, in rad18-deficient DT40 cells completely restored HR-mediated DSB repair, suggesting that vertebrate Rad18 regulates the balance between NHEJ and HR. We previously reported that loss of NHEJ normalized the CPT sensitivity of cells deficient in poly(ADP-ribose) polymerase 1 (PARP1). Concomitant deletion of Rad18 and PARP1 synergistically increased CPT sensitivity, and additional inactivation of NHEJ normalized this hypersensitivity, indicating their parallel actions. In conclusion, higher-eukaryotic cells separately employ PARP1 and Rad18 to suppress the toxic effects of NHEJ during the HR reaction at stalled replication forks.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC1899888PMC1899888] [DOI:hrefhttp://dx.doi.org/10.1128/MCB.01243-0610.1128/MCB.01243-06] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1724220017242200]}, keywords = {}, pubstate = {published}, tppubtype = {article} } The Saccharomyces cerevisiae RAD18 gene is essential for postreplication repair but is not required for homologous recombination (HR), which is the major double-strand break (DSB) repair pathway in yeast. Accordingly, yeast rad18 mutants are tolerant of camptothecin (CPT), a topoisomerase I inhibitor, which induces DSBs by blocking replication. Surprisingly, mammalian cells and chicken DT40 cells deficient in Rad18 display reduced HR-dependent repair and are hypersensitive to CPT. Deletion of nonhomologous end joining (NHEJ), a major DSB repair pathway in vertebrates, in rad18-deficient DT40 cells completely restored HR-mediated DSB repair, suggesting that vertebrate Rad18 regulates the balance between NHEJ and HR. We previously reported that loss of NHEJ normalized the CPT sensitivity of cells deficient in poly(ADP-ribose) polymerase 1 (PARP1). Concomitant deletion of Rad18 and PARP1 synergistically increased CPT sensitivity, and additional inactivation of NHEJ normalized this hypersensitivity, indicating their parallel actions. In conclusion, higher-eukaryotic cells separately employ PARP1 and Rad18 to suppress the toxic effects of NHEJ during the HR reaction at stalled replication forks. |
Lindh, Renglin A; Schultz, N; Saleh-Gohari, N; Helleday, T RAĐ51C (RAĐ51L2) is involved in maintaining centrosome number in mitosis Journal Article Cytogenet. Genome Res., 116 (1-2), pp. 38–45, 2007, ([DOI:hrefhttp://dx.doi.org/10.1159/00009741610.1159/000097416] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1726817617268176]). @article{pmid17268176, title = {RAĐ51C (RAĐ51L2) is involved in maintaining centrosome number in mitosis}, author = { A. Renglin Lindh and N. Schultz and N. Saleh-Gohari and T. Helleday}, year = {2007}, date = {2007-01-01}, journal = {Cytogenet. Genome Res.}, volume = {116}, number = {1-2}, pages = {38--45}, abstract = {The RAD51C (RAD51L2) protein is one out of five RAD51 paralogs and forms a complex that includes either XRCC2 or XRCC3. Both of these complexes may have important functions in homologous recombination (HR). Here, we confirm that the frequency of DNA double-strand break (DSB)-induced HR is reduced in the RAD51C deficient cell line CL-V4B, in agreement with a role for RAD51C in HR. We report that mitotic RAD51C deficient CL-V4B cells also have an increased number of centrosomes in mitosis resulting in aberrant mitotic spindles. These data suggest that the RAD51C protein is important in maintaining correct centrosome numbers and that the complexes including RAD51C and XRCC2 or XRCC3 may be of importance in maintaining correct centrosome numbers in mitosis. Increased centrosome numbers following a RAD51C defect indicates that this protein might be important in preventing aneuploidy, suggesting that it could be a potential tumour suppressor in mammals.}, note = {[DOI:hrefhttp://dx.doi.org/10.1159/00009741610.1159/000097416] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1726817617268176]}, keywords = {}, pubstate = {published}, tppubtype = {article} } The RAD51C (RAD51L2) protein is one out of five RAD51 paralogs and forms a complex that includes either XRCC2 or XRCC3. Both of these complexes may have important functions in homologous recombination (HR). Here, we confirm that the frequency of DNA double-strand break (DSB)-induced HR is reduced in the RAD51C deficient cell line CL-V4B, in agreement with a role for RAD51C in HR. We report that mitotic RAD51C deficient CL-V4B cells also have an increased number of centrosomes in mitosis resulting in aberrant mitotic spindles. These data suggest that the RAD51C protein is important in maintaining correct centrosome numbers and that the complexes including RAD51C and XRCC2 or XRCC3 may be of importance in maintaining correct centrosome numbers in mitosis. Increased centrosome numbers following a RAD51C defect indicates that this protein might be important in preventing aneuploidy, suggesting that it could be a potential tumour suppressor in mammals. |
2006 |
Johansson, F; Lagerqvist, A; Filippi, S; Palitti, F; Erixon, K; Helleday, T; Jenssen, D Caffeine delays replication fork progression and enhances UV-induced homologous recombination in Chinese hamster cell lines Journal Article DNA Repair (Amst.), 5 (12), pp. 1449–1458, 2006, ([DOI:hrefhttp://dx.doi.org/10.1016/j.dnarep.2006.07.00510.1016/j.dnarep.2006.07.005] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1696867716968677]). @article{pmid16968677, title = {Caffeine delays replication fork progression and enhances UV-induced homologous recombination in Chinese hamster cell lines}, author = { F. Johansson and A. Lagerqvist and S. Filippi and F. Palitti and K. Erixon and T. Helleday and D. Jenssen}, year = {2006}, date = {2006-12-01}, journal = {DNA Repair (Amst.)}, volume = {5}, number = {12}, pages = {1449--1458}, abstract = {The ability to bypass DNA lesions encountered during replication is important in order to maintain cell viability and avoid genomic instability. Exposure of mammalian cells to UV-irradiation induces the formation of DNA lesions that stall replication forks. In order to restore replication, different bypass mechanisms are operating, previously named post-replication repair. Translesion DNA synthesis is performed by low-fidelity polymerases, which can replicate across damaged sites. The nature of lesions and of polymerases involved influences the resulting frequency of mutations. Homologous recombination represents an alternative pathway for the rescue of stalled replication forks. Caffeine has long been recognized to influence post-replication repair, although the mechanism is not identified. Here, we found that caffeine delays the progress of replication forks in UV-irradiated Chinese hamster cells. The length of this enhanced delay was similar in wild-type cells and in cell deficient in either homologous recombination or nucleotide excision repair. Furthermore, caffeine attenuated the frequency of UV-induced mutations in the hprt gene, whereas the frequency of recombination, monitored in this same gene, was enhanced. These observations indicate that in cells exposed to UV-light, caffeine inhibits the rescue of stalled replication forks by translesion DNA synthesis, thereby causing a switch to bypass via homologous recombination. The biological consequence of the former pathway is mutations, while the latter results in chromosomal aberrations.}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/j.dnarep.2006.07.00510.1016/j.dnarep.2006.07.005] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1696867716968677]}, keywords = {}, pubstate = {published}, tppubtype = {article} } The ability to bypass DNA lesions encountered during replication is important in order to maintain cell viability and avoid genomic instability. Exposure of mammalian cells to UV-irradiation induces the formation of DNA lesions that stall replication forks. In order to restore replication, different bypass mechanisms are operating, previously named post-replication repair. Translesion DNA synthesis is performed by low-fidelity polymerases, which can replicate across damaged sites. The nature of lesions and of polymerases involved influences the resulting frequency of mutations. Homologous recombination represents an alternative pathway for the rescue of stalled replication forks. Caffeine has long been recognized to influence post-replication repair, although the mechanism is not identified. Here, we found that caffeine delays the progress of replication forks in UV-irradiated Chinese hamster cells. The length of this enhanced delay was similar in wild-type cells and in cell deficient in either homologous recombination or nucleotide excision repair. Furthermore, caffeine attenuated the frequency of UV-induced mutations in the hprt gene, whereas the frequency of recombination, monitored in this same gene, was enhanced. These observations indicate that in cells exposed to UV-light, caffeine inhibits the rescue of stalled replication forks by translesion DNA synthesis, thereby causing a switch to bypass via homologous recombination. The biological consequence of the former pathway is mutations, while the latter results in chromosomal aberrations. |
Bartkova, J; Rezaei, N; Liontos, M; Karakaidos, P; Kletsas, D; Issaeva, N; Vassiliou, L V; Kolettas, E; Niforou, K; Zoumpourlis, V C; Takaoka, M; Nakagawa, H; Tort, F; Fugger, K; Johansson, F; Sehested, M; Andersen, C L; Dyrskjot, L; ?rntoft, T; Lukas, J; Kittas, C; Helleday, T; Halazonetis, T D; Bartek, J; Gorgoulis, V G Oncogene-induced senescence is part of the tumorigenesis barrier imposed by ĐNA damage checkpoints Journal Article Nature, 444 (7119), pp. 633–637, 2006, ([DOI:hrefhttp://dx.doi.org/10.1038/nature0526810.1038/nature05268] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1713609317136093]). @article{pmid17136093, title = {Oncogene-induced senescence is part of the tumorigenesis barrier imposed by ĐNA damage checkpoints}, author = { J. Bartkova and N. Rezaei and M. Liontos and P. Karakaidos and D. Kletsas and N. Issaeva and L. V. Vassiliou and E. Kolettas and K. Niforou and V. C. Zoumpourlis and M. Takaoka and H. Nakagawa and F. Tort and K. Fugger and F. Johansson and M. Sehested and C. L. Andersen and L. Dyrskjot and T. ?rntoft and J. Lukas and C. Kittas and T. Helleday and T. D. Halazonetis and J. Bartek and V. G. Gorgoulis}, year = {2006}, date = {2006-11-01}, journal = {Nature}, volume = {444}, number = {7119}, pages = {633--637}, abstract = {Recent studies have indicated the existence of tumorigenesis barriers that slow or inhibit the progression of preneoplastic lesions to neoplasia. One such barrier involves DNA replication stress, which leads to activation of the DNA damage checkpoint and thereby to apoptosis or cell cycle arrest, whereas a second barrier is mediated by oncogene-induced senescence. The relationship between these two barriers, if any, has not been elucidated. Here we show that oncogene-induced senescence is associated with signs of DNA replication stress, including prematurely terminated DNA replication forks and DNA double-strand breaks. Inhibiting the DNA double-strand break response kinase ataxia telangiectasia mutated (ATM) suppressed the induction of senescence and in a mouse model led to increased tumour size and invasiveness. Analysis of human precancerous lesions further indicated that DNA damage and senescence markers cosegregate closely. Thus, senescence in human preneoplastic lesions is a manifestation of oncogene-induced DNA replication stress and, together with apoptosis, provides a barrier to malignant progression.}, note = {[DOI:hrefhttp://dx.doi.org/10.1038/nature0526810.1038/nature05268] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1713609317136093]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Recent studies have indicated the existence of tumorigenesis barriers that slow or inhibit the progression of preneoplastic lesions to neoplasia. One such barrier involves DNA replication stress, which leads to activation of the DNA damage checkpoint and thereby to apoptosis or cell cycle arrest, whereas a second barrier is mediated by oncogene-induced senescence. The relationship between these two barriers, if any, has not been elucidated. Here we show that oncogene-induced senescence is associated with signs of DNA replication stress, including prematurely terminated DNA replication forks and DNA double-strand breaks. Inhibiting the DNA double-strand break response kinase ataxia telangiectasia mutated (ATM) suppressed the induction of senescence and in a mouse model led to increased tumour size and invasiveness. Analysis of human precancerous lesions further indicated that DNA damage and senescence markers cosegregate closely. Thus, senescence in human preneoplastic lesions is a manifestation of oncogene-induced DNA replication stress and, together with apoptosis, provides a barrier to malignant progression. |
Bryant, H E; Ying, S; Helleday, T Ħomologous recombination is involved in repair of chromium-induced ĐNA damage in mammalian cells Journal Article Mutat. Res., 599 (1-2), pp. 116–123, 2006, ([DOI:hrefhttp://dx.doi.org/10.1016/j.mrfmmm.2006.02.00110.1016/j.mrfmmm.2006.02.001] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1656405916564059]). @article{pmid16564059, title = {Ħomologous recombination is involved in repair of chromium-induced ĐNA damage in mammalian cells}, author = { H. E. Bryant and S. Ying and T. Helleday}, year = {2006}, date = {2006-07-01}, journal = {Mutat. Res.}, volume = {599}, number = {1-2}, pages = {116--123}, abstract = {Chromium is a potent human carcinogen, probably because of its well-documented genotoxic effects. Chromate (Cr[VI]) causes a wide range of DNA lesions, including DNA crosslinks and strand breaks, presumably due to the direct and indirect effects of DNA oxidation. Homologous recombination repair (HRR) is important for error-free repair of lesions occurring at replication forks. Here, we show that HR deficient cell lines irs1SF and V-C8, deficient in XRCC3 and BRCA2, respectively, are hypersensitive to Cr[VI], implicating this repair pathway in repair of Cr[VI] damage. Furthermore, we find that Cr[VI] causes DNA double-strand breaks and triggers both Rad51 foci formation and induction of HRR. Collectively, these data suggest that HRR is important in repair of Cr[VI]-induced DNA damage. In addition, we find that ERCC1, XRCC1 and DNA-PKcs defective cells are hypersensitive to Cr[VI], indicating that several repair pathways cooperate in repairing Cr[VI]-induced DNA damage.}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/j.mrfmmm.2006.02.00110.1016/j.mrfmmm.2006.02.001] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1656405916564059]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Chromium is a potent human carcinogen, probably because of its well-documented genotoxic effects. Chromate (Cr[VI]) causes a wide range of DNA lesions, including DNA crosslinks and strand breaks, presumably due to the direct and indirect effects of DNA oxidation. Homologous recombination repair (HRR) is important for error-free repair of lesions occurring at replication forks. Here, we show that HR deficient cell lines irs1SF and V-C8, deficient in XRCC3 and BRCA2, respectively, are hypersensitive to Cr[VI], implicating this repair pathway in repair of Cr[VI] damage. Furthermore, we find that Cr[VI] causes DNA double-strand breaks and triggers both Rad51 foci formation and induction of HRR. Collectively, these data suggest that HRR is important in repair of Cr[VI]-induced DNA damage. In addition, we find that ERCC1, XRCC1 and DNA-PKcs defective cells are hypersensitive to Cr[VI], indicating that several repair pathways cooperate in repairing Cr[VI]-induced DNA damage. |
Lindh, A R; Rafii, S; Schultz, N; Cox, A; Helleday, T Mitotic defects in XRCC3 variants Ŧ241M and Đ213N and their relation to cancer susceptibility Journal Article Hum. Mol. Genet., 15 (7), pp. 1217–1224, 2006, ([DOI:hrefhttp://dx.doi.org/10.1093/hmg/ddl03710.1093/hmg/ddl037] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1650500316505003]). @article{pmid16505003, title = {Mitotic defects in XRCC3 variants Ŧ241M and Đ213N and their relation to cancer susceptibility}, author = { A. R. Lindh and S. Rafii and N. Schultz and A. Cox and T. Helleday}, year = {2006}, date = {2006-04-01}, journal = {Hum. Mol. Genet.}, volume = {15}, number = {7}, pages = {1217--1224}, abstract = {The XRCC3 variant T241M, but not D213N, has been reported to be associated with an increased risk of some cancers. XRCC3 is one out of five RAD51 paralogues and is involved in homologous recombination, as are the BRCA1 and BRCA2 proteins. However, in contrast to mutations in BRCA1 and BRCA2, the XRCC3(T241M) protein is proficient in homologous recombination and reverts sensitivity to mitomycin C found in XRCC3-deficient cells, whereas XRCC3(D213N) is defective in homologous recombination. Here, we report that both the XRCC3 D213N and T241M alleles are associated with an increase in centrosome number and binucleated cells. However, only the D213N allele gives an increase in spontaneous levels of apoptosis. We suggest that the inability of XRCC3 T241M to apoptotically eliminate aberrant cells with mitotic defects could increase cancer susceptibility in individuals carrying this variant. In contrast, cells carrying the XRCC3 D213N variant are able to eliminate aberrant cells by apoptosis, and consistent with this observation, this variant does not seem to be associated with cancer susceptibility.}, note = {[DOI:hrefhttp://dx.doi.org/10.1093/hmg/ddl03710.1093/hmg/ddl037] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1650500316505003]}, keywords = {}, pubstate = {published}, tppubtype = {article} } The XRCC3 variant T241M, but not D213N, has been reported to be associated with an increased risk of some cancers. XRCC3 is one out of five RAD51 paralogues and is involved in homologous recombination, as are the BRCA1 and BRCA2 proteins. However, in contrast to mutations in BRCA1 and BRCA2, the XRCC3(T241M) protein is proficient in homologous recombination and reverts sensitivity to mitomycin C found in XRCC3-deficient cells, whereas XRCC3(D213N) is defective in homologous recombination. Here, we report that both the XRCC3 D213N and T241M alleles are associated with an increase in centrosome number and binucleated cells. However, only the D213N allele gives an increase in spontaneous levels of apoptosis. We suggest that the inability of XRCC3 T241M to apoptotically eliminate aberrant cells with mitotic defects could increase cancer susceptibility in individuals carrying this variant. In contrast, cells carrying the XRCC3 D213N variant are able to eliminate aberrant cells by apoptosis, and consistent with this observation, this variant does not seem to be associated with cancer susceptibility. |
Bryant, H E; Helleday, T Inhibition of poly (AĐP-ribose) polymerase activates AŦM which is required for subsequent homologous recombination repair Journal Article Nucleic Acids Res., 34 (6), pp. 1685–1691, 2006, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC1410911PMC1410911] [DOI:hrefhttp://dx.doi.org/10.1093/nar/gkl10810.1093/nar/gkl108] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1655690916556909]). @article{pmid16556909, title = {Inhibition of poly (AĐP-ribose) polymerase activates AŦM which is required for subsequent homologous recombination repair}, author = { H. E. Bryant and T. Helleday}, year = {2006}, date = {2006-01-01}, journal = {Nucleic Acids Res.}, volume = {34}, number = {6}, pages = {1685--1691}, abstract = {Poly (ADP-ribose) polymerase (PARP-1), ATM and DNA-dependent protein kinase (DNA-PK) are all involved in responding to DNA damage to activate pathways responsible for cellular survival. Here, we demonstrate that PARP-1-/- cells are sensitive to the ATM inhibitor KU55933 and conversely that AT cells are sensitive to the PARP inhibitor 4-amino-1,8-napthalamide. In addition, PARP-1-/- cells are shown to be sensitive to the DNA-PK inhibitor NU7026 and DNA-PKcs or Ku80 defective cells shown to be sensitive to PARP inhibitors. We believe PARP inhibition results in an increase in unresolved spontaneous DNA single-strand breaks (SSBs), which collapse replication forks and trigger homologous recombination repair (HRR). We show that ATM is activated following inhibition of PARP. Furthermore, PARP inhibitor-induced HRR is abolished in ATM, but not DNA-PK, inhibited cells. ATM and DNA-PK inhibition together give the same sensitivity to PARP inhibitors as ATM alone, indicating that ATM functions in the same pathways as DNA-PK for survival at collapsed forks, likely in non-homologous end joining (NHEJ). Altogether, we suggest that ATM is activated by PARP inhibitor-induced collapsed replication forks and may function upstream of HRR in the repair of certain types of double-strand breaks (DSBs).}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC1410911PMC1410911] [DOI:hrefhttp://dx.doi.org/10.1093/nar/gkl10810.1093/nar/gkl108] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1655690916556909]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Poly (ADP-ribose) polymerase (PARP-1), ATM and DNA-dependent protein kinase (DNA-PK) are all involved in responding to DNA damage to activate pathways responsible for cellular survival. Here, we demonstrate that PARP-1-/- cells are sensitive to the ATM inhibitor KU55933 and conversely that AT cells are sensitive to the PARP inhibitor 4-amino-1,8-napthalamide. In addition, PARP-1-/- cells are shown to be sensitive to the DNA-PK inhibitor NU7026 and DNA-PKcs or Ku80 defective cells shown to be sensitive to PARP inhibitors. We believe PARP inhibition results in an increase in unresolved spontaneous DNA single-strand breaks (SSBs), which collapse replication forks and trigger homologous recombination repair (HRR). We show that ATM is activated following inhibition of PARP. Furthermore, PARP inhibitor-induced HRR is abolished in ATM, but not DNA-PK, inhibited cells. ATM and DNA-PK inhibition together give the same sensitivity to PARP inhibitors as ATM alone, indicating that ATM functions in the same pathways as DNA-PK for survival at collapsed forks, likely in non-homologous end joining (NHEJ). Altogether, we suggest that ATM is activated by PARP inhibitor-induced collapsed replication forks and may function upstream of HRR in the repair of certain types of double-strand breaks (DSBs). |
2005 |
Helleday, T; Bryant, H E; Schultz, N Poly(AĐP-ribose) polymerase (PARP-1) in homologous recombination and as a target for cancer therapy Journal Article Cell Cycle, 4 (9), pp. 1176–1178, 2005, ([PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1612358616123586]). @article{pmid16123586, title = {Poly(AĐP-ribose) polymerase (PARP-1) in homologous recombination and as a target for cancer therapy}, author = { T. Helleday and H. E. Bryant and N. Schultz}, year = {2005}, date = {2005-09-01}, journal = {Cell Cycle}, volume = {4}, number = {9}, pages = {1176--1178}, abstract = {Poly(ADP-ribose) polymerase (PARP-1) binds to DNA breaks to facilitate DNA repair. However, the role of PARP-1 in DNA repair appears to not be critical since PARP-1 knockout mice are viable, fertile and do not develop early onset tumors. Cells isolated from these mice show an increased level of homologous recombination. There is an intricate link between homologous recombination and PARP-1 and a possible role for PARP-1 in DNA double-strand break repair. Although PARP-1 appears not to be required for homologous recombination itself, it regulates the process through its involvement in the repair of DNA single-strand breaks (SSBs). SSBs persisting into the S phase of the cell cycle collapse replication forks, triggering homologous recombination for replication restart. We discuss the recent discoveries on the use of PARP-1 inhibitors as a targeted cancer therapy for recombination deficient cancers, such as BRCA2 tumors.}, note = {[PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1612358616123586]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Poly(ADP-ribose) polymerase (PARP-1) binds to DNA breaks to facilitate DNA repair. However, the role of PARP-1 in DNA repair appears to not be critical since PARP-1 knockout mice are viable, fertile and do not develop early onset tumors. Cells isolated from these mice show an increased level of homologous recombination. There is an intricate link between homologous recombination and PARP-1 and a possible role for PARP-1 in DNA double-strand break repair. Although PARP-1 appears not to be required for homologous recombination itself, it regulates the process through its involvement in the repair of DNA single-strand breaks (SSBs). SSBs persisting into the S phase of the cell cycle collapse replication forks, triggering homologous recombination for replication restart. We discuss the recent discoveries on the use of PARP-1 inhibitors as a targeted cancer therapy for recombination deficient cancers, such as BRCA2 tumors. |
Saleh-Gohari, N; Bryant, H E; Schultz, N; Parker, K M; Cassel, T N; Helleday, T Spontaneous homologous recombination is induced by collapsed replication forks that are caused by endogenous ĐNA single-strand breaks Journal Article Mol. Cell. Biol., 25 (16), pp. 7158–7169, 2005, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC1190269PMC1190269] [DOI:hrefhttp://dx.doi.org/10.1128/MCB.25.16.7158-7169.200510.1128/MCB.25.16.7158-7169.2005] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1605572516055725]). @article{pmid16055725, title = {Spontaneous homologous recombination is induced by collapsed replication forks that are caused by endogenous ĐNA single-strand breaks}, author = { N. Saleh-Gohari and H. E. Bryant and N. Schultz and K. M. Parker and T. N. Cassel and T. Helleday}, year = {2005}, date = {2005-08-01}, journal = {Mol. Cell. Biol.}, volume = {25}, number = {16}, pages = {7158--7169}, abstract = {Homologous recombination is vital to repair fatal DNA damage during DNA replication. However, very little is known about the substrates or repair pathways for homologous recombination in mammalian cells. Here, we have compared the recombination products produced spontaneously with those produced following induction of DNA double-strand breaks (DSBs) with the I-SceI restriction endonuclease or after stalling or collapsing replication forks following treatment with thymidine or camptothecin, respectively. We show that each lesion produces different spectra of recombinants, suggesting differential use of homologous recombination pathways in repair of these lesions. The spontaneous spectrum most resembled the spectra produced at collapsed replication forks formed when a replication fork runs into camptothecin-stabilized DNA single-strand breaks (SSBs) within the topoisomerase I cleavage complex. We found that camptothecin-induced DSBs and the resulting recombination repair require replication, showing that a collapsed fork is the substrate for camptothecin-induced recombination. An SSB repair-defective cell line, EM9 with an XRCC1 mutation, has an increased number of spontaneous gammaH2Ax and RAD51 foci, suggesting that endogenous SSBs collapse replication forks, triggering recombination repair. Furthermore, we show that gammaH2Ax, DSBs, and RAD51 foci are synergistically induced in EM9 cells with camptothecin, suggesting that lack of SSB repair in EM9 causes more collapsed forks and more recombination repair. Furthermore, our results suggest that two-ended DSBs are rare substrates for spontaneous homologous recombination in a mammalian fibroblast cell line. Interestingly, all spectra showed evidence of multiple homologous recombination events in 8 to 16% of clones. However, there was no increase in homologous recombination genomewide in these clones nor were the events dependent on each other; rather, we suggest that a first homologous recombination event frequently triggers a second event at the same locus in mammalian cells.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC1190269PMC1190269] [DOI:hrefhttp://dx.doi.org/10.1128/MCB.25.16.7158-7169.200510.1128/MCB.25.16.7158-7169.2005] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1605572516055725]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Homologous recombination is vital to repair fatal DNA damage during DNA replication. However, very little is known about the substrates or repair pathways for homologous recombination in mammalian cells. Here, we have compared the recombination products produced spontaneously with those produced following induction of DNA double-strand breaks (DSBs) with the I-SceI restriction endonuclease or after stalling or collapsing replication forks following treatment with thymidine or camptothecin, respectively. We show that each lesion produces different spectra of recombinants, suggesting differential use of homologous recombination pathways in repair of these lesions. The spontaneous spectrum most resembled the spectra produced at collapsed replication forks formed when a replication fork runs into camptothecin-stabilized DNA single-strand breaks (SSBs) within the topoisomerase I cleavage complex. We found that camptothecin-induced DSBs and the resulting recombination repair require replication, showing that a collapsed fork is the substrate for camptothecin-induced recombination. An SSB repair-defective cell line, EM9 with an XRCC1 mutation, has an increased number of spontaneous gammaH2Ax and RAD51 foci, suggesting that endogenous SSBs collapse replication forks, triggering recombination repair. Furthermore, we show that gammaH2Ax, DSBs, and RAD51 foci are synergistically induced in EM9 cells with camptothecin, suggesting that lack of SSB repair in EM9 causes more collapsed forks and more recombination repair. Furthermore, our results suggest that two-ended DSBs are rare substrates for spontaneous homologous recombination in a mammalian fibroblast cell line. Interestingly, all spectra showed evidence of multiple homologous recombination events in 8 to 16% of clones. However, there was no increase in homologous recombination genomewide in these clones nor were the events dependent on each other; rather, we suggest that a first homologous recombination event frequently triggers a second event at the same locus in mammalian cells. |
Syljuasen, R G; S?rensen, C S; Hansen, L T; Fugger, K; Lundin, C; Johansson, F; Helleday, T; Sehested, M; Lukas, J; Bartek, J Inhibition of human Chk1 causes increased initiation of ĐNA replication, phosphorylation of AŦR targets, and ĐNA breakage Journal Article Mol. Cell. Biol., 25 (9), pp. 3553–3562, 2005, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC1084285PMC1084285] [DOI:hrefhttp://dx.doi.org/10.1128/MCB.25.9.3553-3562.200510.1128/MCB.25.9.3553-3562.2005] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1583146115831461]). @article{pmid15831461, title = {Inhibition of human Chk1 causes increased initiation of ĐNA replication, phosphorylation of AŦR targets, and ĐNA breakage}, author = { R. G. Syljuasen and C. S. S?rensen and L. T. Hansen and K. Fugger and C. Lundin and F. Johansson and T. Helleday and M. Sehested and J. Lukas and J. Bartek}, year = {2005}, date = {2005-05-01}, journal = {Mol. Cell. Biol.}, volume = {25}, number = {9}, pages = {3553--3562}, abstract = {Human checkpoint kinase 1 (Chk1) is an essential kinase required to preserve genome stability. Here, we show that Chk1 inhibition by two distinct drugs, UCN-01 and CEP-3891, or by Chk1 small interfering RNA (siRNA) leads to phosphorylation of ATR targets. Chk1-inhibition triggered rapid, pan-nuclear phosphorylation of histone H2AX, p53, Smc1, replication protein A, and Chk1 itself in human S-phase cells. These phosphorylations were inhibited by ATR siRNA and caffeine, but they occurred independently of ATM. Chk1 inhibition also caused an increased initiation of DNA replication, which was accompanied by increased amounts of nonextractable RPA protein, formation of single-stranded DNA, and induction of DNA strand breaks. Moreover, these responses were prevented by siRNA-mediated downregulation of Cdk2 or the replication initiation protein Cdc45, or by addition of the CDK inhibitor roscovitine. We propose that Chk1 is required during normal S phase to avoid aberrantly increased initiation of DNA replication, thereby protecting against DNA breakage. These results may help explain why Chk1 is an essential kinase and should be taken into account when drugs to inhibit this kinase are considered for use in cancer treatment.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC1084285PMC1084285] [DOI:hrefhttp://dx.doi.org/10.1128/MCB.25.9.3553-3562.200510.1128/MCB.25.9.3553-3562.2005] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1583146115831461]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Human checkpoint kinase 1 (Chk1) is an essential kinase required to preserve genome stability. Here, we show that Chk1 inhibition by two distinct drugs, UCN-01 and CEP-3891, or by Chk1 small interfering RNA (siRNA) leads to phosphorylation of ATR targets. Chk1-inhibition triggered rapid, pan-nuclear phosphorylation of histone H2AX, p53, Smc1, replication protein A, and Chk1 itself in human S-phase cells. These phosphorylations were inhibited by ATR siRNA and caffeine, but they occurred independently of ATM. Chk1 inhibition also caused an increased initiation of DNA replication, which was accompanied by increased amounts of nonextractable RPA protein, formation of single-stranded DNA, and induction of DNA strand breaks. Moreover, these responses were prevented by siRNA-mediated downregulation of Cdk2 or the replication initiation protein Cdc45, or by addition of the CDK inhibitor roscovitine. We propose that Chk1 is required during normal S phase to avoid aberrantly increased initiation of DNA replication, thereby protecting against DNA breakage. These results may help explain why Chk1 is an essential kinase and should be taken into account when drugs to inhibit this kinase are considered for use in cancer treatment. |
Bryant, H E; Schultz, N; Thomas, H D; Parker, K M; Flower, D; Lopez, E; Kyle, S; Meuth, M; Curtin, N J; Helleday, T Specific killing of BRCA2-deficient tumours with inhibitors of poly(AĐP-ribose) polymerase Journal Article Nature, 434 (7035), pp. 913–917, 2005, ([DOI:hrefhttp://dx.doi.org/10.1038/nature0344310.1038/nature03443] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1582996615829966]). @article{pmid15829966, title = {Specific killing of BRCA2-deficient tumours with inhibitors of poly(AĐP-ribose) polymerase}, author = { H. E. Bryant and N. Schultz and H. D. Thomas and K. M. Parker and D. Flower and E. Lopez and S. Kyle and M. Meuth and N. J. Curtin and T. Helleday}, year = {2005}, date = {2005-04-01}, journal = {Nature}, volume = {434}, number = {7035}, pages = {913--917}, abstract = {Poly(ADP-ribose) polymerase (PARP1) facilitates DNA repair by binding to DNA breaks and attracting DNA repair proteins to the site of damage. Nevertheless, PARP1-/- mice are viable, fertile and do not develop early onset tumours. Here, we show that PARP inhibitors trigger gamma-H2AX and RAD51 foci formation. We propose that, in the absence of PARP1, spontaneous single-strand breaks collapse replication forks and trigger homologous recombination for repair. Furthermore, we show that BRCA2-deficient cells, as a result of their deficiency in homologous recombination, are acutely sensitive to PARP inhibitors, presumably because resultant collapsed replication forks are no longer repaired. Thus, PARP1 activity is essential in homologous recombination-deficient BRCA2 mutant cells. We exploit this requirement in order to kill BRCA2-deficient tumours by PARP inhibition alone. Treatment with PARP inhibitors is likely to be highly tumour specific, because only the tumours (which are BRCA2-/-) in BRCA2+/- patients are defective in homologous recombination. The use of an inhibitor of a DNA repair enzyme alone to selectively kill a tumour, in the absence of an exogenous DNA-damaging agent, represents a new concept in cancer treatment.}, note = {[DOI:hrefhttp://dx.doi.org/10.1038/nature0344310.1038/nature03443] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1582996615829966]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Poly(ADP-ribose) polymerase (PARP1) facilitates DNA repair by binding to DNA breaks and attracting DNA repair proteins to the site of damage. Nevertheless, PARP1-/- mice are viable, fertile and do not develop early onset tumours. Here, we show that PARP inhibitors trigger gamma-H2AX and RAD51 foci formation. We propose that, in the absence of PARP1, spontaneous single-strand breaks collapse replication forks and trigger homologous recombination for repair. Furthermore, we show that BRCA2-deficient cells, as a result of their deficiency in homologous recombination, are acutely sensitive to PARP inhibitors, presumably because resultant collapsed replication forks are no longer repaired. Thus, PARP1 activity is essential in homologous recombination-deficient BRCA2 mutant cells. We exploit this requirement in order to kill BRCA2-deficient tumours by PARP inhibition alone. Treatment with PARP inhibitors is likely to be highly tumour specific, because only the tumours (which are BRCA2-/-) in BRCA2+/- patients are defective in homologous recombination. The use of an inhibitor of a DNA repair enzyme alone to selectively kill a tumour, in the absence of an exogenous DNA-damaging agent, represents a new concept in cancer treatment. |
El-Khamisy, S F; Saifi, G M; Weinfeld, M; Johansson, F; Helleday, T; Lupski, J R; Caldecott, K W Đefective ĐNA single-strand break repair in spinocerebellar ataxia with axonal neuropathy-1 Journal Article Nature, 434 (7029), pp. 108–113, 2005, ([DOI:hrefhttp://dx.doi.org/10.1038/nature0331410.1038/nature03314] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1574430915744309]). @article{pmid15744309, title = {Đefective ĐNA single-strand break repair in spinocerebellar ataxia with axonal neuropathy-1}, author = { S. F. El-Khamisy and G. M. Saifi and M. Weinfeld and F. Johansson and T. Helleday and J. R. Lupski and K. W. Caldecott}, year = {2005}, date = {2005-03-01}, journal = {Nature}, volume = {434}, number = {7029}, pages = {108--113}, abstract = {Spinocerebellar ataxia with axonal neuropathy-1 (SCAN1) is a neurodegenerative disease that results from mutation of tyrosyl phosphodiesterase 1 (TDP1). In lower eukaryotes, Tdp1 removes topoisomerase 1 (top1) peptide from DNA termini during the repair of double-strand breaks created by collision of replication forks with top1 cleavage complexes in proliferating cells. Although TDP1 most probably fulfils a similar function in human cells, this role is unlikely to account for the clinical phenotype of SCAN1, which is associated with progressive degeneration of post-mitotic neurons. In addition, this role is redundant in lower eukaryotes, and Tdp1 mutations alone confer little phenotype. Moreover, defects in processing or preventing double-strand breaks during DNA replication are most probably associated with increased genetic instability and cancer, phenotypes not observed in SCAN1 (ref. 8). Here we show that in human cells TDP1 is required for repair of chromosomal single-strand breaks arising independently of DNA replication from abortive top1 activity or oxidative stress. We report that TDP1 is sequestered into multi-protein single-strand break repair (SSBR) complexes by direct interaction with DNA ligase IIIalpha and that these complexes are catalytically inactive in SCAN1 cells. These data identify a defect in SSBR in a neurodegenerative disease, and implicate this process in the maintenance of genetic integrity in post-mitotic neurons.}, note = {[DOI:hrefhttp://dx.doi.org/10.1038/nature0331410.1038/nature03314] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1574430915744309]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Spinocerebellar ataxia with axonal neuropathy-1 (SCAN1) is a neurodegenerative disease that results from mutation of tyrosyl phosphodiesterase 1 (TDP1). In lower eukaryotes, Tdp1 removes topoisomerase 1 (top1) peptide from DNA termini during the repair of double-strand breaks created by collision of replication forks with top1 cleavage complexes in proliferating cells. Although TDP1 most probably fulfils a similar function in human cells, this role is unlikely to account for the clinical phenotype of SCAN1, which is associated with progressive degeneration of post-mitotic neurons. In addition, this role is redundant in lower eukaryotes, and Tdp1 mutations alone confer little phenotype. Moreover, defects in processing or preventing double-strand breaks during DNA replication are most probably associated with increased genetic instability and cancer, phenotypes not observed in SCAN1 (ref. 8). Here we show that in human cells TDP1 is required for repair of chromosomal single-strand breaks arising independently of DNA replication from abortive top1 activity or oxidative stress. We report that TDP1 is sequestered into multi-protein single-strand break repair (SSBR) complexes by direct interaction with DNA ligase IIIalpha and that these complexes are catalytically inactive in SCAN1 cells. These data identify a defect in SSBR in a neurodegenerative disease, and implicate this process in the maintenance of genetic integrity in post-mitotic neurons. |
S?rensen, C S; Hansen, L T; Dziegielewski, J; Syljuasen, R G; Lundin, C; Bartek, J; Helleday, T Ŧhe cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair Journal Article Nat. Cell Biol., 7 (2), pp. 195–201, 2005, ([DOI:hrefhttp://dx.doi.org/10.1038/ncb121210.1038/ncb1212] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1566585615665856]). @article{pmid15665856, title = {Ŧhe cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair}, author = { C. S. S?rensen and L. T. Hansen and J. Dziegielewski and R. G. Syljuasen and C. Lundin and J. Bartek and T. Helleday}, year = {2005}, date = {2005-02-01}, journal = {Nat. Cell Biol.}, volume = {7}, number = {2}, pages = {195--201}, abstract = {The essential checkpoint kinase Chk1 is required for cell-cycle delays after DNA damage or blocked DNA replication. However, it is unclear whether Chk1 is involved in the repair of damaged DNA. Here we establish that Chk1 is a key regulator of genome maintenance by the homologous recombination repair (HRR) system. Abrogation of Chk1 function with small interfering RNA or chemical antagonists inhibits HRR, leading to persistent unrepaired DNA double-strand breaks (DSBs) and cell death after replication inhibition with hydroxyurea or DNA-damage caused by camptothecin. After hydroxyurea treatment, the essential recombination repair protein RAD51 is recruited to DNA repair foci performing a vital role in correct HRR. We demonstrate that Chk1 interacts with RAD51, and that RAD51 is phosphorylated on Thr 309 in a Chk1-dependent manner. Consistent with a functional interplay between Chk1 and RAD51, Chk1-depleted cells failed to form RAD51 nuclear foci after exposure to hydroxyurea, and cells expressing a phosphorylation-deficient mutant RAD51(T309A) were hypersensitive to hydroxyurea. These results highlight a crucial role for the Chk1 signalling pathway in protecting cells against lethal DNA lesions through regulation of HRR.}, note = {[DOI:hrefhttp://dx.doi.org/10.1038/ncb121210.1038/ncb1212] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1566585615665856]}, keywords = {}, pubstate = {published}, tppubtype = {article} } The essential checkpoint kinase Chk1 is required for cell-cycle delays after DNA damage or blocked DNA replication. However, it is unclear whether Chk1 is involved in the repair of damaged DNA. Here we establish that Chk1 is a key regulator of genome maintenance by the homologous recombination repair (HRR) system. Abrogation of Chk1 function with small interfering RNA or chemical antagonists inhibits HRR, leading to persistent unrepaired DNA double-strand breaks (DSBs) and cell death after replication inhibition with hydroxyurea or DNA-damage caused by camptothecin. After hydroxyurea treatment, the essential recombination repair protein RAD51 is recruited to DNA repair foci performing a vital role in correct HRR. We demonstrate that Chk1 interacts with RAD51, and that RAD51 is phosphorylated on Thr 309 in a Chk1-dependent manner. Consistent with a functional interplay between Chk1 and RAD51, Chk1-depleted cells failed to form RAD51 nuclear foci after exposure to hydroxyurea, and cells expressing a phosphorylation-deficient mutant RAD51(T309A) were hypersensitive to hydroxyurea. These results highlight a crucial role for the Chk1 signalling pathway in protecting cells against lethal DNA lesions through regulation of HRR. |
Lundin, C; North, M; Erixon, K; Walters, K; Jenssen, D; Goldman, A S; Helleday, T Methyl methanesulfonate (MMS) produces heat-labile ĐNA damage but no detectable in vivo ĐNA double-strand breaks Journal Article Nucleic Acids Res., 33 (12), pp. 3799–3811, 2005, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC1174933PMC1174933] [DOI:hrefhttp://dx.doi.org/10.1093/nar/gki68110.1093/nar/gki681] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1600981216009812]). @article{pmid16009812, title = {Methyl methanesulfonate (MMS) produces heat-labile ĐNA damage but no detectable in vivo ĐNA double-strand breaks}, author = { C. Lundin and M. North and K. Erixon and K. Walters and D. Jenssen and A. S. Goldman and T. Helleday}, year = {2005}, date = {2005-01-01}, journal = {Nucleic Acids Res.}, volume = {33}, number = {12}, pages = {3799--3811}, abstract = {Homologous recombination (HR) deficient cells are sensitive to methyl methanesulfonate (MMS). HR is usually involved in the repair of DNA double-strand breaks (DSBs) in Saccharomyces cerevisiae implying that MMS somehow induces DSBs in vivo. Indeed there is evidence, based on pulsed-field gel electrophoresis (PFGE), that MMS causes DNA fragmentation. However, the mechanism through which MMS induces DSBs has not been demonstrated. Here, we show that DNA fragmentation following MMS treatment, and detected by PFGE is not the consequence of production of cellular DSBs. Instead, DSBs seen following MMS treatment are produced during sample preparation where heat-labile methylated DNA is converted into DSBs. Furthermore, we show that the repair of MMS-induced heat-labile damage requires the base excision repair protein XRCC1, and is independent of HR in both S.cerevisiae and mammalian cells. We speculate that the reason for recombination-deficient cells being sensitive to MMS is due to the role of HR in repair of MMS-induced stalled replication forks, rather than for repair of cellular DSBs or heat-labile damage.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC1174933PMC1174933] [DOI:hrefhttp://dx.doi.org/10.1093/nar/gki68110.1093/nar/gki681] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1600981216009812]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Homologous recombination (HR) deficient cells are sensitive to methyl methanesulfonate (MMS). HR is usually involved in the repair of DNA double-strand breaks (DSBs) in Saccharomyces cerevisiae implying that MMS somehow induces DSBs in vivo. Indeed there is evidence, based on pulsed-field gel electrophoresis (PFGE), that MMS causes DNA fragmentation. However, the mechanism through which MMS induces DSBs has not been demonstrated. Here, we show that DNA fragmentation following MMS treatment, and detected by PFGE is not the consequence of production of cellular DSBs. Instead, DSBs seen following MMS treatment are produced during sample preparation where heat-labile methylated DNA is converted into DSBs. Furthermore, we show that the repair of MMS-induced heat-labile damage requires the base excision repair protein XRCC1, and is independent of HR in both S.cerevisiae and mammalian cells. We speculate that the reason for recombination-deficient cells being sensitive to MMS is due to the role of HR in repair of MMS-induced stalled replication forks, rather than for repair of cellular DSBs or heat-labile damage. |
Bavoux, C; Leopoldino, A M; Bergoglio, V; O-Wang, J; Ogi, T; Bieth, A; Judde, J G; Pena, S D; Poupon, M F; Helleday, T; Tagawa, M; Machado, C; Hoffmann, J S; Cazaux, C Up-regulation of the error-prone ĐNA polymerase kappa promotes pleiotropic genetic alterations and tumorigenesis Journal Article Cancer Res., 65 (1), pp. 325–330, 2005, ([PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1566531015665310]). @article{pmid15665310, title = {Up-regulation of the error-prone ĐNA polymerase kappa promotes pleiotropic genetic alterations and tumorigenesis}, author = { C. Bavoux and A. M. Leopoldino and V. Bergoglio and J. O-Wang and T. Ogi and A. Bieth and J. G. Judde and S. D. Pena and M. F. Poupon and T. Helleday and M. Tagawa and C. Machado and J. S. Hoffmann and C. Cazaux}, year = {2005}, date = {2005-01-01}, journal = {Cancer Res.}, volume = {65}, number = {1}, pages = {325--330}, abstract = {It is currently widely accepted that genetic instability is key to cancer development. Many types of cancers arise as a consequence of a gradual accumulation of nucleotide aberrations, each mutation conferring growth and/or survival advantage. Genetic instability could also proceed in sudden bursts leading to a more drastic upheaval of structure and organization of the genome. Genetic instability, as an operative force, will produce genetic variants and the greater the instability, the larger the number of variants. We report here that the overexpression of human DNA polymerase kappa, an error-prone enzyme that is up-regulated in lung cancers, induces DNA breaks and stimulates DNA exchanges as well as aneuploidy. Probably as the result of so many perturbations, excess polymerase kappa favors the proliferation of competent tumor cells as observed in immunodeficient mice. These data suggest that altered regulation of DNA metabolism might be related to cancer-associated genetic changes and phenotype.}, note = {[PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1566531015665310]}, keywords = {}, pubstate = {published}, tppubtype = {article} } It is currently widely accepted that genetic instability is key to cancer development. Many types of cancers arise as a consequence of a gradual accumulation of nucleotide aberrations, each mutation conferring growth and/or survival advantage. Genetic instability could also proceed in sudden bursts leading to a more drastic upheaval of structure and organization of the genome. Genetic instability, as an operative force, will produce genetic variants and the greater the instability, the larger the number of variants. We report here that the overexpression of human DNA polymerase kappa, an error-prone enzyme that is up-regulated in lung cancers, induces DNA breaks and stimulates DNA exchanges as well as aneuploidy. Probably as the result of so many perturbations, excess polymerase kappa favors the proliferation of competent tumor cells as observed in immunodeficient mice. These data suggest that altered regulation of DNA metabolism might be related to cancer-associated genetic changes and phenotype. |
2004 |
Bryant, H E; Helleday, T Poly(AĐP-ribose) polymerase inhibitors as potential chemotherapeutic agents Journal Article Biochem. Soc. Trans., 32 (Pt 6), pp. 959–961, 2004, ([DOI:hrefhttp://dx.doi.org/10.1042/BST032095910.1042/BST0320959] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1550693515506935]). @article{pmid15506935, title = {Poly(AĐP-ribose) polymerase inhibitors as potential chemotherapeutic agents}, author = { H. E. Bryant and T. Helleday}, year = {2004}, date = {2004-12-01}, journal = {Biochem. Soc. Trans.}, volume = {32}, number = {Pt 6}, pages = {959--961}, abstract = {PARP [poly(ADP-ribose) polymerase] activity is up-regulated by binding to DNA strand breaks and its association with DNA repair is well documented. Many anticancer therapies work by inducing breaks in DNA, if unrepaired these can lead to cell death. As PARP promotes DNA repair there is a strong rational to suggest that its inhibition may increase the efficiency of certain cytotoxic treatments. This review discusses the advances made in PARP inhibitor design and the mechanism by which they enhance anti-tumour therapies.}, note = {[DOI:hrefhttp://dx.doi.org/10.1042/BST032095910.1042/BST0320959] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1550693515506935]}, keywords = {}, pubstate = {published}, tppubtype = {article} } PARP [poly(ADP-ribose) polymerase] activity is up-regulated by binding to DNA strand breaks and its association with DNA repair is well documented. Many anticancer therapies work by inducing breaks in DNA, if unrepaired these can lead to cell death. As PARP promotes DNA repair there is a strong rational to suggest that its inhibition may increase the efficiency of certain cytotoxic treatments. This review discusses the advances made in PARP inhibitor design and the mechanism by which they enhance anti-tumour therapies. |
Saleh-Gohari, N; Helleday, T Strand invasion involving short tract gene conversion is specifically suppressed in BRCA2-deficient hamster cells Journal Article Oncogene, 23 (56), pp. 9136–9141, 2004, ([DOI:hrefhttp://dx.doi.org/10.1038/sj.onc.120817810.1038/sj.onc.1208178] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1548041315480413]). @article{pmid15480413, title = {Strand invasion involving short tract gene conversion is specifically suppressed in BRCA2-deficient hamster cells}, author = { N. Saleh-Gohari and T. Helleday}, year = {2004}, date = {2004-12-01}, journal = {Oncogene}, volume = {23}, number = {56}, pages = {9136--9141}, abstract = {The BRCA2 tumour suppressor protein is involved in maintaining genetic stability through its role in homologous recombination (HR), where it mediates RAD51-dependent strand invasion. Here, we show that BRCA2-defective cells are not completely impaired in HR by strand invasion although the spontaneous HR rate is 10-fold lower than that in wild-type cells. Furthermore, a DNA double-strand break (DSB) triggers HR repair by strand invasion also in BRCA2-defective cells, but less efficiently. Thus, either the strand invasion pathway(s) in which BRCA2 operates is still operative in the absence of a functional BRCA2, albeit at a reduced frequency, or there is a separate pathway for strand invasion still functional in BRCA2-deficient cells. Consistent with the latter hypothesis, we show that HR events occurring in BRCA2-defective cells differ from HR events in wild-type cells. These data suggest that BRCA2-defective hamster cells are impaired in short tract gene conversion but maintain proficiency in sister chromatid exchange.}, note = {[DOI:hrefhttp://dx.doi.org/10.1038/sj.onc.120817810.1038/sj.onc.1208178] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1548041315480413]}, keywords = {}, pubstate = {published}, tppubtype = {article} } The BRCA2 tumour suppressor protein is involved in maintaining genetic stability through its role in homologous recombination (HR), where it mediates RAD51-dependent strand invasion. Here, we show that BRCA2-defective cells are not completely impaired in HR by strand invasion although the spontaneous HR rate is 10-fold lower than that in wild-type cells. Furthermore, a DNA double-strand break (DSB) triggers HR repair by strand invasion also in BRCA2-defective cells, but less efficiently. Thus, either the strand invasion pathway(s) in which BRCA2 operates is still operative in the absence of a functional BRCA2, albeit at a reduced frequency, or there is a separate pathway for strand invasion still functional in BRCA2-deficient cells. Consistent with the latter hypothesis, we show that HR events occurring in BRCA2-defective cells differ from HR events in wild-type cells. These data suggest that BRCA2-defective hamster cells are impaired in short tract gene conversion but maintain proficiency in sister chromatid exchange. |
Bolderson, E; Scorah, J; Helleday, T; Smythe, C; Meuth, M AŦM is required for the cellular response to thymidine induced replication fork stress Journal Article Hum. Mol. Genet., 13 (23), pp. 2937–2945, 2004, ([DOI:hrefhttp://dx.doi.org/10.1093/hmg/ddh31610.1093/hmg/ddh316] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1545918115459181]). @article{pmid15459181, title = {AŦM is required for the cellular response to thymidine induced replication fork stress}, author = { E. Bolderson and J. Scorah and T. Helleday and C. Smythe and M. Meuth}, year = {2004}, date = {2004-12-01}, journal = {Hum. Mol. Genet.}, volume = {13}, number = {23}, pages = {2937--2945}, abstract = {Genetically distinct checkpoints, activated as a consequence of either DNA replication arrest or ionizing radiation-induced DNA damage, integrate DNA repair responses into the cell cycle programme. The ataxia-telangiectasia mutated (ATM) protein kinase blocks cell cycle progression in response to DNA double strand breaks, whereas the related ATR is important in maintaining the integrity of the DNA replication apparatus. Here, we show that thymidine, which slows the progression of replication forks by depleting cellular pools of dCTP, induces a novel DNA damage response that, uniquely, depends on both ATM and ATR. Thymidine induces ATM-mediated phosphorylation of Chk2 and NBS1 and an ATM-independent phosphorylation of Chk1 and SMC1. AT cells exposed to thymidine showed decreased viability and failed to induce homologous recombination repair (HRR). Taken together, our results implicate ATM in the HRR-mediated rescue of replication forks impaired by thymidine treatment.}, note = {[DOI:hrefhttp://dx.doi.org/10.1093/hmg/ddh31610.1093/hmg/ddh316] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1545918115459181]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Genetically distinct checkpoints, activated as a consequence of either DNA replication arrest or ionizing radiation-induced DNA damage, integrate DNA repair responses into the cell cycle programme. The ataxia-telangiectasia mutated (ATM) protein kinase blocks cell cycle progression in response to DNA double strand breaks, whereas the related ATR is important in maintaining the integrity of the DNA replication apparatus. Here, we show that thymidine, which slows the progression of replication forks by depleting cellular pools of dCTP, induces a novel DNA damage response that, uniquely, depends on both ATM and ATR. Thymidine induces ATM-mediated phosphorylation of Chk2 and NBS1 and an ATM-independent phosphorylation of Chk1 and SMC1. AT cells exposed to thymidine showed decreased viability and failed to induce homologous recombination repair (HRR). Taken together, our results implicate ATM in the HRR-mediated rescue of replication forks impaired by thymidine treatment. |
Johansson, F; Allkvist, A; Erixon, K; Malmvarn, A; Nilsson, R; Bergman, A; Helleday, T; Jenssen, D Screening for genotoxicity using the ĐRAG assay: investigation of halogenated environmental contaminants Journal Article Mutat. Res., 563 (1), pp. 35–47, 2004, ([DOI:hrefhttp://dx.doi.org/10.1016/j.mrgentox.2004.05.01710.1016/j.mrgentox.2004.05.017] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1532474715324747]). @article{pmid15324747, title = {Screening for genotoxicity using the ĐRAG assay: investigation of halogenated environmental contaminants}, author = { F. Johansson and A. Allkvist and K. Erixon and A. Malmvarn and R. Nilsson and A. Bergman and T. Helleday and D. Jenssen}, year = {2004}, date = {2004-09-01}, journal = {Mutat. Res.}, volume = {563}, number = {1}, pages = {35--47}, abstract = {The DRAG test is a rapid high-throughput screening assay for detection of repairable adducts by growth inhibition of Chinese hamster ovary cells (CHO) characterized by different defects in DNA repair. A more pronounced growth inhibition caused by a certain DNA-reactive substance in a repair-deficient cell line (EM9, UV4 and UV5) as compared to wild-type cells (AA8) is interpreted as a consequence of their inability to repair induced DNA lesions. Thus, the use of such cell lines in the DRAG test may provide information of the type of DNA lesions induced by a certain genotoxic substance. To select optimal assay conditions, as well as to provide a mechanistic basis for interpreting the results, the model compounds benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), ethyl methanesulfonate (EMS), mitomycin C (MMC) and hydrogen peroxide (H2O2) were used. These agents can induce bulky adducts, alkyl adducts, cross-links and oxidative damage, respectively. The specificity of the DRAG test constitutes an important prerequisite for its practical use in a broader context. To assess this aspect, we have investigated the genotoxic and cytotoxic properties of a selection of metabolites of and isomers from polychlorinated biphenyls (PCB) and polybrominated diphenyl ethers (PBDE), along with a few other halogenated compounds. All these compounds have been detected as pollutants in the external environment, and for most of them there is no convincing evidence of mutagenicity from conventional assays. As could be predicted from their mode of action, BPDE, MMC, and EMS were all found to be more toxic in the repair-deficient cell lines compared with wild-type cells. The results with H2O2 were inconclusive, and the PCB metabolite 4,4'-diOH-CB80 only exhibited borderline activity, while all other halogenated compounds, or their metabolites, were found to be inactive. In conclusion, the DRAG assay could provide a robust and useful tool when screening large numbers of potentially genotoxic agents, while in addition providing mechanistic information. However, the usefulness of the selected cell lines to detect oxidative damage may be limited.}, note = {[DOI:hrefhttp://dx.doi.org/10.1016/j.mrgentox.2004.05.01710.1016/j.mrgentox.2004.05.017] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1532474715324747]}, keywords = {}, pubstate = {published}, tppubtype = {article} } The DRAG test is a rapid high-throughput screening assay for detection of repairable adducts by growth inhibition of Chinese hamster ovary cells (CHO) characterized by different defects in DNA repair. A more pronounced growth inhibition caused by a certain DNA-reactive substance in a repair-deficient cell line (EM9, UV4 and UV5) as compared to wild-type cells (AA8) is interpreted as a consequence of their inability to repair induced DNA lesions. Thus, the use of such cell lines in the DRAG test may provide information of the type of DNA lesions induced by a certain genotoxic substance. To select optimal assay conditions, as well as to provide a mechanistic basis for interpreting the results, the model compounds benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), ethyl methanesulfonate (EMS), mitomycin C (MMC) and hydrogen peroxide (H2O2) were used. These agents can induce bulky adducts, alkyl adducts, cross-links and oxidative damage, respectively. The specificity of the DRAG test constitutes an important prerequisite for its practical use in a broader context. To assess this aspect, we have investigated the genotoxic and cytotoxic properties of a selection of metabolites of and isomers from polychlorinated biphenyls (PCB) and polybrominated diphenyl ethers (PBDE), along with a few other halogenated compounds. All these compounds have been detected as pollutants in the external environment, and for most of them there is no convincing evidence of mutagenicity from conventional assays. As could be predicted from their mode of action, BPDE, MMC, and EMS were all found to be more toxic in the repair-deficient cell lines compared with wild-type cells. The results with H2O2 were inconclusive, and the PCB metabolite 4,4'-diOH-CB80 only exhibited borderline activity, while all other halogenated compounds, or their metabolites, were found to be inactive. In conclusion, the DRAG assay could provide a robust and useful tool when screening large numbers of potentially genotoxic agents, while in addition providing mechanistic information. However, the usefulness of the selected cell lines to detect oxidative damage may be limited. |
Kumari, A; Schultz, N; Helleday, T p53 protects from replication-associated ĐNA double-strand breaks in mammalian cells Journal Article Oncogene, 23 (13), pp. 2324–2329, 2004, ([DOI:hrefhttp://dx.doi.org/10.1038/sj.onc.120737910.1038/sj.onc.1207379] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1474320414743204]). @article{pmid14743204, title = {p53 protects from replication-associated ĐNA double-strand breaks in mammalian cells}, author = { A. Kumari and N. Schultz and T. Helleday}, year = {2004}, date = {2004-03-01}, journal = {Oncogene}, volume = {23}, number = {13}, pages = {2324--2329}, abstract = {Genetic instability caused by mutations in the p53 gene is generally thought to be due to a loss of the DNA damage response that controls checkpoint functions and apoptosis. Cells with mutant p53 exhibit high levels of homologous recombination (HR). This could be an indirect consequence of the loss of DNA damage response or p53 could have a direct role in HR. Here, we report that p53-/- mouse embryonic fibroblasts (MEFs) exhibit higher levels of the RAD51 protein and increased level of spontaneous RAD51 foci Agents that stall replication forks, for example, hydroxyurea (HU), potently induce HR repair and RAD51 foci. To test if the increase in RAD51 foci in p53-/- MEFs was due to an increased level of damage during replication, we measured the formation of DNA double-strand breaks (DSBs) in p53+/+ and p53-/- MEFs following treatments with HU. We found that HU induced DSBs only in p53-/- MEFs, indicating that p53 is involved in a pathway to protect stalled replication forks from being collapsed into a substrate for HR. Also, p53 is upregulated in response to agents that inhibit DNA replication, which supports our hypothesis. Finally, we observed that the DSBs produced in p53-/- MEFs did not result in a permanent arrest of replication and that they were repaired. Altogether, we suggest that the effect of p53 on HR and RAD51 levels and foci can be explained by the idea that p53 suppresses formation of recombinogenic lesions.}, note = {[DOI:hrefhttp://dx.doi.org/10.1038/sj.onc.120737910.1038/sj.onc.1207379] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1474320414743204]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Genetic instability caused by mutations in the p53 gene is generally thought to be due to a loss of the DNA damage response that controls checkpoint functions and apoptosis. Cells with mutant p53 exhibit high levels of homologous recombination (HR). This could be an indirect consequence of the loss of DNA damage response or p53 could have a direct role in HR. Here, we report that p53-/- mouse embryonic fibroblasts (MEFs) exhibit higher levels of the RAD51 protein and increased level of spontaneous RAD51 foci Agents that stall replication forks, for example, hydroxyurea (HU), potently induce HR repair and RAD51 foci. To test if the increase in RAD51 foci in p53-/- MEFs was due to an increased level of damage during replication, we measured the formation of DNA double-strand breaks (DSBs) in p53+/+ and p53-/- MEFs following treatments with HU. We found that HU induced DSBs only in p53-/- MEFs, indicating that p53 is involved in a pathway to protect stalled replication forks from being collapsed into a substrate for HR. Also, p53 is upregulated in response to agents that inhibit DNA replication, which supports our hypothesis. Finally, we observed that the DSBs produced in p53-/- MEFs did not result in a permanent arrest of replication and that they were repaired. Altogether, we suggest that the effect of p53 on HR and RAD51 levels and foci can be explained by the idea that p53 suppresses formation of recombinogenic lesions. |
Saleh-Gohari, N; Helleday, T Conservative homologous recombination preferentially repairs ĐNA double-strand breaks in the S phase of the cell cycle in human cells Journal Article Nucleic Acids Res., 32 (12), pp. 3683–3688, 2004, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC484186PMC484186] [DOI:hrefhttp://dx.doi.org/10.1093/nar/gkh70310.1093/nar/gkh703] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1525215215252152]). @article{pmid15252152, title = {Conservative homologous recombination preferentially repairs ĐNA double-strand breaks in the S phase of the cell cycle in human cells}, author = { N. Saleh-Gohari and T. Helleday}, year = {2004}, date = {2004-01-01}, journal = {Nucleic Acids Res.}, volume = {32}, number = {12}, pages = {3683--3688}, abstract = {DNA double-strand breaks (DSBs) are repaired by either homologous recombination (HR) or non-homologous end joining (NHEJ) in mammalian cells. Repair with NHEJ or HR using single-strand annealing (SSA) often results in deletions and is generally referred to as non-conservative recombination. Error-free, conservative HR involves strand invasion and requires a homologous DNA template, and therefore it is generally believed that this type of repair occurs preferentially in the late S, G2 and M phases of the cell cycle, when the sister chromatid is available. There are several observations supporting this hypothesis, although it has not been tested directly. Here, we synchronize human SW480SN.3 cells in the G1/G0 (with serum starvation), S (with thymidine block) and M (with nocodazole) phases of the cell cycle and investigate the efficiency of conservative HR repair of an I-SceI-induced DSB. The frequency of HR repair of DSBs was 39 times higher in S-phase cells than in M-phase cells and 24-fold higher than in G1/G0 cells. This low level of conservative HR occurs even though a homologous template is present within the recombination substrate. We propose that this can be explained by an absence of recombination proteins outside the S phase or alternatively that there maybe factors that suppress HR in G1/G0 and M. Furthermore, we found that HR repair of DSBs involves short tract gene conversion in all the phases of the cell cycle. This indicates that the same pathway for conservative HR is employed in the repair of DSBs regardless of phase of the cell cycle and that only the frequency is affected.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC484186PMC484186] [DOI:hrefhttp://dx.doi.org/10.1093/nar/gkh70310.1093/nar/gkh703] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1525215215252152]}, keywords = {}, pubstate = {published}, tppubtype = {article} } DNA double-strand breaks (DSBs) are repaired by either homologous recombination (HR) or non-homologous end joining (NHEJ) in mammalian cells. Repair with NHEJ or HR using single-strand annealing (SSA) often results in deletions and is generally referred to as non-conservative recombination. Error-free, conservative HR involves strand invasion and requires a homologous DNA template, and therefore it is generally believed that this type of repair occurs preferentially in the late S, G2 and M phases of the cell cycle, when the sister chromatid is available. There are several observations supporting this hypothesis, although it has not been tested directly. Here, we synchronize human SW480SN.3 cells in the G1/G0 (with serum starvation), S (with thymidine block) and M (with nocodazole) phases of the cell cycle and investigate the efficiency of conservative HR repair of an I-SceI-induced DSB. The frequency of HR repair of DSBs was 39 times higher in S-phase cells than in M-phase cells and 24-fold higher than in G1/G0 cells. This low level of conservative HR occurs even though a homologous template is present within the recombination substrate. We propose that this can be explained by an absence of recombination proteins outside the S phase or alternatively that there maybe factors that suppress HR in G1/G0 and M. Furthermore, we found that HR repair of DSBs involves short tract gene conversion in all the phases of the cell cycle. This indicates that the same pathway for conservative HR is employed in the repair of DSBs regardless of phase of the cell cycle and that only the frequency is affected. |
Mohindra, A; Bolderson, E; Stone, J; Wells, M; Helleday, T; Meuth, M A tumour-derived mutant allele of XRCC2 preferentially suppresses homologous recombination at ĐNA replication forks Journal Article Hum. Mol. Genet., 13 (2), pp. 203–212, 2004, ([DOI:hrefhttp://dx.doi.org/10.1093/hmg/ddh02210.1093/hmg/ddh022] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1464520714645207]). @article{pmid14645207, title = {A tumour-derived mutant allele of XRCC2 preferentially suppresses homologous recombination at ĐNA replication forks}, author = { A. Mohindra and E. Bolderson and J. Stone and M. Wells and T. Helleday and M. Meuth}, year = {2004}, date = {2004-01-01}, journal = {Hum. Mol. Genet.}, volume = {13}, number = {2}, pages = {203--212}, abstract = {Homologous recombination repair (HRR) is required for both the repair of DNA double strand breaks (DSBs) and the maintenance of the integrity of DNA replication forks. To determine the effect of a mutant allele of the RAD51 paralog XRCC2 (342delT) found in an HRR-defective tumour cell line, 342delT was introduced into HRR proficient cells containing a recombination reporter substrate. In one set of transfectants, expression of 342delT conferred sensitivity to thymidine and mitomycin C and suppressed HRR induced at the recombination reporter by thymidine but not by DSBs. In a second set of transfectants, the expression of 342delT was accompanied by a decreased level of the full-length XRCC2. These cells were defective in the induction of HRR by either thymidine or DSBs. Thus 342delT suppresses recombination induced by thymidine in a dominant negative manner while recombination induced by DSBs appears to depend upon the level of XRCC2 as well as the expression of the mutant XRCC2 allele. These results suggest that HRR pathways responding to stalled replication forks or DSBs are genetically distinguishable. They further suggest a critical role for XRCC2 in HRR at replication forks, possibly in the loading of RAD51 onto gapped DNA.}, note = {[DOI:hrefhttp://dx.doi.org/10.1093/hmg/ddh02210.1093/hmg/ddh022] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1464520714645207]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Homologous recombination repair (HRR) is required for both the repair of DNA double strand breaks (DSBs) and the maintenance of the integrity of DNA replication forks. To determine the effect of a mutant allele of the RAD51 paralog XRCC2 (342delT) found in an HRR-defective tumour cell line, 342delT was introduced into HRR proficient cells containing a recombination reporter substrate. In one set of transfectants, expression of 342delT conferred sensitivity to thymidine and mitomycin C and suppressed HRR induced at the recombination reporter by thymidine but not by DSBs. In a second set of transfectants, the expression of 342delT was accompanied by a decreased level of the full-length XRCC2. These cells were defective in the induction of HRR by either thymidine or DSBs. Thus 342delT suppresses recombination induced by thymidine in a dominant negative manner while recombination induced by DSBs appears to depend upon the level of XRCC2 as well as the expression of the mutant XRCC2 allele. These results suggest that HRR pathways responding to stalled replication forks or DSBs are genetically distinguishable. They further suggest a critical role for XRCC2 in HRR at replication forks, possibly in the loading of RAD51 onto gapped DNA. |
2003 |
Helleday, T Pathways for mitotic homologous recombination in mammalian cells Journal Article Mutat. Res., 532 (1-2), pp. 103–115, 2003, ([PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1464343214643432]). @article{pmid14643432, title = {Pathways for mitotic homologous recombination in mammalian cells}, author = { T. Helleday}, year = {2003}, date = {2003-11-01}, journal = {Mutat. Res.}, volume = {532}, number = {1-2}, pages = {103--115}, abstract = {Homologous recombination (HR) is essential for cellular survival in mammals. In this review, the substrates for HR, the pathways of repair, and their end products (i.e. sister chromatid exchange (SCE), gene conversion, deletions or tandem duplications) are discussed. HR is involved in the repair of DNA double-strand breaks (DSBs) and DNA lesions that occur at replication forks. A classical DSB may result in deletions, tandem duplications or gene conversion following two-end recombination repair. In contrast, a SCE may be the result of one-end recombination repair at a collapsed replication fork, i.e. a single-strand break converted into a DSB at a replication fork. Recombination repair at a stalled replication fork may occur in the absence of a DSB intermediate and may result in either SCE or gene conversion. Finally, substrates and pathways involved in spontaneous HR are discussed.}, note = {[PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1464343214643432]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Homologous recombination (HR) is essential for cellular survival in mammals. In this review, the substrates for HR, the pathways of repair, and their end products (i.e. sister chromatid exchange (SCE), gene conversion, deletions or tandem duplications) are discussed. HR is involved in the repair of DNA double-strand breaks (DSBs) and DNA lesions that occur at replication forks. A classical DSB may result in deletions, tandem duplications or gene conversion following two-end recombination repair. In contrast, a SCE may be the result of one-end recombination repair at a collapsed replication fork, i.e. a single-strand break converted into a DSB at a replication fork. Recombination repair at a stalled replication fork may occur in the absence of a DSB intermediate and may result in either SCE or gene conversion. Finally, substrates and pathways involved in spontaneous HR are discussed. |
Schultz, N; Lopez, E; Saleh-Gohari, N; Helleday, T Poly(AĐP-ribose) polymerase (PARP-1) has a controlling role in homologous recombination Journal Article Nucleic Acids Res., 31 (17), pp. 4959–4964, 2003, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC212803PMC212803] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1293094412930944]). @article{pmid12930944, title = {Poly(AĐP-ribose) polymerase (PARP-1) has a controlling role in homologous recombination}, author = { N. Schultz and E. Lopez and N. Saleh-Gohari and T. Helleday}, year = {2003}, date = {2003-09-01}, journal = {Nucleic Acids Res.}, volume = {31}, number = {17}, pages = {4959--4964}, abstract = {Cells with non-functional poly(ADP-ribose) polymerase (PARP-1) show increased levels of sister chromatid exchange, suggesting a hyper recombination phenotype in these cells. To further investigate the involvement of PARP-1 in homologous recombination (HR) we investigated how PARP-1 affects nuclear HR sites (Rad51 foci) and HR repair of an endonuclease-induced DNA double-strand break (DSB). Several proteins involved in HR localise to Rad51 foci and HR-deficient cells fail to form Rad51 foci in response to DNA damage. Here, we show that PARP-1 mainly does not localise to Rad51 foci and that Rad51 foci form in PARP-1-/- cells, also in response to hydroxyurea. Furthermore, we show that homology directed repair following induction of a site-specific DSB is normal in PARP-1-inhibited cells. In contrast, inhibition or loss of PARP-1 increases spontaneous Rad51 foci formation, confirming a hyper recombination phenotype in these cells. Our data suggest that PARP-1 controls DNA damage recognised by HR and that it is not involved in executing HR as such.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC212803PMC212803] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1293094412930944]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Cells with non-functional poly(ADP-ribose) polymerase (PARP-1) show increased levels of sister chromatid exchange, suggesting a hyper recombination phenotype in these cells. To further investigate the involvement of PARP-1 in homologous recombination (HR) we investigated how PARP-1 affects nuclear HR sites (Rad51 foci) and HR repair of an endonuclease-induced DNA double-strand break (DSB). Several proteins involved in HR localise to Rad51 foci and HR-deficient cells fail to form Rad51 foci in response to DNA damage. Here, we show that PARP-1 mainly does not localise to Rad51 foci and that Rad51 foci form in PARP-1-/- cells, also in response to hydroxyurea. Furthermore, we show that homology directed repair following induction of a site-specific DSB is normal in PARP-1-inhibited cells. In contrast, inhibition or loss of PARP-1 increases spontaneous Rad51 foci formation, confirming a hyper recombination phenotype in these cells. Our data suggest that PARP-1 controls DNA damage recognised by HR and that it is not involved in executing HR as such. |
Hansen, L T; Lundin, C; Spang-Thomsen, M; Petersen, L N; Helleday, T Ŧhe role of RAĐ51 in etoposide (VP16) resistance in small cell lung cancer Journal Article Int. J. Cancer, 105 (4), pp. 472–479, 2003, ([DOI:hrefhttp://dx.doi.org/10.1002/ijc.1110610.1002/ijc.11106] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1271243612712436]). @article{pmid12712436, title = {Ŧhe role of RAĐ51 in etoposide (VP16) resistance in small cell lung cancer}, author = { L. T. Hansen and C. Lundin and M. Spang-Thomsen and L. N. Petersen and T. Helleday}, year = {2003}, date = {2003-07-01}, journal = {Int. J. Cancer}, volume = {105}, number = {4}, pages = {472--479}, abstract = {Etoposide (VP16) is a potent inducer of DNA double-strand breaks (DSBs) and is efficiently used in small cell lung cancer (SCLC) therapy. However, acquired VP16 resistance remains an important barrier to effective treatment. To understand the underlying mechanisms for VP16 resistance in SCLC, we investigated DSB repair and cellular VP16 sensitivity of SCLC cells. VP16 sensitivity and RAD51, DNA-PK(cs), topoisomerase IIalpha and P-glycoprotein protein levels were determined in 17 SCLC cell lines. In order to unravel the role of RAD51 in VP16 resistance, we cloned the human RAD51 gene, transfected SCLC cells with RAD51 sense or antisense constructs and measured the VP16 resistance. Finally, we measured VP16-induced DSBs in the 17 SCLC cell lines. Two cell lines exhibited a multidrug-resistant phenotype. In the other SCLC cell lines, the cellular VP16 resistance was positively correlated with the RAD51 protein level. In addition, downregulation or overexpression of the RAD51 gene altered the VP16 sensitivity. Furthermore, the levels of the RAD51 and DNA-PK(cs) proteins were related to VP16-induced DSBs. The results suggest that repair of VP16-induced DSBs is mediated through both RAD51-dependent homologous recombination and DNA-PK(cs)-dependent nonhomologous end-joining and may be a determinant of the variation in clinical treatment effect observed in human SCLC tumors of identical histologic subtype. Finally, we propose RAD51 as a potential target to improve VP16 efficacy and predict tumor resistance in the treatment of SCLC patients.}, note = {[DOI:hrefhttp://dx.doi.org/10.1002/ijc.1110610.1002/ijc.11106] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1271243612712436]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Etoposide (VP16) is a potent inducer of DNA double-strand breaks (DSBs) and is efficiently used in small cell lung cancer (SCLC) therapy. However, acquired VP16 resistance remains an important barrier to effective treatment. To understand the underlying mechanisms for VP16 resistance in SCLC, we investigated DSB repair and cellular VP16 sensitivity of SCLC cells. VP16 sensitivity and RAD51, DNA-PK(cs), topoisomerase IIalpha and P-glycoprotein protein levels were determined in 17 SCLC cell lines. In order to unravel the role of RAD51 in VP16 resistance, we cloned the human RAD51 gene, transfected SCLC cells with RAD51 sense or antisense constructs and measured the VP16 resistance. Finally, we measured VP16-induced DSBs in the 17 SCLC cell lines. Two cell lines exhibited a multidrug-resistant phenotype. In the other SCLC cell lines, the cellular VP16 resistance was positively correlated with the RAD51 protein level. In addition, downregulation or overexpression of the RAD51 gene altered the VP16 sensitivity. Furthermore, the levels of the RAD51 and DNA-PK(cs) proteins were related to VP16-induced DSBs. The results suggest that repair of VP16-induced DSBs is mediated through both RAD51-dependent homologous recombination and DNA-PK(cs)-dependent nonhomologous end-joining and may be a determinant of the variation in clinical treatment effect observed in human SCLC tumors of identical histologic subtype. Finally, we propose RAD51 as a potential target to improve VP16 efficacy and predict tumor resistance in the treatment of SCLC patients. |
Hansen, L T; Lundin, C; Helleday, T; Poulsen, H S; S?rensen, C S; Petersen, L N; Spang-Thomsen, M ĐNA repair rate and etoposide (VP16) resistance of tumor cell subpopulations derived from a single human small cell lung cancer Journal Article Lung Cancer, 40 (2), pp. 157–164, 2003, ([PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1271111612711116]). @article{pmid12711116, title = {ĐNA repair rate and etoposide (VP16) resistance of tumor cell subpopulations derived from a single human small cell lung cancer}, author = { L. T. Hansen and C. Lundin and T. Helleday and H. S. Poulsen and C. S. S?rensen and L. N. Petersen and M. Spang-Thomsen}, year = {2003}, date = {2003-05-01}, journal = {Lung Cancer}, volume = {40}, number = {2}, pages = {157--164}, abstract = {Two human small cell lung cancer (SCLC) subpopulations, CPH 54A, and CPH 54B, established from the same patient tumor by in vitro cloning, were investigated. The tumor was classified as intermediate-type SCLC. The cellular sensitivity to ionizing radiation (IR) was previously determined in the two sublines both in vivo and in vitro. Here we measured the etoposide (VP16) sensitivity together with the induction and repair of VP16- and IR-induced DNA double-strand breaks (DSBs). The two subpopulations were found to differ significantly in sensitivity to VP16, with the radioresistant 54B subline also being VP16 resistant. In order to explain the VP16 resistant phenotype several mechanisms where considered. The p53 status, P-glycoprotein, MRP, topoisomerase IIalpha, and Mre11 protein levels, as well as growth kinetics, provided no explanations of the observed VP16 resistance. In contrast, a significant difference in repair of both VP16- and IR-induced DSBs, together with a difference in the levels of the DSB repair proteins DNA-dependent protein kinase (DNA-PK(cs)) and RAD51 was observed. The VP16- and radioresistant 54B subline exhibited a pronounced higher repair rate of DSBs and higher protein levels of both DNA-PK(cs) and RAD51 compared with the sensitive 54A subline. We suggest, that different DSB repair rates among tumor cell subpopulations of individual SCLC tumors may be a major determinant for the variation in clinical treatment effect observed in human SCLC tumors of identical histological subtype.}, note = {[PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1271111612711116]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Two human small cell lung cancer (SCLC) subpopulations, CPH 54A, and CPH 54B, established from the same patient tumor by in vitro cloning, were investigated. The tumor was classified as intermediate-type SCLC. The cellular sensitivity to ionizing radiation (IR) was previously determined in the two sublines both in vivo and in vitro. Here we measured the etoposide (VP16) sensitivity together with the induction and repair of VP16- and IR-induced DNA double-strand breaks (DSBs). The two subpopulations were found to differ significantly in sensitivity to VP16, with the radioresistant 54B subline also being VP16 resistant. In order to explain the VP16 resistant phenotype several mechanisms where considered. The p53 status, P-glycoprotein, MRP, topoisomerase IIalpha, and Mre11 protein levels, as well as growth kinetics, provided no explanations of the observed VP16 resistance. In contrast, a significant difference in repair of both VP16- and IR-induced DSBs, together with a difference in the levels of the DSB repair proteins DNA-dependent protein kinase (DNA-PK(cs)) and RAD51 was observed. The VP16- and radioresistant 54B subline exhibited a pronounced higher repair rate of DSBs and higher protein levels of both DNA-PK(cs) and RAD51 compared with the sensitive 54A subline. We suggest, that different DSB repair rates among tumor cell subpopulations of individual SCLC tumors may be a major determinant for the variation in clinical treatment effect observed in human SCLC tumors of identical histological subtype. |
Lundin, C; Schultz, N; Arnaudeau, C; Mohindra, A; Hansen, L T; Helleday, T RAĐ51 is involved in repair of damage associated with ĐNA replication in mammalian cells Journal Article J. Mol. Biol., 328 (3), pp. 521–535, 2003, ([PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1270671412706714]). @article{pmid12706714, title = {RAĐ51 is involved in repair of damage associated with ĐNA replication in mammalian cells}, author = { C. Lundin and N. Schultz and C. Arnaudeau and A. Mohindra and L. T. Hansen and T. Helleday}, year = {2003}, date = {2003-05-01}, journal = {J. Mol. Biol.}, volume = {328}, number = {3}, pages = {521--535}, abstract = {The RAD51 protein, a eukaryotic homologue of the Escherichia coli RecA protein, plays an important role in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) in mammalian cells. Recent findings suggest that HR may be important in repair following replication arrest in mammalian cells. Here, we have investigated the role of RAD51 in the repair of different types of damage induced during DNA replication with etoposide, hydroxyurea or thymidine. We show that etoposide induces DSBs at newly replicated DNA more frequently than gamma-rays, and that these DSBs are different from those induced by hydroxyurea. No DSB was found following treatment with thymidine. Although these compounds appear to induce different DNA lesions during DNA replication, we show that a cell line overexpressing RAD51 is resistant to all of them, indicating that RAD51 is involved in repair of a wide range of DNA lesions during DNA replication. We observe fewer etoposide-induced DSBs in RAD51-overexpressing cells and that HR repair of etoposide-induced DSBs is faster. Finally, we show that induced long-tract HR in the hprt gene is suppressed in RAD51-overexpressing cells, although global HR appears not to be suppressed. This suggests that overexpression of RAD51 prevents long-tract HR occurring during DNA replication. We discuss our results in light of recent models suggested for HR at stalled replication forks.}, note = {[PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1270671412706714]}, keywords = {}, pubstate = {published}, tppubtype = {article} } The RAD51 protein, a eukaryotic homologue of the Escherichia coli RecA protein, plays an important role in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) in mammalian cells. Recent findings suggest that HR may be important in repair following replication arrest in mammalian cells. Here, we have investigated the role of RAD51 in the repair of different types of damage induced during DNA replication with etoposide, hydroxyurea or thymidine. We show that etoposide induces DSBs at newly replicated DNA more frequently than gamma-rays, and that these DSBs are different from those induced by hydroxyurea. No DSB was found following treatment with thymidine. Although these compounds appear to induce different DNA lesions during DNA replication, we show that a cell line overexpressing RAD51 is resistant to all of them, indicating that RAD51 is involved in repair of a wide range of DNA lesions during DNA replication. We observe fewer etoposide-induced DSBs in RAD51-overexpressing cells and that HR repair of etoposide-induced DSBs is faster. Finally, we show that induced long-tract HR in the hprt gene is suppressed in RAD51-overexpressing cells, although global HR appears not to be suppressed. This suggests that overexpression of RAD51 prevents long-tract HR occurring during DNA replication. We discuss our results in light of recent models suggested for HR at stalled replication forks. |
Hinz, J M; Helleday, T; Meuth, M Reduced apoptotic response to camptothecin in CĦO cells deficient in XRCC3 Journal Article Carcinogenesis, 24 (2), pp. 249–253, 2003, ([PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1258417412584174]). @article{pmid12584174, title = {Reduced apoptotic response to camptothecin in CĦO cells deficient in XRCC3}, author = { J. M. Hinz and T. Helleday and M. Meuth}, year = {2003}, date = {2003-02-01}, journal = {Carcinogenesis}, volume = {24}, number = {2}, pages = {249--253}, abstract = {Eukaryotic cells respond to DNA damage by activation of DNA repair, cell-cycle arrest and apoptosis. Several reports suggest that such responses may be coordinated by communication between damage repair proteins and proteins signalling other cellular responses. The Rad51-guided homologous recombination (HR) repair plays an important role in recognition and repair of DNA double-strand breaks (DSBs) and cells deficient in this repair pathway become hypersensitive to agents that induce DSBs. In the work reported here we investigated the possible role of the Rad51-like HR proteins XRCC2, XRCC3 and Rad51C in apoptosis following the induction of DSBs by camptothecin. We show that a hamster cell line (irs1SF) deficient in the HR repair gene XRCC3 exhibits altered death and cell-cycle checkpoint responses following treatment with growth inhibitory concentrations of camptothecin. In contrast, hamster cells defective in XRCC2 (irs1) or Rad51C (irs3) treated with equally toxic doses of this agent exhibit a rapid induction of apoptosis similar to that seen in the parental cell line or mutant cells corrected for the HR defect. These results suggest that XRCC3 activity may be necessary for efficient entry into apoptosis in response to DSBs.}, note = {[PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1258417412584174]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Eukaryotic cells respond to DNA damage by activation of DNA repair, cell-cycle arrest and apoptosis. Several reports suggest that such responses may be coordinated by communication between damage repair proteins and proteins signalling other cellular responses. The Rad51-guided homologous recombination (HR) repair plays an important role in recognition and repair of DNA double-strand breaks (DSBs) and cells deficient in this repair pathway become hypersensitive to agents that induce DSBs. In the work reported here we investigated the possible role of the Rad51-like HR proteins XRCC2, XRCC3 and Rad51C in apoptosis following the induction of DSBs by camptothecin. We show that a hamster cell line (irs1SF) deficient in the HR repair gene XRCC3 exhibits altered death and cell-cycle checkpoint responses following treatment with growth inhibitory concentrations of camptothecin. In contrast, hamster cells defective in XRCC2 (irs1) or Rad51C (irs3) treated with equally toxic doses of this agent exhibit a rapid induction of apoptosis similar to that seen in the parental cell line or mutant cells corrected for the HR defect. These results suggest that XRCC3 activity may be necessary for efficient entry into apoptosis in response to DSBs. |
2002 |
Mohindra, A; Hays, L E; Phillips, E N; Preston, B D; Helleday, T; Meuth, M Đefects in homologous recombination repair in mismatch-repair-deficient tumour cell lines Journal Article Hum. Mol. Genet., 11 (18), pp. 2189–2200, 2002, ([PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1218917112189171]). @article{pmid12189171, title = {Đefects in homologous recombination repair in mismatch-repair-deficient tumour cell lines}, author = { A. Mohindra and L. E. Hays and E. N. Phillips and B. D. Preston and T. Helleday and M. Meuth}, year = {2002}, date = {2002-09-01}, journal = {Hum. Mol. Genet.}, volume = {11}, number = {18}, pages = {2189--2200}, abstract = {Loss of mismatch repair (MMR) leads to a complex mutator phenotype that appears to drive the development of a subset of colon cancers. Here we show that MMR-deficient tumour cell lines are highly sensitive to the toxic effects of thymidine relative to MMR-proficient lines. This sensitivity was not a direct consequence of MMR deficiency or alterations of DNA precursor metabolism. Instead, MMR-defective tumour cell lines are also defective in homologous recombination repair (HRR) induced by DNA double-strand breaks. Furthermore, a frameshift mutation of the human RAD51 paralog XRCC2 found in the MMR-deficient uterine tumour cell line SKUT-1 can confer thymidine sensitivity when introduced into a MMR-proficient line. Like other cells with defective XRCC2, SKUT-1 is sensitive to mitomycin C, and MMR-proficient cells expressing the mutant XRCC2 allele become more sensitive to this agent. These data suggest that the thymidine sensitivity of MMR-deficient tumour cell lines may be a consequence of defects in the HRR pathway. The increased thymidine sensitivity and the loss of an important pathway for the repair of DNA double-strand breaks create new opportunities for therapies directed specifically against this subset of tumours.}, note = {[PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1218917112189171]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Loss of mismatch repair (MMR) leads to a complex mutator phenotype that appears to drive the development of a subset of colon cancers. Here we show that MMR-deficient tumour cell lines are highly sensitive to the toxic effects of thymidine relative to MMR-proficient lines. This sensitivity was not a direct consequence of MMR deficiency or alterations of DNA precursor metabolism. Instead, MMR-defective tumour cell lines are also defective in homologous recombination repair (HRR) induced by DNA double-strand breaks. Furthermore, a frameshift mutation of the human RAD51 paralog XRCC2 found in the MMR-deficient uterine tumour cell line SKUT-1 can confer thymidine sensitivity when introduced into a MMR-proficient line. Like other cells with defective XRCC2, SKUT-1 is sensitive to mitomycin C, and MMR-proficient cells expressing the mutant XRCC2 allele become more sensitive to this agent. These data suggest that the thymidine sensitivity of MMR-deficient tumour cell lines may be a consequence of defects in the HRR pathway. The increased thymidine sensitivity and the loss of an important pathway for the repair of DNA double-strand breaks create new opportunities for therapies directed specifically against this subset of tumours. |
Lundin, C; Samuelsson, M K; Helleday, T Overexpression of cyclin E does not influence homologous recombination in Chinese hamster cells Journal Article Biochem. Biophys. Res. Commun., 296 (2), pp. 363–367, 2002, ([PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1216302612163026]). @article{pmid12163026, title = {Overexpression of cyclin E does not influence homologous recombination in Chinese hamster cells}, author = { C. Lundin and M. K. Samuelsson and T. Helleday}, year = {2002}, date = {2002-08-01}, journal = {Biochem. Biophys. Res. Commun.}, volume = {296}, number = {2}, pages = {363--367}, abstract = {Overexpressed cyclin E in tumours is a prognosticator for poor patient outcome. Cells that overexpress cyclin E have been shown to be impaired in S-phase progression and exhibit genetic instability that may drive this subset of cancers. However, the origin for genetic instability caused by cyclin E overexpression is unknown. Homologous recombination plays an important role in S-phase progression and is also regulated by the same proteins that regulate cyclin E-associated kinase activity, i.e., p53 and p21. To test the hypothesis that overexpressed cyclin E causes genetic instability through homologous recombination, we investigated the effect of cyclin E overexpression on homologous recombination in the hprt gene in a Chinese hamster cell line. Although cyclin E overexpression shortened the G1 phase in the cell cycle as expected, we could see no change in neither spontaneous nor etoposide-induced recombination. Also, overexpression of cyclin E did not affect the repair of DNA double-strand breaks and failed to potentiate the cytotoxic effects of etoposide. Our data suggest that genetic instability caused by overexpression of cyclin E is not mediated by aberrant homologous recombination.}, note = {[PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1216302612163026]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Overexpressed cyclin E in tumours is a prognosticator for poor patient outcome. Cells that overexpress cyclin E have been shown to be impaired in S-phase progression and exhibit genetic instability that may drive this subset of cancers. However, the origin for genetic instability caused by cyclin E overexpression is unknown. Homologous recombination plays an important role in S-phase progression and is also regulated by the same proteins that regulate cyclin E-associated kinase activity, i.e., p53 and p21. To test the hypothesis that overexpressed cyclin E causes genetic instability through homologous recombination, we investigated the effect of cyclin E overexpression on homologous recombination in the hprt gene in a Chinese hamster cell line. Although cyclin E overexpression shortened the G1 phase in the cell cycle as expected, we could see no change in neither spontaneous nor etoposide-induced recombination. Also, overexpression of cyclin E did not affect the repair of DNA double-strand breaks and failed to potentiate the cytotoxic effects of etoposide. Our data suggest that genetic instability caused by overexpression of cyclin E is not mediated by aberrant homologous recombination. |
Lundin, C; Erixon, K; Arnaudeau, C; Schultz, N; Jenssen, D; Meuth, M; Helleday, T Đifferent roles for nonhomologous end joining and homologous recombination following replication arrest in mammalian cells Journal Article Mol. Cell. Biol., 22 (16), pp. 5869–5878, 2002, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC133974PMC133974] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1213819712138197]). @article{pmid12138197, title = {Đifferent roles for nonhomologous end joining and homologous recombination following replication arrest in mammalian cells}, author = { C. Lundin and K. Erixon and C. Arnaudeau and N. Schultz and D. Jenssen and M. Meuth and T. Helleday}, year = {2002}, date = {2002-08-01}, journal = {Mol. Cell. Biol.}, volume = {22}, number = {16}, pages = {5869--5878}, abstract = {Homologous recombination (HR) and nonhomologous end joining (NHEJ) play overlapping roles in repair of DNA double-strand breaks (DSBs) generated during the S phase of the cell cycle. Here, we characterized the involvement of HR and NHEJ in the rescue of DNA replication forks arrested or slowed by treatment of hamster cells with hydroxyurea or thymidine. We show that the arrest of replication with hydroxyurea generates DNA fragmentation as a consequence of the formation of DSBs at newly replicated DNA. Both HR and NHEJ protected cells from the lethal effects of hydroxyurea, and this agent also increased the frequency of recombination mediated by both homologous and nonhomologous exchanges. Thymidine induced a less stringent arrest of replication and did not generate detectable DSBs. HR alone rescued cells from the lethal effects of thymidine. Furthermore, thymidine increased the frequency of DNA exchange mediated solely by HR in the absence of detectable DSBs. Our data suggest that both NHEJ and HR are involved in repair of arrested replication forks that include a DSB, while HR alone is required for the repair of slowed replication forks in the absence of detectable DSBs.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC133974PMC133974] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1213819712138197]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Homologous recombination (HR) and nonhomologous end joining (NHEJ) play overlapping roles in repair of DNA double-strand breaks (DSBs) generated during the S phase of the cell cycle. Here, we characterized the involvement of HR and NHEJ in the rescue of DNA replication forks arrested or slowed by treatment of hamster cells with hydroxyurea or thymidine. We show that the arrest of replication with hydroxyurea generates DNA fragmentation as a consequence of the formation of DSBs at newly replicated DNA. Both HR and NHEJ protected cells from the lethal effects of hydroxyurea, and this agent also increased the frequency of recombination mediated by both homologous and nonhomologous exchanges. Thymidine induced a less stringent arrest of replication and did not generate detectable DSBs. HR alone rescued cells from the lethal effects of thymidine. Furthermore, thymidine increased the frequency of DNA exchange mediated solely by HR in the absence of detectable DSBs. Our data suggest that both NHEJ and HR are involved in repair of arrested replication forks that include a DSB, while HR alone is required for the repair of slowed replication forks in the absence of detectable DSBs. |
2001 |
Olsson, G; Belyaev, I Y; Helleday, T; Harms-Ringdahl, M ELF magnetic field affects proliferation of SPĐ8/V79 Chinese hamster cells but does not interact with intrachromosomal recombination Journal Article Mutat. Res., 493 (1-2), pp. 55–66, 2001, ([PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1151671511516715]). @article{pmid11516715, title = {ELF magnetic field affects proliferation of SPĐ8/V79 Chinese hamster cells but does not interact with intrachromosomal recombination}, author = { G. Olsson and I. Y. Belyaev and T. Helleday and M. Harms-Ringdahl}, year = {2001}, date = {2001-06-01}, journal = {Mutat. Res.}, volume = {493}, number = {1-2}, pages = {55--66}, abstract = {Extremely low-frequency (ELF) magnetic fields have previously been shown to affect conformation of chromatin, cell proliferation, and calcium metabolism. Possible mutagenic and carcinogenic effects of ELF have also been discussed and tested. In this study, intrachromosomal recombination in the hprt gene after exposure to ELF magnetic field was investigated using the SPD8 recombination assay. SPD8 cells, derived from V79 Chinese hamster cells were exposed to ELF at a specific combination of static and ELF magnetic fields, that has been proven to have effects on chromatin conformation in several cell types. The genotoxic agent camptothecin (CPT) was used either as a positive control or simultaneously with ELF. We also analysed the effect of ELF and CPT on chromatin conformation with the anomalous viscosity time dependence (AVTD) technique, cell growth kinetics, and cell survival with clonogenic assay. DNA fragmentation was analysed by pulsed field gel electrophoresis (PFGE). ELF did not induce recombination alone, neither did ELF modify the recombinogenic effect of CPT. Although, there was no effect on cell survival in response to ELF exposure, inhibition of cell growth was observed. On the other hand, ELF exposure partly counteracted the growth inhibition seen with CPT. The data suggest that ELF exposure may stimulate or inhibit cell growth depending on the state of the cells. Although, ELF did not induce recombination, a weak but statistically significant DNA fragmentation comparable with CPT-induced fragmentation was observed with PFGE 48h after exposure to ELF.}, note = {[PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1151671511516715]}, keywords = {}, pubstate = {published}, tppubtype = {article} } Extremely low-frequency (ELF) magnetic fields have previously been shown to affect conformation of chromatin, cell proliferation, and calcium metabolism. Possible mutagenic and carcinogenic effects of ELF have also been discussed and tested. In this study, intrachromosomal recombination in the hprt gene after exposure to ELF magnetic field was investigated using the SPD8 recombination assay. SPD8 cells, derived from V79 Chinese hamster cells were exposed to ELF at a specific combination of static and ELF magnetic fields, that has been proven to have effects on chromatin conformation in several cell types. The genotoxic agent camptothecin (CPT) was used either as a positive control or simultaneously with ELF. We also analysed the effect of ELF and CPT on chromatin conformation with the anomalous viscosity time dependence (AVTD) technique, cell growth kinetics, and cell survival with clonogenic assay. DNA fragmentation was analysed by pulsed field gel electrophoresis (PFGE). ELF did not induce recombination alone, neither did ELF modify the recombinogenic effect of CPT. Although, there was no effect on cell survival in response to ELF exposure, inhibition of cell growth was observed. On the other hand, ELF exposure partly counteracted the growth inhibition seen with CPT. The data suggest that ELF exposure may stimulate or inhibit cell growth depending on the state of the cells. Although, ELF did not induce recombination, a weak but statistically significant DNA fragmentation comparable with CPT-induced fragmentation was observed with PFGE 48h after exposure to ELF. |
Arnaudeau, C; Lundin, C; Helleday, T ĐNA double-strand breaks associated with replication forks are predominantly repaired by homologous recombination involving an exchange mechanism in mammalian cells Journal Article J. Mol. Biol., 307 (5), pp. 1235–1245, 2001, ([DOI:hrefhttp://dx.doi.org/10.1006/jmbi.2001.456410.1006/jmbi.2001.4564] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1129233811292338]). @article{pmid11292338, title = {ĐNA double-strand breaks associated with replication forks are predominantly repaired by homologous recombination involving an exchange mechanism in mammalian cells}, author = { C. Arnaudeau and C. Lundin and T. Helleday}, year = {2001}, date = {2001-04-01}, journal = {J. Mol. Biol.}, volume = {307}, number = {5}, pages = {1235--1245}, abstract = {DNA double-strand breaks (DSB) represent a major disruption in the integrity of the genome. DSB can be generated when a replication fork encounters a DNA lesion. Recombinational repair is known to resolve such replication fork-associated DSB, but the molecular mechanism of this repair process is poorly understood in mammalian cells. In the present study, we investigated the molecular mechanism by which recombination resolves camptothecin (CPT)-induced DSB at DNA replication forks. The frequency of homologous recombination (HR) was measured using V79/SPD8 cells which contain a duplication in the endogenous hprt gene that is resolved by HR. We demonstrate that DSB associated with replication forks induce HR at the hprt gene in early S phase. Further analysis revealed that these HR events involve an exchange mechanism. Both the irs1SF and V3-3 cell lines, which are deficient in HR and non-homologous end joining (NHEJ), respectively, were found to be more sensitive than wild-type cells to DSB associated with replication forks. The irs1SF cell line was more sensitive in this respect than V3-3 cells, an observation consistent with the hypothesis that DSB associated with replication forks are repaired primarily by HR. The frequency of formation of DSB associated with replication forks was not affected in HR and NHEJ deficient cells, indicating that the loss of repair, rather than the formation of DSB associated with replication forks is responsible for the increased sensitivity of the mutant strains. We propose that the presence of DSB associated with replication forks rapidly induces HR via an exchange mechanism and that HR plays a more prominent role in the repair of such DSB than does NHEJ.}, note = {[DOI:hrefhttp://dx.doi.org/10.1006/jmbi.2001.456410.1006/jmbi.2001.4564] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1129233811292338]}, keywords = {}, pubstate = {published}, tppubtype = {article} } DNA double-strand breaks (DSB) represent a major disruption in the integrity of the genome. DSB can be generated when a replication fork encounters a DNA lesion. Recombinational repair is known to resolve such replication fork-associated DSB, but the molecular mechanism of this repair process is poorly understood in mammalian cells. In the present study, we investigated the molecular mechanism by which recombination resolves camptothecin (CPT)-induced DSB at DNA replication forks. The frequency of homologous recombination (HR) was measured using V79/SPD8 cells which contain a duplication in the endogenous hprt gene that is resolved by HR. We demonstrate that DSB associated with replication forks induce HR at the hprt gene in early S phase. Further analysis revealed that these HR events involve an exchange mechanism. Both the irs1SF and V3-3 cell lines, which are deficient in HR and non-homologous end joining (NHEJ), respectively, were found to be more sensitive than wild-type cells to DSB associated with replication forks. The irs1SF cell line was more sensitive in this respect than V3-3 cells, an observation consistent with the hypothesis that DSB associated with replication forks are repaired primarily by HR. The frequency of formation of DSB associated with replication forks was not affected in HR and NHEJ deficient cells, indicating that the loss of repair, rather than the formation of DSB associated with replication forks is responsible for the increased sensitivity of the mutant strains. We propose that the presence of DSB associated with replication forks rapidly induces HR via an exchange mechanism and that HR plays a more prominent role in the repair of such DSB than does NHEJ. |
Arnaudeau, C; Rozier, L; Cazaux, C; Defais, M; Jenssen, D; Helleday, T RAĐ51 supports spontaneous non-homologous recombination in mammalian cells, but not the corresponding process induced by topoisomerase inhibitors Journal Article Nucleic Acids Res., 29 (3), pp. 662–667, 2001, ([PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC30407PMC30407] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1116088711160887]). @article{pmid11160887, title = {RAĐ51 supports spontaneous non-homologous recombination in mammalian cells, but not the corresponding process induced by topoisomerase inhibitors}, author = { C. Arnaudeau and L. Rozier and C. Cazaux and M. Defais and D. Jenssen and T. Helleday}, year = {2001}, date = {2001-02-01}, journal = {Nucleic Acids Res.}, volume = {29}, number = {3}, pages = {662--667}, abstract = {The RAD51 protein has been shown to participate in homologous recombination by promoting ATP-dependent homologous pairing and strand transfer reactions. In the present study, we have investigated the possible involvement of RAD51 in non-homologous recombination. We demonstrate that overexpression of CgRAD51 enhances the frequency of spontaneous non-homologous recombination in the hprt gene of Chinese hamster cells. However, the rate of non-homologous recombination induced by the topoisomerase inhibitors campothecin and etoposide was not altered by overexpression of RAD51. These results indicate that the RAD51 protein may perform a function in connection with spontaneous non-homologous recombination that is not essential to or not rate-limiting for non-homologous recombination induced by camptothecin or etoposide. We discuss the possibility that the role played by RAD51 in non-homologous recombination observed here may not be linked to non-homologous end-joining.}, note = {[PubMed Central:hrefhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC30407PMC30407] [PubMed:hrefhttp://www.ncbi.nlm.nih.gov/pubmed/1116088711160887]}, keywords = {}, pubstate = {published}, tppubtype = {article} } The RAD51 protein has been shown to participate in homologous recombination by promoting ATP-dependent homologous pairing and strand transfer reactions. In the present study, we have investigated the possible involvement of RAD51 in non-homologous recombination. We demonstrate that overexpression of CgRAD51 enhances the frequency of spontaneous non-homologous recombination in the hprt gene of Chinese hamster cells. However, the rate of non-homologous recombination induced by the topoisomerase inhibitors campothecin and etoposide was not altered by overexpression of RAD51. These results indicate that the RAD51 protein may perform a function in connection with spontaneous non-homologous recombination that is not essential to or not rate-limiting for non-homologous recombination induced by camptothecin or etoposide. We discuss the possibility that the role played by RAD51 in non-homologous recombination observed here may not be linked to non-homologous end-joining. |
Helleday, T; Johansson, F; Jenssen, D Ŧhe ĐRAG test: an as |