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1
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Marple T, Son MY, Cheng X, Ko JH, Sung P, Hasty P. TREX2 deficiency suppresses spontaneous and genotoxin-associated mutagenesis. Cell Rep 2024; 43:113637. [PMID: 38175749 PMCID: PMC10883656 DOI: 10.1016/j.celrep.2023.113637] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2023] [Revised: 09/15/2023] [Accepted: 12/15/2023] [Indexed: 01/06/2024] Open
Abstract
TREX2, a 3'-5' exonuclease, is a part of the DNA damage tolerance (DDT) pathway that stabilizes replication forks (RFs) by ubiquitinating PCNA along with the ubiquitin E3 ligase RAD18 and other DDT factors. Mismatch repair (MMR) corrects DNA polymerase errors, including base mismatches and slippage. Here we demonstrate that TREX2 deletion reduces mutations in cells upon exposure to genotoxins, including those that cause base lesions and DNA polymerase slippage. Importantly, we show that TREX2 generates most of the spontaneous mutations in MMR-mutant cells derived from mice and people. TREX2-induced mutagenesis is dependent on the nuclease and DNA-binding attributes of TREX2. RAD18 deletion also reduces spontaneous mutations in MMR-mutant cells, albeit to a lesser degree. Inactivation of both MMR and TREX2 additively increases RF stalls, while it decreases DNA breaks, consistent with a synthetic phenotype.
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Affiliation(s)
- Teresa Marple
- Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health San Antonio, San Antonio, TX 78229, USA; Greehey Children's Cancer Research Institute, University of Texas Health San Antonio, San Antonio, TX 78229, USA
| | - Mi Young Son
- Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health San Antonio, San Antonio, TX 78229, USA; Department of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, TX 78229, USA; Sam and Ann Barshop Institute for Longevity and Aging Studies, University of Texas Health San Antonio, San Antonio, TX 78229, USA
| | - Xiaodong Cheng
- Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health San Antonio, San Antonio, TX 78229, USA
| | - Jun Ho Ko
- Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health San Antonio, San Antonio, TX 78229, USA
| | - Patrick Sung
- Department of Biochemistry and Structural Biology, University of Texas Health San Antonio, San Antonio, TX 78229, USA; The Mays Cancer Center, University of Texas Health San Antonio MD Anderson Cancer Center, San Antonio, TX 78229, USA; Greehey Children's Cancer Research Institute, University of Texas Health San Antonio, San Antonio, TX 78229, USA
| | - Paul Hasty
- Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health San Antonio, San Antonio, TX 78229, USA; The Mays Cancer Center, University of Texas Health San Antonio MD Anderson Cancer Center, San Antonio, TX 78229, USA; Sam and Ann Barshop Institute for Longevity and Aging Studies, University of Texas Health San Antonio, San Antonio, TX 78229, USA.
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2
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Tichy ED. Specialized Circuitry of Embryonic Stem Cells Promotes Genomic Integrity. Crit Rev Oncog 2023; 27:1-15. [PMID: 36734869 DOI: 10.1615/critrevoncog.2022042332] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Embryonic stem cells (ESCs) give rise to all cell types of the organism. Given the importance of these cells in this process, ESCs must employ robust mechanisms to protect genomic integrity or risk catastrophic propagation of mutations throughout the organism. Should such an event occur in daughter cells that will eventually contribute to the germline, the overall species health could dramatically decline. This review describes several key mechanisms employed by ESCs that are unique to these cells, in order to maintain their genomic integrity. Additionally, the contributions of cell cycle regulators in modulating ESC differentiation, after DNA damage exposure, are also examined. Where data are available, findings reported in ESCs are extended to include observations described in induced pluripotent stem cells (IPSCs).
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Affiliation(s)
- Elisia D Tichy
- Department of Orthopaedic Surgery, Perelman School of Medicine, The University of Pennsylvania, 371 Stemmler Hall, 3450 Hamilton Walk, Philadelphia, PA 19104-6081
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3
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Guervilly JH, Blin M, Laureti L, Baudelet E, Audebert S, Gaillard PH. SLX4 dampens MutSα-dependent mismatch repair. Nucleic Acids Res 2022; 50:2667-2680. [PMID: 35166826 PMCID: PMC8934664 DOI: 10.1093/nar/gkac075] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2021] [Revised: 01/20/2022] [Accepted: 01/25/2022] [Indexed: 12/12/2022] Open
Abstract
The tumour suppressor SLX4 plays multiple roles in the maintenance of genome stability, acting as a scaffold for structure-specific endonucleases and other DNA repair proteins. It directly interacts with the mismatch repair (MMR) protein MSH2 but the significance of this interaction remained unknown until recent findings showing that MutSβ (MSH2-MSH3) stimulates in vitro the SLX4-dependent Holliday junction resolvase activity. Here, we characterize the mode of interaction between SLX4 and MSH2, which relies on an MSH2-interacting peptide (SHIP box) that drives interaction of SLX4 with both MutSβ and MutSα (MSH2-MSH6). While we show that this MSH2 binding domain is dispensable for the well-established role of SLX4 in interstrand crosslink repair, we find that it mediates inhibition of MutSα-dependent MMR by SLX4, unravelling an unanticipated function of SLX4.
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Affiliation(s)
- Jean-Hugues Guervilly
- Centre de Recherche en Cancérologie de Marseille, CRCM, Inserm, CNRS, Aix-Marseille Université, Institut Paoli-Calmettes, Marseille, France
| | - Marion Blin
- Centre de Recherche en Cancérologie de Marseille, CRCM, Inserm, CNRS, Aix-Marseille Université, Institut Paoli-Calmettes, Marseille, France
| | - Luisa Laureti
- Centre de Recherche en Cancérologie de Marseille, CRCM, Inserm, CNRS, Aix-Marseille Université, Institut Paoli-Calmettes, Marseille, France
| | - Emilie Baudelet
- Centre de Recherche en Cancérologie de Marseille, CRCM, Inserm, CNRS, Aix-Marseille Université, Institut Paoli-Calmettes, Marseille, France
| | - Stéphane Audebert
- Centre de Recherche en Cancérologie de Marseille, CRCM, Inserm, CNRS, Aix-Marseille Université, Institut Paoli-Calmettes, Marseille, France
| | - Pierre-Henri Gaillard
- Centre de Recherche en Cancérologie de Marseille, CRCM, Inserm, CNRS, Aix-Marseille Université, Institut Paoli-Calmettes, Marseille, France
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4
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Perne C, Peters S, Cartolano M, Horpaopan S, Grimm C, Altmüller J, Sommer AK, Hillmer AM, Thiele H, Odenthal M, Möslein G, Adam R, Sivalingam S, Kirfel J, Schweiger MR, Peifer M, Spier I, Aretz S. Variant profiling of colorectal adenomas from three patients of two families with MSH3-related adenomatous polyposis. PLoS One 2021; 16:e0259185. [PMID: 34843512 PMCID: PMC8629245 DOI: 10.1371/journal.pone.0259185] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2020] [Accepted: 10/14/2021] [Indexed: 12/12/2022] Open
Abstract
The spectrum of somatic genetic variation in colorectal adenomas caused by biallelic pathogenic germline variants in the MSH3 gene, was comprehensively analysed to characterise mutational signatures and identify potential driver genes and pathways of MSH3-related tumourigenesis. Three patients from two families with MSH3-associated polyposis were included. Whole exome sequencing of nine adenomas and matched normal tissue was performed. The amount of somatic variants in the MSH3-deficient adenomas and the pattern of single nucleotide variants (SNVs) was similar to sporadic adenomas, whereas the fraction of small insertions/deletions (indels) (21-42% of all small variants) was significantly higher. Interestingly, pathogenic somatic APC variants were found in all but one adenoma. The vast majority (12/13) of these were di-, tetra-, or penta-base pair (bp) deletions. The fraction of APC indels was significantly higher than that reported in patients with familial adenomatous polyposis (FAP) (p < 0.01) or in sporadic adenomas (p < 0.0001). In MSH3-deficient adenomas, the occurrence of APC indels in a repetitive sequence context was significantly higher than in FAP patients (p < 0.01). In addition, the MSH3-deficient adenomas harboured one to five (recurrent) somatic variants in 13 established or candidate driver genes for early colorectal carcinogenesis, including ACVR2A and ARID genes. Our data suggest that MSH3-related colorectal carcinogenesis seems to follow the classical APC-driven pathway. In line with the specific function of MSH3 in the mismatch repair (MMR) system, we identified a characteristic APC mutational pattern in MSH3-deficient adenomas, and confirmed further driver genes for colorectal tumourigenesis.
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Affiliation(s)
- Claudia Perne
- Institute of Human Genetics, Medical Faculty, University of Bonn, Bonn, Germany
- Center for Hereditary Tumor Syndromes, University Hospital Bonn, Bonn, Germany
| | - Sophia Peters
- Institute of Human Genetics, Medical Faculty, University of Bonn, Bonn, Germany
| | - Maria Cartolano
- Department of Translational Genomics, Center of Integrated Oncology Cologne-Bonn, Medical Faculty, University of Cologne, Cologne, Germany
- Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany
| | - Sukanya Horpaopan
- Department of Anatomy, Faculty of Medical Science, Naresuan University, Phitsanulok, Thailand
| | - Christina Grimm
- Institute for Translational Epigenetics, Medical Faculty and University Clinic Cologne, University of Cologne, Cologne, Germany
| | - Janine Altmüller
- Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany
- Cologne Center for Genomics (CCG), Faculty of Medicine, University of Cologne, University Hospital Cologne, Cologne, Germany
- Berlin Institute of Health at Charité, Core Facility Genomics, Berlin, Germany
- Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany
| | - Anna K. Sommer
- Institute of Human Genetics, Medical Faculty, University of Bonn, Bonn, Germany
| | - Axel M. Hillmer
- Institute of Pathology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Holger Thiele
- Cologne Center for Genomics (CCG), Faculty of Medicine, University of Cologne, University Hospital Cologne, Cologne, Germany
| | - Margarete Odenthal
- Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany
- Institute of Pathology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Gabriela Möslein
- Zentrum für Hereditäre Tumore, BETHESDA Khs. Duisburg, Duisburg, Germany
| | - Ronja Adam
- Cancer Center Amsterdam, Amsterdam University Medical Centers, Amsterdam, The Netherlands
| | - Sugirthan Sivalingam
- Core Unit for Bioinformatics Data Analysis, Medical Faculty, University of Bonn, Bonn, Germany
- Institute for Genomic Statistics and Bioinformatics, Medical Faculty, University of Bonn, Bonn, Germany
- Institute for Medical Biometry, Informatics and Epidemiology, Medical Faculty, University of Bonn, Bonn, Germany
| | - Jutta Kirfel
- Institute of Pathology, University of Lübeck, Lübeck, Germany
| | - Michal R. Schweiger
- Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany
- Institute for Translational Epigenetics, Medical Faculty and University Clinic Cologne, University of Cologne, Cologne, Germany
| | - Martin Peifer
- Department of Translational Genomics, Center of Integrated Oncology Cologne-Bonn, Medical Faculty, University of Cologne, Cologne, Germany
| | - Isabel Spier
- Institute of Human Genetics, Medical Faculty, University of Bonn, Bonn, Germany
- Center for Hereditary Tumor Syndromes, University Hospital Bonn, Bonn, Germany
| | - Stefan Aretz
- Institute of Human Genetics, Medical Faculty, University of Bonn, Bonn, Germany
- Center for Hereditary Tumor Syndromes, University Hospital Bonn, Bonn, Germany
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5
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Single-Strand Annealing in Cancer. Int J Mol Sci 2021; 22:ijms22042167. [PMID: 33671579 PMCID: PMC7926775 DOI: 10.3390/ijms22042167] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2021] [Revised: 02/18/2021] [Accepted: 02/19/2021] [Indexed: 12/23/2022] Open
Abstract
DNA double-strand breaks (DSBs) are among the most serious forms of DNA damage. In humans, DSBs are repaired mainly by non-homologous end joining (NHEJ) and homologous recombination repair (HRR). Single-strand annealing (SSA), another DSB repair system, uses homologous repeats flanking a DSB to join DNA ends and is error-prone, as it removes DNA fragments between repeats along with one repeat. Many DNA deletions observed in cancer cells display homology at breakpoint junctions, suggesting the involvement of SSA. When multiple DSBs occur in different chromosomes, SSA may result in chromosomal translocations, essential in the pathogenesis of many cancers. Inhibition of RAD52 (RAD52 Homolog, DNA Repair Protein), the master regulator of SSA, results in decreased proliferation of BRCA1/2 (BRCA1/2 DNA Repair Associated)-deficient cells, occurring in many hereditary breast and ovarian cancer cases. Therefore, RAD52 may be targeted in synthetic lethality in cancer. SSA may modulate the response to platinum-based anticancer drugs and radiation. SSA may increase the efficacy of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR associated 9) genome editing and reduce its off-target effect. Several basic problems associated with SSA, including its evolutionary role, interplay with HRR and NHEJ and should be addressed to better understand its role in cancer pathogenesis and therapy.
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6
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Mani C, Reddy PH, Palle K. DNA repair fidelity in stem cell maintenance, health, and disease. Biochim Biophys Acta Mol Basis Dis 2019; 1866:165444. [PMID: 30953688 DOI: 10.1016/j.bbadis.2019.03.017] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2018] [Revised: 12/20/2018] [Accepted: 01/06/2019] [Indexed: 12/13/2022]
Abstract
Stem cells are a sub population of cell types that form the foundation of our body, and have the potential to replicate, replenish and repair limitlessly to maintain the tissue and organ homeostasis. Increased lifetime and frequent replication set them vulnerable for both exogenous and endogenous agents-induced DNA damage compared to normal cells. To counter these damages and preserve genetic information, stem cells have evolved with various DNA damage response and repair mechanisms. Furthermore, upon experiencing irreparable DNA damage, stem cells mostly prefer early senescence or apoptosis to avoid the accumulation of damages. However, the failure of these mechanisms leads to various diseases, including cancer. Especially, given the importance of stem cells in early development, DNA repair deficiency in stem cells leads to various disabilities like developmental delay, premature aging, sensitivity to DNA damaging agents, degenerative diseases, etc. In this review, we have summarized the recent update about how DNA repair mechanisms are regulated in stem cells and their association with disease progression and pathogenesis.
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Affiliation(s)
- Chinnadurai Mani
- Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Centre, Lubbock, TX 79430, United States of America
| | - P Hemachandra Reddy
- Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Centre, Lubbock, TX 79430, United States of America
| | - Komaraiah Palle
- Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Centre, Lubbock, TX 79430, United States of America.
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7
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Gupta D, Heinen CD. The mismatch repair-dependent DNA damage response: Mechanisms and implications. DNA Repair (Amst) 2019; 78:60-69. [PMID: 30959407 DOI: 10.1016/j.dnarep.2019.03.009] [Citation(s) in RCA: 75] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2018] [Revised: 02/25/2019] [Accepted: 03/16/2019] [Indexed: 12/22/2022]
Abstract
An important role for the DNA mismatch repair (MMR) pathway in maintaining genomic stability is embodied in its conservation through evolution and the link between loss of MMR function and tumorigenesis. The latter is evident as inheritance of mutations within the major MMR genes give rise to the cancer predisposition condition, Lynch syndrome. Nonetheless, how MMR loss contributes to tumorigenesis is not completely understood. In addition to preventing the accumulation of mutations, MMR also directs cellular responses, such as cell cycle checkpoint or apoptosis activation, to different forms of DNA damage. Understanding this MMR-dependent DNA damage response may provide insight into the full tumor suppressing capabilities of the MMR pathway. Here, we delve into the proposed mechanisms for the MMR-dependent response to DNA damaging agents. We discuss how these pre-clinical findings extend to the clinical treatment of cancers, emphasizing MMR status as a crucial variable in selection of chemotherapeutic regimens. Also, we discuss how loss of the MMR-dependent damage response could promote tumorigenesis via the establishment of a survival advantage to endogenous levels of stress in MMR-deficient cells.
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Affiliation(s)
- Dipika Gupta
- Center for Molecular Oncology, UConn Health, Farmington, CT 06030, USA
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8
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Jahid S, Sun J, Gelincik O, Blecua P, Edelmann W, Kucherlapati R, Zhou K, Jasin M, Gümüş ZH, Lipkin SM. Inhibition of colorectal cancer genomic copy number alterations and chromosomal fragile site tumor suppressor FHIT and WWOX deletions by DNA mismatch repair. Oncotarget 2017; 8:71574-71586. [PMID: 29069730 PMCID: PMC5641073 DOI: 10.18632/oncotarget.17776] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2017] [Accepted: 04/24/2017] [Indexed: 01/15/2023] Open
Abstract
Homologous recombination (HR) enables precise DNA repair after DNA double strand breaks (DSBs) using identical sequence templates, whereas homeologous recombination (HeR) uses only partially homologous sequences. Homeologous recombination introduces mutations through gene conversion and genomic deletions through single-strand annealing (SSA). DNA mismatch repair (MMR) inhibits HeR, but the roles of mammalian MMR MutL homologues (MLH1, PMS2 and MLH3) proteins in HeR suppression are poorly characterized. Here, we demonstrate that mouse embryonic fibroblasts (MEFs) carrying Mlh1, Pms2, and Mlh3 mutations have higher HeR rates, by using 7,863 uniquely mapping paired direct repeat sequences (DRs) in the mouse genome as endogenous gene conversion and SSA reporters. Additionally, when DSBs are induced by gamma-radiation, Mlh1, Pms2 and Mlh3 mutant MEFs have higher DR copy number alterations (CNAs), including DR CNA hotspots previously identified in mouse MMR-deficient colorectal cancer (dMMR CRC). Analysis of The Cancer Genome Atlas CRC data revealed that dMMR CRCs have higher genome-wide DR HeR rates than MMR proficient CRCs, and that dMMR CRCs have deletion hotspots in tumor suppressors FHIT/WWOX at chromosomal fragile sites FRA3B and FRA16D (which have elevated DSB rates) flanked by paired homologous DRs and inverted repeats (IR). Overall, these data provide novel insights into the MMR-dependent HeR inhibition mechanism and its role in tumor suppression.
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Affiliation(s)
- Sohail Jahid
- Departments of Medicine and Genetic Medicine, Weill Cornell Medicine, 10021, NY, USA
| | - Jian Sun
- Departments of Medicine and Genetic Medicine, Weill Cornell Medicine, 10021, NY, USA
| | - Ozkan Gelincik
- Departments of Medicine and Genetic Medicine, Weill Cornell Medicine, 10021, NY, USA
| | - Pedro Blecua
- Division of Clinical Genetics, Memorial Sloan Kettering Cancer Center, 10065, NY, USA
| | - Winfried Edelmann
- Department of Cell Biology and Department of Genetics, Albert Einstein College of Medicine of Yeshiva University, 10461, NY, USA
| | - Raju Kucherlapati
- Department of Genetics, Harvard Medical School, 02115, Boston, MA, USA
| | - Kathy Zhou
- Department of Biostatistics and Epidemiology, Weill Cornell Medical College, 10021, NY, USA
| | - Maria Jasin
- Developmental Biology Program, Memorial Sloan Kettering Cancer Center, 10065, NY, USA
| | - Zeynep H Gümüş
- Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, 10029, NY, USA.,Icahn Institute for Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, 10029, NY, USA
| | - Steven M Lipkin
- Departments of Medicine and Genetic Medicine, Weill Cornell Medicine, 10021, NY, USA
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9
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Glaire MA, Brown M, Church DN, Tomlinson I. Cancer predisposition syndromes: lessons for truly precision medicine. J Pathol 2017; 241:226-235. [PMID: 27859368 DOI: 10.1002/path.4842] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2016] [Revised: 11/04/2016] [Accepted: 11/05/2016] [Indexed: 02/11/2024]
Abstract
Cancer predisposition syndromes are typically uncommon, monogenic, high-penetrance disorders. Despite their rarity, they have proven to be highly clinically relevant in directing cancer prevention strategies. As such, they share notable similarities with an expanding class of low-frequency somatic mutations that are associated with a striking prognostic or predictive effect in the tumours in which they occur. In this review, we highlight these commonalities, with particular reference to mutations in the proofreading domain of replicative DNA polymerases. These molecular phenotypes may occur as either germline or somatic events, and in the latter case, have been shown to confer a favourable prognosis and potential increased benefit from immune checkpoint inhibition. We note that incorporation of these variants into clinical management algorithms will help refine patient management, and that this will be further improved by the inclusion of other germline variants, such as those that determine the likelihood of benefit or toxicity from anti-neoplastic therapy. Finally, we propose that such integrated patient and tumour profiling will be essential if we are to deliver truly precision medicine for cancer patients, but in a similar way to rare germline mutations, we must ensure that we identify and utilize rare somatic mutations with strong predictive and prognostic effects. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Affiliation(s)
- Mark A Glaire
- Tumour Genomics and Immunology Group, The Oxford Centre for Cancer Gene Research, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK
| | - Matthew Brown
- Tumour Genomics and Immunology Group, The Oxford Centre for Cancer Gene Research, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK
| | - David N Church
- Tumour Genomics and Immunology Group, The Oxford Centre for Cancer Gene Research, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK
| | - Ian Tomlinson
- Molecular and Population Genetics Laboratory, The Oxford Centre for Cancer Gene Research, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK
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10
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Kuhner MK, Kostadinov R, Reid BJ. Limitations of the Driver/Passenger Model in Cancer Prevention. Cancer Prev Res (Phila) 2016; 9:335-8. [PMID: 26932841 DOI: 10.1158/1940-6207.capr-15-0343] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2015] [Accepted: 02/16/2016] [Indexed: 01/04/2023]
Abstract
Mutations detected in cancers are often divided into "drivers" and "passengers." We suggest that this classification is potentially misleading for purposes of early detection and prevention. Specifically, some mutations are frequent in tumors and thus appear to be drivers, but are poor predictors of cancer; other mutations are individually rare and thus appear to be passengers, but may collectively explain a large proportion of risk. The assumptions bundled into the terms "driver" and "passenger" can lead to misunderstandings of neoplastic progression, with unintended consequences including overdiagnosis, overtreatment, and failure to identify the true sources of risk. We argue that samples from healthy, benign, or neoplastic tissues are critical for evaluating the risk of future cancer posed by mutations in a given gene. Cancer Prev Res; 9(5); 335-8. ©2016 AACR.
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Affiliation(s)
- Mary K Kuhner
- Department of Genome Sciences, University of Washington, Seattle, Washington.
| | - Rumen Kostadinov
- Pediatric Oncology, Sidney Kimmel Comprehensive Cancer Center, School of Medicine, Johns Hopkins University, Baltimore, Maryland
| | - Brian J Reid
- Divisions of Human Biology and Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington
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11
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Tokunaga A, Anai H, Hanada K. Mechanisms of gene targeting in higher eukaryotes. Cell Mol Life Sci 2016; 73:523-33. [PMID: 26507245 PMCID: PMC11108335 DOI: 10.1007/s00018-015-2073-1] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2015] [Revised: 10/14/2015] [Accepted: 10/14/2015] [Indexed: 10/22/2022]
Abstract
Targeted genome modifications using techniques that alter the genomic information of interest have contributed to multiple studies in both basic and applied biology. Traditionally, in gene targeting, the target-site integration of a targeting vector by homologous recombination is used. However, this strategy has several technical problems. The first problem is the extremely low frequency of gene targeting, which makes obtaining recombinant clones an extremely labor intensive task. The second issue is the limited number of biomaterials to which gene targeting can be applied. Traditional gene targeting hardly occurs in most of the human adherent cell lines. However, a new approach using designer nucleases that can introduce site-specific double-strand breaks in genomic DNAs has increased the efficiency of gene targeting. This new method has also expanded the number of biomaterials to which gene targeting could be applied. Here, we summarize various strategies for target gene modification, including a comparison of traditional gene targeting with designer nucleases.
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Affiliation(s)
- Akinori Tokunaga
- The Tokunaga Laboratory, Faculty of Medicine, Oita University, 1-1 Idaigaoka, Hasama-machi, Yufu, Oita, 879-5593, Japan
- Section of Physiology, Department of Integrative Aging Neuroscience, National Center for Geriatrics and Gerontology (NCGG), 7-430, Morioka-cho, Obu, Aichi, 474-8511, Japan
| | - Hirofumi Anai
- Clinical Engineering Research Center, Faculty of Medicine, Oita University, 1-1 Idaigaoka, Hasama-machi, Yufu, Oita, 879-5593, Japan
| | - Katsuhiro Hanada
- Clinical Engineering Research Center, Faculty of Medicine, Oita University, 1-1 Idaigaoka, Hasama-machi, Yufu, Oita, 879-5593, Japan.
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12
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Mismatch repair and homeologous recombination. DNA Repair (Amst) 2015; 38:75-83. [PMID: 26739221 DOI: 10.1016/j.dnarep.2015.11.010] [Citation(s) in RCA: 68] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2015] [Revised: 10/26/2015] [Accepted: 11/30/2015] [Indexed: 12/27/2022]
Abstract
DNA mismatch repair influences the outcome of recombination events between diverging DNA sequences. Here we discuss how mismatch repair proteins are active in different homologous recombination subpathways and specific reaction steps, resulting in differential modulation of these recombination events, with a focus on the mechanism of heteroduplex rejection during the inhibition of recombination between slightly diverged (homeologous) DNA sequences.
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13
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Zhang F, Shi J, Chen SH, Bian C, Yu X. The PIN domain of EXO1 recognizes poly(ADP-ribose) in DNA damage response. Nucleic Acids Res 2015; 43:10782-94. [PMID: 26400172 PMCID: PMC4678857 DOI: 10.1093/nar/gkv939] [Citation(s) in RCA: 43] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2015] [Accepted: 09/08/2015] [Indexed: 11/14/2022] Open
Abstract
Following DNA double-strand breaks, poly(ADP-ribose) (PAR) is quickly and heavily synthesized to mediate fast and early recruitment of a number of DNA damage response factors to the sites of DNA lesions and facilitates DNA damage repair. Here, we found that EXO1, an exonuclease for DNA damage repair, is quickly recruited to the sites of DNA damage via PAR-binding. With further dissection of the functional domains of EXO1, we report that the PIN domain of EXO1 recognizes PAR both in vitro and in vivo and the interaction between the PIN domain and PAR is sufficient for the recruitment. We also found that the R93G variant of EXO1, generated by a single nucleotide polymorphism, abolishes the interaction and the early recruitment. Moreover, our study suggests that the PAR-mediated fast recruitment of EXO1 facilities early DNA end resection, the first step of homologous recombination repair. We observed that other PIN domains could also recognize DNA damage-induced PAR. Taken together, our study demonstrates a novel class of PAR-binding module that plays an important role in DNA damage response.
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Affiliation(s)
- Feng Zhang
- College of Life and Environment Sciences, Shanghai Normal University, Guilin Road 100, Shanghai 200234, China Division of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan Medical School, 1150 W. Medical Center Drive, 5560 MSRBII, Ann Arbor, MI 48109, USA
| | - Jiazhong Shi
- Division of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan Medical School, 1150 W. Medical Center Drive, 5560 MSRBII, Ann Arbor, MI 48109, USA Department of Cell Biology, the Third Military Medical University, Chongqing, 400038, China
| | - Shih-Hsun Chen
- Division of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan Medical School, 1150 W. Medical Center Drive, 5560 MSRBII, Ann Arbor, MI 48109, USA Department of Radiation Biology, Beckman Research Institute, City of Hope, Duarte, CA 91773, USA
| | - Chunjing Bian
- Division of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan Medical School, 1150 W. Medical Center Drive, 5560 MSRBII, Ann Arbor, MI 48109, USA Department of Radiation Biology, Beckman Research Institute, City of Hope, Duarte, CA 91773, USA
| | - Xiaochun Yu
- Division of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan Medical School, 1150 W. Medical Center Drive, 5560 MSRBII, Ann Arbor, MI 48109, USA Department of Radiation Biology, Beckman Research Institute, City of Hope, Duarte, CA 91773, USA
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14
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Traver S, Coulombe P, Peiffer I, Hutchins J, Kitzmann M, Latreille D, Méchali M. MCM9 Is Required for Mammalian DNA Mismatch Repair. Mol Cell 2015; 59:831-9. [DOI: 10.1016/j.molcel.2015.07.010] [Citation(s) in RCA: 44] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2015] [Revised: 05/23/2015] [Accepted: 07/15/2015] [Indexed: 10/23/2022]
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15
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Bazrgar M, Gourabi H, Yazdi PE, Vazirinasab H, Fakhri M, Hassani F, Valojerdi MR. DNA repair signalling pathway genes are overexpressed in poor-quality pre-implantation human embryos with complex aneuploidy. Eur J Obstet Gynecol Reprod Biol 2014; 175:152-6. [DOI: 10.1016/j.ejogrb.2014.01.010] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2013] [Revised: 09/25/2013] [Accepted: 01/04/2014] [Indexed: 11/26/2022]
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16
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Koike-Yusa H, Li Y, Tan EP, Velasco-Herrera MDC, Yusa K. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat Biotechnol 2014; 32:267-73. [PMID: 24535568 DOI: 10.1038/nbt.2800] [Citation(s) in RCA: 796] [Impact Index Per Article: 72.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2013] [Accepted: 12/17/2013] [Indexed: 12/23/2022]
Abstract
Identification of genes influencing a phenotype of interest is frequently achieved through genetic screening by RNA interference (RNAi) or knockouts. However, RNAi may only achieve partial depletion of gene activity, and knockout-based screens are difficult in diploid mammalian cells. Here we took advantage of the efficiency and high throughput of genome editing based on type II, clustered, regularly interspaced, short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems to introduce genome-wide targeted mutations in mouse embryonic stem cells (ESCs). We designed 87,897 guide RNAs (gRNAs) targeting 19,150 mouse protein-coding genes and used a lentiviral vector to express these gRNAs in ESCs that constitutively express Cas9. Screening the resulting ESC mutant libraries for resistance to either Clostridium septicum alpha-toxin or 6-thioguanine identified 27 known and 4 previously unknown genes implicated in these phenotypes. Our results demonstrate the potential for efficient loss-of-function screening using the CRISPR-Cas9 system.
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Affiliation(s)
| | - Yilong Li
- 1] Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK. [2]
| | - E-Pien Tan
- Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK
| | | | - Kosuke Yusa
- Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK
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17
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Martin LM, Marples B, Davies AM, Atzberger A, Edwards C, Lynch TH, Hollywood D, Marignol L. DNA mismatch repair protein MSH2 dictates cellular survival in response to low dose radiation in endometrial carcinoma cells. Cancer Lett 2013; 335:19-25. [DOI: 10.1016/j.canlet.2013.01.046] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2012] [Revised: 01/24/2013] [Accepted: 01/24/2013] [Indexed: 11/24/2022]
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18
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The piggyBac transposon displays local and distant reintegration preferences and can cause mutations at noncanonical integration sites. Mol Cell Biol 2013; 33:1317-30. [PMID: 23358416 DOI: 10.1128/mcb.00670-12] [Citation(s) in RCA: 67] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
The DNA transposon piggyBac is widely used as a tool in mammalian experimental systems for transgenesis, mutagenesis, and genome engineering. We have characterized genome-wide insertion site preferences of piggyBac by sequencing a large set of integration sites arising from transposition from two separate genomic loci and a plasmid donor in mouse embryonic stem cells. We found that piggyBac preferentially integrates locally to the excision site when mobilized from a chromosomal location and identified other nonlocal regions of the genome with elevated insertion frequencies. piggyBac insertions were associated with expressed genes and markers of open chromatin structure and were excluded from heterochromatin. At the nucleotide level, piggyBac prefers to insert into TA-rich regions within a broader GC-rich context. We also found that piggyBac can insert into sites other than its known TTAA insertion site at a low frequency (2%). Such insertions introduce mismatches that are repaired with signatures of host cell repair pathways. Transposons could be mobilized from plasmids with the observed noncanonical flanking regions, indicating that piggyBac could generate point mutations in the genome.
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19
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Campo VA, Patenaude AM, Kaden S, Horb L, Firka D, Jiricny J, Di Noia JM. MSH6- or PMS2-deficiency causes re-replication in DT40 B cells, but it has little effect on immunoglobulin gene conversion or on repair of AID-generated uracils. Nucleic Acids Res 2013; 41:3032-46. [PMID: 23314153 PMCID: PMC3597665 DOI: 10.1093/nar/gks1470] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
The mammalian antibody repertoire is shaped by somatic hypermutation (SHM) and class switch recombination (CSR) of the immunoglobulin (Ig) loci of B lymphocytes. SHM and CSR are triggered by non-canonical, error-prone processing of G/U mismatches generated by activation-induced deaminase (AID). In birds, AID does not trigger SHM, but it triggers Ig gene conversion (GC), a ‘homeologous’ recombination process involving the Ig variable region and proximal pseudogenes. Because recombination fidelity is controlled by the mismatch repair (MMR) system, we investigated whether MMR affects GC in the chicken B cell line DT40. We show here that Msh6−/− and Pms2−/− DT40 cells display cell cycle defects, including genomic re-replication. However, although IgVλ GC tracts in MMR-deficient cells were slightly longer than in normal cells, Ig GC frequency, donor choice or the number of mutations per sequence remained unaltered. The finding that the avian MMR system, unlike that of mammals, does not seem to contribute towards the processing of G/U mismatches in vitro could explain why MMR is unable to initiate Ig GC in this species, despite initiating SHM and CSR in mammalian cells. Moreover, as MMR does not counteract or govern Ig GC, we report a rare example of ‘homeologous’ recombination insensitive to MMR.
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Affiliation(s)
- Vanina A Campo
- Institut de Recherches Cliniques de Montréal, Division of Immunity and Viral Infections, Montréal, H2W 1R7 Québec, Canada
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20
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Tichy ED. Mechanisms maintaining genomic integrity in embryonic stem cells and induced pluripotent stem cells. Exp Biol Med (Maywood) 2011; 236:987-96. [PMID: 21768163 DOI: 10.1258/ebm.2011.011107] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
Embryonic stem cells (ESCs) are pluripotent, self-renewing cells that are isolated during the blastocyst stage of embryonic development. Whether these cells are derived from humans, mice or other organisms, all ESCs must employ mechanisms that prevent the propagation of mutations, generated as a consequence of DNA damage, to somatic cells produced by normal programmed differentiation. Thus, the prevention of mutations in ESCs is important not only for the health of the individual organism derived from these cells but also, in addition, for the continued survival and genetic viability of the species by preventing the accumulation of mutations in the germline. Induced pluripotent stem cells (IPSCs) are reprogrammed somatic cells that share several characteristics with ESCs, including a similar morphology in culture, the re-expression of pluripotency markers and the ability to differentiate into defined cell lineages. This review focuses on the mechanisms employed by murine ESCs, human ESCs and, where data are available, IPSCs to preserve genetic integrity.
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Affiliation(s)
- Elisia D Tichy
- Department of Molecular Genetics, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0524, USA.
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21
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Belcheva A, Kolaj B, Martin A. Missing mismatch repair: a key to T cell immortality. Leuk Lymphoma 2011; 51:1777-8. [PMID: 20858090 DOI: 10.3109/10428194.2010.516377] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Affiliation(s)
- Antoaneta Belcheva
- Department of Immunology, University of Toronto, Medical Sciences Building, Toronto, Canada
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22
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Takahashi M, Koi M, Balaguer F, Boland CR, Goel A. MSH3 mediates sensitization of colorectal cancer cells to cisplatin, oxaliplatin, and a poly(ADP-ribose) polymerase inhibitor. J Biol Chem 2011; 286:12157-65. [PMID: 21285347 DOI: 10.1074/jbc.m110.198804] [Citation(s) in RCA: 68] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
The MSH3 gene is one of the DNA mismatch repair (MMR) genes that has undergone somatic mutation frequently in MMR-deficient cancers. MSH3, together with MSH2, forms the MutSβ heteroduplex, which interacts with interstrand cross-links (ICLs) induced by drugs such as cisplatin and psoralen. However, the precise role of MSH3 in mediating the cytotoxic effects of ICL-inducing agents remains poorly understood. In this study, we first examined the effects of MSH3 deficiency on cytotoxicity caused by cisplatin and oxaliplatin, another ICL-inducing platinum drug. Using isogenic HCT116-derived clones in which MSH3 expression is controlled by shRNA expression in a Tet-off system, we discovered that MSH3 deficiency sensitized cells to both cisplatin and oxaliplatin at clinically relevant doses. Interestingly, siRNA-induced down-regulation of the MLH1 protein did not affect MSH3-dependent toxicity of these drugs, indicating that this process does not require participation of the canonical MMR pathway. Furthermore, MSH3-deficient cells maintained higher levels of phosphorylated histone H2AX and 53BP1 after oxaliplatin treatment in comparison with MSH3-proficient cells, suggesting that MSH3 plays an important role in repairing DNA double strand breaks (DSBs). This role of MSH3 was further supported by our findings that MSH3-deficient cells were sensitive to olaparib, a poly(ADP-ribose) polymerase inhibitor. Moreover, the combination of oxaliplatin and olaparib exhibited a synergistic effect compared with either treatment individually. Collectively, our results provide novel evidence that MSH3 deficiency contributes to the cytotoxicity of platinum drugs through deficient DSB repair. These data lay the foundation for the development of effective prediction and treatments for cancers with MSH3 deficiency.
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Affiliation(s)
- Masanobu Takahashi
- Gastrointestinal Cancer Research Laboratory, Division of Gastroenterology, Department of Internal Medicine, Charles A. Sammons Cancer Center and Baylor Research Institute, Baylor University Medical Center, Dallas, Texas 75246-2017, USA
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23
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Wobus AM, Löser P. Present state and future perspectives of using pluripotent stem cells in toxicology research. Arch Toxicol 2011; 85:79-117. [PMID: 21225242 PMCID: PMC3026927 DOI: 10.1007/s00204-010-0641-6] [Citation(s) in RCA: 84] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2010] [Accepted: 12/21/2010] [Indexed: 02/08/2023]
Abstract
The use of novel drugs and chemicals requires reliable data on their potential toxic effects on humans. Current test systems are mainly based on animals or in vitro–cultured animal-derived cells and do not or not sufficiently mirror the situation in humans. Therefore, in vitro models based on human pluripotent stem cells (hPSCs) have become an attractive alternative. The article summarizes the characteristics of pluripotent stem cells, including embryonic carcinoma and embryonic germ cells, and discusses the potential of pluripotent stem cells for safety pharmacology and toxicology. Special attention is directed to the potential application of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) for the assessment of developmental toxicology as well as cardio- and hepatotoxicology. With respect to embryotoxicology, recent achievements of the embryonic stem cell test (EST) are described and current limitations as well as prospects of embryotoxicity studies using pluripotent stem cells are discussed. Furthermore, recent efforts to establish hPSC-based cell models for testing cardio- and hepatotoxicity are presented. In this context, methods for differentiation and selection of cardiac and hepatic cells from hPSCs are summarized, requirements and implications with respect to the use of these cells in safety pharmacology and toxicology are presented, and future challenges and perspectives of using hPSCs are discussed.
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Affiliation(s)
- Anna M Wobus
- In Vitro Differentiation Group, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466 Gatersleben, Germany.
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24
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Martin LM, Marples B, Coffey M, Lawler M, Lynch TH, Hollywood D, Marignol L. DNA mismatch repair and the DNA damage response to ionizing radiation: Making sense of apparently conflicting data. Cancer Treat Rev 2010; 36:518-27. [PMID: 20413225 DOI: 10.1016/j.ctrv.2010.03.008] [Citation(s) in RCA: 57] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2010] [Revised: 03/12/2010] [Accepted: 03/21/2010] [Indexed: 10/19/2022]
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25
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Cooley N, Elder RH, Povey AC. The effect of Msh2 knockdown on methylating agent induced toxicity in DNA glycosylase deficient cells. Toxicology 2009; 268:111-7. [PMID: 20025921 DOI: 10.1016/j.tox.2009.12.008] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2009] [Revised: 11/18/2009] [Accepted: 12/10/2009] [Indexed: 01/17/2023]
Abstract
The DNA structure recognition protein MSH2 is an important protein in DNA mismatch repair due to its role in initiating the repair process. To examine the potential interactions between mismatch repair and base excision repair (BER) we have examined the effect of MSH2 knockdown on 6-thioguanine (6-TG), temozolomide (TMZ) and methylmethane sulphonate (MMS) induced toxicity in BER proficient and deficient cell lines. An shRNA expression vector containing Msh2 target sequences was designed and used to transfect mouse embryonic fibroblasts lacking either alkylpurine DNA N-glycosylase (Mpg) or endonuclease III homologue (Nth1). Significant knockdown of Msh2 gene expression was achieved with three different target sequences, with the highest level being shown by Msh2(283). Clonal selection resulted in differing levels of knockdown in Mpg(-/-) cells: (69.0+/-12.1% from 5 cell clones). Transfection of the Msh2(283) sequence in Mpg+/+, Nth1+/+ and Nth1(-/-) cells resulted in average knockdowns of 45.1+/-40.5% (3 clones), 58.0+/-21.4% (5 clones) and 74.9+/-14.8% (3 clones), respectively. Msh2 knockdown resulted in increased resistance to 6-TG in BER (MPG and NTH1) proficient and deficient cell lines with similar levels of knockdown (84+/-4%) but increased resistance to TMZ only in Mpg+/+ and Nth1(-/-) cell lines and not Mpg(-/-) or Nth1+/+ cells as assessed by an MTT assay. Msh2 knockdown had no effect on sensitivity to MMS induced toxicity. In a clonogenic assay, Msh2 silenced Mpg+/+, Mpg(-/-), Nth1+/+ and Nth1(-/-) cells were more resistant to TMZ. These results confirm previous studies showing that MSH2 is a key protein in influencing 6-TG and O(6)-methylguanine induced toxicity but also suggest that the effect of this protein depends upon the presence of other proteins in different DNA repair pathways.
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Affiliation(s)
- N Cooley
- Centre for Occupational and Environmental Health, School of Community Based Medicine, Faculty of Medical and Human Sciences, University of Manchester, Manchester M139PL, United Kingdom
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26
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Siehler SY, Schrauder M, Gerischer U, Cantor S, Marra G, Wiesmüller L. Human MutL-complexes monitor homologous recombination independently of mismatch repair. DNA Repair (Amst) 2008; 8:242-52. [PMID: 19022408 DOI: 10.1016/j.dnarep.2008.10.011] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2008] [Revised: 09/10/2008] [Accepted: 10/21/2008] [Indexed: 12/19/2022]
Abstract
The role of mismatch repair proteins has been well studied in the context of DNA repair following DNA polymerase errors. Particularly in yeast, MSH2 and MSH6 have also been implicated in the regulation of genetic recombination, whereas MutL homologs appeared to be less important. So far, little is known about the role of the human MutL homolog hMLH1 in recombination, but recently described molecular interactions suggest an involvement. To identify activities of hMLH1 in this process, we applied an EGFP-based assay for the analysis of different mechanisms of DNA repair, initiated by a targeted double-stranded DNA break. We analysed 12 human cellular systems, differing in the hMLH1 and concomitantly in the hPMS1 and hPMS2 status via inducible protein expression, genetic reconstitution, or RNA interference. We demonstrate that hMLH1 and its complex partners hPMS1 and hPMS2 downregulate conservative homologous recombination (HR), particularly when involving DNA sequences with only short stretches of uninterrupted homology. Unexpectedly, hMSH2 is dispensable for this effect. Moreover, the damage-signaling kinase ATM and its substrates BLM and BACH1 are not strictly required, but the combined effect of ATM/ATR-signaling components may mediate the anti-recombinogenic effect. Our data indicate a protective role of hMutL-complexes in a process which may lead to detrimental genome rearrangements, in a manner which does not depend on mismatch repair.
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27
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The rate and spectrum of microsatellite mutation in Caenorhabditis elegans and Daphnia pulex. Genetics 2008; 178:2113-21. [PMID: 18430937 DOI: 10.1534/genetics.107.081927] [Citation(s) in RCA: 59] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
The effective use of microsatellite loci as tools for microevolutionary analysis requires knowledge of the factors influencing the rate and pattern of mutation, much of which is derived from indirect inference from population samples. Interspecific variation in microsatellite stability also provides a glimpse into aspects of phylogenetic constancy of mutational processes. Using long-term series of mutation-accumulation lines, we have obtained direct estimates of the spectrum of microsatellite mutations in two model systems: the nematode Caenorhabditis elegans and the microcrustacean Daphnia pulex. Although the scaling of the mutation rate with the number of tandem repeats is highly consistent across distantly related species, including yeast and human, the per-cell-division mutation rate appears to be elevated in multicellular species. Contrary to the expectations under the stepwise mutation model, most microsatellite mutations in C. elegans and D. pulex involve changes of multiple repeat units, with expansions being much more common than contractions.
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28
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Pineda M, Castellsagué E, Musulén E, Llort G, Frebourg T, Baert-Desurmont S, González S, Capellá G, Blanco I. Non-Hodgkin lymphoma related to hereditary nonpolyposis colorectal cancer in a patient with a novel heterozygous complex deletion in theMSH2 gene. Genes Chromosomes Cancer 2008; 47:326-32. [DOI: 10.1002/gcc.20536] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
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29
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Vaish M. Mismatch repair deficiencies transforming stem cells into cancer stem cells and therapeutic implications. Mol Cancer 2007; 6:26. [PMID: 17407576 PMCID: PMC1851711 DOI: 10.1186/1476-4598-6-26] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2007] [Accepted: 04/02/2007] [Indexed: 01/02/2023] Open
Abstract
For the exceptional self-renewal capacity, regulated cell proliferation and differential potential to a wide variety of cell types, the stem cells must maintain the intact genome. The cells under continuous exogenous and endogenous genotoxic stress accumulate DNA errors, drive proliferative expansion and transform into cancer stem cells with a heterogeneous population of tumor cells. These cells are a common phenomenon for the hematological malignancies and solid tumors. In response to DNA damage, the complex cellular mechanisms including cell cycle arrest, transcription induction and DNA repair are activated. The cells when exposed to cytotoxic agents, the apoptosis lead to cell death. However, the absence of repair machinery makes the cells resistant to tumor sensitizing agents and result in malignant transformation. Mismatch repair gene defects are recently identified in hematopoietic malignancies, leukemia and lymphoma cell lines. This review emphasizes the importance of MMR systems in maintaining the stem cell functioning and its therapeutic implications in the eradication of cancer stem cells and differentiated tumor cells as well. The understanding of the biological functions of mismatch repair in the stem cells and its malignant counterparts could help in developing an effective novel therapies leaving residual non-tumorigenic population of cells resulting in potential cancer cures.
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Affiliation(s)
- Minal Vaish
- Department of Biochemistry, University of Lucknow-226007, UP, India.
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30
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Trouiller B, Schaefer DG, Charlot F, Nogué F. MSH2 is essential for the preservation of genome integrity and prevents homeologous recombination in the moss Physcomitrella patens. Nucleic Acids Res 2006; 34:232-42. [PMID: 16397301 PMCID: PMC1325206 DOI: 10.1093/nar/gkj423] [Citation(s) in RCA: 65] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
MSH2 is a central component of the mismatch repair pathway that targets mismatches arising during DNA replication, homologous recombination (HR) and in response to genotoxic stresses. Here, we describe the function of MSH2 in the moss Physcomitrella patens, as deciphered by the analysis of loss of function mutants. Ppmsh2 mutants display pleiotropic growth and developmental defects, which reflect genomic instability. Based on loss of function of the APT gene, we estimated this mutator phenotype to be at least 130 times higher in the mutants than in wild type. We also found that MSH2 is involved in some but not all the moss responses to genotoxic stresses we tested. Indeed, the Ppmsh2 mutants were more tolerant to cisplatin and show higher sensitivity to UV-B radiations. PpMSH2 gene involvement in HR was studied by assessing gene targeting (GT) efficiency with homologous and homeologous sequences. GT efficiency with homologous sequences was slightly decreased in the Ppmsh2 mutant compared with wild type. Strikingly GT efficiency with homeologous sequences decreased proportionally to sequence divergence in the wild type whereas it remained unaffected in the mutants. Those results demonstrate the role of PpMSH2 in the maintenance of genome integrity and in homologous and homeologous recombination.
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Affiliation(s)
| | - Didier G. Schaefer
- Département de biologie moléculaire végétale, Université de LausanneCH-1015 Lausanne, Switzerland
| | | | - Fabien Nogué
- To whom correspondence should be addressed. Tel: +33 1 30833009; Fax: +33 1 30833319;
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31
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Marple T, Li H, Hasty P. A genotoxic screen: rapid analysis of cellular dose-response to a wide range of agents that either damage DNA or alter genome maintenance pathways. Mutat Res 2004; 554:253-66. [PMID: 15450423 DOI: 10.1016/j.mrfmmm.2004.05.004] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2004] [Revised: 05/04/2004] [Accepted: 05/07/2004] [Indexed: 04/30/2023]
Abstract
SNP analysis has come to the forefront of genomics since the mouse and human genomes have been sequenced. High throughput functional screens are necessary to evaluate these sequence databases. Described here is a genotoxic screen: a rapid method that determines the cellular dose-response to a wide range of agents that either damage DNA or alter basic cellular pathways important for maintaining genomic integrity. Importantly, a single person utilizing standard tissue culture equipment may perform these assays composed of 20 agents that attack genomic integrity or maintenance at many different levels. Thus, a small lab may perform this screen to determine the integrity of a wide range of DNA repair, chromatin metabolism, and response pathways without the limitations of investigator bias. A genotoxic screen will be useful when analyzing cells with either known genetic alterations (generated directly by the investigator or derived from individuals with known mutations) or unknown genetic alterations (cells with spontaneous mutations such as cancer-derived cells). Screening many genotoxins at one time will aid in determining the biological importance of these altered genes. Here we show the dose-response curves of mouse embryonic stem (ES) cells and HeLa cells exposed to 20 genotoxic agents. ES cells were chosen since they are amenable to genetic alteration by the investigator. HeLa cells were chosen since they were derived from cancer and are commonly used. Comparing the dose-response curves of these two cell lines show their relative sensitivity to these agents and helps define their genotoxic profile. As a part of phenomics, a large genotoxic profile database for cancer-derived cells, when integrated with other databases such as expression profiles and comparative genomic hybridization, may aid in maximizing the effectiveness of developing anti-cancer protocols.
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Affiliation(s)
- Teresa Marple
- The Department of Molecular Medicine, The University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, 78245-3207, USA
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32
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Guo G, Wang W, Bradley A. Mismatch repair genes identified using genetic screens in Blm-deficient embryonic stem cells. Nature 2004; 429:891-5. [PMID: 15215866 DOI: 10.1038/nature02653] [Citation(s) in RCA: 124] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2004] [Accepted: 05/13/2004] [Indexed: 01/12/2023]
Abstract
Phenotype-driven recessive genetic screens in diploid organisms require a strategy to render the mutation homozygous. Although homozygous mutant mice can be generated by breeding, a reliable method to make homozygous mutations in cultured cells has not been available, limiting recessive screens in culture. Cultured embryonic stem (ES) cells provide access to all of the genes required to elaborate the fundamental components and physiological systems of a mammalian cell. Here we have exploited the high rate of mitotic recombination in Bloom's syndrome protein (Blm)-deficient ES cells to generate a genome-wide library of homozygous mutant cells from heterozygous mutations induced with a revertible gene trap retrovirus. We have screened this library for cells with defects in DNA mismatch repair (MMR), a system that detects and repairs base-base mismatches. We demonstrate the recovery of cells with homozygous mutations in known and novel MMR genes. We identified Dnmt1(ref. 5) as a novel MMR gene and confirmed that Dnmt1-deficient ES cells exhibit micro-satellite instability, providing a mechanistic explanation for the role of Dnmt1 in cancer. The combination of insertional mutagenesis in Blm-deficient ES cells establishes a new approach for phenotype-based recessive genetic screens in ES cells.
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Affiliation(s)
- Ge Guo
- The Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK
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Wei K, Clark AB, Wong E, Kane MF, Mazur DJ, Parris T, Kolas NK, Russell R, Hou H, Kneitz B, Yang G, Kunkel TA, Kolodner RD, Cohen PE, Edelmann W. Inactivation of Exonuclease 1 in mice results in DNA mismatch repair defects, increased cancer susceptibility, and male and female sterility. Genes Dev 2003; 17:603-14. [PMID: 12629043 PMCID: PMC196005 DOI: 10.1101/gad.1060603] [Citation(s) in RCA: 241] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
Exonuclease 1 (Exo1) is a 5'-3' exonuclease that interacts with MutS and MutL homologs and has been implicated in the excision step of DNA mismatch repair. To investigate the role of Exo1 in mammalian mismatch repair and assess its importance for tumorigenesis and meiosis, we generated an Exo1 mutant mouse line. Analysis of Exo1(-/-) cells for mismatch repair activity in vitro showed that Exo1 is required for the repair of base:base and single-base insertion/deletion mismatches in both 5' and 3' nick-directed repair. The repair defect in Exo1(-/-) cells also caused elevated microsatellite instability at a mononucleotide repeat marker and a significant increase in mutation rate at the Hprt locus. Exo1(-/-) animals displayed reduced survival and increased susceptibility to the development of lymphomas. In addition, Exo1(-/-) male and female mice were sterile because of a meiotic defect. Meiosis in Exo1(-/-) animals proceeded through prophase I; however, the chromosomes exhibited dynamic loss of chiasmata during metaphase I, resulting in meiotic failure and apoptosis. Our results show that mammalian Exo1 functions in mutation avoidance and is essential for male and female meiosis.
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Affiliation(s)
- Kaichun Wei
- Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA
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Pierce EA, Liu Q, Igoucheva O, Omarrudin R, Ma H, Diamond SL, Yoon K. Oligonucleotide-directed single-base DNA alterations in mouse embryonic stem cells. Gene Ther 2003; 10:24-33. [PMID: 12525834 DOI: 10.1038/sj.gt.3301857] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
We have investigated the use of single-stranded oligodeoxy-nucleotides (ssODN) to produce specific single-base alterations in episomal and chromosomal DNA in mouse embryonic stem (ES) cells. Two different reporter genes, EGFP and LacZ, each with a single point mutation that inactivates reporter activity, were used. ssODN homologous to the target sequence, except for a single mismatch at the mutant base, were used to correct the mutant reporter genes. When tested in CHO-K1 cells, the ssODN showed correction rates of 0.5-1.0%, consistent with prior reports. ssODN in the antisense orientation provided higher rates of gene conversion than those in the sense orientation for both reporter genes. Nuclear extracts from mouse ES cells exhibited nearly the same correction activity as extracts from CHO-K1 cells. ssODN corrected the mutant bases of both episomal and chromosomal mutant reporter genes in mouse ES cells. Although the efficiency of gene correction observed in ES cells is low, approximately 10(-4), these results demonstrate that ssODN can produce single-base alterations in the genomic DNA of mouse ES cells. As conversion efficiency is improved by the continued development of oligonucleotide structure and DNA delivery methods, ssODN could be used to produce ES cells with specific mutations in any gene in a single step. The targeted ES cells could in turn be used to create accurate mouse models of inherited diseases.
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Affiliation(s)
- E A Pierce
- FM Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
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Kandel ES, Skeen J, Majewski N, Di Cristofano A, Pandolfi PP, Feliciano CS, Gartel A, Hay N. Activation of Akt/protein kinase B overcomes a G(2)/m cell cycle checkpoint induced by DNA damage. Mol Cell Biol 2002; 22:7831-41. [PMID: 12391152 PMCID: PMC134727 DOI: 10.1128/mcb.22.22.7831-7841.2002] [Citation(s) in RCA: 226] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Activation of Akt, or protein kinase B, is frequently observed in human cancers. Here we report that Akt activation via overexpression of a constitutively active form or via the loss of PTEN can overcome a G(2)/M cell cycle checkpoint that is induced by DNA damage. Activated Akt also alleviates the reduction in CDC2 activity and mitotic index upon exposure to DNA damage. In addition, we found that PTEN null embryonic stem (ES) cells transit faster from the G(2)/M to the G(1) phase of the cell cycle when compared to wild-type ES cells and that inhibition of phosphoinositol-3-kinase (PI3K) in HEK293 cells elicits G(2) arrest that is alleviated by activated Akt. Furthermore, the transition from the G(2)/M to the G(1) phase of the cell cycle in Akt1 null mouse embryo fibroblasts (MEFs) is attenuated when compared to that of wild-type MEFs. These results indicate that the PI3K/PTEN/Akt pathway plays a role in the regulation of G(2)/M transition. Thus, cells expressing activated Akt continue to divide, without being eliminated by apoptosis, in the presence of continuous exposure to mutagen and accumulate mutations, as measured by inactivation of an exogenously expressed herpes simplex virus thymidine kinase (HSV-tk) gene. This phenotype is independent of p53 status and cannot be reproduced by overexpression of Bcl-2 or Myc and Bcl-2 but seems to counteract a cell cycle checkpoint mediated by DNA mismatch repair (MMR). Accordingly, restoration of the G(2)/M cell cycle checkpoint and apoptosis in MMR-deficient cells, through reintroduction of the missing component of MMR, is alleviated by activated Akt. We suggest that this new activity of Akt in conjunction with its antiapoptotic activity may contribute to genetic instability and could explain its frequent activation in human cancers.
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Affiliation(s)
- Eugene S Kandel
- Department of Molecular Genetics, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60607, USA
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Rohwedel J, Guan K, Hegert C, Wobus AM. Embryonic stem cells as an in vitro model for mutagenicity, cytotoxicity and embryotoxicity studies: present state and future prospects. Toxicol In Vitro 2001; 15:741-53. [PMID: 11698176 DOI: 10.1016/s0887-2333(01)00074-1] [Citation(s) in RCA: 111] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Primary cultures or established cell lines of vertebrates are commonly used to analyse the mutagenic, embryotoxic or teratogenic potential of environmental factors, drugs and xenobiotics in vitro. However, these cellular systems do not include developmental processes from early embryonic stages up to terminally differentiated cell types. An alternative approach has been offered by permanent lines of pluripotent stem cells of embryonic origin, such as embryonic carcinoma (EC), embryonic stem (ES) and embryonic germ (EG) cells. The undifferentiated stem cell lines are characterized by nearly unlimited self-renewal capacity and have been shown to differentiate in vitro into cells of all three primary germ layers. Pluripotent embryonic stem cell lines recapitulate cellular developmental processes and gene expression patterns of early embryogenesis during in vitro differentiation, data which are summarized in this review. In addition, recent studies are presented which investigated mutagenic, cytotoxic and embryotoxic effects of chemical substances using in vitro systems of pluripotent embryonic stem cells. Furthermore, an outlook is given on future molecular technologies using embryonic stem cells in developmental toxicology and embryotoxicology.
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Affiliation(s)
- J Rohwedel
- Dept of Medical Molecular Biology, University of Lübeck, D-23538, Lübeck, Germany
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Elliott B, Jasin M. Repair of double-strand breaks by homologous recombination in mismatch repair-defective mammalian cells. Mol Cell Biol 2001; 21:2671-82. [PMID: 11283247 PMCID: PMC86898 DOI: 10.1128/mcb.21.8.2671-2682.2001] [Citation(s) in RCA: 145] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
Chromosomal double-strand breaks (DSBs) stimulate homologous recombination by several orders of magnitude in mammalian cells, including murine embryonic stem (ES) cells, but the efficiency of recombination decreases as the heterology between the repair substrates increases (B. Elliott, C. Richardson, J. Winderbaum, J. A. Nickoloff, and M. Jasin, Mol. Cell. Biol. 18:93-101, 1998). We have now examined homologous recombination in mismatch repair (MMR)-defective ES cells to investigate both the frequency of recombination and the outcome of events. Using cells with a targeted mutation in the msh2 gene, we found that the barrier to recombination between diverged substrates is relaxed for both gene targeting and intrachromosomal recombination. Thus, substrates with 1.5% divergence are 10-fold more likely to undergo DSB-promoted recombination in Msh2(-/-) cells than in wild-type cells. Although mutant cells can repair DSBs efficiently, examination of gene conversion tracts in recombinants demonstrates that they cannot efficiently correct mismatched heteroduplex DNA (hDNA) that is formed adjacent to the DSB. As a result, >20-fold more of the recombinants derived from mutant cells have uncorrected tracts compared with recombinants from wild-type cells. The results indicate that gene conversion repair of DSBs in mammalian cells frequently involves mismatch correction of hDNA rather than double-strand gap formation. In cells with MMR defects, therefore, aberrant recombinational repair may be an additional mechanism that contributes to genomic instability and possibly tumorigenesis.
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Affiliation(s)
- B Elliott
- Cell Biology Program, Memorial Sloan-Kettering Cancer Center and Cornell University Graduate School of Medical Sciences, New York, New York 10021, USA
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Jenab-Wolcott J, Rodriguez-Correa D, Reitmair AH, Mak T, Rosenberg N. The absence of Msh2 alters abelson virus pre-B-cell transformation by influencing p53 mutation. Mol Cell Biol 2000; 20:8373-81. [PMID: 11046134 PMCID: PMC102144 DOI: 10.1128/mcb.20.22.8373-8381.2000] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Defects in DNA mismatch repair predispose cells to the development of several types of malignant disease. The absence of Msh2 or Mlh1, two key molecules that mediate mismatch repair in eukaryotic cells, increases the frequency of mutation and also alters the response of some cells to apoptosis and cell cycle arrest. To understand the way these changes contribute to cancer predisposition, we examined the effects of defective mismatch repair on the multistep process of pre-B-cell transformation by Abelson murine leukemia virus. In this model, primary transformants undergo a prolonged apoptotic crisis followed by the emergence of fully transformed cell lines. The latter event is correlated to a loss of function of the p53 tumor suppressor protein and down-modulation of the p53 regulatory protein p19Arf. Analyses of primary transformants from Msh2 null mice and their wild-type littermates revealed that both types of cells undergo crisis. However, primary transformants from Msh2 null animals recover with accelerated kinetics, a phenomenon that is strongly correlated to the appearance of cells that have lost p53 function. Analysis of the kinetics with which p53 function is lost revealed that this change provides the dominant stimulus for emergence from crisis. Therefore, the absence of mismatch repair alters the molecular mechanisms involved in transformation by affecting a gene that controls apoptosis and cell cycle progression, rather than by affecting these processes directly.
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Affiliation(s)
- J Jenab-Wolcott
- Departments of Pathology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA
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Affiliation(s)
- E Evans
- Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853-2703, USA
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