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1.
J Leukoc Biol ; 99(3): 455-65, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26432900

ABSTRACT

The Fanconi anemia proteins participate in a canonical pathway that repairs cross-linking agent-induced DNA damage. Cells with inactivated Fanconi anemia genes are universally hypersensitive to such agents. Fanconi anemia-deficient hematopoietic stem cells are also hypersensitive to inflammatory cytokines, and, as importantly, Fanconi anemia macrophages overproduce such cytokines in response to TLR4 and TLR7/8 agonists. We questioned whether TLR-induced DNA damage is the primary cause of aberrantly regulated cytokine production in Fanconi anemia macrophages by quantifying TLR agonist-induced TNF-α production, DNA strand breaks, crosslinker-induced chromosomal breakage, and Fanconi anemia core complex function in Fanconi anemia complementation group C-deficient human and murine macrophages. Although both M1 and M2 polarized Fanconi anemia cells were predictably hypersensitive to mitomycin C, only M1 macrophages overproduced TNF-α in response to TLR-activating signals. DNA damaging agents alone did not induce TNF-α production in the absence of TLR agonists in wild-type or Fanconi anemia macrophages, and mitomycin C did not enhance TLR responses in either normal or Fanconi anemia cells. TLR4 and TLR7/8 activation induced cytokine overproduction in Fanconi anemia macrophages. Also, although TLR4 activation was associated with induced double strand breaks, TLR7/8 activation was not. That DNA strand breaks and chromosome breaks are neither necessary nor sufficient to account for the overproduction of inflammatory cytokines by Fanconi anemia cells suggests that noncanonical anti-inflammatory functions of Fanconi anemia complementation group C contribute to the aberrant macrophage phenotype and suggests that suppression of macrophage/TLR hyperreactivity might prevent cytokine-induced stem cell attrition in Fanconi anemia.


Subject(s)
Cross-Linking Reagents/pharmacology , Fanconi Anemia/immunology , Macrophages/immunology , Tumor Necrosis Factor-alpha/biosynthesis , Animals , Cell Polarity , Cells, Cultured , DNA Damage , Fanconi Anemia Complementation Group C Protein/physiology , Histones/analysis , Humans , Imidazoles/pharmacology , Macrophages/drug effects , Mice , Mitomycin/pharmacology , Reactive Oxygen Species/metabolism , Toll-Like Receptors/physiology
2.
Clin Cancer Res ; 21(8): 1962-72, 2015 Apr 15.
Article in English | MEDLINE | ID: mdl-25609062

ABSTRACT

PURPOSE: Fanconi anemia is an inherited disorder associated with a constitutional defect in the Fanconi anemia DNA repair machinery that is essential for resolution of DNA interstrand crosslinks. Individuals with Fanconi anemia are predisposed to formation of head and neck squamous cell carcinomas (HNSCC) at a young age. Prognosis is poor, partly due to patient intolerance of chemotherapy and radiation requiring dose reduction, which may lead to early recurrence of disease. EXPERIMENTAL DESIGN: Using HNSCC cell lines derived from the tumors of patients with Fanconi anemia, and murine HNSCC cell lines derived from the tumors of wild-type and Fancc(-/-) mice, we sought to define Fanconi anemia-dependent chemosensitivity and DNA repair characteristics. We utilized DNA repair reporter assays to explore the preference of Fanconi anemia HNSCC cells for non-homologous end joining (NHEJ). RESULTS: Surprisingly, interstrand crosslinker (ICL) sensitivity was not necessarily Fanconi anemia-dependent in human or murine cell systems. Our results suggest that the increased Ku-dependent NHEJ that is expected in Fanconi anemia cells did not mediate relative ICL resistance. ICL exposure resulted in increased DNA damage sensing and repair by PARP in Fanconi anemia-deficient cells. Moreover, human and murine Fanconi anemia HNSCC cells were sensitive to PARP inhibition, and sensitivity of human cells was attenuated by Fanconi anemia gene complementation. CONCLUSIONS: The observed reliance upon PARP-mediated mechanisms reveals a means by which Fanconi anemia HNSCCs can acquire relative resistance to the ICL-based chemotherapy that is a foundation of HNSCC treatment, as well as a potential target for overcoming chemoresistance in the chemosensitive individual.


Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm/genetics , Fanconi Anemia/complications , Fanconi Anemia/genetics , Head and Neck Neoplasms/etiology , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Animals , Cell Line, Tumor , Cell Proliferation , Cisplatin/pharmacology , DNA Damage/drug effects , DNA End-Joining Repair , DNA Helicases/metabolism , Disease Models, Animal , Enzyme Activation , Fanconi Anemia Complementation Group C Protein/genetics , Fanconi Anemia Complementation Group D2 Protein/deficiency , Fanconi Anemia Complementation Group D2 Protein/metabolism , Gene Knockout Techniques , Head and Neck Neoplasms/drug therapy , Heterografts , Humans , Ku Autoantigen , Mice , Mice, Knockout , Phenotype , Poly(ADP-ribose) Polymerases/metabolism , Spheroids, Cellular , Tumor Cells, Cultured
3.
Cancer Epidemiol Biomarkers Prev ; 23(7): 1421-7, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24740199

ABSTRACT

Survival in epithelial ovarian cancer (EOC) is influenced by the host immune response, yet the key genetic determinants of inflammation and immunity that affect prognosis are not known. The nuclear factor-κB (NF-κB) transcription factor family plays an important role in many immune and inflammatory responses, including the response to cancer. We studied common inherited variation in 210 genes in the NF-κB family in 10,084 patients with invasive EOC (5,248 high-grade serous, 1,452 endometrioid, 795 clear cell, and 661 mucinous) from the Ovarian Cancer Association Consortium. Associations between genotype and overall survival were assessed using Cox regression for all patients and by major histology, adjusting for known prognostic factors and correcting for multiple testing (threshold for statistical significance, P < 2.5 × 10(-5)). Results were statistically significant when assessed for patients of a single histology. Key associations were with caspase recruitment domain family, member 11 (CARD11) rs41324349 in patients with mucinous EOC [HR, 1.82; 95% confidence interval (CI), 1.41-2.35; P = 4.13 × 10(-6)] and tumor necrosis factor receptor superfamily, member 13B (TNFRSF13B) rs7501462 in patients with endometrioid EOC (HR, 0.68; 95% CI, 0.56-0.82; P = 2.33 × 10(-5)). Other associations of note included TNF receptor-associated factor 2 (TRAF2) rs17250239 in patients with high-grade serous EOC (HR, 0.84; 95% CI, 0.77-0.92; P = 6.49 × 10(-5)) and phospholipase C, gamma 1 (PLCG1) rs11696662 in patients with clear cell EOC (HR, 0.43; 95% CI, 0.26-0.73; P = 4.56 × 10(-4)). These associations highlight the potential importance of genes associated with host inflammation and immunity in modulating clinical outcomes in distinct EOC histologies.


Subject(s)
NF-kappa B/genetics , Neoplasms, Glandular and Epithelial/genetics , Neoplasms, Glandular and Epithelial/mortality , Ovarian Neoplasms/genetics , Ovarian Neoplasms/mortality , Signal Transduction/genetics , Adult , Aged , Carcinoma, Ovarian Epithelial , Female , Genotype , Humans , Middle Aged , Neoplasm Invasiveness , Neoplasms, Glandular and Epithelial/pathology , Ovarian Neoplasms/pathology , Polymorphism, Single Nucleotide , Proportional Hazards Models
4.
Blood ; 122(18): 3197-205, 2013 Oct 31.
Article in English | MEDLINE | ID: mdl-24046015

ABSTRACT

Hematopoietic stem and progenitor cells with inactivated Fanconi anemia (FA) genes, FANCA and FANCC, are hypersensitive to inflammatory cytokines. One of these, tumor necrosis factor α (TNF-α), is also overproduced by FA mononuclear phagocytes in response to certain Toll-like receptor (TLR) agonists, creating an autoinhibitory loop that may contribute to the pathogenesis of progressive bone marrow (BM) failure and selection of TNF-α-resistant leukemic stem cell clones. In macrophages, the TNF-α overproduction phenotype depends on p38 mitogen-activated protein kinase (MAPK), an enzyme also known to induce expression of other inflammatory cytokines, including interleukin 1ß (IL-1ß). Reasoning that IL-1ß might be involved in a like autoinhibitory loop, we determined that (1) TLR activation of FANCA- and FANCC-deficient macrophages induced overproduction of both TNF-α and IL-1ß in a p38-dependent manner; (2) exposure of Fancc-deficient BM progenitors to IL-1ß potently suppressed the expansion of multipotent progenitor cells in vitro; and (3) although TNF-α overexpression in FA cells is controlled posttranscriptionally by the p38 substrate MAPKAPK-2, p38-dependent overproduction of IL-1ß is controlled transcriptionally. We suggest that multiple inflammatory cytokines overproduced by FANCA- and FANCC-deficient mononuclear phagocytes may contribute to the progressive BM failure that characterizes FA, and that to achieve suppression of this proinflammatory state, p38 is a more promising molecular therapeutic target than either IL-1ß or TNF-α alone.


Subject(s)
Fanconi Anemia Complementation Group A Protein/metabolism , Fanconi Anemia Complementation Group C Protein/metabolism , Interleukin-1beta/metabolism , Macrophages/metabolism , Toll-Like Receptors/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Blotting, Western , Cell Line, Tumor , Cell Proliferation/drug effects , Cells, Cultured , Fanconi Anemia Complementation Group A Protein/genetics , Fanconi Anemia Complementation Group C Protein/genetics , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/metabolism , Humans , Imidazoles/pharmacology , Inflammasomes/genetics , Inflammasomes/metabolism , Interleukin-1beta/genetics , Interleukin-1beta/pharmacology , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Macrophages/cytology , Macrophages/drug effects , Mice, Knockout , Naphthalenes/pharmacology , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Pyrazoles/pharmacology , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Toll-Like Receptors/agonists , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors
8.
Blood ; 119(9): 1992-2002, 2012 Mar 01.
Article in English | MEDLINE | ID: mdl-22234699

ABSTRACT

Fanconi anemia, complementation group C (FANCC)-deficient hematopoietic stem and progenitor cells are hypersensitive to a variety of inhibitory cytokines, one of which, TNFα, can induce BM failure and clonal evolution in Fancc-deficient mice. FANCC-deficient macrophages are also hypersensitive to TLR activation and produce TNFα in an unrestrained fashion. Reasoning that suppression of inhibitory cytokine production might enhance hematopoiesis, we screened small molecules using TLR agonist-stimulated FANCC- and Fanconi anemia, complementation group A (FANCA)-deficient macrophages containing an NF-κB/AP-1-responsive reporter gene (SEAP). Of the 75 small molecules screened, the p38 MAPK inhibitor BIRB 796 and dasatinib potently suppressed TLR8-dependent expression of the reporter gene. Fanconi anemia (FA) macrophages were hypersensitive to the TLR7/8 activator R848, overproducing SEAP and TNFα in response to all doses of the agonist. Low doses (50nM) of both agents inhibited p38 MAPK-dependent activation of MAPKAPK2 (MK2) and suppressed MK2-dependent TNFα production without substantially influencing TNFα gene transcription. Overproduction of TNFα by primary FA cells was likewise suppressed by these agents and involved inhibition of MK2 activation. Because MK2 is also known to influence production and/or sensitivity to 2 other suppressive factors (MIP-1α and IFNγ) to which FA hematopoietic progenitor cells are uniquely vulnerable, targeting of p38 MAPK in FA hematopoietic cells is a rational objective for preclinical evaluation.


Subject(s)
Fanconi Anemia Complementation Group A Protein/deficiency , Fanconi Anemia Complementation Group C Protein/deficiency , Phagocytes/metabolism , Protein Kinase Inhibitors/pharmacology , Toll-Like Receptors/metabolism , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Animals , Cell Line , Dasatinib , Disease Models, Animal , Enzyme Activation/drug effects , Fanconi Anemia/genetics , Fanconi Anemia/metabolism , Gene Expression Regulation/drug effects , Gene Knockout Techniques , Humans , Macrophages/drug effects , Macrophages/metabolism , Matrix Metalloproteinases/metabolism , Mice , Mice, Knockout , Naphthalenes/pharmacology , Phagocytes/drug effects , Phagocytes/enzymology , Phenotype , Proto-Oncogene Proteins c-jun/metabolism , Pyrazoles/pharmacology , Pyrimidines/pharmacology , RNA Processing, Post-Transcriptional/drug effects , Small Molecule Libraries , Thiazoles/pharmacology , Transcription, Genetic/drug effects , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/genetics , src-Family Kinases/antagonists & inhibitors
9.
Int J Dev Biol ; 56(10-12): 949-58, 2012.
Article in English | MEDLINE | ID: mdl-23417416

ABSTRACT

This review examines the importance of the epithelial origin of granulosa cells and their possible contribution to the development of ovarian cancers in three animal models. We hypothesise that undifferentiated granulosa cells, devoid of their germ cell regulator, retain their embryonic plasticity and may give rise to ovarian cancers of epithelial origin. Dazl-KO and FancD2-KO mice and BMP15-KO sheep are animal models in which germ cells or oocytes are lost at specific stages of follicular formation or growth, leaving behind clusters of residual, but healthy somatic cells. A common feature in Dazl- and Fancd2-KO animals following germ cell/oocyte loss is the presence of sex cords and intraovarian epithelial ducts or tubules. In Dazl-KO mice, cord/tubule-like structures, OSE invaginations and clusters of steroidogenic cells became increasingly prominent with age, but these abnormal structures remained benign. In Fancd2-KO mice, the formation of sex-cords and intraovarian tubules lead to the formation of tumours with multiple phenotypes including luteomas, papillary cysts and malignant carcinomas (e.g. adenocarcinomas). In BMP15-KO sheep, oocytes die as follicles start to grow, leaving 'nodules' containing granulosa cells with a capacity to respond to follicle stimulating hormone and synthesize inhibin. Thereafter, these nodules coalesced and a range of benign solid or semi-solid tumour phenotypes developed. We conclude that premature loss of oocytes, but not granulosa cells, leads to tumour formation with multiple phenotypes. Moreover, the severity of tumour development is linked to both the specificity of the mutation and the timing of oocyte loss relative to that of follicular formation.


Subject(s)
Granulosa Cell Tumor/pathology , Granulosa Cells/cytology , Oocytes/cytology , Ovarian Neoplasms/pathology , Animals , Animals, Genetically Modified , Bone Morphogenetic Protein 15/genetics , Fanconi Anemia Complementation Group D2 Protein/genetics , Female , Granulosa Cell Tumor/genetics , Granulosa Cells/metabolism , Mice , Oocytes/metabolism , Ovarian Neoplasms/genetics , RNA-Binding Proteins/genetics , Sheep
10.
Nat Methods ; 9(1): 78-80, 2011 Nov 27.
Article in English | MEDLINE | ID: mdl-22120468

ABSTRACT

Because mutations are inevitable, the genome of each cell in a multicellular organism becomes unique and therefore encodes a record of its ancestry. Here we coupled arbitrary single primer PCR with next-generation DNA sequencing to catalog mutations and deconvolve the phylogeny of cultured mouse cells. This study helps pave the way toward construction of retrospective cell-fate maps based on mutations accumulating in genomes of somatic cells.


Subject(s)
Cell Lineage/genetics , High-Throughput Nucleotide Sequencing/methods , Mutation , Sequence Analysis, DNA/methods , Animals , Computer Simulation , Genome , Mice , Phylogeny , Polymerase Chain Reaction/methods , Reproducibility of Results
11.
Blood ; 116(12): 2057-60, 2010 Sep 23.
Article in English | MEDLINE | ID: mdl-20554974

ABSTRACT

Fancc suppresses cross-linker-induced genotoxicity, modulates growth-inhibitory cytokine responses, and modulates endotoxin responses. Although loss of the latter function is known to account for endotoxin-induced marrow failure in murine Fancc (mFancc)-deficient mice, some argue that cytokine and endotoxin hypersensitivities devolve simply from genomic instability. Seeking to resolve this question, we planned to ectopically express instructive human FANCC (hFANCC) mutants in murine Fancc-deficient hematopoietic stem cells. To first assure that hFANCC cDNA was competent in murine cells, we compared hFANCC and mFancc in complementation assays for cross-linking agent hypersensitivity and endotoxin hypersensitivity. We found that mFancc complemented murine Fancc-deficient cells in both assays, but that hFANCC fully suppressed only endotoxin hypersensitivity, not cross-linking agent hypersensitivity. These results support the notions that Fancc is multifunctional and that structural prerequisites for its genoprotective functions differ from those required to constrain endotoxin responses known to lead to marrow failure in Fancc-deficient mice.


Subject(s)
Fanconi Anemia Complementation Group C Protein/physiology , Hematopoietic Stem Cells/metabolism , Animals , Endotoxins/pharmacology , Fanconi Anemia Complementation Group C Protein/deficiency , Fanconi Anemia Complementation Group C Protein/genetics , Humans , Hypersensitivity, Immediate/chemically induced , Mice , Mice, Knockout , Transgenes
12.
Blood ; 114(19): 3976-7, 2009 Nov 05.
Article in English | MEDLINE | ID: mdl-19892722
13.
Proc Natl Acad Sci U S A ; 106(40): 17101-4, 2009 Oct 06.
Article in English | MEDLINE | ID: mdl-19805137

ABSTRACT

Organisms require faithful DNA replication to avoid deleterious mutations. In yeast, replicative leading- and lagging-strand DNA polymerases (Pols epsilon and delta, respectively) have intrinsic proofreading exonucleases that cooperate with each other and mismatch repair to limit spontaneous mutation to less than 1 per genome per cell division. The relationship of these pathways in mammals and their functions in vivo are unknown. Here we show that mouse Pol epsilon and delta proofreading suppress discrete mutator and cancer phenotypes. We found that inactivation of Pol epsilon proofreading elevates base-substitution mutations and accelerates a unique spectrum of spontaneous cancers; the types of tumors are entirely different from those triggered by loss of Pol delta proofreading. Intercrosses of Pol epsilon-, Pol delta-, and mismatch repair-mutant mice show that Pol epsilon and delta proofreading act in parallel pathways to prevent spontaneous mutation and cancer. These findings distinguish Pol epsilon and delta functions in vivo and reveal tissue-specific requirements for DNA replication fidelity.


Subject(s)
DNA Polymerase III/genetics , DNA Polymerase II/genetics , Mutation , Neoplasms/pathology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Base Sequence , DNA Polymerase II/metabolism , DNA Polymerase III/metabolism , Female , Gene Expression Profiling , Gene Frequency , Genotype , Kaplan-Meier Estimate , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred Strains , Molecular Sequence Data , MutL Protein Homolog 1 , MutS Homolog 2 Protein/genetics , MutS Homolog 2 Protein/metabolism , Neoplasms/genetics , Neoplasms/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phenotype , Reverse Transcriptase Polymerase Chain Reaction
14.
Cancer Res ; 66(18): 9017-25, 2006 Sep 15.
Article in English | MEDLINE | ID: mdl-16982743

ABSTRACT

Fanconi anemia is an inherited cancer predisposition disease characterized by cytogenetic and cellular hypersensitivity to cross-linking agents. Seeking evidence of Fanconi anemia protein dysfunction in women at risk of ovarian cancer, we screened ovarian surface epithelial cells from 25 primary cultures established from 22 patients using cross-linker hypersensitivity assays. Samples were obtained from (a) women at high risk for ovarian cancer with histologically normal ovaries, (b) ovarian cancer patients, and (c) a control group with no family history of breast or ovarian cancer. In chromosomal breakage assays, all control cells were mitomycin C (MMC) resistant, but eight samples (five of the six high-risk and three of the eight ovarian cancer) were hypersensitive. Lymphocytes from all eight patients were MMC resistant. Only one of the eight patients had a BRCA1 germ-line mutation and none had BRCA2 mutations, but FANCD2 was reduced in five of the eight. Ectopic expression of normal FANCD2 cDNA increased FANCD2 protein and induced MMC resistance in both hypersensitive lines tested. No FANCD2 coding region or promoter mutations were found, and there was no genomic loss or promoter methylation in any Fanconi anemia genes. Therefore, in high-risk women with no BRCA1 or BRCA2 mutations, tissue-restricted hypersensitivity to cross-linking agents is a frequent finding, and chromosomal breakage responses to MMC may be a sensitive screening strategy because cytogenetic instability identified in this way antedates the onset of carcinoma. Inherited mutations that result in tissue-specific FANCD2 gene suppression may represent a cause of familial ovarian cancer.


Subject(s)
Fanconi Anemia Complementation Group D2 Protein/genetics , Ovarian Neoplasms/genetics , Adult , Aged , Chromosome Breakage , DNA Methylation , DNA, Complementary/genetics , Epithelial Cells/pathology , Epithelial Cells/physiology , Fanconi Anemia Complementation Group D2 Protein/biosynthesis , Female , Gene Silencing , Genes, BRCA1 , Genetic Predisposition to Disease , Genomic Instability , Germ-Line Mutation , Humans , Middle Aged , Mitomycin/pharmacology , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Ovary/pathology , Ovary/physiology , Promoter Regions, Genetic , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction
15.
Proc Natl Acad Sci U S A ; 99(24): 15560-5, 2002 Nov 26.
Article in English | MEDLINE | ID: mdl-12429860

ABSTRACT

Mutations are a hallmark of cancer. Normal cells minimize spontaneous mutations through the combined actions of polymerase base selectivity, 3' --> 5' exonucleolytic proofreading, mismatch correction, and DNA damage repair. To determine the consequences of defective proofreading in mammals, we created mice with a point mutation (D400A) in the proofreading domain of DNA polymerase delta (poldelta, encoded by the Pold1 gene). We show that this mutation inactivates the 3' --> 5' exonuclease of poldelta and causes a mutator and cancer phenotype in a recessive manner. By 18 months of age, 94% of homozygous Pold1(D400A/D400A) mice developed cancer and died (median survival = 10 months). In contrast, only 3-4% of Pold1(+/D400A) and Pold1(+/+) mice developed cancer in this time frame. Of the 66 tumors arising in 49 Pold1(D400A/D400A) mice, 40 were epithelial in origin (carcinomas), 24 were mesenchymal (lymphomas and sarcomas), and two were composite (teratomas); one-third of these animals developed tumors in more than one tissue. Skin squamous cell carcinoma was the most common tumor type, occurring in 60% of all Pold1(D400A/D400A) mice and in 90% of those surviving beyond 8 months of age. These data show that poldelta proofreading suppresses spontaneous tumor development and strongly suggest that unrepaired DNA polymerase errors contribute to carcinogenesis. Mice deficient in poldelta proofreading provide a tractable model to study mechanisms of epithelial tumorigenesis initiated by a mutator phenotype.


Subject(s)
DNA Polymerase III/physiology , DNA Repair , Neoplasms, Experimental/genetics , Amino Acid Sequence , Animals , Carcinoma, Squamous Cell/genetics , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Chimera/genetics , Codon/genetics , DNA Polymerase III/deficiency , DNA Polymerase III/genetics , Epithelial Cells/pathology , Female , Gene Targeting , Genetic Predisposition to Disease , Genotype , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , Mutagenesis, Site-Directed , Point Mutation , Skin Neoplasms/genetics
16.
Hum Mol Genet ; 11(18): 2189-200, 2002 Sep 01.
Article in English | MEDLINE | ID: mdl-12189171

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.


Subject(s)
Base Pair Mismatch , DNA Repair , Recombination, Genetic , Amino Acid Sequence , DNA Repair/genetics , DNA Repair/physiology , DNA-Binding Proteins/drug effects , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Deoxyribonucleotides/metabolism , Humans , Mitomycin/pharmacology , Molecular Sequence Data , Mutation , Nucleic Acid Synthesis Inhibitors/pharmacology , Thymidine/metabolism , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/metabolism
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