High rate of
            <i>CAD</i>
            gene amplification in human cells deficient in MLH1 or MSH6

Chen, Sihong; Bigner, Sandra H.; Modrich, Paul

doi:10.1073/pnas.241508098

Cited by 44 publications

(48 citation statements)

References 51 publications

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“…Similarly, we have shown that mitotic recombination is also highly dependent on DNA homology, and is much more so than is meiotic recombination (26). Further, the absence of mismatch repair alleviates a suppression of gene amplification in human cells (27), a process mediated by double-strand DNA breaks, and also renders mammalian cells hypersensitive to camptothecin, a drug that inhibits the DNA breakage-reunion activity of topoisomerase I (28). Thus, it is likely that mismatch repair modulates mitotic recombination and allows it to occur more frequently in MEFs than in ES cells.…”

Section: Discussionmentioning

confidence: 77%

Embryonic stem cells and somatic cells differ in mutation frequency and type

Cervantes

Stringer²,

Shao³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

288

221

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Pluripotent embryonic stem (ES) cells have been used to produce genetically modified mice as experimental models of human genetic diseases. Increasingly, human ES cells are being considered for their potential in the treatment of injury and disease. Here we have shown that mutation in murine ES cells, heterozygous at the selectable Aprt locus, differs from that in embryonic somatic cells. The mutation frequency in ES cells is significantly lower than that in mouse embryonic fibroblasts, which is similar to that in adult cells in vivo. The distribution of spontaneous mutagenic events is remarkably different between the two cell types. Although loss of the functional allele is the predominant mutation type in both cases, representing about 80% of all events, mitotic recombination accounted for all loss of heterozygosity events detected in somatic cells. In contrast, mitotic recombination in ES cells appeared to be suppressed and chromosome loss͞reduplication, leading to uniparental disomy (UPD), represented more than half of the loss of heterozygosity events. Extended culture of ES cells led to accumulation of cells with adenine phosphoribosyltransferase deficiency and UPD. Because UPD leads to reduction to homozygosity at multiple recessive disease loci, including tumor suppressor loci, in the affected chromosome, the increased risk of tumor formation after stem cell therapy should be viewed with concern.T he frequency and types of spontaneous mutation that occur in pluripotent stem cells may have an impact on their future clinical uses. Embryonic stem (ES) cells are intrinsically different from somatic cells in that they retain the capacity to differentiate into multiple cell types (1, 2). Maintenance of genomic stability in these cells is essential for their potential to serve as donor cells for tissue transplants. Here we have examined spontaneous and induced mutagenic events in ES cells. We have taken advantage of a recently described murine model that utilizes mice heterozygous at the selectable marker encoding the purine salvage enzyme adenine phosphoribosyltransferase (APRT) to examine this issue (3-5).The Aprt gene, resident on mouse chromosome 8, is an endogenous marker that supports selection for cells that are APRT deficient and, conversely, for cells that have APRT activity (6). The model uses cells derived from 129 SvEv ϫ C3H͞HeJ mice or embryos in which the strain 129 SvEv Aprt allele has been interrupted by a targeted neo insertion (7). These cells are therefore heterozygous at Aprt, with the neo insertion allowing for rapid discrimination between the targeted and untargeted alleles. Polymorphic microsatellites that span the length of chromosome 8 can be used to confirm loss of heterozygosity (LOH) resulting from either mitotic recombination, or multilocus deletion. Centromeres in mouse strains 129 SvEv and C3H͞HeJ are different sizes. Hence, the parental origin of copies of chromosome 8 can be determined, providing visual determination of uniparental disomy (UPD), defined as the presence of homolo...

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Section: Discussionmentioning

confidence: 77%

Embryonic stem cells and somatic cells differ in mutation frequency and type

Cervantes

Stringer²,

Shao³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

288

221

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“…Interestingly, gene amplification has also been reported to arise in MLH1-deficient cells (10), but a direct connection between MLH1-induced and hypoxia-induced gene amplification has not been tested. Nonetheless, together these observations suggest that hypoxia can constitute a profound cellular stress that can promote genetic instability on several levels.…”

Section: Discussionmentioning

confidence: 99%

Decreased Expression of the DNA Mismatch Repair Gene Mlh1 under Hypoxic Stress in Mammalian Cells

Mihaylova¹,

Bindra²,

Yuan³

et al. 2003

Molecular and Cellular Biology

253

231

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The hypoxic tumor microenvironment has been shown to contribute to genetic instability. As one possible mechanism for this effect, we report that expression of the DNA mismatch repair (MMR) gene Mlh1 is specifically reduced in mammalian cells under hypoxia, whereas expression of other MMR genes, including Msh2, Msh6, and Pms2, is not altered at the mRNA level. However, levels of the PMS2 protein are reduced, consistent with destabilization of PMS2 in the absence of its heterodimer partner, MLH1. The hypoxia-induced reduction in Mlh1 mRNA was prevented by the histone deacetylase inhibitor trichostatin A, suggesting that hypoxia causes decreased Mlh1 transcription via histone deacetylation. In addition, treatment of cells with the iron chelator desferrioxamine also reduced MLH1 and PMS2 levels, in keeping with low oxygen tension being the stress signal that provokes the altered MMR gene expression. Functional MMR deficiency under hypoxia was detected as induced instability of a (CA) 29 dinucleotide repeat and by increased mutagenesis in a chromosomal reporter gene. These results identify a potential new pathway of genetic instability in cancer: hypoxia-induced reduction in the expression of key MMR proteins. In addition, this stress-induced genetic instability may represent a conceptual parallel to the pathway of stationary-phase mutagenesis seen in bacteria.It has been argued that the large number of mutations found in malignant cells cannot be accounted for by the low rate of mutation generally found among somatic cells, leading to the suggestion that there is a mutator phenotype in cancer (26). The basis for this genetic instability has not been fully established. Much work has focused on the role of genetic defects in cancer cells affecting cell cycle regulation and DNA repair that could lead to genomic instability, such as in p53 (18). Hereditary nonpolyposis colon carcinoma is linked to inherited defects in several of the human DNA mismatch repair (MMR) genes, including Msh2, Mlh1, and Pms2 (29). Another cancerprone syndrome, xeroderma pigmentosum, affects individuals with mutations in genes associated with the nucleotide excision repair (NER) pathway (56).As an alternative mechanism by which genetic instability might arise in cancer, we and others have investigated the possible role of the tumor microenvironment (61). Developing tumors form a unique tissue environment because their growth outstrips their blood supply, leading to hypoxia, low pH, and nutrient deprivation (39, 55). Several studies have shown that hypoxia can alter chromosome metabolism, leading to gene amplification and fragile site induction (11, 37, 60). Our prior work found that cells exposed to hypoxia in culture have increased frequencies of point mutations at reporter gene loci (36). In addition, experimental tumors grown from cells implanted into mice show elevated levels of mutations compared to the same cells grown in parallel in normoxic culture, pointing to a deleterious effect of the tumor microenvironment on genome integrity in v...

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“…Our results argue that some mrt mutants are likely to contain mutations that result in increased levels of genomic damage. It also should be pointed out that the recent evidence that human cells with mismatch repair mutations have elevated rates of gene amplification (35) suggests the possibility that msh-2 worms may accumulate multiple types of genomic damage.…”

Section: Discussionmentioning

confidence: 99%

Caenorhabditis elegans DNA mismatch repair gene msh-2 is required for microsatellite stability and maintenance of genome integrity

Degtyareva

Greenwell

Hofmann

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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Mismatch repair genes are important in maintaining the fidelity of DNA replication. To determine the function of the Caenorhabditis elegans homologue of the MSH2 mismatch repair gene (msh-2), we isolated a strain of C. elegans with an insertion of the transposable element Tc1 within msh-2. Early-passage msh-2 mutants were similar to wild-type worms with regard to lifespan and meiotic chromosome segregation but had slightly reduced fertility. The mutant worms had reduced DNA damage-induced germ-line apoptosis after genotoxic stress. The msh-2 mutants also had elevated levels of microsatellite instability and increased rates of reversion of the dominant unc-58(e665) mutation. In addition, serially passaged cultures of msh-2 worms died out much more quickly than those of wild-type worms. These results demonstrate that msh-2 function in C. elegans is important in regulating both short-and long-term genomic stability.apoptosis ͉ mutation ͉ mutator ͉ fertility

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High rate of CAD gene amplification in human cells deficient in MLH1 or MSH6

Abstract: MutS and

Cited by 44 publications

References 51 publications

Embryonic stem cells and somatic cells differ in mutation frequency and type

Embryonic stem cells and somatic cells differ in mutation frequency and type

Decreased Expression of the DNA Mismatch Repair Gene Mlh1 under Hypoxic Stress in Mammalian Cells

Caenorhabditis elegans DNA mismatch repair gene msh-2 is required for microsatellite stability and maintenance of genome integrity

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