Loss Of Mismatch Repair Research Articles

Sensitivity to DNA cross-linking chemotherapeutic agents in mismatch repair-defective cells in vitro and in xenografts.

Together with tolerance to killing induced by methylating agents, loss of mismatch repair (MMR) has previously been found to be associated with hypersensitivity to the DNAcross-linking agent 1-(2-chloroethyl)-3-cyclohexyl-nitrosourea(CCNU) in several human tumor cell lines (Aquilina et al., 1998). Here, we have investigated whether MMR might act as an efficient repair pathway and provide protection against the clastogenicity induced by CCNU and whether the hypersensitivity of MMR-defective cells is extended to other cross-linking agents. An increase in cell killing and in the frequency of micronuclei was observed after CCNU exposure in 2 hPMS2-defective clones (clones 6 and 7) compared with the parental HeLa cells. Introduction of a wild-type copy of chromosome 7 in clone 7 led to re-expression of the hPMS2 protein and brought survival and chromosomal damage upon CCNU exposure to parental levels. Our data indicate that MMR protects against the clastogenic damage induced by this drug. The hPMS2-defective HeLa cells were also hypersensitive to killing by mitomycin C. Mitomycin C sensitivity was confirmed in an hMLH1-defective clone derived from Raji cells and in msh2-defective mouse embryo fibroblasts derived from knock-out mice. hPMS2-defective and parental HeLa cells were transplanted into nude mice, and the animals were treated with mitomycin C. While parental cell growth rate was unaffected, the growth of MMR-defective tumor was significantly reduced. Our results indicate that the in vitro hypersensitivity to mitomycin C conferred by loss of MMR is paralleled in vivo and may have implications for the chemotherapy of MMR-defective tumors.

Genetic reconstruction of individual colorectal tumor histories.

It is difficult to observe human tumor progression as precursor lesions are systematically removed. Alternatives to direct observations, commonly used to reveal the hidden past of species and populations, are sequence comparisons or molecular clocks. Noncoding microsatellite (MS) loci were employed as molecular tumor clocks in 13 human mutator phenotype (MSI(+)) colorectal tumors. Quantitative analysis revealed that specific patterns of somatic MS mutations accumulate with division after loss of mismatch repair (MMR). Tumors had unique patterns of MS mutation, and, therefore, based on this model, each tumor had its own unique history. Loss of MMR occurred very early relative to terminal clonal expansion, with an estimated average of 2,300 divisions since loss of MMR and 280 divisions since expansion. Contrary to the classical adenoma-cancer sequence, MSI(+) adenomas were nearly as old as cancers (2,000 versus 2,400 divisions since loss of MMR). Negative clinical examinations preceded six tumors, independently documenting an absence of visible precursors during early MSI(+) adenoma or cancer progression. These findings further extend a window beyond visible progression since loss of MMR appears to start a genetic phase involving clone sizes or phenotypes below a threshold of clinical detection. This previously occult prologue before visible neoplasia is longer and therefore likely more important than generally appreciated.

HNPCC-like cancer predisposition in mice through simultaneous loss of Msh3 and Msh6 mismatch-repair protein functions.

Cancer predisposition in hereditary non-polyposis colon cancer (HNPCC) is caused by defects in DNA mismatch repair (MMR). Mismatch recognition is attributed to two heterodimeric protein complexes: MutSalpha (refs 2, 3, 4, 5), a dimer of MutS homologues MSH2 and MSH6; and MutSbeta (refs 2,7), a dimer of MSH2 and MSH3. These complexes have specific and redundant mismatch recognition capacity. Whereas MSH2 deficiency ablates the activity of both dimers, causing strong cancer predisposition in mice and men, loss of MSH3 or MSH6 (also known as GTBP) function causes a partial MMR defect. This may explain the rarity of MSH6 and absence of MSH3 germline mutations in HNPCC families. To test this, we have inactivated the mouse genes Msh3 (formerly Rep3 ) and Msh6 (formerly Gtmbp). Msh6-deficient mice were prone to cancer; most animals developed lymphomas or epithelial tumours originating from the skin and uterus but only rarely from the intestine. Msh3 deficiency did not cause cancer predisposition, but in an Msh6 -deficient background, loss of Msh3 accelerated intestinal tumorigenesis. Lymphomagenesis was not affected. Furthermore, mismatch-directed anti-recombination and sensitivity to methylating agents required Msh2 and Msh6, but not Msh3. Thus, loss of MMR functions specific to Msh2/Msh6 is sufficient for lymphoma development in mice, whereas predisposition to intestinal cancer requires loss of function of both Msh2/Msh6 and Msh2/Msh3.

Effect of loss of DNA mismatch repair on development of topotecan-, gemcitabine-, and paclitaxel-resistant variants after exposure to cisplatin.

Loss of DNA mismatch repair (MMR) causes genomic instability by markedly increasing the frequency of sporadic mutations in both coding and noncoding sequences. Little is known about how loss of MMR affects sensitivity to the mutagenic effect of chemotherapeutic agents. We wanted to determine how loss of MMR affects the ability of cisplatin, a known mutagen, to generate human tumor cell variants resistant to other drugs with which cisplatin is commonly combined in treatment regimens. We compared the ability of cisplatin to produce variants resistant to topotecan, gemcitabine, and paclitaxel in two pairs of MMR-proficient and -deficient cells that included sublines of the human colon carcinoma cell line HCT-116 and sublines of the human endometrial adenocarcinoma cell line HEC59. Cells were exposed to increasing concentrations of cisplatin for 1 h, and the surviving population was tested for the frequency of variants resistant to these single molecular target drugs 10 days later. The frequency of variants increased linearly with cisplatin concentration for all three drugs. Cisplatin was 2.6 +/- 0.3- (S.D.), 3.6 +/- 0.9-, and 2.3 +/- 0.1-fold more potent at producing topotecan-, gemcitabine-, and paclitaxel-resistant variants in the MMR-deficient than in the MMR-proficient HCT116 cells (P <.05 for all). Cisplatin was 1.4 +/- 0.3- and 1.4 +/- 0.4-fold more potent at generating topotecan- and gemcitabine-resistant variants in MMR-deficient HEC59 cells than in MMR-proficient HEC59+ch2 cells. Cisplatin was not more potent in generating paclitaxel-resistant variants in the MMR-deficient HEC59 cells. Spontaneous rates of generation of cells resistant to these three drugs were also measured in the HCT116 sublines. MMR-deficient HCT116 cells exhibited rates of generation of resistant variants that were 1.94- and 1.51-fold higher (P <.05) than those in the MMR-proficient cells for topotecan and gemcitabine, respectively; loss of MMR had no effect on the rate of generation of variants resistant to paclitaxel. We conclude that the loss of MMR increases the ability of cisplatin to generate variants resistant to topotecan, gemcitabine, and possibly paclitaxel and that MMR also plays a role in controlling the spontaneous rate of generation of variants resistant to topotecan and gemcitabine.

Decreased cisplatin damage-dependent DNA synthesis in cellular extracts of mismatch repair deficient cells

The proficiency of both nucleotide excision repair (NER) and DNA mismatch repair (MMR) influences cellular sensitivity to cisplatin ( cis-diamminedichloroplatinum). To gain further insight into how MMR may influence platinum drug sensitivity, the effect of loss of MMR on repair synthesis was measured in vitro by a commonly used method that relies on whole-cell extracts to drive [α- 32P]dATP incorporation into cisplatin-damaged plasmid DNA. Extracts evaluated include those from cells with or without functional hMLH1 (HCT116+ch2 versus HCT116+ch3, respectively) and hMSH2 (HEC59 versus HEC59+ch2, respectively). Loss of MMR in the HCT116 system was associated with a 2.8-fold reduction in cisplatin damage-specific DNA synthesis, whereas it was associated with a 3.0-fold reduction in the HEC59 system, suggesting that a decrease in the ability to repair cisplatin-damaged DNA accompanies loss of MMR. An in vitro DNA excision assay that utilized a substrate containing a site-specific cisplatin adduct was performed. Using this highly NER-specific assay, no significant difference was apparent between the extracts derived from NER-proficient versus -deficient cells. These and other data lead us to suggest that the increase in apparent repair synthesis in platinum-damaged plasmids by extracts from MMR-proficient versus -deficient cellular extracts may reflect a distinct and possibly adverse DNA synthetic process rather than productive NER.

Dependence on RAD52 and RAD1 for anticancer drug resistance mediated by inactivation of mismatch repair genes

Mismatch repair (MMR) proteins repair mispaired DNA bases and have an important role in maintaining the integrity of the genome [1]. Loss of MMR has been correlated with resistance to a variety of DNA-damaging agents, including many anticancer drugs [2]. How loss of MMR leads to resistance is not understood, but is proposed to be due to loss of futile MMR activity and/or replication stalling [3,4]. We report that inactivation of MMR genes (MLH1, MLH2, MSH2, MSH3, MSH6, but not PMS1) in isogenic strains of Saccharomyces cerevisiae led to increased resistance to the anticancer drugs cisplatin, carboplatin and doxorubicin, but had no effect on sensitivity to ultraviolet C (UVC) radiation. Sensitivity to cisplatin and doxorubicin was increased in mlh1 mutant strains when the MLH1 gene was reintroduced, demonstrating a direct involvement of MMR proteins in sensitivity to these DNA-damaging agents. Inactivation of MLH1, MLH2 or MSH2 had no significant effect, however, on drug sensitivities in the rad52 or rad1 mutant strains that are defective in mitotic recombination and removing unpaired DNA single strands. We propose a model whereby MMR proteins – in addition to their role in DNA-damage recognition – decrease adduct tolerance during DNA replication by modulating the levels of recombination-dependent bypass. This hypothesis is supported by the finding that, in human ovarian tumour cells, loss of hMLH1 correlated with acquisition of cisplatin resistance and increased cisplatin-induced sister chromatid exchange, both of which were reversed by restoration of hMLH1 expression.

Microsatellite Instability and Mutation of DNA Mismatch Repair Genes in Gliomas

Microsatellite instability (MSI) has been identified in various human cancers, particularly those associated with the hereditary nonpolyposis colorectal cancer syndrome. Although gliomas have been reported in a few hereditary nonpolyposis colorectal cancer syndrome kindred, data on the incidence of MSI in gliomas are conflicting, and the nature of the mismatch repair (MMR) defect is not known. We established the incidence of MSI and the underlying MMR gene mutation in 22 patients ages 45 years or less with sporadic high-grade gliomas (17 glioblastomas, 3 anaplastic astrocytomas, and 2 mixed gliomas, grade III). Using five microsatellite loci, four patients (18%) had high level MSI, with at least 40% unstable loci. Germline MMR gene mutation was detected in all four patients, with inactivation of the second allele of the corresponding MMR gene or loss of protein expression in the tumor tissue. Frameshift mutation in the mononucleotide tract of insulin-like growth factor type II receptor was found in one high-level MSI glioma, but none was found in the transforming growth factor beta type II receptor and the Bax genes. There was no family history of cancer in three of the patients, and although one patient did have a family history of colorectal carcinoma, the case did not satisfy the Amsterdam criteria for hereditary nonpolyposis colorectal cancer syndrome. Three patients developed metachronous colorectal adenocarcinomas, fitting the criteria of Turcot's syndrome. Thus, MSI and germline MMR gene mutation is present in a subset of young glioma patients, and these patients and their family members are at risk of developing other hereditary nonpolyposis colorectal cancer syndrome-related tumors, in particular colorectal carcinomas. These results have important implications in the genetic testing and management of young patients with glioma and their families.

Enrichment for DNA mismatch repair-deficient cells during treatment with cisplatin.

In addition to playing a role in tumorigenesis, loss of DNA mismatch repair results in low-level intrinsic resistance to cisplatin and carboplatin. We used a mismatch repair-deficient (clone B) and -proficient (clone B/rev) pair of Chinese hamster ovary sublines to determine the ability of cisplatin to enrich for repair-deficient cells during growth in vitro and in vivo. Clone B cells were 1.8-fold resistant to cisplatin as measured by a clonogenic assay. These cells were molecularly engineered to express constitutively the green fluorescent protein, and changes in the fraction of these repair-deficient cells were monitored by flow cytometric analysis. A single 1-hr exposure to cisplatin at an IC50 concentration enriched populations initially containing either 5 or 10% clone B cells by 81 and 75%, respectively, when measured at 5 days. Enrichment increased as a function of drug concentration to 158 and 169%, respectively, following an IC90 exposure. When grown as a xenograft, a single LD10 dose of cisplatin enriched the tumors by 48% from 4.6 to 6.8% repair-deficient cells (p = 0.04). To determine whether similar enrichment occurs during the treatment of human ovarian cancer patients, paired tumor samples were obtained from 38 patients before and after treatment with a minimum of 3 cycles of platinum drug-based primary chemotherapy and analyzed immunohistochemically for changes in the fraction of tumor cells expressing hMHL1. Following treatment there was a reduction in hMLH1 staining in 66% of the cases (p = 0.0005). Our results demonstrate that, despite the fact that loss of mismatch repair yields only modest levels of cisplatin resistance, even a single exposure to cisplatin produces quite a marked enrichment for repair-deficient cells in vitro and in vivo. Our results are consistent with the concept that treatment with cisplatin or carboplatin selects for preexisting mismatch repair-deficient cells, and that this contributes to the frequent development of clinical resistance.

Loss of DNA mismatch repair: effects on the rate of mutation to drug resistance.

The loss of the ability of cells to repair mismatches in double-stranded DNA is a common finding in human tumors. This defect results in genomic instability and in increased resistance to several of the drugs used in cancer chemotherapy. The human colon cancer cell line HCT116 is deficient in DNA mismatch repair (MMR) because of a genetic defect in the hMLH1 gene, which is located on chromosome 3. In this study, we investigated whether MMR-deficient HCT116+chr2 cells (i.e., HCT116 cells into which chromosome 2 has been transferred [as a control]) have a higher rate of mutation to resistance to commonly used chemotherapeutic agents (i.e., cisplatin, doxorubicin, paclitaxel [Taxol], and etoposide) than MMR-proficient HCT116+chr3 cells (i.e., HCT116 cells into which chromosome 3 has been transferred to provide a wild-type copy of the hMLH1 gene). Spontaneous mutation rates were calculated from measurements of the mutant fractions of cells before and after their expansion through a known number of generations (also known as the technique of maximum likelihood estimation). Aliquots of 500000 cells were expanded in culture over a period of 2 weeks, and the mutant fractions were determined both before and after expansion of secondary cultures (each also with an initial 500000 cells) in drug concentrations that produced survival fractions of 0.0002%. Mutation rates in MMR-proficient and MMR-deficient cells did not differ on exposure to cisplatin, doxorubicin, or paclitaxel; however, the relative mutation rate was 2.4-fold higher in MMR-deficient cells exposed to etoposide (P=.002). These results suggest that genes involved in the control of cellular sensitivity to etoposide are targets for mutation when the loss of MMR destabilizes the genome. Tumors containing large fractions of MMR-deficient cells may demonstrate more rapid emergence of clinical resistance to etoposide.

Loss of DNA mismatch repair due to knockout of MSH2 or PMS2 results in resistance to cisplatin and carboplatin

Loss of DNA mismatch repair is a common finding in hereditary nonpolyposis colon cancer as well as in many types of sporadic human tumors. The effect of loss of DNA mismatch repair activity on sensitivity to cisplatin and carboplatin was tested using MSH2 and PMS2 knockout cell lines. The knockout dMsh2 embryonic stem cell line was 2.1-fold more resistant to cisplatin and 1.7-fold more resistant to carboplatin when compared to the isogenic wild-type wt-2 cell line. Likewise, the PMS2(-/-) mouse fibroblasts were 1.9-fold more resistant to cisplatin and 1.5-fold more resistant to carboplatin when compared to the isogenic PMS2(+/+) fibroblasts. These findings demonstrate that loss of mismatch repair due to knockout of either MSH2 or PMS2 results in low-level resistance to cisplatin and carboplatin, drugs that form the same types of adducts in DNA. These data validate results previously obtained using non-isogenic mismatch repair-proficient and -deficient cell lines, and indicate that simple recognition of the cisplatin adduct by the MSH2/MSH6 heterodimer is not sufficient for full detector function, but that PMS2 is also required for the pro-apoptotic signal to be generated from this detector.

Clinico-pathological features and p53 expression in left-sided sporadic colorectal cancers with and without microsatellite instability.

Defects in mismatch repair (MMR) can result in the development of a 'mutator phenotype', manifest as an increase in DNA replication errors (RERs). Patients with hereditary non-polyposis colorectal cancer (HNPCC) have germline mutations in MMR genes. These patients develop carcinomas of the colon and other specific sites at a significantly earlier age than patients with sporadic carcinomas. RERs are found in the cancers from patients with HNPCC and have been demonstrated in 10-20 per cent of sporadic colorectal cancers (CRCs). Loss of MMR may simply accelerate tumour development, but it is also possible that these tumours follow a different carcinogenetic pathway from tumours with intact MMR. In particular, it has been suggested that p53 mutations occur less often in RER-positive (RER+) sporadic colorectal cancers. In this study, the clinico-pathological features and frequency of p53 overexpression in 17 left-sided RER+ CRCs were compared with 35 left-sided RER- CRCs. No differences were found in the age and tumour stage at presentation, mucinous differentiation, or Jass prognostic grouping between these two types of CRC. Thirteen out of 17 (76 per cent) RER+ and 19/35 (54 per cent) RER- tumours showed overexpression of p53, a non-significant difference (chi 2 test). Although some previous studies have suggested differences in the clinico-pathological features and p53 expression of RER+ and RER- right-sided CRCs, our results show that these differences do not exist in left-sided cancers.