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PubMed2001

Hypoxia-induced enrichment and mutagenesis of cells that have lost DNA mismatch repair.

Loss of DNA mismatch repair (MMR) increases the risk of spontaneous mutations. We sought to determine whether there was an interaction between hypoxia and MMR deficiency that might contribute to the phenomenon of tumor progression. Human colon carcinoma HCT116+ch2 (MMR-deficient) and HCT116+ch3 (MMR-proficient) sublines were exposed for varying periods of time to an environment of <0.1% O2 and pH as low as 6.1. When a population containing 5% MMR-deficient cells and 95% MMR-proficient cells was subjected to hypoxia for 72 h, the MMR-deficient cells were enriched by a factor of 2-fold in the surviving population, whereas no enrichment was detected in cells maintained under aerobic conditions. The potential of hypoxia to destabilize the genome was determined by measuring the frequency of clones in the surviving population resistant to very high concentrations of 6-thioguanine or cisplatin. A 72-h exposure to hypoxia did not increase the frequency of resistant clones in the MMR-proficient cells but produced a 7.8-fold increase in 6-thioguanine-resistant clones and a 2.5-fold increase in cisplatin-resistant clones in the MMR-deficient cells. Loss of MMR increased the frequency of mutations in a reporter vector sensitive to frameshift mutations in a microsatellite sequence. Exposure to hypoxia for a time period as short as 48 h further increased the number of mutations in both cell types, but the absolute number of mutants was higher in the MMR-deficient cells. These results indicate that hypoxia and its accompanying low pH enrich for MMR-deficient cells and that loss of MMR renders human colon carcinoma cells hypersensitive to the ability of hypoxia to induce microsatellite instability and generate highly drug-resistant clones in the surviving population.

Adenocarcinoma↗
PubMed2001

Expression of hMSH2 and hMLH1 proteins of the human DNA mismatch repair system in ameloblastoma.

The human DNA mismatch repair (hMMR) system plays an important role in reducing mutation and maintaining genomic stability. The MMR system in human cells is composed of at least six genes (hMSH2, hMLH1, hMSH3, hPMS1, hPMS2 and GTBP/hMSH6). In particular, hMSH2 and hMLH1 are expressed in human cells that are undergoing rapid renewal; their reduced expression has been reported in several tumors. We examined the protein expression pattern of hMSH2 and hMLH1 by immunohistochemistry in 25 ameloblastomas. All ameloblastomas expressed hMSH2 and hMLH1 proteins in the outer layer of epithelial cells. The localization of the staining was exclusively nuclear. These data suggest that the development and progression of these tumors do not depend on a defect in the hMMR system.

Ameloblastoma↗
PubMed2002

Maternal effect for DNA mismatch repair in the mouse.

DNA mismatch repair (DMR) functions to maintain genome stability. Prokaryotic and eukaryotic cells deficient in DMR show a microsatellite instability (MSI) phenotype characterized by repeat length alterations at microsatellite sequences. Mice deficient in Pms2, a mammalian homolog of bacterial mutL, develop cancer and display MSI in all tissues examined, including the male germ line where a frequency of approximately 10% was observed. To determine the consequences of maternal DMR deficiency on genetic stability, we analyzed F(1) progeny from Pms2(-/-) female mice mated with wild-type males. Our analysis indicates that MSI in the female germ line was approximately 9%. MSI was also observed in paternal alleles, a surprising result since the alleles were obtained from wild-type males and the embryos were therefore DMR proficient. We propose that mosaicism for paternal alleles is a maternal effect that results from Pms2 deficiency during the early cleavage divisions. The absence of DMR in one-cell embryos leads to the formation of unrepaired replication errors in early cell divisions of the zygote. The occurrence of postzygotic mutation in the early mouse embryo suggests that Pms2 deficiency is a maternal effect, one of a limited number identified in the mouse and the first to involve a DNA repair gene.

Adenosine Triphosphatases↗
PubMed2001

Sequence and stress-response analyses of the DNA mismatch repair gene hexA in Lactococcus lactis.

The DNA mismatch repair gene hexA was identified in Lactococcus lactis by PCR amplification by using a pair of primers homologous to the DNA-binding Dps protein. The gene in its entirety, including the regulatory regions, was sequenced, by using a strategy of chromosomal walking based on two PCR protocols. The open reading frame of 2526 bp was preceded by a strong ribosome-binding site (AGGAAG) and was followed by a potential transcription terminator (hairpin loop structure). The 5' terminus of the hexA mRNA was located 135 bp upstream of the start codon, and putative -10 and -35 regions were identified. The deduced amino acid sequence revealed two motifs, the ATP/GTP-binding site (P-loop) and the "MutS family signature". The hexA promoter was cloned into pMU1327, which contained a promoter-less CAT reporter gene, and the promoter activity was examined under oxidative-stress conditions. It appears that the promoter activity is down-shifted by H2O2 at 4 mM.

Amino Acid Sequence↗
PubMed1999

Roles of the DNA mismatch repair and nucleotide excision repair proteins during meiosis.

Numerous proteins are involved in the nucleotide excision repair (NER) and DNA mismatch repair (MMR) pathways. The function and specificity of these proteins during the mitotic cell cycle has been actively investigated, in large part due to the involvement of these systems in human diseases. In contrast, comparatively little is known about their functioning during meiosis. At least three repair pathways operate during meiosis in the yeast Saccharomyces cerevisiae to repair mismatches that occur as a consequence of heteroduplex formation in recombination. The first pathway is similar to the one acting during postreplicative mismatch repair in mitotically dividing cells, while two pathways are responsible for the repair of large loops during meiosis, using proteins from MMR and NER systems. Some MMR proteins also help prevent recombination between diverged sequences during meiosis, and act late in recombination to affect the resolution of crossovers. This review will discuss the current status of DNA mismatch repair and nucleotide excision repair proteins during meiosis, especially in the yeast S. cerevisiae.

Adenosine Triphosphatases↗
PubMed2002

High-frequency microsatellite instability is associated with defective DNA mismatch repair in human melanoma.

Hereditary nonpolyposis colorectal cancers and a steadily increasing number of sporadic tumors display microsatellite instability. In colorectal tumors, high-frequency microsatellite instability is strictly associated with inactivation of the DNA mismatch repair genes hMSH2, hMLH1, or hPMS2, whereas mutations in the mismatch repair gene hMSH6 have been identified in a subset of tumors with low-frequency microsatellite instability. In addition to epithelial tumors of the colon, endometrium, and ovary, microsatellite instability has been reported to occur also in sporadic melanoma. The relationship between microsatellite instability and mismatch repair in melanoma cells, however, has not been investigated so far. In this study, we analyzed microsatellite instability, mismatch repair activity, and expression of the hMSH2, hMSH6, hMLH1, and hPMS2 proteins in five melanoma cell lines and in tumor specimens from which the cells were derived. Four cell lines displayed normal levels of mismatch repair activity and expressed all the mismatch repair proteins. The extracts of the fifth cell line lacked the hMLH1 and hPMS2 proteins, and were correspondingly deficient in the repair of DNA mismatches. This line displayed high-frequency microsatellite instability, whereas the four mismatch-repair-proficient cell lines displayed either no or low-frequency microsatellite instability. These findings could be confirmed in the tumor specimens, in that only the tumor that did not express hMLH1 and hPMS2 displayed high-frequency microsatellite instability. Our data are consistent with the hypothesis that in melanoma, similarly to epithelial tumors, only the high-frequency microsatellite instability phenotype is strictly dependent on a defective mismatch repair system. Further studies on a large series of tumor specimens are required to establish the frequency of mismatch repair loss in human melanoma.

Base Pair Mismatch↗
PubMed2001

DNA mismatch repair enzyme hMSH2 in malignant melanoma: increased immunoreactivity as compared to acquired melanocytic nevi and strong mRNA expression in melanoma cell lines.

Mutations in the mismatch DNA repair gene human MutS homologen 2 (hMSH2) are causative for microsatellite instability and carcinogenesis in various human tumours, including hereditary nonpolyposis colorectal cancer. Because microsatellite instability has been detected in malignant melanoma, we have investigated hMSH2 in melanocytic tumours. We found strong nuclear immunoreactivity for hMSH2 that was elevated in malignant melanoma and melanoma metastases as compared to acquired nevi. These findings suggest that increased genomic instability in malignant melanoma is associated with elevated protein levels of this DNA repair enzyme. hMSH2 is not exclusively regulated by proliferative activity in melanocytes, because there was no correlation between staining patterns of hMSH2 and the proliferation marker Ki-67. In contrast, immunoreactivity scores for hMSH2 and p53 were both upregulated in malignant melanocytic tumours. These findings support the concept that hMSH2 gene expression may be regulated in melanocytes by the p53 protein, as has been reported previously in other tissues. Using the reverse transcription-polymerase chain reaction, we detected strong hMSH2 mRNA expression in each of 8 melanoma cell lines analysed (highest amounts in SK-MEL-25 cells, lowest amounts in MML-I cells). In conclusion, our findings indicate that hMSH-2 may be of importance for genetic stability, tumorigenesis and progression of malignant melanoma.

Base Pair Mismatch↗
PubMed2004

RAS/RAF mutation and defective DNA mismatch repair in endometrial cancers.

OBJECTIVE: Defective DNA mismatch repair is a common genetic abnormality in both colon cancers and endometrial cancers. Cancers with defective DNA mismatch repair have the so-called mutator phenotype and accumulate genetic errors at an increased rate. An early mutational target in cells with defect DNA mismatch repair may be the RAS/RAF pathway. Colon cancers often have KRAS2 mutations and, if not KRAS2 mutations, may have BRAF mutations. This study investigated the spectrum and frequency of mutations in BRAF and KRAS2 in endometrial carcinomas on the basis of mismatch repair status. STUDY DESIGN: Four hundred forty-one patients with endometrial cancer were staged properly and graded and evaluated for mismatch repair status. These patients were then stratified to groups by the degree of microsatellite instability that was observed in their tumors. One hundred forty-six of the selected tumors were then evaluated for KRAS2 and BRAF mutations on the basis of their microsatellite instability. RESULTS: One hundred forty-six endometrioid endometrial cancers were evaluated for KRAS2 and BRAF mutations. Thirty-five cancers (24%) had activating KRAS2 mutations, but only a single BRAF mutation was identified in an microsatellite instability-positive cancer. Twenty-four of 81 microsatellite instability high cancers (29.6%) in which the MLH1 repair gene was methylated had KRAS2 mutations. When compared with the other groups, this finding approached statistical significance (P=.06). KRAS2 mutation status was associated with increasing age at diagnosis (P=.02). CONCLUSION: Despite many similarities between colon and endometrial cancers, the mechanism of the development of endometrial cancers appears to be different from colon cancers in that BRAF is not affected by a mismatch repair problem, because only KRAS2 mutations were seen. In addition, increasing age appears to lead to an increased likelihood that such a mutation will occur.

Adenocarcinoma, Clear Cell↗
PubMedDate not supplied

Variable MLH1 promoter methylation patterns in endometrial carcinomas of endometrioid subtype lacking DNA mismatch repair.

A lack of DNA mismatch repair (MMR) is observed in approximately 20% of sporadic endometrial tumors, but few of these tumors have mutations in MSH2 or MLH1, the two major MMR genes. Promoter methylation is an important means of silencing transcription, and methylation of the MLH1 promoter has been reported as an important cause of MLH1 inactivation in endometrial cancers. Studies have shown that specific CpG sites within the MLH1 gene promoter are critical for gene expression, but other studies have shown that methylation of both more proximal and more distal sequences are important for MLH1 gene regulation. Here, we used a microsatellite instability assay and MLH1 immunohistochemistry to identify a subset of endometrial carcinomas of the endometrioid subtype lacking MMR. Sequencing of bisulphite-treated DNA from these tumors determined the methylation status of 42 CpG sites across the MLH1 promoter (spanning -204 to -702 bp upstream of the transcriptional start). Unlike the 4 normal endometrial samples that were unmethylated, 17 of 21 MMR-deficient samples showed complete or near-complete methylation and the remaining 4 MMR-deficient samples had a considerable degree of methylation (approximately 50% or greater). Five tumors demonstrated isolated unmethylated CpG sites, despite methylation throughout the rest of the promoter. This underscores the importance of examining the methylation status of at least several CpG sites within the promoter as methylation is not always consistent across DNA. Overall, our findings support the model that density of methylation of CpG sites across the MLH1 promoter is important in determining gene expression.

Adaptor Proteins, Signal Transducing↗
PubMed1999

MED1, a novel human methyl-CpG-binding endonuclease, interacts with DNA mismatch repair protein MLH1.

The DNA mismatch repair (MMR) is a specialized system, highly conserved throughout evolution, involved in the maintenance of genomic integrity. To identify novel human genes that may function in MMR, we employed the yeast interaction trap. Using the MMR protein MLH1 as bait, we cloned MED1. The MED1 protein forms a complex with MLH1, binds to methyl-CpG-containing DNA, has homology to bacterial DNA repair glycosylases/lyases, and displays endonuclease activity. Transfection of a MED1 mutant lacking the methyl-CpG-binding domain (MBD) is associated with microsatellite instability (MSI). These findings suggest that MED1 is a novel human DNA repair protein that may be involved in MMR and, as such, may be a candidate eukaryotic homologue of the bacterial MMR endonuclease, MutH. In addition, these results suggest that cytosine methylation may play a role in human DNA repair.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Promoter hypermethylation and inactivation of hMLH1, a DNA mismatch repair gene, in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a multistage process during which adverse genetic alterations accumulate resulting in loss of cell cycle control, selective cell overgrowth, and ultimately formation of malignancy. Among various genetic alterations in HNSCC is increased microsatellite instability (MSI). hMLH1 is one of the major mismatch DNA repair genes, the inactivation of which caused increased MSI in a variety of human cancers including HNSCC. While somatic mutation is a major mechanism of the hMLH1 gene inactivation in hereditary form of human cancer, promoter hypermethylation appears to be primarily involved in the inactivation of the hMLH1 gene in sporadic form of human cancers. In the current study, we analyzed 78 cases of HNSCC for hMLH1 protein expression and promoter hypermethylation by IHC and methylation-specific PCR (MSP). Twenty-four of 78 cases (31%) of HNSCC contained markedly reduced levels of the hMLH1 protein. Based on the IHC results, 8 cases without and 8 with hMLH1 protein expression (total of 16) were further analyzed by MSP. Seven of 8 cases (88%) that were negative for the hMLH1 protein displayed promoter hypermethylation, whereas 7 of 7 cases (100%) strongly positive for the protein were free of promoter methylation. This study confirms our previous conclusion that promoter hypermethylation represents a major mechanism of the hMLH1 gene inactivation in HNSCC.

Adaptor Proteins, Signal Transducing↗
PubMed2003

DNA mismatch repair: molecular mechanisms and biological function.

DNA mismatch repair (MMR) guards the integrity of the genome in virtually all cells. It contributes about 1000-fold to the overall fidelity of replication and targets mispaired bases that arise through replication errors, during homologous recombination, and as a result of DNA damage. Cells deficient in MMR have a mutator phenotype in which the rate of spontaneous mutation is greatly elevated, and they frequently exhibit microsatellite instability at mono- and dinucleotide repeats. The importance of MMR in mutation avoidance is highlighted by the finding that defects in MMR predispose individuals to hereditary nonpolyposis colorectal cancer. In addition to its role in postreplication repair, the MMR machinery serves to police homologous recombination events and acts as a barrier to genetic exchange between species.

Adenosine Triphosphatases↗
PubMed1998

Physical interaction between components of DNA mismatch repair and nucleotide excision repair.

Nucleotide excision repair (NER) and DNA mismatch repair are required for some common processes although the biochemical basis for this requirement is unknown. Saccharomyces cerevisiae RAD14 was identified in a two-hybrid screen using MSH2 as "bait," and pairwise interactions between MSH2 and RAD1, RAD2, RAD3, RAD10, RAD14, and RAD25 subsequently were demonstrated by two-hybrid analysis. MSH2 coimmunoprecipitated specifically with epitope-tagged versions of RAD2, RAD10, RAD14, and RAD25. MSH2 and RAD10 were found to interact in msh3 msh6 and mlh1 pms1 double mutants, suggesting a direct interaction with MSH2. Mutations in MSH2 increased the UV sensitivity of NER-deficient yeast strains, and msh2 mutations were epistatic to the mutator phenotype observed in NER-deficient strains. These data suggest that MSH2 and possibly other components of DNA mismatch repair exist in a complex with NER proteins, providing a biochemical and genetical basis for these proteins to function in common processes.

Base Pair Mismatch↗
PubMed2004

Mutations in DNA mismatch repair genes: implications for DNA damage signaling and drug sensitivity (review).

The DNA mismatch repair (MMR) system functions in the elimination of biosynthetic errors that arise during DNA replication, in DNA damage surveillance, and in the prevention of recombination between non-identical sequences. It therefore substantially contributes to the maintenance of genome integrity. It is thus not surprising that loss of MMR function may lead to cancer. Inherited defects due to germline mutations in the MMR genes underlie the hereditary non-polyposis colon cancer (HNPCC) syndrome in humans, and epigenetic silencing of the hMLH1 gene accounts for sporadic cancers, including those of the endometrium and ovaries. Another hallmark of MMR is its capacity to elicit DNA damage-induced cell death. Although this might seem to make MMR a useful target for anticancer agents, it has become clear that tumor cells with defective MMR display reduced sensitivity to the cytotoxic effect of DNA damaging agents such as alkylating agents and cisplatin. This is of clinical relevance, as these agents provide MMR-deficient tumor cells a growth advantage. This effect is exacerbated by the potential of some agents to cause the de novo generation of MMR-resistant variants. Thus, the discovery of antitumor agents that retain sensitivity against or specifically target MMR-deficient tumor cells, and the development of strategies to overcome MMR-related drug resistance assumes clinical importance. Efforts have been taken in the past years to come to a better understanding of the molecular mechanisms of MMR, including those underlying MMR-related tumorigenesis, those determining the substrate-specificity of MMR-dependent damage recognition, and those linking damage recognition to cell death responses. This information is also expected to contribute to the establishment of diagnostic methods to screen for MMR gene mutations in tumors and to the development of strategies which enable the oncologists to apply appropriate and specific regimens for tumor treatment.

Apoptosis↗
PubMed2004

Evidence for involvement of HMGB1 protein in human DNA mismatch repair.

Defects in human DNA mismatch repair predispose to cancer, but many components of the pathway have not been identified. We report here the identification and characterization of a novel component required for mismatch repair in human cells. A 30-kDa protein was purified to homogeneity by virtue of its ability to complement a depleted HeLa extract in repair of mismatched heteroduplexes. The complementing activity was identified as HMGB1 (the high mobility group box 1 protein), a non-histone chromatin protein that facilitates protein-protein interactions and recognizes DNA damage. Evidence is also presented that HMGB1 physically interacts with MutSalpha and is required at a step prior to the excision of mispaired nucleotide in mismatch repair.

Amino Acid Sequence↗
PubMed1998

DNA mismatch repair and O6-alkylguanine-DNA alkyltransferase analysis and response to Temodal in newly diagnosed malignant glioma.

PURPOSE: We evaluated the response to Temodal (Schering-Plough Research Institute, Kenilworth, NJ) of patients with newly diagnosed malignant glioma, as well as the predictive value of quantifying tumor DNA mismatch repair activity and O6-alkylguanine-DNA alkyltransferase (AGT). PATIENTS AND METHODS: Thirty-three patients with newly diagnosed glioblastoma multiforme (GBM) and five patients with newly diagnosed anaplastic astrocytoma (AA) were treated with Temodal at a starting dose of 200 mg/m2 daily for 5 consecutive days with repeat dosing every 28 days after the first daily dose. Immunochemistry for the detection of the human DNA mismatch repair proteins MSH2 and MLH1 and the DNA repair protein AGT was performed with monoclonal antibodies and characterized with respect to percent positive staining. RESULTS: Of the 33 patients with GBM, complete responses (CRs) occurred in three patients, partial responses (PRs) occurred in 14 patients, stable disease (SD) was seen in four patients, and 12 patients developed progressive disease (PD). Toxicity included infrequent grades 3 and 4 myelosuppression, constipation, nausea, and headache. Thirty tumors showed greater than 60% cells that stained for MSH2 and MLH1, with three CRs, 12 PRs, three SDs, and 12 PDs. Eight tumors showed 60% or less cells that stained with antibodies to MSH2 and/or MLH1, with 3 PRs, 3 SDs, and 2 PDs. Eleven tumors showed 20% or greater cells that stained with an antibody to AGT, with 1 PR, 2 SDs, and 8 PDs. Twenty-five tumors showed less than 20% cells that stained for AGT, with 3 CRs, 12 PRs, 4 SDs, and 6 PDs. CONCLUSION: These results suggest that Temodal has activity against newly diagnosed GBM and AA and warrants continued evaluation of this agent. Furthermore, pretherapy analysis of tumor DNA mismatch repair and, particularly, AGT protein expression may identify patients in whom tumors are resistant to Temodal.

Adult↗
PubMed2005

DNA mismatch repair deficiency, resistance to cancer chemotherapy and the development of hypersensitive agents.

DNA Mismatch Repair (MMR) deficiency results in resistance to platinating and alkylating agents, DNA minor groove binders, inhibitors of topoisomerases and antimetabolites. The cellular MMR pathway, involving hMLH1 and MSH2, detects and repairs DNA frame shifts replication errors and regulates recombination events. Tumour cells are able to cope with DNA damage caused by chemotherapy as long as the MMR-process is disabled and hence there is a need to develop agents that (i) restore MMR proficiency or (ii) are hypersensitive in cells that are irreversibly MMR deficient. Decitabine is suggested to restore MMR function by reversal of gene promoter hypermethylation of hMLH1. However, when MMR is deficient due to gene mutation it is not feasible to design agents, since the absence of functional proteins that constitute the MMR machinery are not available as targets. The evidence that resistance to chemotherapy is associated with hMSH2 and/or hMLH1 deficiency has revealed a new paradigm for drug discovery of agents that positively exploit this phenotype to therapeutic advantage. Even more attractive is the development of agents that are hypersensitive in the absence of functional MMR to enable even more effective treatment. In this regard, established agents such as mitomycin C, camptothecin or novel hydroxyethylaminoanthraquinones may represent opportunities for exploitation of MMR-deficiency in tumour cells.

Antineoplastic Agents↗