Gene expression profile and mutational analysis of DNA mismatch repair genes in carcinoma prostate in Indian population

DNA mismatch repair (MMR) plays a role in promoting genetic stability by repairing DNA replication errors, inhibiting recombination between nonidentical DNA sequences, and participating in responses to DNA damage. Although the role of MMR in prostate carcinogenesis remains unclear, MMR deficiency in Carcinoma Prostate (Pca) could prove to be clinically significant. Thus, the present study investigated the gene expression profile of six major MMR genes, viz. hMLH1, hMSH2, hPMS1, hPMS2, hMSH3, and hMSH6, and polymorphism in hMLH1 and hMSH2 in Pca in Indian population. Further, correlation with clinicopathological parameters was evaluated to establish their role as a potential prognostic marker. A significant downregulation of hMLH1, hMSH2, and hPMS2 expression was observed in Pca compared to benign prostatic hyperplasia (BPH). A greater loss of hPMS2 protein in poorly differentiated tumors was demonstrated, which was in concordance with a significant inverse correlation of hPMS2 gene expression with the Gleason score indicating its significance as a marker for Pca progression. An important association of hMLH1-93G>A polymorphism with the risk of Pca was also identified. The results of the present study suggest that an altered MMR has important biological and clinical significance in Pca in Indian population.     

The Bloom's syndrome helicase interacts directly with the human DNA mismatch repair protein hMSH6

Bloom's syndrome (BS) is a rare genetic disorder characterised by genome instability and cancer susceptibility. BLM, the BS gene product, belongs to the highly-conserved RecQ family of DNA helicases. Although the exact function of BLM in human cells remains to be defined, it seems likely that BLM eliminates some form of homologous recombination (HR) intermediate that arises during DNA replication. Similarly, the mismatch repair (MMR) system also plays a crucial role in the maintenance of genomic stability, by correcting DNA errors generated during DNA replication. Recent evidence implicates components of the MMR system also in HR repair. We now show that hMSH6, a component of the heterodimeric mismatch recognition complex hMSH2/hMSH6 (hMutS(alpha)), interacts with the BLM protein both in vivo and in vitro. In agreement with these findings, BLM and hMSH6 co-localise to discrete nuclear foci following exposure of the cells to ionising radiation. However, the purified recombinant MutS(alpha) complex does not affect the helicase activity of BLM in vitro. As BLM has previously been shown to interact with the hMLH1 component of the hMLH1/hPMS2 (hMutL(alpha)) heterodimeric MMR complex, our present findings further strengthen the link between BLM and processes involving correction of DNA mismatches, such as in the regulation of the fidelity of homologous recombination events.     

The frequency of hereditary defective mismatch repair in a prospective series of unselected colorectal carcinomas

A comprehensive analysis of somatic and germline mutations related to DNA mismatch-repair (MMR) genes can clarify the prevalence and mechanism of inactivation in colorectal carcinoma (CRC). In the present study, 257 unselected patients referred for CRC resection were examined for evidence of defective DNA MMR. In particular, we sought to determine the frequency of hereditary defects in DNA MMR in this cohort of patients. MMR status was assessed by testing of tumors for the presence or absence of hMLH1, hMSH2, and hMSH6 protein expression and for microsatellite instability (MSI). Of the 257 patients, 51 (20%) had evidence of defective MMR, demonstrating high levels of MSI (MSI-H) and an absence of either hMLH1 (n=48) or hMSH2 (n=3). All three patients lacking hMSH2, as well as one patient lacking hMLH1, also demonstrated an absence of hMSH6. DNA sequence analysis of the 51 patients with defective MMR revealed seven germline mutations-four in hMLH1 (two truncating and two missense) and three in hMSH2 (all truncating). A detailed family history was available for 225 of the 257 patients. Of the seven patients with germline mutations, only three had family histories consistent with hereditary nonpolyposis colorectal cancer. Of the remaining patients who had tumors with defective MMR, eight had somatic mutations in hMLH1. In addition, hypermethylation of the hMLH1 gene promoter was present in 37 (88%) of the 42 hMLH1-negative cases available for study and in all MSI-H tumors that showed loss of hMLH1 expression but no detectable hMLH1 mutations. Our results suggest that, although defective DNA MMR occurs in approximately 20% of unselected patients presenting for CRC resection, hereditary CRC due to mutations in the MMR pathway account for only a small proportion of patients. Of the 257 patients, only 5 (1.9%) appear to have unequivocal evidence of hereditary defects in MMR. The epigenetic (nonhereditary) mechanism of hMLH1 promoter hypermethylation appears to be responsible for the majority of the remaining patients whose tumors are characterized by defective DNA MMR.     

Identification of mismatch repair protein complexes in HeLa nuclear extracts and their interaction with heteroduplex DNA

Deficiencies in DNA mismatch repair (MMR) have been found in hereditary colon cancers (hereditary non-polyposis colon cancer, HNPCC) as well as in sporadic cancers, illustrating the importance of MMR in maintaining genomic integrity. We have examined the interactions of specific mismatch repair proteins in human nuclear extracts. Western blot and co-immunoprecipitation studies indicate two complexes as follows: one consisting of hMSH2, hMSH6, hMLH1, and hPMS2 and the other consisting of hMSH2, hMSH6, hMLH1, and hPMS1. These interactions occur without the addition of ATP. Furthermore, the protein complexes specifically bind to mismatched DNA and not to a similar homoduplex oligonucleotide. The protein complex-DNA interactions occur primarily through hMSH6, although hMSH2 can also become cross-linked to the mismatched substrate when not participating in the MMR protein complex. In the presence of ATP the binding of hMSH6 to mismatched DNA is decreased. In addition, hMLH1, hPMS2, and hPMS1 no longer interact with each other or with the hMutSalpha complex (hMSH2 and hMSH6). However, the ability of hMLH1 to co-immunoprecipitate mismatched DNA increases in the presence of ATP. This interaction is dependent on the presence of the mismatch and does not appear to involve a direct binding of hMLH1 to the DNA.     

Nuclear reorganization of DNA mismatch repair proteins in response to DNA damage

The DNA mismatch repair (MMR) system is highly conserved and vital for preserving genomic integrity. Current mechanistic models for MMR are mainly derived from in vitro assays including reconstitution of strand-specific MMR and DNA binding assays using short oligonucleotides. However, fundamental questions regarding the mechanism and regulation in the context of cellular DNA replication remain. Using synchronized populations of HeLa cells we demonstrated that hMSH2, hMLH1 and PCNA localize to the chromatin during S-phase, and accumulate to a greater extent in cells treated with a DNA alkylating agent. In addition, using small interfering RNA to deplete hMSH2, we demonstrated that hMLH1 localization to the chromatin is hMSH2-dependent. hMSH2/hMLH1/PCNA proteins, when associated with the chromatin, form a complex that is greatly enhanced by DNA damage. The DNA damage caused by high doses of alkylating agents leads to a G₂ arrest after only one round of replication. In these G₂-arrested cells, an hMSH2/hMLH1 complex persists on chromatin, however, PCNA is no longer in the complex. Cells treated with a lower dose of alkylating agent require two rounds of replication before cells arrest in G₂. In the first S-phase, the MMR proteins form a complex with PCNA, however, during the second S-phase PCNA is missing from that complex. The distinction between these complexes may suggest separate functions for the MMR proteins in damage repair and signaling. Additionally, using confocal immunofluorescence, we observed a population of hMSH6 that localized to the nucleolus. This population is significantly reduced after DNA damage suggesting that the protein is shuttled out of the nucleolus in response to damage. In contrast, hMLH1 is excluded from the nucleolus at all times. Thus, the nucleolus may act to segregate a population of hMSH2–hMSH6 from hMLH1–hPMS2 such that, in the absence of DNA damage, an inappropriate response is not invoked.     

Nuclear localization of human DNA mismatch repair protein exonuclease 1 (hEXO1)

Human exonuclease 1 (hEXO1) is implicated in DNA mismatch repair (MMR) and mutations in hEXO1 may be associated with hereditary nonpolyposis colorectal cancer (HNPCC). Since the subcellular localization of MMR proteins is essential for proper MMR function, we characterized possible nuclear localization signals (NLSs) in hEXO1. Using fluorescent fusion proteins, we show that the sequence ⁴¹⁸KRPR⁴²¹, which exhibit strong homology to other monopartite NLS sequences, is responsible for correct nuclear localization of hEXO1. This NLS sequence is located in a region that is also required for hEXO1 interaction with hMLH1 and we show that defective nuclear localization of hEXO1 mutant proteins could be rescued by hMLH1 or hMSH2. Both hEXO1 and hMLH1 form complexes with the nuclear import factors importin β/α1,3,7 whereas hMSH2 specifically recognizes importin β/α3. Taken together, we infer that hEXO1, hMLH1 and hMSH2 form complexes and are imported to the nucleus together, and that redundant NLS import signals in the proteins may safeguard nuclear import and thereby MMR activity.     

Effect of Helicobacter pylori infection on the expression of DNA mismatch repair protein

BACKGROUND: Helicobacer pylori infection is a major gastric cancer risk factor. Deficient DNA mismatch repair (MMR) caused by H. pylori may underlie microsatellite instability (MSI) in the gastric epithelium and may represent a major mechanism of mutation accumulation in the gastric mucosa during the early stages of H. pylori-associated gastric carcinogenesis. In this study, we examined the expression of DNA MMR protein (hMLH1 and hMSH2) in patients with chronic H. pylori infection before and after eradication of the infection. MATERIALS AND METHODS: Gastric tissue samples were collected from 60 patients with H. pylori gastritis and peptic ulcer disease before and after eradication of the infection. The DNA MMR protein expression (hMLH1 and hMSH2) was determined by immunohistochemical staining in 60 patients before and after H. pylori eradication. The percentage of epithelial cell nuclei and intensity of staining were then compared in gastric biopsies before and after eradication. RESULTS: The percentage of hMLH1 (76.60 +/- 20.27, 84.82 +/- 12.73, p=.01) and hMSH2 (82.36 +/- 12.86, 88.11 +/- 9.27, p<.05) positive epithelial cells significantly increased in 53 patients who became H. pylori-negative after eradication therapy. However, the intensity of hMLH1 and hMSH2 staining was not significantly different. In those 7 patients, who did not respond to the eradication therapy and were still H. pylori-positive, the percent positivity and intensity of hMLH1 and hMSH2 staining did not change. CONCLUSIONS: The expression of DNA MMR proteins increased in the gastric mucosa after H. pylori eradication, indicating that H. pylori gastritis may be associated with a reduced DNA MMR system during infection. The effect of H. pylori infection on MMR protein expression appears to be at least partially reversible after H. pylori eradication. These data suggest that H. pylori gastritis might lead to a deficiency of DNA MMR in gastric epithelium that may increase the risk of mutation accumulation in the gastric mucosa cells during chronic H. pylori infection.     

Mutation rates of TGFBR2 and ACVR2 coding microsatellites in human cells with defective DNA mismatch repair

Microsatellite instability promotes colonic tumorigenesis through generating frameshift mutations at coding microsatellites of tumor suppressor genes, such as TGFBR2 and ACVR2. As a consequence, signaling through these TGFbeta family receptors is abrogated in DNA Mismatch repair (MMR)-deficient tumors. How these mutations occur in real time and mutational rates of these human coding sequences have not previously been studied. We utilized cell lines with different MMR deficiencies (hMLH1-/-, hMSH6-/-, hMSH3-/-, and MMR-proficient) to determine mutation rates. Plasmids were constructed in which exon 3 of TGFBR2 and exon 10 of ACVR2 were cloned +1 bp out of frame, immediately after the translation initiation codon of an enhanced GFP (EGFP) gene, allowing a -1 bp frameshift mutation to drive EGFP expression. Mutation-resistant plasmids were constructed by interrupting the coding microsatellite sequences, preventing frameshift mutation. Stable cell lines were established containing portions of TGFBR2 and ACVR2, and nonfluorescent cells were sorted, cultured for 7-35 days, and harvested for flow cytometric mutation detection and DNA sequencing at specific time points. DNA sequencing revealed a -1 bp frameshift mutation (A9 in TGFBR2 and A7 in ACVR2) in the fluorescent cells. Two distinct fluorescent populations, M1 (dim, representing heteroduplexes) and M2 (bright, representing full mutants) were identified, with the M2 fraction accumulating over time. hMLH1 deficiency revealed 11 (5.91 x 10(-4)) and 15 (2.18 x 10(-4)) times higher mutation rates for the TGFBR2 and ACVR2 microsatellites compared to hMSH6 deficiency, respectively. The mutation rate of the TGFBR2 microsatellite was approximately 3 times higher in both hMLH1 and hMSH6 deficiencies than the ACVR2 microsatellite. The -1 bp frameshift mutation rates of TGFBR2 and ACVR2 microsatellite sequences are dependent upon the human MMR background.     

Muir-Torre phenotype has a frequency of DNA mismatch-repair-gene mutations similar to that in hereditary nonpolyposis colorectal cancer families defined by the Amsterdam criteria.

Muir-Torre syndrome (MTS) is an autosomal dominant disease defined by the coincidence of at least one sebaceous skin tumor and one internal malignancy. About half of MTS patients are affected by colorectal cancer. In a subgroup of MTS patients the disease has an underlying DNA mismatch-repair (MMR) defect and thus is allelic to hereditary nonpolyposis colorectal cancer (HNPCC). The purpose of this study was to examine to what extent germ-line mutations in DNA MMR genes are the underlying cause of the MTS phenotype. We ascertained 16 MTS patients with sebaceous skin tumors and colorectal cancer, and we examined their skin and visceral tumors for microsatellite instability. All the patients exhibited high genomic instability in at least one tumor. The search for germ-line mutations in the hMSH2 and hMLH1 genes in 13 of the MTS patients revealed truncating mutations in 9 (69%): eight mutations in the hMSH2 gene and one in the hMLH1 gene. This is the first systematic search for germ-line mutations in patients ascertained on the basis of sebaceous skin tumors. Our results indicate that (1) MTS patients exhibit significantly more mutations in the hMSH2 gene than in the hMLH1 gene; and (2) the subpopulation of MTS patients who are also affected by colorectal cancer, irrespective of family history and age at onset of tumors, may have a likelihood for an underlying DNA MMR defect similar to that for patients with a family history fulfilling the strict clinical criteria for HNPCC.     

The interaction of the human MutL homologues in hereditary nonpolyposis colon cancer

Germline mutations in two human mismatch repair (MMR) genes, hMSH2 and hMLH1, appear to account for approximately 70% of the common cancer susceptibility syndrome hereditary nonpolyposis colorectal cancer (HNPCC). Although the hMLH1 protein has been found to copurify with another MMR protein hPMS2 as a heterodimer, their function in MMR is unknown. In this study, we have identified the physical interaction regions of both hMLH1 with hPMS2. We then examined the effects of hMLH1 missense alterations found in HNPCC kindreds for their interaction with hPMS2. Four of these missense alterations (L574P, K616Delta, R659P, and A681T) displayed >95% reduction in binding to hPMS2. Two additional missense alterations (K618A and K618T) displayed a >85% reduction in binding to hPMS2, whereas three missense alterations (S44F, V506A, and E578G) displayed 25-65% reduction in binding to hPMS2. Interestingly, two HNPCC missense alterations (Q542L and L582V) contained within the consensus interaction region displayed no effect on interaction with hPMS2, suggesting that they may affect other functions of hMLH1. These data confirm that functional deficiencies in the interaction of hMLH1 with hPMS2 are associated with HNPCC as well as suggest that other unknown functional alteration of the human MutL homologues may lead to tumorigenesis in HNPCC kindreds.     

Identification of factors interacting with hMSH2 in the fetal liver utilizing the yeast two-hybrid system. In vivo interaction through the C-terminal domains of hEXO1 and hMSH2 and comparative expression analysis

Mutations in DNA mismatch repair (MMR) genes have been shown to segregate with Hereditary Nonpolyposis Colorectal Cancer (HNPCC). However, because many HNPCC families fail to display mutations in known MMR genes, we argued that changes in other components of the MMR pathway may be responsible. The increasing number of proteins reported to interact in the MMR pathway suggests that larger complexes are formed, the composition of which may differ among cell types and tissues. In an attempt to identify tissue-specific MMR-associated factors, we employed the yeast two-hybrid system, using the human hMSH2 as bait and a human fetal liver library as prey. We demonstrate that hMSH2 interacts with a human 5'-3' exonuclease 1 (hEXO1/HEX1) and that this interaction is mediated through their C-terminal domains. The hMSH6 protein does not interact with hEXO1 in the two-hybrid system. Dot-blot analysis of multiple tissue RNA revealed that hMSH2 and hEXO1 are coexpressed at high levels in fetal liver as well as in adult testis and thymus. Northern blot analysis also revealed that hEXO1/HEX1 is highly expressed in several liver cancer cell lines as well as in colon and pancreas adenocarcinomas, but not in the corresponding non-neoplastic tissue.     

Bi-directional routing of DNA mismatch repair protein human exonuclease 1 to replication foci and DNA double strand breaks

Human exonuclease 1 (hEXO1) is implicated in DNA metabolism, including replication, recombination and repair, substantiated by its interactions with PCNA, DNA helicases BLM and WRN, and several DNA mismatch repair (MMR) proteins. We investigated the sub-nuclear localization of hEXO1 during S-phase progression and in response to laser-induced DNA double strand breaks (DSBs). We show that hEXO1 and PCNA co-localize in replication foci. This apparent interaction is sustained throughout S-phase. We also demonstrate that hEXO1 is rapidly recruited to DNA DSBs. We have identified a PCNA interacting protein (PIP-box) region on hEXO1 located in its COOH-terminal ((788)QIKLNELW(795)). This motif is essential for PCNA binding and co-localization during S-phase. Recruitment of hEXO1 to DNA DSB sites is dependent on the MMR protein hMLH1. We show that two distinct hMLH1 interaction regions of hEXO1 (residues 390-490 and 787-846) are required to direct the protein to the DNA damage site. Our results reveal that protein domains in hEXO1 in conjunction with specific protein interactions control bi-directional routing of hEXO1 between on-going DNA replication and repair processes in living cells.     

Hereditary nonpolyposis colorectal cancer families not complying with the Amsterdam criteria show extremely low frequency of mismatch-repair-gene mutations.

Hereditary nonpolyposis colorectal cancer (HNPCC) is a common autosomal dominant cancer-susceptibility condition characterized by early onset colorectal cancer. Germ-line mutations in one of four DNA mismatch repair (MMR) genes, hMSH2, hMLH1, hPMS1, or hPMS2, are known to cause HNPCC. Although many mutations in these genes have been found in HNPCC kindreds complying with the so-called Amsterdam criteria, little is known about the involvement of these genes in families not satisfying these criteria but showing clear-cut familial clustering of colorectal cancer and other cancers. Here, we applied denaturing gradient-gel electrophoresis to screen for hMSH2 and hMLH1 mutations in two sets of HNPCC families, one set comprising families strictly complying with the Amsterdam criteria and another set in which at least one of the criteria was not satisfied. Interestingly, hMSH2 and hMLH1 mutations were found in 49% of the kindreds fully complying with the Amsterdam criteria, whereas a disease-causing mutation could be identified in only 8% of the families in which the criteria were not satisfied fully. In correspondence with these findings, 4 of 6 colorectal tumors from patients belonging to kindreds meeting the criteria showed microsatellite instability, whereas only 3 of 11 tumors from the other set of families demonstrated this instability. Although the number of tumors included in the study admittedly is small, the frequencies of mutations in the MMR genes show obvious differences between the two clinical sets of families. These results also emphasize the practical importance of the Amsterdam criteria, which provide a valid clinical subdivision between families, on the basis of their chance of carrying an hMSH2 or an hMLH1 mutation, and which bear important consequences for genetic testing and counseling and for the management of colorectal cancer families.     

Loss of Heterozygosity in DNA Mismatch Repair Genes in Human Atherosclerotic Plaques

To detect the incidence of loss of heterozygosity (LOH) in DNA mismatch repair genes (MMR) occurring in atherosclerosis, fifty human autopsy cases of atherosclerosis were examined for LOH using 19 microsatellite markers, in three single and four tetraplex microsatellite assays. The markers used are located on or close to MMR genes. Fourteen specimens (28%) showed allelic imbalance in at least one locus. Loci hMSH2 (2p22.3–p16.1), hPMS1 (2q24.1–q32.1), and hMLH1 (3p21.32–p21.1) exhibited LOH (10, 10, and 12% respectively). We found that loss of heterozygosity on hMSH2, hPMS1, and hMLH1, occurs in atherosclerosis. The occurrence of such genomic alterations may represent important events in the development of atherosclerosis.     

Formation of hMSH4-hMSH5 heterocomplex is a prerequisite for subsequent GPS2 recruitment

Increasing evidence suggests that components of the DNA mismatch repair (MMR) pathway play multifunctional roles beyond the scope of mismatch correction, including the modulation of cellular responses to DNA damage and homologous recombination. The heterocomplex consisting of MutS homologous proteins, hMSH4 and hMSH5, is believed to play essential roles in meiotic DNA repair particularly during the process of meiotic homologous recombination (HR). In order to gain a better understanding of the mechanistic basis underlying the roles of these two human MutS proteins, we have identified G-protein pathway suppressor 2 (GPS2) (i.e., an integral component of a deacetylase complex) as an interacting protein partner specifically for the hMSH4-hMSH5 heterocomplex. The interaction with GPS2 is entirely dependent on the physical association between hMSH4 and hMSH5, as disruption of the interaction between hMSH4 and hMSH5 completely abolishes GPS2 recruitment. Our analysis further indicates that the association with GPS2 is mediated through the interface of hMSH4-hMSH5 complex and the N-terminal region of GPS2. Moreover, these three proteins interact in human cells, and analysis of microarray data suggested a coordinated expression pattern of these genes during the onset of meiosis. Together, the results of our present study suggest that the GPS2-associated deacetylase complex might function in concert with hMSH4-hMSH5 during the process of homologous recombination.     

变性高效液相色谱法筛检hMLH1和hMSH2微小突变技术

目的:建立基于变性高效液相色谱法(DHPLC)的快速筛检错配修复基因hMLH1和hMSH2微小突变的技术平台。方法:自行设计PCR扩增hMLH1和hMSH2各外显子的引物,应用DHPLC检测26个遗传性非息肉病性结直肠癌(HNPCC)家系的先证者hMLH1和hMSH2种系微小突变,并与先前进行的DNA直接测序结果相比较。结果:hMLH1与hMSH2各外显子的PCR扩增引物,均能很好地扩增出相应的外显子及剪接区;DHPLC检出了所有已知突变,突变阳性筛检与阴性筛检的灵敏度和特异性均为100%;hMLH1的扩增子12A和hMSH2的扩增子2、3、7、5中相应外显子的剪接区跨越2个温度,而且相差较大(2.2-8.5℃):与DNA直接测序相比较,DHPLC具有快速、高效、低劳动强度、费用低、人为误差小、灵敏度和特异性高等优点。结论:基于DH- PLC的突变筛检平台,能够有效地筛检hMLH1和hMSH2微小突变,并具有较高的费用效率比。     

Direct association of Bloom’s syndrome gene product with the human mismatch repair protein MLH1

Bloom’s syndrome (BS) is a rare genetic disorder characterised by genomic instability and cancer susceptibility. BLM, the gene mutated in BS, encodes a member of the RecQ family of DNA helicases. Here, we identify hMLH1, which is involved in mismatch repair (MMR) and recombination, as a protein that directly interacts with BLM both in vivo and in vitro, and that the two proteins co-localise to discrete nuclear foci. The interaction between BLM and hMLH1 appears to have been evolutionarily conserved, as Sgs1p, the Saccharomyces cerevisiae homologue of BLM, interacts with yeast Mlh1p. However, cell extracts derived from BS patients show no obvious defects in MMR compared to wild-type- and BLM-complemented BS cell extracts. We conclude that the hMLH1–BLM interaction is not essential for post-replicative MMR, but, more likely, is required for some aspect of genetic recombination.