Silencing-specific methylation and single nucleotide polymorphism of hMLH1 promoter in gastric carcinomas

AIM: To investigate CpG methylation and single nucleotide polymorphism (SNP) of a specific promoter region of hMLH1 in primary gastric carcinoma. METHODS: Primary gastric carcinomas (n=80), their corresponding normal mucosal samples, and gastric mucosal biopsies from normal/gastritis control patients (n=54) were used. Hypermethylation at -253 nt and -251 nt in relation with the translational start site and SNP of a silencing specific region (-339 nt-46 nt) in the hMLH1 promoter were analyzed by Bst UI-combined bisulfite assay (COBRA), denaturing high performance liquid chromatogram (DHPLC), and sequencing. RESULTS: (A) The specific methylation at -253 nt and -251 nt was observed in 2 of 60 primary gastric carcinomas, but neither in all of the corresponding mucosa nor in normal/gastritis samples, by Bst UI-COBRA and DHPLC. (B) The hMLH1 promoter was methylated homogeneously in the xenograft of the primary gastric carcinoma with the methylated and unmethylated hMLH1. (C) The pattern of SNP at -93 nt of the hMLH1 promoter in 54 Chinese patients with gastric carcinoma was the same as that in the control patients: 51 % was A/G heteroalleles, 34 % and 15 % were A/A and G/G homoalleles, respectively. CONCLUSION: Biallelic inactivation of hMLH1 by epigenetic silencing existed in human primary gastric carcinoma homogeneously. Hypermethylation of hMLH1 may play a role in the early stage of development of a few gastric carcinomas. The SNP at -93 nt is not related to the susceptibility of gastric carcinomas.     

Mechanisms of inactivation of mismatch repair genes in human colorectal cancer cell lines: the predominant role of hMLH1.

Fifteen to twenty-five percent of sporadic colorectal carcinomas are replication error (RER) positive. Because the frequency of mutations in the mismatch repair genes (hMLH1 and hMSH2) is low in these tumors, we have investigated the role of mutational inactivation, methylation of the promoter region, and loss of heterozygosity (LOH) as a possible explanation for the mutator phenotype of RER+ colorectal cancer cell lines. Genomic DNA was extracted from a panel of 49 human colorectal cancer cell lines. The RER status was determined by amplification of BAT-26. All exons of hMLH1 and hMSH2 were amplified with the PCR and screened by using single-strand conformational polymorphism and direct sequencing. The methylation status was ascertained by methylation-specific PCR after bisulfite modification of DNA. Western blotting for hMLH1 was performed on methylated cell lines before and after the addition of the demethylating agent 5-azacytidine. LOH was sought by GENESCAN analysis of amplified CA repeat markers and indirectly by determining the number of homozygotes in the cell lines and human random controls. Twelve cell lines from ten tumors (24%) were RER+. Hypermethylation of the hMLH1 promoter occurred in five of ten (50%) RER+ tumors, whereas three of thirty-two (6%) RER tumors showed partial methylation. None of the fully methylated cell lines expressed hMLH1, although all reexpressed hMLH1 after treatment with 5-azacytidine. There was no LOH in the RER+ tumors in either hMLH1 or hMSH2. Our results suggest that mutations of hMLH1 together with hypermethylation of the promoter region, but not LOH, are the cause of the mutator phenotype in the majority (70%) of RER+ tumors.     

Extensive methylation of hMLH1 promoter region predominates in proximal colon cancer with microsatellite instability.

BACKGROUND & AIMS: Methylation of the hMLH1 promoter region has been suggested to cause microsatellite instability (MSI) in sporadic colorectal carcinoma (CRC). We studied the methylation profile in a wide region of the hMLH1 promoter and compared with the hMLH1 protein expression and MSI status in 88 cases of sporadic CRC. METHODS: Na-bisulfite treatment and polymerase chain reaction single-strand conformation polymorphism analysis was performed using 5 sets of polymerase chain reaction primers spanning the promoter region of the hMLH1 to examine methylation status. Results were compared with immunostaining using anti-hMLH1 monoclonal antibody and MSI status of the tumor samples. RESULTS: Methylation status was classified as full or partial methylation. Full methylation indicates the methylation of all CpG sites in the examined regions. Methylation of the hMLH1 promoter was observed in 88.9% (16 of 18) of CRCs showing high frequency MSI (MSI-H), among which 89% (14 of 16) had full methylation with reduced hMLH1 protein expression. All cases showing full methylation were proximal colon tumors with MSI-H. In cases with partial methylation, only the upstream region of the hMLH1 promoter was methylated. Partial methylation was also shown in 33.3% (6 of 18) of the normal mucosa of MSI-H cases. Frequencies of methylation were significantly correlated with female gender (P = 0.0009) and aging (P = 0.007). CONCLUSIONS: Full methylation of the hMLH1 promoter region and subsequent gene inactivation may play a crucial role in the carcinogenesis of MSI-H CRCs in the proximal colon. Methylation upstream of the hMLH1 promoter appears to be an early event in the carcinogenesis of MSI-H tumors.     

Absence or decreased levels of the hMLH1 protein in human gastric carcinoma cell lines: implication of hMLH1 in alkylation tolerance

Defective hMLH1 function has been increasingly associated with acquired cellular resistance to DNA alkylation damage in human colorectal and endometrial cancer cells. To investigate the relationship between the DNA alkylation tolerance and the hMLH1 status in human gastric carcinoma cells, we determined the cellular response to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), the mutational changes, and the expression of hMLH1 in 11 human gastric carcinoma cell lines. Of 11 cell lines, 4 (SNU-5, -16, -620, and -719) were sensitive, whereas 7 (SNU-1, -216, -484, -520, -601, -638, and -668) were resistant to the cytotoxic effect of MNNG. As determined by Western analysis, it was evident that all the MNNG-resistant cell lines except one (SNU-601) produced very low or undetectable levels of hMLH1 protein compared to the MNNG-sensitive cell lines. A homozygous non-sense mutation that resulted in truncated protein was found in one MNNG-resistant cell line (SNU-1). Therefore, to determine whether the sensitivity of cells to MNNG can be restored by exogenous expression of hMLH1 protein, wild-type hMLH1 cDNA was introduced into the MNNG-resistant cells (SNU-1). The cytotoxicity test showed that expression of exogenous wild-type hMLH1 protein caused an increase in sensitivity to the cytotoxic effect of MNNG. This restoration was confirmed by an increase in the cell population containing less than the G1 amount of DNA (cell death) in the wild-type hMLH1-transfected cells, as determined by flow cytometry analysis. Together our results suggest that (1) the absence or decreased level of wild-type hMLH1 protein may be a frequent event in the human gastric carcinoma cell lines, (2) such alterations in the hMLH1 protein are closely associated with the MNNG tolerance in the human gastric carcinoma cell lines, and (3) the hMLH1 protein participates not only in the repair of DNA mismatches but also in the mechanism of escape from the cytotoxic effects of DNA alkylation damage. Received: 26 January 1998 / Accepted: 18 March 1998     

胃癌维甲酸信号通路相关基因启动子区协同高甲基化的研究

背景：启动子区高甲基化与胃癌中多种抑癌基因表达沉默密切相关。目的：探讨维甲酸信号通路相关基因维甲酸受体B（RAR13）、细胞维生素A结合蛋白1（CRBP1）和他扎罗汀诱导基因1（TIG1）启动子区高甲基化与胃癌的关系。方法：以甲基化特异性聚合酶链反应（MSP）检测40例胃癌标本、10例正常胃黏膜标本和6株胃癌细胞株的RAR13、CRBPI和TIG1基因启动子区甲基化状态，分析i者甲基化状态的相关性及其与胃癌1晦床病理特征的关系。以逆转录聚合酶链反应（RT—PCR）检测胃癌细胞株RAR13、CRBP1和TIG1mRNA表达。结果：40例胃癌组织的RAR13、CRBPI和TIG1基因甲基化率分别为45．0％、32．5％和57．5％，10例正常胃黏膜组织均未检测到上述基因甲基化（P〈0．05）。胃癌组织中RAR13的甲基化状态与CRBP1和TIG1的甲基化状态显著相关（P〈0．05），但三者的甲基化状态与胃癌临床病理特征无相关性。启动子区高甲基化胃癌细胞株相应基因mRNA表达缺失或减弱。结论：胃癌组织常发生维甲酸信号通路相关基因RAR13、CRBP1和TIG1启动子区高甲基化，高甲基化可能是相应基因转录失活的重要原因。     

HTRA1 gene expression in gastric epithelial cells

Objective:To explore HtrA1 gene expression aud its regulation in human gastric cancers.Methods:The HtrA1 mRNA levels were examined by QPCR analysis and coufirmed its expression with Northern blot analysis.The HtrA1 protein levels in all six gastric epithelial cell lines were investigated by Western blot analysis.Gene copy number was accessed and then sequenced the coding region from each mRNA in all six cell lines.The HtrA1 promoter region DNA methylation status was detected by using bisulfite sequeucing analysis.Effect of decitabine and TSA on HTRA1 expression in gastric cancer cell line was determined by RTPCR.Results:HIC analysis indicated that HtrA1 was highly expressed in normal epithelium,but dramatically down-regulated in gastric carcinoma tissues and variably expressed in tumor-adjacent tissues.HtrA1 gene expression was dramatically decreased in gastric carcinoma cells compared to nontumorigenic counterparts.The HtrA1 gene loss in any of the 4 breast cancer cell lines was not detected.Total 14 CpGs in this region were all methylated in gastric cancer cells,whereas two normal cells.GES-1 and HFI-145,were having several unmethylated cytosines in this region.HtrA1 showed as~Mr 44,000,Expression of HtrA1 protein was not observed in any of the four gastric caucer cell lines.BGC-823.MKN-45.SGC-7901and MKN-28.HtrA1 expression was observed in the HF1-145and GES-1 cell lines.Conclusions:The epigenetic silencing for HtrA1gene expression could provide a possible strategy for re-activating Htrt1 gene expression in gastric cancer cells.thus facilitating further investigation of HtrA1's role in chemotherapy.     

Effects of dietary intake and genetic factors on hypermethylation of the hMLH1 gene promoter in gastric cancer

AIM: Hypermethylation of the promoter of the hMLH1 gene, which plays an important role in mismatch repair during DNA replication, occurs in more than 30% of human gastric cancer tissues. The purpose of this study was to investigate the effects of environmental factors, genetic polymorphisms of major metabolic enzymes, and microsatellite instability on hypermethylation of the promoter of the hMLH1 gene in gastric cancer. METHODS: Data were obtained from a hospital-based, case-control study of gastric cancer. One hundred and ten gastric cancer patients and 220 age- and sex-matched control patients completed a structured questionnaire regarding their exposure to environmental risk factors. Hypermethylation of the hMLH1 gene promoter, polymorphisms of the GSTM1, GSTT1, CYP1A1, CYP2E1, ALDH2 and L-myc genes, microsatellite instability and mutations of p53 and Ki-ras genes were investigated. RESULTS: Both smoking and alcohol consumption were associated with a higher risk of gastric cancer with hypermethylation of the hMLH1 gene promoter. High intake of vegetables and low intake of potato were associated with increased likelihood of gastric cancer with hypermethylation of the hMLH1 gene promoter. Genetic polymorphisms of the GSTM1, GSTT1, CYP1A1, CYP2E1, ALDH2, and L-myc genes were not significantly associated with the risk of gastric cancer either with or without hypermethylation in the promoter of the hMLH1 gene. Hypermethylation of the hMLH1 promoter was significantly associated with microsatellite instability (MSI): 10 of the 14 (71.4%) MSI-positive tumors showed hypermethylation, whereas 28 of 94 (29.8%) the MSI-negative tumors were hypermethylated at the hMLH1 promoter region. Hypermethylation of the hMLH1 gene promoter was significantly inversely correlated with mutation of the p53 gene. CONCLUSION: These results suggest that cigarette smoking and alcohol consumption may influence the development of hMLH1-positive gastric cancer. Most dietary factors and polymorphisms of GSTM1, GSTT1, CYP1A1, CYP2E1, ALDH2, and L-myc genes are not independent risk factors for gastric cancer with hypermethylation of the hMLH1 promoter. These data also suggest that there could be two or more different molecular pathways in the development of gastric cancer, perhaps involving tumor suppression mechanisms or DNA mismatch repair.     

Effects of dietary intake and genetic factors on hypermethyiation of the hMLH1 gene promoter in gastric cancer

AIM: Hypermethylation of the promoter of the hMLH1 gene, which plays an important role in mismatch repair during DNA replication, occurs in more than 30% of human gastric cancer tissues. The purpose of this study was to investigate the effects of environmental factors, genetic polymorphisms of major metabolic enzymes, and microsatellite instability on hypermethylation of the promoter of the hMLH1 gene in gastric cancer. METHODS: Data were obtained from a hospital-based, case-control study of gastric cancer. One hundred and ten gastric cancer patients and 220 age- and sex-matched control patients completed a structured questionnaire regarding their exposure to environmental risk factors. Hypermethylation of the hMLH1 gene promoter, polymorphisms of the GSTM1, GSTT1, CYP1A1, CYP2E1, ALDH2 and L-myc genes, microsatellite instability and mutations of p53 and Ki-ras genes were investigated. RESULTS: Both smoking and alcohol consumption were associated with a higher risk of gastric cancer with hypermethylation of the hMLH1 gene promoter. High intake of vegetables and low intake of potato were associated with increased likelihood of gastric cancer with hypermethylation of the hMLH1 gene promoter. Genetic polymorphisms of the GSTM1, GSTT1, CYP1A1, CYP2E1, ALDH2, and L-myc genes were not significantly associated with the risk of gastric cancer either with or without hypermethylation in the promoter of the hMLH1 gene. Hypermethylation of the hMLH1 promoter was significantly associated with microsatellite instability (MSI): 10 of the 14 (71.4%) MSI-positive tumors showed hypermethylation, whereas 28 of 94 (29.8%) the MSI-negative tumors were hypermethylated at the hMLH1 promoter region, Hypermethylation of the hMLH1 gene promoter was significantly inversely correlated with mutation of the p53 gene. CONCLUSION: These results suggest that cigarette smoking and alcohol consumption may influence the development of hMLH1-positive gastric cancer. Most dietary factors and polymorphisms of GSTM1, GSTT1, CYP1A1, CYP2E1, ALDH2, and L-myc genes are not independent risk factors for gastric cancer with hypermethylation of the hMLH1 promoter. These data also suggest that there could be two or more different molecular pathways in the development of gastric cancer, perhaps involving tumor suppression mechanisms or DNA mismatch repair.     

hMLH1基因高甲基化在胃癌发生、发展中的作用

基因启动子区CpG岛甲基化使许多基因失活，从而导致恶性肿瘤的发生和发展。目的：检测hMLHl基因启动子区CpG岛甲基化水平，探讨其胃癌发生、发展中的作用。方法：以甲基化特异性聚合酶链反应（MSP）检测41例胃癌、40例癌前病变和38例对照组织中hMLHl基因启动子区CpG岛甲基化状态，并分析其与胃癌患者临床病理特征的关系。结果：胃癌组织中hMLHl基因启动子区CpG岛甲基化阳性率为34．1％，显著高于癌前病变组的5．0％和对照组的0％（P〈0．05）。hMLHl基因甲基化阳性率与胃癌患者的年龄和肿瘤浸润深度有关（阳性率分别为46．4％对7．7％和55．0％对14.3％，P〈0．05），与性别、肿瘤分化程度和淋巴结转移无关（阳性率分别为34．8％对33．3％、28．0％对43．8％和38．1％对30．0％）。结论：胃癌组织中存在hMLHl基因启动子区CpG岛高甲基化，可能与胃癌的发生、发展有关．且可能在老年胃癌患者的肿瘤发生过程中起重要作用。     

Extensive but hemiallelic methylation of the hMLH1 promoter region in early-onset sporadic colon cancers with microsatellite instability.

BACKGROUND AND AIMS: Methylation of the hMLH1 promoter region is frequently observed in microsatellite instability (MSI)-positive sporadic colorectal carcinomas. We studied hMLH1 promoter methylation in peripheral blood lymphocytes of 87 index patients representing 29 cases of hereditary nonpolyposis colorectal cancers (HNPCCs), 28 cases of atypical HNPCCs, and 30 sporadic cases of the development of early-onset colorectal carcinomas or multiple primary cancers. METHODS: Methylation of the hMLH1 promoter region was analyzed by Na-bisulfite polymerase chain reaction/single-strand conformation polymorphism analysis or methylation-specific polymerase chain reaction. MSI, allelic status of the hMLH1 locus, and loss of hMLH1 protein expression were examined in cases for which tumor tissues were available. RESULTS: Extensive methylation of the hMLH1 promoter was detected in peripheral blood lymphocytes of 4 of 30 patients with sporadic early-onset colon cancer, among whom multiple primary cancers (1 colon and 1 endometrial cancer) developed in 2 cases. This methylation was not detected in analyses of HNPCC or atypical HNPCC groups or healthy control subjects. MSI was positive, and extensive methylation was detected in both cancers (colon and endometrial cancer) and normal tissues (colon, gastric mucosa, endometrium, and bone marrow) in all of the examined cases (3 of 3). Analysis of a polymorphic site in the hMLH1 promoter in 2 informative cases showed that methylation was hemiallelic. In 1 case, the unmethylated allele was lost in the colon cancer but not in the metachronous endometrial cancer. CONCLUSIONS: Constitutive, hemiallelic methylation of the hMLH1 promoter region was shown to be associated with carcinogenesis in sporadic, early-onset MSI-positive colon cancers.     

Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma

Inactivation of the genes involved in DNA mismatch repair is associated with microsatellite instability (MSI) in colorectal cancer. We report that hypermethylation of the 5′ CpG island of hMLH1 is found in the majority of sporadic primary colorectal cancers with MSI, and that this methylation was often, but not invariably, associated with loss of hMLH1 protein expression. Such methylation also occurred, but was less common, in MSI− tumors, as well as in MSI+ tumors with known mutations of a mismatch repair gene (MMR). No hypermethylation of hMSH2 was found. Hypermethylation of colorectal cancer cell lines with MSI also was frequently observed, and in such cases, reversal of the methylation with 5-aza-2′-deoxycytidine not only resulted in reexpression of hMLH1 protein, but also in restoration of the MMR capacity in MMR-deficient cell lines. Our results suggest that microsatellite instability in sporadic colorectal cancer often results from epigenetic inactivation of hMLH1 in association with DNA methylation.     

Emerging concepts in colorectal neoplasia

An understanding of the mechanisms that explain the initiation and early evolution of colorectal cancer should facilitate the development of new approaches to effective prevention and intervention. This review highlights deficiencies in the current model for colorectal neoplasia in which APC mutation is placed at the point of initiation. Other genes implicated in the regulation of apoptosis and DNA repair may underlie the early development of colorectal cancer. Inactivation of these genes may occur not by mutation or loss but through silencing mediated by methylation of the gene's promoter region. hMLH1 and MGMT are examples of DNA repair genes that are silenced by methylation. Loss of expression of hMLH1 and MGMT protein has been demonstrated immunohistochemically in serrated polyps. Multiple lines of evidence point to a "serrated" pathway of neoplasia that is driven by inhibition of apoptosis and the subsequent inactivation of DNA repair genes by promoter methylation. The earliest lesions in this pathway are aberrant crypt foci (ACF). These may develop into hyperplastic polyps or transform while still of microscopic size into admixed polyps, serrated adenomas, or traditional adenomas. Cancers developing from these lesions may show high- or low-level microsatellite instability (MSI-H and MSI-L, respectively) or may be microsatellite stable (MSS). The suggested clinical model for this alternative pathway is the condition hyperplastic polyposis. If colorectal cancer is a heterogeneous disease comprising discrete subsets that evolve through different pathways, it is evident that these subsets will need to be studied individually in the future.     

Genetic diagnosis strategy of hereditary non-polyposis colorectal cancer

AIM： To study the characteristics of mismatch repair gene mutation of Chinese hereditary non-polyposis colorectal cancer （HNPCC） and hMLH1 gene promoter methylation, and to improve the screening strategy and explore the pertinent test methods. METHODS： A systematic analysis of 30 probands from HNPCC families in the north of China was performed by immunohistochemistry, microsatellite instability （MSI）, gene mutation and methylation detection. RESULTS： High frequency microsatellite instability occurred in 25 probands （83.3%） of HNPCC family. Loss of hMLH1 and hMSH2 protein expression accounted for 88% of all microsatellite instability. Pathogenic mutation occurred in 14 samples and 3 novel mutational sites were discovered. Deletion of exons 1-6, 1-7 and 8 of hMSH2 was detected in 3 samples and no large fragment deletion was found in hMLH1. Of the 30 probands, hMLH1 gene promoter methylation occurred in 3 probands. The rate of gene micromutation detection combined with large fragment deletion detection was 46.7%-56.7%. The rate of the two methods in combination with methylation detection was 63.3%. CONCLUSION： Scientific and rational detection strategy can improve the detection rate of HNPCC. Based on traditional molecular genetics and combined with epigenetics, multiple detection methods can accurately diagnose HNPCC.