hMLH1 Promoter Region Research Articles

Aberrant methylation detection of the hMLH1 promoter and the IGF2 DMR region using methylation-sensitive high resolution melting (MS-HRM) in colorectal cancer

Epigenetic alterations and gene mutations causing inactivation of tumor suppressor genes and activation of oncogenes can regulate signaling pathways contributing to colon cancer formation. Many genes have been reported to be aberrantly methylated in the CRC genome, and it is likely that only subsets of these genes are important in the pathogenesis of colorectal tumors. We applied the methylation-specific high resolution melting (MS-HRM) technique to study methylation of the IGF2 DMR and the hMLH1 gene promoter in 60 colorectal cancer and adjacent normal tissues of same patients compare to 20 normal tissue samples. In this study, 5 of the 60 colorectal cancer samples (8.3%) were found to be methylated at the hMLH1 promoter region and 32 of the 60 colorectal cancer samples (53.3%) were found to be hypomethylated at the IGF2 DMR region. Adjacent normal tissues were unmethylated for hMLH1 and 5% showed hypomethylation for IGF2 DMR. There was significant correlation between aberrant methylation of hMLH1 and IGF2 DMR with tumor location (p=0.002, p=0.026 respectively). In addition, a tendency of association between IGF2 DMR hypomethylation and age (p=0.06) was observed. We demonstrated effectiveness of the MS-HRM technique to analyze methylation of IGF2 DMR and hMLH1 promoter region and methylation significantly correlated with tumor location in colorectal cancer patients.

5,10-Methylenetetrahydrofolate reductase 677C>T polymorphism and microsatellite instability in sporadic colorectal cancer

Purpose: Hypermethylation of human mut L homologue 1 (hMLH1) promoter region is known to cause sporadic microsatellite instability (MSI) colorectal cancers. 5,10-methylenetetrahydrofolate reductase (MTHFR) is the key enzyme in folate metabolism, acting as a methyl donor for DNA methylation. In this study, we investigate whether the polymorphism of MTHFR 677C>T plays a role in the alteration of the promoter-specific hypermethylation, predisposing to MSI colorectal cancers. Methods: Total of 487 sporadic colorectal cancer patients in CHA Bundang Medical Center were collected. MSI was identified when two or more are positive among five microsatellite markers (BAT25, BAT26, D17S250, D5S346, D2S123). The others were classified as microsatellite stable (MSS). Polymorphism of MTHFR 677C>T was genotyped by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results: MSI was observed in 65 of 487 patients (12.73%). MSI colorectal cancers showed similar clinicopathological features with previously reported; younger age onset, right-sided preponderance, mucinous and poorly differentiated histology, lower stage, fewer lymph node metastases than MSS tumors (each P Conclusion: This study demonstrated higher frequency of MTHFR 677TT genotype in MSI colorectal cancers. Furthermore, individuals with MTHFR 677CT+TT variant type might potentially develop MSI rather than MSS colorectal cancers.

Abstract 3128: Diet folate, DNA methylation and genetic polymorphisms of MTHFR C677T in esophageal squamous cell carcinoma

Abstract Background: To explore the role of aberrant hypermethylation of cancer related genes in the esophageal squamous cell carcinoma (ESCC) as well as its relation to dietary folate intake and MTHFR C677T polymorphism, we conducted a molecular epidemiological study in the Chinese population. Methods: This study was conducted in Yangzhong County, an island located on the middle of Yangtze River in the southeast part of Jiangsu Province, China. Totally, 125 ESCC patients having undergone esophagectomy between January 2005 and March 2006 in the Yangzhong People's Hospital were recruited and followed. Face-to-face interviews were conducted by trained investigators using a structured questionnaire. A food frequency questionnaire was used to estimate the dietary intake. For patients having undergone surgery, tissues in the center of the cancer lesion and remote normal appearing esophagus were excised. The methylation at the promoter region of P16, MGMT and hMLH1 was determined by methylation specific PCR. The effects of diet folate, aberrant DNA methylation of selected genes and methylenetetrahydrofolate reductase (MTHFR) C677T genetic polymorphisms were analyzed. Results: The aberrant CpG island hypermethylation of P16, MGMT and hMLH1 gene could be found in cancer tissues with frequency of about 88.0%, 27.2% and 3.2% respectively, and in remote normal appearing esophageal tissues with frequency of about 36.8%, 11.2% and 0.0% respectively. MGMT gene showed a higher proportion of hypermethylation in cancer tissues while P16 gene showed a higher proportion of hypermethylation in remote normal appearing esophageal tissues in patients with lymph node metastasis. A significant association was found between MTHFR C677T genetic polymorphism and CpG island methylation status of MGMT gene. After adjustment for potential confounders, individuals carrying CT or TT genotype have higher frequency of hypermethylation in MGMT gene in cancer tissues with odds ratio 3.34 (95% CI:1.07-10.39) and 3.83 (95% CI:1.13-12.94) respectively. Based on the follow-up data, our analysis showed an inverse association between diet folate intake and the risk of death after esophagectomy. The median survival time was 2.92 years for low folate consumption, 3.18 years for moderate folate consumption and over 4.59 years for high folate consumption. After adjusting for potential confounders, the hazard ratios (95% confidence interval) were 0.72(0.36-1.46) for moderate and 0.39(0.20-0.78) for high folate intake, respectively. This preventive effect was more evident in patients carrying MTHFR 677CC genotype. Conclusions: This study indicated the aberrant CpG island hypermethylation of cancer related genes were associated with ESCC and might be a promising biomarker in diagnosis. Diet folate intake may have benefits on the prognosis of ESCC after esophagectomy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3128. doi:1538-7445.AM2012-3128

Comparative features of colorectal and gastric cancers with microsatellite instability in Chinese patients

The purpose of this study was to determine the unique and universal features of microsatellite instability-high (MSI-H) colorectal cancer (CRC) and MSI-H gastric cancer (GC) in the Chinese population. A new panel of mononucleotide MSI markers, BAT25, BAT26, NR21, NR24, and MONO-27, was used to define MSI status in 303 CRC and 288 GC subjects. Clinicopathological features of both types of MSI-H tumors were analyzed. Methylation analysis in the hMLH1 promoter region by methylation specific polymerase chain reaction (PCR) and mutation detection of hMSH2/hMLH1 genes by denaturing high-performance liquid chromatography (DHPLC) were carried out simultaneously. MSI-H CRCs and MSI-H GCs account for 11.9% and 8.0% of unselected sporadic CRCs and GCs, respectively. MSI-H CRCs are strongly characterized by early onset, right-side location, low differentiation, mucinous tumor, less infiltration, less lymphatic metastasis, and more often familial tumor. MSI-H GCs only showed site preference for the antrum and less lymphatic metastasis. Genetic and epigenetic analyses were positive in 6/36 MSI-H CRCs and 0/23 MSI-H GCs with pathological mutation in major mismatch repair genes, and in 7/36 MSI-H CRCs and 18/23 MSI-H GCs with methylated hMLH1 promoter (P<0.01), respectively. Although there are many differences in the genetic basis and clinicopathological features between MSI-H CRC and MSI-H GC, when compared with their microsatellite stable (MSS) counterparts, site preference and lymphatic metastasis are features common to both types of MSI-H tumors.

Hereditary and Familial Colon Cancer

Between 2% to 5% of all colon cancers arise in the setting of well-defined inherited syndromes, including Lynch syndrome, familial adenomatous polyposis, MUTYH-associated polyposis, and certain hamartomatous polyposis conditions. Each is associated with a high risk of colon cancer. In addition to the syndromes, up to one-third of colon cancers exhibit increased familial risk, likely related to inheritance. A number of less penetrant, but possibly more frequent susceptibility genes have been identified for this level of inheritance. Clarification of predisposing genes allows for accurate risk assessment and more precise screening approaches. This review examines the colon cancer syndromes, their genetics and management, and also the common familial colon cancers with current genetic advances and screening guidelines.

Methylation status of promoter of mismatch repair gene hMLH1 and hMSH2 in glioma

Objective To explore the methylation status of hMLH1 and hMSH2 promoter region in glioma and its role in the oncogenesis. Methods Methylation status of the hMLH1 and hMSH2 promoter region was assayed in 51 giioma tissues and giioma cell line U251 by methylation-specific polymerase chain reactio (MSP). Results The promoter methylations of hMLH1 and hMSH2 were not found in all of 7 nor-real cerebral tissues. The incidence of methylation in hMLH1 and hMSH2 promoter was 13.7% (7/51), and 35.3% (18/51 ) , respectively in 51 glioma tissues accompanied by unmethytation of hMLH1 and hM-SH2. Methylation frequency of the two genes was not correlated with sex, age and pathological classifica-tion. The incidence of hMLH1 promoter methylations in grade Ⅰ -Ⅱ was 0.0% (0/25), and that was 26. 9% (7/26) in grade Ⅲ-Ⅳ (χ~2 = 5.69,P < 0.05 ). Promoter methylation of the hMSH2 gene was present in cell line U251, but that was not observed for the hMLH1 gene. Conclusion The promoter methylations of hMLH1 and hMSH2 in glioma are not correlated with sex, age and pathological classification. Methyla-tion of hMSH2 promoter exists in glioma with median frequency, which may be an early event and correla-ted with the occurrence of glioma. Methylation of hMLH1 promoter exists in glioma with relatively low fre-quency, and the promoter methylation of hMLH1 in giioma is correlated with pathological grade, hMSH2 promoter (C_PG island) hypermethylation exists in glioma cell line U251. Key words: Glioma; Base pair mismatch; DNA methylation

12LBA A Plasma-based colorectal cancer (CRC) screening assay using DNA methylation markers - first results of multicenter studies

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.GASTROENTEROLOGY 2001;121:1300-1309

Expression and promoter methylation status of mismatch repair gene hMLH1 and hMSH2 in epithelial ovarian cancer

The purpose of this study is to determine the relationship between methylation and loss of hMLH1 and hMSH2 expression in ovarian cancer. We examined the methylation status of hMLH1 and hMSH2 promoter region by methylation-specific polymerase chain reaction (MSP) in 56 primary ovarian cancer tissues and 20 normal ovarian tissues, the relationship between the methylation status of these two genes and clinicopathological characteristics were analysed. We then treated SKOV3 and 3AO ovarian cancer cell lines with the demethylating agent 5-aza-2'-deoxycytidine (5-aza-dc). The hMLH1 and hMSH2 methylation was further assessed by MSP, and their mRNA expression was compared by reverse transcription polymerase chain reaction (RT-PCR) before and after 5-aza-dc treatment in these two cell lines. The methylation frequency of hMLH1 and hMSH2 was 30.4% (17 of 56) and 51.7% (29 of 56) in ovarian cancers, respectively, while no methylation was detected in normal ovarian tissues (P=0.015). There is a significant correlation between hMLH1 promoter hypermethylation and histological grade (P=0.028) as well as lymphatic metastasis (P=0.003). Methylation of hMSH2 correlated with histological grade (P=0.035) and lymphatic metastasis (P=0.015). Besides, the methylation rates of hMSH2 were significantly higher in endometrioid adenocarcinoma tissues than in other pathological types of ovarian cancer. After 5-aza-dc treatment, the expression of hMLH1 and hMSH2 was reversed in two cell lines. Our results indicate that promoter hypermethylation is an important mechanism for loss of hMLH1 and hMSH2 expression in human ovarian cancer and may be a potential prognostic factor in ovarian cancer.

Ulcerative Colitis-Associated Colorectal Cancer is Frequently Associated with the Microsatellite Instability Pathway

Patients with ulcerative colitis have a high risk for the development of colorectal cancer. To understand the molecular mechanisms of the carcinogenesis process of ulcerative colitis-associated colorectal cancer, the genetic alterations in inflamed or neoplastic colon epithelium in ulcerative colitis were analyzed. Fifty-seven patients with ulcerative colitis were enrolled in this study. Specimens were obtained from the patients randomly at six colonic sites. Each patient was histologically classified according to the worst pathologic finding into cancer, dysplasia, indefinite, and normal cases. Microsatellite instability, mutations of target genes, hypermethylation of the hMLH1 promoter region, and mismatch repair protein expression were analyzed. High-microsatellite instability was found in 4 of 11 cancer cases (36 percent), 5 of 15 dysplasia cases (33 percent), 5 of 11 indefinite cases (45 percent), and none of 20 normal cases (0 percent). A significant correlation was found between the malignant potential and high-microsatellite instability. A frameshift mutation of transforming growth factor beta receptor Type II (TGFbetaRII) was significantly correlated with worsening histologic grade. High-microsatellite instability was significantly associated with hMLH1 hypermethylation and loss of hMSH2 expression. The carcinogenesis process in ulcerative colitis-associated colorectal cancer was closely associated with the microsatellite instability pathway through TGFbetaRII mutation by a dysfunction of the mismatch repair system.

Poorly differentiated colorectal adenocarcinomas show higher rates of microsatellite instability and promoter methylation of p16 and hMLH1: A study matched for T classification and tumor location

Extensive genetic and epigenetic analysis of poorly differentiated colorectal adenocarcinomas (Por) has been difficult, as the number of cases is too small. We investigated genetic and epigenetic alterations of 53 cases of Por and 53 cases of well-differentiated colorectal adenocarcinomas (WD) to clarify their differences. The cases of WD were matched with the cases of Por for T classification and tumor location, which influence genetic and epigenetic alterations. We evaluated microsatellite instability (MSI) status and loss of heterozygosity (LOH) of four loci (2p, 5q, 17p, 18q), and defined "MSI tumors" as those that showed MSI-H, and "chromosomal instability (CIN) tumors" as those that showed LOH but not MSI-H. Further, we evaluated the methylation status of the hMLH1 and p16 promoter region. MSI tumors were significantly more frequent in Por (22.6%) than in WD (3.8%; P = 0.0041). CIN tumors were significantly less frequent in Por (64.2%) than in WD (83.0%; P = 0.046). Further, methylation of the p16 and hMLH1 promoter region was significantly more frequent in Por than in WD (P = 0.037, P = 0.047, respectively). Our results indicate that Por tumorigenesis strongly correlates with MSI and methylation of the p16 and hMLH1 promoter region.

Carcinogenesis and microsatellite instability: the interrelationship between genetics and epigenetics

DNA mismatch repair (MMR) deficiency results in a strong mutator phenotype and high-frequency microsatellite instability (MSI-H), which are the hallmarks of tumors arising within Lynch syndrome. MSI-H is characterized by length alterations within simple repeated sequences, microsatellites. Lynch syndrome is primarily due to germline mutations in one of the DNA MMR genes; mainly hMLH1 or hMSH2 and less frequently hMSH6 and rarely hPMS2. Germline hemiallelic methylation of MLH1, termed epimutation, has been reported to be a new cause of Lynch syndrome. MSI-H is also observed in approximately 15% of colorectal, gastric and endometrial cancers and in lower frequencies in a minority of other tumors, where it is associated with the hypermethylation of the promoter region of hMLH1. MSI-H underlies a distinctive tumorigenic pathway because cancers with MSI-H exhibit many differences in genotype and phenotype relative to cancers without MSI-H, irrespective of their hereditary or sporadic origins. Genetic, epigenetic and transcriptomic differences exist between cancers with and those without the MSI-H. The BRAF V600E mutation is associated with sporadic MSI-H colorectal cancers (CRCs) harboring hMLH1 methylation but not Lynch syndrome-related CRCs. The differences in genotype and phenotype between cancers with and those without MSI-H are likely to be causally linked to their differences in biological and clinical features. Therefore, the diagnosis of MSI-H in cancers is thus considered to be of increasing relevance, because MSI-H is a useful screening marker for identifying patients with Lynch syndrome, a better prognostic factor and could affect the efficacy of chemotherapy. This review addresses recent advances in the field of microsatellite instability research.

Microsatellite instability and methylation of the DNA mismatch repair genes in head and neck cancer

BackgroundMethylation in the promoter region of the DNA mismatch repair genes hMLH1 and hMSH2 and microsatellite instability at three loci were analyzed in the tumor tissue from patients with head and neck cancer. MethodsMicrosatellite instability and promoter methylation were investigated by PCR, denaturing-polyacrylamide gel electrophoresis and digestion with methylation-specific restriction enzymes. ResultsMicrosatellite instability was observed in 41% of the patients. hMLH1 and hMSH2 genes were methylated in 47% and 30% of the patients, respectively. BAT25 and BAT26 instability were associated with age and histopathology, respectively. Methylation frequency of the hMLH1 gene promoter was significantly higher in patients displaying a high level of microsatellite instability. Instability at the BAT 26 and D2S123 loci were associated with the MSI-high status. ConclusionsOur results indicate that microsatellite instability and modifications in the hMLH1 and hMSH2 genes are implicated in a significant proportion of the patients with head and neck cancer.

DNA mismatch repair status may influence anti-neoplastic effects of butyrate

HNPCC (hereditary non-polyposis colon cancer) is an autosomal-dominant disorder characterized by early-onset CRC (colorectal cancer). HNPCC is most often associated with mutations in the MMR (mismatch repair) genes hMLH1, hMSH2, hMSH6 or hPMS2. The mutator phenotype of a defective MMR system is MSI (microsatellite instability), which also occurs in approx. 15-25% of sporadic CRC cases, where it is associated with the hypermethylation of the promoter region of hMLH1. Dietary factors, including excessive alcohol consumption, ingestion of red meat and low folate intake, may increase the risk of MSI high tumour development. In contrast, aspirin may suppress MSI in MMR-deficient CRC cell lines. Butyrate, a short-chain-fatty-acid end product of carbohydrate fermentation in the colon, shares a number of anti-neoplastic properties with aspirin, including inhibiting proliferation and inducing apoptosis of CRC cells. Recent in vitro studies suggest that physiological concentrations of butyrate (0.5-2 mM) may have more potent anti-neoplastic effects in CRC cell lines deficient in MMR, but mechanisms for such a differential response remain to be established.

Mucinous carcinomas of the colon and rectum show higher rates of microsatellite instability and lower rates of chromosomal instability

The clinicopathologic significance of mucinous carcinomas (Muc) of the colon and rectum has been widely discussed, but there have been few studies on Muc regarding genetic and epigenetic alterations. The current study analyzed genetic and epigenetic alterations of Muc to clarify their differences from well differentiated adenocarcinomas (WD). Thirty-nine cases of Muc and 39 cases of WD were investigated. Cases of WD were matched with cases of Muc for T classification and tumor location. Microsatellite instability (MSI) status and loss of heterozygosity (LOH) of four loci (2p, 5q, 17p, 18q) were evaluated. The methylation status of the hMLH1 promoter region in Muc was also examined. "MSI tumors" were defined as those that showed MSI-high, and "chromosomal instability (CIN) tumors" were defined as those that showed LOH but not MSI-high. MSI tumors were significantly more frequent in Muc (30.8%) than in WD (5.1%). CIN tumors were significantly less frequent in Muc (53.8%) than in WD (87.2%). In Muc, MSI tumors were significantly more frequent in the proximal colon (55.6%) than in the distal colon (9.5%). Also, methylation of the hMLH1 promoter region in Muc was significantly more frequent in MSI tumors (83.3%) than in CIN tumors (27.8%) (P = 0.0077). When matched for T classification and tumor location, Muc shows higher rates of MSI and lower rates of CIN than WD.. Muc shows different characteristics according to tumor location, and methylation of the hMLH1 promoter region strongly correlates with Muc tumors showing MSI.

Colorectal cancer at a young age

Colorectal cancer at a young age

Effects of dietary intake and genetic factors on hypermethylation of thehMLH1gene promoter in gastric cancer

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. 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. 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. 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.

Frequent microsatellite instability in primary esophageal carcinoma associated with extraesophageal primary carcinoma

Patients with esophageal squamous cell carcinoma (ESCC) frequently develop other primary cancers, such as gastric cancer and head and neck cancer. Details of carcinogenesis in patients with multiple primaries that include esophageal carcinoma with other primary carcinoma (ECOPC) remain uncertain. We examined microsatellite instability (MSI) status, frameshift mutation in target genes of MSI, mismatch repair protein expression and hypermethylation of the hMLH1 promoter region in ECOPC patients to better understand the underlying carcinogenic processes. High frequency MSI (MSI-H) was found in 15 (44.1%) of 34 patients with ECOPC, but in only 6 (14.3%) of 42 patients with esophageal cancer alone (p < 0.01). Frameshift mutations in TGFbetaRII, BAX, MSH3 and MSH6 genes respectively were present in 4, 1, 2 and 2 of 34 ECOPC patients. Immunohistochemical study showed that 12 (80.0%) of 15 MSI-H tumors showed loss of expression of either hMLH1 or hMSH2. In addition, 6 of 9 tumors (66.7%) that showed reduced hMLH1 expression also had hypermethylation of the hMLH1 promoter region. Our findings suggested that carcinogenesis in ECOPC was closely associated with the MSI pathway because of mismatch repair protein deficiency.

Alterations of DNA mismatch repair proteins and microsatellite instability levels in gastric cancer cell lines

Alterations in DNA mismatch repair (MMR) proteins result in microsatellite instability (MSI), increased mutation accumulation at target genes and cancer development. About one-third of gastric cancers display high-level microsatellite instability (MSI-High) and low-level microsatellite instability (MSI-Low) is frequently detected. To determine whether variations in the levels of MMR proteins or mutations in the main DNA MMR genes are associated with MSI-Low and MSI-High in gastric cancer cell lines, the MSI status (MSI-High, MSI-Low or MS-Stable (MSS)) of 14 gastric cancer lines was determined using multiple clone analysis with a panel of five microsatellite markers. Protein levels of hMLH1, hMSH2, hMSH6, hPMS2 and hPMS1 were determined by Western blot. Sequence analysis of hMLH1 and hMSH2 was performed and the methylation status of the hMLH1 promoter was examined. The cell lines SNU1 and SNU638 showed MSI-High, decreased to essentially absent hMLH1 and hPMS2 and reduced hPMS1 and hMSH6 protein levels. The hMLH1 promoter region was hypermethylated in SNU638 cells. The MKN28, MKN87, KATOIII and SNU601 cell lines showed MSI-Low. The MMR protein levels of cells with MSI-Low status was similar to the levels detected in MSS cells. A marked decrease in the expression levels of MutL MMR proteins (hMLH1, hPMS2 and hPMS1) is associated with high levels of MSI mutations in gastric cancer cells. Gastric cancer cell lines with MSI-Low status do not show significant changes in the levels of the main DNA MMR proteins or mutations in the DNA mismatch repair genes hMSH2 and hMLH1. These well-characterized gastric cancer cell lines are a valuable resource to further our understanding of DNA MMR deficiency in cancer development, progression and prognosis.

Effect of eukaryotic expression plasmid DNA methyltransferase gene on methylation status and transcription level of DNA mismatch repair genes in human colon cancer cell line

To analyze the effect of eukaryotic plasmids containing sense or antisense DNA methyltransferase (Dnmt1) genes on the methylation status and transcription level of DNA mismatch repair (MMR) genes and microsatellite instability (MSI) in human colon cancer cell line. Human colorectal cells of the SW1116 line were cultured. Recombinant plasmids containing sense Dnmt1 (HMT) or antisense Dnmt1 (THM) gene, pCMV-HMT and pCMV-THM, were constructed. Then pCMV-HMT, pCMV-THM, and pcMV blank plasmid were transfected into SW1116 cells respectively by using lipofectAMINE. The expression of Dnmt1 protein was examined by Western blotting. The transcription levels of hMLH1 and hMSH2 genes were detected by using real-time (RT-PCR). The status of methylation in promoters of hMLH1 and hMSH2 genes were examined with methylation specific PCR (MSP). The MSI of DNA in SW1116 cells was evaluated by silver-stained polyacrylamide gel electrophoresis. Both the expressions of the hMLH1 and hMSH2 gene mRNAs were remarkably decreased in the SW1116-HMT cells in comparison with those in the untransfected cells. The expression of hMSH2 gene mRNA in the SW1116-THM cells was remarkably increased in comparison with that in the untransfected cells. No significant difference in the expressions of the hMLH1 and hMSH2 gene mRNAs was found between the SW1116 cells transfected with blank pCMV and the untransfected SW1116 cells. MSP showed that the methylation level in the regions of hMLH1 and hMSH2 promoters was remarkably increased in the SW1116 cells transfected with sense Dnmtl plasmid. However, in the SW1116 cells the hMSH2 promoter region was changed from partially-methylated into de-methylated, and the hMLH1 promoter region remained non-methylated. MST test showed that extra bands indicating MSI were seen only in the D2S123 groups. Dnmt1 regulates the expression and methylation status of MMR genes and affects MSI in human colon cancer cell line SW1116.

Geno- and pheno-typic characterization in ten patients with double-primary gastric and colorectal adenocarcinomas.

Although a number of double-primary gastric and colorectal cancers have been known to correlate with the mutator pathway, a possible association with known hereditary cancers regarding geno-pathogenesis has rarely been investigated. This study was intended to identify a possible association of hereditary cancers and the implications of the mutator or tumor-suppressor pathway in double-primary gastric and colorectal cancers. Fresh colorectal tissues and lymphocytes from ten patients with double-primary gastric and colorectal cancers were obtained consecutively. The mutator pathway was evaluated by detecting hMSH2 and hMLH1 mutations, microsatellite instability (MSI) using 12 microsatellite markers, and hMLH1 promoter region methylation. Protein expressions of hMSH2, hMLH1, APC, E-cadherin, and beta-catenin were identified by immune staining. There was no pathogenic mutation in any introns or exons of hMSH2, hMLH1 or CDH1, and in exons 3, 5, 6, and 15 of CTNNB1. Either MSI or methylator phenotype was found in five gastric cancers and in four colorectal cancers. No patients met the Amsterdam criteria of hereditary nonpolyposis colorectal cancer (HNPCC) or its equivalent of hereditary gastric cancer. Two patients with gastric cancers among their first-degree relatives showed no E-cadherin expression. The two of the three patients with rectal cancers among their first-degree relatives showed mutator phenotype either in the gastric or in the colorectal cancer. A subset of double-primary gastric and colorectal cancers may thereby be categorized as variant forms of hereditary gastric or colorectal cancer. Both cytoplasmic and nuclear beta-catenins were expressed in all gastric and colorectal cancers. Among the gastric and colorectal cancers with either MSI or methylator phenotype, four of five gastric cancers showed both APC and E-cadherin expression, whereas one of four colorectal cancers showed them. This may suggest that the mutator pathway and the aberrant tumor suppressor pathway of the APC-E-cadherin may be cooperative or separately activated in the geno-pathogenesis of double-primary gastric and colorectal cancers.