hMLH1 Gene Promoter Research Articles

The relationship between mismatch repair gene 1 gene methylation and adriamycin resistance of osteosarcoma

Objective To investigate whether human mismatch repair gene 1 (hMLH1) gene promoter methylation correlate with adriamycin resistance of osteosarcoma MG63/ADR cell. Methods Methyl thiazol tetrazolium (MTT) method and flow cytometry was used to test the influence of MG63 and MG63/ADR cell proliferation which were dealed with 0, 2, 4, 8, 16, 32 μmol/L of adriamycin for 48 hours, and adriamycin resistance index of MG63/ADR cell, the cytotoxicity of 5-Aza-dc on MG63/ADR, half maximal inhibitory concentration (IC50) and adriamycin resistance reversal index of MG63/ADR cell 48 h after MG63/ADR cell dealed with 0, 5, 10 μmol/L of 5-Aza-dc. Results The IC50 of adriamycin in MG63 cells and MG63/ADR cells were (2.28± 0.12) μmol/L, (11.18±0.11) μmol/L, MG63/ADR cells’ resistance index was 4.90. MTT method and flow cytometry showed that after 48 h intervened by 5-Aza-dc, non-toxic dose and low-toxic dose of MG63/ADR cell were 50 μmol/L and 100 μmol/L. non-toxic dose and low-toxic dose reduced IC50 of adriamycin to (6.18±0.13), (4.42±0.12) μmol/L; adriamycin resistance reversal index were 1.81, 2.53. Conclusion hMLH1 promoter methylation may be involved in the adriamycin resistance of osteosarcoma. Key words: Human mismatch repair gene 1 gene; Methtlation; 5-Aza-dc; Adriamycin; Resistance

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.

LINE-1 hypomethylation in normal colon mucosa is associated with poor survival in Chinese patients with sporadic colon cancer.

Genetic and epigenetic pathways are not independent in colorectal cancer (CRC) carcinogenesis. We aimed to determine the influence of various molecular features on Chinese patients' colon cancer-specific survival (CCSS). Various genetic and epigenetic modifications were detected in paired tumor and normal mucosa tissue samples. The prognostic variables regarding patient CCSS were determined. Overall, 127 patients, including 83 males and 44 females, completed a median follow-up of 65 (3-85) months. A mean LINE-1 methylation rate of 64.62% (range, 9.45-86.93) was observed. Hypermethylation at the hMLH1 gene promoter was detected in 26 (20.47%) patients. KRAS was mutated in 52 (40.94%) patients. Sixteen (12.60%) patients were confirmed as microsatellite instability (MSI)-High, and 76 (59.84%) were found to have loss of heterozygosity at 18q. The LINE-1 methylation level, MSI status, perineural invasion and distant metastases were confirmed as independent prognostic factors for patient CCSS. A stratified survival analysis further revealed that certain subgroups of patients with LINE-1 hypomethylation had significantly worse survival (all p < 0.05). Our data revealed that both genetic and epigenetic abnormalities can concurrently exist during colonic tumorigenesis. As a global epigenetic change, LINE-1 hypomethylation in normal colon mucosa might be associated with a worse outcome in certain Chinese patients with colon cancer.

Effect of methylation of hMLH1 gene promotor on stage tumorigenesis and progression of human gastric cancer

To illustrate the role of methylation level of hMLH1 gene promoter in different stages of gastric carcinogenesis by methylation-specific PCR (MSP) detection of samples from paracancerous tissue and gastric cancer tissue. Methylation status of hMLH1 gene promoter of 40 patients undergoing radical stomach cancer operation in the Tumor Research Institute of China Medical University between January 2006 and August 2006 was detected by MSP. For each patient, 2 samples were chosen from the cancer site, paracancerous tissues of 1 cm, 3 cm, 5 cm away from the cancer site, separately. One sample was used in pathology examination, and the other in methylation detection. Positive rates of hMLH1 gene promoter methylation in the paracancerous tissues of 1 cm, 3 cm, 5 cm away from the cancer site were 10%(4/40), 12.5%(5/40) and 2.5%(1/40) respectively, which were significantly lower than 32.5%(13/40) in cancer site(all P<0.05). Pathological examination showed precancerous lesions in 23 samples of paracancerous 1 cm and 3 cm tissues and normal tissues in 24 samples of paracancerous 5 cm tissues. Positive rates of hMLH1 gene promoter methylation in the cancer site, paracancerous tissue and normal gastric tissue were 32.5%(13/40), 8.7%(2/23) and 0(0/24) (P<0.01). For cancer tissue penetrated the gastric serosa, 8 out of 14 tissue samples were positive methylation (57.1%), which was significantly higher compared with 5 out of 26 tissue samples without penetration of gastric serosa(19.2%). Positive rate of hMLH1 gene promoter methylation in tissue samples with 7 or more of metastatic lymphatic node number was 61.5%(8/13), which was higher compared to that with less than 7(5/27, 18.5%) (P<0.05). No significant differences of positive rate of hMLH1 gene promoter methylation were found between different tumor gross types, tumor grow pattern, tumor differentiation degree, patient age and sex(all P>0.05). Hypermethylation of hMLH1 gene promoter may be associated with the carcinogenesis stages and progression of human gastric cancer.

Relationship between mismatch repair gene hMLH1, hMSH2 and temozolomide-resistance of glioma stem cells

Objective To analyze the status of hMLH1 and hMSH2 of the glioma stem cells (GSC) during TMZ chemotherapy and the relationship between them and temozolomide-resistance of GSC.Methods GSC line U251g and A172g were isolated from the glioma cell line U251 and A172 respectively by suspension culture in the neural stem cell culture medium.CD133,Nestin and GFAP of GSC were examined by Immunofluorescence.The half inhibition concentration (IC50) of TMZ for U251g and A172g were calculated through Cytotoxic Test with CCK-8,then the corresponding two kinds of concentration of TMZ,IC50 and 150％ IC50 as the condition culture mediums,were used to induce U251 g and A172g.At the end,the IC50 of TMZ for U251g and A172g after induction were calculated.Methylation -specific PCR (MSP) and Western blot were used to detect gene promoter methylation and the corresponding protein expressions of hMLH1,hMSH2 of the GSC before and after induction.Results (1) The GSC line U251g and A172g were successfully isolated from the glioma cell line U251 and A172 respectively by suspension culture in the neural stem cell culture medium,which met the definition of cancer stem cells through the identification.(2) The IC50 of TMZ was 1 695.41 μmol / L for U251g,724.63 μmol for A172g,1 913 μmol / L for U251g-1,2 555 μmol / L for U251g-2,754 μmol / L for A172g -1,1 549 μmol / L for A172g-2.(3) After TMZ induction,hMLH1 gene promoter abnormal methylation and the corresponding protein deletion were detected in U251g and A172g,and methylation level promoted by the increase of drug concentration in A172g.hMSH2 gene promoter was found no methylation and protein expression deletion in U251g and A172g.Conclusions There were GSC in the glioma cell line U251 and A172,which had different levels of temozolomide-resistance.During the process of treatment with TMZ,the function of mismatch repair system was abnormalized in U251g and A172g,mainly hMLH1 gene promoter abnormal methylation and the corresponding protein deletion. Key words: Glioma stem cells; Mismatch repair gene; Temozolomide

HMLH1 gene promoter methylation in joint cartilage in patients with osteoarthritis

To investigate the role of Human MutL homologue 1 (hMLH1) gene promoter methylation in the occurrence and development of osteoarthritis (OA). General DNA was dealt with sodium bisulfite. The methylation of hMLH1 promoter was detected by methylation-specific PCR (MSP). hMLH1 protein expression in joint cartilage was detected by immunohistochemical method. The positive percent of hMLH1 promoter methylation in OA patients was higher than that in healthy persons (χ(2)=30.634, P<0.001); the positive percent of hMLH1 protein in OA patients was significantly lower than that in healthy persons (χ(2)=37.724, P<0.001); prmoter methylation and protein expression level of hMLH1 gene showed negative correlation (rs=-0.554, P<0.001). hMLH1 promoter is hypermethlated in joint cartilage cells of OA patients. Hypermethylation may affect the protein expression of hMLH1, which might play a role in the occurrence and development of OA.

Promoter Methylation Status of DNA Repair Gene (hMLH1) in Gastric Carcinoma Patients of the Kashmir Valley

Cancer is a multi-factorial disease and variation in genetic susceptibility, due to inherited differences in the capacity to repair mismatches in the genome, is an important factor in the development of gastric cancer (GC), for example. Epigenetic changes, including aberrant methylation of 5/CpG islands in the promoter regions of mismatch repair (MMR) genes like hMLH1, have been implicated in the development of various types of GC. In the present study we evaluated the role of hMLH1 promoter hypermethylation in Kashmiri GC patients and controls, and assessed correlations with various dietary and lifestyle factors. The study included 70 GC patients (56 males and 14 females; age (mean ± S.D) 50 ± 11.4 years). Distinction between methylated and unmethylated was achieved with MS-PCR and DNA band patterns. The Chi-square test was applied to assess the risk due to promoter hypermethylation. We found a strikingly high frequency of promoter hypermethylation in GC cases than in normal samples (72.9% (51/70) in GC cases vs 20% (14/70) in normal samples (p=0.0001). We also observed a statistically significant association between methylated hMLH1 gene promoter and smoking, consumption of sundried vegetables and hot salted tea with the risk of GC. This study revealed that hMLH1 hypermethylation is strongly associated with GC and suggested roles for epigenetic changes in stomach cancer causation in the Kashmir valley.

HMLH1 promoter methylation is an early event in oral cancer

Promoter methylation is believed to inactivate the expression of hMLH1. This process has been implicated in the tumorigenesis of oral squamous cell carcinoma (OSCC). Thus, the aim of this study was to determine the profile of hMLH1 methylation and protein expression in OSCC. The matched case-control study included 50 OSCC cases and 200 controls, with a median of age 64 (Q₁-Q₃ 54-71) years. Protein expression was determined by immunohistochemical staining, and hMLH1 gene promoter methylation was analyzed by methylation-specific polymerase chain reaction (MSP). A conditional logistic regression model for risk factors was built for OSCC cases and matched controls. Promoter methylation of hMLH1 was detected in 38 (76%) OSCC cases, but in none of the control samples. Of the 38 OSCC samples with promoter methylation, 12 (32%) were negative for hMLH1 protein, and corresponded to early clinical stages (10 in stage II and 2 in stage I). All 12 unmethylated samples showed positive stain for hMLH1. Multiple logistic regression analysis showed an OR of 16.54 (IC 95%: 1.69-161.68, p=0.016) for methylation of the hMLH1 gene and early stages of OSCC, adjusting by gender and tobacco use. This study showed a high frequency of hMLH1 promoter methylation that occurred in most of the early stage cases and in about half of the late stage cases. It is proposed that hMLH1 promoter methylation is an early event that is maintained during tumor progression.

Study the relationship between the gene regulation and microsatellite instability of the cyclooxygen-ase-2,hMLH1 in colon carcinoma

Objective To investigate the cyclooxygenase-2 (COX-2),human mut-lhomologue 1 (hMLH1) of genes promoter methylation and microsatellite instability (MSI) frequency in 42 cases of human colon carcinoma and normal tissues, and to evaluate the relationship between them and occurrence of colon carcinoma. Methods Methylation specific polymerease chain reaction ( MSP) and polymerase chain reaction (PCR) methods were employed in this study in order to investigate the promoter methylation of these genes and 5 loci MSI frequency in human colon carcinoma and normal tissues. Results Of 42 colon carcinoma cases,the total frenquency of MSI was 43.86 % ( 18/42). The MSI frenquency no significant discrepancy was found among the 5 loci (P > 0.05). The number of MSI, MSS was 18 and 24, respectively. Cases with methylation of COX-2 and hMLH1 gene promoter CpG islands in colon carcinoma were 13 and 15 ,and they were not detected in normal tissues. 8 cases with methylation of COX-2 gene promoter CpG islands only occurred in MSI-H colon carcinoma, and 6 cases both with methylation of COX-2 and hMLH1 gene promoter CpG islands was observed in MSI-H. Conclusion The methylation rate of hMLH1 gene promoter CpG islands in MSI colon carcinoma was higher than that in MSS colon carcinoma. It suggested that detecting the methylation of hMLH1 gene promoter CpG islands might be a useful method for determining the colon carcinoma type. Key words: Colon carcinoma; Cyclooxygenase-2; hMLH1; Methylation specific polymerease chain reaction

Comparative evaluation of the effects of 5-Aza-2’-deoxycytidine and Trichostatin A on reactivation of hMLH1 in COC1/DDP ovarian cancer cell line

ObjectivehMLH1 protein serves to detect the DNA damage caused by cisplatin (DDP) and destroys the cell. The absence of hMLH1 expression has been correlated with acquired resistance of ovarian cancer cells to platinum. The aim of this study was to determine the possible role of DNA methylation and histone H3 lysine 9 (H3-K9) acetylation on the loss of hMLH1 expression, and to evaluate the reversal effects of 5-Aza-2′-deoxycytidine (5-Aza-dC) and Trichostatin A (TSA) on DDP-resistance in ovarian cancer cell lines.MethodsTwo human ovarian cancer cell lines, COC1 and its DDP-resistant subline, COC1/DDP were cultured. The two cancer cells were treated with 5-Aza-dC or TSA. Using COC1 cells as a control, we used methylation-specific PCR (MSP) to analyze DNA methylation at hMLH1 gene promoter. hMLH1 mRNA and protein expressions were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot. Chromatin immunoprecipitation assay (ChIP) was used to test the levels of histone H3-K9 acetylation at hMLH1 gene promoter.ResultsIn COC1 cells, there was no DNA methylation at hMLH1 gene promoter, while there were hMLH1 mRNA and protein expression. In COC1/DDP cells, there was DNA hypermethylation at hMLH1 gene promoter, while there was no hMLH1 mRNA or protein expression. The treatment with 5-Aza-dC resulted in DNA demethylation at the promoter region, as well as restoration of hMLH1 expression in COC1/DDP cells. The treatment with TSA had no effects on DNA demethylation or restoration of hMLH1 expression in COC1/DDP cells.ConclusionHypermethylation of DNA at the promoter is related to the silencing of hMLH1 in COC1/DDP ovarian cancer cells. DNA methylation at hMLH1 promoter could play a significant role in determining the sensitivity of ovarian cancer to DDP. The drug resistance mediated by methylation of hMLH1 could be overcome by 5-Aza-dC.

Genetic diagnosis strategy of hereditary non-polyposis colorectal cancer

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. 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. 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%. 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.

MTHFR C677T has differential influence on risk of MSI and MSS colorectal cancer

The majority of colorectal cancer (CRC) exhibiting the micosatellite instability (MSI) phenotype is due to hypermethylation of the hMLH1 gene promoter. We aimed to test the hypothesis that polymorphisms in genes coding for enzymes involved in folate metabolism play a role in altered promoter-specific hypermethylation and thus predispose to MSI CRC. Analysis of MSI was performed in 1685 CRCs, and polymorphism genotypes were determined in germline DNA for all cases and 2692 cancer-free controls. MSI was observed in 171 cancers (10.1%). Compared to homozygous wild-type individuals, those with MTHFR 677TT genotype were more likely to have MSI than microsatellite stable (MSS) CRC [odds ratio (OR) 1.90; 95% confidence interval (CI): 1.09-3.31]. When MTHFR C677T genotype frequencies in MSS CRC cases were compared to controls, individuals with homozygous variant genotype were at 19% reduced risk of cancer compared to wild type (OR = 0.81; 95% CI: 0.65-1.02). Conversely, when MSI CRC cases were compared to controls, individuals with one or two MTHFR 677T alleles were at 42% increased cancer risk (OR = 1.42; 95% CI: 1.02-1.96). Our observations indicate that MTHFR 677TT homozygous individuals are more likely to develop MSI CRC than those with wild-type genotype, and this common polymorphism has differential influences on MSI and MSS CRC risk. Stratification by MSI status should aid future studies investigating the complex relationships between genotype, environmental factors and CRC risk.

The CpG Island Methylator Phenotype and Chromosomal Instability Are Inversely Correlated in Sporadic Colorectal Cancer

The CpG island methylator phenotype (CIMP) is one of the mechanisms involved in colorectal carcinogenesis (CRC). Although CIMP is probably the cause of high-frequency microsatellite instability (MSI-H) sporadic CRCs, its role in microsatellite stable (MSS) tumors is debated. The majority of MSS CRCs demonstrate chromosomal instability (CIN) with frequent loss of heterozygosity (LOH) at key tumor suppressor genes. We hypothesized that the majority of sporadic CRCs without CIN would be associated with CIMP. We tested 126 sporadic CRCs for MSI and LOH and categorized tumors into MSI, LOH, or MSI-/LOH- subgroups. Methylation status was evaluated using 6 CIMP-related markers (MINT1, MINT2, MINT31, p16(INK4alpha), p14(ARF), and hMLH1) and 6 tumor suppressor genes (PTEN, TIMP3, RUNX3, HIC1, APC, and RARbeta2). BRAF V600E mutation analysis was performed using allele-specific polymerase chain reaction and DNA sequencing. We observed frequent methylation at all 12 loci in all CRCs. BRAF V600E mutations correlated with the MSI (P < .0001) and MSI-/LOH- (P = .03) subgroups. MSI and MSI-/LOH- tumors exhibited more promoter methylation than CRCs with LOH (P < .0001). We also found an inverse correlation between the frequencies of methylation and LOH (rho = -0.36; P < .0001). The associations between methylation frequencies at CIMP-related markers and MSI or MSI-/LOH- sporadic CRCs suggest that the majority of these tumors evolve through CIMP. These findings suggest that CIN and CIMP represent 2 independent and inversely related mechanisms of genetic and epigenetic instability in sporadic CRCs and confirm that MSI cancers arise as a consequence of CIMP.

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.

Analysis of inactivation of hMLH1 by promoter hypermethylation and microsatellite instability in gastric carcinogenesis

OBJECTIVE To study the microsatellite instability (MSI) and methylation state of the hMLH1 gene promoter and their mechanisms underlying the development of gastric cancer.

Dna Methylation in Esophageal Diseases Including Cancer: Special Reference to hMLH1 Gene Promoter Status

Chronic inflammation leading to malignancy in the esophagus may be due to errors in mismatch repair (MMR) genes such as hMLH1. Promoter hypermethylation has been suggested as the main cause of hMLH1 silencing. In this study we assessed hMLH1 promoter hypermethylation in a range of esophageal diseases. Further, we evaluated the role of factors affecting the methylation cycle: (1) methylenetetrahydrofolate reductase (MTHFR) C677T mutation and (2) serum homocysteine levels. We endoscopically and histologically categorized 124 paired tissue and blood samples from patients into cancer, precancer, reflux esophagitis, other inflammatory esophagitis and controls (endoscopically normal). Restriction enzyme-based methylation analysis was carried out to assess hMLH1 promoter hypermethylation. hMLH1 promoter hypermethylation in tissue was seen in 63.5% of patients with cancer and 53.8% of those with precancer, which was significantly increased when compared with controls (P < 0.001). There appears to be an increasing degree of hMLH1 hypermethylation with disease progression. Patients with gastroesophageal reflux disease (GERD) showed a high degree of hMLH1 hypermethylation (88.8%), indicating that local environment due to reflux may be promoting hypermethylation. We suggest that GERD is a progressive condition with an increased risk for developing into cancer. Only 14.5% of cases exhibited hypermethylation both in tissue and blood. Hence, we conclude that hMLH1 promoter hypermethylation is a tissue-specific change in the esophagus and blood testing cannot be used as a noninvasive tool to assess it. DNA methylation is dependent on the methylation cycle; MTHFR is a major enzyme in this pathway. MTHFR mutations did not correlate with hypermethylation or clinical pathology (P > 0.5). Elevated homocysteine levels, independent of MTHFR mutation, correlated significantly with hMLH1 hypermethylation in tissue (P < 0.005). Our study shows that hMLH1 hypermethylation in tissue may be the primary event caused by endogenous/exogenous factors in esophageal diseases, aiding disease progression.

Methylation analysis of hMLH1 gene promoter by a bisulfite-sensitive single-strand conformation polymorphism–capillary electrophoresis method

DNA methylation is an important epigenetic modification that alters transcription in those genes containing CpG islands. In this report, a novel DNA methylation analysis method was developed employing bisulfite-single strand conformation polymorphism combined with capillary electrophoresis (bisulfite-SSCP-CE). During the bisulfite treatment of genomic DNA, a high concentration of sodium bisulfite (4.8 mol/L) was preferred in order to shorten reaction time and minimize template degradation. The methylated and unmethylated ssDNA of hMLH1 promoter were simultaneously separated under the optimized CE conditions, including 6% SLPA with 10% glycerol as sieving medium, 25 degrees C as separation temperature and 12 kV as running voltage. The heterogeneous methylation of hMLH1 promoter was identified in 13 of 64 colorectal cancer patients. Moreover, hMLH1 promoter methylation had a significant relationship with protein expression loss and increased with the age of patients. Our results indicated that DNA methylation analysis for a large number of clinical samples would be facilitated by use of the bisulfite-SSCP-CE method.

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.

Epigenetic approaches to cancer therapy

Histone deacetylation and DNA methylation have a central role in the control of gene expression, including transcriptional repression of tumour suppressor genes. Loss of DNA mismatch repair due to methylation of the hMLH1 gene promoter results in resistance to cisplatin in vitro and in vivo. The cisplatin-resistant cell line A2780/cp70 is 8-fold more resistant to cisplatin than the non-resistant cell line, and has the hMLH1 gene methylated. Treatment with an inhibitor of DNA methyltransferase, DAC (2-deoxy-5'-azacytidine), results in a partial reversal of DNA methylation, re-expression of MLH1 (mutL homologue 1) and sensitization to cisplatin both in vitro and in vivo. PXD101 is a novel hydroxamate type histone deacetylase inhibitor that shows antitumour activity in vivo and is currently in phase I clinical evaluation. Treatment of A2780/cp70 tumour-bearing mice with DAC followed by PXD101 results in a marked increase in the number of cells that re-express MLH1. Since the clinical use of DAC may be limited by toxicity and eventual re-methylation of genes, we suggest that the combination of DAC and PXD101 could have a role in increasing the efficacy of chemotherapy in patients with tumours that lack MLH1 expression due to hMLH1 gene promoter methylation.

Defective mismatch repair and the development of recurrent endometrial carcinoma

Objective. To investigate whether defective DNA mismatch repair (MMR) defines a subgroup at risk for recurrence in sporadic endometrial carcinoma patients. Methods. Primary tumors from 44 patients with recurrent stage I endometrial carcinoma were compared after matching, with tumors of 44 patients being free of recurrence for minimal 3 years. Paraffin-embedded primary tumors ( n = 88) and recurrent tumors ( n = 32) were subjected to immunohistochemical analysis for hMSH2 and hMLH1 expression. Subsequently, a staining index (SI = 0–9) was calculated based on staining intensity and quantity. DNA was extracted from paraffin-embedded tissues, and promoter methylation of hMLH1 was determined by nested methylation-specific PCR (MSP). Microsatellite instability (MSI) was assessed by BAT-26 or BAT-25. Results. Low hMSH2 expression was observed in 2% of primary tumors of control patients without recurrence, 14% of primary tumors of patients with recurrence, and 0% of recurrent tumors. Low hMLH1 expression was observed in 32%, 19%, and 22%, respectively. hMLH1 gene promoter methylation was detected in 50%, 47%, and 32%, and MSI was found in 16%, 14%, and 30%, respectively. No significant differences were found between primary tumors of patients with and without recurrence with respect to hMSH2 and hMLH1 expression, hMLH1 promoter methylation, and MSI. When primary and recurrent tumors were compared, there was an increased correlation of hMLH1 methylation with low hMLH1 expression and MSI in recurrent tumors. Conclusion. MSI, hMLH1 promoter methylation, and the expression of hMLH1 and hMSH2 are not predictive for the development of recurrent stage I endometrial carcinoma. In the progression of tumor, “de novo” hMLH1 methylation rarely occurs, instead there is further derailment of the MMR pathway in affected tumors.