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O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme whose expression is controlled by its promoter methylation. A cell that expresses a low amount of MGMT is known to be more sensitive to the antiproliferative effects of alkylating agents. We have previously shown that the colorectal cancer patients treated with 5-fluorouracil (5-FU) as adjuvant chemotherapy had a better prognosis when the tumor revealed hypermethylation in its MGMT promoter. Therefore, we sought to investigate the relationship between the expression levels of MGMT and the anti-tumor effect of 5-FU in vitro by using two colon adenocarcinoma and four oral cancer cell lines with a variety of MGMT expression. We also investigated the effects of MGMT depletion by O6-benzylguanine (O6-BG), a potent inhibitor of MGMT. The 5-FU treatment uniformly depleted protein and mRNA expression of MGMT in all cell lines examined. Cell lines expressing low levels of MGMT were sensitive to 5-FU. On the other hand, cells expressing high levels of MGMT were less sensitive to 5-FU. The 5-FU treatment exhibited a better antiproliferative effect on the cells expressing high levels of MGMT by the pretreatment of O6-BG. Depletion of MGMT by O6-BG enhanced the anti-tumor effect of 5-FU. Assessment of the levels of MGMT expression in cancer cells and the control of its expression could contribute to the effective chemotherapy by 5-FU especially in patients who previously were considered as low-responsive individuals whose tumors have high levels of MGMT.

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is involved in cellular defences against alkylating agents. Alterations in the MGMT gene may result in an increase in the mutation rate and risk of malignant transformation. We have previously shown that MGMT is implicated in colorectal carcinogenesis particularly in cancers which display microsatellite instability, a marker of impaired DNA repair. The aims of the current study were to assess the roles of MGMT and microsatellite instability in hepatocellular carcinomas (HCCs) from Australia and South Africa. DNA was extracted from malignant and non-malignant liver tissue from 37 Australian and 24 South African patients, and histologically normal liver from 20 transplant donors. MGMT promoter hypermethylation and MGMT protein expression were assessed using methylation specific PCR and immunohistochemistry. Microsatellite instability was examined using a panel of 23 microsatellite markers previously used to detect allelic imbalance and two specific markers for the detection of low levels of microsatellite instability. Methylation specific PCR did not detect any methylation of the MGMT promoter in Australian and South African HCCs. Similarly, no hypermethylation of MGMT was observed in the adjacent non-malignant liver or histologically normal liver. MGMT staining was predominantly nuclear with some cytoplasmic staining. Overexpression of MGMT protein was detected in 14 (39%) HCCs, while a reduction in protein expression was evident in 14 (39%) HCCs. In the remaining 8 cases the expression of MGMT was comparable in HCCs and adjacent non-malignant tissue. Interestingly, MGMT expression decreased relative to adjacent non-malignant liver tissue in patients who had aetiologies other than viral hepatitis for their underlying liver diseases (p<0.02). No microsatellite instability was detected in this series of 61 HCCs. This suggests that epigenetic silencing of MGMT and microsatellite instability does not play an important role in this series of HCCs derived from different populations.

O6-alkylguanines are potent mutagenic, pro-carcinogenic and cytotoxic lesions induced by exogenous and endogenous alkylating agents. A facilitated elimination of these lesions by increasing the activity of O6-methylguanine-DNA methyltransferase (MGMT) is likely to be a beneficial chemoprevention strategy, which, however, has not been examined. Because, a marginal enhancement of this protein may be adequate for genomic protection, we studied alterations in MGMT activity and expression in human peripheral blood lymphocytes and cancer cell lines induced by water-soluble and alcohol-soluble constituents of several plants with established antioxidant and medicinal properties. Both the ethanolic and aqueous extracts from neem (Azadirachta indica), holy basil (Ocimum sanctum), winter cherry (Withania somnifera), and oregano (Origanum majorana) increased the levels of MGMT protein and its demethylation activity in a time-dependent manner with a maximum of 3-fold increase after 72-h treatment. The extracts from gooseberry (Emblica officinalis), common basil (Ocimum basilicum), and spearmint (Mentha viridis) were relatively less efficient in raising MGMT levels. Increased levels of MGMT mRNA accounted at least, in part, for the increased activity of the DNA repair protein. The herbal treatments also increased glutathione S-transferase-pi (GSTP1) expression, albeit to a lesser extent than MGMT. These data provide the first evidence for the upregulation of human MGMT by plant constituents and raise the possibility of rational dietary approaches for attenuating alkylation-induced carcinogenesis. Further, they reveal the putative antioxidant responsiveness of the MGMT gene in human cells.

Although the incidence of Barrett's carcinomas (BC) and proximal gastric adenocarcinomas (PGC) is increasing, little is known about different epigenetic changes in these etiopathogenetically distinct entities. Therefore, 29 adenocarcinomas [10 BC, 7 PGC and 12 tumors of the esophagogastric junction (JC)] and corresponding non-tumor controls (NT) were examined using methylation-specific PCR. The most striking result was a significantly higher promoter methylation frequency of O6-methylguanine methyl transferase (MGMT) in BC compared with JC and PGC (0.7 vs. 0.08 vs. 0.29, respectively; p = 0.011; methylation exclusively in tumors), confirmed immunohistochemically by a significant loss of MGMT protein in BC (p = 0.006). Therefore, MGMT might become a prognosticator and key for chemotherapy with alkylating agents in BC. Frequencies of p16INK4a promoter methylation were 0.5 (BC), 0.42 (JC) and 0.29 (PGC; n.s.), but methylation was almost absent in NT controls. As immunonegative tumors slightly outnumber methylation-positive cases, other mechanisms of gene inactivation must be discussed. Methylation of E-cadherin was rarely observed (1/10 BC, 0/12 JC and 2/7 PGC). This is the first report on promoter methylation of death-associated protein kinase (DAPK) and fragile histidine triad gene (FHIT) in BC; both DAPK (BC 0.7, JC 0.92 and PGC 0.86) and FHIT (BC 0.88, JC 1.0 and PGC 1.0) were found to be highly methylated, suggesting that epigenetic silencing of these tumor suppressors is a common event in adenocarcinomas of the upper gastrointestinal tract, including BC. DAPK (0.54 on average) and FHIT methylation (0.77 on average) were also observed in NT samples. This might constitute an early epigenetic precursor lesion in the normally-appearing tissue surrounding the tumor.

The aim of this study was to assess whether combined evaluation of O6-methylguanine methyltransferase (MGMT) and hMLH1 status determines sensitivity to monofuntional alkylating agents such as N-methyl-N-nitrosourea (MNU) and dacarbazine (DTIC) against gallbladder carcinoma cells. The molecular mechanism behind MGMT and hMLH1 status affecting the cell cycle was also addressed. Using 5 gallbladder cancer carcinoma lines and 1 colon carcinoma cell line (SW48), MGMT and hMLH1 expression was analyzed using RT-PCR and Western blotting. MGMT and hMLH1 status in the 6 cell lines was compared with drug sensitivity to MNU. As a result, cell lines that were MGMT-/hMLH1+ had the highest sensitivity to MNU, compared with MGMT+/hMLH1+ and MGMT-/hMLH1- cells. In flow cytometric analysis, G2-M cell cycle arrest was specifically observed in GB-d1 cells with MGMT-/hMLH1+ and expression of cyclin A and Cdc2 in GB-d1 cells was significantly reduced by MNU treatment, but not observed in KMG-C cells with MGMT+/hMLH1+. Finally, we assessed the in vitro and in vivo effect of the clinically used alkylating agent DTIC in these cells. The highest sensitivity to DTIC was also observed in MGMT-/hMLH1+. In conclusion, MNU suppressed cell proliferation of MGMT-/hMLH1+ gallbladder carcinoma cells by arresting the cell cycle at the G2-M phase, accompanied by down-regulation of cyclin A and Cdc2. These results indicated that expression of MGMT and hMLH1 could be used to select candidates for alkylating agent chemotherapy against gallbladder carcinoma.