The N-alkylamides Induces MGMT Gene Hypomethylation In Gastric Epithelium Cells Malignant Transformation

O6-Methylguanine-DNA methyltransferase (MGMT) is a suicide enzyme that repairs pre-mutagenic, pre-carcinogenic and pre-toxic DNA damage O6-methyl-guanine. It can rapidly reverse alkylation at the O6 position of guanine, thereby averting the formation of lethal cross-links. The chemical carsinogen N-methyl-N’-nitro-N-nitrosoguanidine(MNNG)、N-Methyl-N-nitrosourea (MNU), two kinds of alkylating agent, can bind to the DNA and promote the O6-methylguanine-induced point mutations, which is a major cause of tumor initiation.In this study, we observed that MNNG、MNU sostenuto increased MGMT mRNA and protein expression in gastric epithelium cells (GES-1) malignant transformation. But the MGMT promoter luciferase reporter was not transactivated in MNNG、 MNU-induced GES-1 malignant transformed cells. Moreover, we found the reduction of MGMT gene promoter methylation level by methylation-specific PCR (MSP) and buslfite sequencing PCR (BSP) in MNNG、MNU-treated cells. O6-benzylguanine(O6-BG), a specific inhibitor of MGMT, inhibited the MGMT expression and promoted the MNNG、MNU-induced malignant phenotypes. Overexpression of MGMT partially reversed the MNNG、MNU-induced GES-1 malignant transformation process.Then, transfecting MGMT overexpressing plasmid to the MNNG、MNU treated cells can reduce malignant cell proliferation ability. In MNNG、MNU treated cells,we screen some monoclonal cells which express different expression level of MGMT. Malignant cell proliferation test show that MGMT expression level negatively correlated malignant transformed cells.In conclusion, MGMT stably up-regulation in MNNG、MNU-treated cells was induced by its DNA promoter hypomethylation. And the high expressed MGMT prevented the MNNG、MNU-induced GES-1 cells malignant transformation. Our finding could suggest a new mechanism regulation of MGMT in gastric cancer initiation induced by N-nitroso compounds.