| Summary: | 博士 === 中山醫學大學 === 醫學分子毒理學研究所 === 97 === O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation has been demonstrated to associate with the G: C→A: T transition mutation in the p53 gene of lung tumors. The purpose of this study is to clarify whether MGMT promoter methylation is not only associated with the shift from the G: C→A: T mutation in the p53 gene but also whether MGMT increases other mutation patterns in lung tumors. To further verify whether a different prevalence of MGMT promoter methylation is observed in lung tumors with a different tumor histology, gender, and smoking status, 220 lung tumors were collected to evaluate the status of MGMT promoter methylation and p53 mutation using methylation-specific polymerase chain reaction (MSP) and direct sequencing, respectively. The data shows that a higher prevalence of MGMT promoter methylation was observed in tumors with the G: C→A: T transition or other p53 mutation patterns compared with those with p53 wild-type (P < 0.001 for G: C→A: T; P = 0.015 for other mutation patterns), and this prevalence was more pronounced in tumors from male than from female patients. MGMT promoter methylation in p53 mutation patterns had a different effect on squamous cell carcinomas (SCC) and adenocarcinomas (ADC) of the lung. Interestingly, the highest prevalence of MGMT promoter methylation was found in male nonsmokers followed by male smokers and female nonsmokers. This may be a partial explanation for the reason why male nonsmokers had a higher p53 mutation occurrence than female nonsmokers. The study also indicated that MGMT promoter methylation is more common in male, squamous cell carcinomas, and smokers than in female, adenocarcinomas, and nonsmokers. More importantly, MGMT hypermethylation in SCC or ADC was pronouncedly influenced by gender factor, not by smoking status. We questioned whether 17β-estradiol could modulate the machinery of promoter methylation to cause the gender difference of MGMT promoter methylation in lung cancer. Two MGMT promoter methylated CH27 and H1355 lung cancer cells were treated with or without 17β-estradiol and the status of promoter methylation were examined by MSP as compared with both cells to be treated with demethylated agents, 5-AZA-dC (AZA) or TSA. Our data showed that 17β-estradiol, similar with AZA, diminished the MGMT promoter methylation and restored MGMT mRNA expression, which was not observed in the case of TSA. Western blotting demonstrated that 17β-estradiol markedly decreased DNMT1 expression in CH27 and H1355 cells, but slightly reduced HDAC1 expression. Consequently, acetylated H3 and H4 histone levels were slightly increased by 17β-estradiol in both cells. In addition, ChIP analysis revealed that 17β-estradiol simultaneously diminished the binding activity of both proteins on the MGMT promoter of both cells. Collectively, we concluded that MGMT promoter methylation may associate with increased occurrence of p53 mutation including the G: C→A: T transition and other p53 mutation patterns in lung cancer, especially among male nonsmokers. In addition, DNMT1 and HDAC1 protein expression levels and their binding activities on MGMT promoter decreased by 17β-estradiol may partially contribute to the gender difference of MGMT promoter methylation in lung cancer.
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