| Summary: | 博士 === 國防醫學院 === 生命科學研究所 === 102 === Chronic arsenic exposure has been reported to be associated with an increased risk of cancer. Previous studies have shown that the sustained exposure of human urothelial cells (HUCs) to low-dose sodium arsenite induced changes in the gene expression profile and neoplastic transformation. Numerous studies have shown that epigenetic alterations play crucial roles in cellular adaptations to sustained environmental stress and the accumulation of mutations, and hence lead to carcinogenesis. In this study, reduced levels of DNA methyltransferase 1 (DNMT1) and DNMT3A and an increased level of DNMT3B were found in HUCs after exposure to arsenite for an extended period of time (iAs-HUCs). The genome-wide methylation study has shown altered DNA methylation profiles in iAs-HUCs, involving both hypomethylation and hypermethylation. By quantitative real-time PCR analysis, it showed a negative correlation between the level of DNA methylation at a promoter and the mRNA level of that gene. When 5-aza-2'-deoxycytidine (5-aza-dC) was used to examine the involvement of promoter methylation in gene expression, 20 (50%) out of 40 significantly hypomethylated genes (Δβ value < −0.75) showed increased expression in the iAs-HUCs, which was likely due to promoter hypomethylation. However, among the 21 significantly hypermethylated genes (Δβ value > 0.75) in the iAs-HUCs, the expression of only 5 (23.8%) genes was confirmed to be suppressed by promoter hypermethylation. The methylation status of the promoters of several genes was verified using a bisulfite sequencing assay. Because the expression of the oncogene lipocalin-2 (LCN2) was highly enhanced by promoter hypomethylation in the iAs-HUCs as well as in bladder cancer tissues, the promoter activity of LCN2 was further analyzed using a luciferase reporter assay. The results showed that mutations at the binding sequences for NF-κB and C/EBP-α, but not C/EBP-β, NF-Y, or AP-1, significantly reduced LCN2 promoter activity. Chromatin immunoprecipitation (ChIP) assay demonstrated a significant increase in binding of RelA (p65) and NF-κB1 (p50) to the hypomethylated promoter of LCN2 in the iAs-HUCs. These results reveal that the hypomethylation of the LCN2 promoter in iAs-HUCs enhances the binding of NF-κB, which may lead to the enhanced expression of LCN2. This study also demonstrated that LCN2 overexpression was crucial for the neoplastic characteristics of the iAs-HUCs, such as enhanced colony forming ability on soft agar, resistance to serum deprivation, activation of NF-κB signaling and induction of pro-inflammatory genes. Furthermore, enhanced NF-κB activity in iAs-HUCs was via LCN2-mediated elevation of intracellular iron and reactive oxygen species (ROS) levels. Taken together, this study showed that sustained low-dose arsenic exposure results in changes in the profiles of the DNMT isoforms, DNA promoter methylation and gene expression. In addition, arsenic-induced cell transformation is associated with LCN2 overexpression, caused by promoter hypomethylation.
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