Oxidative Stress Induces Hypomethylation of LINE-1 and Hypermethylation of the RUNX3 Promoter in a Bladder Cancer Cell Line

Abstract

Increased oxidative stress and changes in DNA methylation are frequently detected in bladder cancer patients.We previously demonstrated a relationship between increased oxidative stress and hypomethylation of thetransposable long-interspersed nuclear element-1 (LINE-1). Promoter hypermethylation of a tumor suppressorgene, runt-related transcription factor 3 (RUNX3), may also be associated with bladder cancer genesis. In thisstudy, we investigated changes of DNA methylation in LINE-1 and RUNX3 promoter in a bladder cancer cell(UM-UC-3) under oxidative stress conditions, stimulated by challenge with H2O2 for 72 h. Cells were pretreatedwith an antioxidant, tocopheryl acetate for 1 h to attenuate oxidative stress. Methylation levels of LINE-1 andRUNX3 promoter were measured by combined bisulfite restriction analysis PCR and methylation-specific PCR,respectively. Levels of LINE-1 methylation were significantly decreased in H2O2-treated cells, and reestablishedafter pretreated with tocopheryl acetate. Methylation of RUNX3 promoter was significantly increased in cellsexposed to H2O2. In tocopheryl acetate pretreated cells, it was markedly decreased. In conclusion, hypomethylationof LINE-1 and hypermethylation of RUNX3 promoter in bladder cancer cell line was experimentally inducedby reactive oxygen species (ROS). The present findings support the hypothesis that oxidative stress promotesurothelial cell carcinogenesis through modulation of DNA methylation. Our data also imply that mechanisticpathways of ROS-induced alteration of DNA methylation in a repetitive DNA element and a gene promotermight differ.