The Role Of Mismatch Repair Protein HMLH1 In Selenium-induced DNA Damage Response

Animal and epidemiological studies strongly implicate selenium as an effective chemoprevention agent against colon cancer; however, the molecular mechanism by which Se mitigates colorectal tumorigenesis is largely unknown. The majority of colorectal cancers is characterized by microsatellite instability due to a defective mismatch repair system. It's known that hMLHl (human MutL homologue-1) plays an important role in reparing DNA base mismatches. Although hMLHl is acknowledged as a DNA repair protein, whether it functions in DNA damage response pathway is unknown. Here, the MMR-deficient HCT116 colorectal cancer cells and the MMR-proficient HCT116 cells with hMLHl complementation are employed to investigate the role of hMLH1 in selenium induced DNA damage response, a tumorigenesis barrier.The effects of selenium compounds on HCT116 cell viability were detected by colony formation and MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. DNA damages were checked by immunofluorescence analysis. Cell cycle profiles were analyzed by flow cytometry. By using immunoprecipitation and immunoblotting, the association between hMLHl and hPMS2 was studied. Futhermore, the roles of reactive oxygen species (ROS) and DNA damage response protein ATM (Ataxia telangiectasia mutated) in regulating cell cycle progression and mediating hMLH1 and hPMS2 (Post-meiotic segregation protein-2) association were explored. This study was trying to figure out if selenium compounds could activate ATM-dependent DNA damage response via the mismatch repair protein hMLHl in colorectal cancer cells.1. Colony formation and MTT assays showed that the cell viability was substantially decreased by exposure to methylseleninic acid, methylselenocysteine and sodium selenite in a dose-dependent manner (p<0.05); hMLHl complementation makes HCT116+hMLHl cells more sensitive to the three selenium compounds than HCT116 cells (p<0.05). Addition of the antioxidant NAC (N-acetyl cysteine) and Tempo (2,2,6,6-Tetramethylpiperidine-1-oxyl) dramatically suppressed the selenium-induced cell death in colorectal cancer cells, suggesting selenium exerted the cytotoxic effects by generating ROS. 2. Immunofluorescence results indicated that 8-oxoG (oxidative DNA damage) andγH2AX (a marker of DNA breaks) in the nuclei were induced after treatment with Na2SeO3, and the extent was much greater in HCT116+hMLH1 than in HCT116 cells (p<0.05). Pretreating HCT116+hMLH1 cells with NAC or Tempo significantly reduced the Na2SeO3-induced 8-oxoG andγH2AX formation (p<0.05), implying the DNA damages caused by selenium were mediated by hMLH1 and ROS.3. Flow cytometric analyses demonstrated that a time-and dose-dependent G2/M arrest after Na2SeO3 and MSeA exposure was shown in HCT116+hMLH1, but not in HCT116 cells (p< 0.05). Pretreatment of the cells with the ATM kinase inhibitorKU55933, NAC or Tempo attenuated the selenium-induced G2/M arrest, which, therefore, was dependent on hMLH1 and regulated by ATM and ROS.4. Immunofluorescence analyses showed that pATM Ser-1981 (a general marker of ATM pathway activation) and pDNA-PKcs Thr-2647 (a phosphorylation event downstream of ATM kinase activation) expression levels increased marginally in HCT116 cells after selenium exposure. In stark contrast, both the phosphorylation events are substantially induced in HCT116+hMLH1 cells after selenium exposure. So, selenium-induced ATM pathway activation requires hMLH1.5. Treatment of HCT116+hMLH1 cells with Na2SeO3 and MSeA stimulates the association between hMLH1 and hPMS2 proteins, a heterodimer critical for functional MMR; Pretreatment with NAC, Tempo, or KU55933 prevented this association, which, therefore, depended on ATM kinase activity and ROS.Taken together, the results suggest a new role of selenium in mitigating tumorigenesis by targeting the MMR pathway. By employing isogenic cell lines with or without hMLH1 expression, it is shown that hMLH1 is required for selenium-induced DNA damage and G2/M checkpoint response in a manner dependent on ATM kinase and ROS in HCT116 colorectal cancer cells. Lack of hMLH1 renders the HCT116 colorectal cancer cells resistant to selenium-induced DNA damage response.