Involvement of ATM/ATR-p38 MAPK cascade in MNNG induced G1-S arrest.

ABSTRACT

AIM: To understand the effect of low concentration of N-methyl-N'-nitro-nitrosoguanidine (MNNG), which is a widely distributed environmental mutagen and carcinogen especially for human gastric cancer, on DNA damage and to study its possible pathway in regulating cell cycle arrest. METHODS: The DNA damage effect was measured by Comet assay. A specific phospho-(Ser/Thr) ATM/ATR substrate antibody was used to detect the damage sensor by Western blot. p38 kinase activity was measured by direct kinase assay, and immunoprecipitation for the possible connection between ATM/ATR and p38 MAPK. Flow cytometry analysis and p38 MAPK specific inhibitor SB203580 were combined to detect the possible cell cycle arrest by p38 MAPK. RESULTS: With the same low concentration MNNG exposure (0.2 microM 2.5 h), Comet assays indicated that strand breaks accumulated, Western blot and kinase assay showed ATM/ATR and p38 kinase activated, immunoprecipitation showed phospho-ATM/ATR substrate antibody combined with both p38 MAPK antibody and phospho-p38 MAPK antibody. p38 MAPK pathway was involved in the G1-S arrest. CONCLUSION: Activation of ATM/ATR by MNNG induced DNA damage leads to activation of p38 MAPK, which involves in the G1 checkpoint in mammalian cells.