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AIM:To understand the effect of low concentration of N-methyl-N′-nitro-nitrosoguanidine (MNNG),which is a widelydistributed environmental mutagen and carcinogen especiallyfor human gastric cancer,on DNA damage and to study itspossible pathway in regulating cell cycle arrest.METHODS:The DNA damage effect was measured by Cometassay.A specific phospho-(Ser/Thr) ATM/ATR substrateantibody was used to detect the damage sensor by Westernblot.p3.8 kinase activity was measured by direct kinase assay,and immunoprecipitation for the possible connection betweenATM/ATR and p38 MAPK.Flow cytometry analysis and p38MAPK specific inhibitor SB203580 were combined to detectthe possible cell cycle arrest by p38 MAPK.RESULTS:With the same low concentration MNNG exposure(0.2 μM 2.5 h),Comet assays indicated that strand breaksaccumulated,Western blot and kinase assay showed ATM/ATR and p38 kinase activated,immunoprecipitation showedphospho-ATM/ATR substrate antibody combined with bothp38 MAPK antibody and phospho-p38 MAPK antibody,p38MAPK pathway was involved in the Gl-5 arrest.CONCLUSION:Activation of ATM/ATR by MNNG inducedDNA damage leads to activation of p38 MAPK,which involvesin the G1 checkpoint in mammalian cells.
AIM: To understand the effect of low concentration of N-methyl-N’-nitro-nitrosoguanidine (MNNG), which is a widely distributed environmental mutagen and carcinogen for human gastric cancer, on DNA damage and to study inpossible pathway in regulating cell cycle arrest .METHODS: The DNA damage effect was measured by Cometassay. A specific phospho- (Ser / Thr) ATM / ATR substrate antibody was used to detect the damage sensor by Western blot.p3.8 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 μM 2.5 h), Comet assays indicated that strand breaksaccumulated, Western blot and kinase assay showed ATM / ATR and p38 kinase activated, immunoprecipitation showed phospho-ATM / ATR substrate antibody combined with bothp38 MAPK antibody and Activation of ATM / ATR by MNNG induced DNA damage leads to activation of p38 MAPK, which involves in the G1 checkpoint in mammalian cells.