Objective      To elucidate the roles of DNA-dependent protein kinase catalytic subunit

DNA-PKcs and Ataxia-telangiectasia mutated (ATM)

playing in the phosphorylation of H2AX in response to DNA double strand breaks (DSB).        Methods      In HeLa cells

DSBs were induced with neocarzinostatin (Nsc)

a high efficient inducer of DNA DSB. The time course

dose response of phosphorylation of H2AX and the kinetics of dephosphorylation of γ-H2AX were analyzed by Western blotting. The effects of DNA-PKcs inhibitor NU7026 and ATM inhibitor Ku55933 on the phosphorylation of H2AX were also detected by Western blotting. Chromatin immunoprecipitation (CHIP) was applied to detect the enrichment of γ-H2AX on the damaged DNA within α-satellite sequences.        Results      Nsc resulted in increased phosphorylation of H2AX in a time- and dose-dependent manner. γ-H2AX was dephosphorylated after Ncs treatment for short time. The phosphorylation of H2AX was significantly inhibited by DNA-PKcs inhibition. The inhibition of ATM delayed the phosphorylation of H2AX. The enrichment of γ-H2AX after DNA-PKcs inhibition was only 27% of that when DNA-PKcs functioned properly. CHIP indicated that the number of enrichedγ-H2AX was 63% after ATM inhibition.        Conclusion       DNA-PKcs is the predominant kinase involved in the phosphorylation of H2AX in response to DSB.