ATM is the Predominant Kinase Involved in the Phosphorylation of Histone H2AX after Heating

Hyperthermia/γH2AX/ATM/DNA-PK. Heating induces histone H2AX phosphorylation at serine 139 (γH2 AX). Phosphorylated H2AX subsequently forms foci in numerous mammalian cell lines. The aim of this study was to clarify details in the mechanisms involved in the phosphorylation of H2AX after heating. The cell lines used were DNA-PKcs knockout cells, ATM knockout cells, and their parental cell lines. To elucidate mechanisms of induction of phosphorylation of H2AX after heating, ATM/ATR inhibitor (CGK733) and DNA-PK inhibitor (NU7026) were used. The intensity of γH2AX signals was assayed with flow cytometry. The thermal dose-response curve for the fluorescence intensity of γH2AX appearance in DNA-PKcs–/– cells during the heating period was similar to that observed in DNA-PKcs+/+ cells. On the other hand, the slope of thermal dose-response curve for them in ATM–/– cells was lower than that in ATM+/+ cells. Phosphorylation of H2AX after heating was suppressed by a combination of CGK733 and NU7026 in the culture medium in DNA-PKcs–/– cells, ATM–/– cells and in their parental cells. Although the phosphorylation of H2AX after heating was not suppressed by NU7026 in their parental cells, such phosphorylation was suppressed by CGK733 in their parental cells. These results indicate that ATM is the predominant protein which is active in the phosphorylation of histone H2AX after heating.