%0 Journal Article %T DNA-PKcs plays a dominant role in the regulation of H2AX phosphorylation in response to DNA damage and cell cycle progression %A Jing An %A Yue-Cheng Huang %A Qing-Zhi Xu %A Li-Jun Zhou %A Zeng-Fu Shang %A Bo Huang %A Yu Wang %A Xiao-Dan Liu %A De-Chang Wu %A Ping-Kun Zhou %J BMC Molecular Biology %D 2010 %I BioMed Central %R 10.1186/1471-2199-11-18 %X The level of ¦ÃH2AX in HeLa cells increased rapidly with a peak level at 0.25 - 1.0 h after 4 Gy ¦Ã irradiation. SiRNA-mediated depression of DNA-PKcs resulted in a strikingly decreased level of ¦ÃH2AX. An increased ¦ÃH2AX was also induced in the ATM deficient cell line AT5BIVA at 0.5 - 1.0 h after 4 Gy ¦Ã rays, and this IR-increased ¦ÃH2AX in ATM deficient cells was dramatically abolished by the PIKK inhibitor wortmannin and the DNA-PKcs specific inhibitor NU7026. A high level of constitutive expression of ¦ÃH2AX was observed in another ATM deficient cell line ATS4. The alteration of ¦ÃH2AX level associated with cell cycle progression was also observed. HeLa cells with siRNA-depressed DNA-PKcs (HeLa-H1) or normal level DNA-PKcs (HeLa-NC) were synchronized at the G1 phase with the thymidine double-blocking method. At ~5 h after the synchronized cells were released from the G1 block, the S phase cells were dominant (80%) for both HeLa-H1 and HeLa-NC cells. At 8 - 9 h after the synchronized cells released from the G1 block, the proportion of G2/M population reached 56 - 60% for HeLa-NC cells, which was higher than that for HeLa H1 cells (33 - 40%). Consistently, the proportion of S phase for HeLa-NC cells decreased to ~15%; while a higher level (26 - 33%) was still maintained for the DNA-PKcs depleted HeLa-H1 cells during this period. In HeLa-NC cells, the ¦ÃH2AX level increased gradually as the cells were released from the G1 block and entered the G2/M phase. However, this ¦ÃH2AX alteration associated with cell cycle progressing was remarkably suppressed in the DNA-PKcs depleted HeLa-H1 cells, while wortmannin and NU7026 could also suppress this cell cycle related phosphorylation of H2AX. Furthermore, inhibition of GSK3¦Â activity with LiCl or specific siRNA could up-regulate the ¦ÃH2AX level and prolong the time of increased ¦ÃH2AX to 10 h or more after 4 Gy. GSK3¦Â is a negative regulation target of DNA-PKcs/Akt signaling via phosphorylation on Ser9, which leads to its inactivat %U http://www.biomedcentral.com/1471-2199/11/18