DNA DSB induced by gamma irradiation, or other DSB inducers, leads to rapid phosphorylation of H2AX at Ser139 by ATM, ATR and DNA-PKcs, resulting in γH2AX . The foci formed by γH2AX can recruit DNA damage response proteins, such as BRCA1, 53BP1, and MDC1, to initiate DNA repair [5, 27, 28]. In addition, other related proteins such as NBS, CHK2, SMC1, Rad51 and Rad50, are also co-localized to γH2AX foci, but these proteins may be indirectly induced by other DNA damage response proteins . After γH2AX marks sites of DNA damage, the damage recruits repair proteins and dephosphorylation of γH2AX is necessary for release of repair proteins from the damage sites to complete the DNA repair process. The protein phosphatases 2A (PP2A) and PP4 were previously shown to be responsible for dephosphorylation of γH2AX [22–24]. It has been debated as to whether Ser139 of H2AX is phosphorylated by ATM alone or both ATM and DNA-PK after ionizing radiation . Our data indicate that 4 Gy irradiation can rapidly induce H2AX phosphorylation (within 15 min), and that γH2AX then declines to normal levels after 4 h in HeLa cells. Depletion of DNA-PKcs by siRNA significantly abolished the IR-induced phosphorylation of H2AX in HeLa-H1 cells.
It was reported that deficiency of DNA-PKcs causes downregulation of ATM in HeLa cells . Consistent with this, the ATM protein level was also decreased in HeLa-H1 cells in concordance with the reduced DNA-PKcs (data not shown), and this further contributes to the dramatic reduction of γH2AX in HeLa-H1 cells. A dramatic decrease of IR-induced γH2AX was also demonstrated in the DNA-PKcs-depleted HepG2-H1 cells (Figure 2B). In contrast, a dramatic increase in γH2AX was induced by radiation in ATM-deficient AT5BIVA and ATS4 cells (Figure 2D, E). Moreover, ATS4 cells had a very high constitutive level of γH2AX (Figure 2E). In addition, the DNA-PKcs-specific inhibitor NU7026 can effectively abolish IR-induced phosphorylation of H2AX.
Koike et al recently reported that the level of γH2AX also increased in mice lacking either ATM or DNA-PK following X-irradiation , and they suggested that the phosphorylation of H2AX and the elimination of γH2AX following radiation proceeds in both DNA-PK-dependent and independent manner in vivo. Furthermore, they also demonstrated a tissue-specific mechanism of γH2AX level regulation, e.g. the phosphorylation of H2AX at Ser139 after X-irradiation in the spleen is mainly mediated by the DNA-PK . Low doses of replication-inhibitor aphidicolin (APH) induce DSBs in replicating cells, and the formation of these DSBs requires Bloom's syndrome-associated (BLM) helicase and Mus81 nuclease . These APH-induced BLM and Mus81-dependent DSBs activate the phosphorylation of H2AX by ATR kinase, while the DSBs are transient and appear to be rapidly repaired by DNA-PK-dependent non-homologous end joining (NHEJ) . Therefore, it could be more complicated than generally considered regarding the regulatory mechanisms of γH2AX levels after DNA damage induced by ionizing radiation. Our findings further suggest that DNA-PKcs plays no less important role than does ATM, or both are functionally complementary to each other, in modulating of H2AX phosphorylation in response to DNA damage induced by ionizing radiation. Phosphorylated H2AX has also been reported in untreated normal and tumor cells, which can be explained as the consequence of a physiological event that involve DNA recombination  or due to DNA damage induced by reactive oxygen species (ROS) generated by metabolic activity during progression through the cell cycle . The increased constitutive γH2AX seen in ATS4 cells could reflect the high levels of residual DNA damage.
Information regarding the association of H2AX phosphorylation and cell cycle progression is scarce. One report showed ATR-mediated phosphorylation of H2AX generated during DNA replication . Kurise et al. reported that blocking HL-60 cells at the G1/S transition by treatment with inhibitors of DNA replication (thymidine, aphidicolin and hydroxyurea) resulted in H2AX phosphorylation at Ser139, and that this effect is most pronounced in S-phase cells and in cells undergoing induced apoptosis . In this study, we found that a peak of H2AX phosphorylation appeared when synchronized HeLa cells entered the G2/M phases, but not in G1-arrested cells or in cells during S-phase (Figure 4A). These results are consistent with a previous report by Ichijima et al . ATM deficiency did not significantly affect cell cycle progression-associated H2AX phosphorylation (Figure 5A and 5B), but loss of DNA-PKcs by the siRNA strategy (Figure 4A) or chemical inhibitors (Figure 4B and 5C) led to a marked decrease in γH2AX levels. Therefore, we conclude that DNA-PKcs also plays a key role in regulating H2AX phosphorylation associated with cell cycle progression. In addition, DNA-PKcs depletion can lead to some delay in G2-phase entry (Figure 3A and 3B).
The AGC family Ser/Thr kinase protein kinase B (PKB/Akt) was originally identified to be a central regulator of cell metabolism, survival, and proliferation. Following mitogen stimulation, Akt is fully activated through phosphorylation of two key residues, Thr308 in the activation loop and Ser473 in the C-terminal hydrophobic motif. Akt/Thr308 is phosphorylated by 3-phosphoinositide-dependent kinase 1 (PDK1) , while Akt/Ser473 is a target of the mammalian target of rapamycin complex 2 (mTORC2) . Akt/Ser473 has been also identified as one of the downstream substrates of DNA-PKcs in response to DNA damage , and the phosphorylated Akt can inactivate GSK3β by phosphorylating it on Ser9. The direct phosphorylation of Akt on S473 by DNA-PK requires a specific recognition sequence in the C-terminal hydrophobic motif surrounding the Ser-473 phospho-acceptor site in PKB . Bozulic et al have recently shown that PDK1 is responsible for Akt/Thr308 phosphorylation in the DNA damage induced by ionizing radiation, and GSK3 phosphorylation after DNA damage depends on both DNA-PK-mediated and PDK1-mediated activation of Akt . In PDK1-/- cells, phosphorylation of Akt/Thr308 was not detected after ionizing radiation, and phosphorylation of Akt/Ser473 was also much lower than in wild-type cells. This was further reflected by the lack of phosphorylation of GSK3α although DNA-PKcs was active in PDK1-/- cells. Our data demonstrate that inhibition of GSK3β by siRNA or LiCl resulted in increased phosphorylation of H2AX in G2/M phase cells (Figure 7B) and after irradiation (Figure 7A, D). Inhibition of GSK3β prolonged the time of γH2AX elevation after radiation, and which implicates GSK3β in promoting dephosphorylation of γH2AX. Therefore, we suggest another pathway of DNA-PKcs affecting the H2AX phosphorylation level, i.e., DNA-PKcs activates Akt via phosphorylation on Ser473, which in turn inactivates GSK3β via phosphorylating Ser9 . The inactivated GSK3β loses its effect of negatively modulating the γH2AX level. Furthermore, depletion of PDK 1, an upstream regulator of Akt, by RNAi also results in a decrease in radiation-induced (Figure 6C) and cell cycle associated (Figure 6D) phosphorylation of H2AX, This further supports the involvement of Akt/GSK3β in regulating the γH2AX level.