DNA-PKcs plays a dominant role in the regulation of H2AX phosphorylation in response to DNA damage and cell cycle progression

Abstract

BackgroundWhen DNA double-strand breaks (DSB) are induced by ionizing radiation (IR) in cells, histone H2AX is quickly phosphorylated into γ-H2AX (p-S139) around the DSB site. The necessity of DNA-PKcs in regulating the phosphorylation of H2AX in response to DNA damage and cell cycle progression was investigated.ResultsThe level of γH2AX in HeLa cells increased rapidly with a peak level at 0.25 - 1.0 h after 4 Gy γ irradiation. SiRNA-mediated depression of DNA-PKcs resulted in a strikingly decreased level of γH2AX. An increased γH2AX was also induced in the ATM deficient cell line AT5BIVA at 0.5 - 1.0 h after 4 Gy γ rays, and this IR-increased γH2AX 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 γH2AX was observed in another ATM deficient cell line ATS4. The alteration of γH2AX 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 γH2AX level increased gradually as the cells were released from the G1 block and entered the G2/M phase. However, this γH2AX 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 GSK3β activity with LiCl or specific siRNA could up-regulate the γH2AX level and prolong the time of increased γH2AX to 10 h or more after 4 Gy. GSK3β is a negative regulation target of DNA-PKcs/Akt signaling via phosphorylation on Ser9, which leads to its inactivation. Depression of DNA-PKcs in HeLa cells leads to a decreased phosphorylation of Akt on Ser473 and its target GSK3β on Ser9, which, in other words, results in an increased activation of GSK3β. In addition, inhibition of PDK (another up-stream regulator of Akt/GSK3β) by siRNA can also decrease the induction of γH2AX in response to both DNA damage and cell cycle progression.ConclusionDNA-PKcs plays a dominant role in regulating the phosphorylation of H2AX in response to both DNA damage and cell cycle progression. It can directly phosphorylate H2AX independent of ATM and indirectly modulate the phosphorylation level of γH2AX via the Akt/GSK3 β signal pathway.

HIF mediated and DNA damage independent histone H2AX phosphorylation in chronic hypoxia

“…In line with this hypothesis, accumulation of DNA-PKcs, Ku70 and Ku80 following hypoxia and iron chelation have been demonstrated in a number of different cell lines (Ginis and Faller , 2000 ;Lynch et al , 2001 ;Um et al , 2004 ;Bouquet et al , 2011 ). DNA-PK has been shown to phosphorylate H2AX in different cell lines and in vivo in response to DNA damage (Stiff et al , 2004 ;Koike et al , 2008 ;An et al , 2010 ) under hypertonic conditions (Reitsema et al , 2005 ) and during apoptotic DNA fragmentation (Mukherjee et al , 2006 ). Of note, the hypoxic DNA-PK activation resulted in increased HIF-dependent gene expression (Bouquet et al , 2011 ).…”

Phosphorylation of H2AX at Ser139 and a New Phosphorylation Site Ser16 by RSK2 Decreases H2AX Ubiquitination and Inhibits Cell Transformation

“…However, DNA damage also induces cell-cycle checkpoints and g-H2AX is involved in this process. Thus, this response might also account for some of the antiproliferative effects of g-H2AX as well (46,47). Our data, therefore, suggest that both a proliferation signaling pathway (RSK2) and a DNA damage response signaling pathway (DNA-PK) are activated by EGF treatment.…”

Degradation of Human RAP80 is Cell Cycle Regulated by Cdc20 and Cdh1 Ubiquitin Ligases

“…Cells were synchronized at late G 1 phase with the thymidine double blocking method (18). Briefly, the cells were plated in 100 mm diameter Petri dishes and thymidine was added to a final concentration of 2 mmol/L after cell adherence.…”