DNA-PKcs plays a dominant role in the regulation of H2AX phosphorylation in response to DNA damage and cell cycle progression.
DNA-PKcs plays a dominant role in the regulation of H2AX phosphorylation in response to DNA damage and cell cycle progression.
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DNA-PKcs 在响应 DNA 损伤和细胞周期进展的 H2AX 磷酸化调节中起主导作用。
DOI：
10.1186/1471-2199-11-18
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发表时间：
2010-03-06
影响因子：
--
通讯作者：
Zhou PK
中科院分区：
文献类型：
--
作者：
An J;Huang YC;Xu QZ;Zhou LJ;Shang ZF;Huang B;Wang Y;Liu XD;Wu DC;Zhou PK
当电离辐射（IR）诱导细胞DNA双链断裂（DSB）时，组蛋白H2 AX在DSB位点附近迅速磷酸化为γ-H2 AX（p-S139）。研究了DNA-PKcs在响应DNA损伤和细胞周期进程中调节H2 AX磷酸化的必要性。4戈伊γ射线照射后，HeLa细胞γ H2 AX水平迅速升高，在0.25 ~ 1.0 h达高峰。siRNA介导的DNA-PKcs抑制导致γ H2 AX水平显著降低。4戈伊γ射线照射后0.5 ~ 1.0 h，ATM缺陷细胞株AT 5 BIVA中γ H2 AX也被诱导增加，这种IR诱导的ATM缺陷细胞中γ H2 AX的增加被PIKK抑制剂wortmannin和DNA-PKcs特异性抑制剂NU 7026显著消除。在另一个ATM缺陷细胞系ATS 4中观察到γ H2 AX的高水平组成型表达。γ H2 AX水平的变化与细胞周期进程有关。用胸苷双阻断法将siRNA抑制DNA-PKcs的HeLa细胞（HeLa-H1）和正常DNA-PKcs的HeLa细胞（HeLa-NC）同步化于G1期。在同步化细胞从G1阻滞释放后约5 h，S期细胞对于HeLa-H1和HeLa-NC细胞均占优势（80%）。同步化细胞从G1期阻滞释放后8 ~ 9 h，HeLa-NC细胞的G2/M期比例达到56 ~ 60%，高于HeLaH 1细胞的33 ~ 40%。在此期间，DNA PKcs缺失的HeLa-H1细胞S期比例仍维持在26 - 33%的水平，而HeLa-NC细胞S期比例下降至约15%。在HeLa-NC细胞中，随着G1期阻滞解除，γ H2 AX水平逐渐升高，进入G2/M期。然而，在DNA-PKcs缺失的HeLa-H1细胞中，这种与细胞周期进展相关的γ H2 AX的改变被显著抑制，而wortmannin和NU 7026也能抑制这种与细胞周期相关的H2 AX磷酸化。用LiCl或特异性siRNA抑制GSK 3 β活性可上调γ H2 AX水平，并使γ H2 AX水平升高的时间延长至4戈伊后10 h以上。GSK 3 β是DNA-PKcs/Akt信号转导的负调控靶点，通过Ser 9的磷酸化导致其失活。在HeLa细胞中，DNA-PKcs的抑制导致Akt在Ser 473上的磷酸化减少，其靶GSK 3 β在Ser 9上的磷酸化减少，换句话说，这导致GSK 3 β的活化增加。此外，通过siRNA抑制Akt/GSK 3 β的另一上游调节因子PDK也可以减少响应于DNA损伤和细胞周期进展的γ H2 AX的诱导。DNA-PKcs在响应DNA损伤和细胞周期进程的H2 AX磷酸化调节中起主导作用。它可以不依赖ATM直接磷酸化H2 AX，并通过Akt/GSK 3 β信号通路间接调节γ H2 AX的磷酸化水平。
When 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. The 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. DNA-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.
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影响因子：
16
作者：
Chowdhury, Dipanjan;Xu, Xingzhi;Zhong, Xueyan;Ahmed, Fariyal;Zhong, Jianing;Liao, Ji;Dykxhoorn, Derek M.;Weinstock, David M.;Pfeifer, Gerd P.;Lieberman, Judy
通讯作者：
Lieberman, Judy
影响因子：
56.9
作者：
Celeste, A;Petersen, S;Nussenzweig, A
通讯作者：
Nussenzweig, A
影响因子：
11.2
作者：
Peng, YL;Woods, RG;Bedford, JS
通讯作者：
Bedford, JS
影响因子：
9.2
作者：
Paull, TT;Rogakou, EP;Bonner, WM
通讯作者：
Bonner, WM
DOI：
10.1016/j.bbrc.2008.08.095
发表时间：
2008-11-07
影响因子：
3.1
作者：
Koike, Manabu;Sugasawa, Jun;Koike, Aki
通讯作者：
Koike, Aki