Phosphorylation of H2AX Related to the Cell Cycle G2/M Progression

Chinese Journal of Biochemistry and Molecular Biology ›› 2012, Vol. 28 ›› Issue (4) : 339-345.

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Chinese Journal of Biochemistry and Molecular Biology ›› 2012, Vol. 28 ›› Issue (4) : 339-345.
Research Papers

Phosphorylation of H2AX Related to the Cell Cycle G2/M Progression

  • TU Wen-Zhi1), 2) , SHANG Zeng-Fu2) , LI Bing2) , LIU Xiao-Dan2) , WANG-Yu2) ,XU Qin-Zhi2) , RANG Wei-Qing1) * , ZHOU Ping-Kun1), 2) *
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Abstract

γH2AX foci has been generally accepted as a biomarker of DNA double-strand break (DSB). To investigate the feature and characteristics of H2AX phosphorylation (γH2AX) related to cell cycle progression, HeLa cells were synchronized at prometaphase by thymidine double-blocking followed by nocodazole treatment. Cell cycle and protein expression level were analyzed by flow cytometry and Western blotting, respectively. γH2AX foci were detected by fluorescent microscopic observation of immunostaining. The expression level of γH2AX was remarkably increased when the cells entered G2/M and mitotic process. A certain portion of mitotic cells was with a great number of γH2AX foci even without any DNA DSB. As cells exited from M phase, γH2AX expression level was decreased. This G2/M phases-related γH2AX expression pattern was similar to oscilation of G2/M regulators PLK1 and cyclin B1 expression. With 4 Gy of large dose γ- ray irradiation, the cells were arrested in G2/M phases at 8 ~ 12 hours after irradiation. At the 8 hours time point, γH2AX foci number also reached the second peak after the first peak formed at 1 hour postirradiation. However, with 1 Gy of low dose irradiation, G2/M arrest was very weak, and the number of γH2AX foci peaked at 0.5 hour after the irradiation, and then declined with the time. The timing of γH2AX foci formation and disappearance is consistent with the repair kinetics of ionizing radiation-induced DNA DSB. The results indicated that the cell cycle change should be taken into account when γH2AX is considered as DSB biomarker. However, γH2AX foci can be taken as an indicator of DSB when cells are irradiated with a dose of 1 Gy or lower.

Key words

γH2A / Xcell cycle / G2/M arrest / DNA double-strand break(DSB) / Biomarker

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Phosphorylation of H2AX Related to the Cell Cycle G2/M Progression[J]. Chinese Journal of Biochemistry and Molecular Biology, 2012, 28(4): 339-345

References

[1] Downs J A, Lowndes N F, Jackson S P. A role for Saccharomyces cerevisiae histone H2A in DNA repair [J]. Nature, 2000, 408(6815):1001-1004

[2] Downs J A,?Nussenzweig M C,?Nussenzweig A. Chromatin dynamics and the preservation of genetic information [J]. Nature, 2007, 447(7147):951-958

[3] Yang J, Yu Y, Hamrick H E, et al. ATM, ATR and DNA-PK: initiators of the cellular genotoxic stress responses [J].Carcinogenesis, 2003, 24(10):1571-1580

[4] Stiff T, O’Driscoll M, Rief N, et al. ATM and DNA-PK function redundantly to phosphorylate H2AX after exposure to ionizing radiation [J]. Cancer Res, 2004, 64(7):2390-2396

[5] An J, Huang Y C, Xu Q Z, et al. DNA-PKcs plays a dominant role in the regulation of H2AX phosphorylation in response to DNA damage and cell cycle progression [J]. BMC Mol Biol, 2010, 11:18

[6] Celeste A, Fernandez-Capetillo O, Kruhlak M J, et al. Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks [J]. Nat Cell Biol, 2003, 5(7): 675-679

[7] Stucki M, Clapperton J A, Mohammad D, et al. MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks [J]. Cell, 2005, 123(7):1213-1226

[8] Stucki M,?Jackson S P. MDC1/NFBD1: a?key?regulator?of the?DNA?damage response in higher eukaryotes [J]. DNA?Repair (Amst), ?2004, 3(8-9):953-957

[9] Helmink BA,?Tubbs AT,?Dorsett Y,et al. H2AX?prevents?CtIP- mediated?DNA?end?resection and?aberrant repair in G1-phase lymphocytes [J]. Nature, 2011, 469(7329):245-249

[10] Blank M,?Tang Y,?Yamashita M?, et al. A?tumor suppressor?function?of?Smurf2?associated?with controlling chromatin landscape and genome stability through RNF20 [J]. Nat Med, 2012, 18(2):227-234

[11] Celeste A, Petersen S, Romanienko P J, et al. Genomic instability in mice lacking histone H2AX[J]. Science, 2002, 296 (5569):922-927

[12] Revet I, Feeneya L, Bruguera S, et al. Functional relevance of the histone γH2Ax in the response to DNA damaging agents [J]. Proc Natl Acad Sci U S A, 2011, 108(21):8663-8667

[13] Yin B, Yang-lott K S, Chao L H, et al. Cellular context-dependent effects of H2ax and p53 deletion on the development of thymic lymphoma [J]. Blood, 2011, 117(1):175-185

[14] Celeste A, Difilippantonio S, Difilippantonio M J, et al. H2AX haploinsufficiency modifies genomic stability and tumor susceptibility [J]. Cell, 2003, 114(3):371-383

[15] Bassing C H, Chua K F, Sekiguchi J, et al. Increased ionizing radiation sensitivity and genomic instability in the absence of histone H2AX [J]. Proc Natl Acad Sci U S A, 2002, 99 (12): 8173-8178

[16] Redon C E, Dickey J S, Bonner W M, et al. γ-H2AX as a biomarker of DNA damage induced by ionizing radiation in human peripheral blood lymphocytes and artificial skin [J]. Adv Space Res, 2009, 43(8):1171-1178

[17] Bourton E C, Plowman P N, Zahir S A, et al. Multispectral imaging flow cytometry reveals distinct frequencies of γ-H2AX foci induction in DNA double strand break repair defective human cell lines [J]. Cytometry A, 2012, 81(2):130-137

[18] Zlobinskaya O, Dollinger G, Michalski D, et al. Induction and repair of DNA double-strand breaks assessed by gamma-H2AX foci after irradiation with pulsed or continuous proton beams [J]. Radiat Environ Biophys, 2012, 51(1):23-32

[19] Pospelova T V, Demidenko Z N, Bukreeva E I, et al. Pseudo-DNA damage response in senescent cells [J]. Cell Cycle, 2009, 8(24):4112-4118

[20] Wei Y, Yu L, Bowen J, et al. Phosphorylation of histone H3 is required for proper chromosome condensation and segregation [J]. Cell, 1999, 97(1):99-109

Funding

Supported by Outstanding Youth Scientist Foundation of National Natural Science Foundation of China (No.30825011), National Natural Science Foundation of China(No.30970677)

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