Quantitative analysis of γ-H2AX foci formation and dynamic changes in DNA double-strand breaks induced by X-ray radiation

DNA 分子生物学 催化亚单位 免疫荧光 DNA损伤 免疫印迹 细胞 化学 生物 抗体 免疫学 生物化学 基因
作者
Jun Dong,Chengtao Wang,Chun Zhang,Yufeng Ren,Tian Zhang,Bin Ooyang,Zhenyu Wang,Gloria C. Li,Fuqiu He
出处
期刊:Chinese Journal of Radiation Oncology [Chinese Medical Association]
卷期号:27 (3): 303-308
标识
DOI:10.3760/cma.j.issn.1004-4221.2018.03.015
摘要

Objective To quantitatively compare the γ-H2AX foci formation between DNA-PKcs+ /+ and DNA-PKcs-/-mouse embryonic fibroblast (MEF) cells, and to investigate the dynamic changes in DNA double-strand breaks (DSBs) in human nasopharyngeal carcinoma SUNE-1 cells exposed to X-ray radiation. Methods The expression of DNA-PKcs was determined by Western blot. The γ-H2AX foci formation induced by 5 Gy X-ray radiation was detected by cell immunofluorescence. The ImageJ software was used to quantitatively analyze the γ-H2AX foci formation. Results The expression of DNA-PKcs was silenced in DNA-PKcs-/-MEF cells and normal in DNA-PKcs+ /+ MEF cells. According to the dynamic analyses of the numbers of γ-H2AX foci/cell and γ-H2AX foci/mm2, a similar tendency was observed in DSB formation in DNA-PKcs+ /+ MEF cells, DNA-PKcs-/-MEF cells, and SUNE-1 cells exposed to X-ray radiation. A large number of γ-H2AX foci formed at 0.5-1.0 h after radiation. DSBs were repaired at 6 h after radiation in DNA-PKcs+ /+ MEF cells and 24 h after radiation in DNA-PKcs-/-MEF cells and SUNE-1 cells. The peak values of γ-H2AX foci/cell and γ-H2AX foci/mm2 were observed at 1.0 and 0.5 h after radiation, respectively. Compared with DNA-PKcs+ /+ MEF cells, DNA-PKcs-/-MEF cells had different numbers of γ-H2AX foci/cell at 0.5, 1.0, 3.0, 6.0, and 12.0 h after radiation, as well as different numbers of γ-H2AX foci/mm2 at 3.0, 6.0, and 12.0 h after radiation. Conclusions Quantitative measurement of the number of γ-H2AX foci/cell or γ-H2AX foci/mm2 by cell immunofluorescence provides new insights into the quantitative and dynamic study of DSB damage and repair. Key words: Non-homologous terminal connection; DNA dependent protein kinase catalytic subunit; γH2AX foci formation; SUNE-1 cell line
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