510080 广州,中山大学附属第一医院放射治疗科(董隽、王成涛、任玉峰、欧阳斌、张天、王振宇、文碧秀);523059 东莞市人民医院放射治疗科(张纯);10021 New York,Department of Medical Physics and Radiation Oncology,Memorial Sloan-Kettering Cancer Center (Gloria C.Li、Fuqiu He)
Quantitative analysis of γ-H2AX foci formation and dynamic changes in DNA double-strand breaks induced by X-ray radiation
Dong Jun, Wang Chengtao, Zhang Chun, Ren Yufeng, Zhang Tian, Ooyang Bin, Wang Zhenyu, Gloria C. Li, Fuqiu He, Wen Bixiu
Department of Radiation Oncology,First Affiliated Hospital,The Sun Yat-sen University,Guangzhou 510080,China
Abstract：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.
. Quantitative analysis of γ-H2AX foci formation and dynamic changes in DNA double-strand breaks induced by X-ray radiation[J]. Chinese Journal of Radiation Oncology, 2018, 27(3): 303-308.
[1] Morgan MA,Lawrence TS.Molecular pathways:overcoming radiation resistance by targeting DNA damage response pathways[J].Clin Cancer Res,2015,21(13):2898-2904.DOI:10.1158/1078-0432.CCR-13-3229. [2] Pawelczak KS,Bennett SM,Turchi JJ.Coordination of DNA-PK activation and nuclease processing of DNA termini in NHEJ[J].Antioxid Redox Signal,2011,14(12):2531-2543.DOI:10.1089/ars.2010.3368. [3] Pascale RM,Joseph C,Latte G,et al. DNA-PKcs:A promising therapeutic target in human hepatocellular carcinoma[J] DNA Repair (Amst),2016,47:12-20.DOI:10.1016/j.dnarep.2016.10.004. [4] Mah LJ,El-Osta A,Karagiannis TC.gammaH2AX:a sensitive molecular marker of DNA damage and repair[J].Leukemia,2010,24(4):679-686.DOI:10.1038/leu.2010.6. [5] Bonner WM,Redon CE,Dickey JS,et al. GammaH2AX and cancer[J].Nat Rev Cancer,2008,8(12):957-967.DOI:10.1038/nrc2523. [6] 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.DOI:10.1158/0008-5472.CAN-03-3207. [7] Rothkamm K,Lobrich M.Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses[J].Proc Natl Acad Sci U S A,2003,100(9):5057-5062.DOI:10.1073/pnas.0830918100. [8] Dong J,Zhang T,Ren Y,et al. Inhibiting DNA-PKcs in a non-homologous end-joining pathway in response to DNA double-strand breaks[J].Oncotarget,2017,8(14):22662-22673.DOI:10.18632/oncotarget.15153. [9] Chua ML,Wee JT,Hui EP,et al. Nasopharyngeal carcinoma[J].Lancet,2016,387(10022):1012-1024.DOI:10.1016/S0140-6736(15)00055-0. [10] Schaue D,Mcbride WH.Opportunities and challenges of radiotherapy for treating cancer[J].Nat Rev Clin Oncol,2015,12(9):527-540.DOI:10.1038/nrclinonc.2015.120. [11] O′Connor MJ.Targeting the DNA damage response in cancer[J].Mol Cell,2015,60(4):547-560.DOI:10.1016/j.molcel.2015.10.040. [12] He F,Li L,Kim D,et al. Adenovirus-mediated expression of a dominant negative Ku70 fragment radiosensitizes human tumor cells under aerobic and hypoxic conditions[J].Cancer Res,2007,67(2):634-642.DOI:10.1158/0008-5472.CAN-06-1860. [13] Gupta A,Hunt CR,Hegde ML,et al. MOF phosphorylation by ATM regulates 53BP1-mediated double-strand break repair pathway choice[J].Cell Rep,2014,8(1):177-189.DOI:10.1016/j.celrep.2014.05.044. [14] Massey AJ,Stephens P,Rawlinson R,et al.mTORC1 and DNA-PKcs as novel molecular determinants of sensitivity to Chk1 inhibition[J].Mol Oncol,2016,10(1):101-112.DOI:10.1016/j.molonc.2015.08.004. [15] Caron P,Choudjaye J,Clouaire T,et al. Non-redundant Functions of ATM and DNA-PKcs in Response to DNA Double-Strand Breaks[J].Cell Rep,2015,13(8):1598-1609.DOI:10.1016/j.celrep.2015.10.024. [16] Zhang S,Matsunaga S,Lin YF,et al. Spontaneous tumor development in bone marrow-rescued DNA-PKcs (3A/3A) mice due to dysfunction of telomere leading strand deprotection[J].Oncogene,2016,35(30):3909-3918.DOI:10.1038/onc.2015.459. [17] Albarakati N,Abdel-Fatah TM,Doherty R,et al. Targeting BRCA1-BER deficient breast cancer by ATM or DNA-PKcs blockade either alone or in combination with cisplatin for personalized therapy[J].Mol Oncol,2015,9(1):204-217.DOI:10.1016/j.molonc.2014.08.001. [18] Cuneo KC,Morgan MA,Davis MA,et al. Wee1 kinase inhibitor AZD1775 radiosensitizes hepatocellular carcinoma regardless of TP53 mutational status through induction of replication stress[J].Int J Radiat Oncol Biol Phys,2016,95(2):782-790.DOI:10.1016/j.ijrobp.2016.01.028. [19] Zhao H,Traganos F,Darzynkiewicz Z.Kinetics of the UV-induced DNA damage response in relation to cell cycle phase. Correlation with DNA replication[J].Cytometry A,2010,77(3):285-293.DOI:10.1002/cyto.a.20839. [20] Zhao H,Traganos F,Darzynkiewicz Z.Kinetics of histone H2AX phosphorylation and Chk2 activation in A549 cells treated with topotecan and mitoxantrone in relation to the cell cycle phase[J].Cytometry,2008,73(6):480-489.DOI:10.1002/cyto.a.20574. [21] Yu L,Shang Z F,Hsu FM,et al. NSCLC cells demonstrate differential mode of cell death in response to the combined treatment of radiation and a DNA-PKcs inhibitor[J].Oncotarget,2015,6(6):3848-3860.DOI:10.18632/oncotarget.2975. [22] Kuhne M,Riballo E,Rief N,et al. A double-strand break repair defect in ATM-deficient cells contributes to radiosensitivity[J].Cancer Res,2004,64(2):500-508.DOI:10.1158/0008-5472.CAN-03-2384. [23] Zhao Y,Thomas HD,Batey MA,et al. Preclinical evaluation of a potent novel DNA-dependent protein kinase inhibitor NU7441[J].Cancer Res,2006,66(10):5354-5362.DOI:10.1158/0008-5472.CAN-05-4275.
2018, Vol. 27 
