AbstractObjective 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.
. 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.
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