人骨肉瘤细胞DNA双链断裂修复γH2AX分析

γH2AX analysis for monitoring cellular response to DNA Double-Strand Breaks in human osteosarcoma cell

目的:探讨人骨肉瘤细胞U2OS中利用γH2AX分析评价细胞DNA双链断裂(DNA Double-Strand Breaks,DSBs)损伤反应动力学过程。方法:用免疫荧光和蛋白质印迹法结合DNA损伤应答通路抑制剂及siRNA基因沉默技术分析U2OS细胞在遭受DSBs损伤处理前后γH2AX foci和其表达水平的动力学变化,并通过流式细胞术定量分析γH2AX的变化情况。结果:在细胞未经损伤处理时,U2OS细胞仅有极少量的γH2AX foci,而在遭受电离辐射(ionizing radiation,IR)后,γH2AX在损伤部位的募集和表达水平快速升高,1h内达峰值,该过程可被ATM抑制剂和磷酸肌醇激酶3相关激酶抑制剂Wortmannin抑制;而后随着时间的推移,γH2AX表达又逐渐消退,反映了细胞DSBs损伤应答的动力学过程。利用小分子RNA敲除参与DSBs损伤修复的关键因子Mre11表达的细胞及对照siRNA转染细胞,IR处理4h后γH2AX foci数分别为31.68±4.59和21.33±2.08,处理8h分别为21.85±2.49和12.02±4.13,处理12h分别为19.65±2.34和8.51±2.17),处理16h分别为18.05±1.75和6.94±3.19,各时间点Mre11沉默组细胞的γH2AX foci数显著多于对照siRNA处理组细胞,P=0.013,反映了延长的DSBs损伤修复动力学(减缓的γH2AX foci消退)。经γH2AX和PI双染色流式细胞术发现,不同细胞分期γH2AX阳性率差异有统计学意义,G1期细胞在IR处理前为(1.34±0.28)%,处理后为(20.25±3.56)%,P<0.001;S期细胞在IR处理前为(1.26±0.19)%,处理后为(79.48±5.72)%,P<0.001;G2/M期细胞在IR处理前为(8.84±1.25)%处理后为(29.49±4.36)%,P<0.001。结论:γH2AX分析可有效分析U2OS细胞的DSBs损伤应答反应动力学,U2OS细胞具有低γH2AX背景、易于传代培养等优点,可作为研究细胞DSBs损伤应答反应的模式细胞。

OBJECTIVE： To investigate the dynamic changes of cellular response to DNA Double-Strand Breaks （DSBs） in human osteosarcoma （U2OS） cell using yH2AX analysis. METHODS： Using a combination of the inhibitors of DNA damage response pathways and technology of small interfering RNAs,the dynamic changes of γH2AX loci and pro- tein expression level after X-irradiation were determined by immunofluorescence （IF） and western blotting （WB） ,and flu- orescence-activated cell sorting （FACS） was used to analyse the expression level of yH2AX quantitatively. RESULTS： Af- ter treated cells with ionizing radiation （IR）, the recruitment of γH2AX to DNA damage site and the protein level of γH2AX increased rapidly and reached a peak at 1 h, this process was inhibited by ATMi （ATM inhibitor） and wortmannin （a inhibitor of phosphatidyl inositol 3-kinase-related kinases） ~then as time went on, the expression level of γH2AX was e- liminated gradually,which reflected the dynamic process of cellular response to DSBs. At all time point of 4 h, 8 h, 12 h and 16 h after treated cells with IR, the γH2AX loci were significantly increased in Mrell （a key repair factor of the DNA damage response） knockdown cells than in siControl-transfected ceils （P=0. 013） sand the loci number of the two groups were （31. 684±4.59 vs 21.33±2.08） ,（21. 854±2.49 vs 12.02±4.13）,（19. 654±2.34 vs 8. 514±2.17） and （18. 054±1.75 vs 6.94 ± 3.19）, respectively; this result reflected the prolonged DSBs repair kinetics （prolonged existence of the γH2AX loci） of Mrell-defective ceils. Using FACS analysis after cells stained with γH2AX and propidium iodide （PI）, we found that the cells treated with IR had dramatically higher rates of γH2AX-positive in all different cell cycle phases compared with the mock treatment cells,G1 stages were （1.34±0.28）% and （20. 254±3.56）%,P〈0. 001;S stages were （1.26± 0.19）% and （79. 484±5.72）%,P〈0. 001;G2/M stages were （8. 844±1.25）% and （29. 49±4.36）% ,P〈0. 001;respec- tively. CONCLUSIONS： These results suggest that γH2AX analysis can he effectively used to detect the dynamic process of cellular response to DSBs in U2OS cell. And this cell line can be used as an in vitro perfect model for the kinetic analysis of DSBs repair pathways for its low background of γH2AX level with the merit of easy to culture.