摘要
目的 探讨共济失调毛细血管扩张突变基因RAD3相关蛋白（ATR）水平和活性对卵巢癌SKOV3细胞顺铂（DDP）敏感性的影响.方法 以ATR-siRNA转染SKOV3细胞48 h下调ATR蛋白水平,以VE-821预处理SKOV3细胞12 h下调ATR激酶活性.采用CCK8实验检测细胞活性,Western blot法检测ATR、磷酸化组蛋白2A变异体（γ-H2AX）和p-ATR蛋白表达,荧光共聚焦检测细胞γ-H2AX和DNA双链修复蛋白（RAD51）的表达,碱性慧星实验检测细胞内DNA双链损伤情况,流式细胞术检测细胞周期分布.结果 DDP可明显引起SKOV3细胞DNA双链损伤,并激活ATR激酶通路.ATR-siRNA可显著下调ATR蛋白水平.CCK8检测结果显示,NC-siRNA组和ATR-siRNA组经DDP作用48 h后,其半数抑制浓度（IC50）值分别为72.12 μmol/L和41.25 μmol/L（P＜ 0.05）.荧光共聚焦检测结果显示,40 μmol/L DDP处理24 h后,ATR-siRNA组RAD51募集明显减少.碱性彗星实验结果显示,与NC-siRNA组比较,ATR-siRNA组能够引起更长的拖尾效应,DNA双链损伤明显增加.DMSO组和VE-821组经DDP作用48 h后,其IC50值分别为75.32 μmol/L和45.64 μmol/L（P＜0.05）.荧光共聚焦检测结果显示,40 μmol/L DDP处理24 h后,VE-821组RAD51募集明显减少.碱性彗星实验结果显示,与DMSO组比较,VE-821组能够引起更长的拖尾效应,DNA双链损伤明显增加.细胞周期检测结果显示,40 μmol/L DDP作用于卵巢癌SKOV3细胞24h后,其G0/G1期、S期、G2/M期细胞比例分别为71.2％、13.4％和15.4％,而对照组分别为54.2％、21.3％和24.4％,DDP引起明显的G0/G1期阻滞.ATR-siRNA组和VE-821组经DDP作用后,其G0/G1期、S期、G2/M期细胞比例分别为43.2％、20.4％、36.4％和40.2％、22.5％、37.3％,干扰ATR表达和活性后能够逆转DDP引起的细胞周期阻滞。结论 抑制ATR可以影响SKOV3细胞同源重组修复过程,增加DNA双链损伤,缓解G0/G1期周期阻滞,增加其对DDP的敏感性。
Objective To explore the effect of ataxia telanglectasia mutated and RAD3 related protein （ATR） expression and ATR kinase activity on the sensitivity to cisplatin in ovarian cancer SKOV3 cells.Methods SiRNA targeting ATR was transfected into SKOV3 cells for 48 h to reduce the ATR protein level,and ATR kinase inhibitor VE-821 was used for 12 h to inhibit the ATR pathway activity.The alteration of cell viability was examined by CCk-8 assay.Expression levels of ATR,p-ATR and γ-H2AX proteins were detected by Western blot.The DNA double strand breaks （DSB） marker γ-H2AX and homologous recombination repair key protein RAD51 and their co-localization in the cells were examined under the confocal microscope.The status of DNA double strand breaks （DSB） in single cells was visualized by alkaline comet assay.Finally,the cell cycle distribution was assessed using flow cytometry.Results DDP caused evident DNA double strands breaks and activated ATR kinase pathway.ATR-siRNA notably reduced ATR protein level,the 48 h IC50 value of DDP was 72.12 μmol/L and 41.25 μmol/L,respectively,in the NC-siRNA and ATR-siRNA groups （P 〈 0.05）.Confocal microscopic assay presented decreased recruitment of RAD51 at the DSB loci and comet assay showed enhanced DSB in the cells after ATR knocking down.After the inhibition of ATR kinase by VE-821,the 48 h IC50 value of DDP was 75.32 μmol/L and 45.64 μmol/L,respectively,in the DMSO and VE-821 groups （P 〈0.05 for both）,confocal microscopic assay demonstrated reduced RAD51 recruitment,and comet assay showed increased DSB in cells after ATR kinase inhibition.Flow cytometry showed that percentage of cells distributed in G0/G1,S and G2/M phases was 71.2%,13.4% and 15.4%,repectively,after 40 μmol/L DDP treatment for 24 h.Compared with that of control group （G0/G1：54.2%,S：21.3% and G2/M：24.4%）,DDP induced G0/G1 phase arrest.DDP intervention resulted in the cell cycle status （G0/G1：43.2%,S：20.4%,G2/M：36.4%） in the ATR-siRNA group and （G0/G1：40.2%,S：22.5%,G2/M：37.3%） in the VE 821 group,indicating that the inhibition of ATR or ATR kinase could abrogate the effect of G0/G1 phase arrest induced by DDP.Conclusions Suppression of ATR can affect the homologous recombination repair in ovarian cancer cells,leading to accumulation of DNA double strand breaks in the cell nuclei as well as reduction of DDP-caused G0/G1 phase arrest,finally enhances the sensitivity to cisplatin in the ovarian cancer SKOV3 cells.
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2014年第11期805-810,共6页
Chinese Journal of Oncology
基金
国家自然科学基金（81000979）
