中华肿瘤杂志
中華腫瘤雜誌
중화종류잡지
CHINESE JOURNAL OF ONCOLOGY
2014年
11期
805-810
,共6页
杨宗元%孙朝阳%刘毅%龚成%陈刚%翁丹卉
楊宗元%孫朝暘%劉毅%龔成%陳剛%翁丹卉
양종원%손조양%류의%공성%진강%옹단훼
卵巢肿瘤%顺铂%细胞系%共济失调毛细血管扩张突变基因RAD3相关蛋白%DNA双链损伤%同源重组修复%细胞周期
卵巢腫瘤%順鉑%細胞繫%共濟失調毛細血管擴張突變基因RAD3相關蛋白%DNA雙鏈損傷%同源重組脩複%細胞週期
란소종류%순박%세포계%공제실조모세혈관확장돌변기인RAD3상관단백%DNA쌍련손상%동원중조수복%세포주기
Ovarian neoplasms%Cisplatin%Cell line%Ataxia telangiectasia mutated and RAD3 related protein%DNA double strand breaks%Homologous recombination%Cell cycle
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.
目的 探討共濟失調毛細血管擴張突變基因RAD3相關蛋白(ATR)水平和活性對卵巢癌SKOV3細胞順鉑(DDP)敏感性的影響.方法 以ATR-siRNA轉染SKOV3細胞48 h下調ATR蛋白水平,以VE-821預處理SKOV3細胞12 h下調ATR激酶活性.採用CCK8實驗檢測細胞活性,Western blot法檢測ATR、燐痠化組蛋白2A變異體(γ-H2AX)和p-ATR蛋白錶達,熒光共聚焦檢測細胞γ-H2AX和DNA雙鏈脩複蛋白(RAD51)的錶達,堿性慧星實驗檢測細胞內DNA雙鏈損傷情況,流式細胞術檢測細胞週期分佈.結果 DDP可明顯引起SKOV3細胞DNA雙鏈損傷,併激活ATR激酶通路.ATR-siRNA可顯著下調ATR蛋白水平.CCK8檢測結果顯示,NC-siRNA組和ATR-siRNA組經DDP作用48 h後,其半數抑製濃度(IC50)值分彆為72.12 μmol/L和41.25 μmol/L(P＜ 0.05).熒光共聚焦檢測結果顯示,40 μmol/L DDP處理24 h後,ATR-siRNA組RAD51募集明顯減少.堿性彗星實驗結果顯示,與NC-siRNA組比較,ATR-siRNA組能夠引起更長的拖尾效應,DNA雙鏈損傷明顯增加.DMSO組和VE-821組經DDP作用48 h後,其IC50值分彆為75.32 μmol/L和45.64 μmol/L(P＜0.05).熒光共聚焦檢測結果顯示,40 μmol/L DDP處理24 h後,VE-821組RAD51募集明顯減少.堿性彗星實驗結果顯示,與DMSO組比較,VE-821組能夠引起更長的拖尾效應,DNA雙鏈損傷明顯增加.細胞週期檢測結果顯示,40 μmol/L DDP作用于卵巢癌SKOV3細胞24h後,其G0/G1期、S期、G2/M期細胞比例分彆為71.2％、13.4％和15.4％,而對照組分彆為54.2％、21.3％和24.4％,DDP引起明顯的G0/G1期阻滯.ATR-siRNA組和VE-821組經DDP作用後,其G0/G1期、S期、G2/M期細胞比例分彆為43.2％、20.4％、36.4％和40.2％、22.5％、37.3％,榦擾ATR錶達和活性後能夠逆轉DDP引起的細胞週期阻滯.結論 抑製ATR可以影響SKOV3細胞同源重組脩複過程,增加DNA雙鏈損傷,緩解G0/G1期週期阻滯,增加其對DDP的敏感性.
목적 탐토공제실조모세혈관확장돌변기인RAD3상관단백(ATR)수평화활성대란소암SKOV3세포순박(DDP)민감성적영향.방법 이ATR-siRNA전염SKOV3세포48 h하조ATR단백수평,이VE-821예처리SKOV3세포12 h하조ATR격매활성.채용CCK8실험검측세포활성,Western blot법검측ATR、린산화조단백2A변이체(γ-H2AX)화p-ATR단백표체,형광공취초검측세포γ-H2AX화DNA쌍련수복단백(RAD51)적표체,감성혜성실험검측세포내DNA쌍련손상정황,류식세포술검측세포주기분포.결과 DDP가명현인기SKOV3세포DNA쌍련손상,병격활ATR격매통로.ATR-siRNA가현저하조ATR단백수평.CCK8검측결과현시,NC-siRNA조화ATR-siRNA조경DDP작용48 h후,기반수억제농도(IC50)치분별위72.12 μmol/L화41.25 μmol/L(P＜ 0.05).형광공취초검측결과현시,40 μmol/L DDP처리24 h후,ATR-siRNA조RAD51모집명현감소.감성혜성실험결과현시,여NC-siRNA조비교,ATR-siRNA조능구인기경장적타미효응,DNA쌍련손상명현증가.DMSO조화VE-821조경DDP작용48 h후,기IC50치분별위75.32 μmol/L화45.64 μmol/L(P＜0.05).형광공취초검측결과현시,40 μmol/L DDP처리24 h후,VE-821조RAD51모집명현감소.감성혜성실험결과현시,여DMSO조비교,VE-821조능구인기경장적타미효응,DNA쌍련손상명현증가.세포주기검측결과현시,40 μmol/L DDP작용우란소암SKOV3세포24h후,기G0/G1기、S기、G2/M기세포비례분별위71.2％、13.4％화15.4％,이대조조분별위54.2％、21.3％화24.4％,DDP인기명현적G0/G1기조체.ATR-siRNA조화VE-821조경DDP작용후,기G0/G1기、S기、G2/M기세포비례분별위43.2％、20.4％、36.4％화40.2％、22.5％、37.3％,간우ATR표체화활성후능구역전DDP인기적세포주기조체.결론 억제ATR가이영향SKOV3세포동원중조수복과정,증가DNA쌍련손상,완해G0/G1기주기조체,증가기대DDP적민감성.
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.
