ATM对卵巢癌细胞株CaOV3顺铂敏感性的影响

目的:探讨ATM激酶表达水平和活性对卵巢癌细胞Ca OV3顺铂敏感性的影响,以及可能的机制。方法:以ATM-siRNA转染Ca OV3细胞48h下调ATM蛋白水平,以KU-55933预处理Ca OV3细胞12h下调ATM激酶活性。CCK8试验检测细胞活性,Western blot法检测ATM及r-H2AX蛋白表达,荧光共聚焦confocal检测细胞DNA双链损伤标志蛋白r-H2AX表达及同源重组修复关键蛋白RAD51表达及两者细胞核中共定位,应用碱性慧星试验检测细胞内DNA双链损伤情况,流式细胞术检测细胞周期分布。结果:卵巢癌细胞系Ca OV3具备相对完整的同源重组修复能力,下调ATM或抑制ATM激酶活性均可降低DNA双链损伤情况下的同源重组修复能力,增加其对c DDP敏感性,同时减少c DDP引起的G0/G1期周期阻滞。结论:抑制ATM可影响卵巢癌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,增加其对顺铂敏感性。

The effect of ATM cisplatin sensitivity in ovarian cancer cell line CaOV3

Objective: To explore the effect of ATM kinase downregulation on cisplatin sensitivity in ovrian cancer cell line CaOV3,and its possible underlying mechanism. Methods:siRNA targeting ATM was transfected into CaOV3 cells for 48 h to reduce ATM protein level.ATM kinase inhibitor KU-55933 was added for 12 h to inhibit ATM pathway activity.The alteration of cell viability was examined using CCK-8 kit.Expression of ATM and r-H2 AX protein level were detected by Western blot. The expression of DNA double strand breaks marker-H2 AX and homologous recombination repair pivotal protein RAD51,as well as their co-localization inside nucleus were imaged under confocal microscopy. The status of DNA double strand breaks were visualized with the approach of alkaline comet assay.Finally,the cell cycle distribution was tested by flow cytometry. Results: Ovarian cancer cell line CaOV3 had relative intact homologous recombination repair capacity.Repression of ATM protein level or inhibiton of ATM kinase activity in CaOV3 could impair DNA double strand breaks repair,in turn,leads to enhancement of c DDP sensitivity. Meanwhile,ATM inhibition could relieve the effect of G0/ G1 phase arrest exerted by c DDP.Conclusions: Suppression of ATM could affect homologous recombination repair in ovarian cancer cells,which leaded to the accumulation of double DNA breaks inside nucleus.In addition,c DDP-caused G0/ G1 phase cell cycle arrest was partially rescued after ATM inhibition.This comes to a conclusion that suppression of ATM could elevate cisplatin sensitivity in ovarian cancer cell line CaOV3.