CAJ | 학술논문

目的探讨Tip60对细胞辐射敏感性的影响及相关机制.方法采用siRNA和Tip60乙酰转移酶抑制剂漆树酸,抑制U2OS细胞中Tip60的表达或乙酰转移酶活性；用克隆形成率分析细胞对60Co γ射线的敏感性；采用γ-H2AX原位免疫荧光集簇点法,检测DNA双链断裂损伤修复；用免疫共沉淀检测蛋白质的相互作用.结果 siRNA沉默Tip60表达明显提高了U2OS细胞对1、2 Gy中、低剂量γ射线的敏感性(t=3.364、3.979,P＜0.05),但对4 Gy大剂量照射的细胞存活率无明显影响.γ-H2AX集簇点检测结果表明,照射后1、4和8h,Tip60失活导致细胞DNA双链断裂修复能力降低(t=3.875、3.183和3.175,P＜0.05).细胞在受到电离辐射损伤后,Tip60与DNA修复蛋白DNA-PKcs发生相互作用,漆树酸能抑制DNA-PKcs的T2609位点的磷酸化.结论 Tip60通过与DNA-PKcs相互作用,调控细胞DNA双链断裂修复机制,对细胞辐射敏感性产生影响.
목적 탐토Tip60대세포복사민감성적영향급상관궤제.방법 채용siRNA화Tip60을선전이매억제제칠수산,억제U2OS세포중Tip60적표체혹을선전이매활성；용극륭형성솔분석세포대60Co γ사선적민감성；채용γ-H2AX원위면역형광집족점법,검측DNA쌍련단렬손상수복；용면역공침정검측단백질적상호작용.결과 siRNA침묵Tip60표체명현제고료U2OS세포대1、2 Gy중、저제량γ사선적민감성(t=3.364、3.979,P＜0.05),단대4 Gy대제량조사적세포존활솔무명현영향.γ-H2AX집족점검측결과표명,조사후1、4화8h,Tip60실활도치세포DNA쌍련단렬수복능력강저(t=3.875、3.183화3.175,P＜0.05).세포재수도전리복사손상후,Tip60여DNA수복단백DNA-PKcs발생상호작용,칠수산능억제DNA-PKcs적T2609위점적린산화.결론 Tip60통과여DNA-PKcs상호작용,조공세포DNA쌍련단렬수복궤제,대세포복사민감성산생영향.
Objective To investigate the effect of Tip60 on the cellular radiosensitivity,and to explore the related mechanism.Methods siRNA and anacardic acid (AA,an inhibitor of Tip60 acetyltransferase) were used to inhibit Tip60 expression and its acetyltransferase activity,respectively.Radiosensitivity was analyzed by colony-forming ability assay.γ-H2AX foci were detected to analyze the DNA double-strand break (DSB).Immunoprecipitation was used to determine the interaction of proteins.Results siRNA-mediated silencing of Tip60 led to enhanced sensitivity of U2OS cells at 1,2 Gy after γ-ray irradiation,but had no significant effect at 4 Gy post-irradiation ( t =3.364,3.979,P ＜ 0.05 ).γ-H2AX foci detection indicated that Tip60 silencing resulted in a decreased capability of DNA doublestrand break repair at 1,4 and 8 h after irradiation( t =3.875,3.183 and 3.175,respectively,P ＜ 0.05 ).The interaction of Tip60 and DNA-PKcs was prompted by ionizing radiation.Anacardic acid largely abrogated the phosphorylation of DNA-PKcs at T2609 site induced by irradiation.Conclusions Tip60plays a role in the cellular response to ionizing radiation-induced DNA damage through,at least in part,interacting with DNA-PKcs and regulating its phosphorylation.