肿瘤BRCA1亚细胞定位对细胞放射线及PARP抑制剂敏感性的影响

摘要

目的 探讨肿瘤BRCA1细胞内定位对放射线和PARP抑制剂敏感性的影响.方法 采用siRNA干扰抑制乳腺癌细胞株MCF7内源性BRCA1表达,转染BRCA1细胞内定位不同的载体:pCMV-3xFlag-WT-BRCA1、pCMV-3 xFlag-NES-BRCA1、pC-MV-3xFlag-NIS-BRCA1.采用免疫荧光法检测BRCA1的细胞定位及细胞核γ-H2AX和Rad51核焦点形成,应用流式细胞技术检测细胞凋亡,克隆形成实验检测体外细胞存活.结果 转染WT-BRCA1有47％细胞核表达,23％细胞质表达,30％细胞质和细胞核均表达,NES-BRCA1 mutant表达主要定位于细胞核(87％);NLS-BRCA1 mutant定位于细胞质(82％).WT-BRCA1、NES-BRCA1 mutant和NLS-BRCA1 mutant三种载体转染的细胞4 Gy放射处理后2h,Rad51核焦点阳性细胞数分别为87％、84％及13％;放射后24 h,γ-H2AX核焦点阳性细胞分别为22％、25％及59％.NLS-BRCA1 mutant转染细胞较WT-BRCA1和NES-BRCA1 mutant转染细胞ABT-888和放射处理后诱导的凋亡细胞增加,克隆存活减少.结论 BRCA1的细胞内定位影响DNA双链断裂同源重组修复,并可预测肿瘤对放射和PARP抑制剂的敏感性.%Purpose To investigate the effect of subcellular location of tumor BRCA1 on the sensitivity to ionizing radiation (IR) and PARP inhibitor.Methods siRNA of BRCA1 were first used to inhibit endougenous BRCA1 expression in MCF7 cells.Then,plasmids of pCMV-3xFlag-WT-BRCA1,pCMV-3xFlag-NES-BRCA1 and pCMV-3xFlag-NLS-BRCA1 were transfected in MCF7 cells.Immunofluorescence staining was used to detect BRCA1 subcellular location as well as the formation of Rad51 and γ-H2AX foci.Apoptotic cells were analyzed by flow cytometry,and colony formation assay was performed to evaluate the survival of cells.Results There were 47％ cells with nuclear BRCA1,23％ cells with cytoplasmic BRCA1 and 30％ cell with mixed nuclear and cytoplasmic BRCA1 expression in WT-BRCA1 transfected cell.There were 87％ cells with nuclear BRCA1 in NES-BRCA1 transfected cell,and 82％ cells with cytoplasmic BRCA1 in NLS-BRCA1 transfected cell.There were 87％,84％ and 13％ Rad51 foci positive cells at 2 hours after 4 Gy radiation treatment and 22％,25％ and 59％ γ-H2AX foci positive cells at 24 hours after 4Gy radiation treatment in WT-BRCA1,NES-BRCA1 mutant and NLS-BRCA1 mutant transfected cell respectively.ABT-888 and radiation treatment induced more apoptosis and fewer colonies in NLS-BRCA1 transfected cell than WT-BRCA1,NES-BRCA1 mutant transfected cell.Conclusion Subcellular location of BRCA1 might affect homologous recombination repair of DNA double strand breaks and can be used to predict sensitivity to IR and PARP inhibitor.