DNA损伤剂对携带乳腺癌易感基因1功能位点突变细胞的毒性作用研究

Cytotoxic effect of different kinds of DNA damaging agents on cells carrying BRCA1 site mutations

目的通过比较携带乳腺癌易感基因1(BRCA1)的RING或BRCT功能区内位点突变的乳腺癌细胞系(HCC1937)对不同来源DNA损伤的应答反应的敏感性,探讨BRCA1的RING和BRCT两功能区在DNA损伤反应中所起的作用。方法选择RING和BRCT功能区内与肿瘤发生相关的突变位点C64G和P1749R进行研究,应用BRCA1缺欠的HCC1937细胞系建立稳定表达C64G位点突变(C64G)、P1749R位点突变(P1749R)、野生型BRCA1(wt BRCA1)以及vector(空载体空白对照)4种细胞系,并用Western blotting法测定各细胞系BRCA1蛋白的表达。分别对4种细胞系进行电离辐射处理(一次性给予2、4、6 Gy的X射线),丝裂霉素C(mitomycin C,MMC)处理(0.2、1、5μg/ml孵育1 h),紫外线(ultraviolet,UV)处理(一次性给予2、4、6 J/m2)继续培养48 h,并设空白对照。通过3H-Td R掺入实验、MTT实验和细胞克隆形成实验研究细胞3H-Td R掺入率和细胞存活率的变化。结果与空白对照组比较,不同剂量电离辐射、MMC、UV处理后,4种细胞系的3H-Td R掺入率均下降,差异均有统计学意义(P<0.05);且随着暴露剂量的升高,4种细胞系的3HTd R掺入率均呈下降趋势。与wt BRCA1相比,相同剂量电离辐射、MMC、UV处理C64G、P1749R、vector细胞系的3H-Td R掺入率和细胞存活率均较低,差异均有统计学意义(P<0.05),但C64G、P1749R细胞系的3H-Td R掺入率和细胞存活率与vector细胞系的变化趋势基本一致。结论 BRCA1的RING和BRCT功能区的位点突变均可导致细胞对电离辐射、MMC和UV所致DNA损伤的敏感性升高,而且C64位点比P1749对各种损伤应答反应更为敏感。提示这两个功能区可能参与了不同的DNA损伤修复过程并起到关键的调控作用。

Objective To explore the effect of N-terminal RING or C-terminal BRCT domain in breast cancer susceptibility gene 1 （BRCA1） through comparing the sensitivity of breast cancer cells （HCC1937） carrying site mutations in RING or BRCT domain in response to different kinds of DNA damaging agents. Methods Both mutation sites associated with tumorgenesis, C64G in RING domain and P1749R in BRCT domain,were selected for this study. HCC1937 cell line with defective BRCA1 was used to create cell lines stably expressing C64G mutant BRCA1 （C64G）,P1749R mutant BRCA1 （P1749R）,wild-type BRCA1 （wtBRCA1）,or vector-control （vector）. And the expression of BRCAI protein in each cell line was measured with Western blot. Each cell lines was treated by ionizing radiation （IR,2,4,6 Gy at once）, mytomycin C （MMC,0.2,1,5μg/ml for 1 h） or ultraviolet light （UV, 2,4,6 J/m2 at once）, then the cells were further incubated for another 48 h, meanwhile the group of blank control was set up in the experiments. The cytotoxicity was separately detected by 3H-TdR incorporation,MTF and clonogenic assay. Results The percentage of 3H-TdR incorporation in all of four cell lines showed a significant decrease （P〈 0.05） compared with untreated group after the cells were treated with various doses of IR, MMC or UV, and the percentage of 3H-TdR incorporation decreased significantly with dose dependent manner. Importantly,under the same experimental conditions,the percentage of 3H-TdR incorporation and cell survival rate detected by MTF and clonogeinic assay in the cells expressing C64G,P1749R or vector were obviously decreased compared with the cells expressing wtBRCA1 after all of the treatments with IR,MMC or UV （P〈0.O5）,however,3H-TdR incorporation and cell survival rate in the cells expressing C64G or P1749R was similar to vector expressed cell line. Conclusion Both mutation sites of C64G in N-terminal RING and P1749R in C-terminal BRCT, especially C64G, can significantly increase cellular sensitivity to IR, MMC and UV,indicating that both functional domains in BRCA1 might involve in different processes of DNA damage and play key role in DNA repair.