论文部分内容阅读
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 compare the sensitivity of breast cancer cell lines (HCC1937) with mutations in the RING or BRCT functional regions carrying breast cancer susceptibility gene 1 (BRCA1) to DNA damage in different origins and to explore the relationship between BRCA1 RING and BRCT2 Role of functional domains in DNA damage response. Methods The tumor-associated mutation sites C64G and P1749R in RING and BRCT functional regions were selected for study. The stable C64G, P1749R, P1749R and wild type HCC1937 cell lines were established by using BRCA1-deficient HCC1937 cell line. BRCA1 (wt BRCA1) and vector (empty vector control). The expression of BRCA1 protein in each cell line was determined by Western blotting. Four kinds of cell lines were treated with ionizing radiation (2,4,6 Gy x-ray once), mitomycin C (MMC) (0.2,1,5 μg / ml for 1 h) UV (UV, UV treatment (2,4,6 J / m2 given one-time) to continue training for 48 h, and set a blank control. 3H-TdR incorporation assay, MTT assay and cell clone formation assay were used to investigate the 3H-TdR incorporation and cell viability. Results Compared with the blank control group, the 3H-TdR incorporation rates of the four cell lines decreased after different doses of ionizing radiation, MMC and UV treatment, the difference was statistically significant (P <0.05); with the exposure dose The 3HTd R incorporation rate of all four cell lines showed a decreasing trend. Compared with wt BRCA1, 3H-Td incorporation rate and cell survival rate of MMC, UV-treated C64G, P1749R and vector cell lines were all lower than that of wt BRCA1 (P <0.05) However, 3H-TdR incorporation rate and cell viability of C64G and P1749R cell lines were basically the same as that of vector cell line. CONCLUSIONS: Mutations in RING and BRCT functional domains of BRCA1 can cause cell sensitivity to ionizing radiation, DNA damage caused by MMC and UV, and C64 locus is more sensitive than P1749 to various injury response responses. Suggesting that these two functional regions may participate in different DNA damage repair process and play a key regulatory role.