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乳腺癌细胞系HCC1937和MCF7在DNA损伤修复反应时功能的比较
作者:董超 张凤梅 赵锡鹏 郭公社 罗月 凤志慧

摘要:

目的  通过比较DNA损伤修复基因乳腺癌易感基因1(breast cancer susceptibility gene 1,BRCA1)和抑癌基因p53在HCC1937和MCF7两种乳腺癌细胞系中的功能状态及对DNA损伤修复反应的应答特性,研究两种细胞系在功能上的差异。方法  应用western-blotting法检测两种乳腺癌细胞系中BRCA1和p53蛋白的表达,并检测了经过10 Gy电离辐射下1、4、8 h后BRCA1蛋白在MCF7、HCC1937和HCC1937野生型BRCA1(HCC1937 wtBRCA1)细胞系中的表达水平。同时用免疫荧光法观察BRCA1蛋白在MCF7和HCC1937细胞系细胞内的分布和焦点形成情况,并对电离辐射下的BRCA1、Rad51蛋白的焦点形成百分比进行计算。进一步用流式细胞仪检测两种细胞系的细胞周期变化。结果  MCF7细胞中野生型的BRCA1和p53主要分布于细胞核内,这两种蛋白对DNA损伤反应有应答。10 Gy 8 h照射条件下,MCF7细胞系中BRCA1蛋白焦点形成百分比较无电离辐射高[(59.40±3.66)%比(11.80±3.51)%,t=16.26,P<0.05];MCF7细胞系照射组中Rad51蛋白焦点形成百分比较无电离辐射高[(73.90±8.66)%比(16.70±3.76)%,t=10.49,P<0.05],照射组p53蛋白[(82.54±1.04)比(23.75±0.51),t=87.90,P<0.05]和p21蛋白[(90.95±1.13)比(50.19±0.89),t= 49.11,P<0.05]表达水平均较无电离辐射高,细胞在G1期蓄积。与MCF7细胞相比,在HCC1937细胞中的BRCA1和p53都产生了变异,在细胞核内两种蛋白较少。10 Gy 8 h照射条件下HCC1937细胞中无BRCA1蛋白焦点形成、p53和p21几乎无诱导表达以及细胞在G1和G2-M期无明显的蓄积。当恢复野生型BRCA1在HCC1937细胞内表达后,10 Gy 8 h照射条件下Rad51蛋白焦点形成百分比较无电离辐射高[(61.70±4.03)%比(6.22±2.27)%,t=20.78,P<0.05],细胞在G2-M期的蓄积增多。结论  乳腺癌细胞系HCC1937和MCF7在应答DNA损伤修复反应时具有不同的功能特性。

关键词:乳腺肿瘤;BRCA1蛋白质;DNA损伤;HCC1937;MCF7

Abstract:

Objective  The functional characters of MCF7 and HCC1937 cell lines were compared through the activity of BRCA1 and p53 following DNA damage in order to provide more research evidence for the related studies in both breast cancer cell lines.Methods  The protein level of BRCA1 and p53 in two breast cancer cell lines and the protein level of BRCA1 in MCF7, HCC1937 and HCC1937 wtBRCA1 breast cancer cell lines treated with 10Gy after 1 h, 4 h or 8 h were detected by western blotting analysis. The distribution and foci formation of BRCA1 in the cells were observed through immunostaining assay and the percentage of BRCA1 or Rad51 foci formation after ionizing radiation was calculated. Cell cycle profiling was analyzed using flow cytometry.Results  Most of BRCA1 and p53 localized in nucleus, and both proteins responded to DNA damage in MCF7 cells. In MCF7 cells, BRCA1 and Rad51 foci formation respectively increased to (59.40±3.66)% from (11.80±3.51)% (t=16.26, P<0.05) and (73.90±8.66)% from (16.70±3.76)% (t=10.49, P<0.05) after 10 Gy 8 h ; p53 and p21 protein level was further separately induced and enhanced to (82.54±1.04) from (23.75±0.51) (t=87.90, P<0.05) and (90.95±1.13) from (50.19±0.89) ( t=49.11, P<0.05) after 10 Gy 8 h; and the cells were accumulated in G1 phase. In contrast to MCF7, in HCC1937 cell line, both of BRCA1 and p53 were defective in nucleus since both proteins were mutated; in response to DNA damage, BRCA1 foci formation was not found, p53 and p21 was not induced; there was no cell accumulation in both of G1-S and G2-M phases. However, after complementation of wild-type BRCA1 in HCC1937 cells, DNA damage-induced Rad51 foci formation increased to (61.70±4.03)% from (6.22±2.27)% (t=20.78, P<0.05) and accumulation of cells in G2-M phase was also restored after 10 Gy 8h, which was similar to that of in MCF7 cells.Conclusions  We have identified that BRCA1 and p53 have dramatically different functions in MCF7 and HCC1937 cell lines in response to DNA damage.

Key words: Breast neoplasms;BRCA1 protein;DNA damage;HCC1937;MCF7

发表日期:2014/9

引用本文:

图/表:

  • 10.3760/cma.j.issn.0253-9624.2014.09.013.T001:表1 BRCA1蛋白在MCF7和HCC1937细胞系中表达水平(±s,n=3)

    10.3760/cma.j.issn.0253-9624.2014.09.013.T001:表1 BRCA1蛋白在MCF7和HCC1937细胞系中表达水平(±s,n=3)

  • 10.3760/cma.j.issn.0253-9624.2014.09.013.F001:图1 免疫荧光染色观察BRCA1在MCF7和HCC1937细胞中的分布

    10.3760/cma.j.issn.0253-9624.2014.09.013.F001:图1 免疫荧光染色观察BRCA1在MCF7和HCC1937细胞中的分布

  • 10.3760/cma.j.issn.0253-9624.2014.09.013.T002:表2 不同照射时间下BRCA1蛋白在MCF7细胞中表达水平(±s,n=3)

    10.3760/cma.j.issn.0253-9624.2014.09.013.T002:表2 不同照射时间下BRCA1蛋白在MCF7细胞中表达水平(±s,n=3)

  • 10.3760/cma.j.issn.0253-9624.2014.09.013.T003:表3 BRCA1蛋白在HCC1937及HCC1937 wtBRCA1细胞中表达水平(±s, n=3)

    10.3760/cma.j.issn.0253-9624.2014.09.013.T003:表3 BRCA1蛋白在HCC1937及HCC1937 wtBRCA1细胞中表达水平(±s, n=3)

  • 10.3760/cma.j.issn.0253-9624.2014.09.013.T004:表4 电离辐射诱导的BRCA1、Rad51蛋白焦点形成百分比(%,±s,n=3)

    10.3760/cma.j.issn.0253-9624.2014.09.013.T004:表4 电离辐射诱导的BRCA1、Rad51蛋白焦点形成百分比(%,±s,n=3)

  • 10.3760/cma.j.issn.0253-9624.2014.09.013.F002:图2 p53及mp53的功能域示意图

    10.3760/cma.j.issn.0253-9624.2014.09.013.F002:图2 p53及mp53的功能域示意图

  • 10.3760/cma.j.issn.0253-9624.2014.09.013.T005:表5 p53蛋白在MCF7和HCC1937细胞中表达水平(±s,n=3)

    10.3760/cma.j.issn.0253-9624.2014.09.013.T005:表5 p53蛋白在MCF7和HCC1937细胞中表达水平(±s,n=3)

  • 10.3760/cma.j.issn.0253-9624.2014.09.013.F003:图3 免疫荧光染色分析p53在MCF7和HCC1937细胞内的分布

    10.3760/cma.j.issn.0253-9624.2014.09.013.F003:图3 免疫荧光染色分析p53在MCF7和HCC1937细胞内的分布

  • 10.3760/cma.j.issn.0253-9624.2014.09.013.F004:图4 流式细胞术检测MCF7和HCC1937的细胞周期

    10.3760/cma.j.issn.0253-9624.2014.09.013.F004:图4 流式细胞术检测MCF7和HCC1937的细胞周期

  • 10.3760/cma.j.issn.0253-9624.2014.09.013.T006:表6 MCF7、HCC1937及HCC1937 wtBRCA1细胞系中与BRCA1和p53相关的功能状态比较

    10.3760/cma.j.issn.0253-9624.2014.09.013.T006:表6 MCF7、HCC1937及HCC1937 wtBRCA1细胞系中与BRCA1和p53相关的功能状态比较

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