摘要
目的研究抑癌基因p53对丙戊酸(VPA)所致乳腺癌细胞放射增敏性的影响及其与同源重组(HR)修复机制的关系。方法用含PLKO.1或p53 shRNA慢病毒颗粒液感染乳腺癌MCF7细胞,建立p53表达下调的同源配对MCF7细胞系,即MCF7/野生型p53(MCF7/wtp53)和MCF7/缺欠型p53(MCF7/dp53)细胞;感染MCF7/(pDR-GFP)细胞,建立MCF7/pDR-GFP/wtp53和MCF7/pDR-GFP/dp53细胞;各分为细胞对照组、VPA组(VPA 0.5 mmol·L-1处理24 h)、电离辐射(IR,8 Gy)组和VPA+IR组(VPA 0.5 mmol·L-1预处理24 h后联用IR)。通过Western印迹实验检测P53蛋白表达水平,彗星和免疫荧光实验分别检测细胞核拖尾尾距及磷酸化组蛋白(γH2AX)焦点细胞比率,细胞克隆形成实验检测克隆形成率,流式细胞术检测HR修复效率,免疫荧光实验检测含乳腺癌易感基因1(BRCA1)蛋白和重组酶51(Rad51)蛋白焦点细胞比率。结果 Western印迹结果显示,MCF7/wtp53细胞中存在P53蛋白表达,MCF7/dp53细胞中P53蛋白表达显著降低(P<0.05)。彗星和免疫荧光结果显示,在MCF7/wtp53细胞中,与IR组相比,VPA+IR组细胞核拖尾尾距和含γH2AX焦点细胞百分率升高(P<0.05);在MCF7/dp53细胞中,与IR组相比,VPA+IR组细胞核拖尾尾距和含γH2AX焦点细胞百分率均升高(P<0.05),但仍低于MCF7/wtp53细胞VPA+IR组(P<0.05)。细胞克隆形成结果显示,在MCF7/wtp53细胞中,VPA+IR组细胞存活率低于IR组(P<0.05);在MCF7/dp53细胞中,VPA+IR组细胞存活率虽低于IR组(P<0.05),但高于MCF7/wtp53细胞VPA+IR组(P<0.05)。流式细胞术结果显示,在MCF7/pDR-GFP/wtp53细胞中,VPA组HR效率低于细胞对照组(P<0.05);在MCF7/pDR-GFP/dp53细胞中,VPA组HR效率低于细胞对照组(P<0.05),但仍高于MCF7/pDRGFP/wtp53细胞VPA组(P<0.05)。免疫荧光结果显示,在MCF7/wtp53细胞中,VPA+IR组含BRCA1和Rad51焦点细胞百分率均低于IR组(P<0.05);在MCF7/dp53细胞中,VPA+IR组含BRCA1和Rad51焦点细胞百分率均低于IR组(P<0.05),但仍高于MCF7/wtp53细胞VPA+IR组(P<0.05)。结论 VPA对乳腺癌MCF7细胞具有放射增敏性;抑制p53表达可降低VPA的放射增敏作用,与BRCA1-Rad51介导的HR机制过度增强有关。
OBJECTIVE To study the effect of tumor suppressor gene p53 on valproic acid(VPA)-caused radiosensitivity of breast cancer cells MCF7 and its mechanism of homologous recombination(HR) repair. METHODS By infecting breast cancer cells MCF7 with PLKO.1 and p53 shRNA lentiviral particle solution, the isogenic pairing MCF7 cells with down-regulated p53 expression, including MCF7/wild-type p53(MCF7/wtp53) and MCF7/defective p53(MCF7/dp53) cells, were established. By infecting MCF7/pDR-GFP cells, the MCF7/pDR-GFP/wtp53 and MCF7/pDR-GFP/dp53 cells were established.MCF7/wtp53 and MCF7/dp53 cells were divided into control group, VPA group(VPA 0.5 mmol·L-1 treatment for 24 h), ionizing radiation(IR, 8 Gy) group and VPA+IR group(VPA 0.5 mmol·L-1 pretreatment for 24 h combined with IR). The expression level of protein P53 was detected by Western blotting,while the nucleus tail length and percentage of cells containing phosphorylated histone(γH2 AX) focus formation were detected by comet assay and immunofluorescence assay. The clone formation rate was detected by cell clone formation assay. The HR repair efficiency was detected by flow cytometry,and the percentage of cells containing breast cancer susceptibility gene 1(BRCA1) and recombinase51(Rad51) focus formation was detected by immunofluorescence assay. RESULTS Western blotting results showed that the expression of P53 protein was observed in MCF7/wtp53 cells, however, it was decreased in MCF7/dp53 cells significantly(P<0.05). Comet assay and immunofluorescence assay results showed that in MCF7/wtp53 cells, the nucleus tail length and percentage of cells containing γH2 AX focus formation in the VPA+IR group increased compared with the IR group(P<0.05). In MCF7/dp53 cells,the nucleus tail length and percentage of cells containing γH2 AX focus formation in VPA+IR group increased compared with the IR group(P<0.05), but still lower than those of the VPA + IR group in MCF7/wtp53 cells(P<0.05). Cell clone formation assay showed that in MCF7/wtp53 cells, the cell viability of the VPA+IR group was lower than that of the IR group(P<0.05). In MCF7/dp53 cells, the cell viability of the VPA+IR group was lower than that of the IR group, but still higher than that of the VPA+IR group in MCF7/wtp53 cells(P<0.05). Flow cytometry results showed that in MCF7/pDR-GFP/wtp53 cells,compared with cell control group, the HR efficiency of VPA group decreased(P<0.05). In MCF7/pDRGFP/dp53 cells, the HR efficiency of the VPA group was lower than that of the cell control group, but higher than the VPA group in MCF7/pDR-GFP/wtp53 cells(P<0.05). Immunofluorescence assay results showed that in MCF7/wtp53 cells, the percentage of cells containing BRCA1 and Rad51 focus formation in the VPA+IR group was lower than in the IR group respectively(P<0.05). In MCF7/dp53 cells, the percentage of cells containing BRCA1 and Rad51 focus formation in VPA+IR group was lower than in the IR group respectively(P<0.05), but higher than that of VPA+IR group in MCF7/wtp53 cells(P<0.05). CONCLUSION VPA can enhance the sensitivity of breast cancer cells to IR and is capable of radio sensitization. The inhibition of p53 expression can down-regulate the radiosensitization of VPA, which is associated with the BRCA1-Rad51-mediated over-enhancement of the HR mechanism.
引文
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