p73在苯并(a)芘致人肺纤维细胞株MRC-5和人肺腺癌细胞株H1299(p53-null)周期阻滞中的作用

p73-mediated Cell Cycle Arrest in MRC-5 Cells and H1299 Cells Treated with B(a)P

目的研究p73在苯并(a)芘[B(a)P]致人肺纤维细胞株MRC-5和人肺腺癌细胞株H1299(p53-null)周期阻滞中的作用。方法采用0(溶剂对照)、8、16、32、64和128μmol/L的B(a)P分别处理处于对数生长期的MRC-5和H1299细胞24h。采用MTT比色法检测两种细胞系的生长率,采用流式细胞术检测细胞周期各时相的细胞百分比,采用实时荧光定量PCR方法测定p53、p73、p21和Gadd45的mRNA表达水平。结果与溶剂对照组比较,8、16、32、64μmol/LB(a)P染毒组MRC-5细胞和H1299细胞的增殖活性均升高,差异有统计学意义(P<0.05或P<0.01)。16μmol/LB(a)P染毒组MRC-5细胞和32μmol/LB(a)P染毒组H1299细胞的增殖活性达到峰值,与溶剂对照组相比,分别增加了40%和30%。与溶剂对照组比较,8、16、32、64和128μmol/L的B(a)P染毒组MRC-5细胞的p53、p73和p21基因mRNA表达水平上升,差异均有统计学意义(P<0.05或P<0.01),细胞阻滞在G1期。与溶剂对照组比较,8μmol/L的B(a)P染毒组H1299细胞p73和p21基因mRNA表达的表达水平上升,差异均有统计学意义(P<0.05),细胞阻滞在G1期;但各处理组Gadd45基因的mRNA表达水平间比较,差异无统计学意义(P>0.05)。结论一定浓度B(a)P可通过p53介导引起MRC-5细胞周期G1期阻滞,而对于p53缺失的H1299细胞,其细胞周期G1期阻滞可能依赖于p73参与的细胞信号通路的调节。

Objective To understand the effect of p73 on the B（a）P-induced cell cycle arrest in MRC-5 and H1299 cells. Methods Both MRC-5 cells and H 1299 cells in the period of logarithmic growth were treated with B（a）P at the concentrations of 0 μmol/L （the solvent control）, 8,16,32,64 and 128 μmol/L for 24 h. Cell proliferations were detected with the MTr assay. In MRC- 5 cells and H1299 cells, the percentages of cells in the G1, S and G2 phases of the cell cycle were analyzed using the flow cytometry. Expressions ofp53,p73,p21 and Gadd45 genes at mRNA levels were detected by real-time quantitative PCR assay. Results The cell proliferation of MRC-5 cells and H1299 cells were increased at the concentrations of 8, 16,32 and 64 μmol/L （P〈0.05 or P〈0.01）. The proliferation reached to the highest point at the concentration of 16 μmol/L in MRC-5 cells and 32 μmol/L in H1299 cells. It was enhanced by fourty and thirty percent respectively compared with the solvent control group. The increased mRNA levels of p53 ,p73 and p21 genes were determined in the MRC-5 cells treated with B （a）P at the concentrations of 8,16,32,64 and 128 μmol/L （P〈0.05 or P〈0.01）,and cells were arrested in G1 phases in all treatment groups. After H1299 cells were treated with 8 μmol/L B（a）P for 24 h, enhanced mRNA levels of p21 and p73 were observed （P〈0.05）, and cells were also arrested in GI phase. However,for Gadd45 gene no significant differences at mRNA levels were detected in all treatment groups （P〉0.05）. Conclusion When the two kinds of cells are treated with B （a）P at the certain concentrations for 24 h, arrested MRC-5 cells in G1 phase of the cell cycle may be mediated by p53, whereas arrested H1299 cells （053 null） in G1 phase of the cell cycle may be associated with modulations ofp73-mediated signal pathways.