背景与目的:研究p63与p73的mRNA表达与BaP致人肺腺癌细胞(H1299)和人支气管上皮细胞(16HBE)DNA损伤的关系。材料与方法:分别用不同浓度BaP(8、16、32、64和128μmol/L)处理H1299和16HBE两种细胞,在4 h和12 h时,使用相应的生化检测试剂盒分别测定细胞裂解液中MDA的水平和SOD、GSH-Px的活性,用qRT-PCR方法测定处理后细胞的p53、p63、p73、mdm2和mdm4的mRNA水平;用Comet实验评价细胞DNA损伤程度。结果:16、32和64μmol/L BaP处理4 h时,两种细胞MDA水平显著性升高,SOD和GSH-Px活性显著性下降(P〈0.05)。用BaP处理H1299和16HBE细胞4 h和12 h时均观察到DNA损伤随浓度增加而加重,且呈剂量-效应关系(P〈0.01),mdm2、mdm4mRNA表达水平升高(P〈0.01)。不过仅在12 h时p53基因mRNA表达水平较对照组显著增加(P〈0.01)。在4 h和12 h时点,仅在H1299细胞的p63和p73mRNA表达增加(P〈0.05)。结论:在BaP致p53缺失的H1299细胞的DNA损伤中,BaP可能通过不依赖p53信号通路激活了p63和p73 mRNA的表达。
BACKGROUND AND AIM: The associations between the changes of p63 and p73 in mRNA levels and the BaP-induced DNA damage in H1299 cells and in 16HBE cells were investigated. MATERIALS AND METHODS: Both H1299 cells and 16HBE cells were treated with BaP at various concentrations (8, 16, 32, 64 and 128 μmol/L) for 4 h and 12 h.At the two time points, the levels of MDA,SOD and GSH-Px were measured using the test kits. The mRNA levels of p53,p63,p73,mdm2 and mdm4 genes were detected by RT-PCR assay. DNA damage in the cells were evaluated by Comet assay. RESULTS: At 4 h, enhanced MDA level was observed (P 〈 0.05) . However, levels of SOD and GSH-Px were significantly decreased(P 〈 0.05 for both) in both kinds of cells. After 16HBE and H1299 cells were treated for 4 h and 12 h, DNA damage and mRNA expression levels of mdm2 and mdm4 genes increased in a dose-dependent manner(P 〈 0.01 for all) . But enhanced mRNA expression level of p53 was observed only at 12 h(P 〈 0.01) In addition,significant up-regulation of p63 and p73 genes in mRNA levels at the two time points were observed in H1299 cells( P 〈 0.05) . CONCLUSION: The BaP-induced DNA damage may be associated with enhanced mRNA levels of p63 and p73 genes in the p53-null H1299 cells.