腺病毒介导的p53转移对顺铂诱导KB细胞凋亡的影响

The effect of recombinant adenovirus-mediated p53 gene transfer on KB cell apoptosis induced by cisplatin

目的 探讨野生型 p5 3基因对顺铂诱导肿瘤细胞凋亡的影响。方法 用重组p5 3腺病毒 (Ad p5 3)和重组luc腺病毒 (Ad luc)分别感染 p5 3缺失的口腔癌细胞KB ,观察外源p5 3基因对顺铂诱导肿瘤细胞凋亡的影响。 结果 流式细胞术和原位末端标记显示用Ad luc与顺铂联合处理KB细胞不能诱导凋亡发生 ,用Ad p5 3与顺铂联合处理则诱导出较明显的细胞凋亡 ,表明p5 3可增强KB细胞对顺铂的敏感性 ;Northernblot显示 p5 3可下调bcl 2基因表达及上调bax基因的表达。结论 p5 3可能通过调节凋亡相关基因的表达水平而在顺铂诱导的肿瘤细胞凋亡中发挥重要作用 ,重组p5 3腺病毒与顺铂联合应用有助于克服 p5 3失活肿瘤细胞的耐药性。

AIM To explore the effect of wild type p53 gene transfer mediated via recombinant adenovirus vector on tumor cell apoptosis induced by anticancer agent cisplatin. METHOD Eukaryotic expression plasmid pAdCMV p53 or pAdCMV luc, together with pJM17 containing adenovirus genome DNA, was co transfected into human kidney cell 293 with liposome lipofectamine. Recombinant p53 adenovirus(Ad p53) and recombinant luc adenovirus(Ad luc) were generated and identified by dual PCR. Oral cancer cell KB firstly was infected by two recombinant adenoviruses and then treated with cisplatin. The effect of p53 gene on apoptosis induction, and expression level of bcl 2 and bax genes in Ad p53 infected tumor cells were observed. RESULTS E1 deficient and replication defective Ad p53 and Ad luc were generated by homologous recombination in virus packaging cell 293. Western blot indicated that wild type p53 gene was transferred and expressed in Ad p53 infected KB cells. Northern blot showed that bcl 2 mRNA expression level was markedly reduced and bax mRNA level was increased in Ad p53 infected cells, compared with Ad luc infected cells. Flow cytometry and in situ end labeling suggested that the cells treated with Ad luc and cisplatin did not undergo apoptosis; The cells treated with Ad p53 and cisplatin were induced a marked apoptosis. The number of cell death in the latter group was more than the former. CONCLUSION p53 gene plays a key role in the apoptosis of tumor cells induced by cisplatin by down regulating the expression level of bcl 2 gene and up regulating expression level of bax gene. The application of recombinant p53 adenovirus in combination with cisplatin might help to overcome the resistance to chemotherapeutic drug of p53 null tumor cells in clinical anticancer treatment.