Induction of apoptosis by vinblastine via c-Jun autoamplification and p53-independent down-regulation of p21WAF1/CIP1
Induction of apoptosis by vinblastine via c-Jun autoamplification and p53-independent down-regulation of p21WAF1/CIP1
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DOI：
10.1124/mol.108.039750
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发表时间：
2008-01-01
影响因子：
3.6
通讯作者：
Chambers, Timothy C.
中科院分区：
文献类型：
--
作者：
Kolomeichuk, Sergey N.;Bene, Anca;Chambers, Timothy C.
长春碱治疗在所有细胞系检查导致c-Jun蛋白表达和磷酸化的强劲增长，并相应增加激活蛋白-1（AP-1）转录活性。我们在KB-3癌细胞中发现，这是由于c-Jun启动子中近端AP-1位点存在强的自身扩增环，导致c-Jun mRNA和c-Jun蛋白高度增加。RNA转录和蛋白质翻译抑制剂阻断长春碱诱导的c-Jun表达和凋亡性细胞死亡，表明凋亡至少部分依赖于转录/翻译。针对c-Jun的小干扰RNA（siRNA）被用于中断扩增循环，并且被发现是非常有效的，在mRNA和蛋白质水平上将长春碱诱导的c-Jun表达降低了90%。c-Jun siRNA处理的细胞中细胞凋亡和caspase-3活化显著抑制。为了揭示c-Jun介导的细胞死亡和c-Jun siRNA保护的潜在机制，检查了候选靶基因。染色质免疫沉淀显示长春碱处理后c-Jun与p21（细胞周期蛋白依赖性激酶抑制剂）基因启动子的优先关联。在p53功能受损的KB-3细胞中，以及在p53缺失细胞中，而不是在p53野生型细胞中，长春碱导致p21表达下调，同时c-Jun表达增加，表明c-Jun在p21启动子的负调控中的作用独立于p53。这些结果提供了强有力的证据表明，长春碱诱导c-Jun部分通过下调p21，促进有丝分裂受损细胞的循环和随后的细胞死亡而发挥促凋亡作用。
Vinblastine treatment in all cell lines examined causes a robust increase in c-Jun protein expression and phosphorylation and a corresponding increase in activator protein-1 (AP-1) transcriptional activity. We show in KB-3 carcinoma cells that this is due to a strong autoamplification loop involving the proximal AP-1 site in the c-Jun promoter, resulting in highly increased c-Jun mRNA and c-Jun protein. Inhibitors of RNA transcription and protein translation blocked both vinblastine-induced c-Jun expression and apoptotic cell death, suggesting that apoptosis is dependent, at least in part, on transcription/translation. Small interfering RNA ( siRNA) to c-Jun was used to interrupt the amplification cycle and was found to be highly effective, reducing vinblastine-induced c-Jun expression at both the mRNA and protein levels by 90%. Apoptosis and caspase-3 activation were significantly inhibited in c-Jun siRNA-treated cells. To uncover potential mechanisms of c-Jun-mediated cell death and protection by c-Jun siRNA, candidate target genes were examined. Chromatin immunoprecipitation revealed preferential association of c-Jun with the p21 (cyclin-dependent kinase inhibitor) gene promoter after vinblastine treatment. In KB-3 cells, which have compromised p53 function, and in p53-null cells but not in p53 wild-type cells, vinblastine caused down-regulation of p21 expression concomitant with increased c-Jun expression, suggesting a role for c-Jun in negative regulation of the p21 promoter independent of p53. These results provide strong evidence that c-Jun induction in response to vinblastine plays a proapoptotic role in part via down-regulation of p21, promoting cycling and subsequent cell death of mitotically impaired cells.