Kolomeichuk Sergey N, Bene Anca, Upreti Meenakshi, Dennis Richard A, Lyle Christopher S, Rajasekaran Maheswari, Chambers Timothy C
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Mail Slot 516, 4301 W. Markham Street, Little Rock, AR 72205-7199, USA.
Mol Pharmacol. 2008 Jan;73(1):128-36. doi: 10.1124/mol.108.039750.
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.
在所检测的所有细胞系中，长春碱处理均会导致c-Jun蛋白表达和磷酸化显著增加，同时激活蛋白-1（AP-1）转录活性相应增强。我们在KB-3癌细胞中发现，这是由于一个强大的自放大环，该环涉及c-Jun启动子近端的AP-1位点，导致c-Jun mRNA和c-Jun蛋白高度增加。RNA转录抑制剂和蛋白质翻译抑制剂均可阻断长春碱诱导的c-Jun表达和凋亡性细胞死亡，这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，结果发现其非常有效，可使长春碱诱导的c-Jun在mRNA和蛋白水平的表达降低90%。在经c-Jun siRNA处理的细胞中，凋亡和caspase-3激活受到显著抑制。为了揭示c-Jun介导的细胞死亡机制以及c-Jun siRNA的保护作用，我们检测了候选靶基因。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）基因启动子优先结合。在p53功能受损的KB-3细胞以及p53基因缺失的细胞中，但在p53野生型细胞中未出现这种情况，长春碱导致p21表达下调，同时c-Jun表达增加，这表明c-Jun在独立于p53的情况下对p21启动子的负调控中发挥作用。这些结果提供了强有力的证据，即长春碱诱导的c-Jun部分通过下调p21发挥促凋亡作用，促进有丝分裂受损细胞的循环及随后的细胞死亡。
