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Induction of Apoptosis by Vinblastine via c-Jun Autoamplification and p53-Independent Down-Regulation of p21^WAF1/CIP1

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas (S.N.K., A.B., M.U., C.S.L., M.R., T.C.C.); and Geriatric Research, Education, and Clinical Center, Central Arkansas Veterans Healthcare System, North Little Rock, Arkansas (R.A.D.)

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.

Address correspondence to: Dr. Timothy Chambers, Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Mail Slot 516, 4301 W. Markham Street, Little Rock, AR 72205-7199. E-mail: chamberstimothyc{at}uams.edu