Received for publication July 10, 2007.
Revised October 3, 2007.
Accepted for publication October 3, 2007.

Induction of apoptosis by vinblastine via c-Jun auto-amplification and p53-independent downregulation of p21^WAF1/CIP1

Abstract

Vinblastine treatment in all cell lines examined causes a robust increase in c-Jun protein expression and phosphorylation, and a corresponding increase in AP-1 transcriptional activity. We show in KB-3 carcinoma cells that this is due to a strong auto-amplification 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. 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 downregulation 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 pro-apoptotic role in part via downregulation of p21, promoting cycling and subsequent cell death of mitotically impaired cells.

Key words: Jun Kinase, AP-1, Mechanisms of cell killing/apoptosis