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.

Transfection of cells with short double-stranded synthetic DNA molecules that contain a transcription factor binding site, known as decoy oligodeoxynucleotides (ODNs), has been proposed as a novel approach in vitro and in vivo for the study of gene regulation and for gene therapy. Once delivered into cells, decoy ODNs are predicted to bind to nuclear transcription factors, preventing their binding to consensus sequences in target genes. Using a fluorescein-labeled decoy ODN containing a consensus sequence for the AP-1 transcription factor, we show that lipid-complexed decoys were readily transfectable into cells, but were consistently detectable in the cytoplasm and not in the nucleus. The same phenomenon was observed in three different cell lines including KB-3, CHO and MDA-MB-231. The AP-1 decoy ODNs failed to inhibit the transcriptional activity of an AP-1-dependent luciferase reporter. The effect of cytoplasmic AP-1 decoy ODNs on the subcellular localization and function of c-Jun induced by the microtubule inhibitor vinblastine, which strongly induced c-Jun expression, was assessed. No difference in protein level or nuclear localization of vinblastine-induced c-Jun, or of one of its target genes, p53, was noted when cells were transfected with wild-type or mutated forms of the decoy ODNs. We suggest that subcellular localization is an unappreciated and key limiting factor for the use of transcription factor decoy ODNs that must be addressed before meaningful data interpretation can be made.

No abstract

We have shown previously that in KB-3 (HeLa) cells vinblastine causes downregulation of the CDK inhibitor p21 through a c-Jun regulated pathway. To test the hypothesis that p21 downregulation is necessary to alleviate a protective function, we transfected p21 in KB-3 cells and examined the apoptotic response to vinblastine. The results showed that cells overexpressing p21 were apoptosisresistant, not through an ability of p21 to cause cell cycle arrest prior to mitotic arrest, but through altering the fate of mitotically arrested cells after drug treatment. Moreover, p21 null HCT116 cells were more prone to vinblastine-induced apoptosis relative to wild-type cells. The results provide support for a model whereby p21 downregulation promotes vinblastine-induced apoptosis by alleviating its protective function following mitotic arrest.