Abstract
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.
Footnotes
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This work was supported by National Institutes of Health grant CA-5577 (to T.C.C.) and in part through Act 1 of the Arkansas Tobacco Settlement Proceeds Act of 2000 and National Center for Research Resources grants through the Biomedical Research Infrastructure Network Program (P20-RR16460).
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S.N.K. and A.B. contributed equally to this work.
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ABBREVIATIONS: JNK, c-Jun NH2-terminal protein kinase; AP-1, activating protein 1; DMSO, dimethyl sulfoxide; RT, reverse transcription; siRNA, small interfering RNA; PCR, polymerase chain reaction; VBL, vinblastine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]1-propane-sulfonate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ChIP, chromatin immunoprecipitation; ELISA, enzyme-linked immunosorbent assay; AMC, amino-4-methyl coumarin; SP600125, anthra(1,9-cd)pyrazol-6(2H)-one 1,9-pyrazoloanthrone.
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
- Received July 9, 2007.
- Accepted October 3, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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