Microtubule inhibitors including vinblastine inhibit microtubule dynamics and induce mitotic arrest and apoptosis. The long-term goal is to understand the mechanisms of cell death in response to mitotic arrest, to provide insight into the fundamental processes involved, and to aid in the development of more specific therapeutic strategies for cancer. Previous studies in our laboratory showed that a key cellular response to vinblastine is the activation of the c-Jun NH2-terminal kinase (JNK) pathway, which results in induction, phosphorylation and activation of c-Jun, a member of the AP-1 transcription factor family. In order to determine the role of the JNK/AP-1/c-Jun pathway in response to vinblastine, the focus of this dissertation was to investigate means to specifically block c-Jun induction/function. Two methods were investigated and developed: AP-1 decoy oligonucleotides (ODNs), which are predicted to sequester AP-1 complexes in cells and prevent their binding to genomic AP-1 consensus elements; and siRNA against c-Jun mRNA, to reduce or eliminate c-Jun induction. While AP-1 decoy ODNs were readily transfectable into KB-3 carcinoma and other cell lines, they failed to localize to the nucleus, failed to block nuclear localization of c-Jun, and were ineffective at blocking AP-1-dependent transactivation. These results suggested that nuclear exclusion is an underappreciated limiting factor in the use of decoy ODNs. As a second approach, siRNA against c-Jun was successfully used to block vinblastine-induced expression of c-Jun mRNA and protein. Vinblastine-induced apoptosis was significantly decreased in the siRNA transfected cells, establishing a pro-apoptotic role for AP-1/c-Jun in this system. By using chromatin immunoprecipitation, a member of the cyclin-dependent kinase (CDK) inhibitor family, p21WAF1/Cip1 , was identified as an authentic c-Jun target in this system. Vinblastine-induced c-Jun upregulation occurred concomitantly with p21 downregulation, suggesting that c-Jun negatively regulates the p21 promoter. Further, overexpression of p21 protected cells from vinblastine-induced apoptosis. Collectively, these data provide support for a model whereby c-Jun acts, at least in part, to cause p21 downregulation, which alleviate p21's protective function, allowing emergence from mitotic arrest and subsequent apoptosis. Overall, the results provide novel insight into the molecular mechanisms of action of microtubule inhibitors.
|School:||University of Arkansas for Medical Sciences|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 69/12, Dissertation Abstracts International|
|Subjects:||Molecular biology, Pharmacy sciences|
|Keywords:||AP-1, Apoptosis, Decoys, Microtubule inhibitors, Vinblastine, c-Jun, p21|
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