Microtubule inhibiting agents including vinblastine have played a major role in cancer chemotherapy through their ability to block proliferation by inhibiting microtubule dynamics, inducing mitotic arrest and subsequent apoptosis. Understanding the regulation of mitosis arrest induced apoptosis is important for advancing fundamental knowledge and for developing more selective cancer chemotherapeutic drugs and novel drug targets. In this study, the role of Bcl-2 family proteins in apoptosis induced by microtubule inhibiting agents has been investigated in KB-3 cells, a HeLa subline. It was shown that pro-apoptotic Bak, which is mitochondrially localized, undergoes conformational activation and oligomerization in response to vinblastine treatment. Reciprocal immunoprecipitations showed that active Bak physically interacted with active Bax. Overexpression of Bcl-xL but not Bd-2 selectively blocked Bak activation, Bak oligomerization, Bak-Bax interaction, and apoptosis. Co-immunoprecipitation revealed that Bcl-xL interacted with Bax, but no evidence of interaction between Bcl-xL and Bak was found, suggesting that Bcl-xL overexpression may inhibit Bak activation indirectly by inhibiting Bax activation, or by inhibiting an upstream step. In related studies, it was found that the c-Jun NH2-terminal kinase (JNK) inhibitor, SP600125, inhibited Bax mitochondrial translocation and activation, and apoptosis, induced by vinblastine. These results suggested that JNK is a positive regulator of Bax mitochondrial translocation. Studies were also undertaken to examine Mcl-1, a key anti-apoptotic member of the Bd-2 family. Mcl-1 phosphorylation and degradation were observed after treatment of cells with microtubule inhibitors but not DNA damaging agents. Mcl-1 phosphorylation and subsequent proteosome-mediated degradation were inhibited by CDK inhibitors indicating vinblastine induces Mcl-1 phosphorylation and degradation through a CDK1-mediated mechanism. Site-directed mutagenesis of putative CDK phosphorylation sites indicated that alanine replacement of T70, T92, S121, or T163 did not influence Mcl-1 degradation. These results suggest other site(s) may be required for Mcl-1 degradation, or that redundancy exists among these sites. In summary, cell death in response to vinblastine and other microtubule inhibitors involves the complex integration of several signal transduction pathways which regulate the subcellular location, conformational activation, and interaction of key members of the Bcl-2 family of proteins.
|Advisor:||Chambers, Timothy C.|
|School:||University of Arkansas for Medical Sciences|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 71/06, Dissertation Abstracts International|
|Subjects:||Molecular biology, Cellular biology|
|Keywords:||Apoptosis, Bcl-2 proteins, Microtubule inhibitors, Mitotic arrest, Vinblastine|
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