Abstract 14: Role of Cdk1 and Bcl-2 proteins in mitotic cell death
Authors: Sakurikar Nandini , Taylor Stephen S. , Chambers Timothy C. 
Published: June 2012

Tumor cells exhibit variable responses to microtubule inhibitors (MTIs) and an understanding of the molecular basis of this variability and the determinants of resistance and sensitivity to these agents is critical for appropriate patient selection while minimizing associated toxicity. Our studies have focused on elucidation of the mechanism of action of MTIs including vinca alkaloids and taxanes. Using vinblastine-treated KB-3 cells, a HeLa subline, as a model system, we have shown previously that death in mitosis is associated with high levels of phosphorylation of anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-xL and Mcl-1) catalyzed by cyclin dependent kinase-1 (Cdk1)/cyclin B. In this study, we sought to determine whether these results reflected a more general trend, and conversely, whether escape from mitotic arrest (mitotic slippage) was associated with defective Cdk1 activation and/or reduced levels of phosphorylation of anti-apoptotic Bcl-2 proteins. For this purpose, we used DLD-1 and HT-29 colon carcinoma cells lines that stably express GFP-tagged histone H2B which enable cell fate to be studied by examination of nuclear morphology by fluorescent microscopy and time lapse imaging. In confirmation of previous findings, DLD-1 cells on treatment with paclitaxel exhibited extensive mitotic slippage after mitotic arrest and then either survived or died in subsequent interphase. In contrast, the vast majority of HT29 cells upon paclitaxel treatment died in mitosis. To determine if a relationship existed between these different fate profiles and the extent/duration of Cdk1-mediated Bcl-2 protein phosphorylation, cells were synchronized by double thymidine block, treated with paclitaxel, and extracts made at 4-h intervals over a 48-h time-course for analysis by immunoblotting. In DLD-1 cells, both Mcl-1 and Bcl-xL were transiently and partially phosphorylated and PARP cleavage was incomplete, consistent with the majority of the cells surviving through mitotic arrest. In contrast, in HT-29 cells, paclitaxel induced robust phosphorylation and subsequent degradation of Mcl-1 as well as extensive Bcl-xL phosphorylation. In addition, PARP was completely cleaved consistent with widespread mitotic cell death. Interestingly, PARP cleavage was observed to occur in HT-29 cells only under conditions where Bcl-xL was phosphorylated and Mcl-1 was degraded, indicating that mitotic death requires simultaneous functional elimination of both these anti-apoptotic Bcl-2 proteins. (Note that Bcl-2 expression was low to undetectable in both DLD-1 and HT-29 cells compared to KB-3 cells.) These results support the hypothesis that death in mitosis results from sustained Cdk1 activation and high levels of phosphorylation and subsequent inactivation of anti-apoptotic Bcl-2 proteins. Further, our findings suggest that the robustness of these signaling events may be useful as a marker to define tumor cell susceptibility to anti-mitotic drugs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 14. doi:10.1158/1538-7445.AM2011-14