Abstract LB-84: The role of survivin and survivin variants in the mitotic arrest-induced cell death

Abstract

Abstract Eukaryotic cells rely on a mitotic checkpoint in order to preserve their proper ploidy. This mechanism stops mitosis progression before anaphase until each kinetochore is adequately bound to mitotic microtubules, and tension is generated which splits up chromatids towards opposite poles of the mitotic spindle. The resulting paused state of the cell cycle is known as mitotic arrest, and has been long known to induce cell death. However, the mechanisms driving mitotic arrest-mediated cell death have not yet been elucidated. It has been hypothesized that an anti-apoptotic/pro-apoptotic protein interplay might define the mitotic arrest outcome. We are interested in defining the role of survivin and its splicing variants, survivin Δex3 and survivin 2b, in the mitotic arrest-induced cell death. Survivin is a multifaceted member of the IAP family of proteins with functions in the chromosome passenger complex that has been shown to be over expressed in nearly every human tumor type. Noteworthy, Survivin Δex3 retains IAP activity but Survivin 2b has been shown to act as a pro-apoptotic factor. In this study, we defined the mitotic index (MI) of HCT116 cells in response to different concentrations of taxol and vinblastine. The results show that vinblastine induces a wide MI oscillation in contrast to taxol, which produces a homogeneous behavior in the cell population. This difference in population behavior might be explained by the different mechanisms of action of taxol and vinblastine. From the maximum taxol-induced MI point on, the percentage of sub-2n cells increased at the expense of mitotic cells, as assessed by flow cytometry. These results suggest that, in our model, cells die by exposure to taxol in a mitotic arrest-dependent manner. We also determined, by RT-PCR, that survivin variants are endogenously expressed in HCT116 cells. Furthermore, in immunoblot experiments, we found that survivin protein level decreases throughout taxol treatment in contrast to Bub1 levels, a member of the spindle assembly checkpoint. These results suggest that the decreased survivin level might be related to mitotic arrest-dependent cell death. The analysis of the intracellular localization of survivin variants throughout the mitotic arrest showed that survivin is localized in mitotic chromosomes throughout mitosis (before anaphase) independently of the time of treatment with taxol and vinblastine. Noteworthy, we also found that survivin Δex3 localized to the cytosol, and its level decreases throughout mitosis while survivin 2b protein was diffusely localized in the cytosol and mitotic chromosomes, and its level and localization remained unchanged. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-84. doi:1538-7445.AM2012-LB-84