Bcl-2 phosphorylation and proteasome-dependent degradation induced by paclitaxel treatment: consequences on sensitivity of isolated mitochondria to Bid.

Several studies have suggested that Bcl-2 phosphorylation, which occurs during mitotic arrest induced by paclitaxel, inhibits its antiapoptotic function. In the present study, we demonstrated that the level of phosphorylated Bcl-2 was threefold higher in mitochondria than in the nuclear membrane or endoplasmic reticulum. Our results show, in isolated mitochondria, that phosphorylation of Bcl-2 in mitosis does not modify either its integration into the mitochondrial membrane or the ability to release cytochrome c in response to Bid, a cytochrome c releasing agent. In HeLa cells, in which paclitaxel induces apoptosis, the nonphosphorylated form of Bcl-2 is degraded by a proteasome-dependent degradation pathway, whereas the phosphorylated forms of mitochondrial Bcl-2 appear to be resistant to proteasome-induced degradation. We found that low concentrations of recombinant Bid triggered a greater release of cytochrome c from mitochondria isolated from paclitaxel-treated HeLa cells than from mitochondria isolated from control HeLa cells. Taken together, these results show that Bcl-2 phosphorylation does not inhibit its function. On the contrary, Bcl-2 phosphorylation indirectly regulated its antiapoptotic action via protection against degradation. Indeed, in response to paclitaxel treatment, the level of Bcl-2 expression in mitochondria rather than its phosphorylation state could regulate the sensitivity of mitochondria to cytochrome c releasing agents in vitro.

Up-regulation of cdc2 protein during paclitaxel-induced apoptosis.

Microtubule damages induced by paclitaxel inhibit proteasome-dependent degradation of cyclin B, resulting in a sustained activation of cyclin B/cdc2 kinase and a cell cycle arrest in mitosis. It has been previously shown that this kinase activity is also required for paclitaxel-induced apoptosis. We found here that paclitaxel increased cdc2 mRNA and protein levels and led to an accumulation of cdc2 in the active dephosphorylated form in NIH-OVCAR-3 cells. The addition of cycloheximide inhibited the paclitaxel-induced increase in cdc2 protein level, further indicating that paclitaxel stimulates cdc2 synthesis. This increase in cdc2 synthesis is a consequence of paclitaxel-induced arrest in mitosis. Indeed, dual analysis of DNA and cdc2 protein contents indicated that cdc2 up-regulation occurred in cells arrested with a G2/M DNA content. Furthermore, no up-regulation of cdc2 protein was observed when paclitaxel-treated cells were prevented from entering mitosis by treatment with purvalanol A, a cyclin-dependent kinase (CDK) inhibitor, or stimulated to exit mitosis with 2-AP, a non-specific kinase inhibitor. In addition, when paclitaxel-induced apoptosis was inhibited by Bcl-2 over-expression, cdc2 up-regulation did not occur, leading to a lower level of activation of the cyclin B/cdc2 complex. Taken together, these results indicated that paclitaxel-induced cdc2 protein synthesis participates in a positive feedback loop designed to increase the activity of cyclin B/cdc2 kinase and thus may play a role in paclitaxel-induced apoptosis.

Phosphorylation and proteasome-dependent degradation of Bcl-2 in mitotic-arrested cells after microtubule damage.

Treatment of NIH-OVCAR-3 cells with paclitaxel, a microtubule-stabilizing agent, induces mitotic arrest and apoptosis, but also Bcl-2 phosphorylation. We report here that Bcl-2 phosphorylation precedes Bcl-2 down-regulation and that both events are closely associated with mitotic arrest, but are not sufficient for paclitaxel to trigger apoptosis. Indeed, when paclitaxel-treated cells were induced to exit mitosis in the presence of 2-aminopurine, Bcl-2 phosphorylation and Bcl-2 down-regulation were both inhibited. In contrast, when apoptosis was inhibited by a caspase inhibitor or Bcl-2 over-expression, Bcl-2 phosphorylation and down-regulation still occurred. Furthermore, we show that Bcl-2 is degraded in mitosis by the proteasome-dependent pathway since Bcl-2 down-regulation is inhibited by proteasome inhibitors such as MG132, Lactacystin and LLnL. Taken together these results indicate that mitotic spindle damage results in post-translational modifications of Bcl-2 by phosphorylation and degradation.

Vinflunine, a new vinca alkaloid: cytotoxicity, cellular accumulation and action on the interphasic and mitotic microtubule cytoskeleton of PtK2 cells.

Vinflunine, a newly synthesized derivative, possesses marked in vivo antitumor properties and, like other alkaloids, inhibits in vitro tubulin assembly at microM concentrations. However, in contrast to other vinca alkaloids, vinflunine exhibits relatively low in vitro cytotoxic potency. The aim of this report was to investigate whether the action(s) of vinflunine on the microtubule cytoskeleton could account for its cytotoxicity or if its cellular action requires another molecular target. Four vinca alkaloids used in cancer therapy and vinflunine were studied using PtK2 cells. Their activities on the most dynamic microtubules were investigated in mitosis and in interphase by evaluating the disturbance of the metaphase plate and the splitting of the diplosome, respectively. No correlation was observed between the cellular accumulation of these compounds and either their cytotoxicity or their action(s) on the microtubule cytoskeleton. In contrast, cytotoxicity, mitotic disturbance and diplosome splitting were observed in the nM range for vinblastine, vincristine, vindesine and vinorelbine, although these events occurred at 10 times higher concentrations in the case of vinflunine. Hence, dynamic modifications of both the mitotic and interphasic microtubule cytoskeleton are compatible with in vitro cytotoxicity of vinflunine, raising questions about the conventional biochemical screening of these vinca alkaloids.