Mitotic Phosphorylation of Bcl-2 during Normal Cell Cycle Progression and Taxol-induced Growth Arrest

Abstract

There is increasing evidence that prolonged mitotic arrest initiates apoptosis; however, little is known about the signaling pathways involved. Several studies have associated deregulated Cdc2 activity with apoptosis. Herein, we report that the anti-apoptotic protein, Bcl-2, undergoes cell cycle-dependent phosphorylation during mitosis when there is elevated Cdc2 activity. We found that paclitaxel (Taxol ® ) treatment of epithelial tumor cells induced a prolonged mitotic arrest, elevated levels of mitotic kinase activity, hyperphosphorylation of Bcl-2, and subsequent cell death. The Taxol-induced Bcl-2 phosphorylation was dose-dependent. Furthermore, phosphorylated Bcl-2 remained complexed with Bax in Taxol-treated cells undergoing apoptosis. Immunoprecipitation experiments revealed a Bcl-2-associated kinase capable of phosphorylating histone H1 in vitro. However, the kinase was likely not cyclin B1/Cdc2, since cyclin B1/Cdc2 was not detectable in Bcl-2 immunoprecipitates, nor was recombinant Bcl-2 phosphorylated in vitro by cyclin B1/Cdc2. The results of this study further define a link between mitotic kinase activation and the apoptotic machinery in the cell. However, the role, if any, of prolonged Bcl-2 phosphorylation in Taxol-mediated apoptosis awaits further definition of Bcl-2 mechanism of action. Taxol may increase cellular susceptibility to apoptosis by amplifying the normal downstream events associated with mitotic kinase activation.

Growth inhibition of astrocytoma cells by farnesyl transferase inhibitors is mediated by a combination of anti-proliferative, pro-apoptotic and anti-angiogenic effects

“…Although the ratio of Bax to Bcl-2 appears to help determine the ultimate fate of cells (Gross et al, 1998), it is also clear that Bcl-2 and Bax can function independently in regulating cell death (Knudson and Korsmeyer, 1997). Finally, further studies will be needed to determine the functional role of these proteins, as post-translational modifications such as phosphorylation are important in modulating the role of these proteins in apoptosis (Scatena et al, 1998).…”

Kinetic stabilization of microtubule dynamic instability by benomyl increases the nuclear transport of p53

“…Both the floating and attached cells were harvested with the help of a cell scrapper and collected by centrifugation. The total cell lysates were collected as described earlier [15,30,31]. The cell lysates containing polymeric and soluble tubulin (monomeric tubulin) were prepared as described earlier [32,33].…”

Analysis of cyclin B1 and CDK activity during apoptosis induced by camptothecin treatment

“…Cyclin E protein induction has been identified in IM-9, MOLT-4 and RPMI 8226 cell lines undergoing apoptosis (Mazumder et al, 2000), neuronally derived cells (Padmanabhan et al, 1999), as well as in HT29 cells treated with a novel acronycine derivative (Le´once et al, 2001). B-type cyclins associated with CDK1 have been identified in HL60 cells as they enter apoptosis after treatment with the topoisomerase I inhibitor camptothecin (CPT) (Shimizu et al, 1995) and in epithelial-derived cell lines (Scatena et al, 1998), a HeLa syncytia model (Castedo et al, 2002b) as well as neuronal cells (Shirvan et al, 1998) suggesting that this activity might be required for apoptosis in many different cell types (for review see Castedo et al (2002a)). In a similar manner, reports have also described that cyclin A protein levels and kinase activity increase during apoptosis (Meikrantz et al, 1994;Shi et al, 1996;Harvey et al, 1998;Hakem et al, 1999).…”