Phosphorylation of Bcl-2 is a marker of M phase events and not a determinant of apoptosis.
Phosphorylation of Bcl-2 protein is a post-translational modification of unclear functional consequences. We studied the correlation between Bcl-2 phosphorylation, mitotic arrest, and apoptosis induced by the anti-tubulin agent paclitaxel. Continuous exposure of human cervical carcinoma HeLa cells to 50 ng/ml paclitaxel resulted in mitotic arrest with a symmetrical bell-shaped curve over time. The number of mitotic cells was highest at 24 h (82%), then declined as arrested cells progressed into apoptosis, and barely no mitotic cells were present at 48-60 h. The time curves of paclitaxel-induced cyclin B1 accumulation and stimulation of Cdc2/cyclin B1 kinase activity were identical and superimposable to that of M phase arrest. In contrast, apoptosis was first detected at 12 h and steadily increased thereafter until the termination of the experiments at 48-60 h, when about 80-96% of cells were apoptotic. Bcl-2 phosphorylation was closely associated in time with M phase arrest, accumulation of cyclin B1, and activation of Cdc2/cyclin B1 kinase, but not with apoptosis. At 24 h, when about 82% of the cells were in mitosis, almost all Bcl-2 protein was phosphorylated, whereas at 48 h, when 70-90% of the cells were apoptotic, all Bcl-2 protein was unphosphorylated. Similar results were obtained with SKOV3 cells, indicating that the association of paclitaxel-induced M phase arrest and Bcl-2 phosphorylation is not restricted to HeLa cells. We used short exposure to nocodazole and double thymidine to synchronize HeLa cells and investigate the association of Bcl-2 phosphorylation with mitosis. These studies demonstrated that Bcl-2 phosphorylation occurs in tight association with the number of mitotic cells in experimental conditions that do not lead to apoptosis. However, a continuous exposure to nocodazole resulted in a pattern of Bcl-2 phosphorylation, M phase arrest, and apoptosis similar to that observed with paclitaxel. The phosphatase inhibitor okadaic acid was found to inhibit the dephosphorylation of phosphorylated Bcl-2 and to delay the progression of nocodazole M phase-arrested cells into interphase. In contrast, the serine/threonine kinase inhibitor staurosporine, but not the tyrosine kinase inhibitor genistein, led to rapid dephosphorylation of phosphorylated Bcl-2 and accelerated the progression of nocodazole M phase-arrested cells into interphase. Immune complex kinase assays in cell-free systems demonstrated that Bcl-2 protein can be a substrate of Cdc2/cyclin B1 kinase isolated from paclitaxel-treated cells arrested in M phase. Taken together, these studies suggest that Bcl-2 phosphorylation is tightly associated with mitotic arrest and fail to demonstrate that it is a determinant of progression into apoptosis after mitotic arrest induced by anti-tubulin agents.
Department of Thoracic/Head and Neck Medical Oncology, Section of Experimental Therapy, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, 77030, USA.
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Ling
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Tornos
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Perez-Soler
D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D017489:CDC28 Protein Kinase, S cerevisiae / Q000378:metabolism; D019926:Cyclin B / Q000378:metabolism; D056744:Cyclin B1; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D019833:Genistein / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis; D015739:Nocodazole / Q000494:pharmacology; D019319:Okadaic Acid / Q000494:pharmacology; D017239:Paclitaxel / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; C534699:CCNB1 protein, human; D019926:Cyclin B; D056744:Cyclin B1; D004791:Enzyme Inhibitors; D047428:Protein Kinase Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D019319:Okadaic Acid; D019833:Genistein; D017489:CDC28 Protein Kinase, S cerevisiae; D010744:Phosphoric Monoester Hydrolases; D019311:Staurosporine; D017239:Paclitaxel; D015739:Nocodazole