Bcl-2-mediated potentiation of neocarzinostatin-induced apoptosis: requirement for caspase-3, sulfhydryl groups, and cleavable Bcl-2.
ABSTRACT Overexpression of antiapoptotic Bcl-2 family members is thought to contribute to chemotherapeutic resistance of neural crest tumors. Paradoxical potentiation by Bcl-2 of apoptosis induced by the antineoplastic prodrug, neocarzinostatin (NCS), has been observed in PC12 pheochromocytoma cells. Prior studies have indicated that the cleavage of Bcl-2 to its proapoptotic counterpart mediated by caspase-3 is responsible for this potentiation of apoptosis. This has led to the hypothesis that induction of caspase-3 expression in bcl-2-transfected, caspase-3-deficient MCF-7 cells, will result in Bcl-2 cleavage and Bcl-2-dependent potentiation of NCS-induced apoptosis. These studies have further led to the hypothesis that both cleavable Bcl-2 and sulfhydryl groups are required for the activity of caspase-3 in this regard. As hypothesized, co-transfection of bcl-2-transfected MCF-7 cells with a caspase-3 expression construct results in cleavage of Bcl-2 and potentiation of dose-dependent, NCS-mediated cell death. Furthermore, PC12 cells transfected with an expression construct for cleavage-resistant Bcl-2 demonstrated attenuated potentiation of apoptosis relative to their counterparts transfected with wild-type bcl-2. Finally, irreversible oxidative titration of sulfhydryl groups resulted in concentration-dependent attenuation of apoptosis in PC12 cells, along with prevention of caspase-3 activation and Bcl-2 cleavage. These results definitively demonstrate the requirement for caspase-3, cleavable Bcl-2, and available sulfhydryl groups (separate from those required for NCS activation) in potentiation of NCS-induced apoptosis by Bcl-2.
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ABSTRACT: Bcl-2 has been hypothesized to regulate many cellular functions in addition to its well-characterized role in the prevention of programmed cell death. To understand the role of Bcl-2 in regulating cell morphology and to explore the mechanism of this effect, we examined the effects of Bcl-2 overexpression on the morphology of PC12 cells in culture. We demonstrate that the overexpression of Bcl-2 in PC12 cells results in altered cell morphology and reduced actin expression. Analysis of extracellular signal-regulated kinase (ERK) 1/2 phosphorylation reveals that the morphological changes seen after bcl-2 transfection are associated with reduced ERK activation. Treatment of control (mock-transfected) PC12 cells with the mitogen-activated ERK-activating kinase (MEK) inhibitor PD98059 converts their flat, process-bearing morphology into the rounded, process-free morphology of bcl-2-transfected cells, further confirming the association of ERK activation with altered cell shape. In conclusion, the present study describes a novel function of Bcl-2 in regulating cell shape through reduced ERK activation.Brain Research 10/2006; 1112(1):46-55. DOI:10.1016/j.brainres.2006.07.017 · 2.84 Impact Factor
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ABSTRACT: To identify and mathematically model molecular predictors of response to the enediyne chemotherapeutic agent, neocarzinostatin, in nervous system cancer cell lines. Human neuroblastoma, breast cancer, glioma, and medulloblastoma cell lines were maintained in culture. Content of caspase-3 and Bcl-2, respectively, was determined relative to actin content for each cell line by Western blotting and optical densitometry. For each cell line, sensitivity to neocarzinostatin was determined. Brain tumor cell lines were stably transfected with human Bcl-2 cDNA cloned into the pcDNA3 plasmid vector. In human tumor cell lines of different tissue origins, sensitivity to neocarzinostatin is proportional to the product of the relative contents of Bcl-2 and caspase-3 (r (2) = 0.9; P < 0.01). Neuroblastoma and brain tumor cell lines are particularly sensitive to neocarzinostatin; the sensitivity of brain tumor lines to neocarzinostatin is enhanced by transfection with an expression construct for Bcl-2 and is proportional in transfected cells to the product of the relative contents of Bcl-2 and caspase-3 (r (2) = 0.7). These studies underscore the potential of molecular profiling in identifying effective chemotherapeutic paradigms for cancer in general and tumors of the nervous system in particular.Cancer Chemotherapy and Pharmacology 09/2008; 62(4):699-706. DOI:10.1007/s00280-008-0725-7 · 2.77 Impact Factor
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ABSTRACT: Tumors of the nervous system are among the most common and most chemoresistant neoplasms of childhood and adolescence. Malignant tumors of the brain collectively account for 21% of all cancers and 24% of all cancer-related deaths in this age group. Neuroblastoma, a peripheral nervous system tumor, is the most common extracranial solid tumor of childhood, and 65% of children with this tumor have only a 10 or 15% chance of living 5 years beyond the time of initial diagnosis. Novel pharmacological approaches to nervous system tumors are urgently needed. This review presents the role of and current challenges to pharmacotherapy of malignant tumors of the nervous system during childhood and adolescence and discusses novel approaches aimed at overcoming these challenges.Pharmacology [?] Therapeutics 05/2009; 122(1):44-55. DOI:10.1016/j.pharmthera.2009.01.001 · 9.72 Impact Factor