Bcl-2-mediated potentiation of neocarzinostatin-induced apoptosis: requirement for caspase-3, sulfhydryl groups, and cleavable Bcl-2.
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.
Division of Child Neurology, Pediatric Center for Neuroscience, Children's Hospital of Pittsburgh of UPMC, PA 15213, USA.
Zhiping
Z
Mi
Borah
B
Hong
Zeljka Korade
ZK
Mirnics
Yulia Y
YY
Tyurina
Valerian E
VE
Kagan
Ye
Y
Liang
Nina Felice
NF
Schor
D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D053148:Caspase 3; D020169:Caspases / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D009281:Naphthalenes / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D016716:PC12 Cells; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D011758:Pyrroles / Q000494:pharmacology; D051381:Rats; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014162:Transfection; D009353:Zinostatin / Q000494:pharmacology*; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D000903:Antibiotics, Antineoplastic; D009281:Naphthalenes; D019253:Proto-Oncogene Proteins c-bcl-2; D011758:Pyrroles; D013438:Sulfhydryl Compounds; C498335:ThioGlo-1; D051028:bcl-2-Associated X Protein; D009353:Zinostatin; C505407:CASP3 protein, human; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D005978:Glutathione