COOPERATIVE INTERCEPTION OF NEURONAL APOPTOSIS BY BCL-2 AND BAG-1 EXPRESSION - PREVENTION OF CASPASE ACTIVATION AND REDUCED PRODUCTION OF REACTIVE OXYGEN SPECIES

Neuronally differentiated PC12 cells undergo synchronous apoptosis whe n deprived of nerve growth factor(NGF), Here we show that NGF withdraw al induces actinomycin D- and cycloheximide-sensitive caspase (ICE-lik e) activity. The peptide inhibitor of caspase activity, N-acetyl-Asp-G lu-Val-Asp-aldehyde, was more potent than acetyl-Tyr-Val-Ala-Asp-chlor omethyl ketone in preventing NGF withdrawal-induced apoptosis, suggest ing an important role for caspase-3 (CPP32)-like proteases. We observe d a peak of reactive oxygen species (ROS) 6 h after NGF withdrawal. RO S appear to be required for apoptosis, because cell death is prevented by the free radical spin trap, N-tert-butyl-alpha-phenylnitrone, and the antioxidant, N-acetylcysteine. ROS production was blocked by actin omycin D, cycloheximide, and caspase protease inhibitors, suggesting t hat ROS generation is downstream of new mRNA and protein synthesis and activation of caspases. Forced expression of either BCL-2 or the BCL- 2-binding protein BAG-1 blocked NGF withdrawal-induced apoptosis, acti vation of caspases, and ROS generation, showing that they function ups tream of caspases. Coexpression of BCL-2 and BAG-1 was more protective than expression of either protein alone.