Protein synthesis-dependent but Bcl-2-independent cytochrome C release in zinc depletion-induced neuronal apoptosis

Previously, we reported that chelation of intracellular zinc with N,N, N',N ' -tetrakis(2-pyridylmethyl)ethylenediamin (TPEN)-induced macromolecule syn thesis-dependent apoptosis of cultured cortical neurons. According to the c urrent theory of apoptosis, release of mitochondrial cytochrome C into the cytosol is required for caspase activation. In the present study, we examin ed whether cytochrome C release is dependent on macromolecule synthesis. Ex posure of cortical cultures to 2 mu M TPEN for 24 hr induced apoptosis as p reviously described. Fluorescence immunocytochemical staining as well as im munoblots of cell extracts revealed the release of cytochrome C into the cy tosol 18-20 hr after the exposure onset. The cytochrome C release was compl etely blocked by the addition of cycloheximide or actinomycin D. Addition o f the caspase inhibitor zVAD-fmk did not attenuate the cytochrome C release , whereas it blocked TPEN-induced apoptosis. Because Bcl-2 has been shown t o block cytochrome C release potently, we exposed human neuroblastoma cells (SH-SY5Y) to TPEN. Whereas Bcl-2 overexpression completely blocked both cy tochrome C release and apoptosis induced by staurosporine, it attenuated ne ither induced by TPEN. The present results suggest that, in neurons, macrom olecule synthesis inhibitors act upstream of cytochrome C release to block apoptosis and that, in addition to the classical Bcl-2 sensitive pathway, t here may exist a Bcl-2-insensitive pathway for cytochrome C release. (C) 20 00 Wiley-Liss, Inc.