Previously, we reported that chelation of intracellular zinc with N, N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN)-induced macromolecule synthesis-dependent apoptosis of cultured cortical neurons. According to the current theory of apoptosis, release of mitochondrial cytochrome C into the cytosol is required for caspase activation. In the present study, we examined whether cytochrome C release is dependent on macromolecule synthesis. Exposure of cortical cultures to 2 microM TPEN for 24 hr induced apoptosis as previously described. Fluorescence immunocytochemical staining as well as immunoblots of cell extracts revealed the release of cytochrome C into the cytosol 18-20 hr after the exposure onset. The cytochrome C release was completely blocked by the addition of cycloheximide or actinomycin D. Addition of the caspase inhibitor zVAD-fmk did not attenuate the cytochrome C release, whereas it blocked TPEN-induced apoptosis. Because Bcl-2 has been shown to block cytochrome C release potently, we exposed human neuroblastoma cells (SH-SY5Y) to TPEN. Whereas Bcl-2 overexpression completely blocked both cytochrome C release and apoptosis induced by staurosporine, it attenuated neither induced by TPEN. The present results suggest that, in neurons, macromolecule synthesis inhibitors act upstream of cytochrome C release to block apoptosis and that, in addition to the classical Bcl-2 sensitive pathway, there may exist a Bcl-2-insensitive pathway for cytochrome C release.
Copyright 2000 Wiley-Liss, Inc.