NEED FOR CASPASES IN APOPTOSIS OF TROPHIC FACTOR-DEPRIVED PC12 CELLS

PC12 cells are a useful model system for studying neuronal apoptosis, Like neurons, they undergo apoptosis when deprived of trophic support, Involvement of caspases [interleukin 1 beta-converting enzyme (ICE)-r elated proteases] has been implicated in apoptosis induced by various stimuli in many cell types, including neurons, In the present study we investigated the need for caspases participation in apoptosis induced by growth factor deprivation in naive and neuronal PC12 cells, For th is purpose we generated PC12 cell lines that consistently express the viral caspases inhibitor genes p35 or crmA, and analyzed their suscept ibility to trophic factor deprivation, We also examined the effects of cell-permeable peptide inhibitors of caspases. Our results showed tha t broad-spectrum inhibitors of the caspases, namely the baculovirus p3 5 gene and the peptide benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl keto ne, effectively inhibit the death of both naive and neuronal PC12 cell s, However, caspase-1 (ICE)-specific inhibitors, namely the peptides A c-Try-Val-Ala-Asp-chloromethylketone and Ac-Try-Val-Ala-Asp-aldehyde, as well as crmA, were much less effective, These findings demonstrate that caspases, but not caspase-1, are needed for apoptosis induced by trophic factor deprivation in both naive and neuronal PC12 cells, Nort hern and Western blot analyses showed that PC12 cells express caspase- 3, We therefore examined the involvement of caspase-3 in the death pro cess of trophic factor-deprived PC12 cells, Our results showed that th e pro-caspase-3 and its substrate poly(ADP-ribose) polymerase are clea ved at similar rates in serum-deprived PC12 cells, Moreover, cell lysa tes prepared from these cells possess caspase-3-like activity, as dete rmined by their ability to cleave the fluorogenic peptide substrate Ac -Asp-Glu-Val-Asp-7-amino-4-methylcoumarin. These findings strongly sug gest that caspase-3 or caspase-3-like proteases are activated in troph ic factor-deprived PC12 cells. (C) 1997 Wiley-Liss, Inc.