Analysis of redox regulation of cytochrome c-induced apoptosis in a cell-free system

In this study, we investigated the importance of redox and Bcl-2 status on cytochrome c-mediated apoptosis. Two mouse lymphoma cell lines, LYas and LY ar that express Bcl-2 protein at different levels, were used to reconstitut e a cell-free system, Cytoplasmic extracts made from apoptosis-sensitive LY as cells 2.5 h after exposure to 5 Gy gamma-radiation were able to induce a poptosis in isolated nuclei, whereas extracts made from LYas cells at time points earlier than 2.5 h, or from Bcl-2-overexpressing, apoptosis-resistan t LYar cells at all time points after irradiation were inactive, Apoptotic activity was restored to inactive extracts by the addition of oxidized but not reduced cytochrome c. Cytochrome c reductase was able to inhibit apopto sis in extracts made from LYas cells 2.5 h after irradiation and LYar extra cts activated by addition of oxidized cytochrome c, Antioxidants, but not o xidant defensive enzymes, blocked apoptosis implying that antioxidants migh t alter the redox state of factors important in mediating apoptosis, These findings confirm the importance of cellular redox state during apoptosis an d are consistent with a role for Bcl-2 in regulating this redox state.