Cell-specific induction of apoptosis by microinjection of cytochrome c - Bcl-x(L) has activity independent of cytochrome c release

Bcl-x(L), an antiapoptotic member of the Bcl-2 family, inhibits programmed cell death in a broad variety of cell types, Recent reports have demonstrated that cytochrome c is released from mitochondria during apoptosis and have suggested that this release may be a critical step in the activation of proapoptotic caspases and subsequent cell death, Furthermore, it has been demonstrated that Bcl-2 can prevent the release of cytochrome c from mitochondria in cells triggered to undergo apoptosis, This has led to the hypothesis that the antiapoptotic effects of Bcl-2 family members are due specifically to their ability to prevent cytochrome c release thus preventing subsequent cytochrome c-dependent caspase activation, In the present report, we use microinjection techniques to investigate the relationship between cytochrome c release, induction of apoptosis, and Bcl-x(L) activity in intact cells, We demonstrate that microinjection of cytochrome c into the cytosol of human kidney 293 cells results in a dose-dependent induction of apoptosis, In contrast, MCF7 breast carcinoma cells (stably transfected to express the Fas antigen CD95, and denoted MCF7F) that lack detectable levels of caspase 3 (CPP32), are totally resistant to microinjection of cytochrome c, However, transfection of MCF7F cells with an expression plasmid coding for pro-caspase 3, but not other pro-caspases, restores cytochrome c sensitivity, Although MCF7F cells are insensitive to cytochrome c microinjection, they rapidly undergo apoptosis in a caspase dependent manner in response to either tumor necrosis factor or anti-Fas plus cycloheximide, and these deaths are strongly inhibited by Bcl-x(L) expression, Furthermore, microinjection of cytochrome c does not overcome these antiapoptotic effects of Bcl-x(L). Our results support the concept that the release of cytochrome c into the cytoplasm can promote the apoptotic process in cells expressing pro-caspase 3 but that cytochrome c release is not sufficient to induce death in all cells, Importantly, the ability of Bcl-x(L) to inhibit cell death in the cytochrome c-insensitive MCF7F cells cannot be due solely to inhibition of cytochrome c release from mitochondria.