Cytochrome c depletion upon expression of Bcl-XS

We have shown previously that Bcl-XS causes acute cell death in 3T3 cells w ithout activating caspases (Fridman, J. S,, Benedict, M. k, and Maybaum, J. (1999) Cancer Res. 59, 5999-6004). In this study, we determined that the e xplanation for lack of caspase activation is the cellular depletion of cyto chrome c. Electron microscopy revealed gross structural changes in the mito chondria of Bcl-XS-expressing cells; however, cytochrome c was not detected in cytosolic fractions from these cells. Surprisingly, it was determined t hat cellular cytochrome c levels decreased as Bcl-XS expression levels incr eased. Experiments performed to eliminate other possible explanations for t he lack of caspase activation showed that these 3T3 cells have a functional cytoplasmic apoptosome, a complex of proteins that form a functional trigg er capable of activating the proximal caspase in an apoptotic pathway Chinn aiyan, k M. (1999) Neoplasia 1, 5-15, as cytosolic extracts from these cell s were capable of cleaving pro-caspase-9. These cells were also able to rel ease cytochrome c from their mitochondria after appropriate stimulation, ot her than Bcl-XS expression (i.e. withdrawal from serum for 24 h), and initi ate a cell death that is inhibited by a dominant negative caspase-9. We con clude that lack of caspase activation is due to a Bcl-XS-induced depletion of active cytochrome c, a phenomenon that represents an alternative cell de ath effector pathway and/or a novel mechanism for regulating caspase activa tion.