through maintaining the prosurvival function of Bcl-XL, which regulates cytochrome c release from mitochondria. We tested this model by examining whether active Akt can inhibit cytochrome c redistribution in C2-ceramide in- duced apoptosis. Surprisingly, Akt did not block cyto- chrome c release into the cytosol (Fig. 6, A and B), whereas Bcl-2 suppressed cytochrome c release (C and D). This observation is in agreement with the well established role for Bcl-2 to inhibit apoptosis by blocking cytochrome c release from mitochondria (Kluck et al., 1997; Yang et al., 1997). Moreover, it suggests that active Akt inhibits apoptosis at a step after release of cytochrome c into the cytoplasm. Akt, but Not Bcl-2, Inhibits Caspase Activation Induced by Cytochrome c in a Cell-free System To confirm that expressing active Akt inhibits the apop- totic cascade downstream of cytochrome c release, a cell- free system was used to examine PARP cleavage and caspase-3 activation. In extracts prepared from vector- transfected cells, PARP was rapidly cleaved. After a 15- min incubation with cytochrome c and dATP, only a small fraction of intact PARP remained and, by 30 min, all intact PARP disappeared (Fig. 7 A). In contrast, in extracts pre- Figure 8. Overexpressing constitutively active Akt suppresses apoptosis after microinjection of cytochrome c. (A) Fluorescent photomicrographs of FITC-labeled injected cells (left) and Hoechst 33342 nuclear staining (right). Note that cytochrome c but not rabbit IgG induces chromatin condensation in injected cells. Both zVAD pretreatment and expressing active Akt pre- vented cytochrome c–induced chromatin condensation in in- jected cells. (B) Quantification of the protective effects of Akt on inhibiting cytochrome c–induced apoptosis. Cells were injected with cytochrome c and, after 1, 2, or 3 h, apoptosis of injected cells was analyzed as described in Materials and Methods. Data represent the mean 6 SEM from three or more independent ex- periments. pared from Akt stable cell lines, cytochrome c–induced PARP cleavage was markedly inhibited and delayed. The cleaved PARP fragment was detectable only after 60–90 min and a large percentage of intact PARP was still present after 120 min (Fig. 7 A). These data confirm that active Akt inhibits caspase activation downstream of cyto- chrome c. On the other hand, PARP cleavage induced by cytochrome c in extracts prepared from Bcl-2–stable lines proceeded similarly to cleavage in vector-control cell ex- tracts (Fig. 7 B). Taken together, these cell-free studies suggest that Akt can regulate apoptosis at a postmitochon- drial level, whereas Bcl-2 regulates apoptosis primarily at the mitochondrial level. Using an antibody that recognizes cleaved/activated caspase-3, we further analyzed caspase-3 processing induced by cytochrome c in the cell-free assay. Consistent with results for PARP cleavage, caspase-3 cleavage/activation was also inhibited in extracts prepared from Akt-stable lines compared with caspase-3 cleavage/ activation in vector-control cell extracts (Fig. 7 C). Since caspase-9 is the initiator caspase for cleaving and activat- ing caspase-3, we examined whether caspase-9 activation by cytochrome c and dATP was inhibited in extracts pre- pared from Akt-stable lines (Fig. 7 D). Caspase-9 activity assays showed that activation of caspase-9 by cytochrome The Journal of Cell Biology, Volume 151, 2000 490