The pellets were washed once with ice-cold PBS and resuspended with 5 vol of buffer A (20 mM Hepes-KOH, pH 7

The pellets were washed once with ice-cold PBS and resuspended with 5 vol of buffer A (20 mM Hepes-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol, supplemented with 5 g/ml pepstatin, 10 g/ml leupeptin, 2 g/ ml aprotinin, 0.1 mM PMSF) containing 250 mM sucrose for 15 min. Foley and Cooley, 1998; McCall and Steller, 1998). Cytoplasmic dumping and the eventual demise of the nurse cells happens through reproducible phases that provide an excellent model for the analysis of pre-apoptotic events in identifiable cells. We examined the status of cytochrome c in vivo by using this model of programmed cell death. Our studies revealed that a pronounced switch in this protein, evidenced from the exposure of an otherwise hidden epitope (i.e., apoptogenic display of cytochrome c), precedes overt indications of apoptosis. Display of a new cytochrome c epitope was highly specific for pre-apoptotic cells, and did not happen in cells destined to survive. A similar alteration associated with cytochrome c was recognized in the cultured cells expressing the known apoptotic activators ((Chen et al., 1996b). Consistent with our studies of ovarian cells, the altered form of cytochrome c was specific for GDC-0834 Racemate apoptotic cells and was not recognized in a variety of settings. Moreover, this switch was specific for apoptotic death because modified cytochrome c did not Rabbit Polyclonal to ATP5I happen in cells killed by toxic challenge. However, in contrast to reports from studies of mammalian cells, the apoptogenic form of cytochrome c was not released from mitochondria during apoptosis, but instead remained localized to this organelle (as evidenced by cell GDC-0834 Racemate fractionation and cytology). Since apoptosis induced by and is caspase dependent (Chen et al., 1996b; Nordstrom et al., 1996; Pronk et al., 1996; White et al., 1996; Fraser et al., 1997; Kondo et al., 1997) we also examined the influence of caspase function upon the modified display of cytochrome c. The viral caspase inhibitor, p35, and peptide-based caspase inhibitors completely clogged display of the apoptogenic cytochrome c epitope and, GDC-0834 Racemate conversely, expression of an triggered caspase was adequate to result in apoptogenic changes in cytochrome c. Finally, in cell-free studies, we found that mitochondria isolated from apoptotic cells could promote caspase activation whereas identical preparations from healthy cells did not. Taken collectively, we demonstrate that the appearance of an modified form of GDC-0834 Racemate cytochrome c selectively precedes the programmed death of cells in an undamaged, developing organ. This pre-apoptotic alteration uncovers an normally hidden epitope and was provoked inside a caspase-dependent manner by the death activators, or In light of evidence that mitochondrial parts such as cytochrome c can promote caspase activation GDC-0834 Racemate in vitro (Liu et al., 1996; Kluck et al., 1997a; Zou et al., 1997), our data implicate a feed-forward amplification circuit including an apoptogenic form of cytochrome c and caspase activation. Materials and Methods AntiCcytochrome c mAbs The mouse antiCrat cytochrome c mAbs used in this study have been explained (Goshorn et al., 1991; Mueller and Jemmerson, 1996). They were purified from ascites by affinity chromatography using rabbit cytochrome cCcoupled Sepharose beads as with Urbanski and Margoliash (1977). Cytochrome c (or metallothionein promoter and conditional manifestation in Schneider L2 (SL2) cells (Schneider, 1972) can be induced either in stably or transiently transfected cells. After transfection of Mt-rpr and Mt-grim, alone or in combination with the pMt-p35 plasmid (Nordstrom et al., 1996), induction was achieved by exposing the cells to 700 M CuSO4. Transient manifestation assays were carried out as with Chen et al. (1996a) and Nordstrom et al. (1996). Standard transfection efficiencies ranged from 40 to 60%. 48 h after transfection, cells from each well were split.