Figure 2. Caspase-3 activation, PARP cleavage and cytochrome c release from mitochondria during C2-ceramide–induced HMN1 cell death. (A) C2-ceramide induces caspase-3 activation. HMN1 cells were treated with 50 mM C2-ceramide, 50 mM C2-dihydro- ceramide, or 50 mM C2-ceramide plus 100 mM zVAD-fmk for the indicated period. The blot was probed with a polyclonal antibody to caspase-3. Note that zVAD-fmk inhibits caspase-3 processing. (B) C2-ceramide induces PARP cleavage. HMN1 cells were treated as described above for the indicated period. The blot was probed with an mAb to PARP. Note that zVAD-fmk blocks PARP cleavage. (C) C2-ceramide induces cytochrome c release into the cytosol. HMN1 cells were treated as described above for the indicated period. At each time point, cells were fractionated and equal amounts of cytosolic protein were loaded into each lane. The blot was probed with an mAb to cytochrome c (top), or an mAb to a-tubulin (middle). an mAb to cytochrome c oxidase subunit IV (COX IV) was used to show the cytosolic fraction is not contaminated with mitochondria (bottom; M represents mi- tochondria as a positive control for COX IV). Note that zVAD- fmk does not block cytochrome c release. cell-permeable C2-ceramide induces HMN1 cell death and inhibits Akt activation that may represent one mecha- nism by which ceramide promotes apoptosis (Zhou et al., 1998). However, the cellular events involved in ceramide- Figure 3. Establishing stable HMN1 lines overexpressing consti- tutively active Akt or human Bcl-2. (A) Detection of endogenous and transfected Akt in stable HMN1 clones expressing active Akt. HMN1 cells were transfected with either control vector (pCR3.1) or myr-AktD4-129 and selected with hygromycin B (350 mg/ml) for 4 wk. V1 and V2 represent control clones trans- fected with vector. A7, B10, and C10 represent stable HMN1 cell lines expressing myr-AktD4-129. Endogenous Akt runs at z60 kD and myr-AktD4-129 runs at z46 kD. The clones were ana- lyzed using a polyclonal antibody against Akt (A) and an mAb against the HA epitope tag (not shown). (B) Detection of acti- vated/phosphorylated Akt in stable HMN1 clones expressing ac- tive Akt. Blots were probed with a phosphospecific antibody against Akt phosphorylated on Ser-473. (C) Detection of stable HMN1 clones expressing human Bcl-2. HMN1 cells were trans- fected with either control vector (pCMV) or human Bcl-2 and se- lected with hygromycin B (350 mg/ml) for 4 wk. V1 and V2 repre- sent control clones transfected with vector. 1, 2, and 9 represent the stable HMN1 cells expressing human Bcl-2. The clones were analyzed using an mAb specific for human Bcl-2. induced apoptosis remain undetermined. Since caspase-3 activation plays a central role in neuronal apoptosis, we examined caspase-3 activation and cleavage of its sub- strate, PARP, during ceramide-induced apoptosis. Cells treated with C2-ceramide, but not with an inactive cer- amide analogue C2-dihydroceramide exhibited activation of caspase-3 after 3 h based on processing of procaspase-3 into active caspase-3 and the appearance of the p17 sub- unit. Caspase-3 activation was inhibited by the broad caspase inhibitor zVAD-fmk (benzyloxycarbonyl-Val- Ala-Asp-fluoromethylketone) (Fig. 2 A). Consistent with the observed caspase-3 activation, PARP, a known sub- strate for caspase-3, was cleaved with a similar time course, and zVAD-fmk effectively blocked PARP cleav- age (Fig. 2 B). Another important cellular event in the progression of apoptosis is the release of cytochrome c from mitochondria; therefore, we examined the distribu- The Journal of Cell Biology, Volume 151, 2000 486