Figure 1. Inhibition of PI 3-kinase signaling but not MAPK signal- ing induces apoptosis of HMN1 cells. (A) PD 98059 inhibits MAPK signaling (ERK phosphorylation at threonine-202/ty- rosine-204) and LY 294002 inhibits PI 3-kinase signaling (Akt phosphorylation at serine-473). HMN1 cells were grown in serum- free medium for 16 h, followed by stimulation with 10 mg/ml insu- lin or 20% fetal bovine serum in the absence or presence of 30 mM PD 98059 or LY 294002. Samples were blotted with antibodies against phospho-ERK or phospho-Akt. (B) LY 204002, but not PD98059, induces cell death. HMN1 cells were treated with 30 mM LY 294002 (dissolved in ethanol) or 30 mM PD 98059 (dissolved in DMSO), and cell viability was determined after 18 h. 1, DMSO; 2, PD 98059; 3, ethanol; 4, LY 294002. LY 294002 causes a significant decrease in viability. Data represent mean 6 SEM of three inde- pendent experiments. (C) HMN1 cells were treated as described above, and after 18 h soluble DNA was extracted for DNA frag- mentation assay. 1, DMSO; 2, PD 98059; 3, ethanol; 4, LY 294002. Note prominent DNA laddering after treatment with LY 294002. Data are from a representative experiment performed three times. of 120 ml in CEB with protein concentration of 2.5 mg/ml. Apoptosis was induced by addition of bovine heart cytochrome c and dATP to extracts at a final concentration of 50 mg/ml and 1 mM, respectively. The reactions were then incubated at 378C. At the indicated time points, 20 ml of reac- tions were removed and boiled with 10 ml 33 sample buffer for 5 min at 958C. Samples were frozen at 2208C for subsequent SDS-PAGE/Western blot analysis. Addition of cytochrome c and dATP to nonapoptotic HMN1 cell extracts induces rapid cleavage of poly (ADP-ribose) poly- merase (PARP) by activated caspase-3. On the other hand, addition of BSA and dATP does not lead to PARP cleavage (data not shown). These observations suggest that, in this cell free system, caspase activation is strictly dependent on the presence of cytochrome c. Colorimetric Caspase-9 Activity Assay Cell extracts were incubated in the presence or absence of cytochrome c and dATP at 378C for 15 min as described above. Then the lysates were incubated with caspase-9 colorimetric substrate (LEHD-pNA) to measure caspase-9 activity according to the protocol suggested by the manufac- turer. The cleavage of the peptide was quantitated spectrophotometrically at a wavelength of 405 nm. The results are expressed as fold increase in caspase-9 activity of apoptotic reactions (cytochrome c and dATP added) compared with that of control reactions (BSA and dATP added). Immunoprecipitation and Akt Kinase Reaction HMN1 clones expressing constitutively active Akt were lysed in ice-cold ly- sis buffer (20 mM Tris-HCL, pH 7.4, 10 mM MgCl2, 10 mM NaCl, 1 mM EGTA, 5 mM Na3VO4, 1 mM dithiothreitol, 1 mM PMSF, 1 mg/ml leupep- tin, 10 mg/ml aprotinin) as described previously (Zhou et al., 1998). The cell lysates were then incubated with either monoclonal anti–HA antibody (12CA5) or control mouse immunoglobulin for 2 h at 48C. Protein A sepharose was then added and incubated for another hour. The beads were then washed three times with cold lysis buffer, and then incubated with 120 ml vector lysates (CEB, 20 mM ATP) at 308C. After 30 min, bovine heart cy- tochrome c and dATP were added and the reactions were incubated at 378C. Microinjection Cell microinjection was performed as previously described (Cass et al., 1999). HMN1 or HMN1/myr-Akt cells were plated on 35-mm plates 24 h before injection. Rabbit IgG alone or rabbit IgG plus 5 mg/ml cytochrome c (Sigma-Aldrich; bovine heart cytochrome c, diluted in H2O and freshly prepared for each experiment) were injected into the cytoplasm. Injected cells were stained with FITC-conjugated anti–rabbit IgG, and then incu- bated with Hoechst 33258 (4 mg/ml) for nuclear staining. The intracellular concentrations of microinjected proteins are estimated to represent a 10– 100-fold dilution of the pipette concentration based on an estimated HMN1 cell volume of 4–5 pl. We estimate that 0.2–2 pg cytochrome c per cell is delivered after injection of cytochrome c at 5 mg/ml. At various times after injection, injected cells were scored for apoptosis based on condensed chromatin or fragmented nuclei. The percentage of apoptotic cells was calculated as the number of apoptotic nuclei versus total number of injected cells. Data represent the mean 6 SEM from three or more in- dependent experiments. Results PI 3-kinase Is Required for HMN1 Cell Survival Depending on the type of neuron, either PI 3-kinase or MAP kinase can promote cell survival (Pettmann and Henderson, 1998). To determine which pathway (or both pathways) is required for HMN1 cell survival, HMN1 cells were treated with the PI 3-kinase inhibitor, LY 294002, or with the MEK1 inhibitor PD 98059. In HMN1 cells, PD 98059 blocked serum-stimulated ERK phosphorylation at threonine-202/tyrosine-204, indicating effective inhibition of MEK1, and LY 294002 blocked serum- and insulin-stim- ulated Akt phosphorylation at serine-473, indicating effec- tive inhibition of PI 3-kinase (Fig. 1 A). After 18 h, LY 294002 induced z50% cell death, whereas PD 98059 had no obvious effect on cell survival (Fig. 1 B). Consistent with LY 294002 but not PD 98059–inducing apoptosis in HMN1 cells, DNA laddering was observed only in LY 294002- treated cells (Fig. 1 C). Other features characteristic of ap- optosis including cell shrinkage, membrane blebbing, and chromatin condensation were also present in cells treated with LY 294002 but not in cells treated with PD 98059 (data not shown), indicating that PI 3-kinase but not MAP kinase plays an essential role in HMN1 cell survival. Caspase-3 Activation, PARP Cleavage and Cytochrome c Release from Mitochondria during C2-Ceramide Induced HMN1 Cell Death A variety of apoptotic stimuli have been shown to gener- ate ceramide that can serve as a lipid messenger to induce apoptosis (Hannun, 1996). We previously reported that Zhou et al. Akt Inhibits Apoptosis after Cytochrome c Release 485