Ceramide and Reactive Oxygen Species Generated by H2O2 Induce Caspase-3-independent Degradation of Akt/Protein Kinase B

Abstract

This study was designed to elucidate the mechanisms leading to down-regulation of the Akt/protein kinase B (PKB) survival pathway during H2O2-induced cell death. H2O2 produced early activation of Akt/PKB and also DNA damage that was followed by stabilization of p53 levels, formation of reactive oxygen species (ROS), and generation of ceramide through activation of a glutathione-sensitive neutral sphingomyelinase. These events correlated with long term dephosphorylation and subsequent degradation of Akt. A membrane-targeted active Akt version attenuated apoptosis but not necrosis induced by H2O2 and was more resistant to dephosphorylation and proteolysis induced by apoptotic concentrations of H2O2. Proteolysis of Akt was prevented by exogenous addition of glutathione, indicating a role of ROS and ceramide in Akt degradation. However, Akt was degraded similarly in cells transfected with wild type and dominant negative p53 mutant, indicating that degradation of Akt under oxidative injury may be p53-independent. Specific inhibitors of caspase groups I and III prevented proteolysis of Akt/PKB and poly(ADP-ribose) polymerase in cells submitted to apoptotic but not necrotic H2O2 concentrations. Surprisingly, in caspase-3-deficient MCF-7 cells Akt was more sensitive to H2O2-induced degradation than the caspase-3 substrate poly(ADP-ribose) polymerase. Moreover, the Akt/PKB double mutant Akt(D108A,D119A), which is not cleaved by caspase-3, and a triple mutant (D453A,D455A,D456A), which lacks the consensus sequence for caspase-3 cleavage, were also degraded in H2O2-treated cells. Our results suggest that strong oxidants generate intracellular ROS and ceramide which in term lead to down-regulation of Akt by dephosphorylation and caspase-3-independent proteolysis. This study was designed to elucidate the mechanisms leading to down-regulation of the Akt/protein kinase B (PKB) survival pathway during H2O2-induced cell death. H2O2 produced early activation of Akt/PKB and also DNA damage that was followed by stabilization of p53 levels, formation of reactive oxygen species (ROS), and generation of ceramide through activation of a glutathione-sensitive neutral sphingomyelinase. These events correlated with long term dephosphorylation and subsequent degradation of Akt. A membrane-targeted active Akt version attenuated apoptosis but not necrosis induced by H2O2 and was more resistant to dephosphorylation and proteolysis induced by apoptotic concentrations of H2O2. Proteolysis of Akt was prevented by exogenous addition of glutathione, indicating a role of ROS and ceramide in Akt degradation. However, Akt was degraded similarly in cells transfected with wild type and dominant negative p53 mutant, indicating that degradation of Akt under oxidative injury may be p53-independent. Specific inhibitors of caspase groups I and III prevented proteolysis of Akt/PKB and poly(ADP-ribose) polymerase in cells submitted to apoptotic but not necrotic H2O2 concentrations. Surprisingly, in caspase-3-deficient MCF-7 cells Akt was more sensitive to H2O2-induced degradation than the caspase-3 substrate poly(ADP-ribose) polymerase. Moreover, the Akt/PKB double mutant Akt(D108A,D119A), which is not cleaved by caspase-3, and a triple mutant (D453A,D455A,D456A), which lacks the consensus sequence for caspase-3 cleavage, were also degraded in H2O2-treated cells. Our results suggest that strong oxidants generate intracellular ROS and ceramide which in term lead to down-regulation of Akt by dephosphorylation and caspase-3-independent proteolysis. reactive oxygen species 7-aminoactinomycin D acetyl l-buthionine-(S,R)-sulfoximine 4,6-diamidino-2-phenylindole Dulbecco's modified Eagle's medium diethylenetriaminepentaacetic acid enhanced green fluorescent protein mitochondrial transmembrane potential 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide myristoylated nerve growth factor poly(ADP-ribose) polymerase phosphate-buffered saline protein disulfide isomerase phycoerythrin phosphatidylinositol 3 kinase protein kinase B benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone Although molecular oxygen is critically required for aerobic life, mitochondrial respiration in higher organisms constantly generates low levels of potentially dangerous reactive oxygen species (ROS),1 which include superoxide anion. Mitochondrial and cytosolic superoxide dismutases convert superoxide into hydrogen peroxide (H2O2), a non-radical molecule that generates highly toxic hydroxyl radicals via the Fenton reaction (1Fridovich I. Ann. N. Y. Acad. Sci. 1997; 893: 13-18Crossref Scopus (403) Google Scholar). Neurons of the central and peripheral nervous system are particularly sensitive to oxidative damage. Cell death derived from high ROS levels has been associated with a number of neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (2Pettmann B. Henderson C.E. Neuron. 1998; 20: 633-647Abstract Full Text Full Text PDF PubMed Scopus (536) Google Scholar) as well as the general decline of the central nervous system function frequently associated with senescence (3Serrano M. Blasco M.A. Curr. Opin. Cell Biol. 2001; 13: 748-753Crossref PubMed Scopus (346) Google Scholar). In the nervous system, neurotrophins enhance tolerance to oxidative stress in both animal and cellular models (4Huang E.J. Reichardt L.F. Annu. Rev. Neurosci. 2001; 24: 677-736Crossref PubMed Scopus (3333) Google Scholar, 5Miller F.D. Kaplan D.R. Cell. Mol. Life Sci. 2001; 58: 1045-1053Crossref PubMed Scopus (287) Google Scholar). A prominent mechanism involved in neurotrophin-induced cell survival includes activation of the PI3K and Akt/protein kinase B signaling pathway. The survival signal elicited by Akt proceeds through several mechanisms including inactivation of BAD and caspase-9, stimulation of nuclear factor-κB activity, inactivation of forkhead transcription factor, and phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) and glycogen synthase kinase 3 (GSK3) kinases (for recent reviews, see Refs. 6Brazil D.P. Hemmings B.A. Trends Biochem. Sci. 2001; 26: 657-664Abstract Full Text Full Text PDF PubMed Scopus (1039) Google Scholar and 7Brunet A. Datta S.R. Greenberg M.E. Curr. Opin. Neurobiol. 2001; 11: 297-305Crossref PubMed Scopus (1009) Google Scholar). Akt exerts protective actions against oxidative damage in central and peripheral neurons. For instance, neuregulin prevents H2O2 induction of ROS in a PI3K-dependent manner (8Goldshmit Y. Erlich S. Pinkas-Kramarski R. J. Biol. Chem. 2001; 276: 46379-46385Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), and loss of oxidative stress tolerance with aging has been linked in part to reduced Akt kinase activity in old rats (9Ikeyama S. Kokkonen G. Shack S. Wang X.T. Holbrook N.J. FASEB J. 2002; 16: 114-116Crossref PubMed Scopus (115) Google Scholar). Concerning neurodegeneration, we have reported previously the protective effect of active Akt1 against peptides of β-amyolid protein characteristic of senile plaques found in the brains of Alzheimer's disease patients (10Martin D. Salinas M. Lopez-Valdaliso R. Serrano E. Recuero M. Cuadrado A. J. Neurochem. 2001; 78: 1000-1008Crossref PubMed Scopus (134) Google Scholar) and against the Parkinson-inducing toxin 1-methyl-4-phenylpyridinium (11Salinas M. Martin D. Alvarez A. Cuadrado A. Mol. Cell. Neurosci. 2001; 17: 67-77Crossref PubMed Scopus (51) Google Scholar). When cells are exposed to oxidative injury they activate multiple signaling pathways that dictate whether those cells will ultimately tolerate or succumb to the insult. Akt/protein kinase B appears to be one such pathway that becomes activated by ROS-generating agents (10Martin D. Salinas M. Lopez-Valdaliso R. Serrano E. Recuero M. Cuadrado A. J. Neurochem. 2001; 78: 1000-1008Crossref PubMed Scopus (134) Google Scholar,12Tang D. Okada H. Ruland J. Liu L. Stambolic V. Mak T.W. Ingram A.J. J. Biol. Chem. 2001; 276: 30461-30466Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) and by H2O2 (13Konishi H. Matsuzaki H. Tanaka M. Takemura Y. Kuroda S. Ono Y. Kikkawa U. FEBS Lett. 1997; 410: 493-498Crossref PubMed Scopus (236) Google Scholar, 14Shaw M. Cohen P. Alessi D.R. Biochem. J. 1998; 336: 241-246Crossref PubMed Scopus (239) Google Scholar, 15Wang X. McCullough K.D. Franke T.F. Holbrook N.J. J. Biol. Chem. 2000; 275: 14624-14631Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar). Activation of Akt by ROS may reflect an attempt of the damaged cells to survive the oxidative insult. On the other hand, the best characterized signaling pathways that are activated by oxidative injury are those leading to cell death. Stress-triggered apoptosis induces cytochrome crelease from mitochondria and activation of caspase cascades that execute the apoptotic program (16Gross A. McDonell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3249) Google Scholar). In addition, oxidative stress also produces genotoxic damage that results in stabilization of p53 levels and cell death (17Lane D.P. Nature. 1992; 358: 15-16Crossref PubMed Scopus (4487) Google Scholar). The fact that oxidative stress activates contradictory signaling pathways of survival and death implies that there must be sophisticated cross-talk between these opposite signals that dictate cells fate. The regulation of the apoptosis machinery by Akt is being explored in detail and, as stated above, a number of Akt substrates have been identified as elements of the initiation and execution phases of apoptosis. In general, phosphorylation of these proteins leads to their inactivation. However, very little is known about the mechanism(s) employed by the apoptotic machinery to down-regulate the Akt survival signal. We and other groups and have reported the enhanced dephosphorylation of Akt at the critical residues Thr308and Ser473 by a ceramide-activated protein phosphatase/protein phosphatase 2A (18Salinas M. Lopez-Valdaliso R. Martin D. Alvarez A. Cuadrado A. Mol. Cell. Neurosci. 2000; 15: 156-169Crossref PubMed Scopus (174) Google Scholar, 19Zhou H. Summers S.A. Birnbaum M.J. Pittman R.N. J. Biol. Chem. 1998; 273: 16568-16575Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 20Summers S.A. Garza L.A. Zhou H. Birnbaum M.J. Mol. Cell. Biol. 1998; 18: 5457-5464Crossref PubMed Scopus (367) Google Scholar). The putative role of apoptotic proteases in degradation of Akt are more ill defined, but Akt appears to be a substrate of caspase-3 in vitro (21Rokudai S. Fujita N. Hashimoto Y. Tsuruo T. J. Cell. Physiol. 2000; 182: 290-296Crossref PubMed Scopus (98) Google Scholar), and caspase-3-dependent cleavage of Akt may be relevant in detachment-induced and Fas ligand-induced cell death (22Bachelder R.E. Wendt M.A. Fujita N. Tsuruo T. Mercurio A.M. J. Biol. Chem. 2001; 276: 34702-34707Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 23Widmann C. Gibson S. Johnson G.L. J. Biol. Chem. 1998; 273: 7141-7147Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). Because cells submitted to prolonged oxidative stress ultimately die, despite the initial activation of Akt, the long term effects of these insults leading to down-regulation of the PI3K/Akt survival pathway are particularly relevant and require further investigation. We have addressed this question in PC12 cells submitted to H2O2, a well reported activator of Akt which induces oxidative stress, senescence, and cell death. Our findings indicate that Akt is activated rapidly in response to H2O2, and apoptosis resumes at a time when Akt activity has been completely depleted by dephosphorylation and proteolysis. These results are relevant to an understanding of oxidative stress-induced cell death and for devising strategies that might promote cell survival in the nervous system under the presence of oxidative insults. PC12 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 7.5% fetal bovine serum, 7.5% heat-inactivated horse serum, and 80 μg/ml gentamicin. Neuro 2A and MCF-7 cells were grown in DMEM supplemented with 10% fetal bovine serum and 80 μg/ml gentamicin. Transfections of PC12 and Neuro 2A cells were performed with the expression vectors pCEFL(X-)EGFP, pCEFL(X-)EGFP-Akt1, pCEFL(X-)myr-EGFP-Akt1 and pcDNA3HA-Akt1 (11Salinas M. Martin D. Alvarez A. Cuadrado A. Mol. Cell. Neurosci. 2001; 17: 67-77Crossref PubMed Scopus (51) Google Scholar), or with expression vectors pFLAG, pFLAGhAkt1, pFLAGhAkt1(D108A,D119A) and pFLAGhAkt1(D453A,D455A,D456A) (21Rokudai S. Fujita N. Hashimoto Y. Tsuruo T. J. Cell. Physiol. 2000; 182: 290-296Crossref PubMed Scopus (98) Google Scholar), or with expression vectors pcDNA3-p53 and pcDNA3-p53(S15A) (gift of Dr. R. Sánchez-Prieto, Hospital Puerta de Hierro, Madrid; 24) using SuperFect transfection reagent (Qiagen). All reagents were purchased from Sigma except NGF, bacterial sn-1,2 diacylglycerol kinase (Calbiochem), C2-ceramide (Cayman Chemical Company, Ann Arbor, MI), and ZVAD-fmk, Ac-YVAD-cmk, and Ac-DEVD-fmk (RBI, Natick, MA). MTT assays were performed as described previously (25Hansen M.B. Nielsen S.E. Berg K. J. Immunol. Methods. 1989; 119: 203-210Crossref PubMed Scopus (3335) Google Scholar). For trypan blue staining, both detached and attached cells were resuspended in DMEM, mixed with an equal volume trypan blue stain (Biowhittaker, Walkersville, MD), and left at room temperature for 5 min. The percentage of stained cells was analyzed in a Neubauer chamber. In vitro Akt kinase assays were performed as described by Salinas et al. (18Salinas M. Lopez-Valdaliso R. Martin D. Alvarez A. Cuadrado A. Mol. Cell. Neurosci. 2000; 15: 156-169Crossref PubMed Scopus (174) Google Scholar). Reactions were terminated by the addition of SDS loading buffer, resolved in 10% SDS-PAGE, and transferred to Immobilon-P membranes (Millipore). Ponceau-stained histone H2B bands were exposed to autoradiography, excised, and measured individually by Cerenkov counting. Immunoblots of the upper membrane portions were performed routinely using anti-Akt1C20 antibody to confirm equal amounts of kinase/reaction. Cells were processed as described by Salinaset al. (18Salinas M. Lopez-Valdaliso R. Martin D. Alvarez A. Cuadrado A. Mol. Cell. Neurosci. 2000; 15: 156-169Crossref PubMed Scopus (174) Google Scholar), resolved in SDS-PAGE, and transferred to Immobilon-P membranes. Blots were analyzed with the following antibodies: anti-Akt1 (1:500), anti-ERK2 (1:500), anti-p110 PI3K, anti-PARP (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-AktS473, anti-phospho-AktT308 (1:500) (Cell Signaling Technology, Inc, Beverly, MA), anti-PDI (gift of Dr. J. G. Castaño, Instituto de Investigaciones Biomédicas, Madrid). Appropriate peroxidase-conjugated secondary antibodies were used to detect the proteins of interest by enhanced chemiluminescence. Densitometric analyses of representative immunoblots were performed with the NIH Image software. H2O2 concentration in stock solutions was standardized by determining the A 240 nm value (1 A 240 nm = 22.9 mmH2O2). In assays for H2O2 catabolism, 400,000 cells were incubated with 0.5 mm H2O2, and 100 μl of medium was collected at 20-min intervals. Medium from untreated cells was used to prepare a standard curve ranging from 1 mm to 16.75 μm. Samples and standards were diluted 1:10 with DMEM, and 50 μl of each was mixed with 150 μl of a modified ECL mix (0.55 mm p-coumaric acid, 2.5 mmluminol, and 1 μl/ml horseradish peroxidase-conjugated goat anti-rabbit IgG (H+L) (Bio-Rad) as a source of peroxidase. Light emission was measured in a Optocomp I BG-1 luminometer (GEM Biomedical, Inc., Cambridge, UK). Assays were performed as described by Collinset al. (26Collins A.R. Dobson V.L. Dusinska M. Kennedy G. Stetina R. Mut. Res. 1997; 375: 183-193Crossref PubMed Scopus (600) Google Scholar) with slight modifications. 400,000 cells were seeded in P60 dishes, serum starved for 16 h, treated for 30 min with different H2O2 concentrations, washed twice with PBS, detached with 1 ml of PBS, and pelleted at 500 ×g for 1 min at 4 °C. Then the cells were resuspended in 1% low melting point agarose (Biowhittaker) in PBS prewarmed to 37 °C. A 50-μl drop of cell suspension was laid on agarose-precoated slides. Quickly the drop was covered with a coverslip and left at 4 °C for 5 min. Then the coverslip was removed, and the cells were lysed by immersion of the slides in precooled, freshly prepared lysis buffer (2.5 m NaCl, 0.1 m EDTA, 10 mm Tris-HCl, pH 7.5, 1% Triton X-100) for 1 h. After lysis, nuclei were submitted to alkaline pretreatment in electrophoresis buffer (0.3 m NaOH, 1 mm EDTA precooled at 4 °C) for 40 min and electrophoresed for 30 min at 25 V. The samples were neutralized by three washes of 5 min with 0.4m Tris-HCl, pH 7.5. Nuclei were stained by immersion in a solution containing 0.1 μg/ml DAPI in PBS for 5 min and gently washed three times with PBS. The coverslips were dried and mounted with FluorSave (Calbiochem). Analysis was performed in a Nikon Eclipse TE300 using a standard UV filter. The levels of reduced glutathione (GSH) were determined using the GSH-sensitive probe monochlorobimane (Molecular Probes Inc., Eugene, OR) (27Rimpler M.M. Rauen U. Schmidt T. Moroy T. de Groot H. Biochem. J. 1999; 340: 291-297Crossref PubMed Google Scholar). 20,000 cells were seeded on 96-well plates and serum starved for 16 h. 200 μm BSO or H2O2 was added to the medium and incubated for the time indicated in Fig. 2. 100 μm monochlorobimane was added during the last 30 min. Plates were analyzed immediately using a Fluoroscan fluorometer (Labsystems Oy, Helsinki, Finland) (20-ms integration time, 390-nm excitation filter, and 485-nm emission filter). Basal autofluorescence was determined from cells that were not incubated with the probe and was subtracted to all samples. Fluorescence measured from untreated cells was considered as 100% GSH content. For mitochondrial membrane potential analysis cells were detached mechanically, washed once with DMEM at 37 °C, and incubated for 15 min at 37 °C in DMEM containing 100 nm CM-H2XROS (Molecular Probes). Probe incorporation was measured using a 620/22 nm band pass filter (FL3-H). For annexin V-phycoerythrin (PE) and 7-aminoactinomycin D (7-AAD) staining, cells were detached mechanically after treatments and washed twice with annexin-V binding buffer (10 mm Hepes-NaOH, pH 7.4, 140 mm NaCl, 2.5 mm CaCl2). Cells were resuspended in 100 μl of a 1:20 dilution of annexin V-PE (Bender MedSystems Diagnostics, Vienna, Austria) in annexin V binding buffer containing 2 μm 7-AAD and incubated for 15 min at room temperature. Fluorescence was measured using band pass 620/22 nm and 575/24 nm filters. For analysis of ROS levels, 2 μm hydroethidine was added 1 h before Then cells were detached mechanically from the washed twice with PBS, and analyzed immediately using a standard 575/24 nm band pass filter All analyses were performed on a The diacylglycerol kinase was used A. 2000; PubMed Google Scholar). 400,000 cells were serum starved for 16 h and treated with H2O2 or inhibitors for the times indicated in Fig. and detached cells were pelleted at for min. Cells were washed once with PBS, resuspended in μl of PBS, and transferred to were with μl of 1 After and the containing the was transferred to a was under a were resuspended in μl of 0.1 in and incubated for 1 h at 37 °C to μl of PBS was and was as indicated were resuspended in 100 μl of reaction buffer μl μm mm 50 mm NaCl, mm 1 mm EDTA, 2 mm μm 500 μm 5 μl and of diacylglycerol kinase was added to each Reactions were incubated at 30 °C for 30 min and by the addition of μl of buffer mm NaCl, mm 0.5 mm 10 mm pH and 30 μl of 100 mm were with 1 ml of 1 resuspended in 40 μl of plates and resolved using acid as Plates were measured in an of ceramide was by with a standard curve ranging from to of ceramide which was processed in to the samples. was used to in hydroethidine and of ceramide between and H2O2-treated groups and between and was considered all were performed at three times with The in to the of at three samples. indicate standard we analyzed the effect of H2O2 on Akt kinase activity and the signaling mechanisms leading to Akt activation under PC12 cells were with or for 15 min and incubated with H2O2 or for 5 min. Akt activity was measured in in vitro kinase assays using histone H2B as indicated in H2O2 and Akt activity in PC12 and the inhibitors of and prevented the activation of Akt by both results were when Akt activity was as a function of phosphorylation at Ser473 or with antibodies not results indicate that H2O2 activates Akt in a PI3K-dependent Akt activation was with antibodies in cells treated for 10 min and 3 h with H2O2 and in Fig. 1 H2O2 induced a activation of Akt than after a after a was in H2O2-treated cells about the at 10 min. results were when active Akt was with an antibody against not The different of Akt activation may be at in by a down-regulation of Akt activity in H2O2-treated cells or by a degradation of H2O2 by cell that this signal. we determined the of H2O2 degradation under 400,000 PC12 cells were incubated with 0.5 mm H2O2 for time and the H2O2 concentration in the medium was measured by a initial concentration of 0.5 mm H2O2 was reduced to than 0.1 mm in min not We also the of the H2O2 concentration in the medium to activate Akt. The medium from cells treated with 0.5 mm H2O2 was rapidly used to cells for 10 min. a we the effect on Akt activation of medium of samples of H2O2 which was added to in Fig. 1 in medium 0.5 mm H2O2 was degraded in about 2 h from were to of Akt. in the presence of PC12 H2O2 a degradation with of Akt activity in about min. These results indicate that in the cell and a time to term effects that are with the presence of H2O2 and long term effects that into at a time when H2O2 is the of H2O2, cells apoptosis and necrosis in the following h we that in addition to Akt, H2O2 might also a for long term induction of cell such as DNA damage. this PC12 cells were submitted for 30 min to 0.5 and 1 mm H2O2. DNA was analyzed by the as in Fig. D and E. Akt, these also produced strong genotoxic damage as by the long of electrophoresed Moreover, pretreatment with D or with not not enhance the DNA that the PI3K/Akt pathway not the cells from the initial genotoxic produced by H2O2. whether H2O2 the cell we analyzed the intracellular of GSH by the of monochlorobimane (27Rimpler M.M. Rauen U. Schmidt T. Moroy T. de Groot H. Biochem. J. 1999; 340: 291-297Crossref PubMed Google Scholar). The of fluorescent that is by the of intracellular was measured incorporation of the probe was determined in cells treated for h with an of which is the in glutathione M.E. Chem. Biol. 1998; PubMed Scopus Google Scholar). PC12 cells were submitted to 0.5 for the time indicated in A and incubated for the last 30 min with the H2O2 depleted GSH to levels to those with a with Because GSH is the the these results indicate that H2O2 the cell to GSH at a time point when H2O2 was at at a to Akt activation 1 that other secondary of oxidative stress might be for the GSH we oxygen Mitochondrial transmembrane potential was by in cells stained with a reduced that is by the active mitochondria and M. 1999; PubMed Scopus Google Scholar). in Fig. 2 when PC12 cells were submitted to 0.5 mm H2O2, an of the cell was to CM-H2XROS staining, indicating a of the in cells that were not stained by the probe correlated with the drop of GSH We also analyzed the intracellular levels of ROS by of cells stained with a fluorescent probe sensitive for superoxide J. J. Neurosci. 16: PubMed Google Scholar). in Fig. 2 when cells were submitted to 0.5 mm H2O2 for 3 h, they an in intracellular these results suggest that after of DNA a secondary source of ROS including is at in part at the Because ceramide is a of apoptosis in we determined ceramide levels after H2O2 of PC12 cells. in Fig. 3 a in ceramide was during the with H2O2. Although this was not in other groups have reported a in cells treated with other insults PubMed Scopus Google Scholar, S. FASEB J. PubMed Scopus Google Scholar, C. Trends Cell Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). ceramide levels more to that of untreated cells after a We analyzed the of ceramide in the presence of an of ceramide in Fig. 3 the H2O2-induced of this was to that in the of indicating not to the of ceramide The in ceramide to the of GSH levels 2 that the ceramide might from the activation of a neutral as reported previously B. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF