Apoptosis and activation of the sphingomyelin-ceramide pathway induced by oxidized low density lipoproteins are not causally related in ECV-304 endothelial cells

Oxidized low density lipoproteins (oxLDL) are thought to play a central role in the development of atherosclerosis. Toxic concentrations of mildly oxidized LDL elicit massive apoptosis of endothelial cells (Escargueil-Blanc, I., Meilhac, O., Pieraggi, M. T. , Arnal J. F., Salvayre, R., Nègre-Salvayre, A. (1997) Arterioscler. Thromb. Vasc. Biol. 17, 331-339). Since the lipid mediator ceramide emerged as a potent inducer of apoptosis, we aimed at investigating the occurrence of ceramide formation and its potential role in oxLDL-induced apoptosis. In ECV-304 endothelial cells, toxic concentrations of oxLDL triggered an early activation of the sphingomyelin-ceramide pathway, as shown by both sphingomyelin hydrolysis and ceramide formation. N-Tosyl-L-phenylalanyl chloromethyl ketone (TPCK) and dichloroisocoumarin (DCIC), two serine-protease inhibitors (serpins), blocked the oxLDL-induced ceramide generation but, unexpectedly, did not inhibit the oxLDL-induced apoptosis. Conversely, treatment of endothelial cells by bacterial sphingomyelinase, under conditions effectively generating ceramide, did not induce apoptosis. In contrast, short-chain permeant C2- and C6-ceramides elicited apoptosis of ECV-304. However, the mechanisms of apoptosis triggered by C2-ceramide and by oxLDL were (at least in part) different, because C2-ceramide-induced apoptosis was calcium-independent, whereas oxLDL-induced apoptosis was calcium-dependent. In conclusion, it is suggested that oxLDL-induced apoptosis is calcium-dependent but independent of the activation of the sphingomyelin-ceramide pathway and that the toxic effect of short chain permeant ceramides is calcium-independent and does not mimic the effect of natural ceramides induced by oxLDL.     

Oxidized low-density lipoprotein induces apoptosis of human endothelial cells by activation of CPP32-like proteases. A mechanistic clue to the 'response to injury' hypothesis

BACKGROUND: Oxidized LDL (oxLDL) is believed to play a key role as a triggering molecule that causes injury to the endothelium as an early event in atherogenesis. However, the mechanisms by which oxLDL injures endothelial cells are entirely unknown. We speculate that oxLDL may activate a cellular suicide pathway that leads to apoptosis. METHODS AND RESULTS: Human umbilical venous endothelial cells (HUVEC) were incubated with increasing doses of native or oxLDL for 18 hours. Apoptosis of HUVEC was measured with an ELISA specific for histone-associated DNA fragments and confirmed with DNA laddering. Native LDL had no effect, but incubation with oxLDL dose-dependently induced apoptosis of HUVEC. Induction of apoptosis by oxLDL was associated with increased CPP32-like protease activity, which is the major enzyme that initiates the proteolytical cascade leading to cell death. Specific inhibition of CPP32 activity completely abrogated oxLDL-induced apoptosis. The antioxidants N-acetylcysteine and the combination of vitamins C and E prevented oxLDL-induced apoptosis, abrogated the enhancement of CPP32-like protease activity, and inhibited the proteolytic cleavage of CPP32 into its active subunit p17. CONCLUSIONS: oxLDL activates the suicide pathway leading to apoptosis of endothelial cells by enhancing CPP32-like protease activity. The oxLDL-mediated activation of CPP32 appears to involve the elaboration of reactive oxygen species. Activation of the cell death effector CPP32 by oxLDL may provide a mechanistic clue to the "response-to-injury" hypothesis of atherogenesis.     

Caspase-mediated cleavage of the ubiquitin-protein ligase Nedd4 during apoptosis

The onset of apoptosis is coupled to the proteolytic activation of a family of cysteine proteases, termed caspases. These proteases cleave their target proteins after an aspartate residue. Following caspase activation during apoptosis, a number of specific proteins have been shown to be cleaved. Here we show that Nedd4, a ubiquitin-protein ligase containing multiple WW domains and a calcium/lipid-binding domain, is also cleaved during apoptosis induced by a variety of stimuli including Fas-ligation, gamma-radiation, tumor necrosis factor-alpha, C-8 ceramide, and etoposide treatment. Extracts from apoptotic cells also generated cleavage patterns similar to that seen in vivo, and this cleavage was inhibited by an inhibitor of caspase-3-like proteases. In vitro, Nedd4 was cleaved by a number of caspases, including caspase-1, -3, -6, and -7. By site-directed mutagenesis, one of the in vitro caspase cleavage sites in mouse Nedd4 was mapped to a DQPD237 downward arrow sequence, which is conserved between mouse, rat, and human proteins. This is the first report demonstrating that an enzyme of the ubiquitin pathway is cleaved by caspases during apoptosis.     

OxLDL-induced cytotoxicity and its inhibition by API0134 in cultured porcine aortic endothelial cells

Protective effects of API0134 on endothelial cells (EC) damaged by oxidatively modified low density lipoprotein (oxLDL) were studied. The results showed that the content of endothelia (ET) and malondialdehyde (MDA) in the media of porcine aortic EC incubated with oxLDL were increased and the cGMP was decreased significantly, and the activity of superoxide dismutase (SOD) was inhibited. The effect of cytotoxicity of oxLDL can be eliminated by API0134. These results suggest that API0134 may protect EC against damages elicited by oxLDL.     

Role of ceramide in cellular senescence

Recently the sphingomyelin cycle, involving the hydrolysis of membrane sphingomyelin by an activated sphingomyelinase to generate ceramide, has emerged as a key pathway in cell differentiation and apoptosis in leukemic and other cell types. Here we investigate a role for this pathway in the senescence of WI-38 human diploid fibroblasts (HDF). We found that endogenous levels of ceramide increased considerably (4-fold) and specifically (compared with other lipids) as cells entered the senescent phase. Investigation of the mechanism of increased ceramide led to the discovery that neutral sphingomyelinase activity is elevated 8-10 fold in senescent cells. There were no changes in sphingomyelinase activity or ceramide levels as HDF entered quiescence following serum withdrawal or contact inhibition. Thus, the activation of the sphingomyelinase/ceramide pathway in HDF is due to senescence and supports the hypotheses that senescence represents a distinct program of cell development that can be differentiated from quiescence. Additional studies disclosed the ability of ceramide to induce a senescent phenotype. Thus, when exogenous ceramide (15 microM) was administered to young WI-38 HDF, it produced endogenous levels comparable to those observed in senescent cells (as determined by metabolic labeling studies). Ceramide concentrations of 10-15 microM inhibited the growth of young HDF and induced a senescent phenotype by its ability to inhibit DNA synthesis and mitogenesis. These concentrations of ceramide also induced retinoblastoma dephosphorylation and inhibited serum-induced AP-1 activation in young HDF, thus recapitulating basic biochemical and molecular changes of senescence. Sphingomyelinase and ceramide may thus be implicated as mediators of cellular senescence.     

The protein kinase C activators phorbol esters and phosphatidylserine inhibit neutral sphingomyelinase activation, ceramide generation, and apoptosis triggered by daunorubicin

To address the role of protein kinase C (PKC) in the regulation of ceramide production, we evaluated the impact of the PKC activators 12-O-tetradecanoylphorbol-13-acetate and phosphatidylserine on the apoptotic signaling pathway triggered by the chemotherapeutic drug daunorubicin. Treatment of U937 and HL-60 cells with 0.5-1 microM daunorubicin induced a greater than 30% activation of neutral sphingomyelinase activity within 4-10 min with concomitant sphingomyelin hydrolysis and ceramide generation. Activation of PKC by 12-O-tetradecanoylphorbol-13-acetate and phosphatidylserine inhibited daunorubicin-induced neutral sphingomyelinase activation, sphingomyelin hydrolysis, ceramide generation, and apoptosis. The apoptotic response could be restored by the addition of 25 microM cell-permeant C6-ceramide. In conclusion, PKC emerges as a potentially critical negative regulator of the anthracycline-activated sphingomyelin-ceramide apoptotic pathway.     

Glutathione regulation of neutral sphingomyelinase in tumor necrosis factor-alpha-induced cell death

Tumor necrosis factor-alpha (TNFalpha)-induced cell death involves a diverse array of mediators and regulators including proteases, reactive oxygen species, the sphingolipid ceramide, and Bcl-2. It is not known, however, if and how these components are connected. We have previously reported that GSH inhibits, in vitro, the neutral magnesium-dependent sphingomyelinase (N-SMase) from Molt-4 leukemia cells. In this study, GSH was found to reversibly inhibit the N-SMase from human mammary carcinoma MCF7 cells. Treatment of MCF7 cells with TNFalpha induced a marked decrease in the level of cellular GSH, which was accompanied by hydrolysis of sphingomyelin and generation of ceramide. Pretreatment of cells with GSH, GSH-methylester, or N-acetylcysteine, a precursor of GSH biosynthesis, inhibited the TNFalpha-induced sphingomyelin hydrolysis and ceramide generation as well as cell death. Furthermore, no significant changes in GSH levels were observed in MCF7 cells treated with either bacterial SMase or ceramide, and GSH did not protect cells from death induced by ceramide. Taken together, these results show that GSH depletion occurs upstream of activation of N-SMase in the TNFalpha signaling pathway. TNFalpha has been shown to activate at least two groups of caspases involved in the initiation and "execution" phases of apoptosis. Therefore, additional studies were conducted to determine the relationship of GSH and the death proteases. Evidence is provided to demonstrate that depletion of GSH is dependent on activity of interleukin-1beta-converting enzyme-like proteases but is upstream of the site of action of Bcl-2 and of the execution phase caspases. Taken together, these studies demonstrate a critical role for GSH in TNFalpha action and in connecting major components in the pathways leading to cell death.     

Subspecific differentiation of Burkholderia cepacia isolates in cystic fibrosis

While ceramide has emerged as a potent signal transducer, inconsistencies in the kinetics of ceramide generation, or its absence, in response to stimuli have led to confusion and skepticism as to its potential role in apoptosis or proliferation. Here we show that in U937 and HL60 myeloid leukemia cells and in normal skin fibroblasts, cell-permeant ceramides can trigger neutral sphingomyelinase activation, sphingomyelin hydrolysis, and endogenous ceramide generation regardless of Bcl2 overexpression. These observations identify neutral sphingomyelinase as a novel target for ceramide and show that this positive feedback mechanism is responsible for signal propagation, as exemplified by mitogen-activated protein kinase activation in daunorubicin-treated cells. This study provides insight into a fundamental process of cell biology. Indeed, such a sustained ceramide-mediated signal throughout the apoptotic process would ensure self-destruction, perhaps by overriding evolutionary conserved primal cell survival mechanisms.     

Involvement of both caspase-like proteases and serine proteases in apoptotic cell death induced by ricin, modeccin, diphtheria toxin, and pseudomonas toxin

We investigated the involvement of caspases and serine proteases in apoptotic cell death induced by ricin, modeccin, diphtheria toxin, and Pseudomonas toxin in U937 cells. We found that caspase-3- and caspase-6-like activities, but not caspase-1-like activity, increased during toxin-induced apoptosis. Z-D-CH2-DCB, a caspase-like inhibitor, completely inhibited the generation of caspase-3- and caspase-6-like activities and blocked all features of apoptosis induced by toxins: nuclear morphological changes, DNA fragmentation, and cytotoxicity. However, three caspase-specific inhibitors, Ac-YVAD-CHO, Ac-DEVD-CHO, and Ac-VEID-CHO, had no effect, even though Ac-DEVD-CHO and Ac-VEID-CHO inhibited the increased caspase-3- and caspase-6-like activity, respectively. These results suggest that the generation of caspase-3- and caspase-6-like activities is redundant, and other caspases distinct from caspase-3 and -6 may be important in toxin-induced apoptosis. Furthermore, serine protease inhibitor, 3,4-dichloroisocoumarine (DCI), abolished the apoptotic cell death and DNA fragmentation caused by toxins, without affecting the increased caspase-3- and caspase-6-like activities. Our results suggest that multiple proteases with different preferences for apoptotic substrates participate in toxin-induced apoptotic death of U937 cells.     

Caspase-dependent ceramide production in Fas- and HLA class I-mediated peripheral T cell apoptosis

We recently demonstrated that the engagement of HLA class I alpha1 domain induced Fas-independent apoptosis in human T and B lymphocytes. We analyzed the signaling pathway involved in HLA class I-mediated apoptosis in comparison with Fas (APO-1, CD95)-dependent apoptosis. The mouse mAb90 or the rat YTH862 monoclonal antibodies which bind the human HLA class I alpha1 domain induced the production of ceramide which was blocked by addition of the phosphatidylcholine-dependent phospholipase C inhibitor, D609. Furthermore, HLA class I-mediated apoptosis involved at least two different caspases, an interleukin-1 converting enzyme-like protease and another protease inhibited by the CPP32-like protease inhibitor Ac-DEVD-CHO. Despite similarity between Fas and HLA class I signaling pathways, we failed to demonstrate any physical association between these two molecules. We also report that the pan-caspase inhibitory peptide zVAD-fmk, but not Ac-DEVD-CHO and Ac-YVAD-CHO, inhibited decrease of mitochondrial transmembrane potential and generation of ceramide induced by anti-HLA class I and anti-Fas monoclonal antibodies, whereas all three peptides efficiently inhibited apoptosis. Altogether these results suggest that signaling through Fas and HLA class I involve caspase(s), targeted by zVAD-fmk, which act upstream of ceramide generation and mitochondrial events, whereas interleukin-1 converting enzyme-like and CPP32-like proteases act downstream of the mitochondria.     

Effect of antioxidant in endothelial cells exposed to oxidized low-density lipoproteins

Antioxidants such as probucol and alpha-tocopherol have been shown to attenuate the oxidation of low-density lipoproteins (LDL) and atherosclerotic lesions in animal models of atherosclerosis. The purpose of this study is to determine the protection effect of antioxidants on endothelial cells when exposed to oxidized and native LDL. In a cell-free system, we found that probucol, alpha-tocopherol, and ascorbic acid inhibited copper-induced LDL oxidation by a dose-dependent fashion (from 1 microM to 10 mM). In porcine aortic endothelial cells, antioxidants alone did not change basal endothelin-1 (ET-1) secretion. When porcine aortic endothelial cells were exposed to LDL and oxidized-LDL, both of them stimulated ET-1 secretion dose-dependently, whereas oxidized-LDL elicited higher ET-1 secretion. However, probucol, alpha-tocopherol, and ascorbic acid did not prevent LDL or oxidized-LDL induced ET-1 secretion. Furthermore, nimodipine inhibited both of native and oxidized LDL induced ET-1 secretion. Since Ca2+ channel blocker reduced the elevation of induced ET-1 secretion, the [Ca2+]i is possibly involved for the regulation of ET-1 secretion. Our results suggest that antioxidants can only prevent the oxidation of LDL rather than oxidized and native LDL-induced ET-1 secretion in vascular endothelial cells. The increase in the [Ca2+]i of endothelial cells through the opening of voltage-dependent Ca2+ channels may be involved in the LDL-induced ET-1 release.     

Activation of CD95 (APO-1/Fas) signaling by ceramide mediates cancer therapy-induced apoptosis

We report here that anticancer drugs such as doxorubicin lead to induction of the CD95 (APO-1/Fas) system of apoptosis and the cellular stress pathway which includes JNK/SAPKs. Ceramide, which accumulates in response to different types of cellular stress such as chemo- and radiotherapy, strongly induced expression of CD95-L, cleavage of caspases and apoptosis. Antisense CD95-L as well as dominant-negative FADD inhibited ceramide- and cellular stress-induced apoptosis. Fibroblasts from type A Niemann-Pick patients (NPA), genetically deficient in ceramide synthesis, failed to up-regulate CD95-L expression and to undergo apoptosis after gamma-irradiation or doxorubicin treatment. In contrast, JNK/SAPK activity was still inducible by doxorubicin in the NPA cells, suggesting that activation of JNK/SAPKs alone is not sufficient for induction of the CD95 system and apoptosis. CD95-L expression and apoptosis in NPA fibroblasts were restorable by exogenously added ceramide. In addition, NPA fibroblasts undergo apoptosis after triggering of CD95 with an agonistic antibody. These data demonstrate that ceramide links cellular stress responses induced by gamma-irradiation or anticancer drugs to the CD95 pathway of apoptosis.     

Multiple pathways originate at the Fas/APO-1 (CD95) receptor: sequential involvement of phosphatidylcholine-specific phospholipase C and acidic sphingomyelinase in the propagation of the apoptotic signal

The early signals generated following cross-linking of Fas/APO-1, a transmembrane receptor whose engagement by ligand results in apoptosis induction, were investigated in human HuT78 lymphoma cells. Fas/APO-1 cross-linking by mAbs resulted in membrane sphingomyelin hydrolysis and ceramide generation by the action of both neutral and acidic sphingomyelinases. Activation of a phosphatidylcholine-specific phospholipase C (PC-PLC) was also detected which appeared to be a requirement for subsequent acidic sphingomyelinase (aSMase) activation, since PC-PLC inhibitor D609 blocked Fas/APO-1-induced aSMase activation, but not Fas/APO-1-induced neutral sphingomyelinase (nSMase) activation. Fas/APO-1 cross-linking resulted also in ERK-2 activation and in phospholipase A2 (PLA2) induction, independently of the PC-PLC/aSMase pathway. Evidence for the existence of a pathway directly involved in apoptosis was obtained by selecting HuT78 mutant clones spontaneously expressing a newly identified death domain-defective Fas/APO-1 splice isoform which blocks Fas/APO-1 apoptotic signalling in a dominant negative fashion. Fas/APO-1 cross-linking in these clones fails to activate PC-PLC and aSMase, while nSMase, ERK-2 and PLA2 activates are induced. These results strongly suggest that a PC-PLC/aSMase pathway contributes directly to the propagation of Fas/APO-1-generated apoptotic signal in lymphoid cells.     

Superoxide dismutase mimetics inhibit neutrophil-mediated human aortic endothelial cell injury in vitro

In this study, we evaluated the ability of low molecular weight manganese-based superoxide dismutase mimetics to attenuate neutrophil-mediated oxygen radical damage to human aortic endothelial cells in vitro. Human neutrophils, when exposed to tumor necrosis factor-alpha and the complement compound C5a, induced endothelial damage assessed by the release of 51Cr into the medium. This damage correlated with the amount of superoxide generated by neutrophils. Three superoxide dismutase mimetics, with catalytic rate constants for superoxide dismutation ranging from 4 to 9 x 10(7) M-1 S-1, inhibited neutrophil- or xanthine oxidase-mediated endothelial cell injury in a concentration-dependent manner. A similar manganese-based compound with no detectable superoxide dismutase activity was ineffective in inhibiting injury. Fluorescent studies of the neutrophil respiratory burst showed that the superoxide dismutase mimetics were protective without interfering with the generation of superoxide by activated neutrophils. Catalase, elastase inhibitors, and desferrioxamine mesylate (an iron chelator and hydroxyl radical scavenger) were not protective against cell injury. This investigation demonstrates that neutrophil-mediated human aortic endothelial cell injury in vitro is mediated by the superoxide anion and that low molecular weight manganese-based superoxide dismutase mimetics are effective in abrogating this damage.     

Activation of mitochondria and release of mitochondrial apoptogenic factors by betulinic acid

Different classes of anticancer drugs may trigger apoptosis by acting on different subcellular targets and by activating distinct signaling pathways. Here, we report that betulinic acid (BetA) is a prototype cytotoxic agent that triggers apoptosis by a direct effect on mitochondria. In isolated mitochondria, BetA directly induces loss of transmembrane potential independent of a benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone-inhibitable caspase. This is inhibited by bongkrekic acid, an agent that stabilizes the permeability transition pore complex. Mitochondria undergoing BetA-induced permeability transition mediate cleavage of caspase-8 (FLICE/MACH/Mch5) and caspase-3 (CPP32/Yama) in a cell-free system. Soluble factors such as cytochrome c or apoptosis-inducing factor released from BetA-treated mitochondria are sufficient for cleavage of caspases and nuclear fragmentation. Addition of cytochrome c to cytosolic extracts results in cleavage of caspase-3, but not of caspase-8. However, supernatants of mitochondria, which have undergone permeability transition, and partially purified apoptosis-inducing factor activate both caspase-8 and caspase-3 in cytosolic extracts and suffice to activate recombinant caspase-8. These findings show that induction of mitochondrial permeability transition alone is sufficient to trigger the full apoptosis program and that some cytotoxic drugs such as BetA may induce apoptosis via a direct effect on mitochondria.     

A caspase-9 variant missing the catalytic site is an endogenous inhibitor of apoptosis

It is likely that endogenous inhibitors of the apical caspases such as caspase-9 exist to prevent undesirable activation of caspase cascades. A naturally occurring variant of caspase-9 named caspase-9S was cloned from human liver. Caspase-9S is missing most of the large subunit of caspase-9, including the catalytic site, but has the intact prodomain and small subunit. Caspase-9S did not show apoptotic activity in transfection analysis. Overexpression of caspase-9S inhibited apoptosis induced by caspase-9, indicating that caspase-9S is an endogenous dominant-negative of caspase-9. Moreover, caspase-9S inhibited apoptosis induced by tumor necrosis factor(TNF)-alpha, TNF factor-related apoptosis-inducing ligand (TRAIL), Bax, or Fas-associated death domain-containing protein (FADD) as well as the combination of Apaf-1 and caspase-9. In vitro binding assays demonstrated that caspase-9S binds to Apaf-1 and blocks the binding of caspase-9 to Apaf-1. Coexpression of caspase-9 and caspase-9S mRNA was identified in various cell lines. Thus, caspase-9S acts as a dominant-negative inhibitor of caspase-9 activation, at least in part, by blocking Apaf-1-caspase-9 interaction.     

Tumor necrosis factor induces ceramide oscillations and negatively controls sphingolipid synthases by caspases in apoptotic Kym-1 cells

The role, origin, and mode of action of the lipid messenger ceramide in programmed cell death and its linkage to receptor-associated apoptotic signal proteins is still unresolved. We show here in Kym-1 rhabdomyosarcoma cells that tumor necrosis factor (TNF)-induced apoptosis is preceded by a multiphasic increase in intracellular ceramide levels. Distinct enzymes were found to contribute to three waves of ceramide, neutral sphingomyelinase, ceramide synthase, and acid sphingomyelinase, with peak activities at 1-2, 40, and around 200 min, respectively, the latter coinciding with progression to irreversible damage. In parallel with ceramide generation, TNF-mediated inhibition of glucosylceramide and sphingomyelin (SM) synthase prevents the immediate metabolization of this lipid mediator. In the presence of benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-fmk) or benzyloxycarbonyl-Asp-Glu-Val-Asp-chloromethyl ketone (Z-DEVD-cmk), a broad spectrum and a caspase 3-selective inhibitor, respectively, glucosylceramide and SM synthase activity remains unaffected by TNF, and intracellular ceramide accumulation is not observed. Our results show that several lipid enzymes contribute to generation of ceramide in response to TNF and identify glucosylceramide and SM synthase as important regulators of the kinetics and magnitude of intracellular ceramide accumulation. As glucosylceramide and SM synthase activity is caspase-sensitive, our data suggest a novel functional link between caspase(s) and ceramide during apoptotic processes.     

Fas-mediated apoptosis in mouse hepatocytes involves the processing and activation of caspases

The mechanism of Fas antigen-induced hepatocyte apoptosis was investigated. Using a monoclonal antibody directed against the Fas antigen, apoptosis was induced in freshly isolated murine hepatocytes within 90 minutes of antibody addition as assessed by plasma membrane bleb formation, chromatin condensation, and DNA fragmentation. Pretreatment of the cells with the caspase inhibitors, N-acetyl-Asp-Glu-Val-Asp aldehyde (Ac-DEVD-CHO), benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD-FMK), or Z-Asp-2,6-dichlorobenzoyloxymethylketone inhibited anti-Fas-mediated apoptosis. Likewise, the serine protease inhibitors, N-tosyl-L-phenyl chloromethyl ketone (TPCK) and 3,4-dichloroisocoumarin (DCI), prevented apoptosis, whereas N-tosyl-L-lysine chloromethyl ketone (TLCK), Ac-Leu-Leu-L-norleucinal, Ac-Leu-Leu-L-methional, and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane were without effect. Examination of CED-3/caspase-3-related caspases revealed that pro-caspases-3 (CPP32) and -7 (Mch-3alpha) were rapidly processed after Fas antigen stimulation. Caspase-7 was further cleaved to form the catalytically active subunits. In contrast, the p17 subunit of caspase-3 was not detected, indicating slow formation or rapid degradation. The activation of CED-3-related caspases was further confirmed by an increase in the rate of Z-DEVD-7-amino-4-trifluoromethylcoumarin (Z-DEVD-AFC) hydrolysis that was sensitive to Ac-DEVD-CHO and was inhibited by pretreatment of the cells with TPCK but not by DCI. In contrast, no increase in the rates of hydrolysis of Z-YVAD-AFC, a substrate for caspase-1, was detected. Investigation of the in situ proteolytic cleavage of the CED-3 related caspases substrate, poly(ADP-ribose) polymerase, revealed that this protein was not degraded in hepatocytes undergoing Fas-mediated apoptosis. Taken together, our results show that processing of caspases, in particular, caspases-7 and -3, occurs during Fas-induced apoptosis of mouse hepatocytes and suggest a role of these proteases as well as serine protease(s) in the apoptotic response.     

Caspases mediate 6-hydroxydopamine-induced apoptosis but not necrosis in PC12 cells

The neurotoxin 6-hydroxydopamine (6-OHDA) induces apoptosis in the rat phaeochromocytoma cell line PC12. 6-OHDA-induced apoptosis is morphologically indistinguishable from serum deprivation-induced apoptosis. Exposure of PC12 cells to a low concentration of 6-OHDA (25 microM) results in apoptosis, whereas an increased concentration (50 microM) results in a mixture of apoptosis and necrosis. We investigated the involvement of caspases in the apoptotic death of PC12 cells induced by 6-OHDA, using a general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), and compared this with serum deprivation-induced apoptosis, which is known to involve caspases. We show that zVAD-fmk (100 microM) completely prevented the apoptotic morphology of chromatin condensation induced by exposure to either 6-OHDA (25 and 50 microM) or serum deprivation. Furthermore, cell lysates from 6-OHDA-treated cultures showed cleavage of a fluorogenic substrate for caspase-3-like proteases (caspase-2, 3, and 7), acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin, and this was inhibited by zVAD-fmk. However, although zVAD-fmk restored total cell viability to serum-deprived cells or cells exposed to 25 microM 6-OHDA, the inhibitor did not restore viability to cells exposed to 50 microM 6-OHDA. These data show the involvement of a caspase-3-like protease in 6-OHDA-induced apoptosis and that caspase inhibition is sufficient to rescue PC12 cells from the apoptotic but not the necrotic component of 6-OHDA neurotoxicity.     

Cyclosporin A inhibits apoptosis of human endothelial cells by preventing release of cytochrome C from mitochondria

BACKGROUND: Several experimental and clinical studies suggest that cyclosporin A (CSA) treatment reduces transplant atherosclerosis. Because oxidized LDL (oxLDL) is believed to play a key role in the development of atherogenesis, causing injury to the endothelium, and has been shown to induce apoptosis of endothelial cells, we investigated whether CSA inhibits oxLDL-induced apoptosis. METHODS AND RESULTS: Apoptosis was induced in human umbilical venous endothelial cells (HUVECs) by incubation of 10 microg/mL oxLDL for 18 hours. Coincubation with CSA dose dependently decreased oxLDL-induced apoptosis, with a maximal effect at 10 micromol/L. In addition, tumor necrosis factor-alpha- and angiotensin II-induced apoptosis was significantly prevented by CSA treatment, suggesting a general apoptosis-suppressive effect of CSA. CSA has been shown to inhibit disruption of the mitochondrial membrane function, which plays a key role in apoptosis induction. Indeed, oxLDL treatment triggered the release of cytochrome C from the mitochondria into the cytosol, indicating disturbance of the mitochondrial membrane. CSA (10 micromol/L) completely inhibited the oxLDL-induced release of cytochrome C. Moreover, tumor necrosis factor-alpha- and angiotensin II-induced cytochrome C release was prevented by CSA treatment. CONCLUSIONS: OxLDL induces dysfunction of the mitochondrial membrane, leading to cytochrome C release into the cytosol, and thereby stimulates apoptosis of human endothelial cells. Apoptosis suppression by CSA correlates with the prevention of mitochondrial dysfunction and thus indicates the importance of mitochondrial destabilization in oxLDL-induced apoptosis signaling. The inhibition of apoptosis by CSA might preserve the function of the endothelium and may at least in part contribute to the antiatherogenic effects of CSA in transplant atherosclerosis.