Activation of apoptotic caspase-3 and nitric oxide synthase-2 in buccal mucosa with chronic alcohol ingestion

Apoptosis, the process of programmed cell death, involves activation of caspase proteases cascade that remains under the regulatory control of nitric oxide. In this study, we investigated the activity of a key apoptotic protease, caspase-3, and the expression of nitric oxide synthase-2 (NOS-2) associated with buccal epithelial cells apoptosis induced by chronic ethanol diet. The assays revealed that a 7.9-fold enhancement in buccal epithelial cells apoptosis, observed in the alcohol diet group, was accompanied by a 37.6-fold increase in caspase-3 activity and a 10.1-fold increase in NOS-2. Furthermore, the expression of NOS-2 showed a positive correlation (r = 0.92) with the extent of changes induced in caspase-3 activity. These results implicate caspase-3 in the process of alcohol-induced epithelial cells apoptosis, and point towards participation of NOS-2 in the amplification of the cell death signaling cascade.     

Activation of caspase-3-like protease by digitonin-treated lysosomes

Apoptosis, a naturally occurring programmed cell death or cell 'suicide', has been paid much attention as one of the critical mechanisms for morphogenesis and tissue remodeling. Activation of cysteine aspartases (caspases) is one of the critical steps leading to apoptosis. Although a mitochondria-mediated pathway has been postulated to be one of the activation mechanism of caspase-3, another subcellular compartment might be involved in the activation of the enzyme. The present study shows that the supernatant fraction of digitonin-treated lysosomes strongly activates Ac-DEVD-CHO inhibitable caspase-3-like protease. Activation of caspase-3-like protease by digitonin-treated lysosomal fractions was specifically suppressed by leupeptin and E-64, inhibitors of cysteine protease. These results indicate that leakage of lysosomal cysteine protease(s) into the cytosolic compartment might be involved in the activation of caspase-3-like protease.     

Tricyclic antidepressants induce apoptosis in human T lymphocytes

Apoptosis is a form of programmed cell death that is involved in cell turnover. In the present study we show that the tricyclic antidepressants (TCAS) imipramine, clomipramine and citalopram induce apoptosis in human peripheral lymphocytes. Lymphocytes were incubated with these three drugs for up to 48 h. Apoptosis was characterized by typical nucleosomal DNA fragmentation on agarose gel, as well as quantitated using 4'-6-diamidino-2-phenylindole (DAPI) staining and 3'-OH end-labeling of fragmented DNA at the single cell level. Apoptosis induced by TCAs was shown to be dose-dependent and could be detected after a 24 h incubation. The optimal concentrations of the three TCAs found to induce apoptosis were 50 microM imipramine, 20 microM clomipramine and 180 microM citalopram. Furthermore, immunofluorescence and three-color flow cytometry were used to identify the phenotype of apoptotic cells. TCA-induced apoptosis was shown to involve exclusively T-lymphocytes. Cytotoxic T-lymphocytes were more prone to undergo apoptosis than were T-helper cells. In conclusion, the present investigation clearly demonstrates that TCAs exert cell biological effects upon human T-lymphocytes. Further studies are required to determine the possible clinical relevance of these findings.     

Characterization of the gene cassette required for biosynthesis of the (alpha1-->6)-linked N-acetyl-D-mannosamine-1-phosphate capsule of serogroup A Neisseria meningitidis

Apoptosis, the cellular mechanism of ovarian follicular atresia and luteal regression, is triggered by the activation of a proteolytic cascade of cysteine aspartate-specific proteases (caspases). The principle downstream effector of cell death is caspase-3, but little is known about the role or regulation of this enzyme in ovarian apoptosis. Two substrates of caspase-3, actin and poly(ADP-ribose) polymerase (PARP), are inhibitors of DNase I, which is the endonuclease responsible for ovarian apoptotic DNA degradation. We therefore investigated the proteolytic cleavage of actin and PARP as well as the localization of caspase-3 during follicular atresia (induced by gonadotropin withdrawal) and luteal regression (induced by prostaglandin F2alpha) in the rat ovary. Apoptotic DNA degradation was evident during both follicular atresia and luteal regression, but cleavage of PARP and actin was observed only during luteal regression. Caspase-3 was localized in luteal cells of healthy corpora lutea (CL) and in theca, but not in granulosa cells of healthy follicles. However, caspase-3 immunostaining was evident in granulosa cells of atretic follicles in a pattern similar to that of the localization of granulosa cell death. There was no difference between healthy and apoptotic CL in the distribution or intensity of caspase-3 staining. These results demonstrate that the cleavage of actin and PARP are not necessary for activation of apoptotic DNA degradation during ovarian apoptosis. In addition, the presence of caspase-3 in granulosa cells of atretic, but not healthy, follicles suggests that the expression of this enzyme is regulated by gonadotropin and may be up-regulated as part of the apoptotic process in granulosa cells.     

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.     

IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases

Inhibitor of apoptosis (IAP) gene products play an evolutionarily conserved role in regulating programmed cell death in diverse species ranging from insects to humans. Human XIAP, cIAP1 and cIAP2 are direct inhibitors of at least two members of the caspase family of cell death proteases: caspase-3 and caspase-7. Here we compared the mechanism by which IAPs interfere with activation of caspase-3 and other effector caspases in cytosolic extracts where caspase activation was initiated by caspase-8, a proximal protease activated by ligation of TNF-family receptors, or by cytochrome c, which is released from mitochondria into the cytosol during apoptosis. These studies demonstrate that XIAP, cIAP1 and cIAP2 can prevent the proteolytic processing of pro-caspases -3, -6 and -7 by blocking the cytochrome c-induced activation of pro-caspase-9. In contrast, these IAP family proteins did not prevent caspase-8-induced proteolytic activation of pro-caspase-3; however, they subsequently inhibited active caspase-3 directly, thus blocking downstream apoptotic events such as further activation of caspases. These findings demonstrate that IAPs can suppress different apoptotic pathways by inhibiting distinct caspases and identify pro-caspase-9 as a new target for IAP-mediated inhibition of apoptosis.     

Intracellular K+ suppresses the activation of apoptosis in lymphocytes

Little is known about the mechanisms of suppression of apoptosis. We have addressed the novel possibility that the level of intracellular K+ regulates the apoptotic process by controlling the activity of death enzymes. We show that K+, at normal intracellular levels, inhibits both apoptotic DNA fragmentation and caspase-3(CPP32)-like protease activation, suggesting that intracellular K+ loss must occur early during apoptosis. Direct measurement of K+ by inductively coupled plasma/mass spectrometry and flow cytometry indicates a major decrease in intracellular K+ concentration in the apoptotic cell. Flow cytometric analysis revealed that caspase and nuclease activity were restricted to the subpopulation of cells with reduced K+. Disruption of the natural K+ electrochemical gradient suppressed the activity of both caspase and nuclease independent of the mode of activation of the apoptotic inducing agent, demonstrating that a decrease in intracellular K+ concentration is a necessary, early event in programmed cell death.     

Long-term trends in tuberculosis. Comparison of age-cohort data between Hong Kong and England and Wales

Apoptosis is cellular suicide functionally opposite of mitosis. It plays an important role in tissue growth control and removal of damaged and premalignant cells. The decrease in death suppressor Bcl-2 protein level was implicated in the many types of apoptotic cell death. Because Bcl-2 protein was recently found to be cleaved during apoptosis induced by Fas ligation, IL-3 withdrawal, and alphavirus infection, we assessed whether Bcl-2 protein was also cleaved during the anticancer drug (VP-16)-induced apoptotic cell death in U937 cells. We found that Bcl-2 protein was cleaved in vivo and in vitro after the treatment of VP-16. We also found that caspase-3/CPP32, which was activated after VP-16 treatment, was responsible for the direct cleavage of Bcl-2 protein. The overexpression of the cleaved Bcl-2 fragment increased the sensitivity to VP-16 and promoted apoptotic cell death. Therefore, caspase-3/CPP32 accelerates VP-16-induced U937 cell apoptosis by cleaving death suppressor Bcl-2 protein to produce a death promoter Bcl-2 fragment.     

Involvement of interleukin-1beta-converting enzyme in apoptosis of irradiated retinoblastomas

PURPOSE: To investigate whether interleukin-1beta-converting enzyme (ICE), a mammalian homologue of the Caenorhabditis elegans cell death gene ced-3, is involved in gamma-irradiation-induced apoptosis (programmed cell death) of human retinoblastoma cells. METHODS: The induction of apoptotic cell death in human retinoblastoma cell lines WERI-Rb-1 and Y79 by gamma-irradiation was determined with a modified 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide colorimetric assay and the DNA-binding fluorochrome bis (benzimide) trihydro-chloride (Hoechst 33258) staining. The change of ICE protein level in tumor cells during apoptosis was determined by immunoblotting assay. Whether the specific tetrapeptide ICE inhibitor Ac-YVAD-CMK affected gamma-irradiation-induced apoptosis in tumor cells was also examined. The effect of ICE overexpression on tumor cells was evaluated by a transient transfection assay using ICE expression vector. RESULTS: Gamma-irradiation inhibited the cell viability of WERI-Rb-1 and Y79 cells in a dose-dependent manner and induced apoptosis. The protein level of ICE was remarkably enhanced after the treatment. The apoptotic cell death induced by gamma-irradiation was suppressed by the tetrapeptide ICE inhibitor Ac-YVAD-CMK. Moreover, overexpression of ICE induced apoptosis in tumor cells. CONCLUSIONS: These findings suggest that ICE may play an important role in gamma-irradiation-induced apoptosis in retinoblastoma cells. Transfer of the ICE gene induces apoptosis in these cells without gamma-irradiation.     

Unsymmetrically substituted polyamine analogue induces caspase-independent programmed cell death in Bcl-2-overexpressing cells

The polyamine analogue, N1-ethyl-N11-[(cycloheptyl)methyl]-4,8-diazaundecane (CHENSpm)-induced programmed cell death in NCI H157 cells is accompanied by cytochrome c release, the loss of mitochondrial membrane potential, activation of caspase-3, caspase-mediated poly(ADP-ribose) polymerase cleavage, G2-M arrest, and DNA and nuclear fragmentation. Overexpression of Bcl-2 completely inhibits CHENSpm-induced cytochrome c release, caspase-3 activation, and poly(ADP-ribose) polymerase cleavage. However, Bcl-2 does not abrogate CHENSpm-induced programmed cell death. These results suggest that although cytochrome c release and activation of the caspase-3 protease cascade contribute to the rapid and efficient execution of apoptosis, a caspase cascade-independent pathway also exists and can be activated by CHENSpm treatment.     

In situ immunodetection of activated caspase-3 in apoptotic neurons in the developing nervous system

Activation of caspase-3 requires proteolytic processing of the inactive zymogen into p18 and p12 subunits. We generated a rabbit polyclonal antiserum, CM1, which recognizes the p18 subunit of cleaved caspase-3 but not the zymogen. CM1 demonstrated an apparent specificity for activated caspase-3 by specifically immunolabelling only apoptotic but not necrotic cortical neurons in vitro. In the embryonic mouse nervous system, CM1 immunoreactivity was detected in neurons undergoing programmed cell death and was markedly increased in Bcl-xL-deficient embryos and decreased in Bax-deficient embryos. CM1 immunoreactivity was absent in the nervous system of caspase-3-deficient mouse embryos and in neurons cultured from caspase-3-deficient mice. Along with neuronal somata, extensive neuritic staining was seen in apoptotic neurons. These studies indicate that caspase-3 is activated during apoptosis in the developing nervous system in vivo and that CM1 is a useful reagent for its in situ detection.     

Alphavirus-induced apoptosis in mouse brains correlates with neurovirulence

Sindbis virus induces apoptotic cell death in cultured cell lines, raising the possibility that apoptosis of infected neurons and other target cells in vivo may contribute to the resulting disease and mortality. To investigate the role of apoptosis in Sindbis virus pathogenesis, infected mouse brains were assayed by the in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling technique and for DNA ladder formation. Infection with recombinant Sindbis virus strain 633 resulted in widespread apoptosis in newborn mouse brains and spinal cords, but few apoptotic cells were observed following infection of 2-week-old animals. This finding correlates with the age-dependent mortality observed in mice. The more neurovirulent virus TE, which differs from 633 by a single amino acid in the E2 glycoprotein, induced significant apoptosis in brains and spinal cords of 2-week-old animals, consistent with its ability to cause fatal disease in older animals. Double-labeling experiments demonstrated that the apoptotic cells were also infected with Sindbis virus. Thus, Sindbis virus-induced apoptosis appears to be a result of virus infection and is likely to reflect pathogenic mechanisms for other viruses.     

Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis

Interleukin 1beta-converting enzyme-like proteases (caspases) are crucial components of cell death pathways. Among the caspases identified, caspase-3 stands out because it is commonly activated by numerous death signals and cleaves a variety of important cellular proteins. Studies in caspase-3 knock-out mice have shown that this protease is essential for brain development. To investigate the requirement for caspase-3 in apoptosis, we took advantage of the MCF-7 breast carcinoma cell line, which we show here has lost caspase-3 owing to a 47-base pair deletion within exon 3 of the CASP-3 gene. This deletion results in the skipping of exon 3 during pre-mRNA splicing, thereby abrogating translation of the CASP-3 mRNA. Although MCF-7 cells were still sensitive to tumor necrosis factor (TNF)- or staurosporine-induced apoptosis, no DNA fragmentation was observed. In addition, MCF-7 cells undergoing cell death did not display some of the distinct morphological features typical of apoptotic cells such as shrinkage and blebbing. Introduction of the CASP-3 gene into MCF-7 cells resulted in DNA fragmentation and cellular blebbing following TNF treatment. These results indicate that although caspase-3 is not essential for TNF- or staurosporine-induced apoptosis, it is required for DNA fragmentation and some of the typical morphological changes of cells undergoing apoptosis.     

Apoptosis, cancer and cancer therapy

Apoptosis is a specific process that leads to programmed cell death through the activation of an evolutionary conserved intracellular pathway leading to pathognomic cellular changes distinct from cellular necrosis. Apoptosis is essential in the homeostasis of normal tissues of the body, especially those of the gastrointestinal tract, immune system and skin. There is increasing evidence that the processes of neoplastic transformation, progression and metastasis involve alterations in the normal apoptotic pathways. Furthermore, the majority of chemotherapeutic agents as well as radiation utilize the apoptotic pathway to induce cancer cell death. Resistance to standard chemotherapies also seems to be determined by alterations in the apoptotic pathways of cancer cells. Therefore, understanding the signals of apoptosis and the mechanism of apoptosis may allow the development of better chemo- or radiotherapeutic regimens for the treatment of cancer. Finally, components of the apoptotic pathway may represent potential therapeutic targets using gene therapy techniques.     

Apoptosis is abundant in human atherosclerotic lesions, especially in inflammatory cells (macrophages and T cells), and may contribute to the accumulation of gruel and plaque instability

Death of intimal tissue may lead to plaque rupture with thrombosis, which is the basis of the most severe clinical consequences of atherosclerosis. Little is known about the mechanisms that promote intimal cell death or its nature. This work was undertaken to elucidate the extent to which, the cell types in which, and where programmed cell death, apoptosis, might occur in atherosclerotic lesions. The material was fibrous or fibro-fatty non-ulcerated lesions from the human thoracic aorta and coronary arteries. Apoptosis was indicated by the in situ labeling of internucleosomally degraded DNA with the TUNEL technique, which has a preference for apoptosis as compared with cell necrosis and was combined with the immunohistochemical typing of cells. Apoptosis was corroborated by morphological criteria on the light and electron microscope levels and by the presence of an apoptosis-specific protein. It was common in the lesions and virtually absent in non-atherosclerotic regions. It occurred in smooth muscle cells subendothelially, in places of the fibrous cap, and in the underlying media, which may destabilize the plaque and promote rupture. Inflammatory cells, ie, macrophages and T cells, appeared abundantly subendothelially, in the fibrous cap, and in the shoulder regions, and apoptosis was common, maybe reflecting a means for quenching of the inflammatory reaction. Many macrophages contained abundant apoptotic material indicative of phagocytosis of apoptotic cells, but the occurrence of apoptosis, even in some of these cells, and of apoptotic material extracellularly and the very high numbers of apoptotic cells that were encountered may indicate insufficient mechanisms for the removal of apoptotic cells in the atherosclerotic lesion. It is not possible to decide as yet whether this is due to overloading with cellular material by inflammation and cell multiplication, to an increased frequency of apoptosis, to a reduction of the removal/degradation of apoptotic material by macrophages, or a combination of these factors.     

Caspases are activated in a branched protease cascade and control distinct downstream processes in Fas-induced apoptosis

Two novel synthetic tetrapeptides, VEID-CHO and DMQD-CHO, could selectively inhibit caspase-6 and caspase-3, respectively. We used these inhibitors to dissect the pathway of caspase activation in Fas-stimulated Jurkat cells and identify the roles of each active caspase in apoptotic processes. Affinity labeling techniques revealed a branched protease cascade in which caspase-8 activates caspase-3 and -7, and caspase-3, in turn, activates caspase-6. Both caspase-6 and -3 have major roles in nuclear apoptosis. Caspase-6 cleaves nuclear mitotic apparatus protein (NuMA) and mediates the shrinkage and fragmentation of nuclei. Caspase-3 cleaves NuMA at sites distinct from caspase-6, and mediates DNA fragmentation and chromatin condensation. It is also involved in extranuclear apoptotic events: cleavage of PAK2, formation of apoptotic bodies, and exposure of phosphatidylserine on the cell surface. In contrast, a caspase(s) distinct from caspase-3 or -6 mediates the disruption of mitochondrial membrane potential (permeability transition) and the shrinkage of cytoplasm. These findings demonstrate that caspases are organized in a protease cascade, and that each activated caspase plays a distinct role(s) in the execution of Fas-induced cell death.     

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.     

Requirement of caspase-3(-like) protease-mediated hydrogen peroxide production for apoptosis induced by various anticancer drugs

Caspase-3(-like) proteases play important roles in controlling mammalian apoptosis. However, the downstream events from the caspase-3(-like) protease activation to death of cells are still unclear. Previously, we reported that hydrogen peroxide (H2O2) was generated by the activation of caspase-3(-like) proteases in the process of tyrosine kinase inhibitor-induced apoptosis in human small cell lung carcinoma Ms-1 cells. In the present study, we examined whether generation of H2O2 is a critical event for the apoptotic pathway downstream of caspase-3(-like) protease activation by various anticancer drugs. Anticancer drugs such as camptothecin, vinblastine, inostamycin, and adriamycin induced activation of caspase-3(-like) proteases and apoptosis. Generation of H2O2 was commonly detected after treatment with each of the four anticancer drugs, and scavenging of H2O2 caused cells to fail to undergo apoptosis. Moreover, anticancer drug-induced H2O2 production was inhibited not only by an inhibitor of caspase-3(-like) proteases but also by diphenyleneiodonium chloride, an inhibitor of flavonoid-containing enzymes such as NADPH oxidase. However, activation of caspase-3(-like) proteases was not inhibited by diphenyleneiodonium chloride. These findings suggest that activation of caspase-3(-like) proteases by various anticancer drugs causes generation of H2O2 presumably through the activation of NADPH oxidase, thereby inducing apoptosis. Therefore, H2O2 may function as a common mediator for apoptosis induced by various anticancer drugs.     

Histamine does not potentiate cyclic AMP-mediated amylase secretion in the guinea-pig pancreatic acinar cells

Some chemotherapeutic agents, as well as TNF and Fas, induce apoptotic cell death in tumor cells, but the cellular components involved in the process have not yet been identified. Interleukin 1 beta converting enzyme (ICE) is a mammalian homolog of CED-3, a protein required for programmed cell death in nematode Caenorhabditis elegans. We found that a selective inhibitor of ICE/ced 3 family proteases, benzyloxycarbonyl Asp CH2OC(O) 2 6,-dichlorobenzene (Z-Asp-CH2-DCB). completely blocked the apoptotic cell death of human leukemia cells caused by etoposide, camptothecin, 1-beta-D-arabinofuranosyl cytosine (Ara-C) and adriamycin. Moreover, in antitumor agent-treated U937 cells, an ICE-like (CPP32-like) protease was strongly activated. These results indicate that ICE/ ced 3 family proteases are involved in antitumor agent-induced apoptosis. Activation of ICE family proteases plays a key role in apoptosis. However, the subsequent mechanisms resulting in apoptosis are largely unknown. We identified actin as a substrate of ICE family proteases. Cleavage of actin and other substrate proteins by ICE family proteases could be critical in the ongoing process of antitumor agent-induced apoptosis in tumor cells.     

Fas-induced arachidonic acid release is mediated by Ca2+-independent phospholipase A2 but not cytosolic phospholipase A2, which undergoes proteolytic inactivation

Fas-mediated apoptosis of human leukemic U937 cells was accompanied by increased arachidonic acid (AA) and oleic acid release from membrane glycerophospholipids, indicating phospholipase A2 (PLA2) activation. During apoptosis, type IV cytosolic PLA2 (cPLA2), a PLA2 isozyme with an apparent molecular mass of 110 kDa critical for stimulus-coupled AA release, was converted to a 78-kDa fragment with concomitant loss of catalytic activity. Cleavage of cPLA2 correlated with increased caspase-3-like protease activity in apoptotic cells and was abrogated by a caspase-3 inhibitor. A mutant cPLA2 protein in which Asp522 was replaced by Asn, which aligns with the consensus sequence of the caspase-3 cleavage site (DXXD downward arrowX), was resistant to apo-ptosis-associated proteolysis. Moreover, a COOH-terminal deletion mutant of cPLA2 truncated at Asp522 comigrated with the 78-kDa fragment and exhibited no enzymatic activity. Thus, caspase-3-mediated cPLA2 cleavage eventually leads to destruction of a catalytic triad essential for cPLA2 activity, thereby terminating its AA-releasing function. In contrast, the activity of type VI Ca2+-independent PLA2 (iPLA2), a PLA2 isozyme implicated in phospholipid remodeling, remained intact during apoptosis. Inhibitors of iPLA2, but neither cPLA2 nor secretory PLA2 inhibitors, suppressed AA release markedly and, importantly, delayed cell death induced by Fas. Therefore, we conclude that iPLA2-mediated fatty acid release is facilitated in Fas-stimulated cells and plays a modifying although not essential role in the apoptotic cell death process.