Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.22.56 (caspase-3)
35,750 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bcl-xL, an antiapoptotic member of the Bcl-2 family, inhibits programmed cell death in a broad variety of cell types. Recent reports have demonstrated that cytochrome c is released from mitochondria during apoptosis and have suggested that this release may be a critical step in the activation of proapoptotic caspases and subsequent cell death. Furthermore, it has been demonstrated that Bcl-2 can prevent the release of cytochrome c from mitochondria in cells triggered to undergo apoptosis. This has led to the hypothesis that the antiapoptotic effects of Bcl-2 family members are due specifically to their ability to prevent cytochrome c release thus preventing subsequent cytochrome c-dependent caspase activation. In the present report, we use microinjection techniques to investigate the relationship between cytochrome c release, induction of apoptosis, and Bcl-xL activity in intact cells. We demonstrate that microinjection of cytochrome c into the cytosol of human kidney 293 cells results in a dose-dependent induction of apoptosis. In contrast, MCF7 breast carcinoma cells (stably transfected to express the Fas antigen CD95, and denoted MCF7F) that lack detectable levels of caspase 3 (CPP32), are totally resistant to microinjection of cytochrome c. However, transfection of MCF7F cells with an expression plasmid coding for pro-caspase 3, but not other pro-caspases, restores cytochrome c sensitivity. Although MCF7F cells are insensitive to cytochrome c microinjection, they rapidly undergo apoptosis in a caspase-dependent manner in response to either tumor necrosis factor or anti-Fas plus cycloheximide, and these deaths are strongly inhibited by Bcl-xL expression. Furthermore, microinjection of cytochrome c does not overcome these antiapoptotic effects of Bcl-xL. Our results support the concept that the release of cytochrome c into the cytoplasm can promote the apoptotic process in cells expressing pro-caspase 3 but that cytochrome c release is not sufficient to induce death in all cells. Importantly, the ability of Bcl-xL to inhibit cell death in the cytochrome c-insensitive MCF7F cells cannot be due solely to inhibition of cytochrome c release from mitochondria.
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PMID:Cell-specific induction of apoptosis by microinjection of cytochrome c. Bcl-xL has activity independent of cytochrome c release. 937 16

Nitric oxide (NO) has emerged as an important endogenous inhibitor of apoptosis, and here we report that NO prevents hepatocyte apoptosis initiated by the removal of growth factors or exposure to TNFalpha or anti-Fas antibody. We postulated that the mechanism of the inhibition of apoptosis by NO would include an effect on caspase-3-like protease activity. Caspase-3-like activity increased coincident with apoptosis due to all three stimuli, and treatment with the caspase-3-like protease inhibitor N-acetyl-Asp-Glu-Val-Asp-aldehyde inhibited both proteolytic activity and apoptosis. Endogenous or exogenous sources of NO prevented the increase in caspase-3-like activity in hepatocytes. Exposure of purified recombinant caspase-3 to an NO or NO+ donor inhibited proteolytic activity. Dithiothreitol (DTT), but not glutathione, reversed the inhibition of recombinant caspase-3 by NO. When lysates from cells stimulated to express inducible NO synthase or cells exposed to NO donors were incubated in DTT, caspase-3-like activity increased to about 55% of cells not exposed to a source of NO. Similarly, administration of an NO donor to rats treated with TNFalpha and D-galactosamine also prevented the increase in caspase-3-like activity as measured in liver homogenates. The effect of the NO donor was reversed by about 50% if the homogenate was incubated with DTT. TNFalpha-induced apoptosis and caspase-3-like activity were also reduced in cultured hepatocytes exposed to 8-bromo-cGMP, and both effects were inhibited by the cGMP-dependent kinase inhibitor KT5823. The suppression in caspase-3-like activity in hepatocytes exposed to an NO donor was partially blocked by an inhibitor of soluble guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4,3, -a]quinoxalin-1-one, (ODQ), while the incubation of these lysates in DTT almost completely restored caspase-3-like activity to the level of TNFalpha-treated controls. These data indicate that NO prevents apoptosis in hepatocytes by either directly or indirectly inhibiting caspase-3-like activation via a cGMP-dependent mechanism and by direct inhibition of caspase-3-like activity through protein S-nitrosylation.
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PMID:Nitric oxide inhibits apoptosis by preventing increases in caspase-3-like activity via two distinct mechanisms. 938 67

Caspases are a family of cysteine proteases implicated in the biochemical and morphological changes that occur during apoptosis (programmed cell death). The loop domain of Bcl-2 is cleaved at Asp34 by caspase-3 (CPP32) in vitro, in cells overexpressing caspase-3, and after induction of apoptosis by Fas ligation and interleukin-3 withdrawal. The carboxyl-terminal Bcl-2 cleavage product triggered cell death and accelerated Sindbis virus-induced apoptosis, which was dependent on the BH3 homology and transmembrane domains of Bcl-2. Inhibitor studies indicated that cleavage of Bcl-2 may further activate downstream caspases and contribute to amplification of the caspase cascade. Cleavage-resistant mutants of Bcl-2 had increased protection from interleukin-3 withdrawal and Sindbis virus-induced apoptosis. Thus, cleavage of Bcl-2 by caspases may ensure the inevitability of cell death.
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PMID:Conversion of Bcl-2 to a Bax-like death effector by caspases. 939 3

The proliferation and survival of a B cell population is necessarily tightly controlled to prevent the arisal of malignancy or autoimmunity. The expansion or elimination of a B cell clone is determined through a complex interaction of the tumour necrosis factor receptor/nerve growth factor receptor family members CD40 and Fas, which are expressed on the B cell surface, with their respective physiological ligands (CD40L and FasL) expressed on the surface of CD4+ T cells. The regulation of B cell growth by signals transduced through CD40 and Fas contributes to the maintenance of peripheral tolerance and likely takes place and in the germinal centres (GC) of secondary lymphoid tissues. In this study, we investigate the relationship between the expression of Fas and B cell survival following engagement of CD40 and Fas in the Epstein-Barr virus-genome-negative Ramos-Burkitt lymphoma (Ramos-BL) B cell line model of GC B lymphocyte selection during maturation of the humoral immune response. We now present evidence that Ramos-BL B cells constitutively express both Fas and FasL on their surface and that expression of Fas, but not FasL, is enhanced following ligation of CD40. Coligation of CD40 and Fas, triggers for growth inhibition, activation of the interleukin-1 beta-converting enzyme, now caspase, family member CPP32 (caspase-3) but not Ich-1L (caspase-2), cleavage of its death substrate poly(ADP-ribose) polymerase, and apoptosis from the G1 phase of cell cycle; engagement of Fas alone fails to trigger for growth inhibition and apoptosis but enhances AgR-mediated cellular death. This CD40-potentiated Fas-triggered growth inhibition and apoptosis occurs in the presence of CD40-induced expression of the anti-apoptotic proteins Bcl-xL and Bcl-2. Taken together, these data indicate that ligation of CD40 facilitates efficient coupling of Fas to the caspase-mediated pathway of apoptosis.
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PMID:Ligation of CD40 potentiates Fas-mediated activation of the cysteine protease CPP32, cleavage of its death substrate PARP, and apoptosis in Ramos-Burkitt lymphoma B cells. 939 1

It has recently been proposed that doxorubicin (DOX) can induce apoptosis in human T-leukemia cells via the Fas/FasL system in an autocrine/paracrine way. We show here that treatment of Jurkat cells with either anti-Fas antibodies, anthracyclin drugs or actinomycin D induces the activation of CPP32 (caspase-3) and apoptosis. However, DOX treatment did not induce the expression of membrane FasL or the release of soluble FasL and co-incubation with blocking anti-Fas antibodies prevented Fas-induced but not DOX-induced apoptosis. All the morphological and biochemical signs of apoptosis induced by anti-Fas or DOX can be prevented by Z-VAD-fmk, a general caspase inhibitor. DEVD-cho, a specific inhibitor of CPP32-like caspases which completely blocks Fas-mediated apoptosis, prevented drug-induced nuclear apoptosis but not cell death. We conclude that: (i) DOX-induced apoptosis in human T-leukemia/lymphoma is Fas-independent and (ii) caspase-3 is responsible of DOX-induced nuclear apoptosis but other Z-VAD-sensitive caspases are implicated in cell death.
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PMID:Doxorubicin-induced apoptosis in human T-cell leukemia is mediated by caspase-3 activation in a Fas-independent way. 940 52

Fas ligand is a potent inducer of apoptosis in human glioma cells by the Fas/Fas ligand pathway. With comparable efficiency, metalloprotease inhibitors including puromycin and bestatin induce apoptosis in glioma cells. To evaluate the involvement of potential components involved in Fas ligand- and metalloprotease inhibitor-induced apoptosis, we investigated the effect of anti human Fas antibody, soluble Fas ligand and puromycin on cultures of human malignant glioma cell lines (LN-18, LN-229, T98G). Stimulation with Fas ligand lead to apoptotic cell death within 16 h. Costimulation with the translational inhibitor cycloheximide and the transcription blocker actinomycin D did not reduce Fas ligand toxicity. In contrast, apoptosis induced by puromycin was blocked by cycloheximide and decreased by subtoxic doses of actinomycin D in all three gliomas. Whereas inhibition of caspase activity with the general inhibitor zVAD-fmk resulted in a complete block of Fas ligand-induced cell death, puromycin-mediated apoptosis was found to be unaffected by zVAD-fmk as well as by more specific inhibitors for caspase-1 (Interleukin-1 beta converting enzyme) and caspase-3 (CPP32/Yama). Other prominent components involved in many apoptotic pathways as bcl-2 and reactive oxygen intermediates were also examined. Bcl-2 which protects glioma cells from Fas ligand-induced cell death, was shown to have only a small protective effect on puromycin-induced apoptosis. The tested radical scavengers did not reduce Fas- or puromycin-mediated killing of human glioma cells.
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PMID:Differential activity of bcl-2 and ICE enzyme family protease inhibitors on Fas and puromycin-induced apoptosis of glioma cells. 940 14

Various molecules such as cytokines and anticancer drugs, as well as factor deprivation, rapidly induce apoptosis (programmed cell death), which is morphologically characterized by cell shrinkage and the blebbing of plasma membranes and by nuclear condensation. Caspases, particularly caspase 3, are proteases that are activated during apoptosis and which cleave substrates such as poly(ADP-ribose) polymerase, actin, fodrin, and lamin. Apoptosis is also accompanied by the internucleosomal degradation of chromosomal DNA. In the accompanying Article, we have identified and molecularly cloned a caspase-activated deoxyribonuclease (CAD) and its inhibitor (ICAD). Here we show that caspase 3 cleaves ICAD and inactivates its CAD-inhibitory effect. We identified two caspase-3 cleavage sites in ICAD by site-directed mutagenesis. When human Jurkat cells were transformed with ICAD-expressing plasmid, occupation of the receptor Fas, which normally triggers apoptosis, did not result in DNA degradation. The ICAD transformants were also resistant to staurosporine-induced DNA degradation, although staurosporine still killed the cells by activating caspase. Our results indicate that activation of CAD downstream of the caspase cascade is responsible for internucleosomal DNA degradation during apoptosis, and that ICAD works as an inhibitor of this process.
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PMID:Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. 2641 41

Induction of apoptosis in keratinocytes by UV light is a critical event in photocarcinogenesis. Although p53 is of importance in this process, evidence exists that other pathways play a role as well. Therefore, we studied whether the apoptosis-related surface molecule CD95 (Fas/APO-1) is involved. The human keratinocyte cell line HaCaT expresses CD95 and undergoes apoptosis after treatment with UV light or with the ligand of CD95 (CD95L). Incubation with a neutralizing CD95 antibody completely prevented CD95L-induced apoptosis but not UV-induced apoptosis, initially suggesting that the CD95 pathway may not be involved. However, the protease CPP32, a downstream molecule of the CD95 pathway, was activated in UV-exposed HaCaT cells, and UV-induced apoptosis was blocked by the ICE protease inhibitor zVAD, implying that at least similar downstream events are involved in CD95- and UV-induced apoptosis. Activation of CD95 results in recruitment of the Fas-associated protein with death domain (FADD) that activates ICE proteases. Immunoprecipitation of UV-exposed HaCaT cells revealed that UV light also induces recruitment of FADD to CD95. Since neutralizing anti-CD95 antibodies failed to prevent UV-induced apoptosis, this suggested that UV light directly activates CD95 independently of the ligand CD95L. Confocal laser scanning microscopy showed that UV light induced clustering of CD95 in the same fashion as CD95L. Prevention of UV-induced CD95 clustering by irradiating cells at 10 degrees C was associated with a significantly reduced death rate. Together, these data indicate that UV light directly stimulates CD95 and thereby activates the CD95 pathway to induce apoptosis independently of the natural ligand CD95L. These findings further support the concept that UV light can affect targets at the plasma membrane, thereby even inducing apoptosis.
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PMID:Ultraviolet light induces apoptosis via direct activation of CD95 (Fas/APO-1) independently of its ligand CD95L. 942 65

Tumor necrosis factor-alpha (TNF-alpha) is a cytokine that induces apoptosis in various cell systems by binding to the TNF receptor (TNFR). To study TNF-alpha-induced apoptosis, we isolated and characterized a novel TNF-alpha-resistant variant, U937/TNF clone UA, from human monocytic leukemia U937 cells. The UA cells resist apoptosis induced by TNF-alpha and anti-Fas antibody but not by anticancer drugs, such as VP-16 and Ara-C. Somatic cell hybridization between U937 and UA showed that apoptosis resistance to TNF-alpha in UA was genetically recessive. The hybridization analysis also showed that UA and another recessive mutant clone, UC, belong to different complementation groups in TNF-alpha-induced apoptosis signaling. In UA cells, TNF-alpha-induced disruption of mitochondrial membrane potential and CPP32 activation were abrogated. Expression of TNFR, Fas, and Bcl-2 family proteins was not changed in UA cells. These results suggest that the apoptosis resistant UA cells could have a functional defect in apoptosis signaling from the TNFR to mitochondria and interleukin-1beta converting enzyme (ICE) family protease activation. UA cells could be used to study signaling linkage between cell death-inducing receptor and mitochondria.
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PMID:Genetically recessive mutant of human monocytic leukemia U937 resistant to tumor necrosis factor-alpha-induced apoptosis. 942 4

Protein kinase C (PKC) has been implicated in signaling induced by diverse sets of stimuli regulating growth, differentiation, and apoptosis. The present study focused on the fate of PKC isotype proteins during Fas-mediated apoptosis of human leukemic cell lines. Among the PKC isotypes expressed in different cell types, such as Jurkat, HPB-ALL, U937, and HL60, all the nPKC isotypes including nPKCdelta, nPKC epsilon, and nPKCtheta, but not cPKC alpha and betaII and aPKCzeta (n, c, and a represent novel, conventional and atypical, respectively), showed limited proteolytic cleavage during Fas-mediated apoptosis. The limited proteolysis of nPKC isotypes means the disappearance of the intact protein band concomitant with the appearance of two fragments, most likely containing the kinase and regulatory domains, in contrast to the so-called down-regulation known for both cPKC and nPKC isotypes following exposure to stimuli such as 12-O-tetradecanoyl-phorbol 13-acetate (TPA). The time course of Fas-mediated apoptosis in Jurkat cells parallels that of the activation of a 32-kDa cysteine protease (CPP32)-like protease and also closely parallels the proteolytic cleavage of nPKC isotypes. A peptide inhibitor of the CPP32-like protease, Ac-DEVD-CHO, blocked the proteolytic cleavage of nPKC isotypes as well as apoptosis mediated by Fas. Transfection of recombinant protein coding for the catalytic fragment of nPKCdelta to COS1 cells resulted in the apoptotic morphology of cells and nuclei. The effect of TPA on apoptosis depends on the cell type. TPA significantly suppressed Fas-mediated apoptosis in Jurkat, whereas TPA alone caused apoptosis in HPB-ALL, U937, and HL60, only slight apoptosis in Jurkat. The proteolytic fragmentation of nPKC isotypes again closely correlated with the degree of apoptosis even in apoptosis induced by TPA. Separation of TPA-treated cells into apoptotic and non-apoptotic differentiating cells revealed that the proteolytic fragmentation of nPKC isotypes occurs only in apoptotic cells and, in adherent differentiating cells, nPKC isotypes as well as cPKC alpha were down-regulated without the generation of nPKC fragments. These results are consistent with the idea that nPKC isotypes meet two different fates, down-regulation and proteolytic cleavage generating kinase and regulatory fragments, and that the proteolytic cleavage of nPKC isotypes is a step in the signaling pathway involved in Fas-mediated and TPA-induced apoptosis.
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PMID:The proteolytic cleavage of protein kinase C isotypes, which generates kinase and regulatory fragments, correlates with Fas-mediated and 12-O-tetradecanoyl-phorbol-13-acetate-induced apoptosis. 943 85


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