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)

Mitochondrial alterations including permeability transition (PT) constitute critical events of the apoptotic cascade and are under the control of Bcl-2 related gene products. Here we show that induction of PT is sufficient to activate CPP32-like proteases with DEVDase activity and the associated cleavage of the nuclear DEVDase substrate poly(ADP-ribose) polymerase (PARP). Thus, direct intervention on mitochondria using a ligand of the mitochondrial benzodiazepin receptor or a protonophore causes DEVDase activation. In addition, the DEVDase activation triggered by conventional apoptosis inducers (glucocorticoids or topoisomerase inhibitors) is prevented by inhibitors of PT. The protease inhibitor N-benzyloxycabonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk) completely prevents the activation of DEVDase and PARP cleavage, as well as the manifestation of nuclear apoptosis (chromatin condensation, DNA fragmentation, hypoploidy). In addition, Z-VAD.fmk delays the manifestation of apoptosis-associated changes in cellular redox potentials (hypergeneration of superoxide anion, oxidation of compounds of the inner mitochondrial membrane, depletion of non-oxidized glutathione), as well as the exposure of phosphatidylserine residues in the outer plasma membrane leaflet. Although Z-VAD.fmk retards cytolysis, it is incapable of preventing disruption of the plasma membrane during protracted cell culture (12-24 h), even in conditions in which it completely blocks nuclear apoptosis (chromatin condensation and DNA fragmentation). Electron microscopic analysis confirms that cells treated with PT inducers alone undergo apoptosis, whereas cells kept in identical conditions in the presence of Z-VAD.fmk die from necrosis. These observations are compatible with the hypothesis that PT would be a rate limiting step in both the apoptotic and the necrotic modes of cell death. In contrast, it would be the availability of apoptogenic proteases that would determine the choice between the two death modalities.
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PMID:The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death. 938 Apr 9

The tumor suppressor p53 has been implicated in apoptosis induction and is mutated in human T-ALL CCRF-CEM cells. To investigate possible consequences of wild-type p53 loss, we reconstituted CEM-C7H2, a subclone of CCRF-CEM, with a temperature-sensitive p53 allele (p53ts). Stably transfected lines expressed high levels of p53ts and shift to the permissive temperature (32 degrees C) caused rapid induction of p53-regulated genes, such as p21(CIP1/WAF1), mdm-2 and bax. This was followed by extensive apoptosis within 24 h to 36 h, supporting the notion that mutational p53 inactivation contributed to the malignant phenotype. p53-dependent apoptosis was preceded by digestion of poly(ADP-ribose) polymerase, a typical target of interleukin-1beta-converting enzyme (ICE)-like proteases/caspases, and was markedly resistant to the ICE/caspase-1 and FLICE/caspase-8 inhibitor acetyl-Tyr-Val-Ala-Asp.chloromethylketone (YVAD), but sensitive to the CPP32/caspase-3 inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp.fluoromethylketone (DEVD) and benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (zVAD), a caspase inhibitor with broader specificity. This indicated an essential involvement of caspases, but argued against a significant role of ICE/caspase-1 or FLICE/caspase-8. Actinomycin D or cycloheximide prevented cell death, suggesting that, in this system, p53-induced apoptosis depends upon macromolecule biosynthesis. Introduction of functional p53 into CEM cells enhanced their sensitivity to the DNA-damaging agent doxorubicin, but not to the tubulin-active compound vincristine. Thus, mutational p53 inactivation in ALL might entail relative resistance to DNA-damaging, but not to tubulin-destabilizing, chemotherapy.
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PMID:p53-induced apoptosis in the human T-ALL cell line CCRF-CEM. 939 39

The effects of the non-tumor-promoting protein kinase C (PKC) activator bryostatin 1 and the PKC inhibitors staurosporine and UCN-01 were examined with respect to modulation of 1-[beta-D-arabinofuranosyl]cytosine (ara-C)-induced apoptosis in human myeloid leukemia cells (HL-60) overexpressing the antiapoptotic protein Bcl-2. HL-60/Bcl-2 cells displayed a 5-fold increase in Bcl-2 protein compared with empty-vector counter-parts (HL-60/pCEP4) but comparable levels of Bax, Mcl-1, and Bcl-xL. After exposure to an equimolar concentration of ara-C (10 microM for 6 hr), HL-60/Bcl-2 cells were significantly less susceptible to apoptosis, DNA fragmentation, and loss of clonogenicity than HL-60/pCEP4 cells. The protective effect of increased Bcl-2 expression was manifested by a failure of ara-C to induce activation/cleavage of the Yama protease (CPP32; caspase-3) and degradation of one of its substrates, poly(ADP-ribose)polymerase to an 85-kDa cleavage product. When HL-60/Bcl-2 cells were preincubated with bryostatin 1 (10 nM; 24 hr) or coincubated with either staurosporine (50 nM; 6 hr) or UCN-01 (300 nM; 6 hr) after a 1-hr preincubation, exposures that exerted minimal effects alone, ara-C-induced apoptosis and DNA fragmentation were restored to levels equivalent to, or greater than, those observed in empty-vector controls. These events were accompanied by restoration of the ability of ara-C to induce CPP32 cleavage and activation, poly(ADP-ribose) polymerase degradation, and inhibition of colony formation. Western analysis of Bcl-2 protein obtained from overexpressing cells treated with bryostatin 1, staurosporine, or UCN-01 revealed the appearance of a slowly migrating species and a general broadening of the protein band, effects that were insensitive to the protein synthesis inhibitor cycloheximide. Alterations in Bcl-2 protein mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were reversed by treatment of lysates with alkaline phosphatase or protein phosphatase 2A; actions of the latter were blocked by the specific phosphatase inhibitor okadaic acid. In vivo labeling studies of Bcl-2 protein demonstrated increased incorporation of [32PO4]orthophosphate in drug-treated cells. Last, phosphorylated Bcl-2 failed to display decreased binding to the proapoptotic protein Bax. Collectively, these findings indicate that bryostatin 1, which down-regulates PKC, and staurosporine and UCN-01, which directly inhibit the enzyme, circumvent resistance of Bcl-2-overexpressing leukemic cells to ara-C-induced apoptosis and activation of the protease cascade. They also raise the possibility that modulation of Bcl-2 phosphorylation status contributes to this effect.
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PMID:Agents that down-regulate or inhibit protein kinase C circumvent resistance to 1-beta-D-arabinofuranosylcytosine-induced apoptosis in human leukemia cells that overexpress Bcl-2. 939 80

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

Normal lymphocytes are highly sensitive to the damaging effects of ionizing radiation, and undergo cell death by apoptosis. We have investigated the possible involvement of the Interleukin-1 beta-converting enzyme (ICE) (Caspase) protease family, which appears to play an important role as intracellular mediator of apoptosis. Resting B lymphocytes isolated from human peripheral blood were irradiated (6 Gy) and cultured for 24 h, resulting in 25 +/- 5.1% apoptotic cells, as measured by the TUNEL assay (mean +/- SD, n = 6). Addition of the ICE family inhibitor Z-VAD.fmk (50 microM) completely inhibited apoptosis (2.0 +/- 1.5% at 24 h). By using fluorogenic substrates containing the peptide recognition sequences DEVD and YVAD, the type of ICE family protease involved was examined more closely. A marked transient increase in DEVD-, and absent YVAD-cleavage activity indicated the involvement of a CPP32-like protease, not an ICE-like protease. Western blot analysis demonstrated that untreated B lymphocytes expressed the proform of the ICE family members CPP32 and ICH1L, but no detectable ICE. The induction of cell death by radiation was accompanied by the activation of CPP32 as shown by the cleavage of the proform to the active subunit p17, and the cleavage of poly(ADP-ribose) polymerase (PARP), one of the known substrates of CPP32. In contrast, no activation of ICH1L could be detected. These results indicate the involvement of CPP32 and possibly other CPP32-like proteases in radiation-induced apoptosis of resting B lymphocytes.
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PMID:Involvement of ICE (Caspase) family in gamma-radiation-induced apoptosis of normal B lymphocytes. 942 Jun 24

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

The goals of this work were to establish a reproducible and effective model of apoptosis in a cell line derived from advanced prostate cancer and to study the role of the caspase family of proteases in mediating apoptosis in this system. The study involved the use of the prostate cancer cell line LNCaP. Apoptosis was induced using the hydroxymethyl glutaryl CoA reductase inhibitor, lovastatin, and was evaluated by agarose gel electrophoresis of genomic DNA, morphological criteria, and terminal deoxynucleotidyl transferase-mediated nick end labeling. Caspases were studied by catalytic activity, mRNA induction, and protein processing. Lovastatin (30 microM) was an effective inducer of apoptosis, causing changes that were evident after 48 h and essentially complete after 96-120 h of treatment. These effects were prevented by the simultaneous addition of mevalonate (300 microM) to the culture medium. Lovastatin induced a proteolytic activity that was able to cleave the enzyme poly(ADP-ribose) polymerase and the substrate Z-DEVD-AFC, which is modeled after the P1-P4 amino acids of the poly(ADP-ribose) polymerase cleavage site. Caspase-7, but not caspase-3, underwent proteolytic activation during lovastatin-induced apoptosis, an effect prevented by mevalonate. Caspase-7 was the only detected interleukin 1beta converting enzyme family protease with DEVD cleavage activity that exhibited lovastatin-induced mRNA up-regulation. Again, mevalonate blocked this effect. Lovastatin-induced apoptosis also was prevented when the caspase inhibitors Z-DEVD-CH2F or Z-VAD-CH2F (100 microM) where added to the medium. These studies have identified lovastatin as a powerful inducer of apoptosis in the cell line LNCaP. Caspase activation was a necessary event for LNCaP cells to undergo apoptosis during treatment with lovastatin. Of the caspases tested, only caspase-7 underwent proteolytic activation after stimulation with lovastatin. Identification of caspase-7 as a potential mediator of lovastatin-induced apoptosis broadens our knowledge of the molecular events associated with programmed cell death in a cell line derived from prostatic epithelium.
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PMID:Caspase-7 is activated during lovastatin-induced apoptosis of the prostate cancer cell line LNCaP. 942 61

The fibroblast culture 180BR, established from a patient showing an adverse response to radiotherapy, has been shown previously to be hypersensitive to ionizing radiation and to be defective in the repair of DNA double-strand breaks. We demonstrate here that the products of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) and its regulatory subunits (Ku 70 and Ku 80) are present at normal levels and possess functional activity. The product of the gene mutated in the human genetic disorder ataxia-telangiectasia was also detected in these cells. Apoptosis was detected after high-dose ionizing radiation exposure, and this process was accompanied by specific degradation of DNA-PKcs, ATM, and poly(ADP-ribose) polymerase. Activation of CPP32, an interleukin 1beta converting enzyme-like protease implicated in apoptosis, was also observed in 180BR cells in response to radiation damage. The radiosensitivity observed in 180BR cells can be accounted for, at least in part, by radiation-induced apoptosis, and the defect in these cells is not a gross one in DNA-PKcs or ATM.
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PMID:The radiosensitive cell line 180BR is not defective in the major DNA damage-sensing proteins. 942 62

The p34cdc2 kinase is a highly regulated serine-threonine kinase that, when complexed with cyclins A and B, controls cell entry into mitosis. Recently, premature activation of p34cdc2 was shown to be required for apoptosis induced by a wide variety of agents. Here, we show that Taxol induced p34cdc2 kinase activity with a peak at 6 h in human breast carcinoma MCF-7 cells. We subsequently observed that the activation of CPP32/Yama protease as well as the cleavage of its substrate poly(ADP-ribose) polymerase occurred 9 h after Taxol treatment. Olomoucine, a potent p34cdc2 inhibitor, effectively prevented Taxol-induced p34cdc2 kinase activation and subsequent apoptosis. Furthermore, the treatment of cells with cyclin B1-specific antisense oligonucleotide also blocked Taxol-induced apoptosis, suggesting that cyclin B1-associated p34cdc2 kinase plays an important role in the induction of apoptosis by Taxol. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C activator, was found to exert strong protection against Taxol-induced cell death in MCF-7 cells. TPA inhibited Taxol-mediated activation of p34cdc2 kinase by preventing the dephosphorylation of the Tyr-15 residue on p34cdc2 without altering the levels of Cdc2 and cyclin B1. In contrast, the ability of Taxol to enhance tubulin polymerization was not inhibited by TPA. These findings suggest that modulation of protein kinase C signaling can protect against Taxol-induced cell death by inhibiting p34cdc2 kinase activation.
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PMID:Taxol-induced p34cdc2 kinase activation and apoptosis inhibited by 12-O-tetradecanoylphorbol-13-acetate in human breast MCF-7 carcinoma cells. 943 85

Ceramide, a sphingolipid generated by the hydrolysis of membrane-associated sphingomyelin, appears to play a role as a gauge of apoptosis. A further metabolite of ceramide, sphingosine 1-phosphate (SPP), prevents ceramide-mediated apoptosis, and it has been suggested that the balance between intracellular ceramide and SPP levels may determine the cell fate (Cuvillier, O., Pirianov, G, Kleuser, B., Vanek, P. G., Coso, O. A., Gutkind, J. S., and Spiegel, S. (1996) Nature 381, 800-803). Here, we investigated the role of SPP and the protein kinase C activator, phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), in the caspase cascade leading to the proteolysis of poly(ADP-ribose) polymerase (PARP) and lamins. In Jurkat T cells, Fas ligation or addition of exogenous C2-ceramide induced activations of caspase-3/CPP32 and caspase-7/Mch3 followed by PARP cleavage, effects that can be blocked either by SPP or TPA. Furthermore, both SPP and TPA inhibit the activation of caspase-6/Mch2 and subsequent lamin B cleavage. Ceramide, in contrast to Fas ligation, did not induce activation of caspase-8/FLICE and neither SPP nor TPA were able to prevent this activation. Thus, SPP, likely generated via protein kinase C-mediated activation of sphingosine kinase, suppresses the apoptotic pathway downstream of FLICE but upstream of the executioner caspases, caspase-3, -6, and -7.
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PMID:Sphingosine 1-phosphate inhibits activation of caspases that cleave poly(ADP-ribose) polymerase and lamins during Fas- and ceramide-mediated apoptosis in Jurkat T lymphocytes. 944 2


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