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)

We investigated the cytotoxic responsiveness of 40 cell lines derived from representatives of the Ewing's sarcoma family of tumours (ESFT), i.e., Ewing's sarcoma (ES), peripheral primitive neuroectodermal tumour (pPNET) and Askin tumour (AT), to tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Incubation with TRAIL at 100 ng/ml induced cell death at 24 hr in 19 of 26 ES, 11 of 12 pPNET and 2 of 2 AT cell lines. Half-maximal cell death concentrations (IC(50) values) varied from 0.1 to 20 ng/ml. TRAIL displayed potent cytotoxic activity against freshly derived ESFT cell isolates. Cytotoxicity was associated with phosphatidylserine expression and internucleosomal DNA fragmentation, features characteristic of apoptosis. The apoptotic programme in the sensitive ESFT VH-64 cell line revealed TRAIL-induced activation of FLICE/MACH1 (caspase-8) and CPP32/Yama/apopain (caspase-3) and processing of the prototype caspase substrate poly(ADP-ribose) polymerase. In addition, TRAIL provoked a collapse of the mitochondrial transmembrane potential (DeltaPsi(m)), parallelled by a reduction in ATP levels and release of cytochrome c from mitochondria into the cytosol. Inhibition of caspase-8 and caspase-3 by zIETDfmk and zDEVDfmk, respectively, substantially prevented TRAIL-induced apoptosis. However, zIETDfmk, but not zDEVDfmk, reduced TRAIL-mediated DeltaPsi(m) dissipation, indicating that TRAIL causes mitochondrial dysfunction through caspase-8 acting upstream of mitochondria. While macromolecule synthesis inhibitors (actinomycin D, cycloheximide) augmented susceptibility to TRAIL in TRAIL-responsive cell lines, these agents did not render TRAIL-resistant cell lines susceptible to TRAIL. However, the proteasome inhibitor MG132 sensitised to TRAIL in resistant cell lines. Collectively, these results show that TRAIL initiates effective death in the vast majority (80%) of cell lines derived from ESFT. Since TRAIL provoked cell death in ESFT ex vivo, this cytokine may be a promising drug for the treatment of ESFT in vivo.
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PMID:Apoptotic responsiveness of the Ewing's sarcoma family of tumours to tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). 1100 77

A nonmucoid clinical isolate of Pseudomonas aeruginosa, strain 808, elaborated ATP-dependent and ATP-independent types of cytotoxic factors in the growth medium. These cytotoxic factors, active against macrophages, were secreted during the exponential phase of growth in a complex medium. Commensurate with the appearance of the cytotoxic activities in the cell-free growth medium, several ATP-utilizing enzymic activities, such as adenylate kinase, nucleoside diphosphate kinase and 5'-nucleotidase (ATPase and/or phosphatase), were detected in the medium. These ATP-utilizing enzymes are believed to convert external ATP, presumably effluxed from macrophages, to various adenine nucleotides, which then activate purinergic receptors such as P2Z, leading to enhanced macrophage cell death. Pretreatment of macrophages with periodate-oxidized ATP (oATP), which is an irreversible inhibitor of P2Z receptor activation, prevented subsequent ATP-induced macrophage cell death. A second type of cytotoxic factor(s) operated in an ATP-independent manner such that it triggered activation of apoptotic processes in macrophages, leading to proteolytic conversion of procaspase-3 to active caspase-3. This cytotoxic factor(s) did not appear to act on procaspase-3 present in macrophage cytosolic extracts. Intact macrophages, when exposed to the cytotoxic factor(s) for 6-16 h, underwent apoptosis and demonstrated the presence of active caspase-3 in their cytosolic extracts. Interestingly, two redox proteins, azurin and cytochrome c(551), were detected in the cytotoxic preparation. When cell-line-derived or peritoneal macrophages or mast cells were incubated overnight with Q-Sepharose column flow-through fraction or with a mixture of azurin and cytochrome c(551), they underwent extensive cell death due to induction of apoptosis.
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PMID:Secreted products of a nonmucoid Pseudomonas aeruginosa strain induce two modes of macrophage killing: external-ATP-dependent, P2Z-receptor-mediated necrosis and ATP-independent, caspase-mediated apoptosis. 1102 27

Oxidants such as H(2)O(2) can induce a low level of apoptosis at low concentrations but at higher concentrations cause necrosis. Higher concentrations of H(2)O(2) also inhibit the induction of apoptosis by chemotherapy drugs. One theory is that, at higher concentrations, H(2)O(2) causes direct oxidative inactivation of caspase-3 activity, thus preventing the apoptotic pathway from being used. We find that treatment of recombinant caspase-3 with H(2)O(2) can partially reduce its enzymatic activity: However, the following findings show that this does not occur in the cell. (1) The inhibition by H(2)O(2) of VP-16-induced apoptosis and cellular caspase-3 activity can be overcome by adding inhibitors of poly(ADP-ribose) polymerase (PARP) at sub-stoichiometric concentrations. (2) Delayed addition of H(2)O(2) to VP-16-treated cells prevents additional caspase induction but does not inhibit the caspase activity that has already been generated. (3) H(2)O(2) is a poor inhibitor of caspase-3 activity in cell lysates. (4) Addition of H(2)O(2) to cells inhibits activation of caspase-9, which is required for activation of caspase-3. We conclude that inhibition of caspase-3 activity in the cell occurs indirectly at a step located upstream of caspase-3 activation. H(2)O(2) acts in part by inducing DNA strand breaks and activating PARP, thus depleting the cells of ATP. When this pathway is blocked, even high concentrations of H(2)O(2) can induce caspase-9 and -3 activation and cause apoptosis.
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PMID:Hydrogen peroxide inhibits activation, not activity, of cellular caspase-3 in vivo. 1103 21

The 26S proteasome is a non-lysosomal multicatalytic protease complex for degrading intracellular proteins by ATP/ubiquitin-dependent proteolysis. Tightly ordered proteasomal degradation of proteins critical for cell cycle control implies a role of the proteasome in maintaining cell proliferation and cell survival. In this study, we demonstrate that cell-permeable proteasome inhibitors, lactacystin, benzyloxycarbonyl(Z)-leucyl-leucyl-leucinal (ZLLLal; MG-132) and 4-hydroxy-5-iodo-3-nitrophenylacetyl-leucyl-leucyl-leucine vinyl sulfone (NLVS), induce apoptosis abundantly in p53-defective leukemic cell lines CCRF-CEM, U937 and K562 as well as in myelogenic and lymphatic leukemic cells obtained from adult individuals with relapsed acute leukemias. Leukemic cell apoptosis induced by the proteasome inhibitors was dependent on activation of caspase-3 and related caspase family proteases, because caspase-3 inhibitor N-acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspartal (Ac-DEVD-cho) and, more effectively, the general caspase-inhibitor N-benzyloxycarbonyl-L-valyl-L-alanyl-L-aspartate fluoromethylketone (Z-VAD-fmk) were capable of blocking apoptosis induced by lactacystin, ZLLLal or NLVS. Induction of apoptosis by lactacystin or ZLLLal was accompanied by cell cycle arrest at G2/M phase and by accumulation and stabilization of cyclin-dependent kinase inhibitor p21WAF1/Cip and tumor suppressor protein p53. A role of p53 in mediating apoptosis or induction of p21WAF1/Cip1 was ruled out since CCRF-CEM and U937 cells express non-functional mutant p53, and K562 cells lack expression of p53. Viability and hematopoietic outgrowth of human CD34+ progenitor cells treated with lactacystin were slightly reduced, whereas treatment of CD34 + cells with ZLLLal or the cytostatic drugs doxorubicin and gemcitabine resulted in markedly reduced viability and hematopoietic outgrowth. These results demonstrate a basic role of the proteasome in maintaining survival of human leukemic cells, and may define cell-permeable proteasome inhibitors as potently anti-leukemic agents which exhibit a moderate hematopoietic toxicity in vitro.
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PMID:Proteasome inhibitors induced caspase-dependent apoptosis and accumulation of p21WAF1/Cip1 in human immature leukemic cells. 1107 63

Cross-linking of the Ag receptors on the immature B cell lymphoma, WEHI-231, leads to growth arrest and apoptosis. We now show that although commitment to such B cell receptor (BCR)-mediated apoptosis correlates with mitochondrial phospholipase A(2) activation, disruption of mitochondrial function, and ATP depletion, it is executed independently of caspase activation. First, we demonstrate a pivotal role for mitochondrial function in determining B cell fate by showing up-regulation of cytosolic phospholipase A(2) expression, induction of mitochondrial phospholipase A(2) activity, arachidonic acid-mediated collapse of mitochondrial transmembrane inner potential (Delta psi(m)), and depletion of cellular ATP under conditions of apoptotic, but not proliferative, signaling via the BCR. Importantly, disruption of Delta psi(m), ATP depletion, and apoptosis can be prevented by rescue signals via CD40 or by Delta psi(m) stabilizers such as antimycin or oligomycin. Second, we show that commitment and postmitochondrial execution of BCR-mediated apoptosis are not dependent on caspase activation by demonstrating that such apoptotic signaling does not induce release of cytochrome c from the mitochondria or activation of effector caspases, as evidenced by poly(ADP-ribose) polymerase or Bcl-x(L) cleavage. Indeed, apoptotic signaling via the BCR in WEHI-231 B cells does not stimulate the activation of caspase-3 and, consistent with this, BCR-mediated disruption of Delta psi(m) and commitment to apoptosis take place in the presence of caspase inhibitors. In contrast, BCR signaling induces the postmitochondrial activation of cathepsin B, and resultant apoptosis is blocked by the cathepsin B inhibitor, (23,35)trans-epoxysuccinyl-L-leucylamindo-3-methylbutane ethyl ester (EST) suggesting a key role for this executioner protease in Ag receptor-driven apoptosis of WEHI-231 immature B cells.
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PMID:B cell receptor-stimulated mitochondrial phospholipase A2 activation and resultant disruption of mitochondrial membrane potential correlate with the induction of apoptosis in WEHI-231 B cells. 1112 86

The nephrotoxicity of trichloroethylene and dichloroacetylene has previously been linked to mitochondrial dysfunction induced by the metabolite S-(1,2-dichlorovinyl)-l-cysteine (DCVC). In this study, we examined whether key biochemical steps associated with mitochondria occur in DCVC-induced apoptosis in cultured porcine proximal tubular LLC-PK1 cells. DCVC caused a decrease in mitochondrial membrane potential (mt Delta Psi) beginning at 4 h and a release of cytochrome c into the cytoplasm at 6 h. Caspase-3-like activity was detected at 6 h and extensive DNA fragmentation was observed at 8 h. Decreases in cellular ATP were not evident until 8 h and later, even though electron microscopy showed that the mitochondria were extensively swollen. Aminooxyacetic acid (AOAA), an inhibitor of cysteine-conjugate beta-lyase, protected against mitochondrial changes and apoptosis. Overexpression of the antiapoptotic Bcl-2 protein desensitized LLC-PK1 cells to DCVC-induced apoptosis. These results support the interpretation that mitochondrial release of cyt c and cyt c-dependent activation of caspase-3 could have a central role in nephrotoxicity due to haloalkene-derived cysteine S-conjugates.
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PMID:Role of mitochondrial dysfunction in S-(1,2-dichlorovinyl)-l-cysteine-induced apoptosis. 1116 82

Alpha-sarcin is a ribosome-inactivating protein that has been well characterized in vitro, but little is known about its toxicity in living cells. We have analyzed the mechanism of internalization of alpha-sarcin into human rhabdomyosarcoma cells and the cellular events that result in the induction of cell death. No specific cell surface receptor for alpha-sarcin has been found. The toxin is internalized via endocytosis involving acidic endosomes and the Golgi, as deduced from the ATP requirement and the effects of NH4Cl, monensin and nigericin on its cytotoxicity. Specific cleavage of 28S rRNA in cultured rhabdomyosarcoma cells, associated with protein biosynthesis inhibition, has been detected. alpha-Sarcin kills rhabdomyosarcoma cells via apoptosis: incubation of cells with alpha-sarcin at a concentration below its IC50 induces internucleosomal genomic DNA fragmentation, reversion of membrane asymmetry, activation of caspase-3-like activity and cleavage of poly(ADP-ribose)polymerase. Apoptosis is not a general direct consequence of protein biosynthesis inhibition, as deduced from the comparative analysis of the effects of alpha-sarcin and cycloheximide; the latter does not induce apoptosis even at concentrations far beyond its IC50, where protein biosynthesis is null. Experiments with a catalytically inactive alpha-sarcin mutant, neither toxic nor apoptotic, reveal that induced apoptosis is directly related to the effects of catalytic activity of the toxin on the ribosomes. The caspase inhibitor z-VAD-fmk does not suppress protein synthesis inhibition by alpha-sarcin. Together, these data suggest that alpha-sarcin-induced caspase activation is a pathway downstream of the 28S rRNA catalytic cleavage and consequent protein biosynthesis inhibition.
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PMID:Cytotoxic mechanism of the ribotoxin alpha-sarcin. Induction of cell death via apoptosis. 1127 35

The lack of a well-characterized in-vitro cell culture model of load-induced cardiac ischaemia has hampered investigations into the mechanism of ischemic injury. We therefore developed a new in-vitro model of cardiac ischaemia that mimics distinct features of ischaemic injury. Neonatal rat heart cells were cultured in a sealed flask for 24-72 h. In this environment, the cells were exposed to stresses of hypoxia, acidosis and stagnant incubation medium. The pO2 and pH of the medium gradually decreased during the ischaemic insult and ultimately fell to a level of 14 mmHg and pH 6.8, respectively. The model triggered severe cell injury, including morphological degeneration, CPK release, beating impairment and ATP depletion. Apoptosis occurred in some cardiomyocytes as early as 24 h after onset of seal-induced ischaemia. This was evidenced by positive nuclear staining using Hoechst 33258 and by the induction of caspase-3 mRNA. By 72 h, internucleosomal DNA fragmentation was observed in 45% of the myocytes; however, a non-myocyte preparation subjected to the same ischaemic insult exhibited no evidence of DNA fragmentation. These results demonstrate that neonatal cardiomyocytes subjected to the new simulated ischaemia model exhibit several similarities to cardiac ischaemia, including the simultaneous appearance of necrosis, breakdown of cellular ATP, beating cessation and apoptosis. The new model should prove useful in unravelling the molecular alterations underlying ischaemic injury and myocardial apoptosis.
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PMID:Cellular characterization of an in-vitro cell culture model of seal-induced cardiac ischaemia. 1129 53

We have recently shown that nitric-oxide (NO)-induced apoptosis in Jurkat human leukemia cells requires degradation of mitochondria phospholipid cardiolipin, cytochrome c release, and activation of caspase-9 and caspase-3. Moreover, an inhibitor of lipid peroxidation, Trolox, suppressed apoptosis in Jurkat cells induced by NO donor glycerol trinitrate. Here we demonstrate that this antiapoptotic effect of Trolox occurred despite massive release of the mitochondrial protein cytochrome c into the cytosol and mitochondrial damage. Incubation with Trolox caused a profound reduction of intracellular ATP concentration in Jurkat cells treated by NO. Trolox prevented cardiolipin degradation and caused its accumulation in Jurkat cells. Furthermore, Trolox markedly downregulated the NO-mediated activation of caspase-9 and caspase-3. Caspase-9 is known to be activated by released cytochrome c and together with caspase-3 is considered the most proximal to mitochondria. Our results suggest that the targets of the antiapoptotic effect of Trolox are located downstream of the mitochondria and that caspase activation and subsequent apoptosis could be blocked even in the presence of cytochrome c released from the mitochondria.
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PMID:Inhibition of nitric-oxide-mediated apoptosis in Jurkat leukemia cells despite cytochrome c release. 1130 92

In most cases, apoptosis is considered to involve mitochondrial dysfunction with sequential release of cytochrome c from mitochondria, resulting in activation of caspase-3. However, we found that etoposide induced apoptosis in P39 cells, a myelodysplastic syndrome-derived cell line, without the release of cytochrome c. Furthermore, in etoposide-treated P39 cells, no changes in mitochondrial membrane potential (delta psi m) were detected by flow cytometry. Flow cytometry using a pH-sensitive probe demonstrated that lysosomal pH increased during early apoptosis in P39 cells treated with etoposide. A reduction in the ATP level preceded the elevation of lysosomal pH. In addition, specific inhibitors of vacuolar H+-ATPase induced apoptosis in P39 cells but not in HL60 cells. Although etoposide-induced activation of caspase-3 was followed by DNA ladder formation in P39 cells, E-64d, an inhibitor of lysosomal thiol proteases, specifically suppressed etoposide-induced activation of caspase-3. Western blotting analysis provided direct evidence for the involvement of a lysosomal enzyme, cathepsin L. These findings indicate that lysosomal dysfunction induced by a reduction in ATP results in leakage of lysosomal enzymes into the cytosolic compartment and that lysosomal enzyme(s) may be involved in activation of caspase-3 during apoptosis in P39 cells treated with etoposide.
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PMID:Caspase-3 activation by lysosomal enzymes in cytochrome c-independent apoptosis in myelodysplastic syndrome-derived cell line P39. 1130 62


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