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)

Apoptosis is mediated by members of the caspase family of proteases which can be activated by release of mitochondrial cytochrome c. Additional members of the caspase family are activated at the cell surface in response to direct stimulus from the external environment such as by activation of the Fas receptor. It has been suggested that these upstream caspases directly activate the downstream caspases which would obviate a role for cytochrome c in apoptosis induced by the Fas receptor. We demonstrate that cytochrome c is released from mitochondria of Jurkat cells in response to both staurosporine and an agonistic anti-Fas antibody and that only the latter is inhibited by the caspase inhibitor z-VAD-FMK. This suggests that an upstream caspase such as caspase-8 is required for the Fas-mediated release of mitochondrial cytochrome c. The protein phosphatase inhibitor calyculin A prevented cytochrome c release and apoptosis induced by both agents, suggesting that release of cytochrome c is required in both models. Zinc, once thought of as an endonuclease inhibitor, has previously been shown to prevent the activation of caspase-3. We show that zinc prevents the activation of downstream caspases and apoptosis induced by both insults, yet does not prevent release of mitochondrial cytochrome c. The ability of calyculin A and zinc to prevent DNA digestion implies that the mitochondrial pathway is important for induction of apoptosis by both agents. These results do not support an alternative pathway in which caspase-8 directly activates caspase-3. These results also demonstrate that a critical protein phosphatase regulates the release of cytochrome c and apoptosis induced by both insults.
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PMID:The temporal relationship between protein phosphatase, mitochondrial cytochrome c release, and caspase activation in apoptosis. 1006 78

The protein phosphatase inhibitor okadaic acid (OA) dose-dependently induced apoptosis in CHP-100 neuroepithelioma cells when administered for 24 h at concentrations ranging from 10 - 100 nM. Apoptosis was largely, albeit not completely, dependent on cystein protease (caspase) activation. CPP32 processing and poly(ADP-ribose) polymerase (PARP) cleavage started to be observed only at 20 nM OA; moreover, the caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD.fmk) (100 microM) had negligible effect on apoptosis induced by 10 nM OA, but rescued from death an increasing cell fraction as OA concentration was raised from 20 - 100 nM. Cell treatment for 24 h with OA induced ceramide accumulation; the phenomenon started to be evident at 20 nM OA and reached its maximum at 50 - 100 nM OA. In cells exposed to 50 nM OA, ceramide was already elevated by 5 h; at this time, however, PARP cleavage and apoptosis were not yet observed. Z-VAD.fmk (100 microM) had no effect on ceramide elevation induced by 50 nM OA within 5 h, but markedly reduced ceramide accumulation as the incubation was prolonged to 24 h. The latter phenomenon was accompanied by elevation of glucosylceramide levels, thus suggesting that a caspase-dependent reduction of glucosylceramide synthesis might contribute to late ceramide accumulation. Short-chain ceramide (30 microM) induced apoptosis in CHP-100 cells and its effect was additive with that evoked by OA (10 - 20 nM). These results suggest that ceramide generation might be an important mechanism through which sustained protein phosphatase inhibition induces caspase activation and apoptosis in CHP-100 cells.
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PMID:Ceramide accumulation precedes caspase-dependent apoptosis in CHP-100 neuroepithelioma cells exposed to the protein phosphatase inhibitor okadaic acid. 1045 72

Calcineurin is a Ca(2+)/calmodulin-dependent protein phosphatase that is abundantly expressed in several specific areas of the brain, which are exceptionally vulnerable to stroke, epilepsy, and neurodegenerative diseases. In this study, we assessed the effects of high level activity of calcineurin on neuronal cells. Virus-mediated high level constitutive activity of calcineurin rendered neuronal cells susceptible to apoptosis induced by serum reduction or by a brief exposure to calcium ionophore. Adenovirus-mediated, high level forced activity of calcineurin induced cytochrome c/caspase-3-dependent apoptosis in neurons. Preincubation with the calcineurin inhibitors cyclosporin A and FK506 reduced susceptibility to apoptosis. High level constitutive expression of Bcl-2 or CrmA or incubation with a specific caspase-3 inhibitor inhibited the calcineurin-induced apoptosis. These data indicate that high level constitutive activity of calcineurin predisposes neuronal cells to cytochrome c/caspase-3 dependent apoptosis even under sublethal conditions.
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PMID:High level calcineurin activity predisposes neuronal cells to apoptosis. 1056 26

The protein phosphatase (PP) inhibitors nodularin and microcystin-LR induced apoptosis with unprecedented rapidity, more than 50% of primary hepatocytes showing extensive surface budding and shrinkage of cytoplasm and nucleoplasm within 2 min. The apoptosis was retarded by the general caspase inhibitor Z-VAD.fmk. To circumvent the inefficient uptake of microcystin and nodularin into nonhepatocytes, toxins were microinjected into 293 cells, Swiss 3T3 fibroblasts, promyelocytic IPC-81 cells, and NRK cells. All cells started to undergo budding typical of apoptosis within 0.5 - 3 min after injection. This was accompanied by cytoplasmic and nuclear shrinkage and externalization of phosphatidylserine. Overexpression of Bcl-2 did not delay apoptosis. Apoptosis induction was slower and Z-VAD.fmk independent in caspase-3 deficient MCF-7 cells. MCF-7 cells stably transfected with caspase-3 showed a more rapid and Z-VAD.fmk dependent apoptotic response to nodularin. Rapid apoptosis induction required inhibition of both PP1 and PP2A, and the apoptosis was preceded by increased phosphorylation of several proteins, including myosin light chain. The protein phosphorylation occurred even in the presence of apoptosis-blocking concentrations of Z-VAD.fmk, indicating that it occurred upstream of caspase activation. It is suggested that phosphatase-inhibiting toxins can induce caspase-3 dependent apoptosis in an ultrarapid manner by altering protein phosphorylation.
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PMID:Ultrarapid caspase-3 dependent apoptosis induction by serine/threonine phosphatase inhibitors. 1057 79

L-Deprenyl, an irreversible MAO-B (monoamine oxidase B, EC 1.4.3.4) inhibitor, is used for the treatment of Parkinson's disease and to delay the progression of Alzheimer's disease. L-Deprenyl also exhibits protective effects against neuronal apoptosis which are independent of its ability to inhibit MAO-B. The purpose of this study was to compare the antiapoptotic efficacy of L-deprenyl against different types of apoptotic inducers in three neuronal cell culture models. The level of apoptosis was quantified by measuring the activation of caspase-3 enzyme, which is the main apoptotic executioner in neuronal cells. MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] and LDH (lactate dehydrogenase, EC 1. 1.1.27) assays were used to demonstrate the cytotoxic response of apoptotic treatments. Our results showed that okadaic acid, an inhibitor of protein phosphatase 1 and 2A, induced a prominent increase in caspase-3 activity both in cultured hippocampal and cerebellar granule neurons as well as in Neuro-2a neuroblastoma cells. Interestingly, L-deprenyl offered a significant protection against the apoptotic response induced by okadaic acid in all three neuronal models. The best protection appeared at the concentration level of 10(-9) M. L-Deprenyl also provided a protection against apoptosis after AraC (cytosine beta-D-arabinoside) treatment in hippocampal neurons and Neuro-2a cells and after etoposide treatment in Neuro-2a cells. However, L-deprenyl did not offer any protection against apoptosis caused by serum withdrawal or potassium deprivation. Okadaic acid treatment in vivo is known to induce an Alzheimer's type of hyperphosphorylation of tau protein, formation of beta-amyloid plaques, and a severe memory impairment. Our results show that the okadaic acid model provides a promising tool to study the molecular basis of Alzheimer's disease and to screen the neuroprotective capacity of L-deprenyl derivatives.
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PMID:Protective effect of L-deprenyl against apoptosis induced by okadaic acid in cultured neuronal cells. 1079 57

The mechanisms that regulate cardiac myocyte apoptosis are not well understood. To study the role of protein phosphatase 1 (PP1) and 2A (PP2A) in apoptosis, we exposed cultured neonatal rat cardiac myocytes to the phosphatase inhibitor okadaic acid (OA). Exposure (18 h) to 100 nM OA (a concentration which inhibits both PP1 and PP2A) decreased the number of adherent cells, caused genomic DNA fragmentation, and increased the percentage of apoptotic cells. These effects did not occur at a lower concentration of OA (1 nM) which is relatively specific for PP2A. Stimulation of alpha1- or beta-adrenergic receptors with norepinephrine (NE) in the presence of propranolol or prazosin partially blocked OA-induced apoptosis as measured by flow cytometry. Likewise, stimulation of adenylyl cyclase with forskolin reduced OA-induced apoptosis. Conversely, inhibition of protein kinase A with H89 or protein kinase C with chelerethrine potentiated OA-induced apoptosis. OA increased caspase-3 activity, and this effect was reduced by NE. Thus, inhibition of PP1 stimulates apoptosis in NRVM and stimulation of adrenergic receptors protects against OA-induced apoptosis.
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PMID:Inhibition of protein phosphatase 1 induces apoptosis in neonatal rat cardiac myocytes: role of adrenergic receptor stimulation. 1109 66

Docosahexaenoic acid (DHA) is an omega-3 fatty acid under intense investigation for its ability to modulate cancer cell growth and survival. This research was performed to study the cellular and molecular effects of DHA. Our experiments indicated that the treatment of Jurkat cells with DHA inhibited their survival, whereas similar concentrations (60 and 90 microM) of arachidonic acid and oleic acid had little effect. To explore the mechanism of inhibition, we used several measures of apoptosis to determine whether this process was involved in DHA-induced cell death in Jurkat cells. Caspase-3, an important cytosolic downstream regulator of apoptosis, is activated by death signals through proteolytic cleavage. Incubation of Jurkat cells with 60 and 90 microM DHA caused proteolysis of caspase-3 within 48 and 24 h, respectively. DHA treatment also caused the degradation of poly-ADP-ribose polymerase and DNA fragmentation as assayed by flow cytometric TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay. These results indicate that DHA induces apoptosis in Jurkat leukemic cells. DHA-induced apoptosis was effectively inhibited by tautomycin and cypermethrin at concentrations that affect protein phosphatase 1 (PP1) and protein phosphatase 2B (PP2B) activities, respectively, implying a role for these phosphatases in the apoptotic pathway. Okadaic acid, an inhibitor of protein phosphatase 2A, had no effect on DHA-induced apoptosis. These results suggest that one mechanism through which DHA may control cancer cell growth is through apoptosis involving PP1/PP2B protein phosphatase activities.
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PMID:Docosahexaenoic acid induces apoptosis in Jurkat cells by a protein phosphatase-mediated process. 1134 74

The standardized extract from Ginkgo biloba (EGb 761) is used for the treatment of dementia. Because of allergenic and genotoxic effects, ginkgolic acids are restricted in EGb 761 to 5 ppm. The question arises whether ginkgolic acids also have neurotoxic effects. In the present study, ginkgolic acids caused death of cultured chick embryonic neurons in a concentration-dependent manner, in the presence and in the absence of serum. Ginkgolic acids-induced death showed features of apoptosis as we observed chromatin condensation, shrinkage of the nucleus and reduction of the damage by the protein synthesis inhibitor cycloheximide, demonstrating an active type of cell death. However, DNA fragmentation detected by the terminal-transferase-mediated ddUTP-digoxigenin nick-end labeling (TUNEL) assay and caspase-3 activation, which are also considered as hallmarks of apoptosis, were not seen after treatment with 150 microM ginkgolic acids in serum-free medium, a dose which increased the percentage of neurons with chromatin condensation and shrunken nuclei to 88% compared with 25% in serum-deprived, vehicle-treated controls. This suggests that ginkgolic acid-induced death showed signs of apoptosis as well as of necrosis. Ginkgolic acids specifically increased the activity of protein phosphatase type-2C, whereas other protein phosphatases such as protein phosphatases 1A, 2A and 2B, tyrosine phosphatase, and unspecific acid- and alkaline phosphatases were inhibited or remained unchanged, suggesting protein phosphatase 2C to play a role in the neurotoxic effect mediated by ginkgolic acids.
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PMID:Ginkgolic acids induce neuronal death and activate protein phosphatase type-2C. 1169 56

The present investigation explores the role of phosphatidic acid (PA), a specific protein phosphatase-1 (PP1) inhibitor, in cytotoxicity induced by docosahexaenoic acid (DHA). The cytotoxicity of DHA was assayed by quantifying cell survival using the trypan blue exclusion method. A dose-response effect demonstrated that 5 or 10 microM DHA has no effect on Jurkat cell survival; however, 15 microM DHA rapidly decreased cell survival to 40% within 2 h of treatment. Cytotoxicity of 15 microM DHA was prevented by PA. Structurally similar phospholipids (lysophosphatidic acid, sphingosine 1-phosphate, sphingosine, and sphingosine phosphocholine) or metabolites of PA (lyso-PA and diacylglycerol) did not prevent DHA-induced cytotoxicity. PA did not produce micelles alone or in combination with DHA as examined spectrophotometrically, indicating that PA did not entrap DHA and therefore did not affect the amount of DHA available to the cells. Supporting this observation, the uptake or incorporation of [1-14C]DHA in Jurkat cells was not affected by the presence of PA. However, PA treatment reduced the amount of DHA-induced inorganic phosphate released from Jurkat leukemic cells and also inhibited DHA-induced dephosphorylation of cellular proteins. These observations indicate that PA has exerted its anti-cytotoxic effects by causing inhibition of protein phosphatase activities. Cytotoxicity of DHA on Jurkat cells was also blocked by the use of a highly specific caspase-3 inhibitor (N-acetyl-ala-ala-val-ala-leu-leu-pro-ala-val-leu-leu-ala-leu-leu-ala-pro-asp-glu-val-asp-CHO), indicating that the cytotoxic effects of DHA were due to the induction of apoptosis though activation of caspase-3. Consistent with these data, proteolytic activation of procaspase-3 was also evident when examined by immunoblotting. PA prevented procaspase-3 degradation in DHA-treated cells, indicating that PA causes inhibition of DHA-induced apoptosis in Jurkat leukemic cells. Since DHA-induced apoptosis can be inhibited by PA, we conclude that the process is mediated through activation of PP1.
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PMID:Prevention of docosahexaenoic acid-induced cytotoxicity by phosphatidic acid in Jurkat leukemic cells: the role of protein phosphatase-1. 1175 13

Individual subunits of protein phosphatase 2A (PP2A), protein phosphatase 4, and protein phosphatase 5 were knocked out in Drosophila Schneider 2 cells by using RNA interference. Ablation of either the scaffold (A) or catalytic (C) subunits of PP2A caused the disappearance of all PP2A subunits. Treating cells with double-stranded RNA targeting all four of the Drosophila PP2A regulatory subunits caused the disappearance of both the A and C subunits. The loss of PP2A subunits was associated with decreased protein stability indicating that only the heterotrimeric forms of PP2A are stable in intact cells. Ablation of total PP2A by using double-stranded RNA against either the A or C subunit, or specific ablation of the R2/B regulatory subunit, enhanced insulin-induced ERK activation. These results indicated that the R2/B subunit targets PP2A to the mitogen-activated protein (MAP) kinase cascade in Schneider 2 cells, where it acts as a negative regulator. A severe loss of viability occurred in cells in which total PP2A or both isoforms of the Drosophila R5/B56 subunit had been ablated. The reduced viability of these cells correlated with the induction of markers of apoptosis including membrane blebbing and stimulation of caspase-3-like activity. These observations indicated that PP2A has a powerful antiapoptotic activity that is specifically mediated by the R5/B56 regulatory subunits. In contrast to PP2A, ablation of protein phosphatase 4 caused only a slight reduction in cell growth but had no effect on MAP kinase signaling or apoptosis. Depletion of protein phosphatase 5 had no effects on MAP kinase, cell growth, or apoptosis.
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PMID:Actions of PP2A on the MAP kinase pathway and apoptosis are mediated by distinct regulatory subunits. 1190 83


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