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Query: UNIPROT:P42574 (caspase-3)
 45,978 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Apoptotic signaling cascades converge in the activation of caspases (interleukin-1beta converting enzyme like proteases). Treatment of the human promyelocytic leukaemia cell line U937 with actinomycin D resulted in the activation of caspase-3 also known as CPP32. Protease activity was measured in cytosolic extracts by fluorometric analysis of the time-dependent cleavage of acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin (DEVD-AMC), a caspase-3 substrate. Caspase activity was inhibited by thiol modifying agents such as N-ethylmaleimide or iodoacetamide and NO donors such as S-nitrosoglutathione (GSNO), BF4NO, and spermine-NO. NO-mediated enzyme inhibition was fully reversible upon the addition of DTT (dithiothreitol). NO. itself was not primarily responsible for downregulation of caspase-3, as we found no correlation between rates of NO* release and the magnitude of enzyme inhibition. It is likely that S-nitrosation accounts for enzyme inhibition by various NO donors. SIN-1 and peroxynitrite were inhibitory as well. In this case, however, enzyme activity was not restored upon DTT addition, suggesting oxidation as an additional thiol modification mechanism. Our studies provide evidence that caspases are targeted by NO via S-nitrosation and oxidation of critical thiol groups.
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PMID:Inhibition of caspase-3 by S-nitrosation and oxidation caused by nitric oxide. 929 18

The caspase (ICE/CED-3) family of proteases has been implicated to play a crucial role in apoptosis. However, the mechanisms by which caspase activity mediates apoptosis are not fully understood. Progress in this area has been limited due to the lack of a convenient and reliable system to quantify these protease activities. In this report, we describe a quantitative assay for the activity of caspase-3, a member of the caspase family thought to mediate apoptosis in most mammalian cell types. This assay utilizes a synthetic tetrapeptide, Asp-Glu-Val-Asp (DEVD), labeled with either a fluorescent molecule, 7-amino-4-trifluoromethyl coumarin (AFC), or a colorimetric molecule, p-nitroanilide (pNA) as substrates. DEVD-dependent protease activity is assessed by detection of the free AFC or pNA cleaved from the substrates. We demonstrate the utility of the assay for rapid quantification of caspase-3 activity in the onset of apoptosis. Using the assay, we show that apoptosis induced in 32D cells under various conditions is associated with an increase in the DEVD-dependent protease activity. These studies suggest that induction of the DEVD-dependent protease activity is an indicator of apoptosis and demonstrate the utility of the assays for assessment of the role of caspase-family proteases in apoptotic cell progression.
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PMID:Fluorometric and colorimetric detection of caspase activity associated with apoptosis. 930 88

In a cell-free system based on Xenopus egg extracts, Bcl-2 blocks apoptotic activity by preventing cytochrome c release from mitochondria. We now describe in detail the crucial role of cytochrome c in this system. The mitochondrial fraction, when incubated with cytosol, releases cytochrome c. Cytochrome c in turn induces the activation of protease(s) resembling caspase-3 (CPP32), leading to downstream apoptotic events, including the cleavage of fodrin and lamin B1. CPP32-like protease activity plays an essential role in this system, as the caspase inhibitor, Ac-DEVD-CHO, strongly inhibited fodrin and lamin B1 cleavage, as well as nuclear morphology changes. Cytochrome c preparations from various vertebrate species, but not from Saccharomyces cerevisiae, were able to initiate all signs of apoptosis. Cytochrome c by itself was unable to process the precursor form of CPP32; the presence of cytosol was required. The electron transport activity of cytochrome c is not required for its pro-apoptotic function, as Cu- and Zn-substituted cytochrome c had strong pro-apoptotic activity, despite being redox-inactive. However, certain structural features of the molecule were required for this activity. Thus, in the Xenopus cell-free system, cytosol-dependent mitochondrial release of cytochrome c induces apoptosis by activating CPP32-like caspases, via unknown cytosolic factors.
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PMID:Cytochrome c activation of CPP32-like proteolysis plays a critical role in a Xenopus cell-free apoptosis system. 930 8

Six hours after ultraviolet B (UVB) irradiation (11.6 mJ/cm2), the viability of A431 cells decreased, and, at the same time, fragmentation of genomic DNA into nucleosomal units was observed. Z-Asp-CH2-DCB (100 microM), an inhibitor of interleukin-1 beta-converting enzyme (caspase-1) and caspase-1-like proteases, markedly inhibited UVB-induced cell death and DNA fragmentation. Both YVAD-CMK, an inhibitor of caspase-1, and DEVD-CHO, an inhibitor of caspase-3, moderately inhibited the UVB-induced cell death. A combination of YVAD-CMK and DEVD-CHO acted additionally in inhibiting cell death. These observations suggest strongly the cooperative involvement of caspases in the apoptosis induced in A431 cells by UVB.
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PMID:Involvement of caspases in apoptosis induced by ultraviolet B irradiation in A431 human epithelioid tumor cells. 930 45

The current confusion regarding the relevance of endogenous ceramide in mediating CD95/Fas-induced apoptosis is based mainly on (i) discrepancies in kinetics of the ceramide response between different studies using the same apoptotic stimulus and (ii) the observation that late ceramide formation (hours) often parallels apoptosis onset. We investigated CD95-induced ceramide formation in Jurkat cells, using two methods (radiolabeling/thin layer chromatography and benzoylation/high performance liquid chromatography), which, unlike the commonly used diglyceride kinase assay, discriminate between ceramide species and de novo formed dihydroceramide. We demonstrate that ceramide accumulates after several hours, reaching a 7-fold increase after 8 h, kinetics closely paralleling apoptosis induction. No fast response was observed, not even in the presence of inhibitors of ceramide metabolism. The majority ( approximately 70%) of the ceramide response remained unaffected when apoptosis was completely inhibited at the level of caspase-3/CPP32 processing by the inhibitor peptide DEVD-CHO. Exogenous cell-permeable C2-ceramide induced the proteolytic processing of caspase-3, albeit with somewhat slower kinetics than with CD95. DEVD-CHO dose-dependently inhibited C2-ceramide- or exogenous sphingomyelinase-induced apoptosis. The results support the idea that ceramide acts in conjunction with the caspase cascade in CD95-induced apoptosis.
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PMID:CD95/Fas-induced ceramide formation proceeds with slow kinetics and is not blocked by caspase-3/CPP32 inhibition. 930 86

The Caenorhabditis elegans cell death gene, Ced-3, encodes a protein homologous to mammalian interleukin-1beta-converting enzyme (ICE), a cysteine protease implicated in programmed cell death (PCD). CPP32, also known as Yama, apopain, and Caspase-3, is a member of this family, has substrate specificities similar to Ced-3, and has been shown to have an active role in PCD. Evidence suggests that these proteases act downstream of inhibitors of PCD such as Bcl-2 and Bcl-x(L), which are frequently expressed in Reed-Sternberg (RS) cells of Hodgkin's disease (HD). To date there have been no studies examining the role of the ICE/Ced-3 family of proteins, in particular CPP32, in HD. We examined 24 cases of HD with a classical immunophenotype and 6 cases of nodular lymphocyte predominant HD (NLPHD) for the expression of CPP32 in the RS cells and lymphohistiocytic (L&H) cells as detected by immunohistochemistry. Twenty two of 24 cases (92%) of HD expressed the protein in the RS cells, whereas the L&H cells in all 6 cases of NLPHD lacked expression of CPP32. These results provide further evidence that NLPHD is a phenotypically different disease distinct from classical forms of HD. The differential expression of the cell death protein CPP32 may be an important factor contributing to the apparently different clinical behaviour of NLPHD in contrast to classical HD. The lack of expression of CPP32 in NLPHD shares similarities with low-grade B-cell non-Hodgkin's lymphomas and may explain their common clinical course. Further studies are required to elucidate the significance of CPP32 in HD.
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PMID:Immunohistochemical analysis of interleukin-1beta-converting enzyme/Ced-3 family protease, CPP32/Yama/Caspase-3, in Hodgkin's disease. 931 Apr 97

The development of resistance to host defense mechanisms such as tumor necrosis factor (TNF)- and Fas-mediated apoptosis of transformed or virus-infected cells may be a critical component in the development of disease. To find genes that protect cells from apoptosis, we used an expression cloning strategy and identified BHRF1, an Epstein-Barr virus (EBV) early-lytic-cycle protein with distant homology to Bcl-2, as an anti-apoptosis protein. Expression of BHRF1 in MCF-Fas cells conferred nearly complete resistance against both anti-Fas antibody and TNF-mediated apoptosis. In addition, BHRF1 protected these cells from monocyte-mediated killing but failed to protect them from killing mediated by lymphokine-activated killer cells. The ability of BHRF1 to protect MCF-Fas cells from apoptosis induced by various stimuli was identical to that of Bcl-2 and Bcl-xL. Moreover, the mechanism of action of BHRF1 resembled that of Bcl-2 and Bcl-xL as it inhibited TNF- and anti-Fas-induced activation of two enzymes participating in the apoptosis pathway, cytosolic phospholipase A2 and caspase-3/CPP32, but did not interfere with the activation of NF-kappaB-like transcription factors. A putative function of BHRF1 in EBV-infected epithelial cells may be to protect virus-infected cells from TNF- and/or anti-Fas-induced cell death in order to maximize virus production. Surprisingly, expression of neither BHRF1 nor Bcl-2 in a B-cell line, BJAB, protected the cells from anti-Fas-mediated apoptosis even though they increased the survival of serum-starved cells. Thus, the protective role of BHRF1 against apoptosis resembles that of Bcl-2 in being cell type specific and dependent on the apoptotic stimulus.
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PMID:The ability of BHRF1 to inhibit apoptosis is dependent on stimulus and cell type. 931 30

Dissociated cerebellar granule cells maintained in medium containing 25 mM potassium undergo an apoptotic death when switched to medium with 5 mM potassium. Granule cells from mice in which Bax, a proapoptotic Bcl-2 family member, had been deleted, did not undergo apoptosis in 5 mM potassium, yet did undergo an excitotoxic cell death in response to stimulation with 30 or 100 microM NMDA. Within 2 h after switching to 5 mM K+, both wild-type and Bax-deficient granule cells decreased glucose uptake to <20% of control. Protein synthesis also decreased rapidly in both wild-type and Bax-deficient granule cells to 50% of control within 12 h after switching to 5 mM potassium. Both wild-type and Bax -/- neurons increased mRNA levels of c-jun, and caspase 3 (CPP32) and increased phosphorylation of the transactivation domain of c-Jun after K+ deprivation. Wild-type granule cells in 5 mM K+ increased cleavage of DEVD-aminomethylcoumarin (DEVD-AMC), a fluorogenic substrate for caspases 2, 3, and 7; in contrast, Bax-deficient granule cells did not cleave DEVD-AMC. These results place BAX downstream of metabolic changes, changes in mRNA levels, and increased phosphorylation of c-Jun, yet upstream of the activation of caspases and indicate that BAX is required for apoptotic, but not excitotoxic, cell death. In wild-type cells, Boc-Asp-FMK and ZVAD-FMK, general inhibitors of caspases, blocked cleavage of DEVD-AMC and blocked the increase in TdT-mediated dUTP nick end labeling (TUNEL) positivity. However, these inhibitors had only a marginal effect on preventing cell death, suggesting a caspase-independent death pathway downstream of BAX in cerebellar granule cells.
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PMID:Bax deletion further orders the cell death pathway in cerebellar granule cells and suggests a caspase-independent pathway to cell death. 931 40

The caspase-3 (CPP32, apopain, YAMA) family of cysteinyl proteases has been implicated as key mediators of apoptosis in mammalian cells. Gelsolin was identified as a substrate for caspase-3 by screening the translation products of small complementary DNA pools for sensitivity to cleavage by caspase-3. Gelsolin was cleaved in vivo in a caspase-dependent manner in cells stimulated by Fas. Caspase-cleaved gelsolin severed actin filaments in vitro in a Ca2+-independent manner. Expression of the gelsolin cleavage product in multiple cell types caused the cells to round up, detach from the plate, and undergo nuclear fragmentation. Neutrophils isolated from mice lacking gelsolin had delayed onset of both blebbing and DNA fragmentation, following apoptosis induction, compared with wild-type neutrophils. Thus, cleaved gelsolin may be one physiological effector of morphologic change during apoptosis.
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PMID:Caspase-3-generated fragment of gelsolin: effector of morphological change in apoptosis. 932 9

Proteases that are members of the caspase (or interleukin-1beta converting enzyme (ICE)) protease family have been shown to be important mediators of apoptosis induced by Fas activation, neurotrophic factor withdrawal, and detachment from extracellular matrix. In this report we have investigated the potential importance of caspase proteases in apoptosis induced by multiple chemotherapeutic agents. Human T leukemic cells engineered to overexpress the cowpox virus CrmA protein, a direct and specific inhibitor of caspase proteases, were studied for their resistance to 1-beta-D-arabinofurasosyl-cytosine (Ara-C), etoposide (VP-16), doxorubicin (DOX), and cis-dichlorodiammine platinum (CP). Overexpression of CrmA dramatically inhibited drug-induced activation of caspases, as measured by processing of the inactive precursor form of caspase-3 and cleavage of caspase substrate proteins poly(ADP-ribose) polymerase (PARP) and lamin B. CrmA also significantly inhibited the kinetics of cell death induced by each of the four drugs. Moreover, when examined several days or weeks after initial exposure to drug, cultures of CrmA-expressing cells were found to have recovered and repopulated, whereas vector-transfected control cells did not. These studies demonstrate that caspase proteases play an important role in conferring sensitivity to multiple chemotherapy drugs, and that constitutive downmodulation of caspase activities can enhance chemoresistance.
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PMID:Inhibition of caspase proteases by CrmA enhances the resistance of human leukemic cells to multiple chemotherapeutic agents. 932 87


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