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

Intracellular cysteine proteinases (caspases) play key roles in inflammation and apoptosis. Recombinant caspases are typically produced in Escherichia coli expression systems with the attendant problems of solubilization, re-folding and activation of the protease. Here we describe the expression of hexahistidine-tagged caspase-3 (CPP32/Yama/Apopain) in the methylotropic yeast Pichia pastoris, and the purification of soluble enzyme from yeast lysates using cobalt affinity chromatography. The recombinant protease is fully activated, stable, and cleaves the synthetic substrate DEVD-AFC (Km 16.8 microM) but not YVAD-AFC. It mediates the cleavage of the apoptotic death substrate poly(ADP-ribose) polymerase in cell extracts, but does not cleave pro-interleukin-1beta. It is inhibited by the peptide DEVD-CHO (Ki 2.2 nM), far less efficiently by YVAD-CMK (Ki 0.3 microM), and not detectably by CrmA. By these criteria, recombinant caspase-3 is indistinguishable from native caspase-3 purified from apoptotic cell extracts. Activation of recombinant caspase-3 occurs in yeast in the absence of any intrinsic caspase activity, suggesting that caspase-3 can auto-activate. However, the purified enzyme was incapable of cleaving pro-caspase-3 indicating that autoactivation of caspase-3 in vivo is not likely to occur unless very high concentrations are achieved.
Biochem Biophys Res Commun 1997 Sep 29
PMID:Recombinant caspase-3 expressed in Pichia pastoris is fully activated and kinetically indistinguishable from the native enzyme. 932 93

Pim-1 oncoprotein is a serine/threonine kinase that can closely cooperate with c-Myc in lymphomagenesis, as does Bcl-2. Although the molecular mechanism of this cooperative transformation remains unknown, it is speculated that, similar to Bcl-2, Pim-1 contributes to transformation by inhibiting apoptosis. In this study, therefore, we examined the effect of Pim-1 expression on c-Myc-mediated apoptosis of Rat-1 fibroblasts triggered by serum deprivation. Our results showed that, rather than inhibiting apoptosis, Pim-1 expression stimulated c-Myc-mediated apoptosis in Rat-1 fibroblasts. Pim-1 stimulated c-Myc-mediated apoptosis through an enhancement of the c-Myc-mediated activation of caspase-3 (CPP32)-like proteases, since the suppression of this activity by a specific caspase inhibitor abolished the apoptosis stimulation by Pim-1. A kinase-defective Pim-1 mutant failed to stimulate c-Myc-mediated apoptosis, and Pim-1 expression alone in the absence of c-Myc overexpression did not induce apoptosis of serum-deprived Rat-1 cells, indicating that the kinase activity of Pim-1 and the activated c-Myc signaling pathway were required for apoptosis stimulation by Pim-1. Together, these results suggest that Pim-1 oncoprotein stimulates as a serine/threonine kinase the death signaling elicited by c-Myc at a step upstream of caspase-3-like protease activation in Rat-1 fibroblasts. Our results also suggest that Pim-1 kinase might function cooperatively with c-Myc through the phosphorylation of a factor(s) which regulates the common signaling pathway involved in c-Myc-mediated apoptosis and transformation.
Oncogene 1997 Sep 18
PMID:Pim-1 kinase stimulates c-Myc-mediated death signaling upstream of caspase-3 (CPP32)-like protease activation. 933 23

Coxsackievirus B3 (CVB3), an enterovirus in the family Picornaviridae, induces cytopathic changes in cell culture systems and directly injures multiple susceptible organs and tissues in vivo, including the myocardium, early after infection. Biochemical analysis of the cell death pathway in CVB3-infected HeLa cells demonstrated that the 32-kDa proform of caspase 3 is cleaved subsequent to the degenerative morphological changes seen in infected HeLa cells. Caspase activation assays confirm that the cleaved caspase 3 is proteolytically active. The caspase 3 substrates poly(ADP-ribose) polymerase, a DNA repair enzyme, and DNA fragmentation factor, a cytoplasmic inhibitor of an endonuclease responsible for DNA fragmentation, were degraded at 9 h following infection, yielding their characteristic cleavage fragments. Inhibition of caspase activation by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD.fmk) did not inhibit the virus-induced cytopathic effect, while inhibition of caspase activation by ZVAD.fmk in control apoptotic cells induced by treatment with the porphyrin photosensitizer benzoporphyrin derivative monoacid ring A and visible light inhibited the apoptotic phenotype. Caspase activation and cleavage of substrates may not be responsible for the characteristic cytopathic effect produced by picornavirus infection yet may be related to late-stage alterations of cellular homeostatic processes and structural integrity.
J Virol 1998 Sep
PMID:Caspase activation and specific cleavage of substrates after coxsackievirus B3-induced cytopathic effect in HeLa cells. 969 73

To gain insight into the pathogenic mechanisms of Huntington's disease (HD), we have developed a stable cellular model, using a neuroblastoma cell line in which the expression of full-length or truncated forms of wild-type and mutant huntingtin can be induced. While the wild-type forms have the expected cytoplasmic localization, the expression of mutant proteins leads to the formation of cytoplasmic and nuclear inclusions in a time- and polyglutamine length-dependent manner. The inclusions are ubiquitinated, appear more rapidly in cells expressing truncated forms of mutant huntingtin and are correlated with enhanced apoptosis. In lines expressing mutant full-length huntingtin, major characteristics present in Huntington's patients could be modelled. Selective processing of the mutant, but not the wild-type, full-length huntingtin was observed at late time points, with appearance of a breakdown product corresponding to a predicted caspase-3 cleavage product. A more truncated N-terminal fragment of huntingtin is also produced, that appears involved in building up cytoplasmic inclusions at early time points, and later on also nuclear inclusions. This fits with the finding that inclusions in the brain of HD patients are detected only using antibodies directed against epitopes very close to the polyglutamine stretch. This unique model should thus be useful to study the processing mechanism of mutant huntingtin, its role in the formation of intracellular aggregates and the effect of the latter on cellular physiology.
Hum Mol Genet 1998 Sep
PMID:A cellular model that recapitulates major pathogenic steps of Huntington's disease. 970 Jan 87

Exposure of mammalian cells to ultraviolet (UV) light elicits a cellular response and can also lead to apoptotic cell death. In this report, we show that a 36-kDa myelin basic protein (MBP) kinase detected by an in-gel kinase assay can be dramatically activated during the early stages of UV irradiation-triggered apoptosis of A431 cells. Immunoblot analysis revealed that this 36-kDa MBP kinase could be recognized by an antibody against the C-terminal regions of a family of p21Cdc42/Rac-activated kinases (PAKs). By using this antibody and a PAK2-specific antibody against the N-terminal region of PAK2 as studying tools, we further demonstrated that UV irradiation caused cleavage of PAK2 to generate a 36-kDa C-terminal catalytic fragment and a 30-kDa N-terminal fragment in A431 cells. The appearance of the 36-kDa C-terminal catalytic fragment of PAK2 matched exactly with the activation of the 36-kDa MBP kinase in A431 cells upon UV irradiation. In addition, UV irradiation also led to activation of CPP32/caspase-3, but not ICH-1L/caspase-2 and ICE/caspase-1, in A431 cells and the kinetics of activation of CPP32/caspase-3 appeared to correlate well with that of DNA fragmentation and of cleavage/activation of PAK2, respectively. Moreover, blockage of activation of CPP32/caspase-3 by pretreating the cells with two specific tetrapeptidic inhibitors for caspases (Ac-DEVD-cho and Ac-YVAD-cmk) could significantly attenuate the extent of cleavage/activation of PAK2 induced by UV irradiation. Collectively, the results demonstrate that cleavage and activation of PAK2 can be induced during the early stages of UV irradiation-triggered apoptosis and indicate the involvement of CPP32/caspase-3 in this process.
J Cell Biochem 1998 Sep 15
PMID:Proteolytic cleavage and activation of PAK2 during UV irradiation-induced apoptosis in A431 cells. 971 43

We show that mouse sperm die spontaneously within 1-2 days in culture and that treatment with either staurosporine (STS) and cycloheximide (CHX) or a peptide caspase inhibitor does not accelerate or delay the cell death. Chicken erythrocytes, by contrast, are induced to die by either serum deprivation or treatment with STS and CHX, and embryonic erythrocytes are more sensitive than adult erythrocytes to both treatments. Although these erythrocyte deaths display a number of features that are characteristic of apoptosis, they are not blocked, or even delayed, by peptide caspase inhibitors, and most of the cells die without apparently activating caspases. A small proportion of the dying erythrocytes do activate caspase-3, but even these cells, which seem to be the least mature erythrocytes, die just as quickly in the presence of caspase inhibitors. Our findings raise the possibility that both mouse sperm and chicken erythrocytes have a death programme that may not depend on caspases and that chicken erythrocytes lose caspases as they mature. Chicken erythrocytes may provide a useful 'stripped down' cell system to try to identify the protein components of such a death programme, which may serve to back-up the conventional caspase-dependent suicide mechanism in many cell types.
J Cell Sci 1998 Sep
PMID:Are caspases involved in the death of cells with a transcriptionally inactive nucleus? Sperm and chicken erythrocytes. 971 64

Vinorelbine (NVB) is a novel vinca alkaloid FDA approved for use in some advanced carcinomas. However, its role in non-Hodgkin's lymphoma (NHL) is still not well defined. NVB is an antimicrotubule agent, but as yet, it is not known whether it induces apoptosis. By flow cytometry using nuclear staining (propidium iodide) and annexin V, we demonstrated that NVB and vincristine (VCR) induced both mitotic arrest and apoptosis in leukemia and lymphoma cells, in a drug exposure time dependent manner. Cell cycle kinetics in 3 different cell lines varied during vinca alkaloid treatment. The annexin V method showed that apoptosis, as opposed to necrosis, was the dominant mode of cell kill of chemosensitive leukemia and lymphoma cells. Phosphatidylserine expression on the cell surface was detectable as a hallmark of apoptosis at earlier drug exposure when compared to conventional flow cytometry with PI staining. By Western blot analysis, we demonstrated that CPP32 or caspase-3, a critical apoptosis inducer, and its active subunits p20 and p11 were upregulated in chemo- and apoptosis-sensitive lymphoma and leukemia cells treated with NVB. Our data contributes to the emerging hypothesis suggesting that widely divergent exogenous stimuli and chemotherapeutic agents can effect apoptosis in cancer cells via different pathways involving the caspases. We believe that vinorelbine may be a potentially important drug in the treatment of NHL in the future.
Leuk Lymphoma 1998 Sep
PMID:Vinorelbine induces apoptosis and caspase-3 (CPP32) expression in leukemia and lymphoma cells: a comparison with vincristine. 972 Jul 29

The relationship between peripheral lymphocyte apoptosis and human immunodeficiency virus disease progression was studied in infected subgroups with distinct profiles of progression. Long-term nonprogressors (LTNP) and seronegative controls had levels of spontaneous apoptosis significantly lower than those for recent seroconverters who had CD4 cell counts similar to those of nonprogressors but with a high likelihood of disease progression. Lymphocytes from nonprogressors and seronegative controls also showed negligible spontaneous caspase-3 activity, a biochemical indicator for apoptosis, whereas early progressors exhibited substantial activity. In contrast, when activated with mitogens, the lymphocytes from both LTNP and progressors displayed indistinguishable levels of heightened apoptosis. Spontaneous apoptosis and plasma viremia levels correlated positively in progressors, but not in LTNP. These findings demonstrate that increased lymphocyte apoptosis is evident prior to CD4 T cell decline and that LTNP are relatively resistant to the factors that induce accentuated levels of spontaneous but not mitogen-induced cell death.
J Infect Dis 1998 Sep
PMID:Diminished spontaneous apoptosis in lymphocytes from human immunodeficiency virus-infected long-term nonprogressors. 972 34

Apoptosis is a mode of cell death characterized by distinct morphological features and DNA fragmentation. The program that leads to apoptosis has been considered to be predominantly extranuclear, and a signal transduction pathway to the nucleus exists during apoptosis, while characteristic events occur in the nucleus. As for radiation-induced apoptosis, the signal transduction pathway remains unclear, especially the sites where the primary effect of radiation occurs. In this study, we demonstrate that a cytoplasmic extract prepared from irradiated cells has the ability to cause DNA fragmentation and that caspase-3 is activated in this extract. Normal nuclei of HeLa S3 cells were added to a cytoplasmic extract made from HL60 cells which had been irradiated with 30 Gy of 137Cs gamma rays and were incubated. Agarose gel electrophoresis of the added nuclei showed a characteristic DNA laddering pattern. This reaction was blocked by a caspase-3 inhibitor but not by an ICE inhibitor. These observations suggest that a signal transduction pathway from an unknown target of gamma radiation may exist upstream of caspase-3 during radiation-induced apoptosis.
Radiat Res 1998 Sep
PMID:DNA fragmentation induced by a cytoplasmic extract from irradiated cells. 972 55

Murine L929 fibrosarcoma cells were transfected with the human Fas (APO-1/CD95) receptor, and the role of various caspases in Fas-mediated cell death was assessed. Proteolytic activation of procaspase-3 and -7 was shown by Western analysis. Acetyl-Tyr-Val-Ala-Asp-chloromethylketone and benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone++ +, tetrapeptide inhibitors of caspase-1- and caspase-3-like proteases, respectively, failed to block Fas-induced apoptosis. Unexpectedly, the broad-spectrum caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone and benzyloxycarbonyl-Asp(OMe)-fluoromethylketone rendered the cells even more sensitive to Fas-mediated cell death, as measured after 18 h incubation. However, when the process was followed microscopically, it became clear that anti-Fas-induced apoptosis of Fas-transfected L929 cells was blocked during the first 3 h, and subsequently the cells died by necrosis. As in tumor necrosis factor (TNF)-induced necrosis, Fas treatment led to accumulation of reactive oxygen radicals, and Fas-mediated necrosis was inhibited by the oxygen radical scavenger butylated hydroxyanisole. However, in contrast to TNF, anti-Fas did not activate the nuclear factor kappaB under these necrotic conditions. These results demonstrate the existence of two different pathways originating from the Fas receptor, one rapidly leading to apoptosis, and, if this apoptotic pathway is blocked by caspase inhibitors, a second directing the cells to necrosis and involving oxygen radical production.
J Exp Med 1998 Sep 07
PMID:Dual signaling of the Fas receptor: initiation of both apoptotic and necrotic cell death pathways. 973 Aug 93


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