Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P42574 (caspase-3)
 45,978 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have demonstrated that clofilium, a potassium channel blocker, induces apoptosis on human promyelocytic leukemia (HL-60) cells. Cells treated with clofilium led to suppression of viability and proliferation in both time and concentration-dependent manners. Nuclear DAPI staining and electronmicroscopic examination revealed typical nuclear features of apoptosis in cells treated with clofilium that was further verified in DNA fragmentation analysis. Flow cytometry analysis with FITC-annexin V and propidium iodide (PI) revealed that apoptotic cell population with Annexin V+/PI- increased gradually from < 2% at 0 h, to 20% at 4 h and 29% at 16 h after exposure to 10 microM clofilium in HL-60 cells. Furthermore, fluorometric immunosorbent enzyme assay for activity of caspase-3 showed approximately a 10-fold increase of activity in cells treated with 10 microM of clofilium for 2-3 h compared with the basal level of its activity in untreated control cells. Immunoblotting analysis revealed proteolytic cleavage of caspase-3 and subsequent cleavage of PARP. However, there was no significant change of Bcl-2 and Bax proteins. These results indicate that clofilium exerts antiproliferative action and growth inhibition on HL-60 through induction of apoptosis which is mediated via Bcl-2-insensitive activation of caspase-3, and suggest chemotherapeutic and cytostatic potentials of this compound in human leukemias.
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PMID:Clofilium, a potassium channel blocker, induces apoptosis of human promyelocytic leukemia (HL-60) cells via Bcl-2-insensitive activation of caspase-3. 1066 93

In rat hepatocytes and isolated liver mitochondrial fractions, Cyclosporine A (CsA) is often used as a specific inhibitor of mitochondrial Ca(2+) release and as a specific blocker of mitochondrial membrane potential and permeability transition (MPT), which are all processes involved in the inhibition of apoptosis. However, neither inhibition nor induction of apoptosis by CsA has yet been described in the rat hepatocyte primary culture during incubation for 4 and 20 h. It was the purpose of the present study to examine by means of morphological and biochemical criteria the effects of CsA on apoptosis and to characterize the underlying mechanisms. Rat hepatocytes were cultured for 4 or 20 h with CsA at concentrations of 0, 10, 25, and 50 microM. Chromatin condensation and fragmentation, DNA fragmentation (TUNEL), membrane phosphatidylserine distribution (Annexin V), caspase-1, -3, and -6 activity, mitochondrial membrane potential (Rhodamine 123), and cytochrome c release into the cytosol were investigated. Four hours after CsA treatment, chromatin condensation and fragmentation and the number of TUNEL- and Annexin V-positive cells increased dose-dependently without any observable enzyme leakage, which indicated the integrity of the outer cell membrane. After 20 h of CsA incubation apoptosis parameters were further increased and were accompanied by the increased activity of the cysteine protease, caspase-3 (CPP 32), and slightly increased caspase-6 (Mch 2), but not caspase-1 (ICE). The caspase-3 inhibitor, Ac-DEVD-CHO, inhibited caspase-3 activation and attenuated CsA-induced apoptosis and LDH leakage. The caspase-6 inhibitor, Ac-VEID-CHO, only marginally inhibited CsA-induced apoptosis. Decreased mitochondrial membrane potential and cytochrome c release went in parallel with ultrastructural mitochondrial changes and might be regarded as early events that trigger the apoptosis cascade. Transmission electron microscopy confirmed an increase in the number of necrotic cells after 20 h, but not after 4 h, compared with controls.
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PMID:Mechanisms of cyclosporine A-induced apoptosis in rat hepatocyte primary cultures. 1070 60

The immunosuppressive cyclosporine A derivative, O-hydroxyethyl-D(Ser)(8)-cyclosporine (SDZ IMM 125), was examined for its ability to induce apoptosis in rat hepatocytes cultured for 4 or 20 h. Four hours after SDZ IMM 125 treatment, chromatin condensation and fragmentation, and the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeled and Annexin V-positive cells increased dose dependently without any observable lactate dehydrogenase leakage. The activity of the cysteine protease, caspase-3, was increased, but not that of caspase-1 and -6. The specific caspase-3 inhibitor, Ac-Asp-Glu-Val-Asp-aldehyde, inhibited caspase-3 activation and attenuated SDZ IMM 125-induced apoptosis and lactate dehydrogenase leakage. After 20 h of SDZ IMM 125 incubation, the parameters of apoptosis were further increased. Decreased mitochondrial membrane potential (measured by rhodamine 123 uptake) and cytochrome c release went in parallel with ultrastructural mitochondrial changes, and might be regarded as early events that trigger the apoptotic cascade. Transmission electron microscopy showed cytoplasmic blebbing after 4 h of SDZ IMM 125 incubation. As observed by transmission electron microscopy, treatment with SDZ IMM 125 resulted in an increase in the number of necrotic cells after 20 h, but not after 4 h. Our findings suggest that in rat hepatocyte cultures, SDZ IMM 125 is a specific inducer of apoptosis after short-term incubation, and this overlaps with necrosis after longer treatment periods. It is very likely that the necrosis occurring later is the result of the early apoptotic events.
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PMID:Induction of apoptosis by the O-hydroxyethyl-D(Ser)(8)-cyclosporine A derivative SDZ IMM 125 in rat hepatocytes. 1073 49

We investigated the possible roles of mitochondrial manganese superoxide dismutase (MnSOD) and bcl-2 in etoposide-induced cell death in acute myeloblastic leukaemia (AML) using two subclones of the OCI/AML-2 cell line, the etoposide-sensitive (ES) and the etoposide-resistant (ER), as models. Cell death after 24 h exposure to 10 micromol/l etoposide was about 60% and 70% in the ES subclone and about 20% and 25% in the ER subclone, when analysed by trypan blue and annexin V respectively. Cytochrome c efflux from mitochondria to cytosol was observed after 4 h of exposure in both subclones, whereas the activation of caspase-3 was not detectable until after 12 h of exposure in the ES subclone and 24 h of exposure in the ER subclone, using Western blotting. The decrease in mitochondrial membrane potential, when analysed by the JC-1 probe fluorocytometrically, also appeared to take place later in the ER than in the ES subclone. Both subclones showed evident basal expression of MnSOD and bcl-2 by Western blotting. Etoposide caused a potent induction of MnSOD, more than 400% at 12 h, in the ER but not in the ES subclone. No significant change in bcl-2 expression could be observed in either of the subclones during exposure to etoposide when analysed by Western blotting or flow cytometry. In conclusion, we suggest that MnSOD might have a special role in the protection of AML cells against etoposide-induced cell death. Although unable to influence the cytochrome c efflux to cytosol, MnSOD might prevent the disruption of mitochondrial membrane potential, which evidently leads to cell death by releasing various activators of apoptosis.
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PMID:Induction of mitochondrial manganese superoxide dismutase confers resistance to apoptosis in acute myeloblastic leukaemia cells exposed to etoposide. 1075 16

Cyclooxygenase-2 (Cox-2), an enzyme responsible for catalyzing the committed step in prostanoid biosynthesis, is the product of an immediate early gene capable of being up-regulated by diverse stimuli. Significantly Cox-2 mRNA is absent from rat pheochromocytoma (PC12) cells, both basally and following stimulation with a range of agonists. Using PC12 cells engineered to stably express isopropyl-1-thio-beta-D-galactopyranoside-inducible Cox-2 (PCXII-4), we have investigated the putative effects of Cox-2 expression on differentiation, proliferation, and trophic withdrawal apoptosis. Cox-2 bioactivity had no effect on nerve growth factor-induced differentiation, epidermal growth factor-induced proliferation, or aromatic L-amino acid decarboxylase expression. However, trophic withdrawal apoptosis, induced by the removal of nerve growth factor following differentiation, was markedly reduced in the PCXII-4 when compared with control cells, as assessed by annexin V staining, DNA laddering, and Hoechst 33258 staining. The specificity of this effect was confirmed using two pharmacologically distinct nonsteroidal anti-inflammatory drugs, indomethacin and NS398. Investigations showed that the activity of the pro-apoptotic protease caspase-3 was reduced in PCXII cells. This study demonstrates that Cox-2-derived prostaglandins exert cytoprotective effects in trophic factor withdrawal apoptosis and provides evidence that this is, at least in part, due to suppression of caspase-3 activity.
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PMID:Cyclooxygenase-2 expression inhibits trophic withdrawal apoptosis in nerve growth factor-differentiated PC12 cells. 1076 43

Apoptosis is a process of active cell death and is characterized by activation of caspases, DNA fragmentation, and biochemical and morphological changes. To better understand apoptosis, we have characterized the dose- and time-dependent toxic effects of cadmium in Rat-1 fibroblasts. Staining of cells with phosphatidylserine (PS)-annexin V, Hoechst 33258 or Rhodamine 123 and Tunel assays showed that incubating cells with 10 microM cadmium induced a form of cell death exhibiting typical characteristics of apoptosis, including cell shrinkage, externalization of PS, loss of mitochondria membrane potential, nuclear condensation and DNA fragmentation. Expression of Bcl-2 or CrmA each suppressed cadmium-induced cell death although Bcl-2 was somewhat more effective than CrmA. In vitro assay of caspase activity carried out using poly(ADP-ribose) polymerase (PARP) as a substrate as well as intracellular caspase assays using a fluorigenic caspase-3 substrate confirmed that caspase-3 is activated in Rat-1 cells undergoing cadmium-induced apoptosis. Both Asp-Glu-Val-Asp-aldehyde (DEVD-cho) and Tyr-Val-Ala-Asp-chloromethylketone (YVAD-cmk), selective inhibitors of caspase-3 and caspase-1, respectively, suppressed significantly cadmium-induced cell death. However, the nonselective caspase inhibitor, z-Val-Ala-Asp-floromethylketone (zVAD-fmk), was the most efficacious agent, almost completely blocking cadmium-induced cell death. Taken together, these results demonstrate that as in other forms of apoptosis, caspases play a central role in cadmium-induced cell death.
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PMID:Cadmium induces caspase-mediated cell death: suppression by Bcl-2. 1077 Nov 29

Peripheral blood mononuclear cells (PBMCs) obtained from patients with advanced melanoma but not from healthy individuals were found to undergo spontaneous ex vivo apoptosis upon incubation in medium. PBMCs were evaluated for evidence of apoptosis using Annexin V binding, caspase-3 activation, and DNA fragmentation (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling). PBMCs of patients with melanoma contained a significantly higher proportion (P = 0.0027) of spontaneously apoptotic cells than PBMCs of controls after 24-h incubation in medium alone. The relative proportion of activated Fas+ and tumor necrosis factor receptor 1-positive (TNFR1+) PBMCs was significantly higher in patients with melanoma than that observed in controls. To demonstrate that the TNF family of receptors and ligands was involved in this type of apoptosis, PBMCs were incubated in the presence of agonistic anti-Fas antibody (CH-11) or TNF-alpha. The proportion of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive PBMCs undergoing spontaneous apoptosis was found to be comparable with that induced by CH-11 antibody or TNF-alpha. Three-color flow cytometry revealed that CD3+ Fas+ T cells were especially sensitive to apoptosis and were preprogrammed in vivo to die. Apoptosis occurred in all subsets of PBMCs but was significantly higher (P = 0.01) in the CD3+ CD8+ T-cell subset in patients relative to controls. In two patients with melanoma, who responded clinically to dendritic cell-based peptide vaccines, the proportion of apoptotic T cells was decreased by half after therapy. In patients who were treated previously with vaccination-based therapies, levels of T-cell apoptosis were lower than in the other melanoma patients. The observed accelerated death of T cells, which are activated and susceptible to apoptosis in patients with melanoma, may contribute to a rapid turnover of immune cells, resulting in a decreased immunocompetence.
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PMID:Spontaneous apoptosis of CD8+ T lymphocytes in peripheral blood of patients with advanced melanoma. 1077 63

Viral protein R (Vpr) of human immunodeficiency virus type 1 inhibits cell proliferation by arresting the cell cycle at the G(2) phase and inducing to apoptosis after G(2) arrest. We have reported previously that C81, a carboxy-terminally truncated form of Vpr, interferes with cell proliferation via a novel pathway that is distinct from G(2) arrest. However, the mechanism of this effect of C81 is unknown. We demonstrate here that C81 can induce apoptosis via G(1) arrest of the cell cycle. Immunostaining for various markers of stages of the cell cycle and flow cytometry analysis of DNA content showed that most HeLa cells that had been transiently transfected with a C81 expression vector were arrested at the G(1) phase and not at the G(2) or S phase of the cell cycle. Staining for annexin V, which binds phosphatidylserine on the plasma membrane, as an early indicator of apoptosis and measurement of the activity of caspase-3, a signaling molecule in apoptotic pathways, indicated that C81 is a strong inducer of apoptosis. Expression of C81 induced the condensation, fragmentation, and clumping of chromatin that are typical of apoptosis. Furthermore, the kinetics of the C81-induced G(1) arrest were closely correlated with changes in the number of annexin V-positive cells and the activity of caspase-3. Replacement of Ile or Leu residues by Pro at positions 60, 67, 74, and 81 within the leucine zipper-like domain of C81 revealed that Ile60, Leu67, and Ile74 play important roles both in the C81-induced G(1) arrest and in apoptosis. Thus, it appears that C81 induces apoptosis through pathways that are identical to those utilized for G(1) arrest of the cell cycle. It has been reported that Ile60, Leu67, and Ile74 also play an important role in the C81-induced suppression of growth. These results suggest that the suppression of growth induced by C81 result in apoptosis that is independent of G(2) arrest of the cell cycle.
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PMID:A carboxy-terminally truncated form of the human immunodeficiency virus type 1 Vpr protein induces apoptosis via G(1) cell cycle arrest. 1084 89

We investigated the in vitro effect of As2O3 on proliferation, cell cycle regulation, and apoptosis in human myeloma cell lines. As2O3 significantly inhibited the proliferation of all of eight myeloma cell lines examined in a dose-dependent manner with IC50 of approximately 1-2 microM. DNA flow cytometric analysis indicated that As2O3 (2 microM) induced a G1 and/or a G2-M phase arrest in these cell lines. To address the mechanism of the antiproliferative effect of As2O3, we examined the effect of As2O3 on cell cycle-related proteins in MC/CAR cells in which both G1 and G2-M phases were arrested. Western blot analysis demonstrated that treatment with As2O3 (2 microM) for 72 h did not change the steady-state levels of CDK2, CDK4, cyclin D1, cyclin E, and cyclin B1 but decreased the levels of CDK6, cdc2, and cyclin A. The mRNA and protein levels of CDKI, p21 were increased by treatment with As2O3, but those of p27 were not. In addition, As2O3 markedly enhanced the binding of p21 with CDK6, cdc2, cyclin E, and cyclin A compared with untreated control cells. Furthermore, the activity of CDK6-associated kinase was reduced in association with hypophosphorylation of Rb protein. The activity of cdc2-associated kinase was decreased, which was accompanied by the up-regulation of cdc2 phosphorylation (cdc2-Tyr15 phosphorylation) resulting from reduction of cdc25B and cdc25C phosphatases. As2O3 also induced apoptosis in MC/CAR cells as evidenced by flow cytometric detection of sub-G1 DNA content and annexin V binding assay. This apoptotic process was associated with down-regulation of Bcl-2, loss of mitochondrial transmembrane potential (delta psi(m)), and an increase of caspase-3 activity. These results suggest that As2O3 inhibits the proliferation of myeloma cells, especially MC/CAR cells, via cell cycle arrest in association with induction of p21 and apoptosis.
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PMID:Arsenic trioxide-mediated growth inhibition in MC/CAR myeloma cells via cell cycle arrest in association with induction of cyclin-dependent kinase inhibitor, p21, and apoptosis. 1085 Apr 58

Exposure to environmental contaminants, such as polychlorinated biphenyls (PCBs), may severely compromise normal function of vascular endothelial cells (EC). We have previously shown that PCB 77 (3,3',4,4'-tetrachlorobiphenyl), an arylhydrocarbon receptor (AhR) agonist, can induce oxidative stress in cultured EC. We now show that PCB 77 can activate EC and induce a cellular stress response that is reflected by the activation of c-Jun N-terminal/stress-activated protein kinases (JNK/SAPK). Our data also suggest that this PCB 77-mediated stress response can be modulated by the intracellular glutathione content. EC treated with buthionine-sulphoximine (BSO), an inhibitor of glutathione synthesis, further enhanced PCB-induced JNK/SAPK activity. This stress response was sustained only in the presence of BSO plus PCB 77. Media supplementation with the glutathione precursor N-acetyl-cysteine (NAC) reduced PCB 77-induced JNK/SAPK. Intracellular glutathione also may be implicated in PCB-induced EC apoptosis. Individual treatment with PCB, BSO, or linoleic acid induced activation of caspase 3. Compared to PCB 77 alone, annexin V activity was further amplified during combined treatment with BSO and PCB 77. DNA fragmentation was mostly observed when cells were treated with both BSO and PCB 77. The caspase 3-specific inhibitor DEVD-CHO protected cells against PCB 77/BSO-mediated apoptosis and inhibited the caspase activity without affecting JNK/SAPK activation or cellular glutathione levels. These results suggest that AhR ligands, such as PCB 77, cause vascular EC dysfunction by modulating intracellular glutathione, which subsequently leads to activation of stress-specific kinases. Furthermore, inhibition of glutathione synthesis by BSO can further potentiate the PCB 77-induced stress response and ultimately lead to apoptotic cell death.
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PMID:Cellular glutathione status modulates polychlorinated biphenyl-induced stress response and apoptosis in vascular endothelial cells. 1087 16


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