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)

TCR binding to an MHC class I/peptide complex is a central event in CTL-mediated elimination of target cells. In this study, we demonstrate that specific activation of the TCR-binding region of the HLA-A2 class I alpha2 domain induces apoptotic cell death. mAbs to this region rapidly induced apoptosis of HLA-A2-expressing Jurkat E11 cells, as determined by morphologic changes, phosphatidylserine exposure on the cell surface, and propidium iodide uptake. In contrast, apoptosis was not induced following culture with mAbs directed to other regions of the class I molecule. Death signaling by class I molecules is apparently dependent on coreceptor activation, as apoptosis is also signaled by HLA-A2 molecules, where the intracytoplasmic residues were deleted. HLA class I alpha2-mediated cell death appeared to proceed independent of the Fas pathway. Compared with apoptotic signaling by Fas ligation, HLA class I alpha2-mediated responses displayed a faster time course and could be observed within 30 min. Furthermore, class I alpha2-induced cell death did not involve observable DNA fragmentation. The apoptotic response was not affected significantly by peptide inhibitors of IL-1beta converting enzyme (ICE)-like proteases and CPP32. Taken together, activation of the TCR-binding domain of the class I alpha2 helix may result in apoptotic signaling apparently dependent on a novel death pathway. Thus, target HLA class I molecules may directly signal apoptotic cell death following proper ligation by the TCR.
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PMID:The TCR-binding region of the HLA class I alpha2 domain signals rapid Fas-independent cell death: a direct pathway for T cell-mediated killing of target cells? 957 38

Upon transforming growth factor-beta (TGF-beta) treatment, Ramos cells, a B-cell lymphoma cell line, undergo apoptosis, as measured by annexin V labeling, DNA fragmentation, and propidium iodide staining. Apoptosis could be observed by 24 h after TGF-beta exposure and occurred before the development of a significant blockage of cell cycle progression. TGF-beta-mediated apoptosis was also accompanied by a strong induction of caspase-3 subfamily activity. Incubation of cells with the caspase inhibitor Z-VAD.FMK at 20 microM, but not at 10 microM, prevented TGF-beta-induced apoptosis from occurring. By comparison, caspase-3 subfamily activity was 87% inhibited at 10 microM Z-VAD.FMK and completely inhibited at 20 microM. Because of TGF-beta's well-established role of regulating gene transcription, the mRNA levels for proteins associated with apoptosis (Fas- and Fas-associated proteins, Bcl-2 family members, IAP proteins, and I kappa B) were also studied. After 24 h of TGF-beta treatment, the most significant mRNA changes occurred with Bcl-XL (two-fold decrease) and Bik (twofold increase). TGF-beta treatment also resulted after 48 h in a fivefold decrease in Bcl-XL protein levels, based on immunoblotting analysis. Therefore, TGF-beta-mediated apoptosis involves the activation of caspases. In addition, TGF-beta transcriptionally regulates Bcl-2 family members, Bcl-XL and Bik, to further influence the apoptosis process.
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PMID:Transforming growth factor-beta-mediated apoptosis in the Ramos B-lymphoma cell line is accompanied by caspase activation and Bcl-XL downregulation. 966 22

The role of the basal activity of the serine/threonine protein kinase, protein kinase C (PKC) in the regulation of anti-CD95-induced apoptosis in Jurkat T cells was investigated. The PKC-specific inhibitor GF 109203X and the proposed cPKC-specific inhibitor Go 6976, in a concentration-dependent manner, increased the percentage of cells undergoing apoptosis induced by anti-CD95 mAb as demonstrated by propidium iodide (PI) staining, TUNEL assay and DNA fragmentation by gel electrophoresis. Furthermore, Go 6976 and GF 109203X abrogated phorbol myristate acetate-induced inhibition of anti-CD95-induced apoptosis. To examine the molecular mechanism by which PKC modulates anti-CD95-induced apoptosis, the effects of Go 6976 on known effector and regulatory molecules of cell death were studied. Increased recruitment of cells undergoing apoptosis was associated with enhanced anti-CD95-induced proteolytic cleavage of the most receptor-proximal cysteine protease caspase-8, subsequent cleavage and activation of the machinery protease caspase-3, and cleavage of the caspase substrates DNA-dependent protein kinase catalytic subunit, poly-(ADP-ribose) polymerase and lamin B1. CD95 and FADD protein levels in Jurkat T cells were not altered by Go 6976 treatment. In addition, Go 6976 did not alter protein levels and subcellular distribution of the anti-apoptotic molecules Bcl-2 and Bcl-xL. These data suggest indirectly that basal PKC activity acts at an early stage in the anti-CD95-induced caspase pathway to attenuate subsequent activation of downstream effector molecules and associated apoptosis in Jurkat T cells.
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PMID:Inhibition of the protein kinase C pathway promotes anti-CD95-induced apoptosis in Jurkat T cells. 970 Oct 26

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.
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PMID:Vinorelbine induces apoptosis and caspase-3 (CPP32) expression in leukemia and lymphoma cells: a comparison with vincristine. 972 Jul 29

We have characterized the cytotoxic activity of the obligate intracellular bacterium Chlamydia psittaci, which resides within a membrane-bound vacuole during the 2-day infection cycle. We have established that infected epithelial cells and macrophages die through apoptosis, which is measurable within 1 day of infection and requires productive infection by the bacteria. Inhibition of host cell protein synthesis has no effect on cell death, but blocking bacterial entry or bacterial protein synthesis prevents apoptosis, implying that bacterial growth is required for death of the host cell. Apoptosis was confirmed through the use of electron microscopy, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, gel agarose electrophoresis of fragmented DNA, and propidium-iodide labeling of host cell nuclei. Although infected cells died preferentially, both infected and uninfected cells became apoptotic, suggesting that the infected cells may secrete proapoptotic factors. Inhibition of either of two proapoptotic enzymes, caspase-1 or caspase-3, did not significantly affect Chlamydia-induced apoptosis. These results suggest that, as in the case of apoptosis due to Bax expression or oncogene dysregulation, which initiate the apoptotic program within the cell interior, the Chlamydia infection may trigger an apoptotic pathway that is independent of known caspases. As apoptotic cells secrete proinflammatory cytokines, Chlamydia-induced apoptosis may contribute to the inflammatory response of the host.
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PMID:Apoptosis of epithelial cells and macrophages due to infection with the obligate intracellular pathogen Chlamydia psittaci. 978 Jan 96

The antiangiogenic, tubulin-binding drug combretastatin A-4 exhibits a selective toxicity for proliferating endothelial cells in vitro and induces vascular shutdown in tumor models in vivo. The mechanism of combretastatin A-4 cytotoxicity has now been investigated with cultured proliferating human umbilical vein endothelial cells by examining various markers of apoptosis. Incubation of cells with 0.1 mM combretastatin A-4 induced the conversion (first detected after 6 h) of the CPP32 proenzyme to active caspase-3, a cysteine protease that plays an important role in apoptosis in many cell types; the drug also increased caspase-3 activity. Another early event observed was the binding of annexin V to 50% of the cells 8 h after drug treatment. Internucleosomal DNA fragmentation, another hallmark of apoptosis, was detected in cells incubated with 0.1 mM combretastatin A-4 for 24 h. Staining with Hoechst 33258 revealed that about 75% of cells exhibited a nuclear morphology characteristic of apoptosis after incubation with drug for 24 h. Incubation of cells for up to 8 h with combretastatin A-4 did not induce the release of lactate dehydrogenase or increase the uptake of propidium iodide, both indicators of membrane integrity. These results indicate that the selective cytotoxic effect of combretastatin A-4 is mediated by the induction of apoptosis rather than by necrosis and may provide an enhanced clinical strategy in cancer chemotherapy with this new agent.
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PMID:Induction of apoptosis in proliferating human endothelial cells by the tumor-specific antiangiogenesis agent combretastatin A-4. 978 91

Earlier reports on nonneural cells have shown that the normally inner plasma membrane lipid, phosphatidylserine (PS), flip-flops out during the early stages of apoptosis, whereas DNA laddering and plasma membrane permeabilization occur during the late stages. In this study, the applicability of these parameters to CNS-derived neuronal cells was tested using hippocampal HN2-5, cells that undergo apoptosis under anoxia. Because such insults on unsynchronized cells, e.g., undifferentiated HN2-5 cells, result in both early and late apoptotic cells, we mechanically separated these cells into three fractions containing (a) cells that had completely detached during anoxia, (b) cells that remained weakly attached to the tissue culture dish and, once detached by trituration in serum-containing medium, did not reattach, and (c) cells that reattached in 2-3 h. Fractions a and b contained cells that showed pronounced DNA laddering, whereas cells in fraction c did not show any DNA laddering. Double staining with fluorescein isothiocyanate-annexin V (which binds to PS) and propidium iodide (which stains the DNA in cells with a permeable cell membrane) revealed that all cells in fraction a had a permeable cell membrane (propidium iodide-positive) and PS molecules in the outer leaflet of the plasma membrane (fluorescein isothiocyanate-annexin V-positive). By contrast, fractions b and c contained cells with no externalized PS molecules. Cells in fractions a-c also showed, respectively, 50-, 21-, and 5.5-fold higher caspase-3 (CPP32) activity than that in healthy control cells. All these results show that fraction a contained late apoptotic cells, which also had the highest CPP32 activity; cells in fraction b were at an intermediate stage, when DNA laddering had already occurred; and fraction c contained very early apoptotic cells, in which no DNA laddering had yet occurred. Therefore, in the neuronal HN2-5 cells, externalization of PS occurs only during the final stages of apoptosis when the cells have completely lost their adhesion properties. Further experiments showed that ameboid microglial cells isolated from neonatal mouse brain phagocytosed only the cells in fraction a. These results show that in CNS-derived HN2-5 cells, (a) PS externalization is a late apoptotic event and is concomitant with a complete loss of surface adhesion of the apoptotic cells and (b) PS externalization is crucial for microglial recognition and phagocytosis of the apoptotic HN2-5 cells. Thus, PS externalization could be causally linked to the final detachment of apoptotic neuronal cells, which in turn prepares them for rapid phagocytosis by microglia.
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PMID:Externalization of phosphatidylserine may not be an early signal of apoptosis in neuronal cells, but only the phosphatidylserine-displaying apoptotic cells are phagocytosed by microglia. 979 9

Villous trophoblast in the human placenta consists of a population of proliferating stem cells which differentiate and individually fuse into the syncytiotrophoblast. We studied the apoptotic cascade in this complex epithelial layer by immunohistochemical localization of Fas, FasL, Bcl-2, Mcl-1, pro-caspase-3 and caspase-3, T-cell-restricted intracellular antigen-related protein (TIAR), poly(ADP-ribose) polymerase (PARP), lamin B, topoisomerase IIalpha, and transglutaminase II in cryostat and paraffin-fixed tissue sections from normal human first-trimester and term placental villi. The relationship between the apoptotic cascade and syncytial fusion was studied by coincubation of intact villi with FITC-coupled annexin-V, to detect the phosphatidylserine flip, and propidium iodide, to detect plasma membrane permeability. The final events of the apoptotic cascade were studied by the TUNEL reaction and ultrastructural appearance of the trophoblast. The phosphatidylserine flip was identified in some of the villous cytotrophoblastic cells, but the presence of both Bcl-2 and Mcl-1 proteins presumably prevented continuation of the apoptotic cascade. The syncytiotrophoblast demonstrated heterogeneous findings, suggesting variable progression along the apoptotic cascade. In some areas Bcl-2 and Mcl-1 predominated, with preservation of the nuclear proteins PARP, lamin B, and topoisomerase IIalpha; in other areas, especially in and around syncytial sprouts, Bcl-2 and Mcl-1 were absent, accompanied by loss of nuclear proteins, presence of phosphatidylserine flip, and TUNEL positivity. These data suggest that the apoptotic cascade is initiated in the villous cytotrophoblast, which in turn promotes syncytial fusion. Donation of anti-apoptotic proteins into the syncytium, such as Bcl-2 and Mcl-1, focally inhibits further progression along this cascade. Completion of the apoptotic cascade takes place in and around syncytial sprouts, providing further evidence that these are the sites of trophoblast shedding into the maternal circulation.
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PMID:Villous cytotrophoblast regulation of the syncytial apoptotic cascade in the human placenta. 982 29

The mechanism by which radiation induces human peripheral T cell apoptosis is not known. We examined sequential changes in post-irradiated peripheral blood mononuclear cells (PBMC(S)) taken from normal volunteers, by using flow-cytometer and an anti-CD3 monoclonal antibody, annexin V, propidium iodide, anti-Fas antibody, and anti-Fas ligand antibody. After 5 or 10 Gy of irradiation with a 60Co radiation therapy unit, most of the human peripheral T cells showed positivity against annexin V in 15 h, and positivity against propidium iodide in 23 h after irradiation. On a microscopy-video system, approximately 80% of mononuclear cells revealed apoptotic changes in 24 h after irradiation. Because of its proposed role in activation-induced cytotoxicity, we also examined the Fas (CD95/Apo-1) pathway in killing T cells by irradiation. Irradiated PBMC, displayed no increase in surface Fas expression and caspase-3 activity relative to non-irradiated cells. In addition, the anti-Fas ligand failed to eliminate the apoptotic death of PBMC, after irradiation. These results suggest that irradiation induces direct apoptosis of T cells by a Fas-independent mechanism.
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PMID:Radiation kills human peripheral T cells by a Fas-independent mechanism. 985 24

Exposure of the two related human leukemic cell lines U937 and TUR to chemotherapeutic compounds resulted in opposite effects on induction and resistance to apoptosis. Incubation of U937 cells with 1-beta-d-arabinofuranosylcytosine or the etoposide VP-16 was accompanied by growth arrest in G0/G1 of the cell cycle and an accumulation of a population in the sub-G1 phase which exhibited characteristics typical for the apoptotic pathway. In contrast, human TUR leukemia cells demonstrated no significant effects after a similar treatment with Ara-C and VP-16. Thus, TUR cells continued to proliferate in the presence of these anti-cancer drugs and the number of apoptotic cells as evaluated by propidium iodide staining and the detection of internucleosomal DNA fragmentation was significantly reduced when compared to the parental U937 cells. Similar effects were observed upon serum-starvation demonstrating resistance to apoptosis in TUR cells. Whereas induction of apoptosis is regulated by a network of distinct factors including the activation of proteolytically active caspases, we investigated these pathways in both cell lines. U937 cells demonstrated activation of the 32-kDa caspase-3 upon drug treatment by cleavage into the 20-kDa activated form. However, there was no 20-kDa caspase-3 fragment detectable in TUR cells. Simultaneously, the enzymatic activity of caspase-3 was significantly increased in drug-treated U937 cells as measured in vitro by enhanced metabolization of a fluorescence substrate and in vivo by cleavage of an appropriate substrate for caspase-3, namely, protein kinase Cdelta. In contrast, there was little if any caspase-3 activation detectable in drug-treated TUR cells. Taken together, these data suggest a signaling defect in the activation of the caspase-3 proteolytic system in TUR cells upon treatment with chemotherapeutic compounds which is associated with resistance to apoptosis in these human leukemia cells.
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PMID:Signaling defect in the activation of caspase-3 and PKCdelta in human TUR leukemia cells is associated with resistance to apoptosis. 1006 81


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