bongkrekic-acid and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

The role of p53 in apoptosis and the contrasting p53 status in tumors prompted us to investigate the bleomycin-induced apoptosis in p53-null human leukemia HL-60 cells (bleomycin at 160 microM for 7.5 h). Cells with apoptotic phenotype increased from 0.87% in controls to 9.40% in bleomycin-treated cells. Both the enzymes, caspase-3 and -8, were activated. Furthermore, the apoptotic phenotypes totally disappeared with zVAD-fmk, a caspase inhibitor. Besides, cytochrome c release from mitochondria happened simultaneously to apoptotic phenotypes, shrinkage of mitochondria but being independent of the mitochondrial permeability transition, since cyclosporine A and bongkrekic acid were inefficient on induced apoptosis. On the other hand, incubations with bleomycin (BLM) did not result in detectable changes in the expression of Bcl-2- and Bax-mRNA neither Bcl-2- or Bax-proteins. In conclusion, we suggest that BLM can produce apoptosis independently of p53 through three mechanisms: i) at the nuclear level by its endonuclease activities; ii) at the cell membrane, by activating caspases; and iii) at the mitochondria by releasing cytochrome c. These results indicate that BLM-induced apoptosis in HL-60 cells results from the activation of a mitochondria-dependent caspase cascade which includes also the activation of the initiator caspase-8.

Topics: Amino Acid Chloromethyl Ketones; Antimetabolites, Antineoplastic; Apoptosis; bcl-2-Associated X Protein; Bleomycin; Blotting, Western; Bongkrekic Acid; Caspase 3; Caspase 8; Caspases; Cell Line; Cyclosporine; Cytochromes c; DNA Fragmentation; Enzyme Activation; Genes, p53; HL-60 Cells; Humans; Leukemia; Microscopy, Electron; Mitochondria; Phenotype; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Messenger; Time Factors; Tumor Suppressor Protein p53

In normal pancreatic acinar cells, the oxidant menadione evokes repetitive cytosolic Ca(2+) spikes, partial mitochondrial depolarisation, cytochrome c release and apoptosis. The physiological agonists acetylcholine and cholecystokinin also evoke cytosolic Ca(2+) spikes but do not depolarise mitochondria and fail to induce apoptosis. Ca(2+) spikes induced by low agonist concentrations are confined to the apical secretory pole of the cell by the buffering action of perigranular mitochondria. Menadione prevents mitochondrial Ca(2+) uptake, which permits rapid spread of Ca(2+) throughout the cell. Menadione-induced mitochondrial depolarisation is due to induction of the permeability transition pore. Blockade of the permeability transition pore with bongkrekic acid prevents activation of caspase 9 and 3. In contrast, the combination of antimycin A and acetylcholine does not cause apoptosis but elicits a global cytosolic Ca(2+) rise and mitochondrial depolarisation without induction of the permeability transition pore. Increasing the cytosolic Ca(2+) buffering power by BAPTA prevents cytosolic Ca(2+) spiking, blocks the menadione-elicited mitochondrial depolarisation and blocks menadione-induced apoptosis. These results suggest a twin-track model in which both intracellular release of Ca(2+) and induction of the permeability transition pore are required for initiation of apoptosis.

Topics: Acetylcholine; Amino Acid Chloromethyl Ketones; Animals; Anti-Bacterial Agents; Antifibrinolytic Agents; Antimycin A; Apoptosis; Bongkrekic Acid; Calcium; Calcium Signaling; Caspase 3; Caspase 9; Caspases; Cell Nucleus; Cells, Cultured; Chelating Agents; Cysteine Proteinase Inhibitors; Egtazic Acid; Fluoresceins; Fluorescent Dyes; Membrane Potentials; Mice; Mitochondria; Pancreas; Vasodilator Agents; Vitamin K 3

Glucocorticosteroids are potent anti-inflammatory drugs used in the treatment of eosinophilic disorders. These molecules directly promote eosinophil apoptosis, yet the molecular mechanisms regulating this process remain ill-defined. We show here that stimulation of human peripheral blood eosinophils with dexamethasone induced DNA fragmentation, chromatin and cytoplasm condensation, and caspase-3 activation, as assessed by the proteolysis of its zymogen form and by the increase of caspase-3-like activity in eosinophil lysates. These phenomena were accompanied by a reduced uptake of the mitochondrial potential-sensitive marker DiOC(6)(3), suggestive of mitochondrial membrane permeabilization. Eosinophil incubation with the caspase-3 inhibitor, Z-Asp-Glu-Val-Asp-fluromethylketone, or with the broad spectrum caspase inhibitor, Z-Val-Ala-Asp-fluromethylketone, inhibited caspase-3-like activity generation but failed to modify dexamethasone-mediated loss in mitochondrial transmembrane potential and eosinophil apoptosis. In contrast, bongkrekic acid, a ligand of the mitochondrial permeability transition pore component, adenine nucleotide translocator, prevented both dexamethasone-induced mitochondrial disruption and apoptosis. We conclude that the mitochondrial permeability transition pore, rather than the caspase cascade, plays a critical role in the propagation of glucocorticosteroid-mediated apoptotic signals in human eosinophils.

Topics: Amino Acid Chloromethyl Ketones; Anti-Bacterial Agents; Apoptosis; Bongkrekic Acid; Caspase 3; Caspase Inhibitors; Caspases; Cells, Cultured; Dexamethasone; DNA Fragmentation; Enzyme Activation; Enzyme Inhibitors; Eosinophils; Glucocorticoids; Humans; Intracellular Membranes; Ion Channels; Membrane Potentials; Mitochondria; Mitochondrial ADP, ATP Translocases; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Oligopeptides

Apoptosis in response to cellular stress such as treatment with cytotoxic drugs is mediated by effector caspases (caspase-3) which can be activated by different initiator pathways. Here, we report on a cell type specific triggering of death receptor and/or mitochondrial pathways upon drug treatment. In type I cells (BJAB), both the receptor and the mitochondrial pathway were activated upon drug treatment, since blockade of either the receptor pathway by overexpression of dominant negative FADD (FADD-DN) or of the mitochondrial pathway by overexpression of Bcl-X(L) only partially inhibited apoptosis. Drug treatment induced formation of a FADD- and caspase-8-containing CD95 death-inducing signaling complex (DISC) in type I cells resulting in activation of caspase-8 as the most apical caspase. In contrast, in type II cells (Jurkat), apoptosis was predominantly controlled by mitochondria, since overexpression of Bcl-2 completely blocked drug-induced apoptosis, while overexpression of FADD-DN had no protective effect. In these cells, caspases including caspase-8 were activated by mitochondria-driven signaling events and no DISC was detected despite expression levels of CD95, FADD and caspase-8 proteins comparable to type I cells. Likewise, drug-induced CD95 aggregation was predominantly found in type I cells. Bid was cleaved prior to mitochondrial alterations in type I cells providing a molecular link between caspase-8 activation and mitochondrial perturbations, whereas in type II cells, Bid was cleaved downstream of mitochondria. Our findings of a cell type specific response to cytotoxic drugs have implications for the identification of molecular parameters for chemosensitivity or resistance in different tumor cells.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Blotting, Western; Bongkrekic Acid; Carrier Proteins; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Cytochrome c Group; Doxorubicin; Drug Resistance; fas Receptor; Fas-Associated Death Domain Protein; Gene Expression; Humans; Jurkat Cells; Membrane Potentials; Mitochondria; Mutation; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tumor Cells, Cultured

In this study, we examined the relative importance of caspases and mitochondria in Fas-mediated eosinophil apoptosis. Stimulation of human peripheral blood eosinophils with an agonistic anti-human Fas monoclonal antibody, but not with control IgM, induced a time-dependent increase in their apoptosis, which was associated with a loss in mitochondrial transmembrane potential (DeltaPsi(m)) and with caspase-8 and caspase-3 activation. Interleukin (IL)-5 and interferon (IFN)-gamma, two cytokines known to prolong eosinophil survival, inhibited Fas-mediated apoptosis and caspase activation but poorly affected the decrease in DeltaPsi(m). Eosinophil incubation with bongkrekic acid, an inhibitor of the mitochondrial permeability transition pore (MPTP) opening, failed to modify Fas-mediated loss in DeltaPsi(m), caspase activation, and apoptosis. In contrast, caspase inhibitors markedly reduced eosinophil apoptosis without significantly affecting DeltaPsi(m) dissipation. We conclude that caspase-8 and caspase-3 activation, but not MPTP opening, mediate Fas-induced eosinophil apoptosis and are the main targets for the protective effect of IL-5 and IFN-gamma.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antibodies, Monoclonal; Apoptosis; Bongkrekic Acid; Caspase 3; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Cytochrome c Group; Eosinophils; Fas Ligand Protein; fas Receptor; Humans; Hypereosinophilic Syndrome; Immunoglobulin M; Interferon-gamma; Interleukin-5; Intracellular Membranes; Ion Channels; Membrane Glycoproteins; Membrane Potentials; Membrane Proteins; Mice; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Oligopeptides; Pulmonary Eosinophilia; Recombinant Proteins

3-Deazaadenosine (DZA), a cellular methylation blocker was reported to induce the caspase-3-like activities-dependent apoptosis in U-937 cells. In this study, we analyzed the activation pathway of the caspase cascade involved in the DZA-induced apoptosis using specific inhibitors of caspases. In the U-937 cells treated with DZA, cytochrome c release from mitochondria and subsequent activation of caspase-9, -8 and -3 were observed before the induction of apoptosis. zDEVD-Fmk, a specific inhibitor of caspase-3, and zLEHD-Fmk, a specific inhibitor of caspase-9, prevented the activation of caspase-8 but neither caspase-3 nor caspase-9, indicating that caspase-8 is downstream of both caspase-3 and caspase-9, which are activated by independent pathways. zVAD-Fmk, a universal inhibitor of caspases, kept the caspase-3 from being activated but not caspase-9. Moreover, ZB4, an antagonistic Fas-antibody, exerted no effect on the activation of caspase-8 and induction of apoptosis by DZA. In addition, zVAD-Fmk and mitochondrial permeability transition pore (MPTP) inhibitors such as cyclosporin A (CsA) and bongkrekic acid (BA) did not block the release of cytochrome c from mitochondria. Taken together, these results suggest that in the DZA-induced apoptosis, caspase-8 may serve as an executioner caspase and be activated downstream of both caspase-3 and caspase-9, independently of Fas receptor-ligand interaction. And caspase-3 seems to be activated by other caspses including IETDase-like enzyme and caspse-9 seems to be activated by cytochrome c released from mitochondria without the involvement of caspases and CsA- and BA- inhibitory MPTP.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Bongkrekic Acid; Caspase 3; Caspase 8; Caspase 9; Caspases; Cell Line; Cyclosporine; Cytochrome c Group; Enzyme Activation; Fas Ligand Protein; Humans; Leukocytes, Mononuclear; Ligands; Membrane Glycoproteins; Tubercidin; U937 Cells

B cell antigen receptor (BCR)-mediated cell death has been proposed as a mechanism for purging the immune repertoire of anti-self specificities during B cell differentiation in bone marrow. Mitochondrial alterations and activation of caspases are required for certain aspects of apoptotic cell death, but how the mitochondria and caspases contribute to BCR-mediated cell death is not well understood. In the present study, we used the mouse WEHI-231 B cell line to demonstrate that mitochondrial alterations and activation of caspases are indeed participants in BCR-mediated cell death. The peptide inhibitor of caspases, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), blocked cleavage of poly(ADP-ribose) polymerase and various manifestation of nuclear apoptosis such as nuclear fragmentation, hypodiploidy and DNA fragmentation, indicating that signals from the BCR induced the activation of caspases. In addition, z-VAD-fmk delayed apoptosis-associated changes in cellular reduction-oxidation potentials as determined by hypergeneration of superoxide anion, as well as exposure of phosphatidylserine residues in the outer plasma membrane. By contrast, although z-VAD-fmk retarded cytolysis, it was incapable of preventing disruption of the plasma membrane even under the same condition in which it completely blocked nuclear apoptosis. Mitochondrial membrane potential loss was also not blocked by z-VAD-fmk. Bongkrekic acid, a specific inhibitor of mitochondrial permeability transition pores, suppressed not only the mitochondrial membrane potential but also the change of plasma membrane permeability. Overexpression of Bcl-xL prevented mitochondrial dysfunction, nuclear apoptosis and membrane permeability cell death triggered by BCR signal transduction. These observations indicate that death signals from BCR may first cause mitochondrial alterations followed by activation of both necrotic and apoptotic cascades.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antibodies, Anti-Idiotypic; Apoptosis; B-Lymphocytes; bcl-X Protein; Bongkrekic Acid; Cell Death; Cell Membrane Permeability; Cell Nucleus; Cysteine Proteinase Inhibitors; Enzyme Activation; Immunoglobulin M; Intracellular Membranes; Mice; Mitochondria; Necrosis; Phosphatidylserines; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Receptors, Antigen, B-Cell; Signal Transduction; Tumor Cells, Cultured