benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and aloxistatin

Earlier studies showed that the oxidant menadione (MD) induces apoptosis in certain cells and also has anticancer effects. Most of these studies emphasized the role of the mitochondria in this process. However, the engagement of other organelles is less known. Particularly, the role of lysosomes and their proteolytic system, which participates in apoptotic cell death, is still unclear. The aim of this study was to investigate the role of lysosomal cathepsins on molecular signaling in MD-induced apoptosis in U937 cells. MD treatment induced translocation of cysteine cathepsins B, C, and S, and aspartic cathepsin D. Once in the cytosol, some cathepsins cleaved the proapoptotic molecule, Bid, in a process that was completely prevented by E64d, a general inhibitor of cysteine cathepsins, and partially prevented by the pancaspase inhibitor, z-VAD-fmk. Upon loss of the mitochondrial membrane potential, apoptosome activation led to caspase-9 processing, activation of caspase-3-like caspases, and poly (ADP-ribose) polymerase cleavage. Notably, the endogenous protein inhibitor, stefin B, was degraded by cathepsin D and caspases. This process was prevented by z-VAD-fmk, and partially by pepstatin A-penetratin. These findings suggest that the cleaved Bid protein acts as an amplifier of apoptotic signaling through mitochondria, thus enhancing the activity of cysteine cathepsins following stefin B degradation.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Apoptosomes; BH3 Interacting Domain Death Agonist Protein; Caspase 3; Caspase 9; Cathepsin B; Cathepsin C; Cathepsin D; Cathepsins; Cystatin B; Gene Expression Regulation, Neoplastic; Humans; Leucine; Lysosomes; Membrane Potential, Mitochondrial; Mitochondria; Pepstatins; Poly(ADP-ribose) Polymerases; Protease Inhibitors; Proteolysis; Signal Transduction; U937 Cells; Vitamin K 3

Enhancing apoptosis to remove abnormal cells has potential in reversing cancerous processes. Caspase-3 activation generally accompanies apoptosis and its substrates include enzymes responsible for DNA fragmentation and isozymes of protein kinase C (PKC). Recent data, however, question its obligatory role in apoptosis. We have examined whether modulation of PKC activity induces apoptosis in COLO 205 cells and the role of caspase-3. Proliferation ([3H]thymidine) and apoptosis (DNA fragmentation and FACS) of COLO 205 cells were measured in response to PKC activation and inhibition. Caspase-3 activity was assayed and the effects of its inhibition with Ac-DEVD-cmk, and the effect of other protease inhibitors, on apoptosis were determined. PKC activation and inhibition both reduced DNA synthesis and induced DNA fragmentation. As PKC inhibitors induced DNA fragmentation more rapidly than PKC activators and failed to block activator effects, we conclude that it is PKC down-regulation (i.e., inhibition) after activator exposure that mediates apoptosis. Increases in caspase-3 activity occurred during apoptosis but apoptosis was not blocked by caspase inhibition. By contrast, the cysteine protease inhibitor, E-64d, blocked apoptosis. Cysteine proteases not of the caspase family may either act more closely to the apoptotic process than caspases or lie on an alternative, more active pathway.

Topics: Aged; Alkaloids; Amino Acid Chloromethyl Ketones; Aprotinin; Benzophenanthridines; Benzyl Compounds; Caspase 3; Caspases; Cell Division; Cell Transformation, Neoplastic; Colonic Neoplasms; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dipeptides; DNA; DNA Fragmentation; Down-Regulation; Humans; Hydrocarbons, Fluorinated; Leucine; Leupeptins; Male; Pepstatins; Phenanthridines; Protein Kinase C; Pyridines; Tumor Cells, Cultured

Follicular dendritic cells (FDCs) select B cells during germinal center (GC) reactions. The B cells that are able to bind to the FDCs receive a signal that leads to the termination of endonuclease activity in the nuclei of those B cells. This signal must be in addition to the signals transferred through the cross-linkage of the B cell receptors and signals resulting from the interactions of the adhesion molecules lymphocyte function-associated Ag-1 and very late Ag-4 with ICAM-1 and VCAM-1, respectively. In this report, we present evidence that the FDCs silence all apoptotic processes in GC B lymphocytes and additionally switch off pre-present endonuclease activity. We also show that GC B cell apoptosis requires cathepsin activity downstream of caspase-3. This cathepsin activity is directly connected to endonuclease activity and therefore may be an interesting target for the antiapoptotic factors produced by FDCs.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; B-Lymphocytes; Caspase Inhibitors; Caspases; Cathepsins; Cells, Cultured; Cysteine Proteinase Inhibitors; Dendritic Cells; Endonucleases; Enzyme Activation; Germinal Center; Humans; Leucine; Palatine Tonsil; Signal Transduction