benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Leukemia--T-Cell

Photodynamic therapy (PDT) is a cancer treatment based on the interaction of a photosensitizer, light and oxygen. PDT with the endogenous photosensitizer, protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (ALA) or its derivatives is a modification of this treatment modality with successful application in dermatology. However, the mechanism of cell destruction by ALA-PDT has not been elucidated. In this study a human T-cell lymphoma Jurkat cell line was treated with PDT using hexaminolevulinate (HAL, hexylester of ALA). Four hours following treatment nearly 80% of the cells exhibited typical apoptotic features. Mitochondrial pro-apoptotic proteins were evaluated by Western blots in subcellular fractionated samples. PDT caused cytosolic translocation of cytochrome c and nuclear redistribution of apoptosis-inducing factor (AIF), but the release of mitochondrial Smac/DIABLO, Omi/HtrA2 and EndoG was not observed. The release of cytochrome c was followed by the cleavage of caspase-9 and caspase-3 as well as its downstream substrates, together with oligonucleosomal DNA fragmentation. The pan-caspases inhibitor, z-VAD.fmk, prevented oligonucleosomal DNA fragmentation, but failed to inhibit PDT-mediated apoptosis. The apoptotic induction by AIF-mediated caspase-independent pathway was also found after HAL-PDT with large-scale DNA fragmentation in the presence of z-VAD.fmk. These results demonstrate that cytochrome c-mediated caspase-dependent pathway and AIF-induced caspase-independent pathway are simultaneously involved in the apoptotic induction by PDT. When the cytochrome c-induced caspase-dependent pathway is blocked, the cells go into apoptosis via AIF-mediated pathway, clearly demonstrating that the cytochrome c-mediated caspase-dependent pathway is not required for such apoptotic induction. This finding may have an impact on improved PDT effectiveness.

Topics: Amino Acid Chloromethyl Ketones; Aminolevulinic Acid; Apoptosis; Apoptosis Inducing Factor; Caspases; Cytochromes c; Enzyme Inhibitors; Humans; Jurkat Cells; Leukemia, T-Cell; Membrane Potential, Mitochondrial; Mitochondria; Photochemotherapy; Protoporphyrins

We investigated the effect of pressure levels ranging from 80 to 500 bar on the proliferative capacity and viability of Jurkat leukaemic T cells. Pressurization at 360 bar induced apoptotic cell death as shown by apoptotic morphology after Hoechst staining, DNA fragmentation in the TdT-mediated dUTP nick end labelling-assay and cleavage of several caspase substrates. Cell death could be prevented by the general caspase inhibitor zVAD-fmk. Breakdown of the mitochondrial membrane potential and the release of cytochrome c provided strong evidence for an involvement of the mitochondrial pathway, whereas a central role of the death receptor pathway was excluded because caspase-8 was not significantly activated. Pressure incubation led to calcium influx after 5 min, and we hypothesize that calcium influx could be the primary trigger for pressure-induced apoptosis.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Calcium Signaling; Caspase 8; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; DNA Damage; Humans; Hydrostatic Pressure; Jurkat Cells; Leukemia, T-Cell; Mitochondria

The mechanism underlying apoptosis induced by proteasome inhibition in leukemic Jurkat and Namalwa cells was investigated in this study. The proteasome inhibitor lactacystin differentially regulated the protein levels of proapoptotic Bcl-2 family members and Bik was accumulated at the mitochondria. Bik overexpression sufficed to induce apoptosis in these cells. Detailed examination along the respiration chain showed that lactacystin compromised a step after complex III, and exogenous cytochrome c could overcome this compromise. Probably as a result, the succinate-stimulated generation of mitochondrial membrane potential was significantly diminished. Bcl-x(L) interacted with Bik in the cells, and Bcl-x(L) overexpression prevented cytochrome c leakage out of the mitochondria, corrected the mitochondrial membrane potential defect, and protected the cells from apoptosis. These results show that proteasomes can modulate apoptosis of lymphocytes by affecting the half-life of Bcl-2 family members, Bik being one of them.

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytochrome c Group; Electron Transport; Enzyme Activation; Humans; Intracellular Membranes; Jurkat Cells; Leukemia, B-Cell; Leukemia, T-Cell; Membrane Potentials; Membrane Proteins; Mitochondria; Mitochondrial Proteins; Multienzyme Complexes; Peptide Hydrolases; Permeability; Proteasome Endopeptidase Complex; Protein Biosynthesis; Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Tumor Cells, Cultured

Interleukin-1 beta converting enzyme (ICE)-like proteases, which are synthesized as inactive precursors, play a key role in the induction of apoptosis. We now demonstrate that benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK), an ICE-like protease inhibitor, inhibits apoptosis by preventing the processing of CPP32 to its active form. These results suggest that novel inhibitors of apoptosis can be developed which prevent processing of proforms of ICE-like proteases.

Topics: Amino Acid Chloromethyl Ketones; Amino Acid Sequence; Apoptosis; Caspase 3; Caspases; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme Activation; Humans; Leukemia, Monocytic, Acute; Leukemia, T-Cell; Molecular Sequence Data; Oligopeptides; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Protease Inhibitors; Tumor Cells, Cultured