bafilomycin-a and Myelodysplastic-Syndromes

In most cases, apoptosis is considered to involve mitochondrial dysfunction with sequential release of cytochrome c from mitochondria, resulting in activation of caspase-3. However, we found that etoposide induced apoptosis in P39 cells, a myelodysplastic syndrome-derived cell line, without the release of cytochrome c. Furthermore, in etoposide-treated P39 cells, no changes in mitochondrial membrane potential (delta psi m) were detected by flow cytometry. Flow cytometry using a pH-sensitive probe demonstrated that lysosomal pH increased during early apoptosis in P39 cells treated with etoposide. A reduction in the ATP level preceded the elevation of lysosomal pH. In addition, specific inhibitors of vacuolar H+-ATPase induced apoptosis in P39 cells but not in HL60 cells. Although etoposide-induced activation of caspase-3 was followed by DNA ladder formation in P39 cells, E-64d, an inhibitor of lysosomal thiol proteases, specifically suppressed etoposide-induced activation of caspase-3. Western blotting analysis provided direct evidence for the involvement of a lysosomal enzyme, cathepsin L. These findings indicate that lysosomal dysfunction induced by a reduction in ATP results in leakage of lysosomal enzymes into the cytosolic compartment and that lysosomal enzyme(s) may be involved in activation of caspase-3 during apoptosis in P39 cells treated with etoposide.

Topics: Anti-Bacterial Agents; Apoptosis; Caspase 3; Caspase Inhibitors; Caspases; Cathepsin L; Cathepsins; Cell Line; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytochrome c Group; Endopeptidases; Enzyme Activation; Enzyme Inhibitors; Etoposide; Humans; Hydrogen-Ion Concentration; Intracellular Membranes; Leucine; Lysosomes; Macrolides; Membrane Potentials; Mitochondria; Myelodysplastic Syndromes; Proton-Translocating ATPases; Vacuolar Proton-Translocating ATPases