Caspase-3 activation by lysosomal enzymes in cytochrome c-independent apoptosis in myelodysplastic syndrome-derived cell line P39

In mast cases, apoptosis is considered to involve mitochondrial dysfunction with sequential release of cytochrome c from mitochondria, resulting in ac tivation of caspase-3. However, we found that etoposide induced apoptosis i n P39 cells, a myelodysplastic syndrome-derived cell line, without the rele ase of cytochrome c. Furthermore, in etoposide-treated P39 cells, no change s in mitochondrial membrane potential (Delta Psim) were detected by flow cy tometry, Flow cytometry using a pH-sensitive probe demonstrated that lysoso mal pH increased during early apoptosis in P39 cells treated with etoposide . A reduction in the ATP level preceded the elevation of lysosomal pH. In a ddition, specific inhibitors of vacuolar H+-ATPase induced apoptosis in P39 cells but not in HL60 cells, Although etoposide-induced activation of casp ase-3 was followed by DNA ladder formation in P39 cells, E-64d, an inhibito r of lysosomal thiol proteases, specifically suppressed etoposide-induced a ctivation of caspase-3. Western blotting analysis provided direct evidence for the involvement of a lysosomal enzyme, cathepsin L. These findings indi cate that lysosomal dysfunction induced by a reduction in ATP results in le akage of lysosomal enzymes into the cytosolic compartment and that lysosoma l enzyme(s) may be involved in activation of caspase-3 during apoptosis in P39 cells treated with etoposide.