Caspase-3 activation by lysosomal enzymes in cytochrome c-independent apoptosis in myelodysplastic syndrome-derived cell line P39.
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.
Hishita
T
T
Department of Hematology/Oncology, Graduate School of Medicine, Kyoto University, Japan.
Tada-Oikawa
S
S
Tohyama
K
K
Miura
Y
Y
Nishihara
T
T
Tohyama
Y
Y
Yoshida
Y
Y
Uchiyama
T
T
Kawanishi
S
S
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D056668:Cathepsin L; D002403:Cathepsins / Q000096:biosynthesis; D002460:Cell Line; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003574:Cytochrome c Group / Q000502:physiology*; D010450:Endopeptidases; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000502:physiology; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D008247:Lysosomes / Q000201:enzymology; D018942:Macrolides; D008564:Membrane Potentials / Q000502:physiology; D008928:Mitochondria / Q000502:physiology; D009190:Myelodysplastic Syndromes / Q000201:enzymology* / Q000473:pathology*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors; D025262:Vacuolar Proton-Translocating ATPases
D000900:Anti-Bacterial Agents; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D018942:Macrolides; C057620:bafilomycin A; D005047:Etoposide; C036978:concanamycin A; D002403:Cathepsins; D010450:Endopeptidases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; D007930:Leucine; C108192:aloxistatin