COMPARISON OF CASPASE ACTIVATION AND SUBCELLULAR-LOCALIZATION IN HL-60 AND K562 CELLS UNDERGOING ETOPOSIDE-INDUCED APOPTOSIS

Previous studies have shown that K562 chronic myelogenous leukemia cel ls are resistant to induction of apoptosis by a variety of agents, inc luding the topoisomerase II (topo II) poison etoposide, when examined 4 to 24 hours after treatment with an initiating stimulus. In the pres ent study, the responses of K562 cells and apoptosis:proficient HL-60 acute myelomonocytic leukemia cells to etoposide were compared, with p articular emphasis on determining the long-term fate of the cells. Whe n cells were treated with varying concentrations of. etoposide for 1 h our and subsequently plated in soft agar, the two cell lines displayed similar sensitivities, with a 90% reduction in colony formation at 5 to 10 mu mol/L etoposide. After treatment with 17 mu mol/L etoposide f or 1 hour, cleavage of the caspase substrate poly(ADP-ribose) polymera se (PARP), DNA fragmentation, and apoptotic morphological changes were evident in HL-60 cells in less than 6 hours. After the same treatment , K562 cells arrested in G(2) phase of the cell cycle but otherwise ap peared normal for 3 to 4 days before developing similar apoptotic chan ges. When the etoposide dose was increased to 68 mu mol/L, apoptotic c hanges were evident in HL-60 cells after 2 to 3 hours, whereas the sam e changes were observed in K562 cells after 24 to 48 hours. This delay in the development of apoptotic changes in K562 cells was accompanied by delayed release of cytochrome c to the cytosol and delayed appeara nce of peptidase activity that cleaved the fluorogenic substrates Asp- Glu-Val-Asp-aminotrifluoromethylcoumarin (DEVD-AFC) and Val-Glu-IIe-As paminomethylcoumarin (VEID-AMC) as well as an altered spectrum of acti ve caspases that were affinity labeled with benzyloxycarbonylglutamyl- N-epsilon-biotinyllysyl) aspartic acid [(2,6-dimethylbenzoyl)oxy]methy l ketone [z-EK(bio)D-aomk]. On the other hand, the activation of caspa se-3 under cell-free conditions occurred with indistinguishable kineti cs in cytosol prepared from the two cell lines. collectively, these re sults suggest that a delay in the signaling cascade upstream of cytoch rome c release and caspase activation leads to a long latent period be fore the active phase of apoptosis is initiated In etoposide-treated K 562 cells. Once the active phase of apoptosis is initiated, the spectr um and subcellular distribution of active caspase species differ betwe en HL-60 and K562 cells, but a similar proportion of cells are ultimat ely killed in both cell lines. (C) 1997 by The American Society of Hem atology.