Previous studies have shown that K562
chronic myelogenous leukemia cells are resistant to induction of apoptosis by a variety of agents, including the
topoisomerase II (
topo II)
poison etoposide, when examined 4 to 24 hours
after treatment with an initiating stimulus. In the present study, the responses of K562 cells and apoptosis-proficient HL-60
acute myelomonocytic leukemia cells to
etoposide were compared, with particular emphasis on determining the long-term fate of the cells. When cells were treated with varying concentrations of
etoposide for 1 hour 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 for 1 hour, cleavage of the
caspase substrate
poly(ADP-ribose) polymerase (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 G2 phase of the cell cycle but otherwise appeared normal for 3 to 4 days before developing similar apoptotic changes. When the
etoposide dose was increased to 68 mu mol/L, apoptotic changes were evident in HL-60 cells after 2 to 3 hours, whereas the same 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 appearance of
peptidase activity that cleaved the
fluorogenic substrates Asp-Glu-Val-Asp-aminotrifluoromethylcoumarin (
DEVD-AFC) and Val-Glu-Ile-Asp-aminomethylcoumarin (VEID-AMC) as well as an altered spectrum of active
caspases that were affinity labeled with N-(Nalpha-benzyloxycarbonylglutamyl-Nepsilon-
biotin yllysyl)
aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl
ketone [z-EK(bio)D-aomk]. On the other hand, the activation of
caspase-3 under cell-free conditions occurred with indistinguishable kinetics in cytosol prepared from the two cell lines. Collectively, these results suggest that a delay in the signaling cascade upstream of
cytochrome c release and
caspase activation leads to a long latent period before the active phase of apoptosis is initiated in
etoposide-treated K562 cells. Once the active phase of apoptosis is initiated, the spectrum and subcellular distribution of active
caspase species differ between HL-60 and K562 cells, but a similar proportion of cells are ultimately killed in both cell lines.