Cytarabine-induced S-phase accumulation is abrogated by 17-AAG. (A) HL-60 cells treated with diluent (lane 1), 300 nM cytarabine (lane 2), 300 nM cytarabine and 1000 nM 17-AAG (lane 3), or 1000 nM 17-AAG alone (lane 4) were assayed for Cdc25A levels by immunoblotting as previously described.68 The asterisk indicates immunoglobulin G heavy chain. (B-C,E-F) HL-60 cells were treated for 24 hours with diluent (B), 100 nM cytarabine (C), or 300 nM 17-AAG (E). Alternatively, cells pretreated for 24 hours with 100 nM cytarabine (as in panel C) were then treated for an additional 24 hours with 300 nM 17-AAG (F) in the continued presence of cytarabine. At the completion of the incubation, the cell-cycle distributions were determined by flow microfluorimetry. Arrows denote cells with 2N and 4N DNA content determined by Modfit software analysis. (D) HL-60 cells were treated for 24 hours with diluent or the indicated concentrations of cytarabine and analyzed by flow microfluorimetry. Very similar results were obtained when the cytarabine incubation was continued for 48 hours (data not shown). (G) The percentage of S-phase cells detected after treatment with diluent or cytarabine and various concentrations of 17-AAG as illustrated in panels E and F. ⬡ indicates 300 nM cytarabine; ○, no cytarabine.
