Figure 4.
Figure 4. Effect of 17-AAG on cytarabine-induced cytotoxicity. (A) Log-phase HL-60 cells were treated with 0.1% dimethyl sulfoxide (DMSO) containing 0, 10, 30, 100, or 300 nM cytarabine for 24 hours. The indicated concentration of 17-AAG was then added for an additional 24 hours in the continued presence of cytarabine. At the completion of the incubation, cells were sedimented, washed, plated in drug-free medium, and allowed to form colonies for 14 days. Cloning efficiency of diluent-treated cells in multiple experiments ranged from 25% to 35%. Error bars: mean ± standard deviation of quadruplicate samples. (B) Dots represent combination index values calculated from the data in panel A under the assumption that effects of cytarabine and 17-AAG were mutually exclusive.82 Solid line is second-order regression line calculated from the individual data points. (C-D) Log-phase HL-60 cells were treated with 0.1% DMSO containing 0, 100, 300, or 1000 nM cytarabine for 24 hours. The indicated concentration of 17-AAG was then added for an additional 24 hours in the continued presence of cytarabine. At the completion of the incubation, cells were sedimented, incubated in drug-free medium for 72 hours, and examined for apoptotic morphologic changes. (C) Micrographs contain representative fields of cells harvested 72 hours after completion of treatment with diluent (control), 300 nM 17-AAG for 24 hours, 1000 nM cytarabine for 48 hours, or 1000 nM cytarabine for 24 hours followed by the addition of 300 nM 17-AAG for the second 24 hours in the continued presence of the cytarabine (combination). Cells were then incubated for 72 hours and analyzed. (D) Results obtained when more than 500 cells per treatment were examined from the experiment in panel C. (E) Log-phase HL-60 cells were treated with diluent (control or 17-AAG) or 1000 nM cytarabine (Ara-C) for 24 hours. Diluent or 300 nM 17-AAG was then added for 24 hours. At the completion of the drug treatment, cells were washed and cultured in drug-free medium for 3 days prior to staining with APC–annexin V. For these assays, both propidium iodide–negative (early apoptotic) and propidium iodide–positive (late apoptotic) cells were quantitated, a common practice in these prolonged assays.83,84 (F) Log-phase HL-60 cells were treated with 0.1% DMSO containing 0, 100, 300, or 1000 nM cytarabine for 24 hours. The indicated concentration of 17-AAG was then added for an additional 24 hours in the continued presence of cytarabine. At the completion of the incubation, cells were sedimented, incubated in drug-free medium for 72 hours, and stained with APC–annexin V as illustrated in panel E. (G) Combined results obtained in 4 independent experiments performed as described for panels E and F. Error bars: ± 1 standard deviation. *P = .03 and **P = .002 by paired t test.

Effect of 17-AAG on cytarabine-induced cytotoxicity. (A) Log-phase HL-60 cells were treated with 0.1% dimethyl sulfoxide (DMSO) containing 0, 10, 30, 100, or 300 nM cytarabine for 24 hours. The indicated concentration of 17-AAG was then added for an additional 24 hours in the continued presence of cytarabine. At the completion of the incubation, cells were sedimented, washed, plated in drug-free medium, and allowed to form colonies for 14 days. Cloning efficiency of diluent-treated cells in multiple experiments ranged from 25% to 35%. Error bars: mean ± standard deviation of quadruplicate samples. (B) Dots represent combination index values calculated from the data in panel A under the assumption that effects of cytarabine and 17-AAG were mutually exclusive.82  Solid line is second-order regression line calculated from the individual data points. (C-D) Log-phase HL-60 cells were treated with 0.1% DMSO containing 0, 100, 300, or 1000 nM cytarabine for 24 hours. The indicated concentration of 17-AAG was then added for an additional 24 hours in the continued presence of cytarabine. At the completion of the incubation, cells were sedimented, incubated in drug-free medium for 72 hours, and examined for apoptotic morphologic changes. (C) Micrographs contain representative fields of cells harvested 72 hours after completion of treatment with diluent (control), 300 nM 17-AAG for 24 hours, 1000 nM cytarabine for 48 hours, or 1000 nM cytarabine for 24 hours followed by the addition of 300 nM 17-AAG for the second 24 hours in the continued presence of the cytarabine (combination). Cells were then incubated for 72 hours and analyzed. (D) Results obtained when more than 500 cells per treatment were examined from the experiment in panel C. (E) Log-phase HL-60 cells were treated with diluent (control or 17-AAG) or 1000 nM cytarabine (Ara-C) for 24 hours. Diluent or 300 nM 17-AAG was then added for 24 hours. At the completion of the drug treatment, cells were washed and cultured in drug-free medium for 3 days prior to staining with APC–annexin V. For these assays, both propidium iodide–negative (early apoptotic) and propidium iodide–positive (late apoptotic) cells were quantitated, a common practice in these prolonged assays.83,84  (F) Log-phase HL-60 cells were treated with 0.1% DMSO containing 0, 100, 300, or 1000 nM cytarabine for 24 hours. The indicated concentration of 17-AAG was then added for an additional 24 hours in the continued presence of cytarabine. At the completion of the incubation, cells were sedimented, incubated in drug-free medium for 72 hours, and stained with APC–annexin V as illustrated in panel E. (G) Combined results obtained in 4 independent experiments performed as described for panels E and F. Error bars: ± 1 standard deviation. *P = .03 and **P = .002 by paired t test.

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