Adaphostin elevates intracellular peroxide and induces cytotoxicity in BaF3 cells transduced with imatinib-resistant BCR/ABL. (A) BaF3 cells transduced with vector alone, wild-type p210 BCR/ABL, T315I BCR/ABL, or E255K BCR/ABL were exposed to diluent (dark solid line), 10 μM adaphostin (light solid line), or 10 μM imatinib (dotted line) for 1.5 hours. After cells were stained with CMH₂DCF-DA, fluorescence was read on the FL-1 channel of a Becton Dickinson FACSCalibur. These results are representative of 3 independent experiments. (B,C) BaF3 cells transduced with vector, wild-type bcr/abl, T315I BCR/ABL, or E255K BCR/ABL were exposed to either diluent (□), 5 μM (▪), 10 μM (▨), or 20 μM (▦) imatinib (B) or to diluent, 5 μM, or 10 μM adaphostin (C). DNA fragmentation was assessed by PI staining. (D) The degradation of bcr/abl was assessed by Western blotting in BaF3 cells overexpressing wild-type p210BCR/ABL, T315I BCR/ABL, and E255K BCR/ABL after 8 hours of exposure to 0, 1, 2.5, 5, or 10 μM adaphostin. The same membrane was probed with antibody to the caspase substrate PARP³¹  as a marker of cell death and with antibody to actin as a loading control. (E) BaF3 cells overexpressing vector only (□), wild-type p210BCR/ABL (▦), T315I BCR/ABL (▪), or E255K BCR/ABL (▨) were exposed for 24 hours to diluent, 24 mM NAC, 1 μM adaphostin, or 24 mM NAC and 1 μM adaphostin together. DNA fragmentation was assessed by PI staining as described in “Materials and methods.” Decrease in adaphostin-induced cell death by NAC was statistically significant in wild-type p210BCR/ABL (P = .002), T315I BCR/ABL (P < .001), or E255K BCR/ABL (P < .001) as calculated by the Student 2-tailed paired t test. (F) BaF3 cells transduced with vector alone, wild-type p210, T315I bcr/abl, or E255K bcr/abl were exposed to either diluent, 500 nM, or 1 μM adaphostin for 24 hours. Cells were then resuspended in Methocult media and allowed to form colonies for 7 days. Error bars in panels B, C, E, and F indicate standard deviation (SD).