Effects of adaphostin on imatinib-resistant Ph⁺ human leukemia samples. (A) Mononuclear cells from the peripheral blood of 2 CML patients were isolated, incubated with 5 μM adaphostin for the indicated times, stained with CMH₂DCF-DA, and subjected to flow cytometry. Mean fluorescence over the time course is depicted graphically, whereas the inset histogram shows the increased DCF fluorescence after 1 hour of adaphostin exposure. BCR/ABL mutation status was not known for patient 1, but patient 2 was shown to have a previously unreported novel mutation. (B) After mononuclear cells from patient A (left panel) were incubated with increasing doses of imatinib mesylate or adaphostin for 24 hours, DNA fragmentation was assessed. Right panel depicts DNA fragmentation in mononuclear and polymorphonuclear cells from patient B exposed to either diluent (□), 10 μM imatinib (▪), or 10 μM adaphostin (▦) for 24 hours. (C) Isolated mononuclear cells from a pediatric Ph⁺ ALL patient were exposed to diluent (dark solid histogram), 5 μM adaphostin (dotted histogram), or 24 mM NAC and adaphostin (light solid histogram) for 1.5 hours, stained with CMH₂DCF-DA, and read on a FACSCalibur to quantitate intracellular peroxide levels (inset histogram). DNA fragmentation was assessed by PI staining from the same experiment after a 24-hour exposure to indicated treatments and is shown graphically. (D) Mononuclear cells isolated from the peripheral blood of CML patients who progressed on imatinib were exposed for 24 hours to diluent or the indicated concentrations of adaphostin, plated in Methocult methylcellulose as previously described,²³  and scored for myeloid colony formation (CFU-G and CFU-GM) 14 days later according to the instructions of the supplier. (E) The concentration that inhibits colony formation (IC₅₀) was read from each curve in panel D and compared with IC₅₀ values determined in samples from untreated CML patients examined using the same assay over the same time period. Horizontal bars indicate median values. Number in circle represents multiple samples with indistinguishable IC₅₀ values.