Effects of adaphostin on imatinib-resistant human Ph⁺ CML cell lines.(A) The CML-derived cell line KBM5 was treated for 30 minutes with diluent, 24 mM NAC, or 5 μM adaphostin in the absence or presence of a 15-minute pretreatment with NAC. Superoxide levels were assessed by staining cells with 10 μM dihydroethidium for 30 minutes and then analyzing FL3 fluorescence by flow cytometry on a Becton Dickinson FACSCalibur. DNA fragmentation by PI staining and subsequent FACS analysis on the FL-3 channel was also assessed in KBM5 cells after a 24-hour exposure to diluent or 2.5 μM adaphostin (inset). (B) Resistant versions of the KBM5 and KBM7 lines were developed as previously described.29 KBM5R (▪) and KBM7R (▦) cells were exposed for 24 hours to 5 μM imatinib mesylate, 1 μM adaphostin alone, or 1 μM adaphostin after a 15-minute pretreatment with 24 mM NAC. DNA fragmentation was assessed by PI staining and subsequent FACS analysis as described in panel A. Reduction in subdiploid cells by NAC in combination with adaphostin was statistically significant with P = .04 for KBM5R and P = .03 for KBM7R as calculated by the Student t test. Error bars indicate SD.