Bortezomib interacts synergistically with flavopiridol to induce mitochondrial dysfunction and apoptosis in CML cells. (A) K562 and LAMA84 cells were exposed to flavopiridol (FP, K562: 200 nM; LAMA: 150 nM) ± bortezomib (Btzmb or Bz, K562: 8 nM; LAMA: 5 nM) for 24 hours and 48 hours, after which the percentage of apoptotic cells was determined by Annexin V-FITC staining and flow cytometry as described in “Materials and methods.” Annexin V⁺/PI^-corresponds to early apoptosis and Annexin V⁺/PI⁺ to late apoptosis. Numbers reflect the percentage of cells in the corresponding quadrants. Results are representative of 3 separate experiments. (B-C) K562 (B) and LAMA84 (C) cells were treated with a range of FP and Btzmb concentrations alone and in combination for 48 hours at a fixed ratio as indicated. At the end of this period, the percentage of cells exhibiting apoptotic morphology was determined by evaluation of Wright Giemsa-stained cytospin preparations for each condition; fractional effect values were determined by comparing results to those of untreated controls (Ctrl), and Median Dose Effect analysis was used to characterize the nature of the interaction. Combination index (CI) values less than 1.0 denote a synergistic interaction. An additional 2 studies yielded equivalent results. (D) K562 and LAMA84 cells were treated for 24 hours as described for panel A, after which the percentage of cells exhibiting reduced mitochondrial membrane potential (Δψm) was determined by monitoring DiOC₆ uptake as described in “Materials and methods.” Results represent the means ± SDs for 3 separate experiments performed in triplicate. (E) Alternatively, cytosolic (S-100) fractions were prepared as described in “Materials and methods,” and expression of cytochrome c (cyt c) and Smac/DIABLO in cytosol was monitored by Western blot. Each lane was loaded with 30 μg protein; blots were stripped and reprobed with antiactin antibodies to ensure equal loading and transfer of protein. An additional 2 studies yielded equivalent results.