CLL cells exposed to CD154 develop a rapid and sustained resistance to ABT-737 upstream of mitochondrial perturbation. (A,B) CLL cells were cultured on parental (A) or CD154-expressing L cells (B) in the presence of IL-4 (10 ng/mL) for 1, 3, or 6 days in (A) and 1 to 24 hours in (B) before addition of different concentrations of ABT-737 for 4 hours in the absence of L cells. Apoptosis was assessed by PS externalization. Treatment with 10 μM ABT-737 did not result in more than 40% apoptosis upon 24 hours of culture on CD154-expressing cells (data not shown). (C) Freshly isolated CLL cells or CLL cells cultured on either parental or CD154-expressing L cells in the presence of IL-4 (10 ng/mL) for 1 day were exposed to ABT-737 (10 nM) for up to 4 hours, in the absence of L cells and IL-4. Release of cytochrome c from mitochondrial heavy membrane fraction (HM) to the supernatant (SN) was assessed by digitonin lysis and Western blotting. (D-F) CLL cells were cultured on either parental (solid lines) or CD154-expressing L cells (dotted lines) in the presence of IL-4 (10 ng/mL) for 1 day before exposure to ABT-737 for additional 4 hours, in the absence of L cells and IL-4. (D) CLL cells were stained with TMRE (20 nM) to assess the mitochondrial membrane potential (Ψm) by flow cytometry. (E,F) Conformational change of BAX (E) or BAK (F) was detected using conformational specific antibodies and flow cytometric analysis. Data are the mean plus SEM (n = 4).