Proapoptotic effects of CI-1040 in combination with retinoids in retinoid-resistant HL-60R cells and their RARα- and RXRα-overexpressing counterparts. (A) Wild-type HL-60 cells, (B) retinoid-resistant HL-60 (HL-60R), (C) RXRα-overexpressing HL-60R, and (D) RARα-overexpressing HL-60R cells were pretreated with either vehicle (DMSO, □) or the MEK inhibitor CI-1040 (0.5 μM, ⊡) for 30 minutes and subsequently exposed to either ATRA (1 μM) or 9-cis RA (0.1 μM) for 96 hours. Apoptosis was then evaluated by flow cytometric analysis of FITC-conjugated annexin V binding, while simultaneously assessing membrane integrity by PI exclusion. Results are expressed as the percentage of annexin V–positive cells and represent the average ± SD of at least 3 independent experiments for each cell line. (E-F) HL-60R (○) and RARα-overexpressing HL-60R cells (•) were exposed to escalating concentrations of CI-1040 (0.03-1 μM) for 96 hours. (E) Viability was then assessed by measuring the intracellular ATP content using the Vialight assay. Results are expressed as percentage of viable cells relative to vehicle control–treated cells and represent the average ± SD of 3 independent experiments. (F) Apoptosis was evaluated by flow cytometric analysis of annexin V binding, as described for panels A-D. Results are expressed as the percentage of annexin V–positive cells and represent the average ± SD of at least 3 independent experiments.