MEK inhibition potentiates the ATO-induced apoptosis in MM cells. (A) HMCLs seeded at 2.5 × 10⁵ cells/mL were treated sequentially with escalating doses of PD (0.1-20 μM) for 3 hours and subsequently with ATO alone (0.125-10 μM) or in combination with PD at a 1:1 ratio (0.25/0.25, 0.5/0.5, 1.0/1.0, 1.5/1.5, 2.0/2.0 μM). After 48 hours, apoptosis was measured by annexin V labeling. Combination Index plots were then generated using the Calcusyn software. Combination Index values less than 1.0 indicate synergism, Combination Index values equal to 1.0 indicate additive effect, and Combination Index values more than 1.0 indicate an antagonistic effect. (B) CD138-purified plasma cells from 12 patients with MM were seeded at 2.5 × 10⁵ cells/mL in the presence of DMSO (vehicle) or PD (1 μM) for 3 hours and then incubated with ATO (2 μM) for 24 hours. The apoptosis was measured as percentage of cells with hypodiploid DNA content. Results are expressed as the net apoptosis induction (percentage of apoptosis in treated cells − percentage of apoptosis in DMSO [vehicle-treated cells]) and represent the means plus or minus SD of the results obtained in 12 different patient samples (*P < .001, Dunnett test). (C) Normal bone marrow B cells from 3 healthy donors were treated as described in panel B. After 24 hours of treatment, apoptosis was then measured as the percentage of cells with hypodiploid DNA content. CTR indicates control; PD, PD184352 (1 μM); ATO, arsenic trioxide (2 μM). (D,E) Analysis of the effect of PD and ATO on hemopoietic progenitor colony formation studied either using total PBMCs (D) or purified CD34⁺ cells (E). A total of 2 × 10⁵ PBMCs (D) or 2 × 10² CD34⁺ cells (E) have been grown in methylcellulose either in the absence (0) or in the presence of either PD (0.5, 1, or 2 μM) or ATO (0.5, 1, or 2 μM) or both drugs. (F) Study of the effect of PD and ATO on the growth of CD34⁺ cells grown under serum-free conditions allowing the selective growth of either erythroid (E), megakaryocytic (Mk), or granulocytic (G) cells. A total of 5 × 10⁴ CD34⁺ cells have been grown in serum-free liquid suspension cultures under E, Mk, or G cell culture conditions either in the absence (C) or in the presence of PD (1 μM), ATO (1 μM), or PD + ATO (both at 1 μM), and the number of the cell progeny was evaluated at various days of culture. For panels D, E, and F, the results represent the means plus or minus SEM values observed in 3 separate experiments. C indicates control; PD, PD184352; ATO, arsenic trioxide.