Combined exposure of MM cells to PD184352 and ATO changes the balance between Bim and Mcl-1. (A) HMCLs or CD138-purified plasma cells from 2 representative patients were seeded at 2.5 × 105 cells/mL in the presence of DMSO (vehicle) or PD (1 μM) for 3 hours and then were incubated for 48 hours with ATO (2 μM). Endogenous Mcl-1, Bim, Bcl-xL, and Bcl-2 proteins were revealed by immunoblotting analysis using rabbit polyclonal anti-Mcl-1, rabbit polyclonal anti-Bim, and rabbit polyclonal anti-Bcl-xL, all provided by Cell Signaling Technology, and mouse monoclonal anti–Bcl-2 clone 124 (Upstate Biotechnology, Charlottesville, VA). Antiactin immunoblotting was performed as loading control. Bim, Mcl-1, and β-actin bands were subjected to densitometric scanning using the TINA 2 software, and the Bim/Mcl-1 ratio was calculated. (B) Transfection of Bim (004383, Dharmacon RNA Technologies), but not the unrelated nonspecific control siRNA (001206, Dharmacon RNA Technologies), led to a decrease in Bim protein expression in HMCLs without affecting the levels of the unrelated protein actin: 48 hours after siRNA transfection, the HMCLs were treated with PD and/or ATO for 24 hours and then lysed for Western blot analysis to monitor the expression of Bim, caspase-8, -9, -3 activation, and PARP fragmentation. Antiactin immunoblotting was performed as loading control. (C) HMCLs were treated as described in panel B, and the percentages of sub-G1 apoptotic cells were monitored after 48 hours of treatment. Values are mean plus or minus SD of 4 independent experiments (*P < .001 vs PD/ATO cont; Dunnett test). (D) RPMI 8226 cells were pretreated with either DMSO or PD (1 μM) for 3 hours and then treated with ATO (2 μM) for 12 hours, after which cells were lysed in CHAPS lysis buffer and subjected to immunoprecipitation (IP) using rabbit polyclonal anti-Bim (Cell Signaling Technology) or control antibody and then immunoblotted (IB) with either goat polyclonal anti-DR4 (Santa Cruz Biotechnology) or goat polyclonal anti-DR5 (Santa Cruz Biotechnology) or rabbit polyclonal anti-Bim antibodies (Cell Signaling Technology). CTR indicates control; PD, PD184352 (1 μM); ATO, arsenic trioxide (2 μM); cont, nonspecific control siRNA.
