IL-6 and IGF-1 fail to prevent PD184352 from blocking UCN-01–mediated Bim_EL phosphorylation. (A) U266 and RPMI8226 cells were cultured in serum-free medium for 6 hours followed by exposure to IL-6 (100 ng/mL) or IGF-1 (400 ng/mL) for 20 minutes, after which cells were lysed and subjected to Western blot analysis to assess expression of phospho-ERK1/2 and Bim. (B) RPMI8226 cells were starved and preincubated (in some cases) with IL-6 or IGF-1 as described for panel A and then exposed to 150 nM UCN-01 plus or minus 5 μM PD184352 for 4 hours, after which cells were lysed and subjected to Western blot analysis to monitor ERK1/2 phosphorylation and Bim expression. Each lane was loaded with 20 μg protein; blots were stripped and reprobed with antiactin or antitubulin antibodies to ensure equal loading and transfer. Phosphorylated forms of Bim_EL are manifested by slowly migrating species (▼). Two additional studies yielded equivalent results. (C) Hypothetical model for Bim involvement in UCN-01 and MEK1/2 inhibitor interactions in MM cells. UCN-01 triggers death signals (eg, “unscheduled” activation of p34^cdc2 through inhibition of Chk1), which leads to apoptosis. However, the putative proapoptotic actions of UCN-01 may be opposed by a compensatory activation of ERK1/2, which leads to phosphorylation and proteasomal degradation of the proapoptotic BH3 protein Bim. These events can also be stimulated by several MM survival factors, including IL-6 and IGF-1. Blocking ERK1/2 activation by pharmacologic MEK1/2 inhibitors (eg, PD184352 and PD98059) leads to accumulation of Bim, which binds to and disables Bcl-xL/Bcl-2, thus rendering MM cells more vulnerable to UCN-01 lethality, even in the presence of IL-6 or IGF-1. The possibility that increased expression of Bim may directly induce activation of the multidomain proapoptotic proteins Bax/Bak cannot be excluded.