Coadministration of PD184352 and UCN-01 in MM cells results in enhanced cytochrome c and Smac/DIABLO release, PARP degradation, and p34cdc2 dephosphorylation, but diminished ERK activation.
Logarithmically growing 8226, H929, and U266 MM cells were exposed to 10 μM PD184352 ± 150 nM UCN-01 for 24 hours, after which cells were lysed, the proteins separated by SDS-PAGE, and Western blot analysis performed to monitor expression of PARP (A), phospho-ERK (B; p-ERK), or phospho-p34cdc2 (B; p-cdc2) as described in “Materials and methods.” Alternatively, S-100 cytosolic fractions were obtained as described in “Materials and methods,” and expression of cytochrome c (A; cyto c) and Smac/DIABLO (A; Smac) assessed by Western blot analysis. For each condition, lanes were loaded with 30 μg of protein; blots were subsequently stripped and reprobed for expression of β actin to ensure equivalent loading and transfer of protein. The results of a representative experiment are shown; an additional study yielded equivalent results. CF indicates PARP cleavage fragment.
