Synergistic apoptosis induction by proteasome and histone deacetylase inhibitors is dependent on protein synthesis.

Proteasome inhibitors are able to efficiently induce apoptosis in many tumor cells while leaving quiescent, untransformed cells largely unharmed. Here we investigated the further enhancement of proteasome inhibitor-mediated apoptosis induction in Bcr-Abl positive K562 CML cells by simultaneous treatment with different histone deacetylase inhibitors (HDIs). Combining proteasome and HDIs resulted in rapid hyperacetylation of histone H3 and accumulation of polyubiquitinated proteins and the synergistic induction of apoptosis. Apoptosis induction was associated with caspase 8, 3 and 9 activation, Bid processing, destruction of the mitochondrial membrane potential, cleavage of PARP and lamin B and extensive DNA fragmentation. The pan-caspase inhibitor Z-VAD-FMK and the caspase-8 inhibitor Z-IETD-FMK could inhibit K562 cell apoptosis. Apoptosis was also delayed by overexpression of Bcl-xL, as well as by crmA, a known inhibitor of caspases 1 and 8. Caspase 8 activity could still be detected in the presence of ectopic Bcl-xL, but not in crmA transfected cells. The most striking anti-apoptotic effect though was obtained by the translational inhibitor cycloheximide, which abolished caspase 8 processing, blocked Bid cleavage and maintained the mitochondrial transmembrane potential. Apoptosis by the combination treatment occurred independently from CD95/Fas receptor stimulation. These results demonstrated that transcriptional activation by HDIs combined with proteasome inhibitor mediated posttranslational stabilization of protein(s) results in significantly enhanced CML apoptosis which was striktly dependent on uninterrupted protein synthesis.