As shown in Fig. 5A, treatment of PLC5 cells with AR42 had no effect on Csn5 expression (input), but led to a concentration-dependent increase in the association of topoIIα with CK2α and Csn5 (right panel), which is noteworthy in that physical interaction with Csn5 is reported to be a prerequisite

for the degradation of its target proteins.27 This increase in the amount of CK2α associated with the Csn5-topoIIα complex paralleled the increase in total cellular CK2α levels in AR42-treated cells. Moreover, the ectopic expression of Csn5 dose-dependently mimicked SAHA HDAC price the suppressive effect of HDAC inhibitors on topoIIα expression (Fig. 5B), whereas siRNA-mediated knockdown of Csn5 protected against the drug-induced down-regulation of topoIIα in AR42- and MS-275-treated PLC5 cells (Fig. 5C). These results are consistent with the putative role of Csn5 in HDAC inhibitor-mediated topoIIα degradation. The Csn complex facilitates the proteasomal degradation of target proteins by functioning as a docking platform for recruitment of the target’s specific kinase and E3 ligase.29 Consequently, we

sought to identify the E3 ligase that targets topoIIα in the Csn5 complex. Csn5 is known to maintain the stability of a number of the F-box proteins of the Skp1-Cul1-F-box-protein family, including Skp2, Fbw7, Fbx4, and Fbx7, as the silencing of Csn5 led to the down-regulation of these F-box proteins.30 Thus, using these Csn5-interacting F-box proteins as candidates for the topoIIα-targeted E3 ligase, we assessed the concentration-dependent effects of AR42 on GSI-IX order the binding of these F-box proteins to topoIIα. The E3 ligase Bmi1 was also assessed in light of a recent report that Bmi1 controlled topoIIα degradation in response to glucose

starvation.31 PLC5 cells exhibited robust expression of Skp2, Fbw7, and Bmi1, but had low abundance of Fbx4 and Fbx7 (Fig. 6A, input). Coimmunoprecipitation revealed a concentration-dependent increase in the binding of Fbw7 to topoIIα Demeclocycline by AR42 (right panel). This AR42-induced association was highly selective because the other F-box proteins were undetectable or present in extremely low levels, relative to Fbw7, in the complex formation with topoIIα. The functional role of Fbw7 as the topoIIα-targeted E3 ligase was further supported by the protective effect of shRNA-mediated knockdown of Fbw7 on AR42- and MS-275-mediated topoIIα ablation (Fig. 6B). Above, we showed that, in addition to Csn5, CK2α also associated with topoIIα in response to AR42 (Fig. 5A). Thus, we hypothesized that phosphorylation of topoIIα by CK2 facilitated the association of topoIIα with the Csn5-Fbw7 complex in AR42-treated cells. Results in support of this hypothesis are shown in Fig. 6C, where the CK2 inhibitor DMAT abrogated the interaction of topoIIα with Csn5 and Fbw7.