1B). Published reports of the effects of other HDAC inhibitors on topoIIα expression indicate a cell type- and/or context-specificity. For example, treatment of D54 glioblastoma cells with trichostatin A or vorinostat had no effect on topoIIα expression.15 Although sodium butyrate was reported to sensitize leukemia cells to etoposide by increasing topoIIα gene expression,16 treatment of MCF-7 cells with valproic acid led to transcriptional repression of topoIIα.17 check details To clarify this issue, we assessed the concentration-dependent effect of sodium butyrate on topoIIα expression in PLC5 cells. Our data show that treatment with a range of concentrations

of sodium butyrate revealed a biphasic effect on topoIIα expression levels, i.e., up-regulation at low concentrations (≤0.25 mM) and down-regulation at higher concentrations (≥0.5 mM), without disturbing topoIIβ expression (Fig. 1C). These concentrations are consistent with those of sodium butyrate (0.4 mM) and valproic acid (2 mM) that up-regulated and down-regulated topoIIα expression, respectively, in the aforementioned studies. This dichotomous effect may typify the complex mode of action of short-chain fatty

acids in regulating topoIIα expression relative to other HDAC inhibitors examined. The finding that MS-275 was able to suppress topoIIα expression suggests the involvement of class I HDACs in the drug response. selleck kinase inhibitor Thus, we assessed the effect of shRNA or siRNA-mediated knockdown of class I (HDAC1 and 2) vis-à-vis class II isozymes (HDAC4-6) on topoIIα messenger RNA (mRNA) and protein expression in PLC5 cells. Silencing of HDAC1 caused a sharp decrease in the topoIIα protein level, whereas the mRNA expression was not altered (Fig. 2A). However, the knockdown of other isozymes had no effect on the mRNA or protein expression of topoIIα. Evidence indicates that this topoIIα down-regulation was

attributable to proteasomal degradation. First, exposure of PLC5 cells to AR42 or MS-275 did not cause appreciable changes in topoIIα mRNA levels as determined by RT-PCR (Fig. 2B). Second, Edoxaban the proteasome inhibitor MG132 protected cells against the suppressive effect of AR42, MS-275, and vorinostat on topoIIα expression (Fig. 2C). Third, in the presence of cycloheximide, AR42 promoted the elimination of topoIIα relative to the DMSO control (Fig. 2D). Together, these data suggest a pivotal role of HDAC1 in the regulation of topoIIα protein stability. It is well documented that ubiquitin-dependent protein degradation is preceded by phosphorylation.18 As shown in Fig. 3A, concentration-dependent topoIIα repression by AR42 was accompanied by parallel increases in p-Ser/Thr phosphorylation and ubiquitination. However, no appreciable acetylation of topoIIα was noted in response to AR42 treatment, suggesting that topoIIα stability is not influenced by HDAC-regulated acetylation.