Further investigation of the effects of this inhibitor

Further investigation of the effects of this inhibitor on histone acetylation revealed no HAT inhibitory effects at the level of global histone acetylation after 20h of incubation with C646. After 6h of incubation with C646, however, a slight increase in histone H3 acetylation was observed at lysine residues 18 and/or 23. This indicates a time dependent effect of C646 on histone acetylation, which has not been reported before. Evaluation of the ratios between HAT and HDAC activities under the studied conditions may clarify this. It should be noted that previous studies indicated that the effects of C646 on histone acetylation are cell type dependent. Previously, effects of C646 which were in line with HAT inhibition have been demonstrated in cells using immunoblot or ChIP assays [32], [33]. In other studies, inhibiting effects of this compound on histone acetylation were only shown after pre-treatment with an HDACi. HDACi pre-treatments are done more often in studies with HATi to increase the detection window for inhibition of the acetyl transfer reaction. For example, upon pre-treatment with HDACi trichostatin A (TSA) C646 demonstrated a pronounced inhibition of histone acetylation [34], [8]. In contrast, observations similar to ours that C646 increases histone acetylation have been made in a model system employing pancreatic cancer cells [28], also after a short incubation time with C646 (4h). The authors attributed the observed increase in histone H3 acetylation on K18 and K23 to increased cooperative binding of p300 to its substrate acetyl CoA, facilitated by C646 when present at low concentrations. The authors suggest that C646 can be outcompeted by neurokinin 1 receptor under these conditions. Although this model may explain the increased histone acetylation in cells at low concentrations of C646, it would be less suited to explain the increase observed in our system in which we employed relatively high concentrations of C646. The observation that the p300 HATi C646 increases histone acetylation may also be explained through another mechanism. It has recently been reported that the overall histone acetylation levels remain remarkably constant upon depletion of HATs [35]. In Drosophila cells, it was observed that histone acetylation levels were unaffected or in some cases even higher upon siRNA mediated knockdown of specific acetyltransferases. The authors suggest that compensatory modifications occur in the case of a knockdown of specific acetyltransferases in order to compensate structural perturbations, which may be brought about in different ways, including feedback responses to modulate gene expression or enzyme activity of other acetyltransferases or deacetylases. Similar processes could take place when p300 is inhibited by a small molecule inhibitor such as C646. However, the slight increase in histone acetylation prompted us to pose an alternative hypothesis; direct inhibition of HDACs by C646. Upon investigation, we indeed found inhibition of HDAC 2, 3, 6 and 8 by C646 at micromolar concentrations. These observations of HDAC inhibitory activity by C646 place biochemical studies in which C646 has been applied as a HATi in a new perspective. Although the inhibitory potency for HDACs reported here is about 10-fold lower compared to the HAT p300 inhibition, this observation might have important implications. In biochemical experiments, the applied C646 concentrations sometimes exceed the Ki for p300 by a factor of 10. In addition, local concentrations in cells may be even higher. At these concentrations, HDAC inhibition can start playing a role in the observed effects, thus mixing HAT and HDAC inhibitory effects on cellular substrates. Thus, the interpretation of effects connected to presumed inhibition of p300 in the investigated model systems can be hampered by inhibition of HDACs. This clearly calls for a further optimization of C646 with respect to its HDAC inhibitory potency. This is also important in the light of recent studies focusing on applications of this HATi in the treatment of cancer, where HDAC inhibition upon C646 treatment is not presumed [36], [37]. Furthermore, the HDAC inhibitory potency of C646 could explain its ambiguous effects on histone acetylation in cells reported in literature.