Previous reports were mostly supported

Previous reports were mostly supported by chemical inhibition of CDKs, but in our opinion, the observed effects are usually pharmacologically corroborated using non-selective CDK inhibitors or CDK inhibitors with unknown selectivity. The use of inhibitors with unclear selectivity might lead to misleading conclusions, especially when these inhibitors are used as a tool for evaluating certain biological processes that are linked with the function of a particular CDK. Roscovitine is an example, and it was initially believed to be a relatively specific inhibitor of CDK1, 2 and 5. However, subsequent studies have demonstrated that it also blocks transcription through inhibition of CDK7 and 9 [22], [23]. Importantly, at least two groups used roscovitine as a tool in studies related to the function of AR and showed its ability to block androgen-stimulated phosphorylation of S81 of AR in LNCaP lumacaftor [12], [15], but each group pointed to a different CDK as the responsible enzyme. Independently, CGP74514A was shown to explain the role of CDK1 in the phosphorylation of S81-AR [13]. Nevertheless, the available selectivity profile and our own data suggest that this compound is less selective and not suitable as a tool.
Materials and methods
Results
Discussion CDK1 is responsible for the phosphorylation of several cellular substrates that are predominantly important in the progression of mitosis and cytokinesis. Recently, it was suggested that CDK1 may be able to provide a pool of activated AR during mitosis [13]. Protein expression of AR during the cell cycle remains unchanged, but transcription of most AR-responsive genes is suppressed in mitosis [20]. Surprisingly, one study indicated that an increased level of S81-phosphorylation can be observed in nocodazole-G2/M-arrested LNCaP cells independently on androgen addition [13]. Because S81-phosphorylation is connected with transactivation of AR, we investigated whether increased transcriptional activity of AR would be observed in synchronized G2/M cells after nocodazole treatment. In the experiments reported here, we observed neither S81-phosphorylation nor transcriptional activation of AR in synchronized cells, which contrasted with previously published data [13], [16]. Therefore, phosphorylation of S81-AR during mitosis requires further investigation. Possible differences of the abovementioned results may be explained by the different experimental conditions used during LNCaP cultivation, which can clearly affect AR signaling. For example, a recent study revealed that a high cell density, time of cultivation and presence of androgens during cultivation can deregulate expression of several cell cycle regulators and influence the proliferation status of PCa cells [32]. Other reports also showed that the same type of PCa cells can display different patterns of AR expression and that its target PSA is probably influenced by the concentration of androgens in the media or by the duration of cultivation in charcoal-stripped serum at the beginning of the experiment [24], [51].