express tumor-associated antigens, often in the form of an adenoviral vector carrying the transgene coding for the tumor- associated antigen. For example, efficient adenoviral transduction of monocytes is highly warranted for ex vivo generation of T cells against specific antigens [35]. Such T cells could then be used for adoptive transfer to cancer patients. First, we observed that by using Ad5/f35 instead of Ad5 the amount of viral vector needed for transduction of monocytes could be lowered four times compared to Ad5 vectors (from 20 FFU/cell for Ad5 to 5 FFU/ cell for Ad5/f35). We then asked ourselves if FK228 could improve transgene expression even further and we found that addition of FK228 after transduction doubled transgene expres- sion both for Ad5 and Ad5/f35. These data imply that HDACi may increase the effect of adenoviral-based vaccination strategies, importantly also for Ad5/f35-based vectors. The strategy to use adenoviral-mediated CD40L gene therapy to induce anti-tumor immunity has been employed in several cancer models [36] and it has recently been evaluated in a phase I/IIa clinical trial for bladder carcinoma [37]. In a previous study, we demonstrated that administration of Ad[CD40L] can suppress tumor growth and prolong survival of mice with subcutaneous, syngenic TRAMP-C2 tumors [38]. However, relatively high amount of the vector is needed to suppress tumor growth since mouse cells are semi-permissive for infection of human adenoviral vectors. We observed that addition of non-toxic concentrations of FK228 after transduction with Ad[CD40L] enhanced CD40L expression in a dose-dependent manner in prostate cancer cell lines in vitro. For TRAMP-C2 cells, which normally are hard to transduce with Ad5 [38], CD40L-expressing cells increased dramatically from 0.3% without treatment to 62% with FK228 treatment after viral transduction at MOI 3. This suggests that FK228 can be combined with Ad[CD40L] treatment and possibly with any other adenoviral vector when increased expression of a therapeutic gene is desired, i.e., lower amounts of adenoviral vectors would be required to achieve a therapeutic response. The use of tissue-specific promoters is an attractive approach to restrict transgene expression of adenoviral vectors and replication of oncolytic adenoviruses to certain organs [39,40]. We have previously reported on adenoviral vectors with the prostate cell- specific PPT promoter that consists of the PSA enhancer, the PSMA enhancer and the TARP promoter [21]. We have also shown that oncolytic Ad5 viruses with the PPT promoter selectively reduce cell viability of prostate cancer cell lines [20,41] and we therefore wanted to study the putatively additive effect of adding HDAC inhibitors. However, we found that although an adenoviral vector with the CMV promoter yields higher transgene expression (GFP and luciferase) after treatment of LNCaP and PC-346C with the HDACi FK228 and VPA, the opposite was observed for the recombinant PPT promoter. One possible explanation for this is that the PPT promoter is directly affected by HDACi but a more plausible explanation is that HDACi affect gene expression and cell differentiation of LNCaP and PC-346C. Gene expression profiling experiments have revealed that HDACi including FK228 induce changes in genes coding for transcription/translation factors and factors involved in metabolism, differentiation and cell cycle [42,43,44]. Furthermore, VPA and other HDACi have been shown to promote neuroen- docrine-like differentiation of LNCaP cells [26,27]. We speculate that also FK228 induce neuroendocrine differentiation of LNCaP and that the activity of the PPT promoter therefore is decreased since its activity is restricted to normal and neoplastic prostate epithelial cells. In fact, four neuroendocrine-associated genes, b- tubIII, CgA, NSE and SYP, were upregulated in LNCaP and PC- 346C after treatment with either FK228 or VPA. Furthermore, we FK228 Enhances Ad5 Expression also observed that CgA promoter-controlled transgene expression increased in LNCaP and PC-346C upon HDACi treatment after adenoviral vector transduction, again indicating cell differentiation along the neuroendocrine lineage. VPA has previously been shown to hamper the adenovirus major late promoter thus affecting replication in a negative manner [45]. The PPT promoter did not lose its activity completely and we found that the replication of Ad[i/PPT-E1A, E3] in LNCaP and PC-346C was decreased by FK228, although the decrease was small, while VPA decreased the replication to a higher extent. The reason for this observation might be that there is still enough E1A produced by the PPT promoter to ensure efficient replication after FK228 treatment but not after VPA treatment. However, since the reporter gene expression driven by PPT was decreased and the replication was not improved it appears that it is not meaningful to use FK228 or VPA together with an oncolytic adenovirus controlled by this particular promoter. The transgene expression driven by another tissue- specific promoter, the neuroendocrine cell-specific chromogranin A (CgA) promoter [23] was evaluated in the endocrine pancreatic cell line BON. We found that the activity of this promoter behaved as the CMV promoter; the expression of the luciferase transgene increased after treatment of transduced cells with both FK228 and VPA. The replication of Ad[CgA-E1A] was not affected by FK228 but was reduced by VPA. We predict that most promoters will yield higher transgene expression in target tissues when FK228 or VPA are used in conjunction with adenoviral transduction. However, it is unlikely that HDACi will increase replication of viral vectors as neither of the two HDACi increased the replication of wild type Ad5 nor the tissue-restricted viruses investigated in the present study. We conclude from the results in this study that the HDACi FK228 can be used to enhance CMV-driven and CgA-driven transgene expression of Ad5 and Ad5/35 viruses in cell lines and human monocytes. The mechanism for this enhancement seems to be transduction-independent since the highest transgene expres- sion was observed when FK228 was administered after transduc- tion. We therefore discourage from using FK228 as pre-treatment to adenoviral transduction and suggest evaluation of simultaneous administration of vector and FK228 or that FK228 is administered after vector treatment in pre-clinical animal models. HDACi may influence differentiation of cells and can therefore change the activity and selectivity of tissue-specific promoters, as was observed with the prostate cell-specific PPT promoter. For that reason, we propose careful investigation of HDACi effects on target cells and their interaction with viral vectors before usage. Since neither FK228 nor VPA resulted in increased replication of Ad5 wt and the tissue-restricted viruses, the use of HDACi is not likely to be beneficial for oncolytic therapy. Supporting Information Figure S1 FK228 does not improve transgene expression of a lentiviral vector. Cell lines were transduced with LN[CMV-GFP] at 0.3 vp/cell and monocytes at 3 vp/cell followed by incubation for 48 h in medium containing 3 ng/ml FK228. The percentages of GFP positive cells (upper right) and MFI values (lower right) are given. For most cell types as well as monocytes, FK228 did not enhance GFP expression. In TRAMP-C2 however, the GFP expression was doubled in FK228-treated cells. Found at: doi:10.1371/journal.pone.0014700.s001 (1.10 MB TIF) Table S1 Found at: doi:10.1371/journal.pone.0014700.s002 (0.04 DOC) MB PLoS ONE | www.plosone.org 8 February 2011 | Volume 6 | Issue 2 | e14700