FK228 Enhances Ad5 Expression Figure 5. HDAC inhibitors affect tissue-specific promoters differently. A) BON cells were transduced with Ad[CgA-LUC] and Ad[CMV-LUC] and LNCaP and PC-346C were transduced with Ad[i/PPT-LUC], Ad[CgA-LUC] and Ad[CMV-LUC]. Cells were then incubated with 3 ng/ml FK228 or 5 mM VPA for 48 h. Both FK228 and VPA enhanced the transgene expression of the Ad[CgA-LUC] vector in BON cells about 10 times although the increase was not significant. In LNCaP and PC-346C, the expression of Ad[i/PPT-LUC] was significantly decreased while the expression of Ad[CgA-LUC] was significantly increased by HDACi treatment. B) LNCaP and PC-346C cells were transduced with Ad[I/PPT-GFP] and Ad[CMV-GFP] and then incubated with 3 ng/ml FK228 or 5 mM VPA for 48 h followed by flow cytometry analysis. The percentages of GFP positive cells (upper right) and MFI values (lower right) are given. GFP levels were decreased by HDAC inhibitors when the transgene expression was controlled by the PPT promoter. One representative experiment out of three is shown. Significance (p,0.05) is indicated with asterisks. doi:10.1371/journal.pone.0014700.g005 SRD5A1, SRD5A2 and TARP were generally not affected (less than 2-fold up- or down-regulation) by the FK228 and VPA treatment. However, in LNCaP, the expression of AR was slightly decreased to just below a 2-fold down-regulation for both HDACi. In PC-346C, the expression of SRD5A1 was increased after VPA treatment and SRD5A2 expression was increased after both FK228 and VPA treatment while PSMA expression (VPA treatment) and TARP expression (FK228 and VPA treatment) were decreased (Figure 7). We observed that the activity of the CgA promoter increased by treatment of FK228 and VPA in LNCaP and PC-346C. Furthermore, VPA is known to induce neuroendocrine differen- tiation in LNCaP by upregulation of b-tubIII, NSE and SYP [26,27]. RNA levels of these elements were therefore also assessed. Therefore, expressions of the neuroendocrine-associated genes: b- tubIII, CgA, NSE and SYP were evaluated. We found them all to be greatly increased in HDACi-treated LNCaP and PC-346C cells (Figure 7), indicating that HDACi promotes differentiation along the neuroendocrine lineage for these adenocarcinoma cell lines. Discussion HDACi have been shown to increase the effect of adenoviral- mediated gene therapy although the mechanisms are not fully understood [13,14,28]. Upregulation of CAR, the primary receptor for Ad5 infection, has been suggested as a possible mechanism [7,15,16,17,18]. However, in those studies the amount of CAR was determined by measurement of RNA levels by quantitative real-time PCR or protein levels by western blot analysis. Determination of RNA and total protein levels does not determine receptor functionality and the results can therefore be misleading. We measured instead the quantity of CAR and integrins, which are also needed for Ad5 infection on the surface of cells by flow cytometry, since it is the most appropriate technique to address receptor expression. We found that neither CAR nor integrins were upregulated after 48 h of treatment with non-toxic concentrations of FK228. Furthermore, when prostate cancer cells were transduced with Ad[CMV-GFP], the amount of GFP- expressing cells was much higher when FK228 treatment was given after transduction compared to when cells had been pre- PLoS ONE | www.plosone.org 6 February 2011 | Volume 6 | Issue 2 | e14700