Figure 1. FK228 concentrations above 3 ng/ml reduce cell viability in prostate cancer cells. TRAMP-C2, LNCaP and PC-346C cells were treated with various concentrations of FK228 (ranging from 0.1 ng/ml to 300 ng/ml) for 24, 48 and 72 h. Cell viability measured by the MTS assay is expressed in relation to the activity in untreated cells. FK228 concentrations higher than 3 ng/ml reduced viability in all cell lines. doi:10.1371/journal.pone.0014700.g001 anything only marginal after FK228 treatment, further indicating that receptor upregulation is not the main reason for increased transgene expression after adenoviral transduction. FK228 treatment enhances adenoviral-mediated CD40L expression in prostate cancer cells Since FK228 had an effect on the GFP transgene we next wanted to investigate if it can increase also therapeutic transgene expression. An Ad5-based vector with the therapeutic CD40 ligand transgene driven by the CMV promoter, Ad[CD40L], was studied in prostate cancer cell lines. Cells were transduced with the vector for 2 h, washed and then incubated with or without various concentrations of FK228. After 24 h of incubation, the cells were FK228 Enhances Ad5 Expression Figure 2. Addition of FK228 after adenoviral transduction enhances transgene expression. Prostate cancer cell lines were grown in medium with or without 3 ng/ml FK228 for 24 h (before). Cells were then transduced with Ad[CMV-GFP] for 2 h, washed and then cultured in medium with or without FK228 for another 48 h (after). The percentages of GFP positive cells (upper right) and MFI values (lower right) are given. The highest transgene expression was observed when FK228 was administered after transduction. One representative set out of three experiments is shown. doi:10.1371/journal.pone.0014700.g002 stained for CD40L expression and analyzed by flow cytometry. FK228 treatment increased the percentage of CD40L-expressing cells as well as MFI in a dose-dependent manner (Figure 4). The most pronounced effect was observed in TRAMP-C2 cells, which are normally difficult to transduce with Ad5-based vectors. At an MOI as low as 3 FFU/cell, the percentage of CD40L-expressing cells increased from 0.3% for untreated cells to 62% for FK228- treated (3 ng/ml) cells. At MOI 0.3, FK228 treatment increased CD40L expression about 3 times in LNCaP and 2 times in PC- 346C. HDAC inhibitors affect transgene expression driven by the prostate-specific PPT promoter negatively in prostate cancer cell lines After finding out that FK228 increases transgene expression from various viral vectors, we wanted to investigate if FK228 had the same positive effect on adenoviral vectors with tissue-specific promoters as it has on an adenoviral vectors with the CMV promoter. The human chromogranin A (CgA) promoter was positively affected, although not significantly, by FK228 and VPA when the neuroendocrine pancreatic carcinoid cell line BON was transduced with Ad[CgA-LUC] (Figure 5A). A vector with the same transgene driven by the CMV promoter was used for comparison. Next, LNCaP and PC-346C cells were transduced with Ad[i/PPT-LUC and Ad[i/PPT-GFP] and thereafter incu- bated with FK228 or VPA for 48 h. Interestingly, the PPT-driven transgene expression of both luciferase (Figure 5A) and GFP (Figure 5B) was decreased when the cells were incubated with either of the two HDACi while, as before, CMV-driven GFP expression was increased when cells were treated with FK228 after transduction. VPA treatment increased the expression of both luciferase and GFP when the CMV promoter was used (Figure 5A and 5B). Of further interest, when the prostate cancer cell lines LNCaP and PC346-C were transduced with the neuroendocrine- selective Ad[CgA-LUC] vector and then treated with FK228 or VPA, we found that transgene expression was increased. PLoS ONE | www.plosone.org 4 February 2011 | Volume 6 | Issue 2 | e14700