FK228 Enhances Ad5 Expression Figure 3. FK228 enhances transgene expression in various cell types. Cells were transduced with Ad[CMV-GFP] or Ad5/f35[CMV-GFP] for 2 h and then incubated for 48 h in medium containing 3 ng/ml FK228. The percentages of GFP positive cells (upper right) are given as well as MFI values (lower right or left). FK228 enhanced transgene expression of Ad5 virus and Ad5 virus with fiber shaft and knob from serotype 35 in all cell lines. Each cell line was evaluated three times with similar results. Monocytes from five different donors were evaluated. Representative examples are displayed in the figure. doi:10.1371/journal.pone.0014700.g003 Since reporter gene expression of PPT-based vectors was negatively affected by FK228 and VPA, we also wanted to study if replication of a PPT-controlled oncolytic adenovirus was decreased as well. LNCaP and PC-346C cells were transduced with the conditionally replicating Ad[i/PPT-E1A, E3] and Ad5 wt and BON cells were transduced with Ad[CgA-E1A] and Ad5 wt. The cells were then incubated in medium containing either FK228 or VPA for 72 h. Viral copy numbers were determined by quantitative PCR. Replication of the PPT-driven virus was decreased by FK228 in LNCaP (1.6 times) and PC-346C (1.3 times). VPA treatment reduced replication to a higher extent; 7.7 times in LNCaP and 126 times in PC-346C (Figure 6). In BON cells, a decrease (1.7 times) in replication was observed in Ad[CgA-E1A]-transduced cells treated with VPA (Figure 6). Replication of Ad5 wt was not affected in BON cells but reduced in LNCaP and PC-346C. Figure 4. FK228 treatment enhances adenoviral-mediated CD40L expression. Prostate cancer cell lines were transduced with various MOI of Ad[CD40L] and cultured in medium with various concentrations of FK228 for 24 h. Cell surface expression of CD40L was analyzed by flow cytometry. Percentages of CD40L positive cells (upper right) and MFI values (lower right) are given. FK228 increased CD40L expression in a dose-dependent manner. One representative experi- ment out of three is shown. doi:10.1371/journal.pone.0014700.g004 HDACi differentiate virus-transduced prostate cancer cells along the neuroendocrine lineage The increased transgene expression from Ad[CgA-LUC] in the prostate cancer cell lines LNCaP and PC346-C and the decreased expression of Ad[i/PPT-LUC] upon HDACi treatment led us to further investigate the effect of FK228 or VPA on these cell lines. RNA expression of the genes normally controlled by the regulatory elements of PPT: PSA, PSMA and TARP were therefore evaluated along with genes involved in the androgen receptor pathway: AR, ARA24, SRD5A1 and SRD5A2, since the PPT promoter is partly regulated by testosterone. The prostate epithelial cell-associated genes: AR, ARA24, PSA, PSMA, PLoS ONE | www.plosone.org 5 February 2011 | Volume 6 | Issue 2 | e14700