FK228 Enhances Ad5 Expression Figure 6. Virus replication is decreased by treatment of HDAC inhibitors. LNCaP and PC-346C were transduced with 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 with 3 ng/ml FK228 or 5 mM VPA for 72 h. Viral copy numbers were determined by quantitative PCR and related to the values obtained 2 h after transduction. Replication was decreased by FK228 and VPA treatment in most cases. Significance (p,0.0001) is indicated with asterisks. doi:10.1371/journal.pone.0014700.g006 incubated with FK228. These two observations strongly suggest that transgene expression is enhanced by a transduction- independent mechanism. Association of cellular histones with the viral genome DNA in infected cells remains controversial [29,30,31,32]. However, by using chromatin immunoprecipitation (ChIP) assays, Komatsu et al recently showed that viral chromatin is associated with both viral protein VII and cellular histones with post-translational modification (acetylation of histone H3) in infected cells [33]. They also showed that acetylation of histone H3 occurs at the promoter regions of active viral genes. Among a variety of histone modifications, acetylation at K9 and/or K14 of histone H3 is in fact recognized as an active chromatin mark for transcription [34]. It is therefore possible that FK228 affects histones associated with viral DNA and therefore enhances adenoviral transgene expression at the transcrip- tional level. However, this remains to be investigated. Since we observed that FK228 administered directly after adenoviral transduction had a visible effect on transgene expression, we went on to investigate the effect of FK228 in a number of experimental models where it could be therapeutically beneficial. Monocytes are precursors of dendritic cells and as such, highly interesting as cancer vaccines after being modified to Figure 7. Differential gene expression in virus-transduced prostate cancer cells after 48 h of treatment with HDAC inhibitors. LNCaP and PC-346C were transduced with Ad[i/PPT-LUC] and then incubated with 3 ng/ml FK228 or 5 mM VPA for 48 h. RNA was extracted, cDNA was produced and gene expression was evaluated by quantitative PCR (triplicate samples). Gene expression in FK228 and VPA-treated cells was related to untreated cells. A two-fold difference in gene expression was set as a limit for true difference. The expression of prostate-associated genes (AR, ARA24, PSA, PSMA, SRD5A1, SRD5A2 and TARP) was not considerably affected by the HDACi. The expression of neuroendocrine-associated genes (b- tubIII, CgA, NSE and SYP) was increased in HDACi-treated cells. doi:10.1371/journal.pone.0014700.g007 PLoS ONE | www.plosone.org 7 February 2011 | Volume 6 | Issue 2 | e14700