Luciferase reporter gene assay The luciferase reporter gene assay has been described earlier [20]. Briefly, LNCaP cells were transduced with Ad[i/PPT-LUC], Ad[CgA-LUC] and Ad[CMV-LUC] at an MOI of 50 FFU/cell and BON cells were transduced with Ad[CgA-LUC] and Ad[CMV-LUC] at an MOI of 10 FFU/cell in suspension for 2 h. Thereafter, fresh medium alone or fresh medium with FK228 (3 ng/ml) or VPA (5 mM) was added. After 48 h of incubation at 37uC cells were lysed and luciferase activity was measured using a Wallac VICTOR2 multilabel counter (PerkinElmer, Turku, Finland). Background levels were subtracted and luciferase activities were related to total protein concentrations in the samples (relative luciferase units per mg protein, RLU/mg). Replication assay LNCaP and PC-346C cells were transduced with Ad5[i/PPT- E1A, E3] and Ad5 wt in suspension at an MOI of 10 and 1 FFU/ cell respectively. BON was transduced with Ad[CgA-E1A] and Ad5 wt at an MOI of 1 FFU/cell. After 2 h the cells were washed and resuspended in medium with or without FK228 (3 ng/ml) or VPA (5 mM). After 72 h of incubation at 37uC, cells and medium were collected and viral DNA was purified using High Pure Viral Nucleic Acid Kit (Roche, Mannheim, Germany), according to the manufacturer’s instructions. Viral DNA content was analyzed by quantitative real time PCR (qPCR) using the iCycler IQ real-time detection system (Bio-Rad, Hercules, CA). The specific PCR product was continuously measured during 40 cycles (95uC for 15 sec, 60uC for 60 sec) using iQ SYBR Green supermix (Bio- Rad) and primers in the adenovirus E4 region: 5’-CAT CAG GTT GAT TCA CAT CGG-3’ (E4.Forward) and 5’-GAA GCG CTG TAT GTT GTT CTG-3’ (E4.Reverse). All samples were amplified in triplicates. Copy numbers were related to a standard curve made by serial dilutions of a plasmid containing E4 open reading frame 1 (pCR2.1(Ad5E4orf1)). Relative replication was defined as the increase in copy number compared to input virus (also measured by qPCR). Gene expression LNCaP and PC-346C cells were transduced with Ad[i/PPT- LUC] at MOI 10 FFU/cell and then treated with 3 ng/ml FK228 or 5 mM VPA for 48 h. Cells were then collected and RNA was extracted using RNeasy Mini Kit (Qiagen, Hilden, Germany). Three mg of total RNA and 1 ml SuperScript II Reverse Transcriptase (Invitrogen) were used for cDNA synthesis. The expression of prostate epithelial cell-associated genes [androgen receptor (AR), androgen receptor coregulator (ARA24), prostate- specific antigen (PSA), prostate-specific membrane antigen (PSMA), 5-a reductase type 1 and 2 (SRD5A1 and SRD5A2) and T cell receptor c-chain alternate reading frame protein (TARP)] and neuroendocrine-associated genes [b-tubulin III (b- tubIII), chromogranin A (CgA), neuron-specific enolase (NSE) and synaptophysin (SYP)] was analyzed by quantitative PCR with the same protocol as for the replication assay, described above. The primer sequences are listed in Table S1. RNA levels in FK228 and VPA treated cells were related to RNA levels in untreated cells. Statistical analysis Statistical analysis was done using the GraphPad Prism version 4.03 (GraphPad Software, San Diego, CA). One-way analysis of variance followed by Dunnet’s post test was used to compare luciferase activities and replication. The p value is less than 0.05 for the luciferase assay and less than 0.0001 for the replication assay. FK228 Enhances Ad5 Expression Results FK228 above 3 ng/ml reduces viability of prostate cancer cell lines Cell viability studies were performed to determine FK228 toxicity for the human prostate cancer cell lines LNCaP and PC- 346C and the mouse prostate cancer cell line TRAMP-C2. Cells were cultured in medium containing FK228 for 24, 48 and 72 hours before MTS assay analysis. After 72 h of incubation at 3 ng/ml FK228, around 80% of LNCaP and PC-346C cells were viable. TRAMP-C2 appeared to be somewhat less sensitive to FK228 than LNCaP and PC-346C. Reduced cell viability was observed at FK228 concentrations of 3 ng/ml and above for all cell lines and time points (Figure 1). Addition of FK228 after transduction enhances transgene expression of Ad[CMV-GFP] more than addition of FK228 before transduction We next wanted to investigate the effect of FK228 on adenoviral transduction. LNCaP, PC-346C and TRAMP-C2 were grown in medium with or without 3 ng/ml FK228 for 24 h. Subsequently, cells were transduced with Ad[CMV-GFP] for 2 h and then cultured in medium with or without FK228 for another 48 h. GFP expression was analyzed by flow cytometry. Pre-incubation of cells with FK228 enhanced GFP expression to some extent in LNCaP and TRAMP-C2 (Figure 2). Pre- and post-incubation gave an even higher level of GFP expression in all three cell lines. However, the highest increase was observed when FK228 was added after transduction, indicating a post-transductional effect of FK228. The fact that FK228 had the largest effect when given after transduction, both when looking at percentage of GFP positive cells and mean fluorescence intensity (MFI), indicates that the receptors involved in adenoviral transduction may play a less important role for enhanced transgene expression as previously proposed. Next, we used both the serotype 5-based vector Ad[CMV-GFP] and the adenoviral serotype 5 vector with fiber shaft and knob from serotype 35 Ad5/f35[CMV-GFP] to screen cell lines of various origin as well as freshly isolated human monocytes. Treatment of cells with FK228 after transduction enhanced GFP expression (both percentage and MFI) in all cell lines and monocytes for both viruses (Figure 3). The effect was not as pronounced for a lentiviral vector with regard to GFP positive cells as FK228 increased GFP expression of LN[CMV-GFP] in TRAMP-C2 cells but had no effect in the other cell lines tested (Figure S1). The MFI values on the other hand were increased in all cells except for BON and monocytes. FK228 does not upregulate cell surface receptors needed for adenoviral infection To investigate if FK228 upregulate cell surface receptors needed for adenoviral infection (CAR and aVb3 and aVb5 integrins for Ad5 and CD46 for Ad5/f35), cells were treated with FK228 for 48 h and then stained for receptor expression and evaluated by flow cytometry. The receptor expression of FK228-treated cells was related to the expression in untreated cells (set to 1.00). Most cell lines did not show any marked difference in surface receptor expression after treatment of FK228 (values at 1.00625%, Table S2). In the 1064SK fibroblasts the mean fluorescence intensity doubled for CAR and increased by 50% for aVb3 after treatment with FK228. Some increase of CAR was observed also in U343 and the mean fluorescence value for aVb5 in LNCaP and CD46 in PC-346C was reduced by approximately 50% upon treatment with FK228. Overall, the changes in receptor expression was if PLoS ONE | www.plosone.org 3 February 2011 | Volume 6 | Issue 2 | e14700