implying that it has a direct effect on adenoviral transgene expression, probably by a general increment in transcription of the host cell. Materials and Methods Cell lines The prostate adenocarcinoma LNCaP, the colon carcinoma HT-29 and the glioma U343 were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES and 1 mM sodium pyruvate. The prostate adenocarcinoma PC- 346C was cultured in DMEM:F-12 supplemented with 2% FBS, 0.01% bovine serum albumin, 10 mg/ml epidermal growth factor (Sigma Aldrich, St. Louis, MO), 1% insulin-transferrin-selenium, 0.1 nM R1881 (NEN Life Science Products Inc, Boston, MA), 1.4 mM hydrocortisone (Sigma Aldrich), 0.6 ng/ml triiodothyro- nine (Sigma Aldrich), 0.1 mM phosphoetanolamine, 50 ng/ml cholera toxin (Sigma Aldrich), 0.1 mg/ml fibronectin (Sigma Aldrich) and 20 mg/ml fetuin (Sigma Aldrich). The foreskin fibroblast 1064SK and the human embryonic kidney 293FT were cultured in DMEM supplemented with 10% FBS and 1 mM sodium pyruvate. The endocrine pancreatic tumor cell line BON was cultured in DMEM with Glutamax-I and F12 K Nutrient Mixture (Kaighn’s Modification) at a 1:1 ratio, supplemented with 10% FBS and 1 mM sodium pyruvate. The murine prostate adenocarcinoma TRAMP-C2 was cultured in DMEM supple- mented with 5% FBS, 5% NuSerum (Becton Dickinson, Franklin Lakes, NJ), 1 nM dihydrotestosterone (Sigma Aldrich) and 5 mg/ ml insulin (Sigma Aldrich). All medium contained 1% penicillin- streptomycin. The cell culture reagents were from Invitrogen (Carlsbad, CA) except when stated otherwise. All cell lines were purchased from ATCC (Manassas, VA) except for BON that was a kind gift from Dr. CM Townsend (Galveston, TX) and TRAMP- C2 that was a kind gift from Dr. NM Greenberg (Baylor College of Medicine, Houston, TX). Isolation of peripheral blood mononuclear cells Buffy coats from approximately 420 ml of blood were obtained from five healthy volunteers. Peripheral blood mononuclear cells (PBMC) were isolated by density centrifugation over Ficoll-Paque (GE Healthcare, Bucks, UK). The PBMC fraction was obtained, washed twice with PBS and resuspended in culture medium. The cells were divided into five T-75 cell culture flasks and separated by adhesion for 90 min. The non-adherent fraction (mainly lymphocytes) was removed, and the adhesion fraction (mainly monocytes) was washed twice with PBS, trypsinized, counted and used for transduction. Viral vectors The recombinant serotype 5-based adenoviral vectors used in this study have been described previously: Ad[Mock] [19], Ad5 wt [20], Ad[i/PPT-LUC] [20], Ad[CMV-LUC] [21], Ad[i/PPT- E1A, E3] [20], Ad[CMV-GFP] [21], Ad[CD40L] [22], and Ad[CgA-LUC] [23]. One new vector, Ad[I/PPT-GFP] was made by replacing the luciferase transgene in Ad[I/PPT-LUC] [21] with the sequence encoding enhanced green fluorescent protein (GFP) by using HindIII and XbaI. A serotype 5 adenoviral vector with the fiber shaft and knob from serotype 35, Ad5/f35[CMV-GFP], was a kind gift from Dr Fan (Lund University, Lund, Sweden) and has been described and evaluated before [24,25]. The vectors were produced, purified and titrated as described earlier [20]. A vesicular stomatitis virus (VSV)-G protein pseudotyped self- inactivating (SIN) lentiviral vector with a CMV-GFP expression cassette was produced, LN[CMV-GFP]. For lentiviral vector FK228 Enhances Ad5 Expression production, 293FT cells were grown in 15 cm plates to a confluence of 80–90% and transfected with 8.1 mg pRRL-CMV- GFP (kind gift from Dr. F Carlotti, Leiden University Medical Center, Leiden, The Netherlands), 4.05 mg pLP1 (gag/pol), 4.05 mg pLP2 (rev), and 4.05 mg pLP/VSVG using 60 ml Lipofectamine 2000 per plate. Viral supernatants were collected after 48 h, filtered (0.45 mm) and concentrated by ultracentrifu- gation at 75,000 g for 90 min at 4uC using a Sorvall AH629 rotor. The viral pellet was resuspended in PBS and stored at 280uC until further use. Viral titer was determined by the HIV-1 p24 Antigen ELISA from ZeptoMetrix Corporation (Buffalo, NY) according to instructions from the manufacturer. Cell viability assay Prostate cancer cells were treated with various concentrations of FK228 (Fujisawa Pharmaceuticals Co., Ltd., Osaka, Japan) ranging from 0.1 ng/ml to 300 ng/ml and then plated in 96 well plates (7500 cells/well). After 24, 48 and 72 h, cell viability was determined by the MTS Cell Titer 96 Aqueous One Solution Proliferation Assay Kit (Promega, Madison, WI) according to the manufacturer’s instructions. Average enzymatic activity from triplicate samples was expressed in relation to average activity in untreated cells. Green fluorescent protein reporter gene analysis Cells were transduced in suspension for 2 h with Ad[CMV- GFP], Ad5/f35[CMV-GFP] and Ad[I/PPT-GFP] at various MOIs ranging between 0.1 and 300 FFU/cell depending on the cell line. Cells were also transduced with LN[CMV-GFP] using 0.3 vp/cell for all cell lines. Cells were either pre-treated with FK228 (3 ng/ml) for 24 h before transduction and/or incubated with FK228 2 h after transduction. Transduced monocytes were incubated with 1 ng/ml depsi. The HDACi valproic acid (VPA) from Sigma Aldrich was always added 2 h after transduction at a concentration of 5 mM. Cells were harvested 48 h after transduction and analyzed with FACSCalibur Flow Cytometer (San Diego, CA). Analysis of cell surface receptors Cells were incubated with standard medium or medium containing FK228 (3 ng/ml) for 48 h at 37uC. Cells were then harvested and stained with antibodies (30 min, 4uC) for the following surface receptors using mouse monoclonal antibodies: CAR (RmcB hybridoma, ATCC, Manassas, VA), CD46 (BD Biosciences), aVb3 (Chemicon, Temecula, CA) and aVb5 (Chemicon). After washing, the cells were incubated with a FITC-labeled rabbit-anti-mouse antibody (Dako, Glostrup, Den- mark) for 30 min at 4uC followed by washing. A mouse isotype- matched antibody (Dako) and the FITC-labeled secondary antibody were used as controls. The samples were analyzed by FACSCalibur Flow Cytometer. Mean fluorescence intensity (MFI) of receptor expression of FK228 treated cells was related to untreated cells. Analysis of CD40L expression Cells were transduced with Ad[CD40L] for 2 h, washed and then incubated with medium containing various concentrations of FK228. Cells were stained 24 h thereafter with a PE-conjugated antibody against CD40L (Pharmingen, San Diego, CA). An isotype-matched PE-conjugated antibody (Pharmingen) was used as negative control. Cells were stained with antibodies for 15 min at room temperature. Expression of surface CD40L was analyzed by flow cytometry (FACSCalibur). PLoS ONE | www.plosone.org 2 February 2011 | Volume 6 | Issue 2 | e14700