Figure 1
Characterization of bPΔ6 transgene vectors. (a) bPΔ6-hPAP viruses infect and spread in Vero and mouse prostate cancer cells. Vero (African Green Monkey Kidney) cells (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium (DMEM) with glucose (4.5 g l−1; Mediatech Inc., Herndon, VA, USA) supplemented with 10% fetal calf serum (FCS). TRAMP-C2,28, 34 obtained from Dr N Greenberg (Fred Hutchinson Cancer Research Center, Seattle, WA, USA), and RM-1,35 obtained from Dr T C Thompson (Baylor College of Medicine, Houston, TX, USA), were cultured as previously described. Cells were seeded at 80% confluency and 24 h later infected with bPΔ6-hPAP at a multiplicity of infection (MOI) of 1. Cultures were fixed 18 h after infection and stained for β-galactosidase expression with X-Gal (1 mg ml−1), 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM magnesium chloride in phosphate-buffered saline (PBS) for 4 h at 37 °C. Cells were washed with PBS, counterstained with neutral red solution and β-galactosidase expression and viral spread visualized under a light microscope. (b) Virus replication assays. Vero cells were seeded at 1 × 105 cells per well in 12-well plates and 24 h later infected with either bPΔ6-hPAP, bPΔ6-empty or wt viruses at MOI=1. At 2 h after infection the inoculum was removed and replaced with medium (DMEM/1% inactivated FCS). Cells and medium were harvested at indicated times after infection, processed with freeze/thaw cycles and sonication, and titered on Vero cells. Virus yield is plotted as plaque-forming units (PFU) per ml. (c) Cell viability assays. TRAMP-C2 cells were seeded into 96-well plates at 5000 cells per well and 24 h later infected with bPΔ6-hPAP (IC50=2.5) or bPΔ6-empty (IC50=2.9) at threefold serial dilutions (from 0.01 to 10 PFU per cell). Cell viability was assessed 4 days after infection with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma, St Louis, MO, USA) according to the manufacturer's instructions.
