For large-scale preparations, the adenoviruses were amplified in gefitinib mechanism of action a transformed human embryonic kidney cell line, 293, and purified by two steps of caesium chloride density centrifugation. Viral titers were measured in a limiting-dilution bioassay using 293 cells. Cells (1 �� 105cells per dish) were seeded onto a 60mm dish and treated with Ad/EFp-BCD or Ad/5HREp-BCD for 1h. Then, the adenovirus-containing medium was replaced with one without the virus. Western blot analysis The stable transfectants and the virus-infected cells were seeded in 60mm glass dishes (2 �� 105cells per dish), put into pre-warmed aluminium chambers, and flushed with hypoxic gas (95% N2, 5% CO2) for 30min. Then, tightly sealed chambers were incubated at 37��C for 16h for the hypoxic treatment.

The cells were harvested in RIPA lysis buffer (10% SDS, 2M Tris-HCl, pH 7.5, and 1% Triton X) supplemented with a protease inhibitor, Mini complete (Roche, Basel, Switzerland). The lysates were sheared using a syringe with a 23-gauge needle, and the protein concentration was determined using the DC Protein assay kit (Bio-Rad). Twenty micrograms of total protein was electrophoresed on a 12% SDS polyacrylamide gel, transferred onto PVDF membrane (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA) and blocked with 5% non-fat milk in Tris-buffered saline. The BCD protein fused to the myc epitope tag was detected with monoclonal anti myc-tag antibody (Cell Signaling Technology Inc., Danvers, MA, USA) and anti mouse IgG horseradish peroxidase-linked whole antibody (GE Healthcare Bio-Sciences Corp.

) using an ECL-PLUS system (GE Healthcare Bio-Sciences Corp.) according to the manufacturer’s instructions. In vitro cell proliferation assay The stable transfectants and the virus-infected cells were seeded in triplicate into 96-well plates (1 �� 103cells per well) and incubated with various concentrations of 5-fluorocytosine (5-FC) (Sigma Chemical Co., St Louis, MO, USA) for 24h under normoxic or hypoxic conditions. For the hypoxic treatment (<0.02% of oxygen), the cells were treated in a hypoxic chamber, BACTRON-II (Sheldon Manufacturing Inc., Cornelius, OR, USA). The cells were additionally incubated under normoxic conditions for 24h. The culture medium was then changed to one without 5-FC, and the cells were cultured for 48h under the normoxic conditions. Cell growth inhibition was quantified by colorimetric assay using a CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Tumour-bearing mice A suspension of HeLa cells (2 �� 106cells/100��l of PBS) was subcutaneously inoculated into the right hind leg of a 6-week-old Cilengitide nu/nu BALB/c mice (Charles River, Tokyo, Japan).