The cell proliferation was selleck compound significantly inhibited under hypoxic conditions compared to normoxic conditions when the cells were treated with Ad/5HREp-BCD (MOI=100) and the higher concentration of 5-FC. Likewise, MIA PaCa-2 and WiDr cells showed hypoxia-dependent sensitivity to the adenovirus-mediated BCD/5-FC treatment (Figure 3B and C). On the other hand, proliferation was inhibited under both normoxic and hypoxic conditions, when the cells were infected with Ad/EFp-BCD (MOI=100). All of the in vitro experiments clearly indicate that our system functioned as we desired. Figure 3 Ad/5HREp-BCD-mediated cytotoxicity. (A) HeLa, (B) MIA PaCa-2, and (C) WiDr cells were infected with the adenovirus, Ad/EFp-BCD or Ad/5HREp-BCD, and treated with various concentrations of 5-FC under normoxic (open) or hypoxic (solid) .

.. Hypoxia-specific BCD expression after intratumoral Ad/5HREp-BCD injection We examined whether Ad/5HREp-BCD induces the expression of BCD in hypoxic regions of the tumour xenograft. The virus (1 �� 109pfu) was intratumorally injected into HeLa tumour xenografts, and the regions expressing BCD were compared to those stained with a marker of hypoxia, pimonidazole (Durand and Raleigh, 1998). The immunohistochemical analysis showed that the hypoxic cells stained with pimonidazole were located about 100��m from a tumour blood vessel, and a robust expression of BCD was also observed there (Figure 4A�CC). On the other hand, remarkable BCD expression was observed in well-oxygenated viable regions after intratumoral injection of Ad/EFp-BCD (Figure 4D and E).

These results suggest that the trans-gene expression of BCD in hypoxic tumour cells can be achieved by the intratumoral administration of the adenovirus Ad/5HREp-BCD. Figure 4 Immunohistochemical analysis of BCD expression in virus-injected tumour xenografts. The tumour xenograft of HeLa cells was intratumorally injected with Ad/5HREp-BCD (A�CC) or Ad/EFp-BCD (D and E). Serial sections of the xenograft … Improvement of radiotherapy by Ad/5HREp-BCD-mediated gene therapy The in vitro cell proliferation assay (Figure 3) and the immunohistochemical analysis (Figure 4) led us to expect a hypoxia-specific therapeutic effect of the Ad/5HREp-BCD, and 5-FC gene therapy. Actually, we confirmed an advantage of 5HREp concerning side effects on normal tissues. The Ad/5HREp-BCD/5-FC gene therapy caused no obvious side effects, while the Ad/EFp-BCD/5-FC gene therapy, despite the local administration, caused significant weight loss (Figure 5A) and severe diarrhea (data not shown). This result indicates that our system functioned, as we desired. Figure 5 Synergistic antitumour effect of a combination of gene therapy with Cilengitide IR treatment.