To determine whether the specific targeting of tumour hypoxia by the gene therapy strategy improves the efficacy of radiotherapy in a tumour xenograft, we performed growth delay assays. The gene therapy synergistically kept tumour growth suppressed in combination with the single (12.5Gy) and the fractionated (3Gy �� 5 fractions) radiotherapy. These results were consistent with the report that PF01367338 a combination of HRE-driven P450R expression and tirapazamine significantly increased the efficacy of radiotherapy in vivo (Cowen et al, 2004). The data together with ours definitely support that hypoxia-targeting gene therapy combined with radiotherapy is a promising approach to cancer treatment.

Although BCD expression from the 5HREp-BCD gene was not observed under normoxic conditions in the present Western blotting (Figures 1B and and2B),2B), the cells showed slight but clear sensitivity to a high concentration of 5-FC even under normoxic conditions (Figure 3). This sensitivity was observed regardless of infection with the adenovirus in vitro (Figure 3; compare MOI=0�C100 in each cell), indicating that an excess dose of 5-FC itself results in BCD-independent cytotoxicity. In our growth delay assays, a significant difference was not observed in TGDT between the IR group and the IR & 5-FC group (without adenovirus administration) (Figure 5B and C and Table 1), indicating that the dose of 5-FC was not excessive, or rather was moderate in our in vivo studies. In such an experimental setting, tumour growth in the Ad & 5-FC & IR group was significantly delayed compared to that in the IR group (Figure 5B and C and Table 1).

All of these results strongly suggest that the synergistic therapeutic effect of Ad & 5-FC & IR treatment was dependent on the expression of BCD and was caused by the conversion of 5-FC to cytotoxic 5-FU. Hypoxia-inducible factor-1 plays important roles in regulating tumour radiosensitivity, and therefore, it has been recognised as a potentially promising target for tumour radiosensitisation (Moeller et al, 2004; Moeller and Dewhirst, 2006). Because transcription from 5HREp mainly depends on HIF-1 activity, BCD expression from Ad/5HREp-BCD should be induced in the cells with increased HIF-1 activity. In this regard, the gene therapy should have targeted the tumour cells with increased HIF-1 activity and enhanced the therapeutic effect of radiotherapy. We previously used 5HREp to image hypoxic cells in tumour xenografts (Harada et al, 2005; Liu et al, 2005). AV-951 These studies were conducted using tumour xenografts, in which a hypoxia-responsive gene, such as the 5HREp-luciferase or the 5HREp�CGFP gene, had been previously set, but never using vectors responsible for the trans-gene delivery.