Nove| strategies for cancer treatment with tumor-specific gene therapy and radiotherapy

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(1) We are developing new gene therapy vectors whose expression is selectively activated by hypoxia, a unique feature of human solid tumors. In an attempt to achieve higher responsiveness, various combinations of HREs and promoters were examined. We found the combination of 5XHRE and a CMV minimal promoter was exhibited hypoxia responsiveness (over 500-fold) to the similar level to the intact CMV promoter.(2) Using hypoxia-inducible system, we generated vectors expressing a bacterial nitroreductase gene (NTR). In stable transfectants of human tumor cells, hypoxic induction of NTR protein detected by western blotting correlated with increased sensitivity to the prodrug. Growth delay assays were performed with established tumor xenografts. Significant antitumor effects were achieved with i.p. injections of the prodrug both in tumors that express NTR constitutively or with a hypoxia inducible promoter.(3) A modified gene-trap method has been used to detect novel genes induced by-low dose ionizing radiation in human tumor cells. Transfected cells were selected and then we performed X-gal staining to analyze reporter gene expression using ionizing radiation. On database comparison to specify genes in the positive clones, one of the recovered DNA fragments was, confirmed to be identical to the upstream flanking sequences of c-LAP gene. We generated 3.5 kb fragment of c-LAP promoter and constructed luciferase expression vectors. As, the results, these vectors produced a robust induction of the luciferse gene by 2-5 Gy of irradiation. Deletion analysis also revealed that NF-kappaB binding sites could be responsible for the radiation-mediated induction. We generated radiation-inducible gene therapy vector expressing Bax gene. After transfection, a significant augmentation of cell killing effects was observed inresponse to irradiation.Taken together, these results demonstrate that both hypoxia- and radiation-inducible vectors may be useful for tumor selective gene therapy.