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

(1)我们正在开发新的基因治疗载体，其表达可被缺氧选择性激活，这是人类实体瘤的一个独特特征。为了获得更高的响应性，研究了HRE和启动子的各种组合。我们发现，5XHRE和CMV最小启动子的组合表现出与完整CMV启动子相似的低氧响应性(超过500倍)。(2)利用低氧诱导系统，我们构建了细菌硝基还原酶基因(NTR)的表达载体。在稳定的人类肿瘤细胞转染体中，Western blotting检测到低氧诱导NTR蛋白与增加对前药的敏感性相关。对已建立的肿瘤异种移植瘤进行生长延迟分析。腹腔注射有显著的抗肿瘤作用。将该前药注射到组成性表达NTR的肿瘤中或带有缺氧诱导启动子的肿瘤中。(3)改进的基因陷阱方法已用于检测低剂量电离辐射诱导的人肿瘤细胞中的新基因。选择转基因细胞进行X-Gal染色，利用电离辐射分析报告基因的表达。对阳性克隆中的特定基因进行数据库比对，证实其中一个DNA片段与c-LAP基因上游侧翼序列完全一致。我们克隆了3.5kb的c-LAP启动子片段，构建了荧光素酶表达载体。结果表明，这些载体在2-5Gy射线照射下对荧光素基因有较强的诱导作用。缺失分析还表明，核因子-kappaB结合位点可能与辐射诱导有关。我们构建了表达Bax基因的辐射诱导基因治疗载体。结果表明，低氧载体和辐射诱导载体均可用于肿瘤的选择性基因治疗。

(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.