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，其表达被缺氧选择性激活，这是人类实体肿瘤的一个独特特征。为了获得更高的响应性，研究了HREs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启动子相似的缺氧反应性（超过500倍）。(2)利用缺氧诱导系统，制备了细菌硝基还原酶基因（NTR）表达载体。在稳定转染的人肿瘤细胞中，western blotting检测到缺氧诱导的NTR蛋白与前药敏感性增加相关。生长延迟试验对已确定的肿瘤异种移植物进行。在组成性表达NTR或具有缺氧诱导启动子的肿瘤中，腹腔注射前药均取得了显著的抗肿瘤效果。(3)采用改进的基因诱捕法检测低剂量电离辐射诱导的肿瘤细胞新基因。选择转染后的细胞，用电离辐射进行X-gal染色，分析报告基因的表达。通过对阳性克隆中指定基因的数据库比对，证实其中一个DNA片段与c-LAP基因的上游侧翼序列相同。我们生成了3.5 kb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2-5 Gy的辐照下对荧光素基因产生了强烈的诱导作用。缺失分析还显示NF-kappaB结合位点可能负责辐射介导的诱导。我们制备了表达Bax基因的辐射诱导基因治疗载体。转染后，观察到辐照对细胞杀伤作用的显著增强。综上所述，这些结果表明缺氧和辐射诱导的载体可能对肿瘤选择性基因治疗有用。

(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.