Establishment of a new strategy for colorectal cancer gene therapy by controlling Wnt-signal molecules

近年来的研究表明，Wnt信号通路的激活与结直肠癌的发生密切相关，该信号通路包括APC功能丧失、β-Catenin的积累以及转录因子激活下游靶基因c-Myc等一系列反应。通过调控这些Wnt信号分子有可能抑制肿瘤的生长。一是构建载体系统，使β-Catenin在结肠癌细胞中的表达水平降低。另一种是建立毒性基因治疗，即通过上调c-Myc的信号通路来表达一些毒性基因，如DT-A。β-Catenin下调表达载体的构建及其在基因治疗中的应用APC基因全长很大，不利于基因转移。而APC基因的中部1/3区段（1260-2056 a.a.）我 ...更多信息 对β-连环蛋白降解至关重要。这个较小的部分（它被称为APC核心）的功能类似于支架蛋白。APC-核心置于包括CMV和MT启动子的各种启动子的控制下。这些载体在导入大肠癌细胞系中时具有一定的诱导凋亡的潜力。然后，这些载体的体内效用进行了检查。将大肠癌细胞系DLD-1接种于裸鼠体内，分析其体内基因转移效率。采用腺病毒基因转移和电穿孔。结果表明，腺病毒法比电穿孔法更有效。从可治愈性的观点来看，即使通过腺病毒方法，也不能将癌细胞完全从宿主中赶走。腺病毒的重复使用被认为主要由于其免疫原性而降低其有效性。APC-Core具有一定的诱导大肠癌细胞凋亡的潜力，但目前尚需建立有效的基因转移方法。大肠癌特异性毒性基因治疗的构建利用c-Myc激活信号进行该策略。将c-Myc的5'启动子区与最小启动子和DT-A基因连接。这种调控并不严密，在结直肠癌细胞系中不能实现特异性毒性。少

Recent studies indicate that the activation of Wnt-signal pathway, which is composed of the sequential reactions including loss of APC function, resulting accumulation of beta-Catenin and the activation of the down-stream target gene such as c-Myc via transcriptional factors, is deeply involved in the colorectal carcinogenesis. It might be possible to suppress the tumor growth by controlling theses Wnt-signal molecules.For this purpose, two strategies are planned. One is to construct some vector systems, which enable to decrease the level of beta-Catenin in colon cancer cells. The other is to set up the toxic gene therapy, where the up-regulated signal pathway toward c-Myc is connected to express some toxic genes such as DT-A.Strategy 1. Construction of the beta-Catenin down-regulation vector and its application of gene therapyThe entire length of APC gene is very huge and disadvantageous for gene transfer. On the other hand, the central one third portion of APC gene (1260-2056 a.a.) i … More s essential for beta-Catenin degradation. This smaller portion (it is called, APC-Core) functions like a scaffold protein. The APC-Core was put under the control of the various promoters including CMV and MT promoter. These vectors have some potential to induce the apoptosis, when introduced into the colorectal cancer cell lines. Then, in vivo utility of theses vectors were examined. One colorectal cancer cell line, DLD-1 was inoculated in the nude mice to analyze the in vivo gene transfer efficiency. Adenoviral gene transfer and electroporation were adapted. It was indicated that the adenovial method was much more efficient than the electroporation. In the view point of the curability, even by the adenovial method, cancer cell could not be driven away completely from the host. Repetitious use of the adenovirus is thought to diminish its effectiveness mainly due to its immunogenisity. The APC-Core has some potential to induce the apoptosis in the colorectal cancer, but some efficient gene transfer methods into colorectal cancer must be established.Strategy 2. Construction of the colorectal cancer specific toxic gene therapyThe signal to c-Myc activation was utilized to this strategy. The 5' promoter region of c-Myc was connected to the minimum promoter and DT-A gene. This regulation is not so tight and specific toxicicity in the colorectal cancer cell lines could not be achieved. Less