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，是由APC功能丧失、β - catenin积累、转录因子激活下游靶基因c-Myc等一系列反应所组成的，深入参与了结直肠癌的发生。有可能通过控制这些wnt信号分子来抑制肿瘤的生长。为此，规划了两个策略。一是构建一些载体系统，能够降低结肠癌细胞中β -连环蛋白的水平。另一种是建立毒性基因治疗，连接向c-Myc上调的信号通路，表达一些毒性基因，如DT-A。策略1。- catenin下调载体的构建及其在基因治疗中的应用APC基因全长较大，不利于基因转移。另一方面，APC基因的中心三分之一部分（1260-2056 a.a.）对β - catenin的降解至关重要。这个较小的部分（被称为APC-Core）的功能类似于支架蛋白。APC-Core受多种启动子控制，包括CMV和MT启动子。这些载体导入结直肠癌细胞系后，具有一定的诱导细胞凋亡的潜力。然后，对这些载体的体内效用进行了检验。用裸鼠接种大肠癌DLD-1细胞株，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，腺孔法比电穿孔法更有效。从可治愈性的角度来看，即使采用腺瘤法，癌细胞也不能完全从宿主体内赶走。由于腺病毒的免疫原性，反复使用腺病毒被认为会降低其有效性。APC-Core具有一定的诱导结直肠癌细胞凋亡的潜力，但必须建立有效的基因转移到结直肠癌的方法。策略2。构建结直肠癌特异性毒性基因疗法利用c-Myc激活的信号来实现这一策略。c-Myc的5'启动子区与最小启动子和DT-A基因相连。这种调控并不严格，在结直肠癌细胞系中无法实现特异性毒性。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