Development of suicide bomb bectors and its effective transfer for cancer therapy.

自杀式炸弹袭击者的发展及其对癌症治疗的有效转移。

基本信息

批准号：
17591352
负责人：
YAMADA Osamu
金额：
$ 2.24万
依托单位：
Tokyo Women's Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17591352/
关键词：
cancer cell suicide bomb telomerase gene transfection

项目摘要

The promoter of telomerase is active in virtually all types of tumors but is silent in most adult somatic cells. We placed the suicide genes under the control of the telomerase promoter with the aim of restricting their expression to tumor cells. As the suicide genes, we used 1. the herpes simplex virus thymidine kinase gene which may confer ganciclovir sensitivity to all tumor cells and 2. bovine α1-3 galactosyltransferase (α1-3GT) cDNA which produces the αGal epitope. The carbohydrate epitope, αGal epitope, is known as a major xenoantigen and the epitope exists abundantly in non-primate mammals. Human and Old world monkeys have anti-αGal antibody, as a natural antibody. The transfection of the functional α1-3GT gene driven by telomerase promoter into human cancer cells may lead to their transformation, making them susceptible to lysis by natural antibodies.(1) We made the construct of the herpes simplex virus thymidine kinase gene under the control of the telomerase promoter with the … More aim of restrinting its expression to tumor cells. In transfecton experiments, the telomerase promoter driven tymidine kinase gene (hTERTp-TK) conferred ganciclovir sensitivity to leukemic cell line tested, whereas nomal somatic cells remained largely unaffected. Human hTERTp-TK positive K562 cells implanted in nude mice developed into tumors that could be eradicated by ganciclovir treatment.(2) The fragment encompassing the whole coding sequence of bovine α1-3GT cDNA including the tanslation start site was subcloned to PEGFP basic vector which had been inserted with telomerase promoter. Bovine α1-3GT cDNA under the control of telomerase promoter (hTERTp-α1-3GT) was electrophoretically transfected into the human leukemic cell lines. Stable transfomant was obtained by selection with G418 and, limiting dilution technique. The expression of the αGal epitope was confirmed by flow cytometry, using specific binding with IB4 lectin conjugated with fluorescein isothiocyanate. I have been examining whether the leukemic cells expressing the αGal epitope can be lysed by natural antibodies and this is true for another cancer cell lines. Less

端粒酶启动子在几乎所有类型的肿瘤中都有活性，但在大多数成年体细胞中是沉默的。我们将自杀基因置于端粒酶启动子的控制之下，目的是限制它们在肿瘤细胞中的表达。使用 1. 单纯疱疹病毒胸苷激酶基因，该基因可赋予更昔洛韦对所有肿瘤细胞的敏感性，以及 2. 牛 α1-3 半乳糖基转移酶 (α1-3GT) cDNA，其产生αGal表位，碳水化合物表位，αGal表位，被称为主要的异种抗原，并且该表位大量存在于非灵长类哺乳动物中，具有抗αGal抗体，作为天然抗体的转染。由端粒酶启动子驱动的α1-3GT基因进入人类癌细胞可能会导致其转化，使它们易于被天然抗体裂解。（1）我们构建了疱疹病毒单纯病毒胸苷激酶基因受端粒酶启动子控制，其目的是限制其在肿瘤细胞中的表达。在转染实验中，端粒酶启动子驱动的胸苷激酶基因 (hTERTp-TK) 赋予更昔洛韦对测试的白血病细胞系的敏感性。而植入裸鼠体内的人 hTERTp-TK 阳性 K562 细胞基本上不受影响，但会发展成肿瘤。 (2)将包含翻译起始位点的牛α1-3GT cDNA整个编码序列的片段亚克隆到已插入牛α1-3GT cDNA控制下的端粒酶启动子的PEGFP基本载体中。端粒酶启动子（hTERTp-α1-3GT）通过电泳转染到人白血病细胞系中。通过G418选择和有限稀释技术，使用与异硫氰酸荧光素缀合的IB4凝集素的特异性结合，通过流式细胞术确认αGal表位的表达。天然抗体对于另一种癌细胞系来说也是如此。