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Analysis of molecular mechanism for modification of p53 tumor suppressor protein by anti-cancer drugs

抗癌药物修饰p53抑癌蛋白的分子机制分析

基本信息

批准号：
18580289
负责人：
ARAI Katsuhiko
金额：
$ 2.57万
依托单位：
Tokyo University of Agriculture and Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18580289/
关键词：
tumor suppressor gene p53 microtubule tubulin cell culture anti-cancer drug vinca alkaloid vincristine チュブリン

项目摘要

The aim of this research project is to obtain the information about the effects of anti-cancer drugs, especially vinca alkaloid, which play as a inhibitor of polymerization of microtubules, on the function of p53 superfamily. Head investigator, K. Arai found that β-tubulin isotypes transformed during establishment of drug-resistance for vinca alkaloid in mouse melanoma cell line, B16F10. Normally, expression of-tubulin expression was suppressed by p53 protein and decrease of DNA binding activity of Sp1 and p53 was induced by addition of vinca alkaloid to the culture medium. In 2006, to examine whether vinca alkaloid affect interaction between p53 superfamily and Sp1, in vitro translation system for p53 superfamily and Sp1 was established. Briefly, full length cDNAs corresponding to transactivating forms for p63, p73 and Sp1 in addition to wild-type and mutated p53 were amplified, then subcloned into pcDNA3.1 or pcDNA3.1/V5-His and used for in vitro transcription/translation. These enable to analyze interaction between p53 superfamily and Sp1 and their kinetics. In 2007, forced expression of p53 superfamily genes in p53-deficient mouse macrophage cell line was designed. Furthermore, mRNA distribution of p53 superfamily in the lung was examined by in situ hybridization and ribonuclease protection assay. As a result, mRNA corresponding to transactivating form of p73 was expressed in bronchial and type II alveolar epithelium. These findings indicated that p73 participate lung carcinogenesis.

本研究的目的是获得抗癌药物，特别是长春花生物碱，作为微管聚合的抑制剂，对p53超家族功能的影响的信息。首席调查员，K。Arai发现在小鼠黑色素瘤细胞系B16 F10中，β-微管蛋白同种型在对长春花生物碱的耐药性建立期间发生转化。正常情况下，微管蛋白的表达被抑制p53蛋白和减少的DNA结合活性的Sp1和p53的诱导通过添加长春花生物碱的培养基。2006年，为了研究长春花生物碱是否影响p53超家族与Sp1的相互作用，建立了p53超家族与Sp1的体外翻译系统。简言之，扩增对应于p63、p73和Sp1反式激活形式以及野生型和突变型p53的全长cDNA，然后亚克隆到pcDNA3.1或pcDNA3.1/V5-His中，并用于体外转录/翻译。这使得能够分析p53超家族和Sp1之间的相互作用及其动力学。2007年，设计了p53超家族基因在p53缺陷小鼠巨噬细胞系中的强制表达。用原位杂交和核糖核酸酶保护试验检测肺组织中p53超家族mRNA的分布。其结果是，对应于反式激活形式的p73的mRNA表达在支气管和II型肺泡上皮细胞。提示p73参与了肺癌的发生。