Selective elimination mechanism of extrachromosomal autonomously replicating genetic element mediated by the micronucleation

微核介导的染色体外自主复制遗传元件的选择性消除机制

基本信息

批准号：
11440220
负责人：
SHIMIZU Noriaki
金额：
$ 8.58万
依托单位：
HIROSHIMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11440220/
关键词：
Extrachromosomal DNA Gene amplification Double Minutes Homogeneously Staining Region DNA replication Plasmid Gene Delivery Micronucleus Homogeneously Staining Region extrachromosomal DNA Gene Amplification Mitosis plasmid episome

项目摘要

Amplification of oncogene or drug resistance gene plays a pivotal role in human oncogenesis. The amplified genes most likely reside on extrachromosomal autonomously replicating double minute chromatin (DMs). We previously found that the tumor cells revert their malignant phenotype and differentiate if the DMs were eliminated from the cells. The elimination was mediated by the selective incorporation of DMs into the cytoplasmic micronuclei. In this study, we uncovered the following issues. 1) We clarified the question why DMs were incorporated into micronuclei, by examining the intracellular behavior of DMs during the cell cycle progression. 2) We found that the intranuclear motion of DMs was coupled to their replication, by examining the replication timing of DMs. 3) We obtained the results showing that the DMs in the micronuclei were eliminated from the cells by the extrusion of micronuclei from the cells. Furthermore, we tried to extend the understanding on the behavior of DMs to the wide spectrum of extrachromosomal genetic elements. Therefore, we introduced plasmids having various cis-structure to cancer cell, and examined the 4) short-term, or 5) long-term behavior. The former study revealed that the closed circular plasmids were more rapidly eliminated from the cells than the linear molecules, and that the elimination might be mediated by the p53-dependent active mechanism. The latter study revealed that, if the introduced plasmid could autonomously be replicated, the plasmids generated the DMs or chromosomal HSR. The experimental system will be useful for the evaluation of the autonomous replication driven by the mammalian replication origin. The system also will be an excellent model system to uncover the gene amplification phenomenon seen in cancer cells. Furthermore, the system will be utilized to produce valuable proteins by the amplification of the corresponding genes.

癌基因或耐药性基因的扩增在人肿瘤发生中起关键作用。放大的基因最有可能存在于外染色体上自主复制双分钟染色质（DMS）。我们先前发现，肿瘤细胞会恢复其恶性表型，并区分是否从细胞中消除DMS。消除是通过选择性掺入DMS中的细胞质微核中介导的。在这项研究中，我们发现了以下问题。 1）我们通过检查细胞周期进程中DMS的细胞内行为，阐明了为什么将DMS纳入微核中的问题。 2）我们发现，通过检查DMS的复制时间，DMS的核内运动与它们的复制结合。 3）我们获得的结果表明，通过从细胞中挤出微核中，微核中的DMS从细胞中消除了。此外，我们试图将对DM的行为的理解扩展到广泛的肉体外遗传元素。因此，我们引入了具有各种顺式结构的质粒到癌细胞，并检查了4）短期，或5）长期行为。前研究表明，与线性分子相比，闭合的圆形质粒更快地消除了细胞，并且消除可能是由p53依赖性的活性机理介导的。后一项研究表明，如果引入的质粒可以自主复制，则质粒会产生DMS或染色体HSR。实验系统将有助于评估由哺乳动物复制起源驱动的自主复制。该系统还将是一个出色的模型系统，可揭示癌细胞中看到的基因扩增现象。此外，该系统将通过相应基因的扩增来产生有价值的蛋白质。