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Functional analysis of p53-induced apoptosis in glioma cells

p53诱导胶质瘤细胞凋亡的功能分析

基本信息

批准号：
08457369
负责人：
SAYA Hideyuki
金额：
$ 4.61万
依托单位：
Kumamoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08457369/
关键词：
glioma apoptosis p53 gene bcl-2 gene NDP kinase Bcl-2遺伝子発現クローニング EBNA-1 アデノウイルス

项目摘要

It has been known that p53 protein plays an important role in not only cell cycle regulation but also apoptosis. In the present study, we attempted to elucidate molecular mechanisms which negatively regulate p53-induced apoptotic signal. During the period of April, 1991 through March, 1992, we identified the following findings :1)To investigate the effects of the expression of Bcl-2 protein in cancer cells on the apoptosis induced by adenoviral-medicated p53 gene transfer, we transfected the bcl-2 gene into KoTCC-1 cell that does not express the Bcl-2 protein. The Bcl-2-transfected cells exhibited significantly higher resistance to p53-induced apoptosis than did either the parental cell or the vector-only transfected cell both in vitro and in vivo.2)A Hela cDNA library constructed in an episomal plasmid was introduced into EBNA-1 stable expression cells for complementation of the adenoviral p53-induced apoptosis. The cDNA insert of the plasmid which was recovered from the apoptosis-resistant cells encoded a 194 amino acid polypeptide that has significant homology with E.coli nucleoside diphoshate kinase (NDPK). The NDPK is known to catalyze a transfer of the terminal phosphate of triphosphate nucleosides to diphosphate nucleosides by a ping-pong mechanism which regulates balance of nucleoside pool, that may inhibit apoptosis induced by p53 expression.

p53蛋白不仅在细胞周期调控中起重要作用，而且在细胞凋亡中起重要作用。在本研究中，我们试图阐明负调控p53诱导的凋亡信号的分子机制。在1991年4月至1992年3月期间，我们发现了以下发现：1)为了研究肿瘤细胞中Bcl-2蛋白表达对腺病毒药物介导的p53基因转移诱导的凋亡的影响，我们将Bcl-2基因转染到不表达Bcl-2蛋白的KoTCC-1细胞中。在体外和体内实验中，转染bcl -2的细胞对p53诱导的细胞凋亡的抗性明显高于亲本细胞或仅转染载体的细胞。2)将在episomal质粒上构建的Hela cDNA文库导入EBNA-1稳定表达细胞，以补充腺病毒p53诱导的细胞凋亡。从抗凋亡细胞中提取的质粒cDNA插入片段编码了一个194个氨基酸的多肽，该多肽与大肠杆菌核苷二磷酸激酶（NDPK）具有显著的同源性。NDPK通过乒乓机制催化三磷酸核苷末端磷酸向二磷酸核苷的转移，调节核苷池的平衡，从而抑制p53表达诱导的细胞凋亡。