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Functional analysis of p53-target genes.

p53 靶基因的功能分析。

基本信息

批准号：
11138246
负责人：
TOKINO Takashi
金额：
$ 8万
依托单位：
Sapporo Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas (A)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至无数据
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11138246/
关键词：
tumor suppressor gene p53 target gene transcriptional activation apoptosis angiogenesis cell cycle 癌制御遺伝子

项目摘要

We isolated p53-binding sites by genetic approach in yeast and then identified novel p53-target genes. One of these, called BAl1 , which was expressed in brain and repressed in 8/9 glioblastomas, was further characterized to inhibit neovascularization in the rat cornea by basic FGF.To investigate the in vivo anti-angiogenic effect of BAl1 gene on a human glioblastoma cell line, p53-defective human glioblastoma cells U373MG were transduced with the BAl1 gene using a recombinant adenoviral vector. In vivo neovascularization assay of the BAl1-infected glioblastoma cells was then performed using skinfold chamber system transplanted in SCID mice and revealed that BAl1 transduction resulted in reduced neovascurization in SCID mice.Expression of the glycosyl-phosphatidylinositol-anchored molecule-like protein (GML) gene, another p53 target, correlates with the sensitivity of some cancer cell lines to anticancer drugs and ionizing radiation. To investigate the function of GML further, we introduced the GML cDNA into various cancer cell lines under control of the tetracycline-regulated system. When we introduced GML into human glioblastoma cell line T98G, which lacks wild-type p53 and expresses no endogenous GML, we observed significant growth suppression accompanied by G2/M arrest in two independent, stable cell lines. We confirmed introduction of apoptosis by fluorescence-activated cell sorting (FACS) analysis and nuclear staining. Our results indicated that GML could induce apoptosis of T98G without functional p53, and implied that GML plays a crucial role in the apoptosis pathway in some cancer cells.

我们在酵母中通过遗传学方法分离p53结合位点，然后鉴定新的p53靶基因。其中之一，被称为BAl 1，这是在大脑中表达和抑制在8/9胶质母细胞瘤，进一步的特点是抑制新生血管在大鼠角膜碱性FGF.To研究在体内的抗血管生成作用的BAl 1基因对人胶质母细胞瘤细胞系，p53缺陷的人胶质母细胞瘤细胞U373 MG使用重组腺病毒载体转导BAl 1基因。用皮褶腔系统对BAl 1感染的胶质母细胞瘤细胞进行了体内血管生成实验，结果显示BAl 1转导可减少SCID小鼠的血管生成，而另一个p53靶点糖基磷脂酰肌醇锚定分子样蛋白（GML）基因的表达与某些癌细胞系对抗癌药物和电离辐射的敏感性相关。为了进一步研究GML的功能，我们将GML cDNA导入四环素调控系统控制下的各种癌细胞系中。当我们将GML引入缺乏野生型p53且不表达内源性GML的人胶质母细胞瘤细胞系T98 G中时，我们在两个独立的稳定细胞系中观察到显著的生长抑制伴随G2/M停滞。我们通过荧光激活细胞分选（FACS）分析和核染色证实了凋亡的引入。我们的结果表明，GML可以诱导T98 G细胞凋亡，而不需要功能性p53，这意味着GML在某些肿瘤细胞的凋亡途径中起着至关重要的作用。