Functional analysis of p53-target genes.

我们通过遗传方法分离了酵母中的P53结合位点，并鉴定了新的P53靶基因。其中一种名为bal1的基因在脑内表达，在8/9的胶质母细胞瘤中被抑制，并被基础FGF进一步鉴定为抑制大鼠角膜新生血管的作用。为了研究bal1基因对人胶质母细胞瘤细胞系的体内抗血管生成作用，用重组腺病毒载体将bal1基因导入p53基因缺陷的人胶质母细胞瘤细胞U373 MG。用皮肤折叠小室系统移植到SCID小鼠体内，对感染了Bal1的胶质母细胞瘤细胞进行了体内新生血管检测，结果表明，Bal1转导导致SCID小鼠新生血管减少。另一个P53靶点-糖基磷脂酰肌醇锚定分子样蛋白(GML)基因的表达与一些癌细胞对抗癌药物和电离辐射的敏感性有关。为了进一步研究GML的功能，我们在四环素调控系统的控制下，将GML基因导入多种肿瘤细胞系。当我们将GML导入缺乏野生型p53和不表达内源性GML的人胶质母细胞瘤细胞系T98G中时，我们在两个独立的、稳定的细胞系中观察到显著的生长抑制和G2/M期停滞。我们通过荧光激活细胞分类(FACS)分析和核染色证实了细胞凋亡的引入。我们的结果表明，GML可以诱导T98G细胞的凋亡，而不是功能性的P53，提示GML在某些癌细胞的凋亡途径中起着重要的作用。

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.