Clonig and Characterization of a glioma-specific gene

目的：成纤维细胞生长因子-2 （FGF-2）在星形细胞瘤血管生成和肿瘤生长中起重要作用。这项研究的目的是阐明控制FGF-2基因表达的机制。结果：为了寻找调控FGF-2表达的机制，我们重点研究了FGF-2启动子转录起始位点周围的串联重复序列（gccgaac）。以该序列为诱饵，进行酵母- 1杂交筛选，克隆出一个能结合FGF-2启动子区域的24kDa蛋白（p24）。为了确认p24与gccgaac序列的亲和性，我们进行了凝胶迁移位移试验。P24对gccgaac序列具有高度特异性亲和力。逆转录酶(RT)-PCR显示p24在U87MG和U251MG胶质瘤细胞系中均不表达。免疫细胞化学表明，在四环素应答启动子（四环素-on系统）的控制下，p24的表达主要定位于U87MG胶质瘤细胞的细胞核中。荧光素酶检测显示，p24对U87MG细胞系FGF-2启动子活性的抑制作用为10倍，对U251MG细胞系FGF-2启动子活性的抑制作用为5倍（p<0.05）。使用Light Cycler （Roche, Go.）进行定量RT-PCR。有限公司)也发现p24抑制内源性FGF-2基因的表达约10倍（p=0.0010）。结论：我们的数据表明p24作为转录因子抑制胶质瘤细胞中ofFGF-2的基因表达。正在使用手术标本进一步研究p24在体内的意义。

Object : Fibroblast growth factor-2 (FGF-2) is know to play an important role inangiogenesis and in tumor growth of astrocytomas. The goal of the study is theelucidation of a mechanism that controls FGF-2 gene expression.Results : In search of the mechanism that regulates FGF-2 expression, we have focused on a tandem repeat sequence (gccgaac) around the transcription initiation site of the FGF-2 promoter. Using this sequence as bait, we conducted yeast-one-hybrid screening and cloned a 24kDa protein (p24) that could bind the promoter region of FGF-2. To confirm the affinity of p24 to the gccgaac sequence, we performed gel mobility shift assay. P24 shows highly specific affinity to gccgaac sequence. Reverse transcriptase (RT)-PCR showed that p24is expressed neither in U87MG nor in U251MG glioma cell lines. Immunocytochemistry shows that p24 expressing under the control oftetracycline-responsive promoter (tetracyclme-on system) is localized mainly in the nucleus of U87MG glioma cells. Luciferase assay revealed that p24 repressed the FGF-2 promoter activity about 10-fold in the U87MG cell line and about five fold in the U251MG cell line (p<0.05). Quantitive RT-PCR using Light Cycler (Roche, Go. Ltd.) also revealed that p24 repressed the endogenous expression of FGF-2 gene about ten fold (p=0.0010).Conclusion : Our data show that p24 represses the gene expression ofFGF-2 in glioma cells as a transcription factor. Further investigation of the significance of p24 in vivo is going on using surgical specimens.