Clonig and Characterization of a glioma-specific gene

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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.