Cooperative study on molecular mechanism of GFAP expression.

Sueoka教授（本项目的合作研究者）建立了乙基亚硝基脲诱导的大鼠外周神经瘤RT 4的克隆细胞系。RT 4细胞系家族具有干细胞类型RT 4-AC，其中子细胞自发且不可逆地分化成三种衍生细胞类型之一：神经胶质细胞类型4 T4-P和两种神经元类型RT 4-E和RT 4-B。本合作研究的目的是：1）将RT 4家族的细胞系转移到立陶宛医科大学的本实验室，并确认这些细胞中标志蛋白的表达水平，2）分析使用这些细胞系调节GFAP表达的分子基础。本研究取得了以下结果：1）在转基因的4个细胞系中均观察到了稳定的标记蛋白表达模式。2）GFAP的表达受一个sis元件GDR 1的负调控。使用GDR 1序列，我们在非胶质细胞RT 4-E和肝癌HTC中检测到与该序列特异性结合的蛋白质。3）利用cDNA消减法从cDNA文库中特异性地分离出多种转录本。从RT 4-AG减去RT 4-D文库中鉴定GCM和DNMT 1

Clonal cell lines from an ethylnitrosourea-induced rat peripheral neurotumor, RT4, were established by Prof. Sueoka (cooperative investigator in this project). The RT4 cell line family has a stem cell type, RT4-AC, where daughter cells spontaneously and irreversibly differentiate into one of three derivative cell types: a glial cell type, 4T4-P, and two neuronal types, RT4-E and RT4-B. The aims of this cooperative study were: 1) to transfer cell lines of the RT4 family to this laboratory of Fukushinia1 Medical University and confirm expression levels of marker proteins in these cells, 2) to analyze molecular basis on regulation of GFAP expression using these cell lines. Following results were obtained from this research project.1) Stable expression pattern of marker proteins was observed in all of four cell lines transferred. Scince this pattern was similar to that reported previously.2) The expression of GFAP was negatively regulated by a sis-element, GDR1. Using GDR1 seuence, we detected proteins specifically bound to this sequence in non-glial RT4-E and hepatoma HTC. Identification of the proteins has been still on going.3) Using a cDNA subtraction method, kinds of transcripts were specifically isolated from the cDNA libraries. GCM and DNMT1 were identified from the RT4-AG minus RT4-D library