Cooperative study on molecular mechanism of GFAP expression.

乙基亚硝脲诱导的大鼠周围神经肿瘤RT4克隆细胞系由Sueoka教授(本项目的合作研究员)建立。RT4细胞系家族有一种干细胞类型RT4-AC，其中子代细胞自发且不可逆地分化为三种衍生细胞类型之一：神经胶质细胞类型4T4-P和两种神经元类型RT4-E和RT4-B。这项合作研究的目的是：1)将RT4家族的细胞系转移到福岛医科大学的这个实验室，并确定这些细胞中标志蛋白的表达水平；2)利用这些细胞系来分析调控GFAP表达的分子基础。本研究获得了以下结果：1)在所转移的四种细胞系中都观察到了稳定的标记蛋白表达模式。由于这一模式与以前报道的相似。2)GFAP的表达受SIS元件GDR1的负调控。利用GDR1序列，我们在非胶质细胞RT4-E和肝癌HTC中检测到与该序列特异结合的蛋白质。对蛋白质的鉴定仍在进行中。3)采用cDNA消减的方法，从cDNA文库中特异性地分离出各种转录本。GCM和DNMT1是从RT4-AG减去RT4-D文库中鉴定的

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