Takasaki Ichiro, Takarada Satoko, Fukuchi Mamoru, Yasuda Makoto, Tsuda Masaaki, Tabuchi Yoshiaki
Division of Molecular Genetics Research, Life Science Research Center, University of Toyama, Sugitani 2630, Toyama 930-0194, Japan.
J Cell Biochem. 2007 Dec 15;102(6):1472-85. doi: 10.1002/jcb.21369.
The purpose of the present study is to establish and characterize a conditionally immortalized astrocyte cell line and to clarify the genetic networks responsible for the cell growth arrest and differentiation. A conditionally immortalized astrocyte cell line, RCG-12, was established by infecting primary cultured rat cortical glia cells with a temperature-sensitive simian virus 40 large T-antigen. At a permissive temperature of 33 degrees C, the large T-antigen was expressed and cells grew continuously. On the other hand, the down-regulation of T-antigen at a non-permissive temperature of 39 degrees C led to growth arrest and differentiation. The cells expressed astrocyte-expressed genes such as glial fibrillary acidic protein. Interestingly, the differentiated condition induced by the non-permissive temperature significantly elevated the expression levels of several astrocyte-expressed genes. To identify the detailed mechanisms by which non-permissive temperature-induced cell growth arrest and differentiation, we performed high-density oligonucleotide microarray analysis and found that 556 out of 15,923 probe sets were differentially expressed 2.0-fold. A computational gene network analysis revealed that a genetic network containing up-regulated genes such as RB, NOTCH1, and CDKN1A was associated with the cellular growth and proliferation, and that a genetic network containing down-regulated genes such as MYC, CCNB1, and IGF1 was associated with the cell cycle. The established cell line RCG-12 retains some characteristics of astrocytes and should provide an excellent model for studies of astrocyte biology. The present results will also provide a basis for understanding the detailed molecular mechanisms of the growth arrest and differentiation of astrocytes.
本研究的目的是建立并鉴定一种条件永生化星形胶质细胞系，并阐明负责细胞生长停滞和分化的基因网络。通过用温度敏感型猿猴病毒40大T抗原感染原代培养的大鼠皮质神经胶质细胞，建立了一种条件永生化星形胶质细胞系RCG-12。在33℃的允许温度下，大T抗原表达，细胞持续生长。另一方面，在39℃的非允许温度下T抗原的下调导致生长停滞和分化。这些细胞表达星形胶质细胞表达的基因，如胶质纤维酸性蛋白。有趣的是，由非允许温度诱导的分化状态显著提高了几种星形胶质细胞表达基因的表达水平。为了确定非允许温度诱导细胞生长停滞和分化的详细机制，我们进行了高密度寡核苷酸微阵列分析，发现15923个探针组中有556个差异表达2.0倍。计算基因网络分析显示，包含上调基因如RB、NOTCH1和CDKN1A的基因网络与细胞生长和增殖相关，而包含下调基因如MYC、CCNB1和IGF1的基因网络与细胞周期相关。建立的细胞系RCG-12保留了星形胶质细胞的一些特征，应为星形胶质细胞生物学研究提供一个优秀的模型。本研究结果也将为理解星形胶质细胞生长停滞和分化的详细分子机制提供基础。
