Department of Genetic Resources Technology, Kyushu University, Fukuoka, 812-8581, Japan.
Cytotechnology. 2001 Jul;36(1-3):137-44. doi: 10.1023/A:1014016315003.
Serum-free mouse embryo (SFME) cells were established by D. Barnes et al., and are known to be a neural stem cell line, which differentiate into astrocytes upon treatment with TGF-beta. Therefore, SFME cells is thought to be a model well suited to analyze the differentiation mechanism of neural stem cells. Until now, we have investigated the regulation mechanisms of telomerase activity and telomere length in human cancer and normal cells. Telomerase is the enzyme responsible for the synthesis and maintenance of telomere repeats located at chromosomal ends and is normally expressed in embryonic and germline cells, but not in most normal cells. Here, using SFME cells, we attempted to analyze the regulation mechanism of telomerase activity in neural stem cells and to detect a change upon differentiation into astrocytes. When SFME cells were cultured in the presence of TGF-beta, cells showed anelongated morphology and decreased its growth to 50% of control culture. Cells also expressed the glial fibrillary acidic protein (GFAP), a marker for astrocytes,indicating that TGF-beta induced differentiation in SFME cells from neural stem cells into astrocytes. At the same time,TGF-beta also inhibited telomerase activity and repressed the expression of the mouse telomerase reverse transcriptase(mTERT), demonstrating that SFME cells was vested with a finite replicative life span upon treatment with TGF-beta. To understand the mechanisms regulating mTERT levels during differentiation into astrocytes, we have estimated the expression level of c-myc, which is known to be a key molecule in activating the TERT promoter. As a result, TGF-beta-treated SFME cells were shown to repress the expression of c-myc. Furthermore, promoter analysis, using the 5'-region of the mTERT gene, which possess two E-box elements bound to c-Myc/Max, demonstrated that mTERT promoter activity greatly decreased in TGF-beta-treated SFME cells as compared to non-treated SFME cells. These suggest that c-myc might play a critical role in the expression of mTERT, and that down-regulation of c-myc dependent upon the astrocytic differentiation in SFME cells might cause the repression of mTERT in TGF-beta-treated SFME cells.
血清-free 小鼠胚胎 (SFME) 细胞是由 D. Barnes 等人建立的，已知是一种神经干细胞系，经 TGF-β处理后分化为星形胶质细胞。因此，SFME 细胞被认为是一种非常适合分析神经干细胞分化机制的模型。到目前为止，我们已经研究了端粒酶活性和端粒长度在人类癌症和正常细胞中的调节机制。端粒酶是负责合成和维持染色体末端端粒重复序列的酶，通常在胚胎和生殖细胞中表达，但在大多数正常细胞中不表达。在这里，我们使用 SFME 细胞试图分析神经干细胞中端粒酶活性的调节机制，并检测到分化为星形胶质细胞时的变化。当 SFME 细胞在 TGF-β存在下培养时，细胞呈现出伸长的形态，其生长速度降至对照培养的 50%。细胞还表达了神经胶质纤维酸性蛋白 (GFAP)，这是星形胶质细胞的标志物，表明 TGF-β诱导 SFME 细胞从神经干细胞分化为星形胶质细胞。同时，TGF-β还抑制端粒酶活性并抑制小鼠端粒酶逆转录酶 (mTERT) 的表达，表明 SFME 细胞在 TGF-β处理后具有有限的复制寿命。为了了解调节分化为星形胶质细胞过程中端粒酶水平的机制，我们估计了 c-myc 的表达水平，c-myc 是激活 TERT 启动子的关键分子。结果表明，TGF-β处理的 SFME 细胞抑制 c-myc 的表达。此外，使用 mTERT 基因的 5' 区进行启动子分析，该区域包含两个与 c-Myc/Max 结合的 E 盒元件，表明与未经 TGF-β处理的 SFME 细胞相比，TGF-β处理的 SFME 细胞中的 mTERT 启动子活性大大降低。这表明 c-myc 可能在 mTERT 的表达中起关键作用，并且 SFME 细胞中依赖于星形胶质细胞分化的 c-myc 下调可能导致 TGF-β处理的 SFME 细胞中 mTERT 的抑制。
