Hamanaka R, Ono M, Kuratomi Y, Mizoguchi H, Hirai R, Kohno K, Kuwano M
Department of Biochemistry, Oita Medical School, Japan.
Exp Cell Res. 1990 Jan;186(1):83-9. doi: 10.1016/0014-4827(90)90213-t.
Anchorage-independent growth in soft agar of normal rat kidney (NRK) fibroblasts depends on both transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF) (or TGF-alpha). We have isolated two EGF-nonresponsive cell lines, N-3 and N-9, from chemically mutagenized NRK cells, after selection of mitogen-specific nonproliferative variants in the presence of EGF and colchicine. Saturation binding kinetics with 125I-EGF showed one-half or fewer EGF receptors in N-3 and N-9 than in their parental NRK. Cellular uptake of 2-deoxy-D-glucose was enhanced in all NRK, N-3, and N-9 cell lines by TGF-beta treatment, whereas treatment with EGF significantly enhanced the cellular uptake of the glucose analog in NRK cells, but not in N-3 and N-9 cells. DNA synthesis of NRK during the quiescent state, but not that of N-3 and N-9, was stimulated by EGF. Anchorage-independent growth of N-9 could not be observed even in the presence of both EGF and TGF-beta, whereas that of N-3 was significantly enhanced by TGF-beta alone. EGF stimulated phosphorylation of a membrane protein with molecular size 170 kDa of NRK, but not of N-3, when immunoprecipitates reacting with anti-phosphotyrosine antibody were analyzed. Exposure of NRK cells to EGF increased cellular levels of TGF-beta mRNA, but there appeared little expression of TGF-beta mRNA in N-3 and N-9 cells. Exposure of N-3 cells to EGF or TGF-beta enhanced the secretion of EGF into culture medium, but exposure of NRK or N-9 cells did not. Altered response to EGF of N-3 or N-9 might be related to their aberrant growth behaviors.
正常大鼠肾（NRK）成纤维细胞在软琼脂中不依赖贴壁生长依赖于转化生长因子-β（TGF-β）和表皮生长因子（EGF）（或TGF-α）。我们从经化学诱变的NRK细胞中分离出了两种对EGF无反应的细胞系N-3和N-9，这是在EGF和秋水仙碱存在的情况下选择有丝分裂原特异性非增殖变体后得到的。用¹²⁵I-EGF进行的饱和结合动力学显示，N-3和N-9中的EGF受体数量比其亲代NRK细胞减少了一半或更少。TGF-β处理可增强所有NRK、N-3和N-9细胞系对2-脱氧-D-葡萄糖的细胞摄取，而EGF处理可显著增强NRK细胞对葡萄糖类似物的细胞摄取，但对N-3和N-9细胞无此作用。静止状态下NRK细胞的DNA合成受EGF刺激，但N-3和N-9细胞不受影响。即使同时存在EGF和TGF-β，也未观察到N-9细胞的不依赖贴壁生长，而单独的TGF-β可显著增强N-3细胞的不依赖贴壁生长。当分析与抗磷酸酪氨酸抗体反应的免疫沉淀物时，EGF刺激了NRK细胞中分子大小为170 kDa的膜蛋白磷酸化，但对N-3细胞无此作用。NRK细胞暴露于EGF会增加细胞内TGF-β mRNA水平，但N-3和N-9细胞中几乎没有TGF-β mRNA表达。N-3细胞暴露于EGF或TGF-β会增强其向培养基中分泌EGF，但NRK或N-9细胞暴露后则不会。N-3或N-9细胞对EGF反应的改变可能与其异常的生长行为有关。
