Takahashi K, Suzuki K
Department of Biochemistry, Kanagawa Cancer Center Research Institute, 54-2 Nakao-cho, Asahi-ku, Yokohama, 241, Japan.
Exp Cell Res. 1996 Jul 10;226(1):214-22. doi: 10.1006/excr.1996.0221.
Normal human breast epithelial (HBE) cells grown at logarithmic phase and those at the plateau phase were starved of EGF for 3 days then stimulated with EGF. HBE cells which were growth arrested at low density responded to EGF to initiate DNA synthesis 20 h later, whereas cells growth arrested at saturated density lost their responsiveness to EGF. Although the responsiveness to EGF between the two cultures at different densities was distinct, neither the number nor the affinity of EGF receptor (EGFR) in the two cultures significantly differed. In addition, the EGFR mRNA level was not affected by an increase in the cell density. A significant difference between the two cultures was the responsiveness of EGFR to EGF. The intrinsic tyrosine kinase activity and dimerization of the EGFR in cells at low density were induced by EGF, whereas those in cells at saturated density were not. Immunostaining revealed that the EGFR was localized only in the boundary region where adjacent cells are in close contact at low density but in the entire surrounding region of each cell at saturated density. The distribution of EGFR overlapped the region where the cell-cell adhesion protein, E-cadherin, is distributed. The E-cadherin mRNA levels in the two cultures were comparable; however, nearly fivefold more E-cadherin protein accumulated in cells at saturated than at low density. Incubation of cells at saturated density with an antibody to E-cadherin followed by adding EGF resulted in the stimulation of DNA synthesis, which was preceded by autophosphorylation but not dimerization of the EGFR. The results suggested that density-dependent inhibition of growth is achieved through prevention of the EGFR activation by extensive cell-cell adhesion that is mediated by excess E-cadherin.
处于对数生长期和平台期的正常人乳腺上皮（HBE）细胞，在无表皮生长因子（EGF）的条件下饥饿培养3天，然后用EGF刺激。在低密度下生长停滞的HBE细胞在20小时后对EGF作出反应并启动DNA合成，而在饱和密度下生长停滞的细胞则失去了对EGF的反应能力。尽管两种不同密度培养物对EGF的反应性不同，但两种培养物中EGF受体（EGFR）的数量和亲和力均无显著差异。此外，EGFR mRNA水平不受细胞密度增加的影响。两种培养物之间的显著差异在于EGFR对EGF的反应性。低密度细胞中EGFR的内在酪氨酸激酶活性和二聚化由EGF诱导，而饱和密度细胞中的则未被诱导。免疫染色显示，EGFR仅定位于低密度下相邻细胞紧密接触的边界区域，而在饱和密度下则定位于每个细胞的整个周边区域。EGFR的分布与细胞间粘附蛋白E-钙粘蛋白的分布区域重叠。两种培养物中E-钙粘蛋白的mRNA水平相当；然而，饱和密度细胞中积累的E-钙粘蛋白蛋白比低密度细胞中的多近五倍。用E-钙粘蛋白抗体处理饱和密度的细胞，然后添加EGF，可刺激DNA合成，这一过程先于EGFR的自磷酸化，但不先于其二聚化。结果表明，生长的密度依赖性抑制是通过由过量E-钙粘蛋白介导的广泛细胞间粘附来防止EGFR激活而实现的。
