Gardner D P, Shimizu N
Department of Molecular and Cellular Biology, University of Arizona, Tucson 85721.
J Cell Physiol. 1994 Feb;158(2):245-55. doi: 10.1002/jcp.1041580206.
The biological activity of epidermal growth factor (EGF) is mediated through the intrinsic tyrosine kinase activity of the EGF receptor (EGFR). In numerous cell types, binding of EGF to the EGFR stimulates the tyrosine kinase activity of the receptor eventually leading to cell proliferation. In tumor-derived cell lines, which overexpress the EGFR, however, growth inhibition is often seen in response to EGF. The mechanism for growth inhibition is unclear. To study the relationship between growth inhibition and EGFR kinase activity, we have used a cell line (PC-10) derived from a human squamous cell carcinoma that overexpresses EGFR. When exposed to 25 ng/ml EGF at low cell densities (1,300 cells/cm2), PC-10 cells exhibit cell death. In contrast, if EGF is added to high density cultures, no EGF mediated cell death is seen. When PC-10 cells were maintained at confluency in the presence of 25 ng/ml EGF for a period of 1 month, they were subsequently found competent to proliferate at low density in the presence of EGF. We designate these cells APC-10. The APC-10 cells exhibited a unique response to EGF, and no concentration of EGF tested could produce cell death. By 125I-EGF binding analysis and [35S]methionine labeling of EGFR, it was found that the total number of EGFR on the cell surface of APC-10 was not decreased relative to PC-10. No difference between PC-10 and APC-10 was seen in EGF binding affinity to the EGFR. Significantly, EGF stimulated autophosphorylation of the EGFR of APC-10 was 8-10-fold lower than that of PC-10. This reduced kinase activity was also seen in vitro in membrane preparations for EGFR autophosphorylation as well as phosphorylation of an exogenously added substrate. No difference between PC-10 and APC-10 in the overall pattern of EGFR phosphorylation in the presence or absence of EGF was detectable. However, the serine and threonine phosphorylation of the EGFR of APC-10 cells was consistently 2-3-fold lower than that seen in PC-10 cells. These results suggest a novel mechanism for EGFR overexpressing cells to survive EGF exposure, one that involves an attenuation of the tyrosine kinase activity of the EGFR in the absence of a change in receptor levels or receptor affinity.
表皮生长因子（EGF）的生物活性是通过EGF受体（EGFR）的内在酪氨酸激酶活性介导的。在众多细胞类型中，EGF与EGFR的结合会刺激受体的酪氨酸激酶活性，最终导致细胞增殖。然而，在过表达EGFR的肿瘤衍生细胞系中，常常会观察到EGF导致的生长抑制。生长抑制的机制尚不清楚。为了研究生长抑制与EGFR激酶活性之间的关系，我们使用了一种源自人鳞状细胞癌且过表达EGFR的细胞系（PC-10）。当在低细胞密度（1300个细胞/cm²）下暴露于25 ng/ml EGF时，PC-10细胞会出现细胞死亡。相反，如果将EGF添加到高密度培养物中，则看不到EGF介导的细胞死亡。当PC-10细胞在25 ng/ml EGF存在下保持汇合状态1个月后，随后发现它们能够在EGF存在下以低密度增殖。我们将这些细胞命名为APC-10。APC-10细胞对EGF表现出独特的反应，所测试的任何EGF浓度都不会导致细胞死亡。通过¹²⁵I-EGF结合分析和EGFR的[³⁵S]甲硫氨酸标记发现，相对于PC-10，APC-10细胞表面EGFR的总数并未减少。在EGF与EGFR的结合亲和力方面，PC-10和APC-10之间没有差异。值得注意的是，EGF刺激的APC-10细胞EGFR自磷酸化比PC-10细胞低8至10倍。在用于EGFR自磷酸化以及外源添加底物磷酸化的膜制剂中，体外也观察到了这种降低了的激酶活性。在有无EGF的情况下，PC-10和APC-10在EGFR磷酸化的总体模式上没有可检测到的差异。然而，APC-10细胞EGFR的丝氨酸和苏氨酸磷酸化始终比PC-10细胞低2至3倍。这些结果表明，EGFR过表达细胞在EGF暴露下存活的一种新机制，该机制涉及在受体水平或受体亲和力没有变化的情况下，EGFR酪氨酸激酶活性的减弱。
