Oval J, Hershberg R, Gansbacher B, Gilboa E, Schlessinger J, Taetle R
Department of Medicine, University of California, San Diego.
Cancer Res. 1991 Jan 1;51(1):150-6.
To test the feasibility of using the human epidermal growth factor receptor (EGFR) as a model for growth factor receptor action in human hematopoietic cells, we infected Burkitt lymphoma cells (Namalwa) with a recombinant amphotrophic retrovirus containing a thymidine kinase promoter-driven human EGFR complementary DNA and the neomycin resistance gene. Neomycin-resistant cells expressing surface EGFR were selected by cell sorting using anti-EGFR monoclonal antibody 225. The selected cells expressed a Mr 170,000 protein immunoprecipitated by monoclonal antibody 225 and apparently identical to EGFR from A431 carcinoma cells. Infected Namalwa cells expressed 42,000 epidermal growth factor (EGF) binding sites/cell, and Scatchard analysis showed two affinities (Kd approximately 5 nM and approximately 0.5 nM). EGFR autophosphorylation was detected using antiphosphotyrosine antibodies after 5 min exposure to EGF. EGF binding induced rapid EGFR internalization (t1/2 = 9 min) and mobilization of transferrin receptors to the cell surface within 1 min. In fetal bovine serum-containing and serum-free cultures, EGF did not stimulate Namalwa cell proliferation. However, in the presence of 1.25% dimethyl sulfoxide (DMSO), EGF caused a dose-dependent increase in DNA synthesis. DMSO induced a cell cycle block in G1, which was partially reversed by EGF. DMSO induced changes in some B-cell markers suggesting cellular differentiation and increased surface EGF receptor number. Cells grown in DMSO and EGF were established as an EGF-dependent cell line for greater than 12 weeks, whereas cells grown in DMSO without EGF died within 1-2 weeks. Namalwa cells expressing EGFR demonstrated more rapid tumor growth in athymic mice. These studies demonstrate expression of functional EGFR mediating early biochemical and growth responses in a human hematopoietic cell, and indicate that EGFR can be used as an effective model in human hematopoietic cells. Results using DMSO are consistent with studies in other human leukemia cells indicating that agents inducing differentiation can restore growth factor dependence in previously factor-independent leukemia cells.
为了测试将人表皮生长因子受体（EGFR）用作人造血细胞中生长因子受体作用模型的可行性，我们用一种重组嗜双嗜性逆转录病毒感染伯基特淋巴瘤细胞（Namalwa），该病毒含有胸苷激酶启动子驱动的人EGFR互补DNA和新霉素抗性基因。使用抗EGFR单克隆抗体225通过细胞分选选择表达表面EGFR的新霉素抗性细胞。所选细胞表达一种由单克隆抗体225免疫沉淀的分子量为170,000的蛋白质，且明显与A431癌细胞中的EGFR相同。感染的Namalwa细胞每个细胞表达42,000个表皮生长因子（EGF）结合位点，Scatchard分析显示有两种亲和力（Kd约为5 nM和约0.5 nM）。在暴露于EGF 5分钟后，使用抗磷酸酪氨酸抗体检测到EGFR自身磷酸化。EGF结合诱导EGFR快速内化（t1/2 = 9分钟），并在1分钟内将转铁蛋白受体转运至细胞表面。在含胎牛血清和无血清培养中，EGF不刺激Namalwa细胞增殖。然而，在存在1.25%二甲基亚砜（DMSO）的情况下，EGF导致DNA合成呈剂量依赖性增加。DMSO诱导细胞周期在G1期阻滞，EGF可部分逆转该阻滞。DMSO诱导一些B细胞标志物发生变化，提示细胞分化并增加表面EGF受体数量。在DMSO和EGF中生长的细胞被建立为EGF依赖性细胞系超过12周，而在无EGF的DMSO中生长的细胞在1 - 2周内死亡。表达EGFR的Namalwa细胞在无胸腺小鼠中显示出更快的肿瘤生长。这些研究证明了功能性EGFR在人造血细胞中介导早期生化和生长反应的表达，并表明EGFR可作为人造血细胞中的有效模型。使用DMSO的结果与其他人类白血病细胞的研究一致，表明诱导分化的药物可恢复先前不依赖因子的白血病细胞对生长因子的依赖性。
