Kaufmann A, Khazaie K, Wiedemuth M, Rohdeschulz B, Ullrich A, Schirrmacher V, Lichtner R
SCHERING AG,RES LABS,D-13342 BERLIN,GERMANY. GERMAN CANC RES CTR,DIV CELLULAR IMMUNOL,D-69120 HEIDELBERG,GERMANY. MAX PLANCK INST BIOCHEM,DEPT MOLEC BIOL,D-82152 MARTINSRIED,GERMANY.
Int J Oncol. 1994 Jun;4(6):1149-55. doi: 10.3892/ijo.4.6.1149.
Increased expression of EGFR in metastases of human mammary carcinoma as compared to cells of the primary cancer suggests a contribution of EGFR to mammary carcinoma metastasis. To test for a positive correlation, we investigated 13762NF rat mammary adenocarcinoma cloned tumor cell lines of high (MTLn3) or low (MTC) metastatic potential. While MTC cells expressed barely detectable amounts of EGFR, MTLn3 cells expressed readily detectable levels of receptor. This was demonstrated in Northern blot analysis, in immunoprecipitation studies using metabolically labeled whole cell lysates and in Western blot analysis of membrane fractions. Cross-linking of radiolabeled ligand to intact cells identified on both cell types specific binding to a 170 kd protein, however, at much lower levels on low-metastatic MTC cells and not in sufficient amounts to estimate receptor numbers by Scatchard analysis. In contrast, Scatchard plot analysis of I-125-EGF binding to MTLn3 cells revealed the expression of about 10,000 high and 46,000 low affinity sites. Both cell lines expressed the ligand in comparable amounts as was demonstrated by using a specific rat TGFalpha cDNA probe in Northern blot and an antibody recognising membrane bound TGF in FACS analysis. Adhesion of MTC cells to immobilized collagen or fibronectin was rapid reaching 50% after 30 min while control MTLn3 cells demonstrated lower adhesion to collagen. Addition of 10 ng/ml EGF increased the rate and the maximal adhesion of MTLn3 cells to collagen G, while the adhesion kinetics of MTC cells to collagen G or fibronectin were unaffected.
与原发性乳腺癌细胞相比，人乳腺癌转移灶中表皮生长因子受体（EGFR）表达增加，提示EGFR在乳腺癌转移中发挥作用。为检测二者的正相关性，我们研究了具有高转移潜能（MTLn3）或低转移潜能（MTC）的13762NF大鼠乳腺腺癌克隆肿瘤细胞系。MTC细胞几乎检测不到EGFR表达，而MTLn3细胞则表达易于检测到的受体水平。这在Northern印迹分析、使用代谢标记全细胞裂解物的免疫沉淀研究以及膜组分的Western印迹分析中得到证实。将放射性标记配体与完整细胞交联，在两种细胞类型上均鉴定出与170kd蛋白的特异性结合，然而，低转移MTC细胞上的结合水平要低得多，且数量不足以通过Scatchard分析估计受体数量。相比之下，对I-125-EGF与MTLn3细胞结合的Scatchard作图分析显示，约有10,000个高亲和力位点和46,000个低亲和力位点。通过Northern印迹中使用特异性大鼠转化生长因子α（TGFα）cDNA探针以及FACS分析中使用识别膜结合TGF的抗体表明，两种细胞系表达的配体数量相当。MTC细胞与固定化胶原蛋白或纤连蛋白的黏附迅速，30分钟后达到50%，而对照MTLn3细胞对胶原蛋白的黏附较低。添加10ng/ml EGF可提高MTLn3细胞与胶原蛋白G的黏附速率和最大黏附程度，而MTC细胞与胶原蛋白G或纤连蛋白的黏附动力学不受影响。
