| Popis: |
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. |
