Cellular growth response to epidermal growth factor in colon carcinoma cells with an amplified epidermal growth factor receptor derived from a familial adenomatous polyposis patient

Abstract

The receptor binding and cellular growth responses to exogenous epidermal growth factor (EGF) were studied using the DiFi cell line established from a familial adenomatous polyposis patient. The number of cell membrane EGF receptors on DiFi cells, as measured by competitive radioligand binding assays and Scatchard analysis of 125I-EGF binding isotherms, was calculated to be 4.8 x 10(6) receptors/cell. An acid prewash step performed prior to ligand binding assays did not reveal additional receptor numbers. A single, low-affinity receptor population was identified by Scatchard analysis, with an apparent Kd of 4.6 nM. This result was confirmed by radioligand binding studies performed in the presence and absence of the receptor-antagonist monoclonal antibody 528 IgG that binds predominantly to the low-affinity form of the EGF receptor. DiFi cells at 50-60% confluence, when exposed to 50 nM exogenous EGF, exhibited a rapid but partial (30%) reduction in their cell membrane-associated receptor, characteristic of sequestration. Exposure of DiFi cells to 50 nM EGF for longer periods of time (4 h) did not result in any further reduction in EGF-receptor number. The cellular growth response of DiFi cells to exogenous EGF was studied in monolayer cultures as well as in a soft agarose assay. Inhibition of soft agar colony formation was observed at exogenous EGF concentrations greater than 1.7 nM, and inhibition of monolayer growth occurred at EGF concentrations greater than 1 nM. In immune complex kinase assays, the DiFi receptor showed similar specific activity to that from the well-characterized A431 cell line. Additionally, phosphorylation of the receptor on tyrosine was qualitatively similar to that of A431 cells, further suggesting that the DiFi receptors identified by EGF-binding studies were biologically functional.