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Cellular Growth Response to Epidermal Growth Factor in Colon Carcinoma Cells with an Amplified Epidermal Growth Factor Receptor Derived from a Familial Adenomatous Polyposis Patient¹

Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030 [M. E. G., M. A. Z., Y. J. D., M. G. B., L. C. Y.]; Department of Tumor Biology, The M. D. Anderson Cancer Center, Section of Gastrointestinal Oncology and Digestive Diseases, University of Texas System Cancer Center, Houston, Texas 77030 [R. G., G. E. G., M. O.]; and Creighton Cancer Center, Creighton University School of Medicine, Omaha, Nebraska 68131-2197 [B. M. B.]

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 10⁶ 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 K_d 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.

¹ Support for these studies was provided by NIH grants CA45967, CA53617, the Texas Higher Education Coordinating Board Grant No. 15048, and funds provided by Bristol-Myers Squibb Company.

² To whom requests for reprints should be addressed, at Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.

Received 1/ 8/90. Accepted 12/20/90.

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