JOURNAL OF CELLULAR PHYSIOLOGY 130:397-409 (1987) Loss of Growth Responsiveness to Epidermal Growth Factor and Enhanced Production of Alpha-Transforming Growth Factors in ras-Transformed Mouse Mammary Epithelial Cells DAVID S. SALOMON,* ISABELLE PERROTEAU, WILLIAM R. KIDWELL, JAMES TAM, AND RIK DERYNCK Laboratory of Tumor Immunology and Biology (D.S.S., I.P., W.R.K.), National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, Department o f Biochemistry (].lJ, The Rockefeller University, New York, New York 10202, Department of Molecular Biology (R. D.), Genentech, Inc., South San Francisco, California 94080 A mouse mammary epithelial cell line, NMuMG, exhibits a low capacity to grow in semisolid medium as colonies and it is not tumorigenic in nude mice. In contrast, NMuMG cells which have been transformed by an activated c- Harvey ras proto-oncogene, NMuMGlrasH, or by the polyoma middle T-trans- forming gene, NMuMGlpyt, are able to grow in soft agar and, when injected into nude mice, produce undifferentiated carcinomas. Human epidermal growth factor (EGF) or human alpha-transforming growth factor (aTGF) can stimulate, in a dose-dependent fashion, the anchorage-independent growth of NMuMG and NMuMGlpyt cells in soft agar but fail to enhance the anchor- age-independent growth of the NMuMCrasH cells. Likewise, human EGF or human aTGF is also able to stimulate the anchorage-dependent growth of normal NMuMG cells and NMuMGlpyt cells in a serum-free medium supple- mented with insulin, transferrin, fetuin, and laminin, or in medium containing low concentrations of serum, whereas these same growth factors under comparable culture conditions have little or no effect upon the anchorage- dependent growth of the ras-transformed NMuMG-rasH cells. The biological refractoriness of the NMuMGlras" cells to human EGF or human a TGF is reflected by a reduction in the total number of cell surface receptors for EGF and by an absence of a high-affinity population of binding sites for mouse ['2s1]EGF on these cells as compared to the NMuMG or NMuMGlpyt cells. In addition, concentrated conditioned medium (CM) obtained from NMuMGl rasH and NMuMGlpyt cells contains a relatively higher amount of biologically active TGFs than CM obtained from comparably treated NMuMG cells as measured by the ability to induce the anchorage-independent growth of normal rat kidney cells in soft agar. The higher levels of biologically active TGFs found in the CM from the transformed cells relative to the NMuMG cells is paralleled by a corresponding increase in the CM from these cells in the amount of immunoreactive aTGF, by an increase in the amount of EGF receptor-competing activity, and by an increase in the levels of aTGF mRNA in the NMuMGlrasH cells. These results demonstrate that mammary epithelial cells which have been transformed by an activated ras proto-oncogene, but not by the polyoma middle T-transforming gene, become unresponsive to exogenous ECF or aTGF. The growth refractoriness of the NMuMGlrasH cells to exogenous ECF may be due to the reduction in the number and affinity of EGF receptors on these cells and because of an increased capacity of these cells to synthesize and secrete their own aTGFs. Received June 12, 1986; accepted October 27, 1986. rium dissociation constant; MOPS, 3-[N-morpholine]propane- sulfonic acid; NBT, nitroblue-tetrazolium; NRK, normal rat kid- Abbreviations used: BSA, bovine serum albumin; CHO, Chinese ney; PBS, phosphate-buffered saline; PDGF, platelet-derived hamster ovary; CM, conditioned medium; DMEM, Dulbecco's growth factor; RIA, radioimmunoassay; RRA, radioreceptor as- modified ~ ~ ~ l ~ ' ~ medium; ~m, dithiothreitol; EGF, epidermal say; SDS, sodium dodecyl sulfate; SSC, standard saline citrate; growth factor; FCS, fetal calf serum; IGFs, insulinlike growth TGFs, transforming growth factors. factors; IMEM, improved minimal essential medium; Kd, equilib- *To whom reprint requestskorrespondence should be addressed. 0 1987 ALAN R. LISS, INC