Loss of growth responsiveness to epidermal growth factor and enhanced production of alpha‐transforming growth factors in ras‐transformed mouse mammary epithelial cells

Abstract

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, NMuMG/rasH, or by the polyoma middle T-transforming gene, NMuMG/pyt, 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 (alpha TGF) can stimulate, in a dose-dependent fashion, the anchorage-independent growth of NMuMG and NMuMG/pyt cells in soft agar but fail to enhance the anchorage-independent growth of the NMuMGrasH cells. Likewise, human EGF or human alpha TGF is also able to stimulate the anchorage-dependent growth of normal NMuMG cells and NMuMG/pyt cells in a serum-free medium supplemented 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 NMuMG/rasH cells to human EGF or human alpha 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 [125l]EGF on these cells as compared to the NMuMG or NMuMG/pyt cells. In addition, concentrated conditioned medium (CM) obtained from NMuMG/rasH and NMuMG/pyt 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 alpha TGF, by an increase in the amount of EGF receptor-competing activity, and by an increase in the levels of alpha TGF mRNA in the NMuMG/rasH 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 EGF or alpha TGF.(ABSTRACT TRUNCATED AT 400 WORDS)

Transformation of mouse mammary epithelial cells with the Ha‐ras but not with the neu oncogene results in a gene dosage‐dependent increase in transforming growth factor‐α production

“…This phenomenon is probably due in large part to a chronic occupation of these receptors by endogenously produced TGFa~, which results in a down-regulation of these receptors [1,7,8,22]. Fig.4 The results of this study demonstrate that the amount of TGFa produced in Ha-ras-transformed mammary epithelial cells is dependent on the levels of p21 ras expression in these cells.…”

“…Enhanced production and secretion of TGFoe are frequently associated with rodent fibroblasts and epithelial cells that have been transformed with an activated c-Ha-ras proto-oncogene and v-Ki-ras or v-Ha-ras oncogenes [7,8,22,23]. To ascertain if there was any relationship among the level of v-Haras expression, soft agar growth and TGFa production, CM from these cell lines was analyzed for immunoreactive and biologically active TGFot in a RIA and RRA, respectively.…”

“…One consequence of an increase in TGFt~ production and secretion in a number of different rastransformed cells is a reduction in the number of EGF receptors that can be measured on the surface of these cells [7,8,24]. This phenomenon is probably due in large part to a chronic occupation of these receptors by endogenously produced TGFa~, which results in a down-regulation of these receptors [1,7,8,22].…”

“…TGFa expression can be enhanced by estrogens in estrogen-responsive rat and human breast tumor cell lines [4][5][6]. In addition, we have demonstrated previously that transformation of two different mouse mammary epithelial cell lines with an activated c-Ha-ras proto-oncogene induces an increase in both TGFcr mRNA expression and TGF~ protein production [7,8]. In fact, the increase in TGF~ synthesis in ras-transformed NOG-8 mouse mammary epithelial cells is coordinately and temporally coupled to a change in ras gene expression [8].…”

“…act. 100/zCi//~g, Amersham, Arlington Heights, IL) as previously described [7]. The number of EGF-binding sites and the Ads for binding were determined by Scatchard analysis of the specific-binding isotherms [8].…”