Induction of Transforming Growth Factor α Expression in Mouse Mammary Epithelial Cells after Transformation with a Point-Mutated c-Ha-rasProtooncogene

Abstract

NOG-8 ras cells are a normal mouse mammary epithelial cell line transfected with a plasmid containing a glucocorticoid-inducible mouse mammary tumor virus long terminal repeat linked to the activated c-Ha-ras protooncogene. After addition of dexamethasone, there is a rapid induction (within 1-3 h) of p21ras protein that is concomitant with a parallel induction of the c-Ha-ras specific mRNA. After 4-6 days of dexamethasone treatment, NOG-8 ras cells are able to grow as colonies in semisolid medium. Between 9 and 12 days of dexamethasone treatment, there is a 5- to 6-fold increase of transforming growth factor alpha (TGF alpha) activity in the conditioned medium from NOG-8 ras cells. A 60-65% reduction in epidermal growth factor cell surface receptors on NOG-8 ras cells also occurs during this time interval. A 3- to 4-fold increase of the expression of a specific TGF alpha mRNA can be detected within 2 days of dexamethasone treatment, preceding the increase in TGF alpha protein found in the conditioned medium. Exogenous TGF alpha is able to stimulate in a dose-dependent fashion the anchorage-dependent and anchorage-independent growth of NOG-8 ras cells to a level comparable to that observed in dexamethasone treated ras-transformed NOG-8 ras cells. These results suggest that the enhanced expression of TGF alpha after induction of an activated ras protooncogene may be necessary for the anchorage-independent growth and subsequent morphological changes and the enhanced growth rate observed in ras-transformed mammary epithelial cells.

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

“…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].…”

“…Preparation of conditioned medium (CM) that was collected over a 48-h period from cultured ceils and determination of TGF~ protein by radioimmunoassay (RIA) and by radioreceptor assay (RRA) were performed as previously described [5,8].…”

“…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].…”

“…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]. These results collectively suggest that TGFa may be functioning as an autocrine growth factor for breast cancer cells and that TGFcr may be acting as an intermediary in the transformation pathway for an activated ras proto-oncogene in mammary epithelial cells [9,10].…”

“…brane (ICN Biomedicals, Costa Mesa, CA) [18] and hybridized sequentially as previously described [8] to the following 32p_ labeled, nick-translated eDNA probes: a 6.6-kb BamHI fragment of a human c-Ha-ras eDNA probe [19]; a 406-bp EcoRIApaI restriction fragment derived from a human TGFa eDNA clone, pTGF-CI [20]; a 6.0-kb EcoRI/HindlII fragment of the human c-myc eDNA probe [21]; and a 770-bp human ~-actin eDNA probe (Oncor, Gaithersburg, MD).…”