Stimulation of Mammary Epithelial Cell Growthin Vitro: Interaction of Epidermal Growth Factor and Mammogenic Hormones*

Abstract

A serum-free primary cell culture system was used to examine the direct effects and interactions of mammogenic hormones and epidermal growth factor (EGF) on the growth of mouse mammary epithelial cells. Epithelial cells were isolated by collagenase dissociation followed by Percoll gradient centrifugation and cultured within collagen gels in a mixture of Ham's F-12-Dulbecco's Minimum Essential Medium (1:1) containing insulin (10 micrograms/ml), crude soybean lecithin, trace elements, trypsin inhibitor, and antioxidants. Progesterone (P; 10(-6) - 10(-8) M) or ovine PRL (1 microgram/ml), in the absence of EGF, stimulated the growth of cells from mature virgin mice 2- to 4-fold over that of controls cultured in basal medium only. P and PRL synergized in stimulating growth 3- to 17-fold. 17 beta-Estradiol (10(-7) - 10(-10) M) alone did not stimulate growth or synergize with P and/or PRL. This lack of growth stimulation by 17 beta-estradiol was also observed in medium containing a low concentration of insulin (0.1 microgram/ml). EGF (10 ng/ml) alone stimulated growth to the same extent as the combination of P and PRL. EGF at 1, but not 10, ng/ml when combined with P and PRL could additively stimulate growth. Cells from midpregnant mice were less responsive than cells from virgin mice to the growth-stimulating effects of the combination of P and PRL (2-fold stimulation at most), but not to EGF (3- to 6-fold stimulation). Corticosterone, deoxycorticosterone, and aldosterone, but not cortisol, could synergize with PRL in stimulating the growth of cells from mature virgin mice. However, only deoxycorticosterone could stimulate growth in the absence of PRL. These results suggest that PRL, P, and adrenal corticoids may directly stimulate the growth of mouse mammary epithelial cells. The physiologically relevent adrenal corticoids, corticosterone and aldosterone, only potentiate the stimulatory effect of PRL. The hormonal stimulation of growth in vitro can be obscured by an optimum concentration (10 ng/ml) of EGF. The relative growth responses to mammogenic hormones and EGF may depend on the degree of differentiation of the cells.

Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities.

“…For example, previous studies have shown that although estradiol alone can induce DNA synthesis in mammary glands of ovariectomized mice (Bresciani 1968;Shyamala and Ferenczy 1984;Daniel et al 1987), this mitogenic response can be augmented further by coadministration of progesterone (Bresciani 1968;Shyamala 1987). In addition, studies have implied that progesterone may initiate proliferative responses in the normal mammary epithelium in virgin animals (Haslam 1988) as well as contributing to the growth and development of the lobular-alveolar system in mammary glands of pregnant animals (Imagawa et al 1985;Haslam 1988).…”

Linoleic acid, but not cortisol, stimulates accumulation of casein by mouse mammary epithelial cells in serum-free collagen gel culture.

“…This is probably not the case, however, as cortisol directly stimulates the secretory process in the epithelial cells (42,43). The culture system used in this investigation is not useful for examining this point since it would be expected that secreted casein would accumulate in the lumina of the colonies inside the collagen gel (27) (49). A similar effect is seen in prolactin-treated rat mammary explants (50).…”

“…Collagen gel culture of mammary epithelial cells has been extensively described and reviewed elsewhere (24,30). The serum-free medium, which has also been described (27,28), was a 1:1 (vol/vol) mixture of Ham's F12 medium/Dulbecco's modified Eagle's medium (both from GIBCO) containing NaHCO3 at 1.2 g/liter and buffered with Hepes at 20 mM. This medium was supplemented with transferrin, soybean trypsin inhibitor, D-a-tocopherol (all from Sigma), trace element mixture, antibiotics, 10 pg of insulin per ml (Sigma), and fatty acid-free bovine serum albumin (BSA) (Armour, Tarrytown, NY) alone or conjugated to the sodium salt of linoleic acid ref.…”

“…Toward this end we have attempted to determine the minimal requirement for casein accumulation by mammary epithelial cells from adrenalectomized and ovariectomized virgin mice using the serum-free collagen gel culture system developed in this laboratory (24)(25)(26)(27)(28). Since DNA synthesis has been shown to be a prerequisite to hormone-induced casein accumulation by mammary epithelial cells from virgin mice (29), we have used a two-step system in our studies.…”

“…The cell dissociation was essentially as described (24,30 (24,30). The serum-free medium, which has also been described (27,28) 160 ,g/ml in all cultures, and, when used, the concentration of fatty acid was 10 ttg/ml. The medium was changed every 2 days.…”