Identification and partial characterization of mesenchyme-derived growth factor that stimulates proliferation and inhibits functional differentiation of mouse mammary epithelium in culture

The effect of mesenchyme on both proliferation and differentiation of mammary epithelial cells was investigated in a primary cell culture system. Mammary cells cultured on collagen gel for 4 days produced casein in response to the synergistic action of insulin, cortisol, and prolactin. When mammary epithelial cells were co-cultured with fibroblasts derived from three different kinds of fetal mesenchymal tissues, casein production was suppressed. The addition of conditioned media obtained from cultures of these mesenchymal cells stimulated DNA synthesis and reduced casein synthesis in a dose-dependent fashion in the cultured mammary cells. Although such biological actions are similar to those of epidermal growth factor (EGF), the capability to compete with EGF for EGF receptor was not found in this conditioned medium. Sephadex G-200 column chromatography revealed that molecular weight of the peak which has these biological activities was around 100,000. These results indicate that fetal mesenchymal cells secrete a substance(s) which has a stimulatory effect on proliferation and an inhibitory effect on differentiation of mammary epithelial cells.     

Serum-free primary culture of human normal mammary epithelial cells in collagen gel matrix

Collagen gel matrix has been used successfully to promote sustained growth of human normal mammary epithelial cells in primary culture using serum-containing medium supplemented with hormones and growth factors (Nandi et al., 1932). Sustained growth can now be accomplished in a serum-free medium consisting of a 1:1 mixture of Ham's F12 and DMEM supplemented with insulin, transferrin, epidermal growth factor, cholera toxin, cortisol, and BSA. Human normal mammary epithelial cells derived from reduction mammoplasties can be routinely propagated in this serum-free medium. The extent of growth and the resulting three-dimensional outgrowths in this serum-free medium, using the collagen gel matrix system, are comparable to those seen in serum-containing medium. This is the first demonstration of sustained growth of human normal mammary epithelial cells in serum-free primary culture.     

Comparative analysis of casein synthesis during mammary cell differentiation in collagen and mammary gland development in vivo

Substrata upon which epithelial cells are cultured modulate their morphology,growth, and ability to differentiate. Mouse mammary epithelial cells cannot be induced to synthesize caseins, a marker of cell differentiation, when grown on a plastic surface. An analysis was made of the effect of time within a collagen matrix on the ability of normal mammary epithelial cells to be induced to synthesize caseins and that response was compared to mammary gland development in vivo. Primary cultures of mammary cells from unprimed virgin BALB/c mice were embedded in rat-tail collagen gel mixtures and maintained in growth medium. Induction medium containing lactogenic hormones was added at various times. The cells were monitored every 3-7 days over a period of 8 weeks for cell growth, casein synthesis, and ability to grow in vivo in cleared mammary fat pads. Casein accumulation was assayed quantitatively by an ELISA competition assay and qualitatively by the immunoblot procedure using specific antisera prepared against purified mouse caseins. No marked differences in cell numbers and transplantability potential were observed among cells cultured for various times in collagen. Mammary cells grown in collagen for up to 8 weeks retained the capacity to grow in vivo as normal ductal outgrowths. The duration of culture within collagen prior to hormonal stimulation did influence the kinetics of casein synthesis. Cells cultured for 1 week in growth medium did not accumulate detectable levels of casein until after 3 weeks of induction, whereas cells cultured for 2 or 4 weeks responded by accumulating caseins after 2 weeks and 3 days of induction, respectively. While the levels of total caseins that accumulated under optimal conditions of induction in culture approached levels found during lactation in vivo, the relative proportion of specific casein polypeptides synthesized in culture was altered from alpha casein (43K) in favor of the beta casein (30K) species. These results suggest that a period of culture within collagen is required to permit mammary epithelial cells to become responsive for hormone-induced differentiation. It is possible that during growth within the collagen the cells synthesize and deposit extracellular matrix components important in modulating gene expression.     

Hormonal regulation of the synthesis of casein and alpha-lactalbumin in a primary mammary cell culture system

The addition of 5 micrograms/ml of both insulin and prolactin, 3 microM cortisol and 5% fetal bovine serum stimulated casein synthesis during a 5 day culture of mammary epithelium from lactating mice using a floating collagen gel as a culture substratum. Omission of any of the three hormones or serum decreased casein synthesis substantially. The use of 10% serum or the attached gel culture system also decreased casein synthesis. Cells cultured with the combination of the three hormones and 5% serum contained a low level of casein mRNA on day 2, but it increased to much higher levels on day 4 and 5, amounting to over 30% of total mRNA on day 5. In contrast to casein synthesis, the maximal increase in alpha-lactalbumin synthesis required the presence of 0.03 microM cortisol. The combination of insulin, prolactin and 3 microM cortisol or insulin and prolactin elicited smaller increases. The translatable mRNA for alpha-lactalbumin in cells cultured with insulin, cortisol and prolactin for 5 days was detected, but not in cells with insulin and cortisol. Both a high and low concentration of cortisol in combination with insulin increased prolactin binding capacity of cultured cells to the same extent, whereas cells cultured with insulin alone contained much lower levels of prolactin binding. The difference in the capacity of prolactin binding between cells cultured with insulin alone and those cultured with insulin and cortisol correlated well with their ability to synthesize casein in response to prolactin.     

Serum-free culture of enriched mouse anterior and ventral prostatic epithelial cells in collagen gel

Summary Sustained growth of mouse ventral and anterior prostatic epithelial cells embedded within collagen gel matrix was achieved in a serum-free medium composed of Dulbecco's modified Eagle's medium and Ham's F12 medium, 1∶1 (vol/vol), supplemented with bovine serum albumin fraction V, epidermal growth factor, transferrin, cholera toxin, prolactin, 5α-dihydrotestosterone, cortisol, putrescine, fibroblast growth factor, and a trace element mixture. Three-dimensional growth of prostatic epithelial cells occurred inside the collagen gel matrix. This serum-free medium allowed cell growth greater than sevenfold over 10 d in culture. Tissue recombination and cell culture techniques were integrated to demonstrate that cultured cells retained prostatic characteristics. Following 10 d of culture, epithelial colonies from mouse ventral and anterior prostatic epithelial cell cultures were isolated and combined with rat fetal urogenital sinus mesenchyme and grown for 4 wk under the renal capsule of intact athymic male mice. These tissue recombinants showed distinctive prostatic histologic characteristic (alveoli and ducts lined with cuboidal or columnar epithelium surrounded by stroma). When histologic sections of recombinants were stained with the Hoechst 33258, epithelial cells of mouse origin were distinguishable from stromal cells of rat origin. Aided by grants CA-05388 and CA-09041 from the National Institutes of Health, Bethesda, MD, and by M. A. R. C. fellowship GM08730 to T. T.     

Prolactin sensitivity of mammary epithelial cells from mice exposed neonatally to diethylstilbestrol

We examined the responsiveness to prolactin and growth hormone of mammary epithelial cells from mice exposed neonatally to diethylstilbestrol (DES) and from control mice. The mammary epithelial cells were cultured inside collagen gels with serum-free medium containing insulin, epidermal growth factor, and linoleic acid. This produces prolactin-sensitive cells with low levels of casein production, as measured in cellular homogenates with a specific enzyme-linked immunosorbent assay for alpha-casein. The collagen gels containing these cells were then released and the medium supplements changed to insulin, linoleic acid, and prolactin at concentrations from 10 to 1000 ng/ml and growth hormone at 0, 10, or 100 ng/ml. This second phase of the culture, the differentiation phase, allows the cells to accumulate casein if they have this capacity. When cultured with prolactin only (no growth hormone), the cells from DES-exposed mice consistently accumulated 50-100% of the casein content of normal cells, but never more. Growth hormone, when added to prolactin-containing medium, increased casein accumulation above the levels seen with prolactin alone. Combinations of prolactin and growth hormone enhanced the difference between casein accumulation in DES-exposed and control cells, and DES-exposed cells were much less responsive to growth hormone. In our studies, the isolated mammary epithelial cells of estrogen-exposed mice are not more sensitive to prolactin than cells from normal animals as previous reports reports had suggested, but rather are generally less sensitive to hormonal stimulants.     

Effect of polypeptide hormones on stimulation of casein secretion by mouse mammary epithelial cells grown on floating collagen gels

The in vitro effects of protein hormones on the stimulation of casein secretion by mouse mammary epithelial cells were studied. Mouse mammary glands were enzymatically dissociated and used immediately or were stored frozen and thawed just before use. Cells were cultured on floating collagen gels in the presence of insulin, cortisol and a pituitary or placental polypeptide hormone. Casein, released into the medium, was assayed by a radioimmunoassay against one of the components of mouse casein. Mammary cells released casein into the medium in the presence of as little as 10 ng of ovine prolactin per ml of medium. Human growth hormone stimulated the casein secretion to the same extent as prolactin. Human placental lactogen, ovine and bovine growth hormones were less stimulatory. Luteinizing hormone, follicle-stimulating hormone and thyroid-stimulating hormone had no effect on the stimulation of casein secretion.     

Effect of polypeptide hormones on stimulation of casein secretion by mouse mammary epithelial cells grown on floating collagen gels

Summary The in vitro effects of protein hormones on the stimulation of casein secretion by mouse mammary epithelial cells were studied. Mouse mammary glands were enzymatically dissociated and used immediately or were stored frozen and thawed just before use. Cells were cultured on floating collagen gels in the presence of insulin, cortisol and a pituitary or placental polypeptide hormone. Casein, released into the medium, was assayed by a radioimmunoassay against one of the components of mouse casein. Mammary cells released casein into the medium in the presence of as little as 10 ng of ovine prolactin per ml of medium. Human growth hormone stimulated the casein secretion to the same extent as prolactin. Human placental lactogen, ovine and bovine growth hormones were less stimulatory. Luteinizing hormone, follicle-stimulating hormone and thyroid-stimulating hormone had no effect on the stimulation of casein secretion. This investigation was supported by Grant No. CA 05388 awarded by the National Cancer Institute, DHEW, and by Cancer Research Funds of the University of California.     

Growth and Differentiation of Primary Cultures of Mouse Mammary Epithelium Embedded in Collagen Gel

Mammary glands from BALB/cfC3H midpregnant (9–11 days) mice were dissociated with collagenase and pronase, separated on a Percoll gradient, and the epithelial cells were cultured inside collagen gel. The cell number increased three-to five-fold when cultured for 6–8 days in DME/F12 (1: 1) medium containing 3% swine serum, insulin (10 μg/ml), cortisol (1.0 μg/ml), prolactin (10 μg/ml), transferrin (10 μg/ml), and epidermal growth factor (0.01 μg/ml). The casein level, as determined by radioimmunoassay, at the end of this growth phase, was much lower than that present in freshly dissociated cells. In order to stimulate casein production, the gels were released from the sides of the plastic dish and allowed to float for eight days in Waymouth's medium, containing insulin (10 μg/ml), cortisol (5 μg/ml), prolactin (10 μg/ml), and 0.25% bovine serum albumin. The casein level at the end of this differentiation phase was found to be comparable to that seen in the original freshly dissociated cells. Cells grown in DME/F12 (1: 1) medium containing 3% swine serum, insulin (10 μg/ml), and transferrin (10 μg/ml) were still capable of undergoing casein production, indicating that the presence of exogenous lactogenic hormones such as cortisol and prolactin, as well as exogenous growth factors such as epidermal growth factor, is not necessary during the growth phase for subsequent casein production during the differentiation phase. Two factors that seemed more important for subsequent casein stimulation were: (1) releasing collagen gels at the beginning of the differentiation phase, and (2) switching to'differentiation' medium. This present two-step protocol has allowed primary cultures of dissociated midpregnant mouse mammary epithelial cells to undergo several rounds of division inside a collagen gel matrix and to be subsequently stimulated to produce the mammary-specific protein, casein.     

Morphological and functional differentiation of cryopreserved lactating bovine mammary cells cultured on floating collagen gels

Cryopreserved bovine mammary epithelial cells prepared from lactating mammary tissue synthesize and secrete the milk proteins alpha_s1-casein, lactoferrin (Lf), and alpha-lactalhumin during in vitro culture on collagen gels in serum-free medium. Each milk protein is differently regulated by detachment and thickness of the collagen substratum, fetal calf scrum, and prolactin in the medium. Collagen detachment did not modulate lactoferrin secretion but strongly induced casein secretion, with detachment on day 6 (after formation of cell sheets) inducing casein secretion to 3 μg/ml medium, which was 2–3-fold higher than for cells on collagen detached on day 2 (prior to cell spreading to form sheets), and ten-fold higher than for cells grown on collagen not detached. Alpha-lactalbumin secretion was also induced, but only to low levels, in cells grown on detached but not on attached collagen. Cells grown on thin collagen gels secreted lower levels of lactoferrin and casein compared to cells on thick collagen. Lactoferrin but not casein secretion was increased in cells grown in the presence of fetal calf serum. Casein but not lactoferrin secretion was completely dependent on prolactin. Cells grown serum-free on collagen gels detached on day 6 of culture showed a polarized epithelial cell layer with high differentiation evidenced by the apical microvilli, tight junctions, and fat droplets surrounded by casein-containing secretory vesicles. An underlying layer of myoepithelial-like cells was also evident. These studies show for eryopreserved primary bovine mammary cells prepared from lactating mammary tissue the induction of highly differentiated and polarized cell morphology and ultrastructure with concomitant induction of the secretion of casein, lactoferrin. and alpha-lactalbumin in vitro, and that the non-coordinate regulation of milk protein secretion by substratum, prolactin, and serum likely involves alternate routing and control of secretion pathways for casein and lactoferrin.     

Cortisol 21-mesylate exerts glucocorticoid action on the induction of milk protein synthesis in cultured mammary gland

Cortisol 21-mesylate, an alkylating derivatives of cortisol, was previously shown to exert an anti-glucocorticoid action in rat hepatoma cell culture (Simons, Thompson and Johnson 1980). In this study the effect of cortisol 21-mesylate on milk protein synthesis induced in cultured mouse mammary gland by glucocorticoid, insulin, and prolactin was investigated. Addition of cortisol 21-mesylate at concentrations ranging from 10(-8) M to 10(-6) M produced no inhibition of casein synthesis that was induced by glucocorticoid, insulin and prolactin in mammary explants from midpregnant mice. On the other hand, cortisol 21-mesylate in combination with insulin and prolactin stimulated casein synthesis in cultured tissue. The potency of cortisol mesylate was about 1/10 to 1/30th of that of cortisol. Cortisol 21-mesylate, like cortisol, also augmented the accumulation of alpha-lactalbumin in midpregnant rat mammary tissue cultured in the presence of insulin and prolactin. A cell-free competition study of glucocorticoid receptors using cytoplasmic extracts from mouse mammary tissue showed that cortisol 21-mesylate competitively inhibited the binding of dexamethasone on glucocorticoid receptors. The apparent affinity of cortisol 21-mesylate for glucocorticoid receptors is about 1/10th of that of cortisol. These results indicate that cortisol 21-mesylate acts as a glucocorticoid but not as an antiglucocorticoid in the mammary gland.     

Effect of TGF-Alpha on growth of normal human breast epithelial cells in serum-free primary culture using 3-dimensional collagen gels.

The mitogenic effect of TGF-alpha, acidic-FGF, basic-FGF and lithium on normal human breast epithelial cells was studied in a collagen gel culture system using a serum-free 1:1 mixture of Ham's F12 and DME medium containing insulin, cholera toxin and bovine serum albumin. TGF-alpha elicited a strong mitogenic response in a dose dependent manner. Addition of cortisol to TGF-alpha stimulated growth over and above that achieved with TGF-alpha alone. A consistent observation has been the effect of a combination of TGF-alpha and cortisol on growth stimulation of normal human breast epithelial cells resulting in 3-12 fold growth after 11-13 days in culture. Acidic-FGF, basic-FGF and lithium were not growth promoting.     

Growth effect of lithium on mouse mammary epithelial cells in serum-free collagen gel culture

The effect of lithium on the growth of mammary epithelial cells from adult virgin and midpregnant BALB/c or BALB/cfC3H mice was tested in a serum-free collagen gel culture system. The serum-free medium consisted of a 1:1 mixture of Ham's F12 and Dulbecco's Modified Eagle's medium supplemented with insulin, transferrin, cholera toxin, epidermal growth factor (EGF), and bovine serum albumin fraction V (BSA V). A multifold increase in cell number occurred during 10–12 days of culture in this medium. In dose-response studies in which the concentration of each component of this serum-free medium was varied in turn, the addition of LiCL (10 mM) enhanced growth at most concentrations of each factor. However, LiCL could not enhance growth in the absence of insulin or BSA V, but could replace EGF. The optimal concentration of LiCl was 5–10 mM; higher concentrations (20–80 mM) were toxic. KCl (1–10 mM) when added to the serum-free medium slightly stimulated growth; the addition of NaCl to the medium had little effect on growth. LiCl did not enhance the growth of cells from spontaneous mammary tumors of BALB/cfC3H mice.     

Studies of hormonal regulation of osteocalcin synthesis in cultured fetal rat calvariae

The synthesis of osteocalcin, the major non-collagenous protein of adult bone, was examined in cultures of 21-day fetal rat calvariae. Osteocalcin was measured by a sensitive and specific radioimmunoassay. Osteocalcin concentration in unincubated calvariae was 14.5 +/- 0.5 ng/calvaria. After incubation, there was a continuous increase in bone and medium osteocalcin, and by 96 h the values were about 100% higher than in unincubated calvariae. 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) at 10(-11) to 10(-8)M increased osteocalcin synthesis. The effect appeared as early as 6 h after treatment and was primarily observed in the culture medium, and 1,25-(OH)2D3 stimulated osteocalcin up to 9-fold by 96 h. Concomitant with the effect on osteocalcin synthesis, 1,25-(OH)2D3 inhibited collagen synthesis. Cycloheximide markedly decreased osteocalcin concentrations in control and 1,25-(OH)2D3-treated calvariae. The stimulatory effect on osteocalcin synthesis was specific to 1,25-(OH)2D3 since 24,25-dihydroxyvitamin D3, parathyroid hormone, epidermal growth factor, and prostaglandin E2 did not stimulate osteocalcin synthesis, and parathyroid hormone and epidermal growth factor opposed the 1,25-(OH)2D3 stimulatory effect. Insulin did not alter osteocalcin concentration by itself but enhanced the effect of 1,25-(OH)2D3. In conclusion, 1,25-(OH)2D3 stimulates osteocalcin synthesis in cultures of normal calvariae, but this effect is not shared by other hormones known to affect bone metabolism.     

Growth of mouse mammary epithelium in response to serum-free media conditioned by mammary adipose tissue

Normal mouse mammary epithelial cells, isolated from female Balb/c mice, were cultured as multicellular organoids either on or within collagen gel matrices. Cultures were maintained in either serum-free control medium or the same medium conditioned by mammary adipose tissue. A significant proliferative response above that observed in control cultures (2.5-3.5 fold increase) was induced by conditioned medium derived from either mammary fat-pad explants or isolated adipocytes. In addition, scanning electron microscopy revealed epithelial morphology to be preserved in a more in vivo-like state in the conditioned medium. We conclude that diffusible factors derived from the mouse mammary fat pad influence the proliferative activity and morphology of mammary epithelial cells in culture.     

A permissive role for extracellular Ca2+ in regulation of prolactin production by 1,25-dihydroxyvitamin D3 in GH3 pituitary cells

A clonal strain of rat pituitary tumor cells (GH3) that spontaneously synthesizes and secretes prolactin (PRL) and growth hormone (GH) was used as model system to study the mechanism of action of 1,25-(OH)2D3. We have previously demonstrated that these cells possess specific cytosol binding proteins for 1,25-(OH)2D3 (Haug and Gautvik, 1985). When the GH3 cells were incubated in a serum-free, chemically defined medium of low extracellular Ca2+ concentration, 1,25-(OH)2D3 stimulated PRL production in a dose-dependent manner. The stimulation was detectable at 10(-11) M, and the maximum effect (2-fold increase) was observed at 10(-9) M (ED50 = 2 x 10(-11) M). The dose-response curve was bell-shaped, and at 10(-6) M 1,25-(OH)2D3 even suppressed PRL production to about 75% of controls. The stimulatory effect was first seen after 2 days and was maximal after 4 days. On a molar basis 25-OHD3 and 1-OHD3 were at least 100 times less potent than 1,25-(OH)2D3, while 24,25-(OH)2D3 had no effect on PRL production. At an extracellular concentration of Ca2+ as low as 4 x 10(-5) M the stimulatory effect of 1,25-(OH)2D3 was small (1.3-fold). Increasing extracellular Ca2+ to 1.5 x 10(-4) M increased the 1,25-(OH)2D3-induced PRL response to 2.1-fold. In contrast to the biphasic effect of 1,25-(OH)2D3 on PRL production, GH production was decreased to about 60% of controls at 10(-8) M and above. These findings indicate that in serum-free medium the stimulatory effect of 1,25-(OH)2D3 on PRL production is critically dependent on the concentration of extracellular Ca2+.     

The study of differentiative potential of the lactating mouse mammary gland in organ culture

The organ culture of the mammary gland of lactating mice was used to examine the response of the differentiated gland to lactogenic stimuli, insulin, cortisol, and prolactin. Time course studies showed that casein synthesis in cultured tissue decreased rapidly during the first 2 d despite the presence of the three hormones, but on the 3rd d tissue cultured with either insulin and prolactin or all three hormones regained the ability to synthesize milk proteins, casein, and alpha-lactalbumin: a greater increase occurred in the three hormone system. The delayed addition of prolactin on Day 2 to the culture system containing insulin and cortisol also stimulated casein synthesis. The addition of cytarabine, which inhibited insulin-dependent cell proliferation in cultured explants, did not block the rebound of milk protein synthesis. These results indicate that in the presence of insulin, cortisol, and prolactin mammary epithelial cells in culture first lose and then regain the ability of synthesizing milk protein without requiring the formation of new daughter cells.     

Differential response of normal rat mammary epithelial cells to mammogenic hormones and EGF

A simple dissociation procedure and the collagen gel culture system have been utilized to determine the effects of mammogenic hormones and epidermal growth factor (EGF) on the proliferation of normal rat mammary epithelial (RME) cells in serum-free culture. Epithelial fragments, isolated from normal virgin F344 rat mammary glands by enzyme digestion followed by Percoll density gradient centrifugation, were embedded within a rat tail collagen matrix. A three- to four-fold increase in cell number was observed when ovine prolactin (PRL) and progesterone (P) were present in the basal medium during 7 days of culture. Mouse EGF stimulated one cell doubling during the same culture period. Isolated mammary organoids produced a 'stellate' type colony when PRL + P were present in the culture medium. These colonies were composed of small, tightly packed cuboidal cells. The addition of EGF to the basal medium produced a diffuse 'basket' type colony which was composed of large, elongate cells. When the complete hormonal and growth factor combination (PRL + P + EGF) was present, a 'mixed' type colony was observed which contained both the large and small epithelial cell types. Immunocytochemical analysis revealed that both the cuboidal and elongate cells present in the two colony types stained with antibodies to keratin indicating that these cells were epithelial in nature. The small cuboidal cells also expressed thioesterase II and alpha-lactalbumin, both specific for secretory mammary epithelial cells. The large, elongate cell type, however, was positive for actin but did not stain for either secretory epithelial specific marker. The results reported here suggest that normal rat mammary tissue may contain two epithelial populations, one which responds to PRL + P and the other which responds to EGF.