Estrogen enhances epidermal growth factor-induced DNA synthesis in mammary epithelial cells

Estradiol (E₂) priming (1 nM for 48 h) of normal murine mammary gland epithelial cells significantly increased the response of those cells to epidermal growth factor (EGF)-induced DNA synthesis. The synergism between E₂ and EGF was evident in two aspects: After serum-free synchronization for 24 h, more cells entered the S-phase of the cell cycle after E₂ priming and when treated with 0.17 nM EGF (13%) than did control cells (1.3%) or cells treated with EGF (4%) or E₂ (3.5%) alone; further, the dose of EGF required to elicit maximal response was reduced an order of magnitude in estrogen-primed cells (0.17 nM) compared to controls (1.7 mM). Estrogen alone, however, did not increase DNA synthesis in these cells. Ligand binding studies indicate that these effects of estrogen on proliferating mammary epithelial cells may be explained, at least in part, by a 3.7-fold increase in the number of high affinity EGF-receptors observed in estrogen primed cells (7,300 receptors per cell) compared to estrogen deprived cells (1,960 receptors/cell). © 1993 Wiley-Liss, Inc.     

Regulation of differentiation and polarized secretion in mammary epithelial cells maintained in culture: extracellular matrix and membrane polarity influences

Several previous studies have demonstrated that mammary epithelial cells from pregnant mice retain their differentiated characteristics and their secretory potential in culture only when maintained on stromal collagen gels floated in the culture medium. The cellular basis for these culture requirements was investigated by the monitoring of milk protein synthesis and polarized secretion from the mouse mammary epithelial cell line, COMMA-1-D. Experiments were directed towards gaining an understanding of the possible roles of cell-extracellular matrix interactions and the requirements for meeting polarity needs of the epithelium. When cells are cultured on floating collagen gels they assemble a basal lamina-like structure composed of laminin, collagen (IV), and heparan sulfate proteoglycan at the interface of the cells with the stromal collagen. To assess the role of these components, an exogenous basement membrane containing these molecules was generated using the mouse endodermal cell line, PFHR-9. This matrix was isolated as a thin sheet attached to the culture dish, and mammary cells were then plated onto it. It was found that cultures on attached PFHR-9 matrices expressed slightly higher levels of beta-casein than did cells on plastic tissue culture dishes, and also accumulated a large number of fat droplets. However, the level of beta-casein was approximately fourfold lower than that in cultures on floating collagen gels. Moreover, the beta-casein made in cells on attached matrices was not secreted but was instead rapidly degraded intracellularly. If, however, the PFHR-9 matrices with attached cells were floated in the culture medium, beta-casein expression became equivalent to that in cells cultured on floating stromal collagen gels, and the casein was also secreted into the medium. The possibility that floatation of the cultures was necessary to allow access to the basolateral surface of cells was tested by culturing cells on nitrocellulose filters in Millicell (Millipore Corp., Bedford, MA) chambers. These chambers permit the monolayers to interact with the medium and its complement of hormones and growth factors through the basal cell surface. Significantly, under these conditions alpha 1-, alpha 2-, and beta-casein synthesis was equivalent to that in cells on floating gels and matrices, and, additionally, the caseins were actively secreted. Similar results were obtained independently of whether or not the filters were coated with matrices.(ABSTRACT TRUNCATED AT 400 WORDS)     

Large-scale expansion of mammary epithelial stem cell aggregates in suspension bioreactors

Mutations in the pathways regulating mammary epithelial stem cell (MESC) self-renewal and differentiation are currently hypothesized to result in uncontrolled cell division and, in turn, breast tumor formation. Although research is aggressively being pursued to understand how such pathways result in breast cancer formation, current studies have been greatly limited by MESC scarcity. To address this issue, this study has successfully developed large-scale expansion protocols for MESC through the subculture of murine mammary epithelial tissue aggregates, called mammospheres, in suspension bioreactors. Growth kinetics of mammospheres cultured in 125 mL suspension bioreactors and T-flasks were found to be comparable, achieving cell densities of 3.10 x 10(5) and 2.75 x 10(5) cells/mL, respectively. This corresponded to a 4-fold expansion over 8 days. Yields were also found to be strongly affected by liquid shear forces, where high agitation rates reduced overall cell numbers. Bioreactor cultures were scaled up to 1000 mL operating volumes, resulting in the production of 4.21 x 10(8) total cells (5.6-fold expansion) from a single passage. Furthermore, intermittent replacement of culture medium with fresh medium dramatically improved maximum cell densities, resulting in an 11-fold expansion, thereby enabling the generation of stem cells in quantities sufficient for standard biochemical and genetic analyses. After being cultured in suspension bioreactors for several passages, analysis by flow cytometry of Ki-67 revealed that 85% of the population was composed of proliferating cells. The successful development of expansion protocols for MESC aggregates in suspension bioreactors makes available experimental avenues that were not previously accessible for breast cancer research, thereby facilitating future investigations into elucidating the role of MESCs in breast cancer tumorigenesis.     

Multiple mechanisms are responsible for transactivation of the epidermal growth factor receptor in mammary epithelial cells

The number of distinct signaling pathways that can transactivate the epidermal growth factor receptor (EGFR) in a single cell type is unclear. Using a single strain of human mammary epithelial cells, we found that a wide variety of agonists, such as lysophosphatidic acid (LPA), uridine triphosphate, growth hormone, vascular endothelial growth factor, insulin-like growth factor-1 (IGF-1), and tumor necrosis factor-alpha, require EGFR activity to induce ERK phosphorylation. In contrast, hepatocyte growth factor can stimulate ERK phosphorylation independent of the EGFR. EGFR transactivation also correlated with an increase in cell proliferation and could be inhibited with metalloprotease inhibitors. However, there were significant differences with respect to transactivation kinetics and sensitivity to different inhibitors. In particular, IGF-1 displayed relatively slow transactivation kinetics and was resistant to inhibition by the selective ADAM-17 inhibitor WAY-022 compared with LPA-induced transactivation. Studies using anti-ligand antibodies showed that IGF-1 transactivation required amphiregulin production, whereas LPA was dependent on multiple ligands. Direct measurement of ligand shedding confirmed that LPA treatment stimulated shedding of multiple EGFR ligands, but paradoxically, IGF-1 had little effect on the shedding rate of any ligand, including amphiregulin. Instead, IGF-1 appeared to work by enhancing EGFR activation of Ras in response to constitutively produced amphiregulin. This enhancement of EGFR signaling was independent of both receptor phosphorylation and PI-3-kinase activity, suggestive of a novel mechanism. Our studies demonstrate that within a single cell type, the EGFR autocrine system can couple multiple signaling pathways to ERK activation and that this modulation of EGFR autocrine signaling can be accomplished at multiple regulatory steps.     

Interactions between normal mammary epithelial cells and mammary tumour cells in a model system

Abstract. Normal mammary epithelial (NME) cells and MCF-7 cells aggregate and grow as spheroids when cultured on extracellular matrix derived from Engelbreth/ Holmes/Swarth (EHS) tumour. NME cells stop dividing and differentiate but MCF-7 cells continue to proliferate, although growth is counterbalanced by cell death. In mixed cultures of NME cells and MCF-7 cells, the two cell types form mixed aggregates but then segregate to form well separated domains, often joined by only a narrow neck of cells. In these mixed cultures the growth of MCF-7 cells is inhibited by a factor secreted by NME cells into the medium.     

Oncogene expression in mammary epithelial cells.

Mouse strains which develop tumors at a high incidence with characteristics very similar to human cancers have been derived over the last 8 years. The tumors are caused by defined genetic alterations in the mouse genome. Three areas of research have contributed to the derivation of these mouse strains: (1) Molecular analysis of human tumors has shown that distinct oncogenes and tumor suppressor genes are consistently involved in a high percentage of primary tumors. (2) Regulatory enhancer-promoter sequences have been identified which direct gene expression to specific target cells, preferentially mammary epithelial cells. (3) The introduction of recombinant DNA molecules into fertilized mouse eggs by microinjection and integration of the injected DNA into the genome of injected cells has given rise to mutant mouse strains with unique and defined genetic alterations. Studies with different promoter-oncogene combinations introduced into transgenic mouse strains have led to the following general conclusions: (1) Oncogenes expressed in mammary gland cells predispose transgenic mice to mammary tumors. (2) The oncogenic potential of individual oncogenes in mammary epithelial cells differs. (3) Oncogene expression initially often causes a preneoplastic state affecting growth and differentiation parameters of cells. (4) The expression of different oncogenes synergizes to reduce tumor latency. Synergism can also be observed with physiological growth signals like estrogen or growth hormone. The oncogenes with a role in mammary carcinomas which have been investigated in transgenic mice will be described here. The phenotypic consequences of oncogene expression and the implications for the multistep carcinogenesis model will be discussed.     

Linoleate metabolites enhance the in vitro proliferative response of mouse mammary epithelial cells to epidermal growth factor

Linoleic acid, arachidonic acid, prostaglandin E1, and prostaglandin E2 stimulated the proliferation of mammary epithelial cells in serum-free primary cultures only in the presence of epidermal growth factor. Linoleate-stimulated growth was manifest later in culture when proliferation, initiated by epidermal growth factor only, reached a plateau while linoleate-supplemented epidermal growth factor cultures continued to proliferate. The cultures in the plateau phase of growth could be restimulated to grow by adding either linoleic acid or prostaglandin E2 to the media. While the linoleate response could be abolished by the cyclooxygenase inhibitor, indomethacin, prostaglandin E2-stimulated growth remained unaffected. Linoleic acid was metabolized to arachidonic acid and prostaglandin E2, both in the growing and resting cultures. Proliferating cells metabolized linoleate and prostaglandin E2 extensively so that neither the fatty acid nor prostaglandin E2 accumulated in large quantities in the proliferating cultures. The concentrations of prostaglandin E2 in growing cultures supplemented with linoleic acid were much higher than in cultures without it. These results suggest that the metabolism of linoleic acid leading to prostaglandin production, not its contribution to membrane polyunsaturation, is necessary for sustained growth of mammary epithelial cells in the presence of epidermal growth factor.     

Relationship between epidermal growth factor receptor levels, autophosphorylation and mitogenic-responsiveness in normal mouse mammary epithelial cells in vitro

Mammary epithelial cells were isolated from mid-pregnant BALB/c mice, grown within collagen gels and maintained on DME/F12 (1:1) media containing 10% bovine calf serum and 10 μ/ml insulin. Initial time-course and dose-response studies showed that epidermal growth factor (EGF)-induced autophosphorylation of the EGF-receptor (EGF-R) in these cells was maximal 5 min after exposure to 75 ng/ml EGF. Mammary epithelial cells displaying little or no growth during their first 2 days in primary culture cells were found to contain low levels of EGF-R. However, EGF-induced autophosphorylation of the EGF-R in these cells was extremely intense. Subsequent studies demonstrated that during the proliferative and plateau phases of growth, EGF-R levels progressively increased, while conversely EGF-induced autophosphorylation of the EGF-R decreased over time in primary culture. These results demonstrate that EGF-R levels and autophosphorylation do not show a direct correlation with mammary epithelial cell mitogen-responsiveness. Intense EGF-R autophosphorylation appears to be required for initiating growth, but sustained mammary epithelial cell proliferation occurs when EGF-R autophosphorylation is low. This inverse relationship between EGF-R levels and autophosphorylation may reflect changes in receptor affinity and function during the various phases of mammary epithelial cell growth in primary culture.     

Cultured proliferating rat mammary epithelial cells

Summary Normal epithelial cells from the rat mammary gland proliferated in culture when plated with lethally irradiated cells of the LA7 rat mammary tumor line. Proliferation of the normal rat cells occured as the LA7 cells slowly died from the radiation. By labeling the cultures with³H-thymidine it was determined that most of the proliferating rat cells were those adjacent to the LA7 feeder cells. The epithelial cells from the primary culture proliferated after subsequent passages if the cells were plated at each subculture with newly irradiated LA7 cells. If the cells were plated at a ratio of ∼1:8 rat:LA7 a confluent layer of normal rat cells covered the plastic substrate after 6 to 7 wk. The cells have so far been carried up through Passage 7, which amounted to ∼19 doublings in cell number, and still proliferate vigorously. The growth medium for this culture system was Dulbecco’s modified Eagle’s medium:Ham’s F12 1:1 supplemented with fetal bovine serum, insulin, and antibiotics. The presence in the cells of keratin, desmosomes, and cell junctions attested to their epithelial origin. The cultures were composed of cells with diploid or near diploid chromosome numbers. Samples of the cultured cells were implanted into the cleared fat pads of nude mice. Most of the implants from Passage 2 formed normal mammary ductal structures, but the incidence of outgrowths decreased significantly with later passages until no out-growths resulted from the implantation of cells from Passage 5. The one unusual, feeder-independent cell line that arose from a primary culture seemed to be immortal in culture, contained a hyperdiploid chromosome complement, and formed abnormal structures when implanted into cleared fat pads. This work was supported by the Veterans Administration, Washington, DC, and by CA grant 05388 from the U.S. Public Health Service, Washington, DC.     

Transforming growth factor alpha production and epidermal growth factor receptor expression in normal and oncogene transformed human mammary epithelial cells

We have characterized the expression of transforming growth factor alpha (TGF alpha) and its receptor, the epidermal growth factor receptor (EGF-R), in normal and malignantly transformed human mammary epithelial cells. Human mammary epithelial cells were derived from a reduction mammoplasty (184), immortalized by benzo-a-pyrene (184A 1N4), and further transformed by the oncogenes simian virus 40 T (SV40 T), v-Ha-ras, and v-mos alone or in combination using retroviral vectors. 184 and 184A 1N4 cells require EGF for anchorage-dependent clonal growth. In mass culture, they secrete TGF alpha at high concentrations and exhibit an attenuated requirement for exogenous EGF/TGF alpha. SV40 T transformed cells have 4-fold increased EGF-R, have acquired the ability to clone in soft agar with EGF/TGF alpha supplementation, but are not tumorigenic. Cells transformed by v-mos or v-Ha-ras are weakly tumorigenic and capable of both anchorage dependent and independent growth in the absence of EGF/TGF alpha. Cells transformed by both SV40 T and v-Ha-ras are highly tumorigenic, are refractory to EGF/TGF alpha, and clone with high efficiency in soft agar. The expression of v-Ha-ras is associated with a loss of the high (but not low) affinity binding component of the EGF-R. Malignant transformation and loss of TGF alpha/EGF responsiveness did not correlate with an increase in TGF alpha production. Thus, TGF alpha production does not appear to be a tumor specific marker for human mammary epithelial cells. Differential growth responses to EGF/TGF alpha, rather than enhanced production of TGF alpha, may determine the transition from normal to malignant human breast epithelium.     

Regulation of autophagy in bovine mammary epithelial cells

The bovine mammary gland undergoes intensive remodelling during the lactation cycle, and the escalation of this process is observed during dry periods. The main type of cell death responsible for bovine mammary gland involution is apoptosis; however, there are also a lot of cells exhibiting morphological features of autophagy during drying off. Our in vitro and in vivo studies of bovine mammary gland physiology suggest that the enhanced process of autophagy, observed at the end of lactation and during dry periods, is the result of: (1) decreased level of lactogenic hormones (GH, IGF-I), (2) decreased GH-R and IGF-IR alpha expression, (3) increased expression of auto/paracrine apoptogenic peptides (IGFBPs, TGFbeta), (4) increased influence of sex steroids (17beta-estradiol and progesterone) and (5) enhanced competition between the between the intensively developing fetus and the mother organism for nutritional and bioactive compounds. The above conditions may create a state of temporary malnutrition of mammary epithelial cells, which forces the cells to the induction of autophagy, as a mechanism for stabilizing intracellular supplies of energy and amino acids, especially during the enhanced activity of apoptogenic factors.     

Transferrin receptor activity in rat mammary epithelial cells

The binding of 125I-transferrin to rat mammary cells isolated by collagenase and hyaluronidase digestion has been investigated. Surface binding was determined at 4 degrees C and total binding also at 4 degrees C but in the presence of 0.1% w/v saponin. KD values between 20 and 25 nM were obtained. Binding assays at 37 degrees C showed the internalisation of the receptor and the bound transferrin was occurring but also provided evidence for an impaired recycling of the receptors to the cell surface in the freshly isolated cells. No differences in total binding were observed in cells prepared at different stages of lactation with a mean value of 29 fmol transferrin bound/micrograms cellular DNA, equivalent to 180,000 receptors per cell.     

Regulation of clusterin expression in mammary epithelial cells

Mammary epithelial cells undergo changes in growth, invasion, differentiation, and dedifferentiation throughout much of adult hood, and most strikingly during pregnancy, lactation, and involution. Clusterin is a multifunctional glycoprotein that is involved in the differentiation and morphogenesis of epithelia, and that is important in the regulation of postnatal mammary gland development. However, the mechanisms that regulate clusterin expression are still poorly understood. Here, we show that clusterin is up-regulated twice during mouse mammary gland development, a first time at the end of pregnancy and a second time at the beginning of the involution. These points of clusterin up-regulation coincide with the dramatic phenotypic and functional changes occurring in the mammary gland. Using cell culture conditions that resemble the regulatory microenvironment in vivo, we determined that the factors responsible for the first up-regulation of clusterin levels can include the extracellular matrix component, laminin, and the lactogenic hormones, prolactin and hydrocortisone. On the other hand, the second and most dramatic up-regulation of clusterin can be due to the potent induction by TGF-beta1, and this up-regulation by TGF-beta1 is dependent on beta1 integrin ligand-binding activity. Moreover, the level of expression of beta-casein, a marker of mammary epithelial cell differentiation, was decreased upon treatment of cells with clusterin siRNA. Overall, these findings reveal several novel pathways for the regulation of clusterin expression during mammary gland development, and suggest that clusterin is a morphogenic factor that plays a key role during differentiation.     

To grow mouse mammary epithelial cells in culture

Normal mouse mammary epithelial cells from Balb/c mice were successfully cultivated on tissue culture plastic with lethally irradiated LA7 feeder cells. The feeder cells also promoted colony formation from single mouse mammary cells, and the fraction of cells that formed colonies was proportional to the density of feeder cells. The mouse mammary cells could be passaged at least 8-12 times as long as new feeder cells were added at each passage. The cells now in culture have doubled in number at least 30 times, but the in vitro lifespan is not yet known. The cultures of mouse cells maintained by this technique never became overgrown with fibroblasts and numerous domes formed in the cultures.     

Characteristics of rat normal mammary epithelial cells and dimethylbenzanthracene-induced mammary adenocarcinoma cells grown in monolayer culture

Summary The characteristics of normal mammary epithelial and 7,12-dimethylbenz[a]anthracene (DMBA)-induced adenocarcinoma cells derived from rats and grown in monolayer culture were compared. Normal mammary epithelial cells exhibited different morphology and agglutinability by plant lectins, slower growth rate, and lower saturation density and cloning efficiency. In addition, the normal cells were sensitive to the toxic effect of DMBA, and were unable to grow in soft agar or to form tumors, when inoculated into newborn Sparague-Dawley rats. The converse was true in each case for the adenocarcinoma cells. Supported by Public Health Service Research Grant CA 01237603 from the National Cancer Institute Portions of this paper were presented at the 65th Annual Meeting of the American Association for Cancer Research at Houston, Texas, 1974.     

Viability maintenance of mammary epithelial cells in vitro

Mammary epithelial cells were isolated from mice lactating for 6 to 10 days and incubated in plastic culture dishes for 10 days. Viability of the cells was tested daily for 8 different treatment regimens including control (Minimum Essential Medium and antibiotics). Tested in cultures were horse serum, a collagen gel matrix, the lactogenic hormones prolactin, insulin, cortisol and all combinations of the above. Effectiveness of treatment was compared each day using the Duncan's New Multiple Range Test (DNMRT) and over the entire 10 day experimental period using regression analysis. After 1 day the collagen gel matrix was the most effective treatment followed by lactogenic hormones and horse serum. On days 2, 4 and 5, horse serum alone was the best treatment while day 3 demonstrated a slight superiority for hormones only. By day 6, and until day 10, a combination of horse serum and hormones maintained viability most successfully. The second and third most effective treatments during this portion of the experimental period were a combination of all three components and hormones alone, respectively. These data support the concept of complex support for mammary epithelial cell viability by a collagen gel matrix accompanied by three known hormones and unknown factors in horse serum.     

Collagen processing in ras-transfected mouse mammary epithelial cells

A mouse mammary epithelial cell line (NMuMG), after transfection with the c-rasH oncogene, forms invasive tumors in nude mice. NMuMG and NMuMG/p-rasH cells produce similar amounts of collagen (mostly type IV) when grown on plastic. NMuMG cells respond to growth on collagen gels by increasing the rate of collagen synthesis and deposition by 100%, unlike NMuMG/p-rasH cells which synthesize similar amounts of collagen whether grown on plastic or collagen gels. These results suggest that ras transformation partially inhibits the interaction between epithelial cells and the surrounding stroma that is necessary for basement membrane deposition in vivo and consequently may facilitate the invasion of the stroma by transfected cells.     

EGF stimulates Mg influx in mammary epithelial cells

Magnesium is well established as a fundamental factor that regulates cell proliferation. However, the molecular mechanisms linking mitogenic signals, extracellular magnesium availability and intracellular effectors are still largely unknown. In the present study we sought to determine whether EGF regulates magnesium homeostasis in normal HC11 mammary epithelial cells. To this end, we measured Mg²⁺ and Ca²⁺ fluxes by confocal imaging in live cells loaded with specific fluorescent ion indicators (Mag-Fluo-4 and Fluo-4, respectively).