Hydrocortisone and progesterone regulation of the proliferation,morphogenesis, and functional differentiation of normal rat mammary epithelial cells in three dimensional primary culture

The mechanisms of action of, and resistance to, the steroidal regulators of normal mammary epithelial and breast cancer cell development are only partially understood. A major obstacle to research progress has been the difficulty in supporting physiologically relevant development of normal mammary epithelial cells (MEC) under defined serum-free conditions. A primary culture system was developed in our laboratory that permits nonfunctional rat MEC to undergo extensive proliferation, functional differentiation, as well as multilobular and lobuloductal branching alveolar morphogenesis. In the studies reported here, the contributions of hydrocortisone and progesterone during the coordinate induction of cellular proliferation, organoid morphogenesis, and functional capacity were assessed. Hydrocortisone (0.1–10 μg/ml) induced alveolar and multilobular branching morphogenesis, suppressed lobuloductal branching morphogenesis, and enhanced casein accumulation. Hydrocortisone also played a role in maintaining alveolar as well as multilobular branching morphogenesis and casein levels. Progesterone (0.01–1 μg/ml) induced cellular proliferation as well as multilobular and lobuloductal branching morphogenesis, and suppressed casein accumulation. At a supraphysiological concentration (10 μg/ml), progesterone inhibited cell growth, alveolar branching morphogenesis, and casein accumulation. MEC cultured without progesterone for up to 1 week retained the ability to respond when subsequently exposed to this steroid. Reversibility studies suggested that progesterone was required for the induction, but not the maintenance of the mitogenic, morphogenic, and lactogenic effects. This physiologically relevant primary culture system can be used to study the factors that regulate steroid responsiveness as well as the cross-talk between steroid and growth factor receptor signaling pathways in normal MEC and breast cancer cells. © 1995 Wiley-Liss, Inc.     

Phorbol 12-myristate 13-acetate stimulates proliferation and ductal morphogenesis and inhibits functional differentiation of normal rat mammary epithelial cells in primary culture

The effect of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on proliferation and differentiation of normal mammary epithelial cells from 50-day-old virgin rats was investigated using a model system that allows for full morphological and functional development of the cells. In this model, mammary epithelial cells are grown within a reconstituted basement membrane in a defined serum-free medium. PMA at a concentration of 10^?6 M effected translocation of protein kinase C from cytosol to membrane. At the same concentration, it stimulated cell proliferation both in the presence and absence of EGF, and this stimulation was observed even when PMA exposure was limited to 15 min at the time of each media change. In contrast to the effect on proliferation, PMA at concentrations of 10^?7 and 10^?6 M inhibited functional differentiation as assessed by casein accumulation. Phorbol 12,13-dibutyrate at 10^?6 M also stimulated proliferation and inhibited casein accumulation and was more effective than PMA in both cases. In contrast, the nonactive tumor promoter 4-α PMA had no effect on either proliferation or differentiation. One of the most striking effects of PMA was its ability to stimulate an atypical ductal morphogenesis, as manifested by the formation of intricate web-like colonies, and to inhibit the development of the well-differentiated alveolar-like multilobular colonies. PMA was also shown to completely suppress the growth of the squamous-like colonies that develop when EGF is absent or deficient. These effects of phorbol esters in mammary epithelial cells to stimulate proliferation, inhibit functional differentiation, and stimulate the development of ductal colonies are consistent with the suggestion that the signal transduction pathways evoked by PMA could act to stimulate the growth of initiated cells or render normal cells more sensitive to carcinogen. © 1994 Wiley-Liss, Inc.     

Mammary fibroblasts stimulate growth, alveolar morphogenesis, and functional differentiation of normal rat mammary epithelial cells

Summary Stromal-epithelial interactions play a profound role in regulating normal and tumor development in the mammary gland. The molecular details of these events, however, are incompletely understood. A novel serum-free transwell coculture system was developed to study the natural paracrine interactions between mammary epithelial cells (MEC) and mammary fibroblasts (MFC) isolated from normal rats during puberty. The MEC were cultured within a reconstituted basement membrane (RBM) in transwell inserts with or without MFC in the lower well. The presence of MFC stimulated epithelial cell growth, induced alveolar morphogenesis, and enhanced casein accumulation, a marker of the functional differentiation of MEC, but did not induced ductal morphogenesis. Potent mitogenic, morphogenic, and lactogenic effects were observed after 1 wk in serum-free medium, fibroblast survival was enhanced significantly when the MFC were cultured within the RBM. Taken together, this in vitro model effectively reconstitutes a physiologically relevant three-dimensional microenvironment for MEC and MFC, and seems ideal for studying the locally derived factors that regulate the developmental fate of the epithelial and fibroblast compartments of the mammary gland.     

Serum-free culture conditions for the growth of normal rat mammary epithelial cells in primary culture

Summary A monolayer culture system has recently been developed for the extended growth and serial passage of normal rat mammary epithelial (RME) cells. In this system the cells undergo greater than 20 population doublings when grown on type I collagen-coated tissue culture dishes in Ham's F12 medium supplemented with insulin, hydrocortisone, epidermal growth factor, prolactin, progesterone, cholera toxin, and 5% fetal bovine serum (FBS). The purpose of the present studies was to define additional growth factors that would allow equivalent RME cell proliferation in serum-free medium. Ethanolamine (EA) was effective at reducing the FBS requirements for RME cell proliferation and at its optimum concentration did so by greater than 20-fold. Even with optimum levels of EA there was essentially no cell proliferation in the absence of FBS. However, addition of bovine serum albumin (BSA) to the hormone, growth factor, and EA-supplemented medium resulted in substantial proliferation in the absence of serum, and the further addition of transferrin (T) potentiated this effect. Thus, in this culture system, replacement of FBS with EA, BSA, and T resulted in RME cell proliferation in primary culture which was equivalent to that obtained in the 5% FBS-containing medium. This work was supported by grant RR-05529 from the Division of Research Resources, National Institutes of Health, Bethesda, MD, and by Public Health Service grant CA40064-01 from the National Cancer Institute, Bethesda, MD.     

Conjugated linoleic acid inhibits proliferation and induces apoptosis of normal rat mammary epithelial cells in primary culture.

The trace fatty acid conjugated linoleic acid (CLA) inhibits rat mammary carcinogenesis when fed prior to carcinogen during pubertal mammary gland development or during the promotion phase of carcinogenesis. The following studies were done to investigate possible mechanisms of these effects. Using a physiological model for growth and differentiation of normal rat mammary epithelial cell organoids (MEO) in primary culture, we found that CLA, but not linoleic acid (LA), inhibited growth of MEO and that this growth inhibition was mediated both by a reduction in DNA synthesis and a stimulation of apoptosis. The effects of CLA did not appear to be mediated by changes in epithelial protein kinase C (PKC) since neither total activity nor expression nor localization of PKC isoenzymes alpha, beta II, delta, epsilon, eta, or zeta were altered in the epithelium of CLA-fed rats. In contrast, PKCs delta, epsilon, and eta were specifically upregulated and associated with a lipid-like, but acetone-insoluble, fibrillar material found exclusively in adipocytes from CLA-fed rats. Taken together, these observations demonstrate that CLA can act directly to inhibit growth and induce apoptosis of normal MEO and may thus prevent breast cancer by its ability to reduce mammary epithelial density and to inhibit the outgrowth of initiated MEO. Moreover, the changes in mammary adipocyte PKC expression and lipid composition suggest that the adipose stroma may play an important in vivo role in mediating the ability of CLA to inhibit mammary carcinogenesis.     

Synthesis of basic fibroblast growth factor upon differentiation of rat mammary epithelial to myoepithelial-like cells in culture

Acidic fibroblast growth factor (aFGF) mRNA was detected in a rat mammary fibroblastic cell line, but not in rat mammary epithelial cell lines or myoepithelial-like cell lines. Basic FGF (bFGF) mRNA was detected in both the fibroblasts and the myoepithelial-like cells, but was absent from the epithelial cells. A series of cell lines representing stages in the differentiation pathway of epithelial cells to a myoepithelial-like morphology showed an increase in the amount of bFGF mRNA and activity present and the FGF from the myoepithelial-like rat mammary 29 cells was able to displace [125I]-bFGF specifically bound to rat mammary fibroblasts. FGF activity was also present in an extract of rat mammary gland. Analysis of cell extracts and conditioned medium indicated that FGF activity was cell-associated. The cell-associated bFGF was resistant to degradation by trypsin. Extraction of myoepithelial-like cells with Triton X-100 and 2 M NaCl showed that 50-65% of the cell-associated bFGF was in a detergent-resistant but 2 M NaCl-labile structure. Thus, the synthesis of bFGF is developmentally regulated in rat mammary cell lines, and at least 50% is present in the extracellular matrix.     

Calcium regulation of normal human mammary epithelial cell growth in culture

Summary The concentration of Ca⁺⁺ in culture media profoundly affected the growth and differentiation properties of normal human mammary epithelial cells in short-term culture. In media where Ca⁺⁺ was above 0.06 mM, longevity was limited to an average of three to four cell divisions. The extended growth fraction (those cells able, to divide more than once) was only approximately 50% and diminished to zero quickly with time. Stationary cells inhibited from dividing appeared differentiated in the formation of lipid vacuoles and accumulation of α-lactalbumin. Growth of stationary cultures could be reinstituted in about half the cells, either by disruption and transfer or by a reduction in Ca⁺⁺ to less than 0.08 mM. The reduction of Ca⁺⁺ to levels below 0.08 mM extended the longevity of normal cells to eight to nine divisions. The extended growth fraction was 100%. Under these conditions, cells did not differentiate. The effects of Ca⁺⁺ on growth and differentiation were specific (Mg⁺⁺ and Mn⁺⁺ variations were without effect) and reversible and in many respects resembled Ca⁺⁺ effects on epidermal cells. One major difference is that the dual pathways of growth and differentiation in mammary cells were controlled by glucocorticoid and insulin. Based on the kinetics of the reversible Ca⁺⁺-induced coupling and uncoupling of proliferation and the program of differentiation, we propose that Ca⁺⁺ may be an essential trigger for cell divisions that commit a mammary cell to differentiate progressively in a permissive hormonal milieu. This study was supported by grants NIH-CA18175 and CA36399 and an institutional grant from the United Foundation of Greater Detroit.     

Appearance of basic fibroblast growth factor receptors upon differentiation of rat mammary epithelial to myoepithelial-like cells in culture

The binding of [125I]-epidermal growth factor (EGF) and [125I]-basic fibroblast growth factor (bFGF) to a number of single-cell cloned rat mammary cell lines was measured using a saturation assay. Similar numbers of high-affinity [125I]-EGF binding sites (KD 1.3 nM) were found in epithelial and myoepithelial-like cell lines. In contrast, high-affinity (KD 35-276 pM) [125I]-bFGF binding sites were present on fibroblastic and myoepithelial-like cell lines but were not detectable on epithelial cell lines. A series of cell lines representing stages in the differentiation pathway of epithelial cells to an elongated myoepithelial-like morphology showed a graded increase in the number of bFGF receptors. The sensitivity of a cell line to stimulation of DNA synthesis by bFGF correlated with the level of expression of bFGF receptors on the cellular surface. Complexes of cell surface receptors affinity-cross-linked to [125I]-bFGF were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In each case two distinct complexes having apparent molecular weights of 180 kDa and 160 kDa were observed.     

Epidermal growth factor stimulates proliferation of mouse uterine epithelial cells in primary culture

Epidermal growth factor (EGF) is one of growth factors that are thought to mediate the stimulatory effects of estrogen on the proliferation of uterine epithelial cells. The present study was attempted to obtain direct evidence for the mitogenic effects of EGF on uterine epithelial cells, and to prove that EGF and EGF receptors are expressed in these cells. Mouse uterine epithelial cells were isolated from immature female mice and cultured with or without EGF for 5 days. EGF (1 to 100 ng/ml) significantly increased the number of uterine epithelial cells, and the maximal growth (141.9+/- 8.3% of controls) was obtained at a dose of 10 ng/ml. In addition, EGF (0.1 to 100 ng/ml) increased the number of DNA-synthesizing cells immunocytochemically detected by bromodeoxyuridine uptake to the nucleus. Northern blot analysis revealed that the uterine epithelial cells expressed both EGF mRNA (4.7 kb) and EGF receptor mRNAs (10.5, 6.6, and 2.7 kb) These results suggest that the proliferation of uterine epithelial cells is regulated by the paracrine and/or autocrine action of EGF. Our previous study demonstrated the mitogenic effect of IGF-I on uterine epithelial cells. To examine whether the EGF- and IGF-I signaling act at the same level in the regulation of the proliferation of uterine epithelial cells, the cultured cells were simultaneously treated with IGF-I and EGF. IGF-I was found to additively stimulate the mitogenic effects of EGF, suggesting that the EGF-induced growth of uterine epithelial cells is distinct from IGF-I-induced growth.     

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.     

Influence of epidermal growth factor and cyclic AMP on growth and differentiation of palatal epithelial cells in culture

A serum-free, hormonally defined medium was developed which supports growth and differentiation in primary culture of epithelial cells from prefusion embryonic mouse palatal shelves. Using this culture system, medial epithelial programmed cell death was investigated. In the absence of EGF, medial epithelial cells undergo cell death and detach from the substratum by 24 hr of culture. The addition of EGF alone or in combination with various agents which increase intracellular cyclic AMP levels prevented medial epithelial cell death in both cell and organ culture. EGF appeared to exert its most dramatic effect in cell culture on growth and differentiation of the squamous oral epithelial cells. In addition, EGF and agents such as 8-bromo-cyclic AMP, dibutyryl cyclic AMP, or cholera toxin synergistically stimulated the appearance of a long-lived, rapidly proliferating cell type by Day 4 of culture. Our results suggest that both EGF and cyclic AMP together may be important in regulating proliferation of embryonic palatal epithelial cells.     

Serum-free primary culture of normal mouse mammary epithelial and stromal cells

Summary An in vitro serum-free culture system provides an important approach to the understanding of local hormonal regulation of mammary epithelial and fibroblast cells, avoiding the complexity of the in vivo environment and the influence of undefined serum factors. The substratum conditions and medium components have been examined for the basal growth of epithelial cells, fibroblasts, and combined epithelial and fibroblast cells in monolayer cultures. Epithelial cells and mixed cells exhibit good attachment and maintenance on a collagen-coated surface in a minimal medium supplemented with fetuin and insulin. In contrast, fibroblast-enriched cultures require a plastic substratum and a medium supplemented with insulin, fetuin, and hydrocortisone. In mixed cell culture, fibroblasts are maintained well in the minimal media which supports the maintenance of epithelial cells. These results indicate that the presence of epithelial cells in mixed cell cultures can influence fibroblast function. The media developed in the present study can be used in future studies of fibroblast and epithelial cell interactions with regard to hormone and growth factor regulation of their growth and differentiation.     

Pyruvate regulation of growth and differentiation in primary cultures of rat tracheal epithelial cells

These studies examined the effect of exogenous pyruvate on the growth and differentiation of primary cell cultures of rat tracheal epithelial cells. The cell cultures were derived from outgrowths of tracheal explants, and require pyruvate for survival and growth in the presence of 10% FBS. In pyruvate-supplemented (2 mM) medium, the number of cells attached to the dish increased rapidly, while exfoliation of cells into the medium as well as formation of cornified envelopes were relatively low. The growth response to pyruvate was concentration-dependent in these cell cultures. In the absence of pyruvate, the extent of terminal differentiation to keratinization gradually increased. This was characterized by a cessation of growth after one week, and an increase in exfoliation until all cells had sloughed from the dish. Accompanying these changes was a marked increase in the formation of cornified envelopes. Cells undergoing DNA synthesis were present throughout 2 weeks of culture in pyruvate-deprived medium, even as the total number of cells was diminishing. Several compounds, including other 2-oxocarboxylic acids, were ineffective growth substitutes for pyruvate. These results indicate that the requirement for pyruvate is quite stringent in these cultures and that one way pyruvate promotes the growth of tracheal epithelial cells is by inhibiting terminal differentiation.     

Maintenance of normal rat mammary epithelial cells by insulin and insulin-like growth factor 1

Normal rat mammary epithelial cells were cultured within a rat tail collagen gel matrix formed under improved conditions for controlling pH and osmolarity. Under these conditions, growth can be maintained for up to 3 weeks with a 10- to 15-fold increase in cell number. The cells grow in response to prolactin, progesterone, epidermal growth factor, and cholera toxin, in a medium of DME: Ham's F12 supplemented with BSA and insulin at 10 micrograms/ml. When the insulin concentration was reduced to more physiological levels (10 ng/ml) the cells did not grow. However, at these more physiological concentrations it could be shown that insulin had a concentration-dependent effect on the maintenance of the cells with an optimum concentration around 25 ng/ml. The cells could be maintained in hormone-supplemented medium with low levels of insulin in a quiescent state for up to 14 days. The high levels of insulin needed for optimal growth could be replaced by insulin-like growth factor 1 (IGF-1) at much lower concentrations (25-50 ng/ml). The superphysiological level of insulin required for optimum growth is probably due to its acting weakly through an IGF-1-mediated growth-promoting mechanism. Insulin's effect on cell maintenance occurs at physiological levels and may better reflect its role in mammary cell growth.     

Development of insulin and epidermal growth factor receptors during the differentiation of rat preadipocytes in primary culture

An homogeneous cell population isolated from the inguinal tissue of 3-day-old rats is able to proliferate in primary culture. In the presence of a physiological concentration of insulin (1.5 nM) it converts into cells exhibiting the morphology and the biochemical characteristics of adipocytes. Insulin and epidermal growth factor (EGF) receptors were studied during both the exponential growth and the adipose conversion phases of these cells. Binding experiments with 125I-labelled peptides were performed directly in the culture dishes. The number of high affinity insulin binding sites increased, during the entire culture period studied, reaching 18 days after plating the value of 10,600 x 2360. Control cells (cultured in the presence of anti-insulin antibody) exhibited an increase of the concentration of insulin binding sites from no more than 500 sites/cell to 6880 +/- 1710 sites/cell between dat 0 and 9 (corresponding to the exponential growth phase); this increase was followed by a rapid reduction in insulin receptors during the stationary phase. The density of EGF binding sites increased between day 0 and 4 (one cell cycle), whether the cells were maintained or not with insulin, and plateaued thereafter. Mature adipocytes freshly isolated from the inguinal tissue of 3-day-old rats had no detectable EGF binding sites, but their content in high affinity binding sites for insulin was similar to that of cells after complete adipocyte conversion in primary culture.     

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.     

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.     

Calcium regulation of growth and differentiation of mouse epidermal cells in culture

Modification of the ionic calcium concentration in the culture medium markedly alters the pattern of proliferation and differentiation in cultured mouse epidermal cells. When medium calcium is lowered to 0.05--0.1 mM, keratinocytes proliferate rapidly with a high growth fraction and do not stratify, but continue to synthesize keratin. The cells grow as a monolayer for several months and can be subcultured and cloned in low Ca++ medium. Ultrastructural examination of cells cultured under low Ca++ conditions reveals widened intercellular spaces, abundant microvilli and perinuclear organization of tonofilaments and cellular organelles. Desmosomes are absent. Epidermal cells growing as a monolayer in low Ca++ can be induced to terminally differentiate by adding calcium to the level normally found in the culture medium (1.2 mM). Cell-to-cell contact occurs rapidly and desmosomes form within 2 hr. The cells stratify by 1--2 days and terminally differentiate with cell sloughing by 3--4 days. After Ca++ addition, DNA synthesis decreases with a lag of 5--10 hr and is totally inhibited within 34 hr. In contrast, RNA and protein synthesis continue at 40--50% of the low Ca++ level at day 3, a time when many cells are detaching from the culture dish. Keratin synthesis is unaffected by the Ca++ switch.     

The mitogenic action of insulin-like growth factor I in normal human mammary epithelial cells requires the epidermal growth factor receptor tyrosine kinase

The signals used by insulin-like growth factor I (IGF-I) to stimulate proliferation in human mammary epithelial cells have been investigated. IGF-I caused the activation of both ERKs and Akt. Activation of ERKs was slower and more transient than that of Akt. ZD1839, a specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, prevented activation of ERKs but not Akt by IGF-I. Inhibition of the EGFR with function-blocking monoclonal antibodies also specifically blocked IGF-I-induced ERK activation. These effects occurred in primary mammary epithelial cells and in two cell lines derived from normal mammary epithelium but not in mammary fibroblasts or IGF-I-responsive breast carcinoma cell lines. Although IGF-I stimulated the proliferation of both normal and carcinoma cell lines, ZD1839 blocked this only in the normal line. ZD1839 had no effect on IGF-I receptor (IGF-IR) autophosphorylation in intact cells. IGF-I-induced ERK activation was insensitive to a broad spectrum matrix-metalloproteinase inhibitor and to CRM-197, an inhibitor of the EGFR ligand heparin-bound epidermal growth factor. EGFR was detectable within IGF-IR immunoprecipitates from normal mammary epithelial cells. Treatment of cells with IGF-I led to an increase in the amount of tyrosine-phosphorylated EGFR within these complexes. ZD1839 had no effect on complex formation but completely abolished their associated EGFR tyrosine phosphorylation. These findings indicate that IGF-I utilizes a novel EGFR-dependent signaling pathway involving the formation of a complex between the IGF-IR and the EGFR to activate the ERK pathway and to stimulate proliferation in normal human mammary epithelial cells. This form of regulation may be lost during malignant progression.     

Interaction of epidermal growth factor with adult rat liver parenchymal cells in primary culture

Adult rat liver parenchymal cells in primary culture exhibit specific saturable binding of ¹²⁵I-labeled murine epidermal growth factor (EGF). The Scatchard plot of the binding data obtained at 36 °C was curvilinear yielding two apparent dissociation constants of 1.5 × 10^?10m and 1.2 × 10^?9m with 27,000 and 57,000 sites per cell, respectively. The binding data obtained at 2 °C yielded a linear Scatchard plot with an apparent dissociation constant of 4.4 × 10^?9m and 78,000 sites per cell. Exposure of the hepatocytes to EGF at 36 °C resulted in a loss of EGF binding capacity due to down regulation of receptors. The cells recovered the capacity to bind EGF upon incubation in medium which did not contain EGF; this recovery was inhibited by cycloheximide. The cultures appeared to internalize and degrade bound EGF at 36 °C but not at 2 °C. The degradation of EGF was inhibited by chloroquine, an inhibitor of lysosomal enzymes. These data indicate that liver specifically binds and further processes EGF, and therefore, may be a physiological target tissue for this growth factor.