Effects Of EGF Research Articles

EGF and TGF alpha influence in vitro lung development by the induction of matrix-degrading metalloproteinases.

Remodeling of the extracellular matrix by matrix-degrading metalloproteinases (MMPs) has been implicated in the early morphogenesis of branched organs. Growth factors such as EGF and TGF alpha are known to regulate the expression of MMPs in a variety of systems. We therefore examined the effects of EGF, TGF alpha, and collagenase upon in vitro branching of the embryonic lung. Lung rudiments from 11.5 day post coitum mice underwent extensive growth and repetitive branching during a 3-day period in organ culture. Lungs treated with EGF or TGF alpha were larger than controls, yet displayed fewer branches along with markedly dilated end buds which lacked clefts, indicating that these growth factors inhibit normal lung branching. Addition of purified mammalian collagenase to lung cultures similarly inhibited epithelial branching and produced end bud enlargement. In addition, gelatin-substrate enzymography of the conditioned medium from EGF- and TGF alpha-treated lungs revealed a marked induction of a metalloproteinase activity which most likely corresponds to the 72kDa type IV collagenase/gelatinase which degrades basement membrane collagens. Lungs maintained in the presence of both TGF alpha and TIMP, a specific inhibitor of MMPs, branched repeatedly and displayed normal, narrow end buds as seen with controls, suggesting that TIMP is capable of preventing or reversing the observed growth factor mediated effects upon lung branching. Taken together, these results provide evidence that the growth factors EGF and TGF alpha guide lung development, at least in part, by inducing the expression of matrix-degrading metalloproteinases.

An in Vitro Model for the Study of Human Hair Growth

Human anagen hair follicles were isolated by microdissection from human scalp skin. Isolation of the hair follicles was achieved by cutting the follicle at the dermosubcutaneous fat interface using a scalpel blade. Intact hair follicles were then removed from the fat using watch makers' forceps. Isolated hair follicles maintained free floating in supplemented Williams E medium in individual wells of 24-well multiwell plates showed a significant increase in length over 4 days. The increase in length was seen to be attributed to the production of a keratinized hair shaft, and was not associated with the loss of hair follicle morphology. [Methyl-3H]thymidine autoradiography confirmed that in vitro the in vivo pattern of DNA synthesis was maintained; furthermore, [35S]methionine labeling of keratins showed that their patterns of synthesis did not change with maintenance. Serum was found to inhibit hair follicle growth in vitro; and when follicles were maintained in serum-free medium, they grew for up to 10 days, suggesting that in vitro the hair follicles are able to regulate their own growth, possibly by the production of relevant growth factors. This may prove useful in identifying the autocrine/paracrine mechanisms that operate in the hair follicle. The importance of this model to hair follicle biology is further demonstrated by the observations that TGF-beta 1 has a negative growth regulatory effect on hair follicles in vitro and that EGF and its other receptor ligand TGF-alpha mimic the in vivo depilatory effects of EGF that have been reported for sheep and mice.

Effect of epidermal and insulin-like growth factors on vectorial secretion of transferrin by rat Sertoli cells in vitro

Within the seminiferous tubules, the Sertoli cells create an impermeable blood-testis barrier and an unique intratubular microenvironment that fosters the development of spermatozoa. The functional differentiation of spermatozoa therefore requires vectorial secretion by Sertoli cells of substances that cannot cross the blood-testis barrier. We investigated the role of epidermal (EGF) and insulin-like growth factors I and II (IGF-I and IGF-II) in the regulation of vectorial secretion of transferrin by Sertoli cells. In order to study the regulation of vectorial transferrin secretion, we modified culture conditions in the twin chamber culture system to maximise gradients of transferrin secretion. Sertoli cells were plated at high density (3−4 × 10 6 cells/well) into chambers of near equal volume, cultured at 37°C and maintained in simple, fully defined media omitting standard supplements (insulin, EGF, FSH) which affect vectorial transferrin secretion. Using this optimised culture system, maximum gradients of transferrin secretion occurred between days 2 and 3 of culture with preferential secretion (mean ratio 3.7 ± 0.2) directed towards the apical compartment. The transferrin ratio (ratio of transferrin secreted into the upper over the lower chamber) was decreased by insulin and FSH but not by retinoic acid or testosterone, yet all four stimuli increased total transferrin secretion. IGF-I and IGF-II were effective at physiological concentrations (ED 50 = 1 ng/ml) in lowering transferrin ratio and were 100-fold more potent than insulin suggesting that insulin effects on vectorial transferrin secretion by Sertoli cells is mediated through type 1 IGF receptors. EGF also reduced the transferrin ratio (ED 50 = 50 ng/ml) as well as stimulating total transferrin secretion. The hormonally mediated reduction in transferrin ratio was consistently due to enhanced secretion of transferrin into the lower chamber. In the first demonstration of a highly polarised response of Sertoli cells to hormonal stimuli, the effects of insulin, FSH and EGF on vectorial transferrin secretion were effected primarily via the basal membrane of the Sertoli cell and operated independent of mechanisms controlling total transferrin secretion. These results establish a potential role for epidermal and insulin-like growth factors in the paracrine regulation of vectorial secretion by the Sertoli cell, in particular the developmental regulation of vectorial transferrin secretion by Sertoli cells. These findings also indicate that previous studies which included insulin and EGF routinely in culture media have systematically underestimated apically directed transferrin secretion.

Proliferation of fetal brown adipocyte primary cultures: Relationship with the genetic expression of glucose 6 phosphate dehydrogenase

Fetal brown adipocyte primary cultures increase DNA synthesis; cell number; and DNA, RNA, and protein contents in response to 10% fetal calf serum, IGF-I, and EGF plus vasopressin plus bombesin when added for 64 h to quiescent cells. IGF-I is a complete growth factor in this system while EGF needs the presence of vasopressin plus bombesin for its maximal proliferative effects. These mitogens induce the genetic expression of G6P dehydrogenase, increasing its mRNA content as well as its specific activity and amount of immunoreactive protein. The presence of cAMP elevating agents prevents the stimulatory effect of EGF plus vasopressin plus bombesin on DNA synthesis, cell number, and DNA content as well as on the induction of G6P dehydrogenase expression. Thus, changes on the proliferative state of these cells are associated with the level of expression of G6P dehydrogenase.

Synergistic stimulatory effect of glucocorticoid, EGF and insulin on the synthesis of ribosomal RNA and phosphorylation of nucleolin in primary cultured rat hepatocytes

Effects of dexamethasone, EGF and insulin on the synthesis of rRNA and phosphorylation of nucleolin in primary cultures of adult rat hepatocytes were studied. Hepatocytes were incubated for 8 h with EGF (20 ng/ml) plus insulin (0.1 μM) and/or for 20 h with dexamethasone (1 μM) before the end of incubation. The incorporation of [ 3H]uridine into acid-insoluble materials and the nuclear activity of RNA polymerase I were stimulated approx. 2-fold with EGF plus insulin and these were further enhanced 2–3-times by dexamethasone, although dexamethasone alone exerted no stimulation. When hepatocytes were incubated with [ 32P]orthophosphate, similar enhancement by these hormones was also observed in the phosphorylation of a nucleolar protein, nucleolin, which was detected by immunoprecipitation with anti-nucleolin antibodies. The amount of nucleolin was slightly increased by EGF plus insulin in the presence of dexamethasone, but scarcely changed by treatment with EGF plus insulin or dexamethasone alone. Cycloheximide inhibited RNA synthesis to a greater or lesser degree in the case of all hepatocytes which were cultured with or without these hormonal treatments. These results indicate that the in vivo effect of glucocorticoid on rRNA synthesis and nucleolin phosphorylation in liver is primarily a direct action on parenchymal cells and requires other growth factors such as EGF and insulin.

Lack of correlation between changes in polyphosphoinositide levels and actin/gelsolin complexes in A431 cells treated with epidermal growth factor.

The polyphosphoinositides, PIP and PIP2, have been proposed to regulate actin polymerization in vivo because they dissociate actin/gelsolin complexes in vitro. We tested this hypothesis by comparing the ability of EGF and bradykinin to affect PI metabolism and the actin cytoskeleton in A431 cells. EGF, but not bradykinin, was found to induce ruffling and dissociation of actin/gelsolin complexes in these cells. However, both EGF and bradykinin stimulated the accumulation of inositol phosphates in [3H]inositol-labeled cells indicating that stimulation of PI turnover is not sufficient for the induction of changes in actin/gelsolin complex levels. EGF stimulated a twofold increase in the level of PIP in A431 cells. Other phosphoinositide levels were not markedly altered. Treatment of the cells with cholera toxin abrogated the EGF-induced rise in PIP levels without altering the dissociation of actin from gelsolin. These data indicate that increases in PIP and/or PIP2 are not necessary for dissociation of actin/gelsolin complexes. Overall, these experiments suggest that in A431 cells, the effects of EGF on the actin cytoskeleton are unlikely to be mediated through changes in PIP or PIP2 levels.

Inhibition of T47D human breast cancer cell growth by the synthetic progestin R5020: effects of serum, estradiol, insulin, and EGF

The mechanism of the antiproliferative effects of progestins on human breast cancer cells is not known. In view of the ability of estrogen to stimulate human breast cancer cell production of peptide growth factors, and since previous studies have suggested that the inhibitory action of progestins is dependent on estrogen-stimulated growth, the present study examined the interaction of growth factors and the synthetic progestin R5020 on the proliferation of T47D human breast cancer cells. In this study, the concentrations of estradiol, insulin, and EGF for optimal stimulation of T47D cell growth in 3% dextran-charcoal treated fetal bovine serum (DCC-FBS) were determined to be 1 nM, 100 nM, and 1 nM, respectively. Furthermore, incubation with these optimal concentrations of estradiol, insulin, and EGF in various combinations produced additive effects on T47D cell proliferation, suggesting that these agents act, at least in part, by different mechanisms. In contrast, in a chemically defined medium (DM), both estradiol and EGF were unable to stimulate T47D cell proliferation. In the case of estradiol, the inability to demonstrate stimulation of T47D cell growth in DM was not due to down-regulation of the estrogen receptor. R5020 inhibited the growth of T47D cells, although its effect was more marked in the presence of 3% DCC-FBS than in DM. Stimulation of T47D cell growth by either estradiol or insulin in 3% DCC-FBS was effectively inhibited by R5020. In contrast, growth of T47D cells stimulated by EGF in the absence of estradiol was not markedly inhibited by R5020, the growth being comparable to that of untreated control cells. These findings suggest that the inhibitory effect of R5020 on T47D cell proliferation is dominant over the action of some, but not all, breast cancer mitogens.

Comparative biochemistry of mouse and chick secondary-palate development in vivo and in vitro with particular emphasis on extracellular matrix molecules and the effects of growth factors on their synthesis

A biochemical study analysing the wet weight, dry weight, water, protein and DNA content, collagen and GAG composition of all stages of the developing secondary palate in vivo and in vitro was undertaken to investigate differences between a species in which the palatal shelves elevate (mouse) and one in which they do not (chick). The effects of EGF, bFGF, PDGF and TGF- β 1 on collagen and GAG synthesis by cultured mouse and chick palatal shelves of different embryonic stages were also studied. The total GAG content of developing mouse palatal shelves decreased with developmental time; heparan sulphate proteoglycan formed the major species in early palates but hyaluronan was the major species in mid-late palates. There was a peak of hyaluronan synthesis in embryonic palatal shelves in vitro at day 13 (T-21), i.e. immediately before shelf elevation. By contrast the total GAG content of chick palates increased with development; chondroitin-6-sulphate formed the major GAG species and there was no peak in hyaluronan synthesis. The water content of developing murine palates rose rapidly at day 14 (T.22), i.e. the time of shelf elevation. No such peak was seen in the chick, where the water content rose exponentially with developmental time. Mouse palates synthesized chondroitin-4-sulphate and novel proteins around the time of shelf elevation; chick palates synthesized chondroitin-6-sulphate and no novel proteins at any developmental stage. Collagen synthesis also peaked in vitro in T.21 murine palates. EGF markedly stimulated murine palatal collagens and GAG synthesis between stages T.20–T.22, but had no effect thereafter. Basic FGF had similar but smaller stage-related effects. PDGF had no effect on mouse palatal collagen and GAG synthesis whilst TGF- β 1 inhibited GAG synthesis at T.21. The ratios of collagens I, III and V produced by mouse palates were unaltered by the growth factors. All the growth factors had no effect on chick palatal collagen synthesis at any stage and minimal effect on GAG synthesis; TGF- β 1 stimulated it in early but inhibited it in mid- to late-stage chick palates. These data indicate that extracellular matrix molecule metabolism within the palate is markedly different in the two species studied and suggest that the differing profiles of such molecules may be regulated at certain developmental stages by specific growth factors.

Epidermal Growth Factor Induces the Functional Expression of Dopamine Receptors in the GH3 Cell Line*

GH3 cells are a clonal strain from a rat pituitary tumor that synthesizes and secretes both PRL and GH. The peculiarity of these cells is that they do not express receptors for dopamine; thus the hormone release is insensitive to the inhibitory effect of dopamine and D2 receptor agonists. Exposure of GH3 cells to epidermal growth factor for 4 consecutive days markedly altered the cell morphology, from a spherical appearance to an elongated flattened shape, and increased the cell size. These morphological changes were accompanied by the functional expression of D2 dopamine receptors as shown by the presence of a specific, saturable, and stereoselective high affinity binding for [3H]spiroperidol in epidermal growth factor-treated cells and by the fact that the selective D2 agonist quinpirole recovered the property to inhibit PRL secretion in the cell cultures exposed to the neurotrophic factor. The effect of EGF on the functional expression of D2 receptors was dose dependent (EC50 = 8 pM) and reversible. These data suggest that EGF elicits major effects on the expression of specific genes leading to the differentiation of GH3 cells into lactotroph-like cells endowed with dopamine D2 receptors.

Studies on the Effect of Thyroid Hormone and Epidermal Growth Factor on the Cultured Human Cytotrophoblast.

We have previously reported that human placental cytotrophoblasts (C-cells) contain nuclear 3,5,3'-triiodo-L-thyronine (T3) receptors. Using a C-cell culture system, the present study was undertaken to clarify some of the effects of T3 and EGF on trophoblastic cells. C-cells were purified from human term placenta by treatment with trypsin-DNAse and percoll gradient centrifugation aggregated, then fused, differentiating into multinuclear syncytiotrophoblasts (S-cells) with incubation times up to 96 h in vitro. As the incubation time increased, the number of immunocytochemically reactive cells with antibodies to hCG-alpha, hCG-beta and hPL increased. Anti-EGF antibody reacted only with the initial C-cells, while anti-EGF receptor antibody reacted only with fused S-cells. Maximum secretion of hCG and hCG-alpha by the cultured cells was evident only when the cells were cultured in T3 (10(-8)M) or EGF (10 ng/ml) containing medium. When the initial cells were exposed to 10(-8) M T3 from 0 to 48 h of incubation, the secretion in 48-96 h was significantly accelerated. However, exposure from 48 to 96 h had no effect on peptide excretion. Although an exposure of these cells to 10 ng/ml EGF during 48-96 h of incubation stimulated the secretion of hCG and hCG-alpha, 0-48 h exposure did not produce any positive effect regardless of incubation time. These results indicated that the main target cell of T3 is the C-cell, while that of EGF is the S-cell. Furthermore, it is suggested that the interaction between T3 and its receptor facilitated functional cell differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of the Alzheimer Amyloid Precursor in Brain Tissue and Effects of NGF and EGF on Its Metabolism

Alzheimer's disease (AD) is the major cause of serious dementia and memory loss in several million elderly Americans. The most prominent lesions in the brains of these patients are the depositions of two types of abnormal filaments: the predominantly intraneuronal neurofibrillary tangles (NFT), which consist of paired helical filaments (PHF), and the extracellular amyloid fibers. These changes are characteristic of AD, and a final diagnosis of this disease is based on the presence of large numbers of these abnormal filamentous structures in the patient's brain. The amyloid fibers consist of a peptide subunit termed beta-protein or A4 peptide, which derives from a larger precursor protein. In this study we review the structural characteristics, regulation of expression, and metabolism of the Alzheimer amyloid precursor in brain tissue and cell cultures.

Cooperative enhancement of hyaluronic acid synthesis by combined use of IGF-I and EGF, and inhibition by tyrosine kinase inhibitor genistein, in cultured mesothelial cells from rabbit pericardial cavity

We examined the combined effects of IGF-I and EGF on hyaluronic acid synthesis using cultured pericardial mesothelial cells from rabbit pericardial cavity. Combined exposure of pericardial cells to IGF-I and EGF cooperatively increased hyaluronic acid synthesis and the level of hyaluronic acid synthase activity over that seen with or without IGF-I or EGF alone, but did not significantly stimulate the sulfated glycosaminoglycan synthesis. Increase in the level of hyaluronic acid synthesis and hyaluronic acid synthase activity induced by these growth factors was blocked after pretreatment with a tyrosine-specific protein kinase inhibitor, genistein. Genistein had no direct inhibitory effect on hyaluronic acid synthase activity. These results suggest that cooperative enhancement of hyaluronic acid synthesis induced by combined use of IGF-I and EGF could be mediated by a receptor tyrosine kinase-involved transmembrane signaling process that is responsive to IGF-I and EGF.

Regulation of growth of LNCaP human prostate tumor cells by growth factors and steroid hormones

The mitogenic activity of several growth factors on androgen responsive LNCaP human prostate tumor cells was studied. A two-fold stimulation of cell proliferation was observed after a culture period of 6 days in 1 ng EGF/ml, 10 ng TGF-α/ml or 20 ng basic FGF/ml. TGF-β (0.02 ng/ml), which did not affect cell proliferation when added alone to the culture medium, inhibited the EGF- and TGF-α-induced growth. The synthetic androgen R1881 (0.1 nM) stimulated cell proliferation three-fold and increased the number of EGF receptors from 11500 to 28500 sites/cell. One of the mechanisms involved in androgen action on these cells in therefore an increased EGF receptor expression and increased sensitivity to EGF. TGF-β did not directly affect androgen-responsive growth but inhibited the synergistic effect of EGF. A considerable expression of TGFα (precursors) could be demonstrated on the cells by immunohistochemical staining. However the staining intensity was not affected by androgens. These results make it less likely that androgen-responsive growth is mediated by regulation of secretion of an EGF- or TGFga-likely activity, which in turn acts in an autocrine manner to stimulate growth. Estrogens, progestagens and antiandrogens do not inhibit androgen responsive growth of LNCaP cells but have striking growth stimulatory effects, increase EGF receptor level and increase acid phosphatase secretion. LNCaP cells contain a modified androgen receptor system with respect to both steroid specificity and antiandrogen sensitivity. It has recently been shown that the stimulatory effects are due to a mut of the androgen receptor.

Effect of EGF on polarized iodide efflux and pseudopod formation in thyroid follicular epithelium

Effect of EGF on polarized iodide efflux and pseudopod formation in thyroid follicular epithelium

Differentiation expression during proliferative activity induced through different pathways: in situ hybridization study of thyroglobulin gene expression in thyroid epithelial cells.

In canine thyrocytes in primary culture, our previous studies have identified three mitogenic agents and pathways: thyrotropin (TSH) acting through cyclic AMP (cAMP), EGF and its receptor tyrosine protein kinase, and the phorbol esters that stimulate protein kinase C. TSH enhances, while EGF and phorbol esters inhibit, the expression of differentiation. Given that growth and differentiation expression are often considered as mutually exclusive activities of the cells, it was conceivable that the differentiating action of TSH was restricted to noncycling (Go) cells, while the inhibition of the differentiation expression by EGF and phorbol esters only concerned proliferating cells. Therefore, the capacity to express the thyroglobulin (Tg) gene, the most prominent marker of differentiation in thyrocytes, was studied in proliferative cells (with insulin) and in quiescent cells (without insulin). Using cRNA in situ hybridization, we observed that TSH (and, to a lesser extent, insulin and insulin-like growth factor I) restored or maintained the expression of the Tg gene. Without these hormones, the Tg mRNA content became undetectable in most of the cells. EGF and 12-0-tetradecanoyl phorbol-13-acetate (TPA) inhibited the Tg mRNA accumulation induced by TSH (and/or insulin). Most of the cells (up to 90%) responded to both TSH and EGF. Nevertheless, the range of individual response was quite variable. The effects of TSH and EGF on differentiation expression were not dependent on insulin and can therefore be dissociated from their mitogenic effects. Cell cycling did not affect the induction of Tg gene. Indeed, the same cell distribution of Tg mRNA content was observed in quiescent cells stimulated by TSH alone, or in cells approximately 50% of which had performed one mitotic cycle in response to TSH + insulin. Moreover, after proliferation in "dedifferentiating" conditions (EGF + serum + insulin), thyrocytes had acquired a fusiform fibroblast-like morphology, and responded to TSH by regaining a characteristic epithelial shape and high Tg mRNA content. 32 h after the replacement of EGF by TSH, cells in mitosis presented the same distribution of the Tg mRNA content as the rest of the cell population. This implies that cell cycling (at least 27 h, as previously shown) did not affect the induction of the Tg gene which is clearly detectable after a time lag of at least 24 h. The data unequivocally show that the reexpression of differentiation and proliferative activity are separate but fully compatible processes when induced by cAMP in thyrocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential effects of NGF, FGF, EGF, cAMP, and dexamethasone on neurite outgrowth and sodium channel expression in PC12 cells

PC12 cells are a pheochromocytoma cell line that can be made to differentiate into sympatheticlike neurons by nerve growth factor (NGF). An essential component of the NGF-induced differentiation is the development of action potentials and sodium channels. Using whole-cell clamp we have confirmed that NGF produces a 5- to 6-fold increase in sodium channel density. The sodium channels induced by NGF are not different from those in cells not treated with NGF and are similar to those in other cell types. Basic fibroblast growth factor (FGF), another growth factor that causes PC12 cells to differentiate into sympathetic-like neurons, also produces a 5- to 6-fold increase in sodium current density with channels indistinguishable from those in PC12 cells treated and not treated with NGF. Basic FGF produces the same or somewhat larger increase in sodium channel density but much less neurite outgrowth. In contrast, epidermal growth factor does not produce neurite outgrowth but induces a small, reproducible increase in sodium channel density. Cyclic AMP produces spike-like processes but not neurites and results in a decrease in sodium current and sodium current density. Dexamethasone, a synthetic glucocorticoid, inhibits the increase in sodium current and sodium current density but does not antagonize the neurite outgrowth induced by NGF. Thus, although the increase in sodium channel expression induced by NGF and basic FGF parallels the changes in morphology that lead to neurite outgrowth, it clearly does not depend on them. The results show that different aspects of neuronal differentiation might be independently regulated by the microenvironment.

Activation of Cancer Cell Proteases and Cytotoxicity by EGF and PDGF Growth Factors

The biological effects of EGF and PDGF growth factors on A172 and hEGFr-3T3 cell lines were studied using RBC induced cytolysis and polyacrylamide-gelatin gel electrophoresis assays. The authors report that growth factor-induced cytotoxicity in these cells is mediated by proteolytic enzymes. Treatment of A172 cells with either EGF or PDGF resulted in marked increase of their cytotoxicity (Release Index = 150%). Similarly, RBC induced release index by hEGFr-3T3 cells was elevated to 420% in the presence of 3.4 pM of EGF. However, in A172 cells, PDGF did not have a significant effect on DNA and protein synthesis indicating that stimulation of proteolytic activity is independent of the growth factor signaling pathway. Growth factor induced cytotoxicity was significantly reduced by protease inhibitors in both cell lines. Using EDTA and leupeptin several proteolytic species were identified and localized to cellular membranes as evidenced by polyacrylamide-gelatin electrophoresis assay. These data suggest that growth factors regulate the activation or secretion of proteolytic enzymes in cancer cells and may mediate the invasive and metastatic behavior of these cells.

Effect of EGF on the proliferation of human epithelial cancer cell lines: Correlation with the occupation of EGF receptors

Effect of EGF on the proliferation of human epithelial cancer cell lines: Correlation with the occupation of EGF receptors

Inhibition of mouse mammary ductal morphogenesis and down-regulation of the EGF receptor by epidermal growth factor

EGF, initially demonstrated to be a potent mitogen for a variety of cell types, has more recently been shown to inhibit proliferation of several cell lines. Few studies, however, have addressed the effects of EGF on growth and morphogenesis of tissues in vivo, particularly with regard to EGF as a possible inhibitor. We now demonstrate that EGF treatment of vigorously growing mammary ducts, administered directly to the glands by slow release plastic implants, inhibited normal ductal growth. Inhibition was restricted to the region around the implant and untreated glands in the same animal were normal, indicating direct effects of EGF. EGF-treated end buds were smaller and demonstrated reduced levels of DNA synthesis, although remnants of a stem (cap) cell layer persisted. Full inhibition of growth occurred within 3 days of implantation and required extended exposure to EGF, since treatment of 5 hr or less had no effect on ductal growth. At the lower inhibitory doses tested, growth resumed within 8 days, indicating reversibility of inhibition. No lobuloalveolar or hyperplastic response was seen. 125I-EGF autoradiography revealed that ductal growth inhibition was preceded by the disappearance of EGF receptors located in the cap cell layer of the end bud epithelium and in stromal cells adjacent to the buds. These results, in conjunction with our previous evidence demonstrating the growth-stimulatory effect by EGF on nonproliferating mammary ducts, suggest a growth regulatory role for EGF in mouse mammary ductal morphogenesis.

Role of transforming growth factor-α-related peptides in the autocrine/paracrine control of experimental breast cancer growthin vitro by estradiol, prolactin, and progesterone

We have recently suggested that estradiol (E2), prolactin (oPrl), and progesterone (Pg) support the growth of the hormone-responsive N-nitrosomethylurea (NMU) rat mammary tumor in soft agar through autocrine/paracrine mechanisms. To gain insight into the nature of these hormonally regulated growth factors, we tested the ability of two monoclonal antibodies (MAb-425 and 528) directed against the epidermal growth factor receptor (EGF-R) to inhibit the colony-stimulating effects of conditioned media (CM) obtained from E2, oPrl, and Pg-treated NMU rat mammary tumors. Since both MAbs are specific for human EGF-R, MCF-7 breast cancer cells grown in soft agar in the absence of serum were used as our indicator system. Both MAb-425 and 528 totally abolished the colony-stimulating effect of genuine EGF, while having no agonistic/antagonistic action when added alone. Both MAb-425 and 528 markedly inhibited the colony-stimulating effect of rat mammary tumor E2-CM in a dose-dependent fashion. MAb-425 was also found to inhibit the growth-promoting action of Pg-CM, although this effect appeared to be somewhat less consistent and pronounced than that observed with E2-CM. In contrast, the colony-stimulating effect of Prl-CM was only rarely and, usually, modestly affected by the addition of either MAb-425 or 528. Our data suggest that in the NMU mammary tumor grown in soft agar, EGF/TGF alpha-related peptides are produced upon exposure to E2 and possibly Pg but only rarely following Prl administration. The possible role of these growth factors as mediators of hormonal effects in our experimental system remains to be established.