Transformed growth phenotype of mouse mammary epithelium in primary culture induced by specific fetal mesenchymes.

When mesenchyme from fetal mammary or salivary gland is implanted into adult mouse mammary gland, adjacent epithelium responds with intense hyperplasia. The hyperplastic cells are more vulnerable than are non-stimulated cells to transformation in vivo by a chemical carcinogen or by mammary tumor virus. This system provides a potentially useful model for determining how stroma contributes to mammary tumorigenesis. We have developed co-culture systems and used them to investigate in more detail the nature of the signal produced by the mesenchyme cells. Monolayers of mesenchyme cells were prepared on tissue-culture wells. The mesenchyme cells were trapped on the surface by a thin overlay of agarose. Primary mammary epithelial cells were cultured atop this barrier layer, either as organoids in collagen gels for assessment of anchorage-dependent growth, or as single-cell dispersions in soft agarose for assessment of anchorage-independent growth. Our procedures for assay of anchorage-independent growth allow us for the first time to detect and measure this transformation-defining characteristic in non-immortalized mammary epithelial cells in primary culture. Fetal mammary fat pad precursor tissue and fetal salivary mesenchyme both stimulated anchorage-dependent growth of mammary epithelium, with cell number increasing as much as fifteenfold during a 6-day culture period. These same fetal tissues also stimulated anchorage-independent growth of the mammary epithelial cells, with colony-forming efficiencies of up to 40% in co-cultures with salivary mesenchyme. No colonies formed in the absence of mesenchyme. Cells of colonies contained keratin, which indicates that the colonies grew from epithelial cells and not from a contaminant of another cell type. When co-cultured epithelial cells were subsequently re-cultured in the absence of mesenchyme, they lost their ability to grow independent of anchorage. No colonies grew in co-cultures with fetal cells from heart, kidney, or lung, which is consistent with the lack of stimulation by these tissues in the mammary gland in vivo. A tumor promoter, 12-O-tetradecanoylphorbol acetate (TPA), also caused anchorage-independent growth of the dispersed mammary epithelial cells. Culture medium conditioned by primary or early-passage salivary mesenchyme cells was capable of stimulating growth under both anchorage-dependent and anchorage-independent conditions, confirming that these effects are mediated by a paracrine factor. The results indicate that stimulatory fetal mesenchymes produce soluble molecules that act analogously to transforming growth factors.     

Secretion of an epidermal growth factor-like growth factor by epidermal growth factor-independent rat mammary carcinoma cells.

Rat mammary carcinoma (RMC) cells derived from serially transplantable mammary tumors are independent of epidermal growth factor (EGF) for long-term growth in serum-free medium. This phenotype is in contrast to that of normal mammary epithelial cells or cells derived from nontransplantable tumors that express an absolute requirement for EGF for growth in culture. The results of the experiments reported here indicate that EGF-independent RMC cells secrete a growth factor with potent EGF-like mitogenic activity. Conditioned media obtained from these cells can substitute for EGF for the growth of the EGF-dependent cell line MCF-10. This growth factor is neither EGF nor transforming growth factor alpha and does not compete with 125I-EGF for binding to EGF receptors. Phosphotyrosine Western blot analysis of lysates obtained from EGF-independent RMC cells revealed the presence of a 190 kilodalton (kDa) protein that was distinct from the EGF receptor. Similarly, growth of MCF-10 cells to confluence in serum-free medium supplemented with conditioned medium growth factor in place of EGF resulted in the disappearance of the EGF receptor band and appearance of the 190 kDa band in phosphotyrosine Western blots. The 190 kDa tyrosine-phosphorylated protein detected in cells stimulated by the conditioned medium factor is unlikely to be the c-erbB-2 protein, as indicated by negative results in immunoprecipitation experiments and in vitro kinase assays. In summary, EGF-independent RMC cells secrete a factor with potent EGF-like mitogenic activity. This suggests that an autocrine loop involving this growth factor mediates EGF independence in these cells.     

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.     

Localization and quantitation of 125I-epidermal growth factor binding in mouse embryonic tooth and other embryonic tissues at different developmental stages

We have shown earlier that epidermal growth factor (EGF) inhibits morphogenesis and cell differentiation in mouse embryonic teeth in organ culture. This inhibition depends on the stage of tooth development so that only teeth at early developmental stages respond to EGF (A-M. Partanen, P. Ekblom, and I. Thesleff (1985) Dev. Biol. 111, 84-94). We have now studied the quantity and pattern of EGF binding in teeth at various stages of development by incubating the dissected tooth germs with 125I-labeled EGF. Although the quantity of 125I-EGF binding per microgram DNA stays at the same level, localization of 125I-EGF binding by autoradiography reveals that the distribution of binding sites changes dramatically. In bud stage the epithelial tooth bud that is intruding into the underlying mesenchyme has binding sites for EGF, but the condensation of dental mesenchymal cells around the bud does not bind EGF. At the cap stage of development the dental mesenchyme binds EGF, but the dental epithelium shows no binding. This indicates that the dental mesenchyme is the primary target tissue for the inhibitory effect of EGF on tooth morphogenesis during early cap stage. During advanced morphogenesis the binding sites of EGF disappear also from the dental papilla mesenchyme, but the dental follicle which consists of condensed mesenchymal cells surrounding the tooth germ, binds EGF abundantly. We have also studied EGF binding during the development of other embryonic organs, kidney, salivary gland, lung, and skin, which are all formed by mesenchymal and epithelial components. The patterns of EGF binding in various tissues suggest that EGF may have a role in the organogenesis of epitheliomesenchymal organs as a stimulator of epithelial proliferation during initial epithelial bud formation and branching morphogenesis. The results of this study indicate that EGF stimulates or maintains proliferation of undifferentiated cells during embryonic development and that the expression of EGF receptors in different organs is not related to the age of the embryo, but is specific to the developmental stage of each organ.     

Loss of growth responsiveness to epidermal growth factor and enhanced production of alpha-transforming growth factors in ras-transformed mouse mammary epithelial cells

A mouse mammary epithelial cell line, NMuMG, exhibits a low capacity to grow in semisolid medium as colonies and it is not tumorigenic in nude mice. In contrast, NMuMG cells which have been transformed by an activated c-Harvey ras proto-oncogene, NMuMG/rasH, or by the polyoma middle T-transforming gene, NMuMG/pyt, are able to grow in soft agar and, when injected into nude mice, produce undifferentiated carcinomas. Human epidermal growth factor (EGF) or human alpha-transforming growth factor (alpha TGF) can stimulate, in a dose-dependent fashion, the anchorage-independent growth of NMuMG and NMuMG/pyt cells in soft agar but fail to enhance the anchorage-independent growth of the NMuMGrasH cells. Likewise, human EGF or human alpha TGF is also able to stimulate the anchorage-dependent growth of normal NMuMG cells and NMuMG/pyt cells in a serum-free medium supplemented with insulin, transferrin, fetuin, and laminin, or in medium containing low concentrations of serum, whereas these same growth factors under comparable culture conditions have little or no effect upon the anchorage-dependent growth of the ras-transformed NMuMG-rasH cells. The biological refractoriness of the NMuMG/rasH cells to human EGF or human alpha TGF is reflected by a reduction in the total number of cell surface receptors for EGF and by an absence of a high-affinity population of binding sites for mouse [125l]EGF on these cells as compared to the NMuMG or NMuMG/pyt cells. In addition, concentrated conditioned medium (CM) obtained from NMuMG/rasH and NMuMG/pyt cells contains a relatively higher amount of biologically active TGFs than CM obtained from comparably treated NMuMG cells as measured by the ability to induce the anchorage-independent growth of normal rat kidney cells in soft agar. The higher levels of biologically active TGFs found in the CM from the transformed cells relative to the NMuMG cells is paralleled by a corresponding increase in the CM from these cells in the amount of immunoreactive alpha TGF, by an increase in the amount of EGF receptor-competing activity, and by an increase in the levels of alpha TGF mRNA in the NMuMG/rasH cells. These results demonstrate that mammary epithelial cells which have been transformed by an activated ras proto-oncogene, but not by the polyoma middle T-transforming gene, become unresponsive to exogenous EGF or alpha TGF.(ABSTRACT TRUNCATED AT 400 WORDS)     

Epidermal growth factor receptor, platelet-derived growth factor receptor, and c-erbB-2 receptor activation all promote growth but have distinctive effects upon mouse mammary epithelial cell differentiation.

Three different receptor tyrosine kinases, epidermal growth factor (EGF), c-erbB-2/neu, and platelet-derived growth factor (PDGF) receptors, have been found to be present in the mouse mammary epithelial cell line HC11. We have investigated the consequences of receptor activation on the growth and differentiation of HC11 cells. HC11 cells are normal epithelial cells which maintain differentiation-specific functions. Treatment of the cells with the lactogenic hormones glucocorticoids and prolactin leads to the expression of the milk protein beta-casein. Activation of EGF receptor has a positive effect on cell growth and causes the cells to become competent for the lactogenic hormone response. HC11 cells respond optimally to the lactogenic hormone mixture and synthesize high levels of beta-casein only if they have been kept previously in a medium containing EGF. Transfection of HC11 cells with the activated rat neuT receptor results in the acquisition of competence to respond to the lactogenic hormones even if the cells are grown in the absence of EGF. The activation of PDGF receptor, through PDGF-BB, also stimulates the growth of HC11 cells. Cells kept only in PDGF do not become competent for lactogenic hormone induction. The results show that activation of the structurally related EGF and c-erbB-2/neu receptors, but not the PDGF receptor, allows the HC11 cells to subsequently respond optimally to lactogenic hormones.     

Expression of the epidermal growth factor receptor in developing fetal mouse palates: an immunohistochemical study

Epidermal growth factor (EGF) stimulates the growth of various tissues and, therefore, EGF receptor expression in fetal tissues may play a key role in organogenesis. We have examined immunohistochemically the ontogeny and localization of the EGF receptor in the fetal mouse palate during in vivo and in vitro palatogenesis using the anti-human EGF receptor rabbit antibody. Immunoreactive products against the EGF receptor were observed in the palatal tissue examined on days 12, 13, and 14 of gestation. On days 12 and 13, the immunoreactive products were predominantly positive on the oral and medial edge epithelia but were minimal on the epithelium of the vertical shelf. The EGF receptor immunoreactivity was less intense in the posterior palate as compared with the midpalatal region. In the fusing palate of day 14 fetuses, the cells forming the midline epithelial seam were continuously positive for EGF-R immunoreactivity. The mesenchyme of palatal shelves also showed regional heterogeneity and temporal sequence in EGF receptor expression. The localization of the EGF receptor in fetal mouse palates cultured in a serumless medium generally simulated that observed in vivo.     

Identification of alpha transforming growth factor as a possible local trophic agent for the mammary gland

Biologically active alpha-transforming growth factor (alpha-TGF) has been identified in medium conditioned by rat mammary myoepithelial and, to a lesser extent, by epithelial cell lines in culture and in the rat mammary gland. The alpha-TGF has been identified by its wide spectrum of activity in promoting growth of mammary-derived cells in vitro, by its chromatographic behaviour on reversed-phase high-performance liquid chromatography (HPLC), by its competition with epidermal growth factor (EGF) for the EGF receptor, and by the presence of messenger RNA for alpha-TGF in the secreting cells. In vivo the amount of alpha-TGF isolated is sixfold greater from the mammary glands of lactating than from those of virgin female rats. It is proposed that alpha-TGF is produced by the myoepithelial cells of the mammary gland, as a local trophic agent that stimulates growth of the various cell types of the gland.     

Development and growth of mouse embryonic kidney in organ culture and modulation of development by soluble growth factor

Differentiation of the metanephrogenic mesenchyme is triggered by an inductive tissue interaction between an inducer tissue and the mesenchyme. It is generally believed that the epithelial ureter bud acts as an inducer during in vivo development. In response to the inductive stimulus most of the mesenchymal cells convert into epithelial cells, while a small fraction differentiates into stromal cells. In vitro, differentiation of isolated mesenchyme to epithelium can be induced by a variety of embryonic tissues, but nothing is known about the molecular nature of the inducing stimulus. In recent years, large numbers of polypeptide growth factors have been described, which in addition to proliferative effects were shown to exert effects on a variety of biological phenomena such as chemotaxis, inflammation, tissue repair, or induction of embryonic development. We therefore analyzed whether growth factors in the absence of inducer tissue can induce isolated kidney mesenchyme to differentiate into epithelium or interstitium. As expected, both growth and differentiation into epithelium were stimulated by an inducer tissue, the spinal cord. We found that none of the various growth factors tested (including epidermal growth factor, transforming growth factors alpha and beta, insulin-like growth factors I and II, fibroblast growth factor, platelet-derived growth factor, and retinoic acid) could mimick the effect of an inducer tissue, although we tested the factors over a wide concentration range. One of the tested factors, epidermal growth factor (EGF) stimulated the mesenchymal cells to become stromal cells, although it could not stimulate development into epithelium. EGF could stimulate stromal development both when the mesenchyme was cultured in isolation and when the mesenchyme was stimulated by an inducer tissue to become epithelium. The expansion of the stromal compartment in response to EGF treatment occurred at the expense of the epithelial cells, but EGF could not completely suppress the formation of epithelium. These data suggest the presence of EGF receptors in the developing kidney, but since application of soluble EGF leads to abnormal development, soluble EGF cannot be the natural ligand. We suggest that locally produced mitogens with an EGF-like structure may regulate the relative amounts of stroma (interstitium) and epithelium in the developing kidney.     

Substitution for mesenchyme by basement-membrane-like substratum and epidermal growth factor in inducing branching morphogenesis of mouse salivary epithelium

Mouse salivary epithelium cannot undergo branching morphogenesis in the absence of the surrounding mesenchyme. To clarify the nature of the mesenchymal influence on the epithelium, we have investigated the culture conditions in which the epithelium could normally branch in the absence of mesenchymal cells. Combination of basement-membrane-like substratum (Matrigel) and epidermal growth factor (EGF) could substitute for the mesenchyme, the epithelium showing typical branching morphogenesis. Transforming growth factor alpha had the same effect as EGF. Matrigel plus basic fibroblast growth factor or transforming growth factor beta 1 and collagen gel plus EGF were not sufficient to support the branching of the epithelium. These results clearly reveal that the role of mesenchyme in salivary morphogenesis is both to provide the epithelium with an appropriate substratum and to accelerate growth of the epithelium.     

Epidermal growth factor inhibits morphogenesis and cell differentiation in cultured mouse embryonic teeth

Although local epithelial-mesenchymal tissue interactions which are presumably mediated by extracellular matrix molecules are important regulators of tooth morphogenesis and differentiation, our studies have indicated that these developmental processes also depend on circulating molecules. The iron-carrying serum protein transferrin is necessary for the early morphogenesis of mouse tooth in organ culture (A-M. Partanen, I. Thesleff, and P. Ekblom, 1984, Differentiation 27, 59-66). In the present study we have examined the effects of other growth factors on mouse tooth germs grown in a chemically defined medium containing transferrin. Fibroblast growth factor and platelet derived growth factor had no detectable effects but epidermal growth factor (EGF) inhibited dramatically the morphogenesis of teeth, and prevented odontoblast and ameloblast cell differentiation. EGF stimulated cell proliferation in the explants measured as [3H]thymidine incorporation in DNA. However, when the distribution of dividing cells was visualized in autoradiographs, it was observed that cell proliferation was stimulated in the dental epithelium but was inhibited in the dental mesenchyme. The inhibition of cell proliferation in the dental mesenchyme apparently caused the inhibition of morphogenesis. We do not know whether the dental epithelium or mesenchyme was the primary target for the action of EGF in the inhibition of morphogenesis. It is, however, apparent that the response of the dental mesenchymal cells to EGF (inhibition of proliferation) is regulated by their local environment, since EGF enhanced proliferation when these cells were disaggregated and cultured as monolayers. This indicates that the organ culture system where the various embryonic cell lineages are maintained in their original environment corresponds better to the in vivo situation when the roles of exogenous growth factors during development are examined.     

Secretion of polypeptides related to epidermal growth factor and insulinlike growth factor I by a human teratocarcinoma cell line

Summary To identify polypeptide growth factors for human teratocarcinoma cells, we studied the malignant ovarian teratoma-derived cell line, PA-1, that grew autonomously in serum-free medium. Medium conditioned by undifferentiated PA-1 cells strongly stimulated proliferation of the mouse mammary tumor cell line, GR 2H6, which is responsive to epidermal growth factor (EGF) and insulinlike growth factor-I (IGF-I). After ammonium sulfate precipitation, PA-1 conditioned medium was analyzed by anion exchange chromatography and bioassay of elution fractions on GR 2H6 cells that were grown in medium deficient in either EGF or insulin. The results demonstrated that PA-1 CM contained factors that can substitute for EGF and IGF-I in stimulating growth of GR 2H6 cells. Western blots of peak mitogenic fractions revealed low molecular weight polypeptides that were immunoreactive with either anti-EGF or anti-IGF-I antibodies. Indirect immunofluorescence staining of PA-1 cells with monoclonal antibodies localized receptors for each growth factor, and binding of human EGF and IGF-I to these cells was quantified by radioreceptor assays. Secretion of factors closely related to EGF and IGF-I by PA-1 cells under serum-free conditions may provide a novel model system to study molecular mechanisms of autocrine growth stimulation in teratocarcinomas.     

Platelet-derived growth factor receptor regulates salivary gland morphogenesis via fibroblast growth factor expression

A coordinated reciprocal interaction between epithelium and mesenchyme is involved in salivary gland morphogenesis. The submandibular glands (SMGs) of Wnt1-Cre/R26R mice have been shown positive for mesenchyme, whereas the epithelium is beta-galactosidase-negative, indicating that most mesenchymal cells are derived from cranial neural crest cells. Platelet-derived growth factor (PDGF) receptor alpha is one of the markers of neural crest-derived cells. In this study, we analyzed the roles of PDGFs and their receptors in the morphogenesis of mouse SMGs. PDGF-A was shown to be expressed in SMG epithelium, whereas PDGF-B, PDGFRalpha, and PDGFRbeta were expressed in mesenchyme. Exogenous PDGF-AA and -BB in SMG organ cultures demonstrated increased levels of branching and epithelial proliferation, although their receptors were found to be expressed in mesenchyme. In contrast, short interfering RNA for Pdgfa and -b as well as neutralizing antibodies for PDGF-AB and -BB showed decreased branching. PDGF-AA induced the expression of the fibroblast growth factor genes Fgf3 and -7, and PDGF-BB induced the expression of Fgf1, -3, -7, and -10, whereas short interfering RNA for Pdgfa and Pdgfb inhibited the expression of Fgf3, -7, and -10, indicating that PDGFs regulate Fgf gene expression in SMG mesenchyme. The PDGF receptor inhibitor AG-17 inhibited PDGF-induced branching, whereas exogenous FGF7 and -10 fully recovered. Together, these results indicate that fibroblast growth factors function downstream of PDGF signaling, which regulates Fgf expression in neural crest-derived mesenchymal cells and SMG branching morphogenesis. Thus, PDGF signaling is a possible mechanism involved in the interaction between epithelial and neural crest-derived mesenchyme.     

A uterine cell mitogen distinct from epidermal growth factor in porcine uterine luminal fluids: characterization and partial purification

Uterine luminal fluids (ULF) from early (Days 10 and 12)-pregnant sows contain factors that stimulate DNA synthesis in a variety of cell lines. The major growth factor component in these fluids has been partially purified 200-fold by heat treatment, anion-exchange chromatography, and gel filtration using mouse embryo-derived AKR-2B fibroblasts as an indicator cell line. The ULF mitogen (ULFM) is a polypeptide with an apparent molecular weight of 4800; it is extremely heat stable and resistant to treatment with urea. This mitogen is also present in ULF from cycling sows but is not detectable in uterine cytosolic extracts or in serum isolated from pigs at Day 12 of pregnancy. The addition of this factor to medium containing 0.5% calf serum results in a 50% increase in final cell density of AKR-2B cells. ULFM appears biologically distinct from mouse and human epidermal growth factor (EGF), since its activity is not inhibited by antibody to mouse EGF and it does not compete for binding to human (A431) EGF receptors. In addition, the ULF factor stimulates DNA synthesis in human A431 epidermoid carcinoma cells, whereas EGF is inhibitory. Partially purified ULFM also stimulates DNA synthesis in primary cultures of pig uterine stromal cells. This mitogen activity is dose-dependent and is not inhibited by antibody to mouse EGF. Thus ULFM may act in concert with other peptide growth factors in regulating uterine growth and/or differentiation.     

Study of the growth factors for the mammary gland: Epidermal growth factor and mesenchyme-derived growth factor

The physiological importance of EGF in the development of the mouse mammary gland during pregnancy and in spontaneous mammary tumorigenesis has been documented by a series of experimental results presented herein. In our study, we have taken a variety of experimental approaches including radioimmunoassay of EGF in the submandibular gland and plasma, mammary gland organ/cell culture, EGF receptor assay, sialoadenectomy and treatment with EGF and EGF antibodies to assess the role of EGF in the mammary gland. In particular, studies employing sialoadenectomy and EGF replacement have provided valuable information concerning the function of EGF in the body. These studies are possible in the mouse system because the submandibular gland serves as a major source of circulating EGF and also because purified mouse EGF is available commercially. Our work on the biological, endocrinological, and physiological aspects of EGF in normal and neoplastic growth of the mammary gland should be useful for the study of the regulation of mammary gland growth at the molecular level as well as for clinical investigations of mammary tumors. Finally, our findings of a mammary growth factor in embryonic mesenchymal cultures suggest the possible involvement of paracrine growth factor(s) in mammary cell growth. Further progress in this area is needed for better understanding of the complex process of mammary gland growth and development.     

Cripto: a novel epidermal growth factor (EGF)-related peptide in mammary gland development and neoplasia

Growth and morphogenesis in the mammary gland depend on locally derived growth factors such as those in the epidermal growth factor (EGF) superfamily. Cripto-1 (CR-1, human; Cr-1, mouse)--also known as teratocarcinoma-derived growth factor-1--is a novel EGF-related protein that induces branching morphogenesis in mammary epithelial cells both in vitro and in vivo and inhibits the expression of various milk proteins. In the mouse, Cr-1 is expressed in the growing terminal end buds in the virgin mouse mammary gland and expression increases during pregnancy and lactation. Cr-1/CR-1 is overexpressed in mouse and human mammary tumors and inappropriate overexpression of Cr-1 in mouse mammary epithelial cells can lead to the clonal expansion of ductal hyperplasias. Taken together, this evidence suggests that Cr-1/CR-1 performs a role in normal mammary gland development and that it might contribute to the early stages of mouse mammary tumorigenesis and the pathobiology of human breast cancer.     

Autocrine epidermal growth factor signaling stimulates directionally persistent mammary epithelial cell migration.

Cell responses to soluble regulatory factors may be strongly influenced by the mode of presentation of the factor, as in matrix-bound versus diffusible modes. The possibly diverse effect of presenting a growth factor in autocrine as opposed to exogenous (or paracrine) mode is an especially important issue in cell biology. We demonstrate here that migration behavior of human mammary epithelial cells in response to stimulation by epidermal growth factor (EGF) is qualitatively different for EGF presented in exogenous (paracrine), autocrine, and intracrine modes. When EGF is added as an exogenous factor to the medium of cells that express EGF receptor (EGFR) but not EGF, cell migration speed increases while directional persistence decreases. When these EGFR-expressing cells are made to also express via retroviral transfection EGF in protease-cleaveable transmembrane form on the plasma membrane, migration speed similarly increases, but directional persistence increases as well. Addition of exogenous EGF to these cells abrogates their enhanced directional persistence, reducing their directionality to a level similar to wild-type cells. If the EGFR-expressing cells are instead transduced with a gene encoding EGF in a soluble form, migration speed and directional persistence were unaffected. Thus, autocrine presentation of EGF at the plasma membrane in a protease-cleavable form provides these cells with an enhanced ability to migrate persistently in a given direction, consistent with their increased capability for organizing into gland-like structures. In contrast, an exogenous/paracrine mode of EGF presentation generates a "scattering" response by the cells. These findings emphasize the functional importance of spatial restriction of EGFR signaling, and suggest critical implications for growth factor-based therapeutic treatments.     

Density dependent proliferation of human glia cells stimulated by epidermal growth factor.

Epidermal growth factor (EGF) isolated from mouse salivary glands, enhanced the multiplication and [³H]TdR incorporation of human normal glia cells in serum-free medium supplemented with human serum albumin. Optimal dose was 2 ng/ml for both dense and sparse cultures but dense cultures were stimulated by EGF to a much less extent than sparse cultures. Data are presented that make the possibility unlikely that the density dependent inhibition of the EGF response is due to depletion of EGF in the medium or a local, juxtacellular starvation for the factor.     

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.