Epidermal growth factor receptor regulates normal urothelial regeneration

Members of the epidermal growth factor (EGF) family and their receptors are involved in many cellular processes, including proliferation, migration, and differentiation. We have previously reported that these growth factors are expressed and have specific regulatory functions in an organ-like culture model of normal human urothelial cells. Here, we used this model to investigate the involvement of EGF receptor (EGFR) in human urothelial regeneration. Three 4-mm-diameter damaged areas were made in confluent normal human urothelial cell cultures with a biopsy punch. Regeneration was measured, on fixed stained cultures, with an image analyzer, at 4, 24, and 48 hours after injury. Cell proliferation was assessed by 5-bromo-2-deoxyuridine incorporation. To identify EGF family factors potentially involved in the healing process, we studied the effect of these factors on damaged confluent cultures and the level of expression of mRNAs extracted from these cultures. EGFR inhibition of the proliferation and migration of urothelial cells was tested with (1). a specific tyrosine kinase inhibitor (AG1478) and (2). a blocking anti-EGFR antibody (LA22). Exogenously added amphiregulin, EGF, transforming growth factor-alpha and heparin-binding EGF (HB-EGF) stimulated urothelial regeneration. The damaged areas were repaired by regrowth within 48 hours. Both AG1478 and LA22 inhibited the repair (by 50% and 30%, respectively), as well as proliferation and migration. This regeneration was accompanied by increased HB-EGF mRNA expression in cultures of cells from four of six subjects, but no corresponding change in EGFR protein level was observed. These results indicate that the EGFR signaling pathway is involved in urothelial regeneration. Our data support an autocrine role of HB-EGF in this process and suggest that the EGFR pathway is a potential therapeutic target for modulating urothelial cell proliferation.     

Effects of transforming growth factor-beta, transforming growth factor-alpha, and other growth factors on renal proximal tubule cells

Transforming growth factor (TGF)-alpha, epidermal growth factor (EGF), and insulin-like growth factor-1 (IGF-1) addition to quiescent, confluent monolayers of rabbit renal proximal tubule cells in primary culture stimulated [3H]thymidine incorporation. TGF-alpha and EGF promoted a 14-fold rise in thymidine incorporation over control levels with half-maximal responses at 2 x 10(-9) M. IGF-1 only promoted a 4-fold rise in thymidine incorporation compared with control values with a half-maximal response of 10(-8) M. Platelet-derived growth factor alone did not stimulate [3H]thymidine incorporation and did not potentiate the effects of EGF or IGF-1 on DNA synthesis, suggesting that platelet-derived growth factor is neither a competence nor a progression growth factor for renal proximal tubule cells. TGF-beta inhibited both baseline and EGF-stimulated [3H]thymidine incorporation after 48 hours of exposure but enhanced EGF-stimulated DNA synthesis at 24 hours. Morphologic evaluation with phase contrast microscopy, scanning, and transmission electron microscopy demonstrated that TGF-beta promoted a dramatic phenotypic transformation of the epithelial monolayer with migration and adhesion of the cells to form solid clusters of adherent cells. Quantitative morphometry demonstrated that this transformation developed 24 hours after TGF-beta exposure, was nearing completion after 48 hours of TGF-beta treatment, and correlated to TGF-beta related inhibition of EGF-induced DNA synthesis (r = -0.82, p less than 0.01). These results demonstrate that EGF and TGF-alpha are the most potent growth promoters for renal proximal tubule cells. IGF-1 is only a modest growth promoter, whereas platelet-derived growth factor has no effect either as a competence or progressive growth factor. TGF-beta inhibited EGF-induced DNA synthesis but only after observable phenotypic transformation of the cells. The degree of TGF-beta promoted transformation on renal tubule cells was highly correlative to th e antiproliferative effect of TGF-beta, suggesting that similar molecular components which promote this phenotypic transformation may also be critical in the antiproliferative effect of TGF-beta.     

Simultaneous visualization of the binding of nerve growth factor and epidermal growth factor to single rat pheochromocytoma (PC12) cells through indirect immunohistofluorescence

The rat pheochromocytoma cell line, PC12, which has receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF), was used to develop a technique for the simultaneous visualization of separate growth factor receptors by indirect immunohistofluorescence. The cells were incubated with saturating concentrations of nerve growth factor and epidermal growth factor. After fixation, the cells were treated with anti-NGF sheep antiserum and then with antisheep rabbit IgG conjugated with fluorescein; they also were treated with anti-EGF rabbit antiserum and then with anti-rabbit sheep IgG conjugated with rhodamine. Fluorescence microscopy showed that a single PC12 cell bound both NGF and EGF. The fluorescence due to EGF binding was reduced when the cells were grown in the presence of NGF. A similar reduction of fluorescence was observed after addition of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Both manipulations are known to reduce the specific binding of 125I-EGF to these cells. Subclones of PC12 cells, NR11 and NR20, reported not to have NGF receptors, did not demonstrate NGF binding when tested with this indirect immunohistofluorescence method. Thus, the binding of growth factors which is demonstrable by indirect immunohistofluorescence method seems to reflect the presence of the specific cell surface receptors for both peptides on individual PC12 cells.     

Heparin-binding growth factors stimulate DNA synthesis in rat alveolar type II cells

The proliferation of alveolar type II cells is important for repair of the alveolar epithelium after lung injury. We have previously reported that epidermal growth factor (EGF), insulin, cholera toxin, and endothelial cell growth supplement (ECGS) stimulate DNA synthesis of rat alveolar type II cells in culture. ECGS is a crude extract from bovine neural tissue that contains heparin-binding growth factors, and in this report we have compared the effect of ECGS to purified heparin-binding growth factors. ECGS stimulated [3H]thymidine incorporation into type II cells by 3-fold with half-maximal stimulation at 50 micrograms/ml. The purified acidic, class I heparin-binding growth factors, alpha-endothelial cell growth factor (-ECGF) and beta-ECGF stimulated type II cell DNA synthesis by 10-fold and 5-fold, respectively, with half-maximal stimulation at 40 ng/ml. Acidic fibroblast growth factor (FGFa) stimulated [3H]thymidine incorporation by 16-fold with half-maximal stimulation at 20 ng/ml, whereas basic FGF (FGFb) only stimulated type II cell DNA synthesis by 3-fold. Heparin potentiates the mitogenic effect of the acidic heparin-binding growth factors for both endothelial cells and fibroblasts but was found to inhibit FGFa- and FGFb-induced [3H]thymidine incorporation in type II cells by 80% with half-maximal inhibition occurring with 0.4 micrograms/ml and 1.3 micrograms/ml, respectively. When type II cells were cultured in the absence of serum, the heparin-binding growth factors had very little effect on [3H]thymidine incorporation. Only rat high density lipoprotein (HDL), but not insulin, EGF, or transferrin, was found to act synergistically with FGFa in stimulating [3H]thymidine incorporation in type II cells cultured in serum-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of transforming growth factor-beta actions in rat osteoblast-like cells: the effects of bFGF and EGF

The present study examines how the mitogenic and differentiation functions of transforming growth factor-beta (TGF-beta) are modulated by basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) in primary cultures of rat osteoblast-like (ROB) cells. TGF-beta, bFGF, and EGF individually stimulated [3H]thymidine incorporation and cell proliferation in a dose range of 0.01-10 ng/ml. When studied in combination, high doses of bFGF and EGF were additive to low doses of TGF-beta. The additive effects of bFGF and EGF on mitogenesis diminished with increasing doses of TGF-beta. These three factors also decreased alkaline phosphatase activity individually within the same dose range. When cells were treated with the combined factors, only high doses of bFGF and EGF were additive to the TGF-beta inhibition. We were unable to detect any change in collagen synthesis with each individual factor or in combined treatments. In addition, TGF-beta or bFGF alone or in combination did not affect fibronectin synthesis. Our studies showed that the biological functions of TGF-beta can be modulated by bFGF and EGF in ROB cells. The pattern of modulation is varied depending on the specific function examined.     

Similar transforming growth factors (TGFs) produced by cells transformed by different isolates of feline sarcoma virus

Fisher rat embryo cells transformed by each of three independent isolates of feline sarcoma virus (FeSV) are shown to release transforming growth factors (TGFs) into cell culture medium. These acid- and heat-stable peptides compete for binding to, and stimulate phosphorylation of, EGF membrane receptors and promote anchorage-independent cell growth. Cells transformed by the Gardner and Snyder-Theilen strains of FeSV produce high titers of TGF (60-200 ng eq EGF/liter) while cells transformed by McDonough FeSV produce TGF at only low levels (<10 ng eq EGF/liter). Growth factors produced by cells transformed by each of the three FeSV isolates functionally and biochemically resemble each other, mouse sarcoma growth factor (SGF), and TGFs produced by human tumor cells.     

Sequential changes in epidermal growth factor receptor/ligand function in cultured rat liver epithelial cells transformed chemically in vitro

Diploid WB rat liver epithelial cells contain abundant, rapidly internalized epidermal growth factor receptors, and respond pleiotropically to ligand binding. Signal transduction pathways downstream from the EGF receptor involve activation of elements that are both dependent on and independent of protein kinase C activation. Neoplastic transformation of wild-type WB rat liver epithelial cells by exposure to N-methyl-N'-nitro-N-nitrosoguanidine is associated with progressive alterations in the responses of affected cells to binding of EGF to EGF receptors, including heightened cell proliferation and the expression of several other phenotypic properties. Tumorigenic rat liver epithelial cells acquire the ability to express transforming growth factor-alpha (TGF-alpha), and to secrete this growth factor in a regulated and then unregulated manner. TGF-alpha expression, together with the presence of abundant EGF receptors, provides affected cells with an autocrine growth cycle. The ability of transformed WB rat liver epithelial cells to produce tumors cosegregates clonally with TGF-alpha expression and with heightened expression of c-myc, c-Ha-ras and c-Ki-ras proto-oncogenes.     

A 34-kd protein with strong homology to ras-like proteins inhibits epidermal growth factor activity.

Epidermal growth factor (EGF) and its analog, transforming growth factor-alpha, are felt to be important in oncogenesis. When malignant rabbit fibroma virus infects RK-13 rabbit kidney cells, a 34-kd protein that inhibits the effects of EGF on certain target cell lines is produced. We have purified this protein using high-pressure liquid chromatography and gel electrophoresis. This purified protein abolishes EGF-induced cellular proliferation. It also causes the EGF receptor-bearing A431 carcinoma cell line to stop proliferating in vitro. This purified 34-kd EGF inhibitor (EGFI) redirects cellular protein phosphorylation in the presence or absence of EGF. Whereas EGF increases phosphorylation of cellular proteins in normal rat kidney cells, clone 49F, and A431 EGFI generally decreases it. Both EGF and EGFI cause increased protein production in A431 and normal rat kidney cells. The major species of protein synthesized by cells seem invariant to EGFI, with or without EGF. The partial protein sequence of two fragments of EGFI shows striking similarity to two ras like proteins. Possible means by which such a ras-like protein might inhibit EGF-induced cellular proliferation are discussed. Therefore, a purified 34-kd ras-like protein inhibits EGF-induced cellular proliferation and changes the targets for cellular protein phosphorylation. Studies are in progress to characterize this protein further, both structurally and functionally.     

Presence of immunoglobulin M antibodies around the glomerular capillaries and in the mesangium of normal and passive Heymann nephritis rats

Summary Diffuse distribution of small, faintly staining, beaded deposits of rat immunoglobulin M (IgM) around the glomerular capillary blood vessels, and a more intensely staining larger deposition in the mesangium, were observed on the kidney sections of normal rats. As glomerular-fixed nephritogenic antigens are known to be present on the epithelial aspect of the glomerular basement membrane (GBM), especially at the soles of foot processes and at the slit pores, it was assumed that the IgM antibodies were directed against these antigens. Investigation by immunofluorescent antibody double-staining techniques of rat kidney sections obtained from normal and rabbit anti-FX1A-injected rats stained for the nephritogenic antigen showed that a number of antigenic sites in the glomeruli and in the mesangium shared antibody hits by heterologous rabbit IgG and autologous rat IgM antibodies. Most sites in the glomeruli stained specifically for rat IgM or rabbit IgG, but preferentially for the latter. The intensely fluorescent mesangial deposits stained mainly for rat IgM, indicating that at these sites the antigenic material was virtually saturated, while areas at the entry to the mesangial space also stained for rabbit IgG, indicating that at these locations free nephritogenic epitopes were still available for reaction with the anti-FX1A antibody. Western blot analysis have shown that the rabbit anti-rat FX1A IgG and the rat anti-rat KF3 IgM antibodies are directed against the same renal tubular-derived antigen with a molecular weight of 70,000. These experimental findings collectively demonstrate that the heterologous IgG and autologous IgM antibodies are directed against the same nephritogenic antigen, which is found in the glomeruli, the mesangium and the proximal convoluted tubules. Thus, the IgM autoantibody has a possible physiological role but, in addition, there is evidence of active immunophagocytic events, manifested in a rapid and continuous entrapment and expulsion of macromolecules after their processing by the mesangial cells of normal and passive Heymann nephritis rats.     

Epidermal growth factor transcription, translation, and signal transduction by rat type II pneumocytes in culture.

Epidermal growth factor (EGF) is known to induce fetal lung maturation and its receptor is present in the lungs of several species. Recently, EGF has been immunolocalized in type II pneumocytes in rat lung. We postulated that EGF is synthesized in type II pneumocytes and that, because of its position-restricted distribution within the alveolus, EGF might act as an autocrine regulator of type II pneumocyte function. Herein, we have tested the hypothesis using adult rat type II pneumocytes in primary culture. In situ hybridization, using an oligonucleotide probe corresponding to amino acid residues 1070 to 1081 of mouse EGF precursor, demonstrated the presence of EGF precursor mRNA. Upon S-200 Sephacryl gel chromatography of type II pneumocyte extracts, EGF-reactive protein eluted as a high-molecular-weight form (greater than 100 kD). EGF immunoreactivity was localized within type II pneumocytes in the periphery of groups of 10 to 15 cells in culture. The type II pneumocytes bound [125I]EGF in a specific manner, indicating the presence of EGF receptors. Scatchard plots gave an apparent affinity constant (Ka) of 1 x 10(9) liters/mol, and the number of receptors was estimated to be 4.8 x 10(11) mg protein (50 per cell). EGF receptor binding specificity was confirmed by the absence of an autoradiographic signal for cells incubated in the presence of a 100-fold excess concentration of transforming growth factor-alpha. Binding of [125I]EGF could also be downregulated 95% by incubation with 0.2 nM transforming growth factor-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mitogenic activity of hepatocyte growth factor on rat hepatocytes is dependent upon endogenous transforming growth factor-alpha

Both transforming growth factor-alpha (TGF-alpha) and hepatocyte growth factor (HGF) induce DNA synthesis in hepatocytes in vitro and in vivo. Hepatic and circulating levels of HGF have been reported to increase before an increase in TGF-alpha levels in several rat models of liver regeneration. In addition, serum TGF-alpha levels increase after an increase in serum HGF levels in patients with either partial hepatectomy or acute hepatitis. In this study, we investigate the significance of TGF-alpha in hepatocyte proliferation. TGF-alpha contents and DNA synthesis in cultured rat hepatocytes increased in response to HGF addition to the culture medium in a dose-related manner. These increases were suppressed by the addition of anti-sense TGF-alpha mRNA oligonucleotide. Furthermore, the addition of anti-TGF-alpha rabbit IgG suppressed the increase in DNA synthesis. When the anti-TGF-alpha antibody was administered to rats after partial hepatectomy, the number of mitotic hepatocytes was reduced in comparison to rats treated with normal rabbit IgG. These results were observed even though hepatic HGF levels were increased equally in rats given either anti-TGF-alpha antibody or normal rabbit IgG. Our results suggest that HGF stimulates TGF-alpha production in rat hepatocytes, and that the mitogenic activity of HGF depends on endogenous TGF-alpha activity.     

Involvement of mitogen-activated protein kinase in transforming growth factor alpha-stimulated cell proliferation in the cultured granulosa cells of the Japanese quail

The avian granulosa cells proliferate during follicular growth phase and differentiate to produce progesterone in response to luteinizing hormone (LH) when the follicle becomes the largest. In order to study the involvement of mitogen-activated protein (MAP) kinase in proliferation of the granulosa cells in avian species, quail granulosa cells were cultured for 66 h with various hormones (follicle stimulating hormone (FSH), LH, progesterone, estradiol-17beta, testosterone), or growth factors (transforming growth factor alpha (TGF alpha), epidermal growth factor (EGF), insulin-like growth factor I (IGF-I), IGF-II), and the presence of immunodetectable MAP kinase was examined in the cell lysates. When the granulosa cells were cultured with TGF alpha, the cell number as well as the incorporation of [3H]thymidine was increased. Other hormones or growth factors caused no significant increase in cell numbers. Stimulation of the cells with TGF alpha for 10 min caused a retarded mobility of MAP kinase in the gel of SDS-PAGE. Both the increases in [3H]thymidine incorporation and the retarded mobility were inhibited by the presence of a tyrosine kinase inhibitor, genistein, indicating the importance of phosphorylation of protein during the TGF alpha-stimulation.     

Chemically induced transplantable malignant fibrous histiocytoma of the rat. Analyses with immunohistochemistry, immunoelectron microscopy and [3H]thymidine autoradiography

Malignant fibrous histiocytoma was produced in rats by injection of 9,10-dimethyl-1,2-benzanthracene into their knee joints. The original tumors consisted mainly of fibroblast-like cells and histiocyte-like cells, often intermixed with bizarre giant cells, and they frequently showed the storiform-pleomorphic pattern. By immunohistochemistry, anti-rat macrophage monoclonal antibodies, TRPM-3, RM-1, and Ki-M2R, and anti-rat leukocyte common antigen reacted to the histiocyte-like cells but not to the fibroblast-like cells. By the single cell cloning method, we established six tumor cell lines, none of which reacted with the anti-rat macrophage monoclonal antibodies, possessed any Fc receptors, or conducted immune phagocytosis and Latex particle phagocytosis. The ultrastructure of the cloned tumor cells resembled that of long-term cultured dermal fibroblasts. Collagen production by the tumor cells was demonstrated immunohistochemically with a monoclonal antibody for type I collagen. Inoculation of the cloned tumor cells into rats produced tumors with the histology of malignant fibrous histiocytoma and induced prominent macrophage infiltration. In the rat tumors produced by the inoculation of [3H]thymidine labeled cells, no reactivity of tumor cells with the anti-rat macrophage monoclonal antibodies was observed. Transplantation of the cultured rat tumor cells into nude mice produced tumors similar in histology to the original rat malignant fibrous histiocytoma. Tumor cells in nude mice induced marked macrophage infiltration as detected by immunohistochemistry with the anti-mouse macrophage monoclonal antibody F4/80. No differentiation of tumor cells into macrophages was detected, since no cells were stained with biotinylated anti-rat macrophage monoclonal antibody TRPM-3. By the flash labeling method with [3H]thymidine, infiltrating macrophages in the nude mouse tumors were proved to derive from the bone marrow of the host animals. These results indicate a possible experimental reproduction of malignant fibrous histiocytoma by proliferation of malignant fibroblasts or their related cells in combination with macrophage infiltration.     

Effect of Anti Light-Chain Antibodies on Rat Leukocytes in Vitro

The effect of the IgG fraction of rabbit anti rat L-chain antibodies (aL) and their Fab fragments (Fab aL) on rat blood leukocytes cultured in vitro has been examined. Undigested aL increased the thymidine uptake in both mixed and unmixed leukocyte cultures over a very wide dose range. Fab aL had no effect on either mixed or unmixed leukocyte cultures. The nature of T lymphocyte receptors is briefly discussed.     

Mode of action of anti-lymphocyte globulin: II. Changes in the lymphoid cell population in rats treated with anti-lymphocyte globulin

Lymphopenia in rats which had been made tolerant to normal rabbit immunoglobulin G(IgG) was induced by the administration of rabbit anti-rat immunoglobulin G(IgG). The rats were injected with tritiated thymidine ([³H] thymidine) and the labelling pattern in lymphoid tissues was studied. The thymus weight decreased with continued lymphopenia and this could be explained by the release of small lymphocytes into the circulation. Lymphopoiesis in the thymus and spleen was not inhibited by anti-lymphocyte globulin. Plasmacytosis was noted in the lymph nodes. These findings support the idea that anti-lymphocyte globulin acts mainly on peripheral lymphocytes and suppresses the immune function mediated by these cells.     

Isolation and growth of smooth muscle-like cells derived from tuberous sclerosis complex-2 human renal angiomyolipoma: epidermal growth factor is the required growth factor

Tuberous sclerosis complex (TSC) is a tumor suppressor gene disorder characterized by mutations in the TSC1 or TSC2 genes. These mutations lead to the development of benign tumors involving smooth muscle cells, causing life-threatening lymphangioleiomyomatosis. We isolated and characterized two types of cells bearing a mutation in TSC2 exon 18 from a renal angiomyolipoma of a TSC patient: one population of alpha-actin-positive smooth muscle-like cells with loss of heterozygosity for the TSC2 gene (A(+) cells) and another of nonloss of heterozygosity keratin 8/18-positive epithelial-like cells (R(+) cells). Unlike control aortic vascular smooth muscle cells, A(+) cells required epidermal growth factor (EGF) to grow and substituting EGF with insulin-like growth factor (IGF)-1 failed to increase the cell number; however, omission of EGF did not cause cell loss. The A(+) cells constantly released IGF-1 into the culture medium and constitutively showed a high degree of S6K phosphorylation even when grown in serum-free medium. Exposure to antibodies against EGF and IGF-1 receptors caused a rapid loss of A(+) cells: 50% by 5 days and 100% by 12 days. Signal transduction mediated by EGF and IGF-I receptors is therefore involved in A(+) cell survival. These results may offer a novel therapeutic perspective for the treatment of TSC complications and lymphangioleiomyomatosis.     

Modulation of Transforming Growth Factor-β Actions in Rat Osteoblast-like Cells: The Effects of bFGF and EGF

Abstract

The present study examines how the mitogenic and differentiation functions of transforming growth factor-β (TGF-β) are modulated by basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) in primary cultures of rat osteoblast-like (ROB) cells. TGF-β, bFGF, and EGF individually stimulated [³H]thymidine incorporation and cell proliferation in a dose range of 0.01-10 ng/ml. When studied in combination, high doses of bFGF and EGF were additive to low doses of TGF-β. The additive effects of bFGF and EGF on mitogenesis diminished with increasing doses of TGF-β. These three factors also decreased alkaline phosphatase activity individually within the same dose range. When cells were treated with the combined factors, only high doses of bFGF and EGF were additive to the TGF-β inhibition. We were unable to detect any change in collagen synthesis with each individual factor or in combined treatments. In addition, TGF-β or bFGF alone or in combination did not affect fibronectin synthesis. Our studies showed that the biological functions of TGF-β can be modulated by bFGF and EGF in ROB cells. The pattern of modulation is varied depending on the specific function examined.     

Hyperplasia of epithelium adjacent to transitional cell carcinoma can be induced by growth factors through paracrine pathways

Hyperplasia of transitional cell epithelium adjacent to human transitional cell carcinomas (TCC) is a common finding in pathology. This hyperplasia may be a precancerous aberration. Alternatively, it has been suggested that the hyperplasia is due to paracrine action of tumour-derived growth factors. In this study we tested the latter hypothesis using the mouse tumorigenic TCC cell line NUC-1. Transplantation of NUC-1 tumour cells into the urinary bladder submucosa of syngeneic mice in vivo induced hyperplasia of normal adjacent urothelium in all tested mice. Implantation of normal mouse bladder mucosa did not induce urothelial hyperplasia. In vitro, conditioned medium of NUC-1 cells induced the proliferation of the mouse urothelial cell line g/G, which closely resembles normal urothelial cells. This induction was inhibited by transforming growth factor ₁ (TGF₁). Similarly, TGF₁ inhibited the fibroblast growth factor-1 (FGF-1) and FGF-2 induced proliferation of g/G cells. Chemico-physical examination, bioassays with conditioned media, and RNA analysis of NUC-1 cells revealed that these cells secreted a growth factor with FGF-like properties. These results indicate that epithelial hyperplasia surrounding carcinomas is not necessarily a precancerous aberration, but may result from direct paracrine action of tumour-derived growth factors.     

The effects of continuous exposure to epidermal growth factor on the spontaneous transformation of cultured rat liver epithelial cells.

A long-term continuous exposure to epidermal growth factor (EGF) enhanced the tumorigenicity of spontaneously transformed cells arising in a clonal population of normal cultured rat liver epithelial cells propagated in a selective growth condition. Lengthy EGF exposure also induced the expression of several phenotypes that differed from the phenotypes of rat liver epithelial cells transformed spontaneously in the absence of EGF. Epidermal growth factor treatment caused consistently an enhancement of the constitutive mRNA expression of transforming growth factor-alpha (TGF-alpha), but not of the EGF receptor and transforming growth factor-beta. The overexpression of TGF-alpha persisted in cell lines derived from tumors formed by the EGF-treated transformed cells. These tumors also exhibited high metastatic incidence and ductal cell differentiation. In contrast, untreated spontaneously transformed cells formed non-metastatic tumors with hepatocellular differentiation. These results suggest that long-term, continuous exposure to EGF/TGF-alpha may modulate the phenotypic expressions of neoplastic transformation.