Role of TGF alpha and its receptor in proliferation of immortalized rat tracheal epithelial cells: studies with tyrphostin and TGF alpha antisera

We have shown in the present study and in studies reported previously that preneoplastic and neoplastic rat tracheal epithelial (RTE) cell lines express TGF alpha and do so regardless of the mechanism by which they were transformed. In order to determine whether TGF alpha is an autocrine growth regulator of immortalized RTE cells, we have examined the function of TGF alpha/EGF receptors and the growth requirements for TGF alpha in these cells. The level of immunoprecipitated TGF alpha/EGF receptor protein in immortalized RTE cells was similar to or less than levels in primary RTE cells, indicating that chemically induced transformation of RTE cells does not involve overexpression of TGF alpha/EGF receptors. Scatchard analysis of TGF alpha/EGF receptors in the neoplastic EGV5T cell line revealed the presence of high-affinity (Kd = 0.4 nM) and low-affinity (Kd = 9.8 nM) binding sites. A tyrphostin TGF alpha/EGF receptor tyrosine kinase inhibitor decreased in a dose-dependent manner the proliferation as well as EGF-induced autophosphorylation of the TGF alpha/EGF receptor of transformed RTE cells. The inhibitory effect of tyrphostin on proliferation and receptor kinase activity was attenuated in late log and plateau phase cultures. The phosphotyrosine content of several other EGF-dependent and independent phosphoproteins was also decreased by the tyrphostin. Proliferation of transformed RTE cells was also inhibited when TGF alpha antisera was added to the media of growing cells. These data are consistent with the hypothesis that proliferation of transformed RTE cells involves autocrine regulation by TGF alpha and its receptor.     

The proliferative and morphologic responses of a colon carcinoma cell line (LIM 1215) require the production of two autocrine factors.

The role of autocrine growth factors in tumor cell growth has been difficult to prove. Our results indicate that more than one autocrine factor is required for the autonomous growth of the LIM 1215 colonic carcinoma cell line. Furthermore, the morphologic changes induced by epidermal growth factor (EGF) are also density dependent and appear to require a synergistic autocrine factor. The serum-free proliferation of the colonic carcinoma cell line LIM 1215 depends on cell density and the presence of EGF (A. Sizeland, S. Bol, and A.W. Burgess, Growth Factors 4:129-143, 1991). At cell densities below 10(4)/cm2, conditioned medium (from cells at a density of 10(5)/cm2) was required for the cells to elicit a mitogenic response to exogenous EGF. At higher cell densities (10(5)/cm2), the cells were independent of both exogenous EGF and conditioned medium. In addition, the EGF receptor was found to be phosphorylated on tyrosine in LIM 1215 cells proliferating at high density, suggesting that the autocrine production of transforming growth factor alpha (TGF alpha) and subsequent ligation to the EGF receptor was occurring. The proliferation of cells at high density was partly inhibited by TGF alpha antibodies but was almost completely inhibited by an antisense oligonucleotide to TGF alpha. The antisense inhibition could be overcome by the addition of EGF, indicating that the effect of the antisense TGF alpha oligonucleotide was on the production of autocrine TGF alpha. LIM 1215 cells were also observed to undergo morphologic changes (spreading and actin cable organization) in response to EGF. These changes were density dependent, but they occurred with a cell density dependence different from that of the proliferative response. These results suggest two possibilities: that the morphologic changes and proliferative responses have different sensitivities to the autocrine factors or that the actions of the autocrine factors are mediated through different signal transduction pathways.     

Monoclonal antibody 425 inhibits growth stimulation of carcinoma cells by exogenous EGF and tumor-derived EGF/TGF-alpha

Carcinoma cells frequently coexpress transforming growth factor (TGF)-alpha and its receptor, the epidermal growth factor (EGF) receptor, implicating an autocrine function of carcinoma-derived TGF-alpha. Using a monoclonal antibody (425) to the EGF-receptor, we investigated the role of exogenous and tumor cell-derived EGF/TGF-alpha mitogenic activities in proliferation of cell lines derived from solid tumors. Monoclonal antibody 425 was chosen for these studies because it inhibits binding of EGF/TGF-alpha to the EGF-receptor and effectively blocks activation of the EGF-receptor by EGF/TGF-alpha. Seven malignant cell lines originating from carcinomas of colon, pancreas, breast, squamous epithelia, and bladder expressed surface EGF-receptor and secreted EGF/TGF-alpha-like mitogenic activities into their tissue culture media. All cell lines were maintained in a defined medium free of exogenous EGF/TGF-alpha. EGF and TGF-alpha added to the culture medium stimulated proliferation of five cell lines to comparable levels. EGF/TGF-alpha-dependent proliferation was significantly reduced by addition of MAb 425 to culture media. In addition, monoclonal antibody 425 reduced proliferation of the five EGF/TGF-alpha responsive cell lines in the absence of exogenous EGF/TGF-alpha. Antiproliferative effects induced by monoclonal antibody 425 were reversible and could be overcome by addition of EGF to culture media. Our results indicate that tumor-derived EGF-receptor-reactive mitogens can promote proliferation of carcinoma cells in an autocrine fashion.     

Anti-sense transforming growth factor alpha oligonucleotides inhibit autocrine stimulated proliferation of a colon carcinoma cell line.

Many carcinoma cells secrete transforming growth factor alpha (TGF alpha). A 23 base anti-sense oligonucleotide that recognizes the TGF alpha mRNA inhibits both DNA synthesis and the proliferation of the colon carcinoma cell line LIM 1215. The effects of the anti-sense TGF alpha oligonucleotide are reversed by epidermal growth factor (EGF) at 20 ng/ml. When the LIM 1215 cells are grown under serum free conditions, the anti-sense TGF alpha oligonucleotides have their greatest effects at high cell density (2 x 10(5) cells/cm2), indicating that the secreted TGF alpha is acting as an exogenous growth stimulus. In addition, at higher cell densities, the kinase activity of the EGF receptor is activated and the receptor is down-modulated. The cell density dependent activation of the EGF receptor is inhibited by the application of the antisense TGF alpha oligonucleotides.     

Growth regulation of ovarian cancer cells by epidermal growth factor and transforming growth factors alpha and beta 1.

Regulation of ovarian cancer growth is poorly understood. In this study, the effects of EGF, TGF alpha and TGF beta 1 on two ovarian cancer cell lines (OVCAR-3 and CAOV-3) were investigated. The results showed that EGF/TGF alpha stimulated cell growth and DNA synthesis in OVCAR-3 cells, but inhibited cell proliferation and DNA synthesis in CAOV-3 cells. TGF beta 1 invariably inhibited cell proliferation and DNA synthesis in both cell lines. These effects on growth factors are dose dependent. The interaction of TGF beta 1 and EGF/TGF alpha was antagonistic in OVCAR-3 cells. In contrast, EGF/TGF alpha and TGF beta 1 had an additive inhibitory effect on CAOV-3 cells. Our results demonstrated that mature and functional EGF receptors are present in both cell lines and that they are capable of ligand binding, internalization, processing and ligand-enhanced autophosphorylation. Both high- and low-affinity binding are present in these cell lines, with CAOV-3 cells having about 2-3-fold higher total receptors than OVCAR-3 cells. These results together with those from our previous studies show that these cells express TGF alpha, TGF beta 1 and EGF receptors and that cell growth may be modulated by these growth factors in an autocrine and paracrine manner. This report presents evidence supporting the important roles of growth factors in ovarian cancer growth and provides a foundation for further study into the mechanism of growth regulation by growth factors in these cell lines.     

Transforming growth factor-beta (TGF beta) inhibits TGF alpha expression in bovine anterior pituitary-derived cells.

Transforming growth factor-beta 1 (TGF beta 1) is a multifunctional regulator of cell growth and differentiation. We report here that TGF beta 1 decreased the proliferation of nontransformed bovine anterior pituitary-derived cells grown in culture. We have previously demonstrated that these cells express both TGF alpha and its receptor [the epidermal growth factor (EGF) receptor] and that expression can be stimulated by phorbol ester (TPA) and EGF. TGF beta 1 treatment over a 2-day period decreased the proliferation of pituitary cells. This decreased growth rate was accompanied by a decrease in the TGF alpha mRNA level. The effect of TGF beta 1 on TGF alpha mRNA down-regulation was both dose dependent (maximal effect observed at 1.0 ng/ml TGF beta 1) and time dependent (minimum of 2-day treatment with TGF beta 1 was required before a decrease in TGF alpha mRNA was observed). Studies on TGF alpha mRNA stability indicated that TGF beta 1 did not alter the TGF alpha mRNA half-life. Treatment of the TGF beta 1 down-regulated cells with EGF resulted in the stimulation of TGF alpha mRNA levels; thus, the TGF beta 1-treated cells remained responsive to EGF. The decreased proliferation in response to TGF beta 1 could be only partially reversed by simultaneous treatment of the cells with EGF (10(-9)M) and TGF beta 1 (3.0 ng/ml). Qualitatively, the TGF beta 1-induced reduction of TGF alpha mRNA content was independent of cell density. TGF beta 1 treatment of the anterior pituitary-derived cells also reduced the levels of c-myc and EGF receptor mRNA. These results represent the first demonstration of the down-regulation of TGF alpha synthesis by a polypeptide growth factor and suggest that TGF beta 1 may be a physiological regulator of TGF alpha production in vivo.     

Growth regulation of primary rat tracheal epithelial cell cultures by endogenous transforming growth factor-βs

Primary rat tracheal epithelial (RTE) cell cultures have previously been shown to secrete transforming growth factor-β (TGFβ) and to be growth inhibited by exogenous TGFβ. The purpose of the present studies was to determine whether the endogenous TGFβ(s) were regulating the growth of RTE cell cultures and, if so, which isoforms were involved. Neutralizing antibodies specific to TGFβ1 and TGFβ2 were added to cultures, and their effects on several growth parameters were measured. Addition of antibodies to early cultures (day 1), resulted in 1.8-and 3-fold increases in colony formation and cell number, respectively, above control IgG-treated cultures. Antibody dose-response experiments revealed that TGFβ2 was the predominant isoform inhibiting early RTE cell growth. The antibody treatments resulted in similar stimulation of early growth at low and high seeding densities, suggesting that the endogenous TGFβs were acting locally. Anti-TGFβ1 treatment of cultures at various stages of growth resulted in 1.2–1.7-fold increases in DNA synthesis above controls, whereas anti-TGFβ2 treatment resulted in increased DNA synthesis only in early and late cultures (1.7- and 2.5-fold, respectively), but not during midlogarithmic growth. Continuous treatment with a combination of both antibodies resulted in increased growth and decreased exfoliation in early cultures, but had no effect on the slow down of growth in late cultures. Thus endogenous TGFβs inhibited primarily early growth and contributed to, but did not appear to be responsible for, plateau of growth in late stage cultures. Antibody treatment of secondary cultures resulted in 4–70-fold increases in colony formation, depending on the age of the primary cultures when replated, indicating that endogenous production of both TGFβ1 and TGFβ2 greatly inhibits the subculturability of primary RTE cells. Other experiments suggested that cholera toxin enhances RTE cell growth in part by counteracting the inhibitory effects of endogenous TGFβs. © 1993 Wiley-Liss, Inc.     

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.     

Human keratinocyte growth factor activity on proliferation and differentiation of human keratinocytes: differentiation response distinguishes KGF from EGF family.

Human keratinocyte growth factor (KGF) is an epithelial cell specific mitogen which is secreted by normal stromal fibroblasts. In the present studies, we demonstrate that KGF is as potent as EGF in stimulating proliferation of primary or secondary human keratinocytes in tissue culture. Exposure of KGF- or EGF-stimulated keratinocytes to 1.0 mM calcium, an inducer of differentiation, led to cessation of cell growth. However, immunologic analysis of early and late markers of terminal differentiation, K1 and filaggrin, respectively, revealed striking differences in keratinocytes propagated in the presence of these growth factors. With KGF, the differentiation response was associated with expression of both markers whereas their appearance was retarded or blocked by EGF. TGF alpha, which also interacts with the EGF receptor, gave a similar response to that observed with EGF. These findings functionally distinguish KGF from the EGF family and support the role of KGF in the normal proliferation and differentiation of human epithelial cells.     

Growth regulation of ovarian cancer cells by epidermal growth factor and tranforming growth factors α and β1

Regulation of ovarian cancer growth is poorly understood. In this study, the effects of EGT, TGFα and TGFβ1 on two ovarian cancer cell lines (OVCAR-3 and CAOV-3) were investigated. The results showed that EGF/TGFα stimulated cell growth and DNA synthesis in OVCAR-3 cell, but inhibited cell proliferation and DNA synthesis in CAOV-3 cells. TGFβ1 invariably inhibited cell proliferation and DNA synthesis in both cell lines. These efefects on growth factors are dose dependent. The interaction of TGFβ1 and EGF/TGFα was antagonistic in OVCAR-3 cells. In contrast, EGF/TGFα and TGFβ1 had an additive inhibitory effect on CAOV-3 cells. Our results demonstrated that mature and functional EGF receptors are present in both cell lines and that they are capable of ligand binding, internalization, processing and ligand-enhanced autophosphorylation. Both high- and low-affinity binding are present in these cell lines, with CAOV-3 cells having about 2–3 fold higher total receptors than OVCAR-3 cells. These results together with those from our previous studies show that these cells express TGFα, TGFβ1 and EGF receptors and that cell growth may be modulated by these growth factors in an autocrine can paracrine manner. This report presents evidence supporting the important roles of growth factors in ovarian cancer growth and provides a foundation for futher study into the mechanism of growth regulation by growth factors in these cell lines.     

Changes in responsiveness of rat tracheal epithelial cells to transforming growth factor-β1 with time in culture

Primary rat tracheal epithelial (RTE) cell cultures have previously been shown to be highly sensitive to growth inhibition by transforming growth factor-β1 (TGF-β1) when treated within 1–2 days after plating. The purpose of the present studies was to examine the effects of TGFβ1 on the growth of RTE cells as a function of time in culture. We found that the sensitivity of RTE cells to growth inhibition by TGFβ1 decreased dramatically as the cultures aged. The IC₅₀ for inhibition of colony forming efficiency was 0.18 pM when TGFβ1 was added 24 h after cell plating. When TGFβ1 treatment was begun on day 5 of culture, the IC₅₀ was 3–4 pM as measured by inhibition of growth (cell number) and DNA synthesis. However, when TGFβ1 was begun on day 19, the IC₅₀ was 65 pM or > 500 pM, depending on whether inhibition of growth or DNA synthesis, respectively, was measured. TGFβ1 accelerated cell death, as measured by exfoliation of cells, and inhibited cell proliferation. The decrease in responsiveness to TGFβ1 in late cultures was shown to be dependent on culture age as well as on cell density. No evidence was found for inactivation or degradation of the added TGFβ1 by the late stage cultures. Cells subcultured from late stage primary cultures remained less responsive to TGFβ1 than subcultured cells from early cultures. Similar to its effect on proliferation, TGFβ1 down-regulated the expression of two proliferation-related genes, c-myc and transforming growth factor-α, in early but not late RTE cell cultures. On the other hand, fibronectin expression was increased by TGFβ1 by about twofold at both early and late times in culture. This indicates that the changes in TGFβ1 responsiveness with time in culture are selective, apparently affecting primarily proliferation-related events. © 1992 Wiley-Liss, Inc.     

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)     

Transforming growth factor alpha and a PC12-derived growth factor induce neurites in PC12 cells and enhance the survival of embryonic brain neurons.

We have identified and characterized a 5000-Da protein that induces neurite outgrowth from PC12 pheochromocytoma cells, enhances the survival of embryonic rat brain neurons in primary culture, and induces the multiplication of embryonic rat brain astrocytes in primary culture. The factor is produced by a flat cell PC12 variant that expresses the activated ras oncogene after transfection of the gene. The factor resembles transforming growth factor alpha (TGF alpha) and epidermal growth factor (EGF) in that it induces anchorage-independent colony formation of normal rat kidney cells in soft agar and competes with EGF for binding to the EGF receptor. Rat TGF alpha and human TGF alpha also induce neurite outgrowth from PC12 and enhance the survival of embryonic brain neurons. The PC12 variant-derived factor can be distinguished from TGF alpha and EGF immunologically and by migration rates on reversed-phase high-performance liquid chromatography.     

Bidirectional effects of Kupffer cells on hepatocyte proliferation in vitro.

The control of rat hepatocyte DNA synthesis in vitro by Kupffer cells and transformed perisinusoidal lipocytes, i.e. myofibroblast-like cells was studied. Conditioned media from Kupffer cells inhibit the replicative (hydroxyurea-sensitive) DNA synthesis dose-dependently in primary cultures of hepatocytes stimulated by epidermal growth factor (EGF). The cytokine responsible for the inhibition was identified as transforming growth factor beta (TGF beta). After neutralization of activated TGF beta in these media, DNA synthesis is stimulated in quiescent hepatocytes via transforming growth factor alpha (TGF alpha) demonstrated by competitive TGF alpha/EGF-receptor blocking on hepatocytes. Results similar to those obtained with Kupffer cells were found with conditioned media of myofibroblast-like cells. Northern blot hybridization confirms the expression of both TGF beta and TGF alpha in Kupffer cells and myofibroblast-like cells, respectively. These findings support the notion that Kupffer cells and myofibroblast-like cells might regulate in both directions liver regeneration depending on the proportion of secreted TGF alpha and TGF beta and on the activation status of TGF beta, of which a significant fraction is secreted in the latent form.     

Modulation of transforming growth factor alpha-dependent expression of epidermal growth factor receptor gene by transforming growth factor beta, triiodothyronine, and retinoic acid

We have investigated the actions of transforming growth factor (TGF) type alpha on epidermal growth factor (EGF) receptor mRNA expression in MDA-468 human mammary carcinoma cells in serum-free media. We found that exposure of MDA-468 cells to TGF alpha results in elevated levels of EGF receptor mRNA. This increase in mRNA accumulation showed time and dose dependence. Addition of TGF beta 1 enhanced the accumulation of EGF receptor mRNA induced by TGF alpha in a time- and dose-dependent manner. We also found that triiodothyronine at physiological concentrations exerts synergistic control on the action of TGF alpha alone, or in association with TGF beta 1, on EGF receptor mRNA expression. Similarly, retinoic acid treatment also enhanced in a time- and dose-dependent manner the TGF alpha-dependent response of EGF receptor mRNA and acted synergistically with TGF beta 1. The results described here suggest that optimum regulation of EGF receptor gene expression by TGF alpha is a complex process involving synergistic interactions with heterologous growth factors and hormones.     

Estrogen stimulates growth of mammary tumor cells ZR-75 without activation of S6 kinase and S6 phosphorylation. Difference from epidermal growth factor and alpha-transforming growth-factor-induced proliferation

Growth of the human mammary tumor cell line ZR-75-1 is stimulated by epidermal growth factor (EGF) and alpha-type transforming growth factor (alpha TGF), as well as by estradiol (E2). The role of activation of S6 kinase and S6 phosphorylation in the EGF(alpha TGF)-induced and E2-induced growth was investigated. Maximal effects on growth are observed at 10 nM EGF or alpha TGF. EGF as well as alpha TGF treatment of serum-starved cells leads to rapid activation of S6 kinase; the activity is increased about tenfold after 30 min of EGF treatment and declines with the time reaching about 25% of the maximal activity after 2 h of EGF treatment. Similar to the growth response, S6 kinase is activated at lower doses of EGF than alpha TGF and shows a maximal response at 10 nM for both growth factors. In contrast to this finding the incubation of serum-starved cells with E2 over a concentration range between 1 pM and 10 nM and times from 30 min to 4 h does not lead to increased S6 kinase activity. On investigating whether this lack of response to E2 is due to desensitization of the system by induction of alpha TGF it was found that preincubation of cells with alpha TGF for 2-6 h desensitizes them to reactivation of S6 kinase by alpha TGF, whereas preincubation with E2 does not. When S6 phosphorylation is monitored over times from 1 h to 6 h, it is observed that EGF leads to increased S6 phosphorylation, whereas E2 does not. The rate of onset of protein synthesis in the first 2 h of stimulation, when EGF-induced S6 phosphorylation is maximal, is more rapid with EGF than with E2. The results suggest that different pathway are involved in E2-induced and EGF(alpha TGF)-induced proliferation.     

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-alpha/ml or 20 ng basic FGF/ml. TGF-beta (0.02 ng/ml), which did not affect cell proliferation when added alone to the culture medium, inhibited the EGF- and TGF-alpha-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 is therefore an increased EGF receptor expression and increased sensitivity to EGF. TGF-beta did not directly affect androgen-responsive growth but inhibited the synergistic effect of EGF. A considerable expression of TGF alpha (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 TGF alpha-like 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 mutated amino acid in the steroid binding domain of the androgen receptor.