Phorbol ester or epidermal growth factor (EGF) stimulates the concurrent accumulation of mRNA for the EGF receptor and its ligand transforming growth factor-alpha in a breast cancer cell line

We have previously reported that both 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) can stimulate the synthesis rate of EGF receptors. We now show that the MDA468 breast cancer cells express the mRNA for the EGF-like molecule, transforming growth factor-alpha (TGF-alpha), and demonstrate that TPA or EGF cause an accumulation of both EGF receptor and TGF-alpha mRNA. The levels of EGF receptor mRNA paralleled our earlier protein data, with peak accumulations of 2-3-fold with 10(-9) M EGF and 3-5-fold with 100 ng/ml TPA seen between 6 and 8 h. A 7-fold accumulation of TGF-alpha mRNA was seen following 4 h of treatment with TPA, and a 2-fold accumulation was seen after 8 h with EGF. These changes in EGF receptor and TGF-alpha mRNAs were observed in the absence of any change in the mRNA level of the alpha-subunit of hexosaminidase A (a lysosomal enzyme), demonstrating some degree of specificity. Detectable quantities of immunoreactive TGF-alpha accumulated in the cell culture medium of MDA468 cell treated with the blocking anti-EGF receptor monoclonal antibody B1D8 while no immunoreactive TGF-alpha was detected in the medium of cells with unblocked receptors. The concentration of B1D8 used was sufficient to block the binding of exogenously added 125I-EGF to undetectable levels but had only minor effects on cell growth and no effect on the expression of the TGF-alpha and EGF receptor mRNA.     

Characterization of transforming growth factors produced by the insulin-independent teratoma-derived cell line 1246-3A

The 1246-3A cell line is an insulin-independent variant derived from the adipogenic cell line 1246. Data presented in this paper indicate that the 1246-3A cell line releases in its culture medium two types of transforming growth factors, TGF-alpha- and TGF-beta-like polypeptides, and a growth inhibitor. TGF-alpha like polypeptide eluted from Biogel P60 column into two fractions with an apparent molecular weight of 50 kDa and 13 kDa. These high-molecular-weight TGF-alpha-like factors competed with 125I-EGF for binding to epidermal growth factor (EGF) receptors and were specifically immunoprecipitated by incubation with antirat TGF-alpha antibody, not by incubation with anti-EGF antibody. Both fractions promoted anchorage-independent growth of normal rat kidney NRK cells in the absence of EGF and stimulated DNA synthesis in quiescent Balb/c-3T3 cells in a fashion similar to EGF and synthetic TGF-alpha. In addition to secreting TGF-alpha-like polypeptides, 1246-3A cells produce TGF-beta. This polypeptide, eluted from Biogel P60 chromatography with an apparent molecular weight of 25 kDa, promoted anchorage-independent growth of NRK cells in the presence of EGF and was growth inhibitory for Chinese hamster lung fibroblasts CCL 39 cells. Interestingly, another growth inhibitory activity was detected in Biogel P60 fractions and eluted with an apparent molecular weight of between 9.5-11 kDa. This fraction was different from TGF-beta and TGF-alpha as determined by specific radioreceptor competition assays. TGF-alpha and TGF-beta-like polypeptides could represent autocrine growth stimulators for the insulin-independent 1246-3A cells and act in synergy with insulin-related factor (IRF) for an optimal stimulation of 1246-3A cell proliferation in serum-free medium.     

Transforming growth factor-beta modulates the high-affinity receptors for epidermal growth factor and transforming growth factor-alpha

The epidermal growth factor (EGF) receptor mediates the induction of a transformed phenotype in normal rat kidney (NRK) cells by transforming growth factors (TGFs). The ability of EGF and its analogue TGF-alpha to induce the transformed phenotype in NRK cells is greatly potentiated by TGF-beta, a polypeptide that does not interact directly with binding sites for EGF or TGF-alpha. Our evidence indicates that TGF-beta purified from retrovirally transformed rat embryo cells and human platelets induces a rapid (t 1/2 = 0.3 h) decrease in the binding of EGF and TGF-alpha to high-affinity cell surface receptors in NRK cells. No change due to TGF-beta was observed in the binding of EGF or TGF-alpha to lower affinity sites also present in NRK cells. The effect of TGF-beta on EGF/TGF-alpha receptors was observed at concentrations (0.5-20 pM) similar to those at which TGF-beta is active in promoting proliferation of NRK cells in monolayer culture and semisolid medium. Affinity labeling of NRK cells and membranes by cross-linking with receptor-bound 125I-TGF-alpha and 125I-EGF indicated that both factors interact with a common 170-kD receptor structure. Treatment of cells with TGF-beta decreased the intensity of affinity-labeling of this receptor structure. These data suggest that the 170 kD high-affinity receptors for EGF and TGF-alpha in NRK cells are a target for rapid modulation by TGF-beta.     

Differential effects of epidermal growth factor, transforming growth factor-alpha, and vaccinia virus growth factor in the positive regulation of IFN-gamma production

We have recently shown that epidermal growth factor (EGF) is capable of positive regulation of IFN-gamma production, thus establishing a functional relationship between nonhemopoietic growth factors and the immune system. In order to study this relationship further, EGF and the EGF-related growth factors transforming growth factor-alpha (TGF-alpha) and vaccinia virus growth factor (VGF), which stimulate cellular proliferation via binding to the EGF receptor, were studied for their functional and physicochemical effects on IFN-gamma production. In contrast to the positive signal of purified murine EGF and recombinant human EGF (both at 1 nM), neither synthetic TGF alpha nor recombinant VGF were capable of restoring competence for IFN-gamma production by Th cell-depleted spleen cell cultures. TGF-alpha and VGF, in molar excess, also failed to block the helper signal of EGF for IFN-gamma production. Thus TGF-alpha and VGF failed to functionally compete for the EGF receptor in the murine spleen cell system. Both TGF-alpha and VGF stimulated murine 3T3 cell proliferation at concentrations similar to those of EGF, and thus their failure to provide help for IFN-gamma production was not due to a general lack of biologic activity. Binding studies with 125I-EGF suggest that the EGF receptor on murine lymphocytes is not constitutively expressed, but inducible by the T cell mitogen staphylococcal enterotoxin A. TGF-alpha did not compete with 125I-EGF for the induced receptor. The data suggest that lymphocytes express a novel inducible EGF receptor that differs from that expressed on cells such as 3T3 fibroblasts.     

Down-modulation of EGF receptors in cells transformed by the src oncogene

The effects of src oncogene expression on epidermal growth factor (EGF) receptors have been investigated in mouse 3T3 and rat-1 fibroblasts. Transformation of both cell types with src resulted in marked reductions in cellular EGF receptor levels, as assayed by either 125I-EGF binding or immunoprecipitation of receptor protein from radiolabeled cell lysates. In contrast to cells transformed by other types of retroviral oncogenes, the loss of EGF receptors in the src-transformed cells did not appear to be due to secreted transforming growth factor-alpha (TGF-alpha), since such factors were undetectable in culture fluids from the src-transformed cells. By several criteria of transformation, an EGF-receptorless cell line infected with src was shown to be transformed, suggesting that EGF receptors themselves are not obligatory to the src transformation process. We suggest that pp60src down-modulates EGF receptors by an intracellular mechanism and that the loss of the receptors is symptomatic of more general effects of pp60src on the machinery of growth regulation.     

Growth modulation by epidermal growth factor (EGF) in human colonic carcinoma cells: constitutive expression of the human EGF gene

The functional role of epidermal growth factor (EGF) in epithelium-derived human colonic carcinoma cells was investigated by transfection with plasmid pUCDS3, which contained synthetic human EGF encoding sequences, into two human colonic carcinoma cell types with dissimilar phenotypic properties: the moderately differentiated and growth factor-responsive Moser and the highly metastatic KM12SM cells. The Moser cells exhibited a proliferative response to treatment with exogenous EGF, while the KM12SM cells did not. The constitutive expression of the human EGF gene in these colonic carcinoma cell types resulted in elevated expression of EGF mRNA, with concurrent production and secretion of a large amount of EGF, and downmodulation of transforming growth factor-alpha (TGF-alpha) secretion. Growth stimulation and down-modulation of both high and low affinity EGF receptors were observed in the EGF-transfected Moser clones. Results of experiments using anti-EGF and anti-EGF-receptor antibody to block the proliferation of EGF-transfected Moser clones suggested that autocrine stimulatory mechanisms involving both EGF and TGF-alpha were operative in these cells. By comparison, a growth-inhibitory effect, with no apparent EGF receptor modulation, was observed in the EGF-transfected KM12SM clones. Both the parental and EGF-transfected KM12SM clones possessed fewer EGF receptors than the Moser cells, and anti-EGF or anti-EGF-receptor antibody did not affect the cells' growth properties. These results suggested that the mechanisms of growth inhibition in the EGF-transfected KM12SM clones were non-autocrine or intracellular in nature. Thus, constitutive expression of the human EGF gene in two phenotypically different, epithelium-derived human colonic carcinoma cells resulted in divergent altered growth characteristics.     

Organ culture of suckling rat intestine: comparative study of various hormones on brush border enzymes

Jejunal mucosa of 6 d-old rats were cultured for 24 and 48 h in the presence of thyroxine, insulin, pentagastrin, glucagon, epidermal growth factor (EGF) or dibutyryl-A-3:5-MP cyclic with or without dexamethasone (DX). The enzymes were assayed on the purified brush borders. The various agents added alone to the basic culture medium had no effect with the exception of DX on the levels of enzyme activities. Dexamethasone alone induced sucrase, stimulated maltase, and protected other brush border enzyme activities (aminopeptidase, lactase, and alkaline phosphatase). When added to DX-supplemented medium, only the following factors modified the levels of enzymatic activities observed with DX alone. Insulin (10(-6) M) increased maltase, alkaline phosphatase, and lactase activity to a greater extent than DX at 24 h culture, the effect being maintained at 48 h on alkaline phosphatase only. At 48 h culture, both EGF (10(-8) M) and dbcAMP (10(-3) M) decreased DX-induced sucrase activity. The latter agent also depressed DX-stimulated aminopeptidase activity.     

Schlafen-3 decreases cancer stem cell marker expression and autocrine/juxtacrine signaling in FOLFOX-resistant colon cancer cells

We have previously demonstrated that expression of the novel gene schlafen-3 (Slfn-3) correlates with intestinal epithelial cell differentiation (Patel VB, Yu Y, Das JK, Patel BB, Majumdar AP. Biochem Biophys Res Commun 388: 752-756, 2009). The present investigation was undertaken to examine whether Slfn-3 plays a role in regulating differentiation of FOLFOX-resistant (5-fluorouracil + oxaliplatin) colon cancer cells that are highly enriched in cancer stem cells (CSCs). Transfection of Slfn-3 in FOLFOX-resistant colon cancer HCT-116 cells resulted in increase of alkaline phosphatase activity, a marker of intestinal differentiation. Additionally, Slfn-3 transfection resulted in reduction of mRNA and protein levels of the CSC markers CD44, CD133, CD166, and aldehyde dehydrogenase 1 in both FOLFOX-resistant HCT-116 and HT-29 cells. This was accompanied by decreased formation of tumorosphere/colonosphere (an in vitro model of tumor growth) in stem cell medium and inhibition of expression of the chemotherapeutic drug transporter protein ABCG2. Additionally, Slfn-3 transfection of FOLFOX-resistant HCT-116 and HT-29 cells reduced Hoechst 33342 dye exclusion. Finally, Slfn-3 transfection inhibited the expression of transforming growth factor-α in both FOLFOX-resistant colon cancer cells, but stimulated apoptosis in response to additional FOLFOX treatment. In summary, our data demonstrate that Slfn-3 expression inhibits multiple characteristics of CSC-enriched, FOLFOX-resistant colon cancer cells, including induction of differentiation and reduction in tumorosphere/colonosphere formation, drug transporter activity, and autocrine stimulation of proliferation. Thus Slfn-3 expression may render colon CSCs more susceptible to cancer chemotherapeutics.     

Epidermal growth factor and transforming growth factor-alpha: differential intracellular routing and processing of ligand-receptor complexes.

Two structurally related but different polypeptide growth factors, epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), exert their activities after interaction with a common cell-surface EGF/TGF-alpha-receptor. Comparative studies of the effects of both ligands have established that TGF-alpha is more potent than EGF in a variety of biological systems. This observation is not explained by differences in affinities of the ligands for the receptor, because the affinity-constants of both factors are very similar. We have compared the intracellular processing of ligand-receptor complexes using either EGF or TGF-alpha in two different cell systems. We found that TGF-alpha dissociates from the EGF/TGF-alpha-receptor at much higher pH than EGF, which may reflect the substantial difference in the calculated isoelectric points. After internalization, the intracellular TGF-alpha is more rapidly cleared than EGF, and a substantial portion of the released TGF-alpha represents undegraded TGF-alpha in contrast to the mostly degraded EGF. In addition, TGF-alpha did not induce a complete down-regulation of cell surface receptors, as observed with EGF, which is at least in part responsible for a much sooner recovery of the ligand-binding ability after down-regulation, in the case of TGF-alpha. These differences in processing of the ligand-receptor complexes may explain why TGF-alpha exerts quantitatively higher activities than EGF.     

Attenuated processing of epidermal growth factor in the face of marked degradation of transforming growth factor-alpha

T3M4 human pancreatic carcinoma cells avidly bound and internalized 125I-labeled epidermal growth factor (EGF) but did not readily degrade the ligand. Pulse-chase experiments in which the cell-bound radioactivity was allowed to dissociate into the incubation medium in the presence of unlabeled EGF indicated that the majority of the released 125I-EGF consisted of intact EGF and a slightly processed species that readily bound to the cell. Omission of unlabeled EGF during the chase period markedly decreased the amount of radioactivity in the incubation medium, mainly as a result of the rebinding of EGF to the cells. In contrast, T3M4 cells readily degraded 125I-labeled transforming growth factor-alpha (TGF-alpha), and the released radiolabeled products did not rebind to the cells. Both ligands were released from T3M4 cells under acidic conditions, complete dissociation occurring at a pH of 4.5 for EGF, and a pH of 6.5 for TGF-alpha. A 431 human epidermoid carcinoma cells and ASPC-1 human pancreatic carcinoma cells also failed to extensively degrade 125I-EGF, whereas Rat-1 fibroblasts markedly degraded the growth factor. As in the case of T3M4 cells, ASPC-1 cells extensively degraded 125I-TGF-alpha. Degradation of either ligand was blocked by the lysosomotropic compound methylamine in all the tested cell lines. Immunoprecipitation of the EGF receptor with specific polyclonal antibodies and Western blot analysis revealed the anticipated 170-kDa protein in T3M4 cells. Both EGF and TGF-alpha enhanced EGF receptor degradation, but TGF-alpha was less effective than EGF. These findings indicate that in certain cell types EGF and TGF-alpha may be differentially processed.     

Characterization of epidermal growth factor receptor induction by retinoic acid in a chemically transformed rat liver cell line

Levels of epidermal growth factor (EGF) receptor expression vary widely among cell lines derived clonally from a chemically transformed population of rat liver epithelial cells. Retinoic acid (RA), a derivative of vitamin A that stimulates differentiation in a number of embryonal cell lines, increases the level of 125I-EGF binding in several clones of the transformed cell lines. One such cell line, GP6ac, which reverts to a less transformed phenotype when treated with RA, exhibited a 3-4-fold increase in surface EGF receptors with prolonged (2-5-day) RA exposure. The increase persisted as long as the cells were treated with RA. The increase in surface EGF receptors was due to induction of receptor biosynthesis, which occurred within 4 h at both the mRNA and protein levels and persisted until the RA was withdrawn. Paradoxically, the RA response was accompanied by an initial 40-50% decrease in 125I-EGF binding during the first 12 h of RA treatment. The decrease was due primarily to a reduction of receptor affinity. Since the phorbol ester 12-O-tetradecanoylphorbol-13-acetate also decreases 125I-EGF binding and increases EGF receptor biosynthesis in GP6ac cells, we tested the effect of RA in cells depleted of protein kinase C by prolonged treatment (18 h) with 10 microM 12-O-tetradecanoylphorbol-13-acetate. The absence of protein kinase C did not affect the induction of receptor mRNA and protein or the decrease in binding during the early period of RA exposure. This indicates that RA induction of EGF receptor synthesis in GP6ac cells involves signaling pathways distinct from those utilized by phorbol esters.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epidermal growth factor and transforming growth factor-alpha decrease gamma interferon receptors and induction of intercellular adhesion molecule (ICAM-1) on cultured keratinocytes.

The link between the epidermal keratinocytes of the skin and the activated T lymphocytes of the immune system is mediated by a variety of cytokines, including gamma interferon (IFN-gamma). We studied the influence of keratinocyte mitogens such as transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF), and somatomedin-C (SM-C) on the ligand binding of 32P-labeled IFN-gamma to cultured keratinocytes derived from normal appearing adult human skin. Keratinocytes placed in a medium devoid of mitogens become growth arrested, and these quiescent cells expressed 2.4 times (28,900 versus 12,200 sites/cell) as many high affinity IFN-gamma receptors (Kd = 0.22 nM) compared to keratinocytes which were actively growing in medium containing TGF-alpha (25 ng/ml) or EGF (10 ng/ml). The reduction in IFN-gamma receptor sites by TGF-alpha/EGF was mitogen specific, as adding SM-C (500 ng/ml) did not have any effect on ligand binding, although it similarly stimulated keratinocyte growth. The reduction in IFN-gamma receptors was time dependent, occurring primarily after 24-48 hours of change in tissue culture conditions. The reduction in the number of high affinity IFN-gamma receptors by TGF-alpha/EGF had immunobiological consequences, because quiescent keratinocytes in basal medium had an increased expression of HLA-DR and intercellular adhesion molecule-1 (ICAM-1) induced by IFN-gamma, compared to actively growing TGF-alpha/EGF treated keratinocytes. These results suggest that rapidly proliferating keratinocytes exposed to TGF-alpha/EGF but not SM-C are capable of altering their response to IFN-gamma by decreasing their number of cell surface high affinity receptors for IFN-gamma.     

Inhibition of epidermal growth factor binding to mouse cultured cells by fibroblast-derived growth factor. Evidence for an indirect mechanism

Fibroblast-derived growth factor (FDGF), a basic, heat- and acid-stable polypeptide partially purified from the serum-free conditioned medium of BHK cells transformed by simian virus 40, is a potent mitogen for Swiss 3T3 cells and causes a marked reduction in 125I-labeled epidermal growth factor (125I-EGF) binding to these cells. The activity which inhibits EGF binding coelutes with the growth-stimulating activity after gel filtration, ion exchange chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Both cellular responses are elicited by the same range of FDGF concentration in several murine cell types. The inhibition of EGF binding is rapid and results from a decrease in the apparent affinity of cellular receptors for 125I-EGF. FDGF does not affect the rate of cell-mediated 125I-EGF degradation. Several lines of evidence suggest that FDGF does not bind directly to EGF receptor. First, the effect of FDGF is dependent on the temperature of the assay; furthermore, treatment of cells with EGF results in loss of EGF receptors while exposure to FDGF for up to 24 h does not induce "down-regulation" of EGF receptors. Further, in A431 cells which display a large number of specific EGF receptors, 125I-EGF binding is not sensitive to FDGF. Finally, the effect of FDGF on 125I-EGF binding is not observed with isolated plasma membranes. Taken together, these findings suggest that FDGF binds to sites which are separate from EGF receptors. The results show a novel mechanism whereby a growth-promoting factor produced by a tumor cell line can rapidly modulate the affinity of the cellular receptors for EGF in an indirect manner.     

Epidermal growth factor (EGF)-nonresponsive variants of normal rat kidney cell line: response to EGF and transforming growth factor-beta

Anchorage-independent growth in soft agar of normal rat kidney (NRK) fibroblasts depends on both transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF) (or TGF-alpha). We have isolated two EGF-nonresponsive cell lines, N-3 and N-9, from chemically mutagenized NRK cells, after selection of mitogen-specific nonproliferative variants in the presence of EGF and colchicine. Saturation binding kinetics with 125I-EGF showed one-half or fewer EGF receptors in N-3 and N-9 than in their parental NRK. Cellular uptake of 2-deoxy-D-glucose was enhanced in all NRK, N-3, and N-9 cell lines by TGF-beta treatment, whereas treatment with EGF significantly enhanced the cellular uptake of the glucose analog in NRK cells, but not in N-3 and N-9 cells. DNA synthesis of NRK during the quiescent state, but not that of N-3 and N-9, was stimulated by EGF. Anchorage-independent growth of N-9 could not be observed even in the presence of both EGF and TGF-beta, whereas that of N-3 was significantly enhanced by TGF-beta alone. EGF stimulated phosphorylation of a membrane protein with molecular size 170 kDa of NRK, but not of N-3, when immunoprecipitates reacting with anti-phosphotyrosine antibody were analyzed. Exposure of NRK cells to EGF increased cellular levels of TGF-beta mRNA, but there appeared little expression of TGF-beta mRNA in N-3 and N-9 cells. Exposure of N-3 cells to EGF or TGF-beta enhanced the secretion of EGF into culture medium, but exposure of NRK or N-9 cells did not. Altered response to EGF of N-3 or N-9 might be related to their aberrant growth behaviors.     

Characterization of binding and receptors for epidermal growth factor in smooth muscle

Summary The mitogenic and differentiation-inducing activities of epidermal growth factor (EGF) in epithelial tissues have been well described. Since non-mitogenic effects of EGF, especially in mesenchymal tissues such as smooth muscle are not well-known (Nanney et al. 1984), we have examined EGF-binding and receptors in smooth muscle from many sites. Specific EGF binding sites were detected by incubating small pieces of tissue with ¹²⁵I-EGF; immunoreactive EGF receptors were detected by immunohistochemistry. In-situ localization of ¹²⁵I-EGF binding sites and immunoreactive EGF receptors of smooth muscle cells in intact mammalian tissues were identical using either ¹²⁵I-EGF autoradiography or anti-EGF receptor antibody in an immunoperoxidase method. Cultured rat aortic smooth muscle also contained specific EGF receptors as detected by their biological response to EGF-binding and internalization of ¹²⁵I-EGF, as well as EGF-stimulated phosphorylation of a 170K protein. The presence of EGF receptors in a well-differentiated smooth muscle cell indicates that EGF may play a physiological, but non-mitogenic role in mammalian tissues in vivo.     

Ligand-induced activation of A431 cell epidermal growth factor receptors occurs primarily by an autocrine pathway that acts upon receptors on the surface rather than intracellularly

A431 cells express high numbers of epidermal growth factor (EGF) receptors and produce a ligand for these receptors, transforming growth factor-alpha (TGF-alpha). We have obtained evidence that the EGF receptors on these cells may be activated through an "autocrine" pathway by ligand and have investigated whether activation of phosphorylation of the receptor by the endogenously produced TGF-alpha occurs intracellularly or at the cell surface. When A431 cells were cultured under serum-free conditions, in the absence of exogenous ligand, EGF receptors were found to have a basal level of phosphorylation. When cells were labeled by culturing with 32Pi in the continuous presence of monoclonal antibodies that block binding of TGF-alpha to the EGF receptor, phosphorylation decreased to 30 +/- 10% of the basal level. This reduction could not be accounted for by the decrease in receptor content attributable to down-regulation and catabolism of EGF receptors that resulted from the binding of anti-receptor monoclonal antibodies. The reduction in receptor phosphorylation mediated by antibody was accompanied by the accumulation of increased levels of secreted TGF-alpha species in the culture medium. We also pulse-labeled A431 cells for 15 min with [35S]cysteine and immunoprecipitated the cell lysate with anti-phosphotyrosine antibody after various chase periods. Tyrosine-phosphorylated EGF receptor became detectable after 40 min of chase and reached a maximum after 4-6 h; these times are in agreement with the intervals required for EGF receptors to reach the cell surface after synthesis and then to achieve maximal expression. In addition, only the 170-kDa, mature EGF receptor species, and not the 160-kDa intracellular precursor, was immunoprecipitated with the anti-phosphotyrosine antibody. The results of these pulse-chase experiments and the finding that anti-receptor monoclonal antibody can block receptor phosphorylation suggest that activation of EGF receptors can result from the binding of an endogenous ligand (presumably TGF-alpha), which occurs at the cell surface and not during receptor biosynthesis and intracellular processing.     

Loss of epidermal growth factor receptors and release of transforming growth factors do not correlate with sarcoma virus-transformation in clonally-related NIH/3T3-derived cell lines.

Transformation of NIH/3T3 cells by Kirsten murine sarcoma virus (MSV) caused a dramatic reduction in the number of cell-surface receptors for epidermal growth factor (EGF). However, the number of EGF receptors remained at a very low level in a non-tumourigenic revertant cell line isolated from the virus-transformed cells, indicating that an increase in EGF receptors is not a requirement for the phenotypic reversion of Kirsten MSV-transformed 3T3 cells. Serum-free conditioned medium from normal and virus-transformed cell lines contained similar amounts of cell growth-promoting activity as assayed by the ability to stimulate DNA synthesis in quiescent Swiss 3T3 cell cultures. However, the concentrated conditioned medium from these cell lines showed no evidence of beta-transforming growth factor (TGF) activity as assayed by promotion of anchorage-independent growth of untransformed normal rat kidney (NRK) fibroblasts in agarose. The cellular release of alpha-TGF activity was assayed by measuring the ability of concentrated conditioned medium to inhibit the binding of 125I-EGF to Swiss 3T3 cells. Conditioned medium protein from the virus-transformed cell line inhibited 125I-EGF binding but only to the same extent as conditioned medium protein prepared from the untransformed cell line. The alpha-TGF secretion by these cell lines was estimated to be 30-45-fold lower than the level of alpha-TGF released by a well-characterized alpha-TGF-producing cell line (3B11). These results suggest that the induction of TGF release is not a necessary event in the transformation of NIH/3T3 cells by Kirsten MSV.     

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.     

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.     

Growth factor production by a human megakaryocytic tumor cell line

A recently described human megakaryocytic tumor cell line was analyzed for the presence of growth factor activity and was found to produce large quantities of transforming growth factor beta-like (TGF-beta) and basic fibroblast growth factor-like (bFGF) activities. Growth factor activities were identified using a radioreceptor assay for the TGF-beta-like activity, a heparin-binding assay for the b-FGF-like activity, and a demonstration of distinct biological activities for each type of factor. Tumor poly-A+ RNA revealed strong signals when probed with complementary DNA corresponding to bovine basic FGF and human TGF-beta and weak signals when probed with cDNA corresponding to epidermal growth factor (EGF) and TGF-alpha. The levels of EGF and TGF-alpha produced in the tumor line were too low to be detected by radioreceptor assays. Relative levels of messenger RNA encoding each of the growth factors reflected the relative levels of each of the respective factors tested. These data represent the first definitive identification of FGF-like activities in megakaryocytic-like cell lines. Interestingly, the line displayed little activity similar to platelet-derived growth factor (PDGF) when assayed either biochemically or by poly-A+ RNA analysis.