Mitogenic action of tumor necrosis factor in human fibroblasts: interaction with epidermal growth factor and platelet-derived growth factor

We have previously shown that tumor necrosis factor (TNF) can increase the number of epidermal growth factor (EGF) receptors on human FS-4 fibroblasts and that this increase may be related to the mitogenic action of TNF in these cells. Here we show that TNF stimulated the growth of FS-4 fibroblasts in a chemically defined, serum-free medium in the absence of EGF. Anti-EGF receptor antibody, which blocked the mitogenic effects of EGF in FS-4 cells, did not inhibit the mitogenic action of TNF in serum-free or serum-containing medium, indicating that EGF or an EGF-like molecule was not responsible for the mitogenic effects of TNF. However, the simultaneous addition of TNF and EGF to cells grown in serum-free medium resulted in a synergistic stimulation of DNA synthesis and cell growth. The actions of TNF and EGF were also examined in growth-arrested FS-4 cells and were compared with the action of platelet-derived growth factor (PDGF). In the absence of other growth factors, TNF was a relatively weak mitogen in growth-arrested cells, compared with EGF or PDGF. Nevertheless, TNF synergized with EGF or high doses of PDGF in stimulating DNA synthesis. Furthermore, antibodies specific for TNF or the EGF receptor were used to selectively inhibit the actions of these two factors, after specific incubation periods, in growth-arrested cells treated concurrently with EGF and TNF. To produce an optimal stimulation of DNA synthesis, EGF had to be present for a longer period of time than TNF. We conclude that in their synergistic action on growth-arrested FS-4 cells, EGF was responsible for driving the majority of the cells into S phase, while TNF appeared to make the cells more responsive to the mitogenic action of EGF. The findings indicate that TNF can cooperate with, and enhance the actions of, EGF in promoting DNA synthesis and cell division.     

Clonal growth of human keratinocytes with small amounts of dialyzed serum

Summary A survey of commercially availabla media revealed that medium F-12 was superior to medium 199 for clonal growth of human epidermal keratinocytes (HK) when supplemented with 10 μg/ml hydrocortisone (HC) plus dialyzed fetal bovine serum protein (FBSP), rather than the whole serum used in previous studies. Qualitative and quantitative adjustment of the medium composition for optimal clonal growth with minimal amounts of FBSP generated a new medium, MCDB 151, which supports clonal growth of HK with 10 μg/ml HC and as little as 1 mg/ml FBSP (equivalent in protein concentration to 2.0% whole serum). MCDB 151 differs significantly from MCDB 105, previously developed in this laboratory for normal human fibroblasts, and each medium selectively favors growth of its own type of cell in primary cultures of disaggregated human neonatal foreskin cells. Differences in the amounts of calcium and adenine in the two media appear to be among the most influential factors mediating the selective growth. Optimal growth of HK occurs at a very low level of Ca²⁺ that causes the colonies to remain as monolayers rather than stratifying as they do in the presence of higher levels of calcium. However, keratin synthesis, which was examined through use of highly specific fluorescent antibodies, is not affected by the Ca²⁺ concentration. Agents that increase intracellular cyclic AMP levels appear to have no effect on HK growth in MCDB 151 with 10 μg/ml HC and 1.0 mg/ml FBSP. This paper contains material from a thesis submitted to the Graduate School of the University of Colorado, Boulder, by Donna M. Peehl in partial fulfillment of the requirements for the Ph.D. degree. This work was supported by Grant CA 15305 from the National Cancer Institute and Grant AG 00310 from the National Institute on Aging.     

Inhibition of epidermal growth factor-stimulated DNA synthesis by a bovine sialoglycopeptide inhibitor occurs at an intracellular level

The control of cell proliferation involves the complex interaction between growth factors and growth inhibitors. We have examined this interaction with the mitogen epidermal growth factor (EGF) and a recently purified 18 kD, pI 3, sialoglycopeptide that reversibly inhibits cellular metabolism of a variety of cells. The sialoglycopeptide was a very potent inhibitor of EGF action; 0.22 nM of the inhibitor completely blocked the mitogenic effect of 1.60 nM of EGF. The sialoglycopeptide, however, did not affect the binding of EGF to 3T3 cells. Neither the mixed affinities (0.11-1.9 nM) of binding nor the total number of receptors (50,000 receptors/cell) for EGF were altered by the addition of the sialoglycopeptide. In addition, competitive binding experiments demonstrated the specificity of inhibitor binding to 3T3 cells and also showed that EGF and the sialoglycopeptide did not share the same receptor, suggesting that the inhibitor blocked EGF action at a postreceptor, intracellular event in the signal cascade. We further demonstrated that the sialoglycopeptide had to be added within 2.5 hr after EGF to block effectively the stimulation of DNA synthesis by the growth factor, suggesting that the inhibitor blocked EGF stimulation at a relatively early step in the signal transduction mechanism.     

Epidermal growth factor induces tyrosine hydroxylase in a clonal pheochromocytoma cell line, PC-G2

We have previously described the isolation of a clonal cell line (PC-G2) in which the level of tyrosine hydroxylase (TH), the rate-limiting step in the synthesis of the catecholamine neurotransmitters, is induced by nerve growth factor (NGF). We now report that epidermal growth factor (EGF) also induces TH in the PC-G2 cell line. Although EGF has been shown to be mitogenic for many cultured cells, no neuronal function has been previously reported for this protein. The TH response to EGF is elicited in a dose-dependent fashion at concentrations as low as 0.1 ng/ml and is maximal at 10 ng/ml EGF. The maximal response is observed after 3--4 d of exposure to 10 ng/ml EGF. The induction by NGF and EGF is inhibited by their respective antisera. Dexamethasone, a synthetic glucocorticoid which we have previously shown modulates the response of PC-G2 cells to NGF, also modulates the TH induction elicited by EGF.     

Inhibition of epidermal growth factor/transforming growth factor-alpha-stimulated cell growth by a synthetic peptide

Estrogen-stimulated growth of the human mammary adenocarcinoma cell line MCF-7 is significantly inhibited by monoclonal antibodies to the epidermal growth factor (EGF) receptor that act as antagonists of EGF's mitogenic events by competing for high-affinity EGF receptor binding sites. These antibodies likewise inhibit the EGF or transforming growth factor-alpha (TGF-alpha)-stimulated growth of these MCF-7 cells. An analogous pattern of specific EGF or TGF-alpha growth inhibitory activity was obtained using a synthetic peptide analog encompassing the third disulfide loop region of TGF-alpha, but containing additional modifications designed for increased membrane affinity [( Ac-D-hArg(Et)2(31),Gly32,33]HuTGF-alpha(31-43)NH2). The growth factor antagonism by this synthetic peptide was specific in that it inhibited EGF, TGF-alpha, or estrogen-stimulated growth of MCF-7 cells but did not inhibit insulin-like growth factor-1 (IGF-1)-stimulated cell growth. Altogether, these results suggest that a significant portion of the estrogen-stimulated growth of these MCF-7 cells is mediated in an autocrine/paracrine manner by release of EGF or TGF-alpha-like growth factors. The TGF-alpha peptide likewise inhibited EGF- but not fibroblast growth factor (FGF)- or platelet-derived growth factor (PDGF)-stimulated growth of NIH-3T3 cells in completely defined media; but had no effect on growth or DNA synthesis of G0-arrested cells, nor did it effect growth of NR-6 cells, which are nonresponsive to EGF. Although this synthetic peptide did not directly compete with EGF for cell surface receptor binding, it exhibited binding to a cell surface component (followed by internalization), which likewise was not competed by EGF. The peptide did not directly inhibit EGF-stimulated phosphorylation of the EGF receptor, nor did it inhibit phosphorylation of an exogenous substrate, angiotensin II, by activated EGF receptor. The TGF-alpha peptide did, however, affect the structure of laminin as manifested by laminin self-aggregation; this affect on laminin may, in turn, have a modulatory effect on EGF-mediated cell growth.     

Epidermal growth factor-dependent growth of human KB cells in a defined medium and altered growth factor requirements of KB mutants resistant to EGF-Pseudomonas exotoxin conjugates

A serum-free culture system was established for human KB carcinoma (HeLa) cells that consisted of a chemically defined medium and several growth factors including epidermal growth factor (EGF), insulin, transferrin, hydrocortisone, and ethanolamine. EGF and insulin showed the greatest effects on the growth rate of KB cells. Insulin-like growth factor I (IGF-I) at the same concentration as insulin stimulated cell growth less than insulin. Transferrin, hydrocortisone, or ethanolamine had no growth-stimulatory effects alone but were stimulatory when combined with EGF and/or insulin. Transforming growth factor-beta inhibited growth and triiodothyronine stimulated growth. The growth factor requirements were established for several KB mutants with low EGF receptor levels that had been selected for resistance to a conjugate of EGF with Pseudomonas exotoxin (EGF-PE). Three of five KB mutants did not respond to EGF; two other mutants responded to a lesser extent than the parental KB cells. Four mutants had a reduced response to insulin and responded to T3; one mutant (ET-30) responded to neither. These results indicate that KB cells selected for EGF-PE resistance have lost their growth response to EGF and illustrate the usefulness of serum-free medium for studying the growth factor requirements of mutants with altered receptor levels.     

Mitogenesis in fetal rat bone cells simultaneously exposed to type beta transforming growth factor and other growth regulators

Type beta transforming growth factor (TGF-beta) is found in large amounts in bone tissue, and is a potent mitogen for osteoblast-enriched cell cultures obtained from fetal rat parietal bone. Because other local and systemic factors may be presented to bone cells simultaneously with TGF-beta, it is important to understand the effects of this complex growth regulator in such circumstances. Unlike the effects observed in many tissue systems, TGF-beta does not invariably inhibit the mitogenic response of bone cells to other growth promoters. In contrast, other factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and type alpha tumor necrosis factor (TNF-alpha) limit the response of osteoblastic bone cells to TGF-beta. TGF-beta is a much weaker mitogen for fibroblastic cells obtained from fetal rat bone, whereas fetal bovine serum, EGF, bFGF, and TNF-alpha are more potent stimulators. In addition, TGF-beta does not significantly impair the response of the fibroblastic bone cells to the other tested agents. These findings reinforce a role of TGF-beta as an anabolic bone growth regulator, and suggest that its function may be modified by other local or systemic agents that can also affect bone cells.     

Transforming growth factors and control of neoplastic cell growth

Transforming growth factors (TGFs) are peptides that affect the growth and phenotype of cultured cells and bring about in nonmalignant fibroblastic cells phenotypic properties that resemble those of malignant cells. Two types of TGFs have been well characterized. One of these, TGF alpha, is related to epidermal growth factor (EGF) and binds to the EGF receptor, whereas the other, TGF beta, is not structurally or functionally related to TGF alpha or EGF and mediates its effects via distinct receptors. TGF beta is produced by a variety of normal and malignant cells. Depending upon the assay system employed, TGF beta has both growth-inhibitory and growth-stimulating properties. Many of the mitogenic effects of TGF beta are probably an indirect result of the activation of certain growth factor genes in the target cell. The ubiquitous nature of the TGF beta receptor and the production of TGF beta in a latent form by most cultured cells suggests that the differing cellular responses to TGF beta are regulated either by events involved in the activation of the factor or by postreceptor mechanisms. The combined effects of TGF beta with other growth factors or inhibitors evidently play a central role in the control of normal and malignant cellular growth as well as in cell differentiation and morphogenesis. Since transforming growth factor as a concept has partially proven misleading and insufficient, there is a need to find a new nomenclature for these regulators of cellular growth and differentiation.     

Regulation of human amnion cell growth and morphology by sera,plasma, and growth factors

Summary The requirements of human epithelial cells derived from the amnion membrane for serum factors were investigated. The growth promoting effects of human whole blood serum (WBS), platelet-poor defibrinogenated plasma, and plasma-derived serum (PDS) were examined in primary cultures of these ectodermal cells. The numbers of population doublings recorded after 10 days in the presence of 10% WBS, defibrinogenated plasma, or PDS were 2.3, 2.0 or 1.5, respectively. Although dialysis of sera or plasma had little effect on growth promotion, it markedly decreased the capacity of plasma to maintain cells in culture beyond 10 days. The differences in growth activities could not be attributed to the presence of anticoagulant in plasma and PDS or to the presence of excess calcium in PDS. Platelet lysates and purified platelet-derived growth factor had no effect on growth. Amnion cell growth was enhanced by epidermal growth factor (EGF) or hydrocortisone, but the glucocorticoid did not condition cells to respond to growth factors. Insulin and fibroblast growth factor singly or in combination had no effect on cell replication. Giant cell formation accompanied maintenance in hydrocortisone with defibrinogenated plasma and PDS. Discrete regions of dense population appeared in the presence of hydrocortisone, EGF, and undialyzed supplements.Supported in part by ACS grant PDT-140     

Bloom''s syndrome cells have an abnormal serum growth response

Certain growth responses of Bloom's syndrome (BS) dermal fibroblasts have been compared to those of normal human fibroblasts. By applying the principles of Michaelis-Menton kinetics to clonal dose-response data, serum and epidermal growth factor (EGF) requirements of the two cell types were found to be similar. However, the maximal clonal growth rate of BS cells was significantly lower than that of their normal counterparts. Although specific EGF binding by BS cells was marginally higher than in normal cells, EGF's growth-promoting activity was only half of that seen in normal cells. These observations indicate that the abnormally low growth rate of BS cells is not attributable to excessive requirements for serum-derived growth factors and suggest instead that the genetic defect in some way impairs the cells' ability to respond fully to growth stimulation.     

Regulation of rat ovarian cell growth and steroid secretion

A cultured rat ovarian cell line (31 A-F(2)) was used to study the effect of growth factors (epidermal growth factor [EGF] and fibroblast growth factor [FGF]), a survival factor (ovarian growth factor [OGF]), a hormone (insulin), and an iron-binding protein (transferring) on cell proliferation and steroid production under defined culture conditions. EGF and insulin were shown to be mitogenic (half-maximal response at 0.12 nM and 0.11 muM, respectively) for 31A-F(2) cells incubated in serum-free medium. EGF induced up to three doublings in the cell population, whereas insulin induced an average of one cell population doubling. FGF, OGF, and transferrin were found not to have any prominent effect on cell division when incubated individually with 31A-F(2) cells in serum-free medium. However, a combination of EGF, OGF, insulin, and transferrin stimulated cell division to the same approximate extent as cells incubated in the presence of 5 percent fetal calf serum. EGF or insulin did not significantly affect total cell cholesterol levels (relative to cells incubated in serum-free medium) when incubated individually with 31A-F(2) cells. However, cell cholesterol levels were increased by the addition of OGF (250 percent), FGF (370 percent), or a combination of insulin and EGF (320 percent). Progesterone secretion from 31A-F(2) cells was enhanced by EGF (25 percent), FGF (80 percent), and insulin (115 percent). However, the addition of a mitogenic mixture of EGF, OGF, insulin, and transferrin suppressed progesterone secretion 150 percent) below that of control cultures. These studies have permitted us to determine that EGF and insulin are mitogenic factors that are required for the growth of 31A-F(2) cells and that OGF and transferrin are positive cofactors that enhance growth. Also, additional data suggest that cholesterol and progesterone production in 31A-F(2) cells can be regulated by peptide growth factors and the hormone insulin.     

Inhibition of parathyroid hormone-responsive adenylate cyclase in clonal osteoblast-like cells by transforming growth factor alpha and epidermal growth factor

The effects of transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) on parathyroid hormone (PTH)-responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR-106) with the osteoblast phenotype. Recombinant TFG-alpha and EGF incubated with UMR-106 cells for 48 h each produced concentration-dependent inhibition of PTH-responsive adenylate cyclase, with maximal inhibition of 38-44% at 1-3 ng/ml of either growth factor. TGF-alpha and EGF also inhibited beta-adrenergic agonist (isoproterenol)-stimulated adenylate cyclase by 32%, but neither growth factor affected enzyme response to prostaglandin or basal (unstimulated) activity. Nonreceptor-mediated activation of adenylate cyclase by forskolin and cholera toxin was inhibited 18-20% by TGF-alpha and EGF. Pertussis toxin augmented PTH-stimulated adenylate cyclase, suggesting modulation of PTH response by a functional inhibitory guanine nucleotide-binding regulatory component of the enzyme. However, pertussis toxin had no effect on TGF-alpha inhibition of PTH response. Growth factor inhibition of PTH response was time-dependent, with maximal inhibition by 4-12 h of TGF-alpha exposure, and was reduced by prior treatment of UMR-106 cells with cycloheximide. TGF-alpha was not mitogenic for UMR-106 cells. The results indicate that TGF-alpha and EGF selectively impair PTH- and beta-adrenergic agonist-responsive adenylate cyclase of osteoblast-like cells. Growth factor inhibition of adenylate cyclase may be exerted at the receptor for stimulatory agonist and at nonreceptor components excluding pertussis toxin-sensitive guanine nucleotide-binding regulatory proteins. The inhibitory action of growth factors may also require protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bombesin and the C-terminal tetradecapeptide of gastrin-releasing peptide are growth factors for normal human bronchial epithelial cells

Bombesin and the C-terminal portion of gastrin-releasing peptide (GRP14-27) each increase clonal growth rate and colony-forming efficiency of normal human bronchial epithelial cells. These effects occur in the presence or absence of an optimal concentration (5 ng/ml) of epidermal growth factor (EGF). In contrast to EGF bombesin and GRP14-27 do not stimulate cell migration. Thus, bombesin and the C-terminal fragment of gastrin-releasing peptide represent a new class of peptides mitogenic for normal human epithelial cells.     

Rat Sertoli cells secrete a growth factor that blocks epidermal growth factor (EGF) binding to its receptor

The conditioned medium from Sertoli cells contains a potent mitogen(s) that can markedly stimulate the proliferation of 4 different cell lines of endoderm or mesoderm origin in the presence or absence of serum. With A431 cells, conditioned medium produced in a dose-dependent manner up to a 5.2-fold increase in cell number after 5 days in culture. Addition of follicle-stimulating hormone (FSH), testosterone, retinol, and insulin to the Sertoli cells increased the secretion of the mitogenic activity. The ability of Sertoli cell conditioned medium (SCCM) to displace 125I-labeled epidermal growth factor (125I-EGF) from formalin-fixed A431 cells was also examined. The SCCM from Sertoli cells incubated with insulin contained 1.42 ng eq of EGF/ml; testosterone, retinol, and FSH (in the presence of insulin) further increased the secretion of this EGF competing activity to 2.09, 2.56, and 3.22 ng eq/ml, respectively. The amount of EGF competing activity was positively correlated with mitogenic activity. Separation of SCCM by gel filtration on Bio-Gel P-10 produced three major peaks of EGF-competing activity at apparent Mr = 1800-2100, 3800-4200, and 8000-9500. Chromatographing SCCM (in the presence of protease inhibitors) on size exclusion high performance liquid chromatography revealed two peaks of EGF competing activity at Mr about 8000 and 2000 coincident with and proportional to peaks of mitogenic activity. This activity was heat-sensitive and resistant to reducing agents, and addition of an equivalent amount of EGF as that present in SCCM produced an inhibition in growth of the A431 cells compared to a 3-fold stimulation with SCCM. Thus, the Sertoli cells secrete a potent mitogen that is distinct from EGF and alpha TGF. This factor that we have termed Sertoli cell-secreted growth factor is hormonally regulated by FSH, testosterone, and retinol and may play an important role in controlling spermatogenesis.     

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

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

Biological actions of the epidermal growth factors-like domain peptides of mouse schwannoma-derived growth factor and human amphiregulin.

Several members of the epidermal growth factor (EGF) family of growth factors that contain EGF-like units at their carboxy portion have been isolated and characterized. Schwannoma-derived growth factor (SDGF) and amphiregulin (AR) are members of this family. SDGF has high sequence homology to AR, and is known to be not only a potent mitogen for astrocytes and fibroblasts but also a neurotrophic factor. We previously confirmed that the synthetic EGF-like peptides SDGF(38-80) and AR(44-84), corresponding to the EGF-like domain of mouse SDGF and human AR, respectively, formed similar disulfide bond patterns to that of EGF. In the present study, we further investigated the biological actions of these two EGF-like peptides on several cultured cell lines. We found that SDGF(38-80) and AR(44-84) have weak mitogenic activity in NIH/3T3 cells and weak binding affinity to the EGF receptor on the surface of A431 cells compared with EGF. However, SDGF(38-80) and EGF induced short neurite outgrowth in PC12 h cells, a subclone of PC12 cells, at 100 nM. In addition, a significant increase in acetylcholinesterase (AChE) activity induced by SDGF(38-80) was observed at a concentration similar range to that of EGF, which is known as a differentiation marker of these cells. The effect of AR(44-84) in PC12 h cells was weaker than those of SDGF(38-80) and EGF, but the AChE activity was significantly increased by the addition of 100 nM AR(44-84), which did not stimulate NIH/3T3 cell growth. These results also suggest that SDGF(38-80) and AR(44-84) may be effective for neuronal differentiation rather than proliferation.     

Effects of retinoic acid on the binding and mitogenic activity of epidermal growth factor

In this study the effects of retinoic acid on the binding and mitogenic activity of epidermal growth factor (EGF) in mouse fibroblast Balb/c 3T6 cells are further examined. Retinoic acid treatment of 3T6 cells results in a sixfold enhancement of 125I-labeled mouse EGF binding when assayed at 37 degrees C. In both retinoic acid-treated and control cells, cell-associated 125I-EGF is rapidly internalized, degraded, and secreted. Retinoic acid treatment does not seem to have a significant effect on the rate of internalization and degradation of EGF. At 0 degrees C, internalization of EGF is strongly inhibited in both retinoic acid-treated and control cells. Under these conditions retinoic acid-treated cells still exhibit a tenfold higher level of EGF binding compared to control cells. When exposed to high concentrations of EGF both retinoic acid-treated and control cells "down-regulate" their EGF receptors. And although the growth rate of retinoic acid-treated cells is about half that of control cells, the rate at which EGF binding capacity is restored after down-regulation is about three times as fast as in control cells. No direct antagonism on EGF binding was observed between the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and retinoic acid. EGF is a potent mitogen for 3T6 cells in serum-free medium; retinoic acid inhibits the mitogenic activity of EGF even though it increases EGF binding. Retinoic acid also inhibits cell proliferation induced by sarcoma growth factor (SGF) and insulin.     

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.     

Different protein kinase C isoforms determine growth factor specificity in neuronal cells

Although mitogenic and differentiating factors often activate a number of common signaling pathways, the mechanisms leading to their distinct cellular outcomes have not been elucidated. In a previous report, we demonstrated that mitogen-activated protein (MAP) kinase (ERK) activation by the neurogenic agents fibroblast growth factor (FGF) and nerve growth factor is dependent on protein kinase Cdelta (PKCdelta), whereas MAP kinase activation in response to the mitogen epidermal growth factor (EGF) is independent of PKCdelta in rat hippocampal (H19-7) and pheochromocytoma (PC12) cells. We now show that EGF activates MAP kinase through a PKCzeta-dependent pathway involving phosphatidylinositol 3-kinase and PDK1 in H19-7 cells. PKCzeta, like PKCdelta, acts upstream of MEK, and PKCzeta can potentiate Raf-1 activation by EGF. Inhibition of PKCzeta also blocks EGF-induced DNA synthesis as monitored by bromodeoxyuridine incorporation in H19-7 cells. Finally, in embryonic rat brain hippocampal cell cultures, inhibitors of PKCzeta or PKCdelta suppress MAP kinase activation by EGF or FGF, respectively, indicating that these factors activate distinct signaling pathways in primary as well as immortalized neural cells. Taken together, these results implicate different PKC isoforms as determinants of growth factor signaling specificity within the same cell. Furthermore, these data provide a mechanism whereby different growth factors can differentially activate a common signaling intermediate and thereby generate biological diversity.     

Role of epidermal growth factor-stimulated protein kinase in control of proliferation of A431 cells

Epidermal growth factor (EGF), which stimulates tyrosine-specific protein kinase activity both in vivo and in vitro, inhibits proliferation of A431 human epidermoid carcinoma cells. After mutagenesis clonal cell lines that were resistant to the growth inhibitory effects of EGF were selected. All six variants examined contained decreased EGF-stimulated protein kinase. The number of EGF receptors in variant cells decreased in parallel with EGF-stimulated protein kinase activity so that the specific activity of EGF-stimulated protein kinase per EGF receptor remained constant in variant cell lines with up to tenfold reductions in both activities. This result suggests that both EGF binding and kinase activities reside in the same or closely coupled molecules. The effect of EGF on growth of two resistant variants was examined in detail. Clone 29 contains approximately 50% and clone 4 contains approximately 20% of the EGF-stimulated protein kinase activity of the parental A431 cell line. In serum-supplemented medium, EGF stimulated proliferation of clone 29 but did not affect growth of clone 4. In a 1:1 mixture of DME and F-12 medium without serum, EGF caused both clone 29 and clone 4 to grow as well as in 10% serum. These variants, which were selected for resistance to the growth inhibitory effects of EGF, thus exhibit a strong mitogenic response to EGF. This result suggests that resistance to the growth inhibitory effect of EGF may involve both a decrease in EGF-stimulated protein kinase and an alteration in the response pathway.