Effects of platelet-contained growth factors (PDGF, EGF, IGF-I, and TGF-β) on DNA synthesis in porcine aortic smooth muscle cells in culture

Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and transforming growth factor-β (TGF-β) are potent mitogens present in human platelets. Since they are likely to be released simultaneously at the site of vessel injury, their combined effects on vascular smooth muscle cells are more relevant physiologically than their individual actions. Therefore, we added various concentrations of growth factors to quiescent porcine aortic smooth muscle cells cultured in lowserum (0.5%) medium and measured the amount of [³H]thymidine incorporated into DNA. Effect of TGF-β alone was concentration-dependent: stimulatory (1.5-fold increase over the basal) at 0.025 ng/ml and inhibitory at 0.1 ng/ml. Effects of the other three growth factors on DNA synthesis were only stimulatory; their maximally effective concentrations were 20 ng/ml for PDGF (eightfold over the basal), 40 ng/ml for EGF (sixfold increase), and 20 ng/ml for IGF-I (fourfold increase). When PDGF, EGF, and IGF-I were added at submaximally effective concentrations, their effects were additive. TGF-β at 1 ng/ml inhibited at least 50% of the effects of 20 ng/ml EGF and of 10 ng/ml IGF-I, whereas inhibition of the effect of 10 ng/ml PDGF required 10 ng/ml of TGF-β. The concentration of TGF-β needed to inhibit 50% of the combined effect of EGF, IGF-1, and PDGF was 5 ng/ml. These results show complex interrelationships between the growth factors contained in the α-granules of human platelets in their effects on porcine aortic smooth muscle cells.     

Epidermal growth factor inhibits growth of A431 human epidermoid carcinoma in serum-free cell culture

A medium consisting of a rich basal nutrient mixture supplemented with bovine insulin (10 micrograms/ml), human transferrin (10 micrograms/ml), human cold-insoluble globulin (5 micrograms/ml), and ethanolamine (0.5 mM) supported the growth of the A431 human epidermoid cell line in the absence of serum with a generation time equal to that of cells in serum-containing medium. Addition of epidermal growth factor (EGF) to this culture medium at concentration mitogenic for other cell types resulted in a marked inhibition of A431 cell growth. Inhibitory effects of EGF were observed at 1 ng/ml and near-maximal effects were observed at 10 ng/ml. The inhibitory effect of EGF could be reversed by the omission of EGF in subsequent medium changes and could be prevented by the addition of anti-EGF antibody to the culture medium. Inhibition of A431 cell growth by EGF also could be demonstrated in serum-containing medium.     

Effects of epidermal growth factor and transforming growth factor-beta 1 on rat heart endothelial cell anchorage-dependent and -independent growth

This report describes the effects of epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGF-beta 1) on the anchorage-dependent and -independent growth of rat heart endothelial cells (RHE-1A). When RHE-1A cells were grown in monolayer culture with medium containing 10% fetal bovine serum (FBS) supplemented with epidermal growth factor (0.1-100 ng/ml), growth was stimulated fivefold when compared to that of cells grown in medium containing 10% FBS alone. The stimulatory effect of EGF on RHE-1A cell monolayer growth was dose-dependent and half-maximal at 5 ng/ml. The addition of TGF-beta 1 in the range 0.1-10 ng/ml had no effect on RHE-1A cell monolayer growth when added to medium containing 10% FBS alone or 10% FBS supplemented with EGF (50 ng/ml). RHE-1A cells failed to grow under anchorage-independent conditions in 0.3% agar medium containing 10% FBS. In the presence of EGF, however, colony formation increased dramatically. The stimulatory effect of EGF was dose-dependent in the range 0.1-100 ng/ml and was half-maximal at 5 ng/ml. In contrast to its effects under anchorage-dependent conditions, TGF-beta 1 (0.1-10 ng/ml) antagonized the stimulatory effects of EGF on RHE-1A cell anchorage-independent growth. The inhibitory effect of TGF-beta 1 was dose-dependent and half-maximal at 0.1 ng/ml. EGF-induced RHE-1A soft agar colonies were isolated and reinitiated in monolayer culture. They retained the cobblestone morphology and contact-inhibition characteristic of normal vascular endothelial cells. Each of the clones continued to express Factor VIII antigen. These findings suggest that TGF-beta may influence not only endothelial cell proliferation but also anchorage dependence. These effects may in turn be of relevance to endothelial cell growth and angiogenesis in vivo.     

Additive effect of oestradiol-17 beta and serum on synthesis of deoxyribonucleic acid in guinea-pig endometrial cells in culture.

Normal guinea-pig endometrial cells, grown in primary culture, were made quiescent by serum depletion. Quiescent cells cultured in the control medium (containing 1% fetal calf serum treated with dextran-coated charcoal, DCC-FCS) showed a steady and weak rate of [3H]thymidine incorporation, but the addition of 15% fetal calf serum (FCS) or 10% DCC-FCS to the control medium induced a significant increase of DNA synthesis, demonstrating the responsiveness of the quiescent cells to stimulation. A lower but significant increase in [3H]thymidine incorporation was elicited by epidermal growth factor (EGF, 100 ng/ml) or insulin (10 micrograms/ml) added to the basal medium. Oestradiol-17 beta added to the control medium at concentrations ranging from 10(-10) to 10(-5) mol/l not only failed to increase but even inhibited [3H]thymidine incorporation at the highest concentrations tested. An additive effect was noticed when quiescent cells were incubated with oestradiol-17 beta (10(-9) mol/l) in the presence of 10% DCC-FCS, but no synergistic effect occurred when 2 x 10(-9) mol oestradiol-17 beta/l was combined with either EGF (100 ng/ml) or insulin (10 micrograms/ml). Oestradiol-17 beta appears unable alone to stimulate DNA synthesis in normal endometrial cells, but requires factor(s) present in fetal calf serum.     

The effects of gastrin, epidermal growth factor, and somatostatin on DNA synthesis in a small intestinal crypt cell line (IEC-6)

Exposure of IEC-6 cells for 24 hr to either gastrin (50-500 ng/ml) or EGF (100-500 ng/ml) significantly stimulated (100-165%) the rate of [3H]thymidine incorporation into DNA (referred to as DNA synthesis) when compared with the corresponding basal levels. Somatostatin (10-500 ng/ml) produced no apparent change in DNA synthesis in IEC cells. On the other hand, somatostatin completely inhibited the EGF-induced rise in DNA synthesis. The gastrin-mediated stimulation in DNA synthesis was not affected by somatostatin. The rate of DNA synthesis in IEC cells in the presence of both gastrin and EGF was found to be greater (additive) than that caused by either of the peptides alone. A similar but less dramatic change in the actual number of cells (assessment of cell replication) was observed when the IEC cells were exposed for 24 hr to gastrin, EGF, and somatostatin, either alone or in combination. Whereas gastrin (250 ng/ml) and EGF (250 ng/ml) by themselves increased the number of cells significantly by 29 and 37%, respectively, together they caused a 72% stimulation, when compared with the basal levels. Somatostatin by itself caused no apparent change in IEC cell population, but it significantly inhibited the EGF- but not the gastrin-induced stimulation in IEC cell replication. It is concluded that both gastrin and EGF exert a direct proliferative effect on IEC cells, and the EGF action is regulated by somatostatin.     

Regulation of baboon fetal adrenal androgen production by adrenocorticotropic hormone, prolactin, and growth hormone

Factors other than adrenocorticotropic hormone (ACTH) are thought to influence fetal adrenal steroidogenesis during primate pregnancy. Therefore, we determined the effects of prolactin (Prl), growth hormone (GH), and human chorionic gonadotropin (hCG) as well as ACTH on steroid secretion by collagenase-dispersed baboon fetal adrenal cells. Adrenal glands were obtained from seven baboon (Papio anubis) fetuses following cesarean section at Day 100-107 of gestation (term = Day 184). Tissue was minced with a fine scissors and cells were dispersed with 0.2% collagenase, then washed with Medium 199 containing penicillin/streptomycin. Cells (0.5 X 10(4)) were placed in 4 ml Medium 199 with or without 10 nmol ovine Prl, ovine GH, or ACTH, or 50 nmol hCG. After 18 h incubation (37 degrees C), cells were separated by centrifugation and the quantities of cortisol (F), dehydroepiandrosterone (DHA), and DHA-sulfate (DHAS) secreted into the medium were determined. In controls, DHA secretion [224 +/- 96 ng/(24 h X 10(5) cells] was greater (P less than 0.05) than that of DHAS (20 +/- 12) and F (14 +/- 12). Adrenocorticotropic hormone, Prl, and GH stimulated (P less than 0.05) DHA secretion by 370% +/- 71%, 215% +/- 61%, and 292% +/- 73%, respectively; hCG was not effective. Due primarily to the relatively low secretion rates, DHAS and F secretion were not altered by hormonal treatment. Moreover, addition of 20 nmol progesterone to the medium in the presence or absence of ACTH did not influence F production. These findings indicate that the baboon fetal adrenal at midgestation does not utilize placental progesterone for F synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

FSH and growth factors affect the growth and endocrine function in vitro of granulosa cells of bovine preantral follicles

The hypothesis was tested that bovine preantral follicles can be stimulated to grow in vitro by FSH and by the mitogens, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), but not by transforming growth factor-beta (TGFbeta), which generally inhibits EGF and bFGF action. Preantral follicles, 60 to 179 mum in diameter, were isolated from fetal ovaries by treatment with collagenase and DNase and cultured for 6 d in serum-free medium, with or without FSH and growth factors. Basic FGF (50 ng/ml), and to a lesser extent FSH (100 ng/ml) and EGF (50 ng/ml), stimulated thymidine incorporation by granulosa cells in bovine preantral follicles compared to control cultures (8-, 4- and 2.5-fold the labeling index of the controls; P < 0.05). Alone TGFbeta (10 ng/ml) had no effect on (3)H-thymidine incorporation, but it completely inhibited the bFGF- but not the FSH-stimulated increase in the labeling index and mean follicular diameter of preantral follicles (P < 0.05). By the end of the culture period oocytes in most treatments had degenerated, and the few surviving oocytes were in preantral follicles cultured with FSH or bFGF. Progesterone accumulation was greater (P < 0.05) in the presence of FSH (100 ng/ml) or EGF (50 ng/ml) than with bFGF, TGFbeta or control medium. Basic FGF strongly inhibited the effect of FSH on progesterone secretion (P < 0.05). Only FSH stimulated the conversion of exogenous testosterone to estradiol and both bFGF and TGFbeta markedly inhibited FSH-stimulated estradiol accumulation. These results indicate that proliferation of granulosa cells of bovine preantral follicles can be stimulated by bFGF, FSH and EGF, whereas TGFbeta inhibits growth, and that they are steroidogenically active in culture. Basic FGF and TGFbeta antagonize FSH-stimulated steroid production by granulosa cells of cultured bovine preantral follicles.     

Epidermal growth factor partially reverses the inhibitory effects of antiestrogens on T 47D human breast cancer cell growth

When T 47D human breast cancer cells were treated with 10 nM of the potent antiestrogen, 4-hydroxyclomiphene, growth rate was reduced to about 50% of control. Simultaneous treatment with epidermal growth factor (EGF) and 4-hydroxyclomiphene led to a partial reversal of the growth inhibitory effect of the antiestrogen. The effect of EGF was concentration-dependent being half-maximal at 0.10 ng/ml (0.02 nM) and maximal at concentrations greater than 0.5 ng/ml (greater than 0.08 nM). Furthermore, EGF partially reversed the growth inhibitory effects of several other antiestrogens including tamoxifen, 4-hydroxytamoxifen, and LY 117018. These results are compatible with the hypothesis that part of the growth inhibitory effects of antiestrogens on breast cancer cell proliferation are mediated by inhibition of autocrine secretion of growth stimulatory peptides acting through the EGF receptor.     

Effects of transforming growth factor beta and epidermal growth factor on cell proliferation and the formation of bone nodules in isolated fetal rat calvaria cells

When cells enzymatically isolated from fetal rat calvaria (RC cells) are cultured in vitro in the presence of ascorbic acid and Na beta-glycerophosphate, discrete three-dimensional nodules form with the histologic, immunohistochemical, and ultrastructural characteristics of bone (Bellows et al; Calcified Tissue International 38:143-154, 1986; Bhargava et al., Bone, 9:155-163, 1988). Quantitation of the number of bone nodules that forms provides a colony assay for osteoprogenitor cells present in the RC population (Bellows and Aubin, Develop. Biol., 133:8-13, 1989). Continuous culture with either epidermal growth factor (EGF) or transforming growth factor beta (TGF-beta) results in dose-dependent inhibition of bone nodule formation; however, the former causes increased proliferation and saturation density, while the latter reduces both parameters. Addition of EGF (48 h pulse, 2-200 ng/ml) to RC cells at day 1 after plating results in increased proliferation and population saturation density and an increased number of bone nodules formed. Similar pulses at confluence and in postconfluent multilayered cultures when nodules first begin forming (approx. day 11) inhibited bone nodule formation and resulted in a smaller stimulation of cell proliferation. Forty-eight hour pulses of TGF-beta (0.01-1 ng/ml) reduced bone nodule formation and proliferation at all times examined, with pulses on day 1 causing maximum inhibition. The effects of pulses with TGF-beta and EGF on inhibition of nodule formation are independent of the presence of serum in the culture medium during the pulse. The data suggest that whereas EGF can either stimulate or inhibit the formation of bone nodules depending upon the time and duration of exposure, TGF-B inhibits bone nodule formation under all conditions tested. Moreover, these effects on osteoprogenitor cell differentiation do not always correlate with the effects of the growth factors on RC cell proliferation.     

Propagation of fetal human RPE cells: preservation of original culture morphology after serial passage

The permissive effects of extracellular matrix (ECM) on in vitro growth and differentiation of fetal human retinal pigment epithelial (RPE) cells have been studied. Factors which enhanced the effect of ECM to support cell division were also examined, including growth factors, culture media, and serum requirement. Under the specific culture conditions we have defined, it is possible to propagate these RPE cells at low density (less than 20 cells/mm2) with excellent growth properties for greater than 72 doublings (fourteen passages) in serial culture. Later-passaged cells maintained the morphological appearance of early-passaged cultures. ECM produced by bovine corneal endothelial cells was by far the most predominant factor in promoting rapid cell proliferation and viability over repeated passaging. Basic fibroblast growth factor (bFGF) exerted a substantial effect on the rate of cell division at different serum concentrations on plastic dishes. In addition, this factor showed profound synergistic effect when RPE cells were maintained on ECM, both in the preservation of cell morphology and also in long term viability. Other growth factors, such as epidermal growth factor (EGF) and transforming growth factor-beta (TGF-B), were also tested, but EGF effects were less prominent than those observed with bFGF, and TGF-B had an inhibitory effect at high concentrations. The ability to obtain a relatively large number of human RPE cells in vitro which preserve the appearance of early passage cells may provide useful opportunities to study the physiological properties and pathological alterations involving this important cell type.     

Epidermal growth factor (EGF)-induced morphological changes in the basement membrane of chick embryonic skin

Summary The effect of epidermal growth factor (EGF) on the basement membrane structure of chick embryonic skin cultured in a chemically defined medium (BGJb) containing 20 mM hydrocortisone, and EGF at 10, 50, or 100 ng/ml supplemented with 5% delipidized fetal calf serum, was examined by electron microscopy. During development of the epidermis in vitro, EGF (100 ng/ml) caused striking changes to occur in the basement membrane structure and in the keratinization process. The basement membrane frequently became discontinuous with many gaps apparent in section, and occasionally became folded following detachment from the basal surface of the epidermis and protruded into the underlying dermis. In the basal and intermediate cells of EGF-treated epidermis, tonofilament bundles were decreased in number, while desmosomes and hemidesmosomes revealed no significant changes in morphology.     

ErbB2 and ErbB3 receptors mediate inhibition of calcium-dependent chloride secretion in colonic epithelial cells

We have previously demonstrated that epidermal growth factor (EGF) inhibits calcium-dependent chloride secretion via a mechanism involving stimulation of phosphatidylinositol 3-kinase (PI3-K). The muscarinic agonist of chloride secretion, carbachol (CCh), also stimulates an antisecretory pathway that involves transactivation of the EGF receptor (EGFR) but does not involve PI3-K. Here, we have examined if ErbB receptors, other than the EGFR, have a role in regulation of colonic secretion and if differential effects on ErbB receptor activation may explain the ability of the EGFR to propagate diverse signaling pathways in response to EGF versus CCh. Basolateral, but not apical, addition of the ErbB3/ErbB4 ligand alpha-heregulin (HRG; 1-100 ng/ml) inhibited secretory responses to CCh (100 microM) across voltage-clamped T(84) epithelial cells. Immunoprecipitation/Western blot studies revealed that HRG (100 ng/ml) stimulated tyrosine phosphorylation and dimerization of ErbB3 and ErbB2, but had no effect on phosphorylation of the EGFR. HRG also stimulated recruitment of the p85 subunit of PI3-K to ErbB3/ErbB2 receptor dimers, while the PI3-K inhibitor, wortmannin (50 nM), completely reversed the inhibitory effect of HRG on CCh-stimulated secretion. Further studies revealed that, while both EGF (100 ng/ml) and CCh (100 microM) stimulated phosphorylation of the EGFR, only EGF stimulated phosphorylation of ErbB2, and neither stimulated ErbB3 phosphorylation. EGF, but not CCh, stimulated the formation of EGFR/ErbB2 receptor dimers and the recruitment of p85 to ErbB2. We conclude that ErbB2 and ErbB3 are expressed in T(84) cells and are functionally coupled to inhibition of calcium-dependent chloride secretion. Differential dimerization with other ErbB family members may underlie the ability of the EGFR to propagate diverse inhibitory signals in response to activation by EGF or transactivation by CCh.     

Competition of a growth stimulating-/cholecystokinin (CCK) releasing-peptide (monitor peptide) with epidermal growth factor for binding to 3T3 fibroblasts

The growth stimulating-/cholecystokinin (CCK) releasing-peptide (monitor peptide) is a peptide purified from rat bile-pancreatic juice on the basis of its stimulatory activity toward pancreatic enzyme secretion. Its multiple functions and peptide sequence suggested that it is distinct from epidermal growth factor (EGF). However, we found that the peptide competes with [125I]-EGF in the binding to Swiss 3T3 fibroblast cells to almost the same extent as unlabeled EGF does. [125I]-EGF binding was inhibited by 50% by the peptide at 82.8 ng/ml and by unlabeled EGF at 71.4 ng/ml. This suggests that the growth stimulating effect of the peptide on 3T3 fibroblasts is mediated via the EGF receptor, and also suggests that the partial homologous sequence between monitor peptide and EGF is required for the receptor binding, or that the EGF receptor has a broad ligand specificity.     

Characterization of the growth of murine fibroblasts that express human insulin receptors. II. Interaction of insulin with other growth factors

The effects of insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and insulin on DNA synthesis were studied in murine fibroblasts transfected with an expression vector containing human insulin receptor cDNA (NIH 3T3/HIR) and the parental NIH 3T3 cells. In NIH 3T3/HIR cells, individual growth factors in serum-free medium stimulated DNA synthesis with the following relative efficacies: insulin greater than or equal to 10% fetal calf serum greater than PDGF greater than IGF-1 much greater than EGF. In comparison, the relative efficacies of these factors in stimulating DNA synthesis by NIH 3T3 cells were 10% fetal calf serum greater than PDGF greater than EGF much greater than IGF-1 = insulin. In NIH 3T3/HIR cells, EGF was synergistic with 1-10 ng/ml insulin but not with 100 ng/ml insulin or more. Synergy of PDGF or IGF-1 with insulin was not detected. In the parental NIH 3T3 cells, insulin and IGF-1 were found to be synergistic with EGF (1 ng/ml), PDGF (100 ng/ml), and PDGF plus EGF. In NIH 3T3/HIR cells, the lack of interaction of insulin with other growth factors was also observed when the percentage of cells synthesizing DNA was examined. Despite insulin's inducing only 60% of NIH 3T3/HIR cells to incorporate thymidine, addition of PDGF, EGF, or PDGF plus EGF had no further effect. In contrast, combinations of growth factors resulted in 95% of the parental NIH 3T3 cells synthesizing DNA. The independence of insulin-stimulated DNA synthesis from other mitogens in the NIH 3T3/HIR cells is atypical for progression factor-stimulated DNA synthesis and is thought to be partly the result of insulin receptor expression in an inappropriate context or quantity.     

Hormonal control of the replication of human fetal fibroblasts: role of somatomedin C/insulin-like growth factor I

Sparse cultures of fetal and postnatal human fibroblasts were equivalent in their responsiveness to the mitogenic action of somatomedin C/insulin-like growth factor I (SM-C/IGF-I). At both developmental stages, the addition of SM-C/IGF-I (100 ng/ml) increased cell number at day 3 1.4-fold in serum-free medium and 2-fold in the presence of 0.25% human hypopituitary serum. Furthermore, dose-response curves indicated that there was no difference in the sensitivity of fetal and postnatal fibroblasts to the growth-promoting effects of SM-C/IGF-I, with a half-maximal response occurring at 6 ng/ml SM-C/IGF-I. This biological action of SM-C/IGF-I correlated with SM-C/IGF-I binding to fetal and postnatal fibroblast monolayers. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) also stimulated replication of fetal and postnatal fibroblasts. The mitogenic effects of SM-C/IGF-I, EGF, and PDGF were additive. Dexamethasone, which alone had no effect, was synergistic with SM-C/IGF-I in stimulating replication of postnatal fibroblasts. The combination of SM-C/IGF-I (100 ng/ml), dexamethasone (10(-7) M), EGF (10 ng/ml), and PDGF (5 ng/ml) had the same mitogenic effectiveness as 10% calf serum (CS) in postnatal cells. In marked contrast, there was no mitogenic interaction between SM-C/IGF-I and dexamethasone in fetal fibroblasts. In fetal cells, SM-C/IGF-I + EGF + PDGF +/- dexamethasone could only account for 50% of the activity of 10% CS. Moreover, fetal cells were 50-100% more responsive than postnatal cells to the proliferative effect of serum.     

Lysophosphatidic acid stimulates nuclear and cytoplasmic maturation of golden hamster immature oocytes in vitro via cumulus cells

Lysophosphatidic acid (LPA), a member of the phospholipid autacoid family, is induced in incubated human follicular fluid by lysophospholipase D. It is well known that LPA functions as a growth factor and the hypothesis that LPA in human follicular fluid takes a part in meiosis of oocytes is quite plausible. We studied the effects of LPA on the maturation of golden hamster immature oocytes in vitro. Hamster oocytes with a germinal vesicle were cultured in Tyrode's albumin lactate pyruvate (TALP) medium with 10(-5) M LPA, 10 ng/ml epidermal growth factor (EGF), 30 ng/ml insulin-like growth factor-1, 1 ng/ml tumor growth factor-alpha or 1 ng/ml basic fibroblast growth factor. The nuclear maturation rates in the LPA and EGF groups were significantly higher than in the control group and the other growth factors did not show any stimulatory effect (LPA group; 74.3% [75/101], EGF group; 82.4% [89/108] vs. control group; 60.2% [59/98], p < 0.05, p < 0.01, respectively). When the cells of cumulus were removed, EGF and LPA did not increase the nuclear maturation rates. Cotreatment EGF and LPA did not significantly enhance the stimulatory effect observed with LPA alone on maturation in vitro. The penetration rate determined by the zona-free hamster oocyte test was significantly higher in the LPA group than in the control group (26.7% vs. 13.2%, p < 0.05) and was comparable with that of oocytes matured in vivo. In conclusion, LPA stimulates the nuclear and cytoplasmic maturation of hamster immature oocytes via cumulus cells.     

Effects of estrogen, epidermal growth factor, and transforming growth factor-alpha on the growth of human breast epithelial cells in primary culture.

Since 17 beta-estradiol (E2)-stimulated growth in human breast cancer cell lines has been shown to be accompanied by increased production of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) and their receptor, we investigated the effects of E2 and these growth factors on the growth of human breast epithelial cells (HBEC) in primary culture. HBEC from normal, benign, and malignant tissues were cultured in serum-free medium [DME:F12(1:1), 5 mg/ml BSA, 10 ng/ml cholera toxin, 0.5 micrograms/ml cortisol, 10 micrograms/ml insulin] in the presence and absence of E2, EGF, and TGF-alpha. Tritiated-thymidine ([3H]TdR) incorporation into DNA was used as a measure of cell growth. E2 did not stimulate growth of any of the cultures at all concentrations examined (10(-9) to 10(-6) M). In contrast, EGF ranging from 1 to 100 ng/ml consistently increased the growth of cells of all three breast tissue types in a dose-dependent manner. The EGF stimulation was inhibited by MAb 528, a monoclonal antibody against the EGF receptor. TGF-alpha was equally or more effective in stimulating proliferation, although its dose-response range was different than that of EGF. E2 and EGF together acted in a synergistic manner in 50% of the samples examined. These studies suggest that E2 can exert effects on HBEC growth via modulation of the cells' response to EGF.     

A role for protein kinase cepsilon in the inhibitory effect of epidermal growth factor on calcium-stimulated chloride secretion in human colonic epithelial cells

Epidermal growth factor (EGF) inhibits carbachol-induced chloride secretion in T(84) colonic epithelial cells and has been shown to activate phosphatidylinositol (PI) 3-kinase, leading to inhibition of a basolateral potassium conductance. We asked whether the inhibitory effect of EGF on secretion is due to activation of specific isoforms of protein kinase C (PKC) by PI 3-kinase. Western analysis revealed that PKCalpha, gamma, epsilon, eta, mu, lambda/iota, and zeta were expressed in T(84) cells. Ro318220 (an inhibitor active against PKCepsilon, 10 micrometer) but not G?6983 (an inhibitor active against PKCzeta, 10 micrometer) reversed the inhibitory effect of EGF (100 ng/ml) on carbachol-stimulated chloride secretion. EGF induced the rapid translocation of PKCepsilon from the cytoplasm to the membrane. Wortmannin (50 micrometer) and LY294002 (20 nm), which are PI 3-kinase inhibitors that by themselves had no effect on PKCepsilon activity, significantly suppressed PKCepsilon translocation activated by EGF. LY294002 also reversed the inhibitory action of EGF on chloride secretion. PI (3,4)P(2) increased membrane-associated PKCepsilon and reduced carbachol-induced (86)Rb(+) efflux. Antisense oligonucleotides against PKCepsilon decreased PKCepsilon mass and prevented the inhibitory effect of EGF on carbachol-induced (86)Rb(+) efflux. Thus, the inhibitory effect of EGF on carbachol-induced chloride secretion involves the activation of PKCepsilon mediated by PI 3-kinase. Our findings contribute to the understanding of the cellular mechanisms that control chloride secretion.     

Comparison of metabolic effects of EGF,TGF-α, and TGF-β1 in primary culture of fetal bovine myoblasts and rat L6 myoblasts

• 1.1. Comparative studies of EGF, TGF-α, and TGF-βl action on the synthesis of DNA and cellular proteins in rat L6 myogenic cells and fetal bovine myoblasts demonstrated considerable differences between particular growth factors, dependent on dose and target cells.
• 2.2. Among examined growth factors only EGF exerted mitostimulatory action, more pronounced at lower concentrations. EGF, progressively with dose, stimulated protein synthesis much more effectively in fetal bovine myoblasts than in L6 cells.
• 3.3. The dynamics of stimulation of protein synthesis by TGF-α was greater than by EGF in both examined types of cell cultures.
• 4.4. The maximal response of fetal bovine myoblasts to TGF-α in a concentration of 100 ng/ml reached 370%, whereas EGF in a 10 times higher concentration stimulated protein synthesis only to 123% of control.
• 5.5. In contrast to EGF, TGF-α significantly inhibits DNA synthesis. Inhibition of the mitogenic response with simultaneous stimulation of protein synthesis by TGF-α may indicate changes toward cell differentiation.
• 6.6. TGF-β 1 in smallest concentration inhibits both DNA and protein synthesis. The suppressive action of TGF-β 1 was more distinct in fetal bovine myoblasts than in the L6 cell line.
• 7.7. Increasing concentrations of TGF-β l diminished its inhibitory effect, even leading to stimulation of protein synthesis at higher doses in L6 myoblasts.

     

EFFECTS OF ADENOSINE AND EPIDERMAL GROWTH FACTOR ON THE GROWTH OF MOUSE MAMMARY EPITHELIAL CELLS

The objective of this study was to determine if adenosine alters growth of mammary epithelium. Mouse mammary epithelial cells (NMuMG) were cultured in DMEM supplemented with 10% fetal calf serum. After serum starvation for 24h, EGF (0–100ng/ml) and/or adenosine (0–100μm ) was added. Adenosine at concentrations of 1, 10 or 100μm increased DNA synthesis significantly, when compared to control. Addition of epidermal growth factor (EGF) (10ng/ml) into 1 or 10μm adenosine showed the interaction in DNA synthesis between EGF and adenosine. A similar result was observed when 100μm adenosine added to various concentrations of EGF (0–100ng/ml). In the second mammary gland (thoracic) organ culture studies, mammary development scores were increased by adenosine (100μm ), EGF (100ng/ml) and adenosine plus EGF. These results indicate that the purine nucleoside adenosine stimulates mammary epithelial cell growth and interacts with EGF in DNA synthesis of mouse mammary epithelial cells.