Epidermal growth factor-like proteins in breast fluid and human milk

Epidermal growth factor (EGF), and the transforming growth factor-alpha (TGF-alpha) family of proteins, which also bind to the EGF receptor, have been associated with human breast cancer. The total EGF-like proteins were determined by a radioreceptor assay, and TGF-alpha by radioimmunoassay, in human milk and breast fluid samples. The breast fluids were collected by nipple aspiration from healthy premenopausal women. Both the 24 milks and 18 breast fluids assayed contained EGF-like proteins, at concentrations ranging from 32-600 ng/ml (median, 140 ng/ml), and 62-654 ng/ml (median, 205 ng/ml) respectively. Immunoreactive TGF-alpha proteins were detected at higher levels in 21 breast fluids (range, 0-50.0; median 5.1 ng/ml) than in 24 milk samples (range, 0-8.4; median, 0.8 ng/ml).     

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.     

Stimulatory effect of epidermal growth factor on the development of mouse early embryos in vitro.

Effects of epidermal growth factor (EGF) on the development of mouse 2-cell embryos cultured in vitro were investigated. The addition of EGF at a concentration of 0.5 ng/ml enhanced the development of 2-cell embryos during 24 h of incubation. As expected, EGF stimulated the synthesis of DNA in the 2-cell embryos about 4-fold over the control. The growth-promoting effect of EGF seemed to be specific in that other growth factors, such as transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta (TGF-beta), insulin-like growth factor-1 (IGF-1), platelet-derived growth factor (PDGF), nerve growth factor (NGF) and fibroblast growth factor (FGF) had no effect on the embryonal development. The addition of EGF also increased the rate of RNA synthesis in a dose-related manner between 0.1 and 50 ng/ml. However, protein synthesis was unaffected by EGF. These results raise the possibility that EGF may participate in the process of early embryogenesis in vivo.     

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.     

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.     

Inhibition of stimulus-dependent epidermal growth factor receptor and transforming growth factor-alpha mRNA accumulation by the protein kinase C inhibitor staurosporine

The ability of staurosporine, a potent inhibitor of protein kinase C, to block certain cellular events initiated by 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) was examined. Treatment of MDA468 breast cancer cells with TPA decreases EGF binding to the cell surface and this effect is blocked by pretreatment with staurosporine with an IC50 of 30 nM. Either 10(-9) M EGF or 100 ng/ml TPA stimulated the accumulation of both EGF receptor and TGF-alpha mRNA and staurosporine (50 nM) completely abolished these mRNA accumulations. Staurosporine did not block EGF-stimulated tyrosine phosphorylation of its receptor as measured by immunoblotting with anti-phosphotyrosine antibodies. The ability of staurosporine to block the mRNA responses of either EGF or TPA suggests that these two agents have common signaling pathways and it implies a role for protein kinase C in the control of EGF receptor and TGF-alpha expression.     

Regulation of TGF-alpha expression in human keratinocytes: PKC-dependent and -independent pathways.

Transforming growth factor-alpha (TGF-alpha) is an autocrine growth factor for epidermal keratinocytes that can induce its own expression (autoinduction). Because the regulation of this process may be important for the control of epidermal growth, we examined the roles of EGF receptor tyrosine kinase and protein kinase C (PKC) in TGF-alpha autoinduction in cultured human keratinocytes. Antiphosphotyrosine immunoblot analysis demonstrated that EGF and TGF-alpha rapidly and markedly stimulated tyrosine phosphorylation of a 170 kDa protein in growth factor-deprived keratinocytes. This protein was identified as the EGF receptor by immuno-precipitation using anti-EGF receptor mAbs. Tyrosine phosphorylation and TGF-alpha mRNA accumulation in response to EGF and TGF-alpha were both inhibited by a monoclonal antibody against the EGF receptor and by the EGF receptor tyrosine kinase inhibitor RG50864, demonstrating the involvement of the tyrosine kinase activity of the receptor in TGF-alpha autoinduction. The monoclonal antibody inhibited keratinocyte growth and TGF-alpha autoinduction with similar potency (IC50 approximately 0.1 microgram/ml). TGF-alpha and the PKC activator tetradecanoyl phorbol 12-myristyl, 13-acetate (TPA) had similar effects on TGF-alpha steady-state mRNA levels, suggesting that PKC activation might be a downstream mediator of TGF-alpha autoinduction. However, down-regulation of more than 90% of keratinocyte PKC activity by bryostatin pretreatment abrogated the induction of TGF-alpha mRNA in response to TPA without affecting the autoinductive response or EGF-stimulated tyrosine phosphorylation. These results indicate that EGF receptor and PKC stimulate TGF-alpha gene expression by different pathways, and suggest that PKC is not required for TGF-alpha autoinduction in this system. Moreover, the fact that EGF-stimulated tyrosine phosphorylation and TGF-alpha autoinduction were not potentiated after PKC down-regulation suggests that PKC does not exert a tonic inhibitory influence on EGF receptor tyrosine kinase activity in normal human keratinocytes.     

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.     

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)     

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.     

Transforming growth factor-alpha binds to the epidermal growth factor receptor in gastric mucosa.

The ability of transforming growth factor-alpha (TGF-alpha) to interact with the gastric mucosal epidermal growth factor (EGF) receptor was investigated using a mucosal membrane preparation. TGF-alpha inhibited specific binding of [125I]EGF to its receptor, but the IC50 for TGF-alpha was at least 100 fold greater than that observed for unlabeled EGF. Cross-linking studies revealed no attachment of [125I]TGF-alpha to EGF-receptor size components, and the unlabeled TGF-alpha was only weakly effective in inhibiting cross-linking of [125I]EGF to the 170 kDa receptor. However, when the cytosolic fraction was reconstituted with the membrane preparation, an enhancement in binding of [125I]TGF-alpha to the EGF receptor occurred in a manner dependent on the concentration of cytosolic protein. Hence the binding characteristics of TGF-alpha to the EGF receptor in gastric mucosa are different from those for EGF.     

Intrapituitary regulatory system of mammotrophs in the mouse

Estrogen stimulates the proliferation of pituitary cells, in particular mammotrophs. The present study was designed to clarify involvement of transforming growth factor alpha (TGF-alpha) in the estrogen-induced growth of mouse pituitary cells in vitro. Anterior pituitary cells obtained from ICR male mice were cultured in a primary, serum-free culture system. Proliferation of pituitary cells was detected by monitoring the cellular uptake of a thymidine analogue, bromodeoxyuridine. Secretory cell types were immunocytochemically determined. Treatment with TGF-alpha (0.1 and 1 ng/ml) for 5 days stimulated cell proliferation. Since TGF-alpha binds to the epidermal growth factor (EGF)-receptor, this action may be exerted through this receptor. Estradiol-17beta (E2, 10(-9) M) stimulated proliferation of mammotrophs. RG-13022, an EGF receptor inhibitor, reduced the cell proliferation induced by EGF or E2, showing that the EGF receptor was involved in this induction of mammotroph growth. Treatment with TGF-alpha antisense oligodeoxynucleotide (ODN) inhibited the cell proliferation induced by E2, but treatment with EGF antisense ODN did not. Dual detection of TGF-alpha mRNA and growth hormone by in situ hybridization and fluorescence-immunocytochemistry demonstrated that TGF-alpha mRNA was detected in most somatotrophs. Our recent RT-PCR analysis revealed that E2 stimulated TGF-alpha-mRNA and EGF-receptor mRNA expression. These results indicate that TGF-alpha produced in somatotrophs mediates the stimulatory effect of estrogen on pituitary cell proliferation in a paracrine manner, and that EGF-receptor expression is stimulated by estrogen. These findings indicate that intrapituitary cell-to-cell interaction plays an important role in the control of pituitary secretory cells.     

Influences of epidermal growth factor and insulin-like growth factor-I on bovine blastocyst development in vitro

Experiments were carried out to investigate putative beneficial effects of adding epidermal growth factor (EGF) or insulin-like growth factor-I (IGF-I) for bovine embryo culture in chemically defined media. Presumptive zygotes (18 h post-insemination) were randomly assigned to culture treatments. In experiment 1, treatments involved additions of recombinant human EGF to provide concentrations of 0 ng (control), 1, 5, and 25 ng/ml. No differences were seen in numbers of 4-cell stage embryos between groups. A concentration of 5 ng/ml EGF but not 1 or 25 ng/ml during embryo culture improved percentages of 4-cell stage embryos reaching blastocysts compared to the control (P<0.05). Numbers of inner cell mass (ICM) cells and trophoblast cells of day 8 blastocysts were similar for the control and 5 ng/ml EGF-treated groups. In experiment 2, culture with recombinant human IGF-I in concentrations of 0 ng (control), 2, 10, and 50 ng/ml resulted in no differences in numbers of 4-cell stage embryos between groups. When compared to controls, IGF-I treatments at 10 and 50 ng/ml improved proportions of 4-cell stage embryos that reached blastocysts (P<0.05). In experiment 3, numbers of ICM cells of day 8 blastocysts were significantly higher after being cultured with 50 ng/ml of IGF-I compared to those of the controls (P<0.05). No additive effect of combining EGF (5 ng/ml) and IGF-I (50 ng/ml) was seen when results were compared to those following supplementation of the media with either EGF or IGF-I alone. In conclusion, both EGF and IGF-I could independently enhance bovine preimplantational development in chemically defined media and IGF-I but not EGF may play a mitogenic role during early bovine development.     

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.     

Genistein represses the induction of prostatic buds by testosterone]

Genistein, a phytoestrogen and a kind of endocrine disrupters, inhibits tyrosine-specific protein kinase activity of the epidermal growth factor (EGF) receptor. It is also effective both in the suppression of the prostatic cell proliferation and the prostate carcinogenesis. We have recently demonstrated that several growth factors, like EGF, transforming growth factor-alpha (TGF-alpha), or keratinocyte growth factor (KGF), can induce prostatic bud formation in the absence of androgen. The present study was performed to investigate whether genistein can suppress testosterone-induced prostatic bud formation. Urogenital sinuses of 16.5-day male rat fetuses were cultured organotypically for 5 days in a serum-free medium containing 10 or 100 ng/ml genistein and 50 ng/ml testosterone. The number and total volume of prostatic buds were analyzed by laser scanning microscopy and computerized. We found that genistein inhibits significantly testosterone-induced prostatic bud formation. In the presence of genistein, cell proliferation of the sinus epithelium was suppressed and the number of prostatic buds and total volume of the buds were reduced as compared with those in the sinuses cultured with testosterone alone. Genistein did not appear to cause necrosis of the sinus. These results support our hypothesis that growth factors like EGF secreted from the sinus mesenchyme activated by testosterone are involved in the induction and stimulation of growth of the prostatic buds.     

Transforming growth factor-beta down-regulates apolipoprotein M in HepG2 cells

Apolipoprotein M (apoM) is a novel apolipoprotein presented mostly in high-density lipoprotein (HDL) in human plasma, and is exclusively expressed in liver and in kidney. The pathophysiological function of apoM has not yet been elucidated. Apolipoprotein B (apoB), the characteristic apolipoprotein of low-density lipoprotein (LDL), is like apoM, a very hydrophobic protein, and thereafter they both must co-circulate with lipoprotein particles in plasma. The cytokine, transforming growth factor-beta (TGF-beta), has been shown to decreased apoB secretion in HepG2 cells, and we hypothesized that TGF-beta may have the same effects on apoM expression in HepG2 cells. In the present study, we used real-time RT-PCR to analyze apoM and apoB mRNA levels during administration of TGF-beta, as well as TGF-alpha, epidermal growth factor (EGF) and hepatic growth factor (HGF). TGF-beta significantly inhibited both apoM and apoB mRNA expression in HepG2 cells. The inhibitory effects of TGF-beta were dose-dependent, i.e. 1 ng/ml of TGF-beta decreased apoM mRNA levels by 30%, and 10 or 100 ng/ml of TGF-beta decreased apoM mRNA levels more than 65%. The effect of TGF-beta on apoB mRNA expression was slightly weaker than that of apoM, with a maximum effect at 10 or 100 ng/ml TGF-beta where apoB mRNA levels decreased about 55%. The inhibitory effects of TGF-beta on apoM and apoB mRNA levels also increased with increasing incubation time, where the maximum effect was obtained at 24 h. Moreover TGF-alpha, EGF and HGF all decreased both apoM and apoB mRNA levels, but to a less extent than TGF-beta. Further, all four cytokines had more pronounced effects on apoM mRNA expression than apoB mRNA expression. The present study suggested that apoM, like apoB, may be involved in the hepatic lipoprotein assembly in vivo.     

Expression of several growth factors and their receptor genes in human colon carcinomas

We have examined the expression of mRNAs for epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), EGF receptor (EGFR), PDGF-A chain (PDGFA), PDGF-B chain (PDGFB) and PDGF receptor (PDGFR) genes in seven human colorectal carcinoma cell lines and 18 human colorectal carcinomas. In surgically resected specimens of the 18 colorectal tumors, TGF-alpha, EGFR, PDGFA, PDGFB and PDGFR mRNAs were detected at various levels in 15 (83%), 9 (50%), 18 (100%), 8 (44%) and 12 (67%), respectively. They were also detected in normal tissues. Interestingly, EGF mRNA was detected in only five (28%) of the tumors, but not in normal mucosa. Expression of EGF was also confirmed immunohistochemically in tumor cells. Of the five tumors expressing EGF, four expressed EGFR mRNA and showed a tendency to invade veins and lymphatics. All the colorectal carcinoma cell lines expressed TGF-alpha mRNA, and five cell lines expressed EGFR mRNA simultaneously. Production of TGF-alpha protein by DLD-1 and CoLo320DM cells was confirmed by TGF-alpha specific monoclonal antibody binding assay. The spontaneous 3H-thymidine uptake by DLD-1 was suppressed by an anti-TGF-alpha monoclonal antibody. PDGFA and PDGFB mRNA were also expressed in four cell lines, but PDGFR and EGF mRNA was not detected. These results suggest that human colorectal carcinomas express multi-loops of growth factors and that TGF-alpha produced by tumor cells functions as an autocrine growth factor in human colonic carcinoma.     

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.     

Growth of normal human amnion epithelial cells in serum-free medium

The serum-free growth of primary cultures of normal human epithelial-like cells from amniotic membranes was accomplished. The synthetic medium consists of a 1 : 1 basal nutrient mixture of Dulbecco's modified Eagle medium (DMEM) and Ham's F-12 supplemented with 2.5 μg/ml insulin, 50 ng/ml epidermal growth factor (EGF), 5 μg/ml transferrin, and 0.1 ng/ml triiodothyronine (T₃). EGF is the primary mitogen and is essential for cell proliferation in this system.