Low Concentrations Of Epidermal Growth Factor Research Articles

Regulation of estrogen receptor-alpha gene expression by epidermal growth factor.

The role of epidermal growth factor (EGF) in the regulation of estrogen receptor-alpha (ER-alpha) gene expression in the human breast cancer cell line MCF-7 was investigated. Treatment of cells with 0.4 ng/ml EGF resulted in an approximately 60% decrease in ER-alpha protein concentration by 6 h and the amount of receptor remained suppressed for 24 h. Ligand binding assays demonstrated that the decrease in ER-alpha protein corresponded to a similar decrease (approximately 50%) in estradiol binding sites. Although EGF treatment resulted in a decrease in the number of binding sites, it had no effect on the binding affinity of ER-alpha. The dissociation constant of the estradiol-ER-alpha complex in the presence or absence of EGF was the same (K(d)=2.3x10(-)(10) M in control cells versus K(d)=1.98x10(-)(10) M in EGF-treated cells). The decrease in ER-alpha protein concentration paralleled a decrease in the steady-state amount of ER-alpha mRNA. By 9 h there was an approximately 60% decrease in ER-alpha mRNA. The amount of ER-alpha mRNA remained suppressed for 48 h. Transcription run-on experiments demonstrated that there was a decrease of approximately 70% in ER-alpha gene transcription upon EGF treatment, suggesting that the mechanism by which EGF regulates ER-alpha gene expression is transcriptional. In addition to regulating the amount of ER-alpha, EGF affected the activity of the receptor. At high concentrations, EGF induced progesterone receptor. Estradiol and high concentrations of EGF had an additive effect on progesterone receptor. In contrast to high concentrations, low concentrations of EGF had no effect on progesterone receptor and blocked estradiol induction. The effects of EGF on ER-alpha expression were inhibited by tyrophostins and wortmannin, suggesting that the effects of the growth factor are mediated by the EGF receptor and protein kinase B. When the cells were placed in serum-free medium and then treated with EGF, there was no effect on ER-alpha protein concentration or activity. However, increasing concentrations of serum restored the effects of EGF on ER-alpha, suggesting that an additional serum factor was required for the EGF-mediated effect on the decrease in ER-alpha protein concentration.

Serum and correspondent tissue measurements of epidermal growth factor (EGF) and epidermal growth factor receptor (EGF-R). Clinical relevance in pancreatic cancer and chronic pancreatitis.

The results of this study show that routine measurements of epidermal growth factor (EGF) and epidermal growth factor receptor (EGF-R) cannot improve screening for pancreatic cancer despite the frequently present tissue overexpression. Both values fail to reveal this malignancy in a serum test. Patients with chronic pancreatitis exhibit no or very low concentrations of EGF. In cases where preoperative diagnosis is difficult the noninvasive EGF and EGF-R serum measurements may be helpful in discriminating between pancreatic cancer and chronic pancreatitis. EGF and EGF-R are frequently overexpressed in the tissue of patients suffering from ductal pancreatic cancer and to lesser degree in patients with chronic pancreatitis. The aim of this study was to determine the value of serum measurements in these patients to detect malignant pancreatic disease. In cases of pancreatic cancer, the tissue expression of EGF and EGF-R was evaluated by immunohistochemistry. Thirty-five patients with chronic pancreatitis and 31 patients with pancreatic cancer were evaluated; 71 patients admitted for routine surgery (hernia repair, cholecystectomy, goiter surgery) served as controls. EGF and EGF-R values were not significantly different in pancreatic cancer as compared to controls and did not correlate with other tumor markers (CA 19-9, carcinoembryonic antigen [CEA], tumor polypeptide antigen [TPA]) or with the stage of the disease. Fourteen patients (67%) with pancreatic cancer displayed tissue overexpression for EGF and 11 patients for EGF-R (52%). These patients, however, also failed to exhibit any significant pathological changes in serum concentration. In chronic pancreatitis, EGF and EGF-R were significantly decreased as compared to pancreatic cancer and controls. This was an unexpected finding. There was a positive correlation to clinical exocrine insufficiency.

Prevention of EGF-modulated adhesion of tumor cells to matrix proteins by specific EGF receptor inhibition.

The adhesion of tumor cells to various extracellular matrix (ECM) proteins is influenced by epidermal growth factor (EGF). Maximal effects are obtained at low EGF concentrations, at which mostly the cytoskeleton-associated high-affinity EGF receptors (EGFRs) are saturated. Tumor cells expressing EGFR either endogenously (MDA MB 231, MTLn3) or, for the human EGFR, ectopically (MTC HER1/1) in intermediate amounts exhibited, upon EGF addition, increased cellular adhesion to various ECM proteins, such as fibronectin, collagens and vitronectin. In contrast, human A431 and MDA MB 468 cells, over-expressing EGFR, demonstrated reduced attachment in similar experimental conditions. Both increased as well as reduced EGF-dependent adhesion could be blocked using either ligand-blocking monoclonal antibody 14E1 or the potent EGFR tyrosine kinase inhibitor PD 153035. Our data indicate that signals downstream of EGFR activation are responsible for the opposing effects of EGF on cellular adhesion since both can be prevented by EGFR inhibition. Thus, the integration of EGFR- and integrin-dependent signals can be different in carcinoma cell lines and might be influenced by EGFR numbers.

Regulation of p42 mitogen-activated-protein kinase activity by protein phosphatase 2A under conditions of growth inhibition by epidermal growth factor in A431 cells.

Epidermal growth factor (EGF), which plays an important role in the growth regulation of a large variety of normal and tumor cells, has been shown to display an ambivalent dose-dependent effect on the proliferation of epithelial cells overexpressing EGF receptor. In a previous study aimed at dissecting the biochemical events leading to this dual action in A431 cells which over express EGF receptor, we have reported a relationship between the dual stimulator/inhibitor effect of EGF and the activity of the serine/threonine p42 mitogen-activated protein (MAP) kinase. Indeed, a growth stimulatory concentration of EGF is shown to lead to a moderate but persistent activation of p42 MAP kinase. Conversely, an early peak of MAP kinase activation, that rapidly falls below the basal level, is observed in the presence of a growth-inhibitory concentration of EGF. To assess the mechanism of the p42 MAP kinase inactivation under circumstances of negative growth regulation by EGF, we have investigated the role of the serine/threonine phosphatase 2A in this process. A constitutive phosphatase 2A activity was observed in untreated cells, that decreases rapidly in response to both high and low EGF concentrations. However, after this early inactivation, the phosphatase 2A activity was completely reversed concurrently with MAP kinase inactivation, after 40 min of treatment with 10 nM EGF. Conversely, in cells treated with 1 pM EGF, phosphatase 2A activity remained below the control level during all the time of the treatment, in association with a sustained MAP kinase activation. These results suggest that MAP kinase inactivation is closely related to phosphatase 2A activation. We then investigated the effect of the serine/threonine phosphatase inhibitor okadaic acid on the MAP kinase inactivation and observed that okadaic acid, at a concentration reported to specifically inhibit phosphatase 2A activity, totally reverses the MAP kinase inactivation induced by long-term treatment with 10 nM EGF. Additionally, we have shown that the protein synthesis inhibitor cycloheximide fails to affect the EGF-induced MAP kinase regulation, indicating that mitogen-induced protein phosphatases are not, or are only slightly, required in this regulation. In conclusion, our data demonstrate that the ambivalent action of EGF on the proliferation of A431 cells is associated with differential mechanisms of p42 MAP kinase regulation catalysed by the serine/threonine phosphatase 2A.

Structure and biochemistry of normal and mutant ( [formula omitted]) mouse mammary tissue during gestation and lactation

Mammary gland structure and function in mutant hub hub (hyper-unconjugated bilirubinemia) mice were compared to controls (+/ hub). Pups from normal and mutant mice have decreased weight gain when suckled by mutant dams. Samples examined by light microscopy showed no apparent differences when excised at mid- (day 9–11) and late- (day 17–18) gestation, and early (day 3–4) lactation. There was some evidence that early involution may be occurring in the hub mice at midlactation (day 10–11). Total DNA was greater (33%) when sampled at midgestation in the normal mice but lower (20%) at late gestation when compared to the hub mice. Total protein concentration was higher (132%) during late gestation in hub mice but was lower (34%) by early lactation when compared to normal mice. Epidermal growth factor (EGF) in normal mice was 79% and 71% higher in early and midlactation, respectively, than in mutant mice. There were no differences in serum concentrations of progesterone between strains at any stage. From these results, there is a suggestion of premature production of proteins, possibly followed by early involution in hub mice. Milk yield was less in mutant mice than in normal mice. Low concentrations of EGF during lactation in mutant mice may partially account for the decreased growth observed in mutant and normal pups suckling mutant dams.

Epidermal Growth Factor Receptor Signaling in Rat Pancreatic Acinar Cells

Epidermal growth factor (EGF) regulates pancreatic acinar enzyme secretion. The mechanism of action of EGF in pancreatic acinar cells is not clear. In the present study we investigated the role of heterotrimeric GTP-binding proteins (G proteins) in EGF receptor signal transduction. Pancreatic acini were isolated from rat pancreas by collagenase digestion and permeabilized by digitonin. Activation of phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PLC) was assessed using a radioreceptor assay specific for inositol 1,4,5-trisphosphate [IP3(1,4,5)]. For measurement of amylase secretion isolated pancreatic acini were incubated with secretagogues for 30 min at 37 degrees C. Amylase released into the medium was assessed by monitoring the hydrolysis rate of p-nitrophenyl-alpha,D-maltohepatoside. The weakly hydrolyzable GTP analogue guanosine 5'-[3-O-thio]triphosphate (GTP gamma S) and guanosine 5'-diphosphate (GDP) were used to activate and inhibit G protein-mediated signal transduction, respectively. EGF (90 nM) stimulated amylase release in isolated pancreatic acini. This effect was enhanced by guanosine 5'-[3-O-thio]triphosphate (0.1 mM), which stimulates G proteins. Guanosine 5'-diphosphate (1 mM), which inhibits the activity of heterotrimeric G proteins, had no effect on basal and EGF-induced amylase release. Lower EGF concentrations (20 nM) inhibited COOH-terminal cholecystokinin octapeptide (CCK8)-induced IP3(1,4,5) production and amylase release in pancreatic acini). However, in the presence of GDP, EGF had no significant effect on CCK8-stimulated amylase release. Furthermore, coincubation of the acini with CCK8, EGF, and GDP revealed that GDP reduces the inhibitory effect of EGF on CCK8-induced IP3(1,4,5) production.(ABSTRACT TRUNCATED AT 250 WORDS)

THE GROWTH-RESPONSE OF HUMAN TUMOR-CELL LINES EXPRESSING THE EGF RECEPTOR TO TREATMENT WITH EGF AND OR MABS THAT BLOCK LIGAND-BINDING

We have investigated the effect of treatment with epidermal growth factor (EGF) and/or monoclonal antibodies (Mabs) to the epidermal growth factor receptor (EGFR) on the growth in vitro of a number of human tumour cell lines. Mabs ICR10, ICR11, and ICR16 that prevent the binding of both I-125-EGF and I-125-TGFalpha to the EGF receptor were found to inhibit the growth of human tumour cell lines overexpressing the EGF receptor. At high concentrations (5 nM), EGF was also found. to inhibit the growth of HN5, A431 and MDA MB-468 cells whereas the proliferation of SKBR3 and HN6 cells was stimulated. While some of the cell lines (e.g. HN5 and HN6) were very susceptible to growth inhibition by antibodies to the EGFR others, known to secrete autocrine growth factors (e.g. A431 and MDA MB-468), were less affected by these antibodies. Indeed, growth of the latter cell lines was inhibited more effectively by the addition of 5 nM of exogenous EGF than by treatment with 156 nM of the anti-EGFR Mabs ICR16, ICR11, and ICR10. In addition, we show that the growth of HN5 cells, which express very large numbers of EGF receptors (1.4x10(7)/cell), was stimulated at picomolar concentrations of EGF but inhibited at concentrations of EGF in the nanomolar range. Maximal stimulation (25% above control) was observed with the addition of 78 pM EGF to the cultures. The mitogenic effect of low concentrations of EGF and the growth inhibitory effect of nanomolar concentrations of EGF on HN5 cells could be reversed by the addition of 156 nM of the anti-EGFR antibodies. We conclude that growth inhibition can follow from either inhibition of signal transduction by blocking ligand binding to the EGFR or from excess binding of ligand. With tumours that produce significant quantities of autocrine growth factors the latter will compete with the Mabs for binding to the receptor and reduce their effectiveness.

Effect of Epidermal Growth Factor on Cholecystokinin-Induced Amylase Release and Inositol 1,4,5-Trisphosphate Production in Pancreatic Acinar Cells

In the present study we investigated the role of epidermal growth factor (EGF) in cholecystokinin-octapeptid (CCK8)-induced inositol 1,4,5-trisphosphate (IP3) production and amylase release. The data show that high EGF concentrations (90 nM) increased the basal amylase release, but did not increase the acinar IP3 content. High EGF concentrations shifted the dose-response curve for CCK8-induced amylase release to lower CCK8 concentrations, whereas low EGF concentrations (17 nM) shifted this dose-response curve to higher CCK8 concentrations. 17 nM EGF inhibited CCK8-induced IP3 production during the whole period of observation of 5 min, whereas 90 nM EGF inhibited only the initial component (15 s) of CCK8-induced IP3 production. At later time points, 90 nM EGF increased CCK8-induced IP3 production. We conclude that low concentrations of EGF are inhibitory on CCK8-induced IP3 production and amylase release, whereas high EGF concentrations stimulate CCK8-induced amylase release.

Existence of two pathways for the endocytosis of epidermal growth factor by rat liver: phenylarsine oxide-sensitive and -insensitive pathways.

The effect of phenylarsine oxide (PAO) on the internalization rate of epidermal growth factor (EGF) was investigated using perfused rat liver and isolated rat hepatocytes. In perfused liver, a tracer concentration of 125I-EGF alone or with excess unlabeled EGF (20 nM) was perfused and the internalization rate constants (kint) were measured. In the absence of PAO, kint values did not differ significantly for either dose condition. However, with the addition of PAO to the perfusate, the kint value dropped to 4% of that of the control at the low concentration of EGF, while dropping to only 40% of that of the control at the high concentration of EGF. These results suggest the existence of a PAO-insensitive internalization pathway having a kint value comparable with that of the other pathway. Similar EGF concentration-dependent inhibition of 125I-EGF internalization caused by PAO was ascertained using isolated rat hepatocytes. PAO also decreased the cellular ATP content in isolated hepatocytes. However, when we lowered the cellular ATP content with rotenone, the cell-surface binding and internalization of EGF were comparable with the control levels. We concluded that there exist dual pathways for the internalization of EGF and that excess doses of EGF lead to EGF internalization not only through a PAO-sensitive pathway but also through a PAO-insensitive pathway, whereas at a tracer dose of EGF, the internalization occurs mainly via the PAO-sensitive pathway.

Epidermal growth factor in urine from the patients with urothelial tumors.

Epidermal growth factor (EGF) concentration in urine was measured with a radioimmunoassay in 40 untreated patients with urothelial tumors and a comparable control group of 25 normals. The concentration of EGF in urine from the patients with bladder, renal pelvic or ureter tumors was lower than normal (p less than 0.05). Patients with T4 tumors had lower concentrations of EGF compared with those of other stages. EGF concentrations after transurethral resection of bladder tumors were significantly elevated compared with the values before operation. These findings suggest that urothelial tumors are related to a low concentration of EGF and that a decrease of EGF is associated with invasion of tumors.

The effects of epidermal growth factor and chondroitin sulfate on the animal corneal endothelial cell culture.

In order to investigate the effects of different culture media containing various concentrations of epidermal growth factor (EGF) and chondroitin sulfate (CDS) on the growth of cultured corneal endothelial cell, pig corneal endothelial cells were used for this study. The cells were divided into 7 groups and each group was cultured with a medium containing: 1. Eagle's minimal essential medium with Earle's salt (EMEM) only; 2. EMEM + EGF (10 ng/ml); 3. EMEM + EGF (100 ng/ml); 4. EMEM + CDS (1 mg/ml); 5. EMEM + CDS (25 mg/ml); 6. EMEM + EGF (10 ng/ml) + CDS (1 mg/ml); 7. EMEM + EGF (100 ng/ml) + CDS (25 mg/ml), respectively. The results are shown below: (1) High concentration of EGF (100 ng/ml) stimulated the growth of pig corneal endothelial cells, shortened doubling time and the cells reached confluence earliest in group 3. But a low concentration of EGF (10 ng/ml) showed no effects on the growth of pig corneal endothelial cells. (2) A high concentration of CDS (25 mg/ml) might retard the growth of pig corneal endothelial cell, but a low concentration of CDS (1 mg/ml) has no retarding effects on the growth of pig corneal endothelial cells. (3) A high concentration of EGF could "antagonize" the CDS induced growth retarding effects of endothelial cells. (4) Those cells cultured with a medium containing high concentrations of EGF had more mitotic activity, including many prominent binuclear and polynucleolar cells. (5) The cells cultured with a medium containing high concentrations of CDS showed a more flat-shaped morphology under observation with a phase contrast microscope.(ABSTRACT TRUNCATED AT 250 WORDS)

Conditioned Medium from Cultured Human Keratinocytes Has Growth Stimulatory Properties on Different Human Cell Types

Evidence for growth-stimulatory properties of keratinocyte-conditioned medium (KCM) on human fibroblasts, endothelial cells, keratinocytes, smooth muscle cells, and a mouse fibroblast cell line (3T3 cells) is presented. On human fibroblasts KCM caused an increase of over 400% in DNA synthesis as revealed by 3H-thymidine incorporation and autoradiography. The proliferative effect was comparable to that of platelet-derived growth factor (PDGF), but was not inhibited by PDGF antibodies and exceeded that of transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and basic fibroblast growth factor (bFGF). Furthermore, KCM was found to stimulate smooth muscle cells, keratinocytes, and endothelial cells more potently than PDGF, EGF/TGF-alpha, and bFGF, respectively. KCM was also potent in stimulating thymidine incorporation in 3T3 cells, whereas EGF showed a twenty-fold weaker stimulatory effect. Because keratinocytes have been shown to secrete TGF-alpha, which binds to the EGF receptor, binding of factors in KCM to the EGF receptor was assayed. The displacement of radiolabeled EGF by KCM corresponded to a low concentration of EGF (0.5 ng/ml), implying that the growth-stimulatory effect of KCM was not mediated via activation of EGF receptors. Taken together, these results suggest the presence of hitherto unidentified growth-stimulatory factor(s), expressed and secreted by cultured human keratinocytes.

The Design of Analytical Methods for Use in Topical Epidermal Growth Factor Product Development

Data from a reverse phase gradient elution HPLC assay for human epidermal growth factor (EGF) was compared and correlated with data obtained from a competitive heterogeneous radioimmunoassay (RIA). The RIA was established to measure very low concentrations of EGF in formulation drug release and compatibility studies. The HPLC assay, capable of resolving parent and possible modified or fragmented forms of EGF isolated from human urine, was studied as a potential development tool for stability and final product evaluation. As independent analytical methods, the HPLC and the RIA procedures produced correlated results when quantifying freshly prepared and certain degraded samples of EGF. The capacity of the HPLC method to serve as a stability indicating assay was examined. Degradation of EGF was induced by storage in 0.05 M phosphate buffer pH 7.4 at 25, 37 or 50 degrees C. The same three degradation products were detected at each temperature by the HPLC method, one of which was identified as L-isoaspartyl EGF.

泌尿器悪性腫瘍における尿中 Epidermal growth factor の検討

We measured epidermal growth factor (EGF) in the urine of 54 untreated patients with malignant tumors (7 prostatic cancer, 11 renal tumor, 31 bladder cancer, 3 renal pelvic tumor, 2 ureter tumor) in the Toyama Medical & Pharmaceutical University Hospital. We also measured EGF in the urine of 77 normal subjects (43 males, 34 females). Urinary concentration of EGF in normal subjects decreased with increasing age. It was significantly higher in females than males (p less than 0.05). Urinary concentration of EGF in patients with prostatic cancer or renal tumor was similar to that in normal subjects. The patients with prostatic cancer controlled by estrogen showed a slightly high level of EGF in the urine. In patients with renal tumor, urinary concentration of EGF decreased after nephrectomy. Patients with bladder cancer showed a significant decrease of EGF in the urine compared with normal subjects (p less than 0.05), and urinary concentration of EGF in the patients with bladder cancer of high stage was remarkably low. Low concentration of EGF in the urine was recognized in patients with renal pelvic tumor or ureter tumor. However, the relationship between the decrease of urinary concentration of EGF in these urothelial tumors and the growth of these tumors remains to be elucidated.

Effect of epidermal growth factor on rat pleural mesothelial cell growth

We recently reported that the growth of normal rat pleural mesothelial cells (RPMCs) is inhibited by conditioned media from either in vivo or in vitro transformed RPMCs. In this study we report that the growth of normal RPMCs is inhibited by epidermal growth factor (EGF). This was demonstrated by using three methods of investigation. Two types of studies were carried out with growing cells. First, cell counts indicated that the number of cells was reduced in EGF-treated cultures when compared with untreated cultures. Second, the percentage of S cells detected by flow cytometry following treatment with EGF was lower than without EGF. In other experiments, incorporation of tritiated thymidine in confluent cells was decreased by EGF treatment, either in the presence or absence of fetal calf serum; these effects were dose dependent and were observed from 2 ng/ml EGF. Lower EGF concentrations did not significantly modify thymidine incorporation when compared with untreated cells. Analysis of 125I EGF binding experiments by the Scatchard method indicated that RPMCs posses EGF receptors (about 10(5) per cell) with low ligand binding affinity (Kd = 1.7 +/- 0.4 nM). These results indicate that EGF might modulate the growth of RPMCs.

Anti-epidermal growth factor receptor antibodies inhibit the autocrine-stimulated growth of MDA-468 human breast cancer cells.

The response of malignant and nonmalignant human breast cell lines to the growth inhibitory effects of monoclonal antibodies against the epidermal growth factor (EGF) receptor was studied. A series of human breast cell lines, which express EGF receptor, were used: MDA-468, MDA-231, and Hs578T human breast cancer cells and the transformed human mammary epithelial cell lines 184A1N4 and 184A1N4-T that have been benzo[a]pyrene immortalized and further transformed with SV40T, respectively. Four antibodies of two different classes were tested: 225 immunoglobulin G (IgG), 108.4 IgG, 96 immunoglobulin M (IgM), and 42 IgM. All four antibodies inhibited the anchorage-dependent and -independent, EGF-stimulated growth of 184A1N4 and 184A1N4-T cells, respectively, and this growth inhibition could be reversed by the addition of increasing concentrations of EGF. In contrast, the antibodies inhibited the anchorage-dependent and -independent growth of MDA-468 cells in the absence of exogenous EGF suggesting that the antibodies were acting to block access of an endogenously produced ligand to the EGF receptor. In the presence of antibody and increasing concentrations of EGF, MDA-468 cell growth was first stimulated then inhibited as the EGF concentration increased, thus, uncovering the growth stimulatory potential of low concentrations of EGF in these cells. Data is presented that indicates MDA-468 cells secrete a transforming growth factor with autocrine growth stimulatory capabilities. The growth of MDA-231 and Hs578T cells, which contain activated ras oncogenes, was not inhibited by the antibodies and the growth of these cell lines was not stimulated by EGF. Of the cell lines studied only MDA-468 cells appear to possess an autocrine growth stimulatory capacity.

Epidermal growth factor (EGF) prevents methylprednisolone-induced inhibition of wound healing

Subcutaneously implanted cylindrical hollow viscose cellulose sponges were used to study the effect of locally applied epidermal growth factor (EGF) on methylprednisolone-induced inhibition of granulation tissue formation in rats. In in vivo studies the sponges were treated immediately after implantation with a single injection of 2 mg (≈1.7 × 10 −3 M) of depot methylprednisolone or with its carrier solution only. Thereafter the implants were injected daily with 5 μg of EGF or with its carrier solution 0.1% albumin for 7 days. Methylprednisolone treatment decreased the accumulation of nucleic acids, collagen, and glycosaminoglycans in the developing granulation tissue. After daily injections of EGF the concentrations of these tissue components returned close to the control values. In cultures of rat granulation tissue fibroblasts, 10 −4 M and 10 −3 M methylprednisolone decreased collagen synthesis by 41 and 81% from the control level, respectively. In the presence of methylprednisolone EGF treatment could not increase collagen synthesis of fibroblasts. Methylprednisolone treatment resulted in a dose-dependent reduction in pro←(I) collagen mRNA levels, which was partially inhibited by low EGF concentrations (1 and 10 ng/ml). In the presence of methylprednisolone all concentrations of EGF increased fibronectin mRNA levels in a dose-dependent manner. It is concluded that EGF treatment can prevent the inhibitory effect of methylprednisolone on wound healing by stimulating fibroblast proliferation but does not stimulate collagen synthesis per cell.

Androgens stimulate both growth rate and epidermal growth factor receptor activity of the human prostate tumor cell LNCaP.

LNCaP cells (derived from a lymph node carcinoma of the human prostate) contain androgen receptors and show androgen-responsive growth in vitro. Maximal effects on growth were seen at 0.1 nM of the synthetic androgen R1881 or 0.2 nM of epidermal growth factor (EGF); both compounds independently increased the growth rate 2-3 times. EGF-receptors were measured after 6 days culture in the presence or absence of 0.1 nM R1881. A 2.3-fold increase in receptor number/cell was found when binding was measured at 0 degrees C (from 12,500 to 28,900 sites/cell in stimulated cells). The kD value (0.45 nM) was not affected by androgen treatment. The increase of EGF-receptor activity was first observed between 6 and 12 h after exposure to androgen. It is concluded that LNCaP cells are sensitive to low concentrations of EGF (or EGF-like compounds) and that one of the mechanisms involved in androgen action on these cells is an increase of EGF-receptor expression at the cell surface.

Sn-1,2-Dioctanoylglycerol. A cell-permeable diacylglycerol that mimics phorbol diester action on the epidermal growth factor receptor and mitogenesis.

The cell-permeable diacylglycerol, sn-1,2-dioctanoylglycerol (DiC8), is shown to mimic the effect of tumor promoting phorbol diesters on epidermal growth factor (EGF) binding and action in intact cells. DiC8 inhibited the binding of [3H]phorbol dibutyrate to A431 cell monolayers indicating that the diacylglycerol interacts with the phorbol diester receptor. At 0.3 microM, DiC8 half-maximally inhibited the high affinity binding of 125I-EGF to A431 human epidermoid carcinoma cells. Scatchard analysis indicated that the inhibition of 125I-EGF binding was very similar to that observed in the presence of 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). DiC8 also mimicked the action of PMA to increase the phosphorylation state of the EGF receptor in 32P-labeled cells. Phosphoamino acid analysis demonstrated that DiC8 and PMA caused an increase in the level of EGF-receptor phosphoserine and phosphothreonine, whereas EGF caused an increase in the level of phosphoserine, phosphothreonine, and phosphotyrosine. Phosphopeptide mapping of the EGF receptor showed that DiC8 and PMA enhanced the phosphorylation of the same tryptic peptides. DiC8 inhibited the EGF-dependent tyrosine phosphorylation of the EGF receptor in A431 cells in a similar manner to that observed with PMA. In further experiments with quiescent Swiss 3T3 fibroblasts, DiC8 mimicked the ability of PMA to stimulate the incorporation of [methyl-3H]thymidine synergistically with low concentrations of EGF. This result indicates that DiC8 will mimic the long-term effects of PMA to regulate mitogenesis and raises the possibility that it may be active in two stage carcinogenesis. As both DiC8 and PMA stimulate the Ca2+- and phospholipid-dependent protein kinase (C-kinase) in vitro, the results support the hypothesis that the activation of C-kinase is a critical component of phorbol diester action on EGF receptor modulation and cell proliferation.

Epidermal growth factor initiates DNA synthesis after a time-dependent sequence of regulatory events in Swiss 3T3 cells--interactions with hormones and growth factors.

Epidermal growth factor (EGF) stimulates the initiation of DNA synthesis in Swiss 3T3 cells after a constant prereplicative period of 14-15 hours. The final rate of initiation follows apparent first-order kinetics and can thus be quantified by a rate constant k. The value of k can be changed by later additions during the prereplicative period: When cells stimulated by a very low concentration of EGF, alone or with insulin, which results in a relatively low value of k, receive a saturating amount of EGF at 15 hours, then k is markedly increased after 4-6 hours. Insulin alone (up to 200 ng/ml) is unable to set the lag phase, but does have a synergistic effect on the value of k given by EGF. When added at 15 hours, insulin also increases K, but after a delay of 4-6 hours. In contrast, both hydrocortisone and prostaglandin E1 (PGE1) inhibit the stimulation of DNA synthesis by EGF only during the first 8 hours of the prereplicative period of decreasing the value of k. Prostaglandin F2 alpha), which stimulates DNA synthesis in a similar mode as EGF, when added with EGF has a synergistic effect on DNA synthesis. This suggests that EGF and PGF2 alpha, nevertheless, act through different regulatory events.