High Concentrations Of Epidermal Growth Factor Research Articles

A retrospective analysis of characteristic features of responder patients to autologous serum eye drops in routine care

PurposeAutologous serum eye drops (ASEDs) are used worldwide to treat dry eye disease (DED). However, the biological composition of ASEDs has not been well investigated, and effectiveness predictive factors remain to be identified. The main objective of this study was to compare the response of patients treated with ASEDs biologically characterized and used for DED routine care. MethodsThis retrospective observational study was conducted in a single university hospital, and included 50 patients (87 eyes) with DED refractory to conventional treatment and resulting from various etiologies with Ocular Surface Disease Index (OSDI) ≥ 20. Each patient used eight drops a day per treated eye with 20% diluted ASEDs. Undiluted serum extensive biological characterizations were performed, and symptoms were recorded before the initiation of ASEDs and closer to the sixth month of treatment. Responders were defined as presenting an improvement from baseline ≥14 points in OSDI and/or ≥1 grade in corneal fluorescence staining for all eyes treated. ResultsThe OSDI and the Oxford scale were significantly reduced from 68.7 ± 23.2 to 54.8 ± 25.7 and 3.2 ± 1.5 to 2.1 ± 1.3 (p ≤ 0.0001), respectively. A total of 68% of the patients were responders. Nonresponding patients had significantly higher epidermal growth factor concentrations in the serum compared to responders (p = 0.017). ConclusionsASED administration resulted in significant clinical improvement in the management of DED. Biological differences observed between responders and nonresponders suggested that a better understanding of the biological activity of ASEDs is still required.

The positional identity of iPSC-derived neural progenitor cells along the anterior-posterior axis is controlled in a dosage-dependent manner by bFGF and EGF

Neural rosettes derived from human induced pluripotent stem cells (iPSCs) have been claimed to be a highly robust in vitro cellular model for biomedical application. They are able to propagate in vitro in the presence of mitogens, including basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). However, these two mitogens are also involved in anterior-posterior patterning in a gradient dependent manner along the neural tube axis.Here, we compared the regional identity of neural rosette cells and specific neural subtypes of their progeny propagated with low and high concentrations of bFGF and EGF. We observed that low concentrations of bFGF and EGF in the culturing system were able to induce forebrain identity of the neural rosettes and promote subsequent cortical neuronal differentiation. On the contrary, high concentrations of these mitogens stimulate a mid-hindbrain fate of the neural rosettes, resulting in subsequent cholinergic neuron differentiation. Thus, our results indicate that different concentrations of bFGF and EGF supplemented during propagation of neural rosettes are involved in altering the identity of the resultant neural cells.

Assessment of the Developmental Toxicity of Epidermal Growth Factor using Embryonic Stem Cell Test

Purpose: To determine whether epidermal growth factor (EGF) is involved in reproductive developmental toxicity, using the embryonic stem cell test (EST), as well as ascertain how EGF influences embryonic development. Methods: To predict developmental toxicity on the basis of reducing cell viability and inhibition of differentiation of embryonic stem cells, EST was used to assess changes in different blastodermic genes and expression of proteins including ectodermal-specific genes Pax6, NF-H and glial fibrillary acidic protein (GFAP), mesodermal-specific genes BMP4, GATA4, and MyoD, and endodermal-specific genes, viz, α-fetoprotein, transthyretin (TTR), and albumin, as well as undifferentiated genes, Nanog and Oct4. Results: The results indicate that EGF was weakly embryotoxic with IC50ESC (i.e., the concentration that reduced ESC viability by 50 %), IC 50 3T3 (the concentration that reduced 3T3 cell viability by 50 %), and ID 50 ESC (the concentration that inhibited differentiation of ESC by 50 %) of 6.773, 10.531, and 1.793 μg/mL, respectively. The expression levels of tissue-specific genes of the three germ layers were mainly promoted by 0.01 - 1 μg/mL EGF. Distinctively, relatively high concentrations of EGF caused a discordant effect on the three germ layers. High concentrations of EGF promoted differentiation of the ectoderm and mesoderm, and either inhibited or had mostly no impact on the endoderm. Conclusion : The imbalance of the three layer-specific genes and expression of proteins, as a result of EGF, might be responsible for its weak level of developmental toxicity. The sensitivity of TTR means that further investigation is required to determine whether it can be used as an embryotoxicity biomarker for growth factors. Keywords : Embryonic stem cell test, Epidermal growth factor, Developmental toxicity, Germ layers, Blastodermic genes, Protein expression

Downregulation of galectin-3 by EGF mediates the apoptosis of HepG2 cells

Epidermal growth factor (EGF) in high concentrations induces apoptosis of the tumor cells which express high levels of epidermal growth factor receptor. However, the precise mechanism for this induction is not clear. Galectin-3 is the most probable candidate for mediating this effect, as it is known to induce anti-apoptotic activity in a variety of tumor cells exposed to diverse apoptotic stimuli. In this study, we determined whether galectin-3 plays a role in high concentrations of EGF-induced apoptosis of HepG2 cells. We found that EGF in high concentrations led to the growth inhibition of HepG2 cells, which were associated with promotion of cell death. High concentrations of EGF suppressed cytoplasmic expression of galectin-3. Moreover, we demonstrated overexpression of galectin-3 could reduce EGF-induced apoptosis in HepG2 cells. Our study demonstrated for the first time that downregulation of cytoplasmic galectin-3 was essential for high concentrations of EGF-induced apoptosis in HepG2 cells.

And what about epidermal growth factor (EGF) as the bridge between survivin and cardiac remodelling?

Neonates of diabetic mothers are at increased risk for hypertrophic cardiomyopathy whose functional significance is variable. Regardless of severity, cardiac hypertrophy is transient lasting at six months of age as shown by echocardiographic measurements [1]. These findings emphasize the concept that the heart is capable of rapid anatomic remodelling in response to physiologic and pathologic stimuli. However, the molecular mechanism regulating the postnatal regression of hypertrophic cardiomyopathy in infants of diabetic mothers is currently unknown. It has been recently demonstrated that activation of apoptotic cell death involving activation of the MAPK and caspases is correlated with the development and resolution of hypertrophic cardiomyopathy in offspring of diabetic rats [1]. Survivin is a recently characterized member of the inhibitors of apoptosis that probably exerts its action by blunting apoptotic pathways also involving caspases. Survivin is over-expressed during fetal development and in cancer cells but is undetectable in terminally differentiated normal adult tissues [2]. It has been shown that survivin concentrations in cord blood do not reflect the disturbances of feto-placental apoptosis expected in the offspring of diabetic mothers compared to controls [2]. The regression of hypertrophic cardiomyopathy implies that cardiomyocytes survive in spite of the initiation of apoptosis. In this respect, we hypothesized that fetal survivin by balancing cardiomyocyte proliferation and death may drive the reversibility of apoptosis [3]. As a result of this phenomenon the identification of signalling molecules regulating survivin expression in fetal cardiomyocytes, both upstream and downstream, may provide important insights into the machinery driving myocardial regeneration. EGF is regarded as a major component responsible for the effects of the swallowed amniotic fluid on the development of various organs [4]. Human amniotic fluid contains high concentrations of EGF [4]. It is a potent growth factor that plays a key role in fetoplacental development. It is well established that EGF plays an important role in human cardiac differentiation and development including fetal cardiomyocyte proliferation [5]. In addition, it has been recently described that EGF regulates survivin stability [6]. Fetal macrosomia, representing one of the complications of gestational diabetes, is not associated with changes in amniotic fluid EGF levels. All together these observations strongly suggest that EGF may be a critical molecule for the upregulation of prenatal survivin gene expression, promoting the increased resistance to apoptosis with the escape of cardiomyocytes from the point-of-no-return of apoptotic signals. More in-depth studies are needed to better clarify this issue because the interaction between EGF and survivin may be important targets of potential therapeutic interventions aimed at reversing unfavourable cardiac remodelling at any stage of human cardiomyopathies. The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology [7].

Ram Sperm Motility Parameters under The Influence of Epidermal Growth Factor

Epidermal growth factor (EGF) is one of the important cytokines that play a role in fertility. It is known that EGF affects both male and female reproduction, but its effect on sperm parameters is not fully understood. Up to the present, the effect of EGF on ram sperm motility parameters has not been published. We analyzed motility parameters of ejaculates after 24, 48, and 72 hours from the EGF addition. EGF was added to chilled ram sperm at concentrations of 0, 100, 200, and 400 ng·ml−1. Analyses were realized using computer, assisted semen analyzer (CASA)—Hamilton Thorn motility analyzer (version 7). The effect of EGF was already visible after 30 min of incubation. Significant effect on ram sperm total motility and progressive movement was observed at higher EGF concentrations after 48 h of incubation. Our results show that EGF affects sperm motility parameters depending on concentration and time of exposure.

206 THE IMPORTANCE OF CUMULUS EXPANSION FOR MONOSPERMIC FERTILIZATION OF PORCINE CUMULUS - OOCYTE COMPLEXES

In pigs, the exact role of the cumulus oöphurus during IVF still needs to be clarified. Indirect evidence exists that the rate of cumulus expansion is positively correlated with the defense against polyspermy. Epidermal growth factor (EGF) acts synergistically with FSH in the synthesis of hyaluronic acid, the deposition of which in the extracellular matrix is a prerequisite for cumulus expansion. Therefore, we aimed to evaluate the importance of cumulus expansion for fertilization results of porcine cumulus–oocyte complexes (COC) by using different EGF concentrations in the maturation medium. Cumulus–oocyte complexes were matured in vitro in NCSU23 medium supplemented with 10% follicular fluid (FF, obtained from 6- to 10-mm follicles) or 10% serum and 10, 20, or 50 ng mL–1 of EGF (n = 480 per experiment). In vitro maturation (IVM) in the presence of 10% FF and 10 ng mL–1 of EGF served as the control group. At 0, 22, 36, and 44 h of IVM, 20 COC of each group were selected for evaluation of cumulus expansion by measuring the maximum distance across the cumulus matrix (3 replications). Matured COC were co-incubated with frozen–thawed semen (6000 spermatozoa per oocyte) for 6 h. Subsequently, oocytes were cultured for 18 h. Zygotes were stained with 10 μg mL–1 of bis-benzimide (Hoechst) to assess the fertilization rate, polyspermy, and sperm penetration index (sp index, mean number of penetrated spermatozoa per fertilized oocyte; 2 replications). Differences in cumulus diameter were analyzed by one-way ANOVA. Fertilization parameters were analyzed by applying a logistic regression model to the results. Cumulus–oocyte complexes selected for IVM had a mean diameter of 240 μm. After 22 h of IVM in 10% FF, the mean diameter of COC was 336, 313, and 300 μm for 10, 20, and 50 ng mL–1 of EGF, respectively. After 44 h of IVM, these diameters had increased to 425, 388, and 397 μm. Twenty-two hours of IVM in 10% serum resulted in a COC diameter of 296, 305, and 276 μm for 10, 20, and 50 ng mL–1 of EGF. After 44 h of IVM, these diameters reached 330, 325, and 275 μm, respectively. Only 10% serum with 50 ng mL–1 of EGF proved unfavorable for cumulus expansion (P < 0.05). In vitro maturation for 44 h in 10% serum resulted in a smaller rate of cumulus expansion compared with IVM in 10% FF (P < 0.05), irrespective of EGF concentration. Penetration rate fluctuated between 84 and 100%, with no significant differences. Monospermic fertilization was lower in COC matured in the presence of 50 ng mL–1 of EGF compared with the control group (P < 0.05). The sp index increased in parallel with EGF concentrations and was higher after IVM in 10% serum than in 10% FF. Oocytes were penetrated by 3.1, 4.3, and 6.0 spermatozoa after IVM in serum with 10, 20, and 50 ng mL–1 of EGF, respectively. Results showed a tendency toward a lower rate of cumulus expansion concomitant with higher EGF concentrations. Follicular fluid was superior to serum in supporting cumulus expansion. Oocytes were penetrated by more spermatozoa when matured in 10% serum and 20 or 50 ng mL–1 of EGF compared with 10 ng mL–1 of EGF. Thus, the degree of cumulus expansion appears to be related to the sp index, confirming that the cumulus matrix may play a role in the polyspermy defense. This study was supported by Research Foundation-Flanders.

Epidermal Growth Factor Stimulates Human Trophoblast Cell Migration through Rho A and Rho C Activation

This study investigated the roles of Rho protein in epidermal growth factor (EGF)-induced trophoblast cell migration and its mechanism. Using choriocarcinoma cell lines JEG-3 and JAR and first-trimester human chorionic villus explant cultures on matrigel, we examined EGF-mediated stimulation of trophoblast migration. EGF is shown to have a dose-dependent effect on trophoblast migration. A low concentration of EGF (1 ng/ml) has a stimulatory effect on cell migration, whereas high concentrations of EGF (100 ng/ml) shows an inhibitory effect. EGF (1 ng/ml) activates RhoA and RhoC, but not RhoB, through elevated protein levels and activity. EGF-induced migration was shown to be inhibited by either cell-permeable C3 exoenzyme transferase or selective RhoA or RhoC small interfering RNAs. The inhibition was not mitigated by the addition of EGF, suggesting that RhoA and RhoC play an important role in trophoblast migration and are obligatory for EGF action. Treatment of JEG-3 and JAR cells with RhoA small interfering RNA induced F-actin cytoskeleton disruption and cell shrinkage, which is consistent with the effect of C3 exoenzyme transferase, and this action was not mitigated by EGF treatment. RhoC small interfering RNA had no apparent effect on the F-actin arrangement, suggesting that RhoA but not RhoC takes part in the EGF-induced migration through F-actin rearrangement. These results indicate that RhoA and RhoC play more important roles than RhoB in EGF-mediated migration of trophoblast cells, and RhoA but not RhoC regulates this migration through F-actin cytoskeleton reorganization.

Breast Milk Jaundice Correlates With High Levels of Epidermal Growth Factor

Maternal milk plays an important role in breast milk jaundice (BMJ) development and is the major source of epidermal growth factor (EGF) for neonates. The aim of this study was to investigate whether there is a relationship between EGF levels in the infant serum and in the milk of nursing mothers and BMJ. Two groups were defined: study group (n = 30), newborns who were followed up for BMJ without any identifiable pathologic cause; control group, healthy newborns whose serum total bilirubin levels were <10 mg/dL. Milk and infant plasma samples were collected between the third and the fourth postpartum week. EGF concentrations in all of the samples were determined by using ELISA. The infants with BMJ had higher concentrations of EGF in the serum and in the breast milk compared with that of the infants without BMJ. The milk concentrations of EGF were significantly correlated with neonatal bilirubin and blood EGF concentrations. The degree of BMJ was associated with the increased levels of milk borne EGF. Although the exact mechanisms of the hyperbilirubinemic action of EGF are not completely known, the inhibition of gastric motility, increased absorption, and activation of bilirubin transport have been suggested as possible mechanisms.

A High Concentration of Epidermal Growth Factor Increases the Growth and Survival of Neurogenic Radial Glial Cells Within Human Neurosphere Cultures

Human neural progenitor cells (hNPC) isolated from the fetal cortex can be expanded as aggregates of cells termed neurospheres. Traditional methods have used 20 ng/ml epidermal growth factor (EGF) to drive the proliferation of these cells. Here, we show that 100 ng/ml EGF can significantly increase growth rates of hNPC at later passages. This was through increased survival of dividing cells rather than increased proliferation and associated with prolonged activation of ErbB2 and phosphorylated Akt. High EGF also resulted in a larger proportion of elongated "radial glial"-like cells within the growing neurospheres and increased expression of the radial glial markers. The number of new neurons generated from cultures maintained in 100 ng/ml EGF was significantly higher than from 20 ng/ml EGF. Thus, high concentrations of EGF increase the survival of a highly neurogenic human radial glial cell.

Role of Epidermal Growth Factor and ErbB2 Receptors in 3T3‐L1 Adipogenesis

Epidermal growth factor (EGF) stimulates proliferation in 3T3-L1 preadipocytes, but EGF action in differentiation is less clear. EGF promotes differentiation at concentrations <1 nM but inhibits differentiation at higher concentrations, suggesting a dual role in adipogenesis. We hypothesized that differences in EGF receptor activation and downstream signaling mediate distinct biological effects of EGF at low vs. high abundance. We compared the effects of low (0.1 nM) vs. high (10 nM) EGF on the activation of EGF receptors, proximal signaling molecules Src and Shc, and the downstream mitogen-activated protein kinase (MAPK) pathways extracellular regulated kinase (ERK) and p38 in proliferating and differentiated 3T3-L1 cells. Both low and high EGF activated ERK and p38 in preadipocytes. Src inhibitors PP1 and PP2 blocked ERK and p38 activation by low but not high EGF, and only high EGF increased Shc phosphorylation. Selective inhibition of the EGF receptor (EGFR) with AG1478 blocked ERK and p38 activation at both concentrations; however, selective inhibition of the ErbB2 receptor (EB2R) with AG825 or small interfering RNA (siRNA) blocked low but not high EGF activation of ERK and p38. Coimmunoprecipitation of EGFR with EB2R and Src was observed with low EGF in preadipocytes but at both concentrations in adipocytes. EB2R inhibition during differentiation decreased p38 activity and peroxisome proliferator-activated receptor gamma (PPARgamma) abundance. Our results show that EGFR homodimers mediate action of EGF at high abundance, but at low abundance, EGF promotes differentiation through EGFR/EB2R heterodimer activation of Src and p38. These results may partially explain the observations that high EGF concentrations inhibit, whereas low concentrations support, preadipocyte differentiation.

The effect of epidermal growth factor on matrix metalloproteinases and tissue inhibitors of metalloproteinase gene expression in cultured human gingival fibroblasts

Objective Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) play a role in the breakdown of the extracellular matrix during normal physiological processes, and in pathological processes, including periodontitis. The aim of this study was to evaluate the effect of epidermal growth factor (EGF) on the expression of MMPs and TIMPs in cultured human gingival fibroblasts. Methods Fibroblasts were stimulated with 10 −3, 10 −6 or 10 −12 M EGF for 24 h; untreated fibroblasts served as controls. Alterations in the expression of MMP-1, 2, 3, 7, 11, TIMP-1 and 2 were evaluated using real-time PCR and Western blotting. β-Actin expression was used as a reference to normalize gene expression. Results Increased MMP-1, 3, 7 and 11 expressions were observed at all EGF concentrations ( p < 0.05). At the lowest EGF concentration, MMP-1, 3 and 7 presented the lowest expression and MMP-11 presented the greatest expression; at higher EGF concentrations, MMP-1, 3 and 7 presented greater up-regulation, and MMP-11 lower up-regulation ( p < 0.05). Protein expression was similarly regulated by EGF: increased up-regulation of MMP-1, 3 and 7 was observed with increasing EGF concentrations, except for MMP-11 that exhibited greater up-regulation at the lower EGF concentration. The gene expression of MMP-2, TIMP-1 and 2 was not affected by EGF ( p < 0.05). Conclusions We conclude that EGF regulates expression for MMP-1, 3, 7 and 11 in a dose-dependent manner, suggesting that EGF may play a role in periodontal destruction and wound repair.

Epidermal growth factor (EGF) induces apoptosis in a transfected cell line expressing EGF receptor on its membrane

Interaction between epidermal growth factor (EGF) and EGF receptor (EGFR) promotes cell growth in most cell lines, but in a number of cell lines, EGF paradoxically inhibits proliferation. In the present study, we established a cell line expressing full-length human EGFR on membrane with a GFP fluorescence reporter at the C-terminal and studied the effects of EGF on cell proliferation in the transfected cell line. Our results suggested that low concentrations of EGF promoted proliferation, while high concentrations of EGF induced loss of adhesion, cell cycle arrest, apoptosis, and inhibition of proliferation. The effects of EGF on cell proliferation correlated well with the expression levels of EGFR. High concentrations of EGF induced both EGFR expression and apoptosis in a dose-dependent manner. Our study reported, for the first time, a relationship between the effects of EGF on cell proliferation and levels of EGFR expression in one cell line expressing different levels of EGFR caused by different concentrations of EGF treatment. The study should provide considerable insight into the effects of EGF on cell proliferation and tumor cell metastasis.

EGF mediates multiple signals: dependence on the conditions

Epidermal growth factor (EGF) has been reported to both increase and decrease proliferation and apoptosis. In KB cells, EGF-induced alteration in proliferation and apoptosis are concentration and time dependent. High concentrations of EGF (10(-7) and 10(-8) M) stimulated proliferation and inhibited apoptosis at 24 h, with apoptosis increasing with prolonged exposure. Low concentrations of EGF (10(-10)-10(-11) M) inhibited apoptosis without affecting proliferation. EGFR protein expression was downregulated at high and upregulated at low EGF concentrations. Significant inverse correlation was shown between EGFR expression level and apoptosis. These results suggest the EGF receptor involvement in the modulation of apoptosis and the role of EGF anti-apoptotic effects in EGF-induced apoptosis.

Intracystic epidermal growth factor level is predictive of breast‐cancer risk in women with gross cystic disease of the breast

Women affected by gross cystic disease of the breast have an increased risk of breast cancer. We report here the incidence of breast cancer by cyst type and intracystic epidermal growth factor (EGF) concentration. Our retrospective study included 504 women who had at least 1 cyst aspiration between 1985 and 1993. Cyst fluids were processed for electrolyte concentration (n = 378), EGF concentration (n = 347) or both (n = 337). Age-standardized incidence ratios (SIRs) were estimated using the population of the Genoa Cancer Registry. A multivariate Poisson regression model was used to estimate relative risks (RRs) when the study groups were compared directly. By June 1999, 19 invasive breast cancers had developed in the cohort of women. The age SIR of breast cancer calculated for the whole cohort was 3.32 (95% confidence interval 2.00–5.18). The ratio was not affected by age and was only moderately increased in women with a positive family history of breast cancer and type I cysts (i.e., those with a Na+/K+ ratio <3). However, it was significantly increased in women with high EGF concentrations. Direct comparisons confirmed that age, cyst type and family history only moderately increased the RR, whereas EGF concentration was a strong predictor of risk. Our results confirm that women affected by palpable cysts have an increased risk of developing breast cancer and suggest that the risk is higher in women with high intracystic EGF concentrations. © 2001 Wiley-Liss, Inc.

Regulation of mucous differentiation and mucin gene expression in the tracheobronchial epithelium

The goal of our studies is to elucidate mechanisms that control and modulate mucous differentiation and mucin gene expression in the conducting airways. We used cultures of normal human tracheobronchial epithelial (NHTBE) cells that were shown to secrete two major airway mucins, namely MUC5AC and MUC5B as well as several other secretory products. Mucous differentiation and expression of MUC2, MUC5AC, MUC5B and MUC7, but not MUCi, MUC4, and MUC8 mucin genes, were shown to be retinoic acid- (RA) or retinol-dependent. We found that RA control of mucin genes was mediated by the retinoid acid receptors RAR α and, to a lesser extent, by RAR γ. Our studies also showed that other important bioregulators such as thyroid hormone (T 3) and epidermal growth factor (EGF) modulate basal expression of mucin genes, interacting with RA in a concentration-dependent manner. T 3, which binds to thyroid receptors (TRs) belonging to the same superfamily of steroid hormone nuclear receptors as the RARs, inhibits mucin gene expression, particularly MUC5AC. One possible mechanism of this T 3 effect is downregulation of RAR proteins, which are critical for mucin gene expression. However, we also found that T 3 inhibits MUC5AC transcription. EGF, which had previously been shown to stimulate mucin expression and mucin secretion in cultured rat tracheal epithelial (RTE) cells, inhibited mucin secretion in human bronchial epithelial cell cultures. This effect was EGF concentration- and time-dependent and was progressively abolished by increasing the RA concentration. Subsequent studies suggested that the inhibitory effects of high concentrations of EGF may result from selective reduction of MUC5AC expression. These studies thus point to potentially important species differences in the mechanisms regulating mucous production, and they also confirm previous findings indicating differential regulation of MUC5AC and MUC5B gene expression.

Intracystic epidermal growth factor level is predictive of breast-cancer risk in women with gross cystic disease of the breast.

Women affected by gross cystic disease of the breast have an increased risk of breast cancer. We report here the incidence of breast cancer by cyst type and intracystic epidermal growth factor (EGF) concentration. Our retrospective study included 504 women who had at least 1 cyst aspiration between 1985 and 1993. Cyst fluids were processed for electrolyte concentration (n = 378), EGF concentration (n = 347) or both (n = 337). Age-standardized incidence ratios (SIRs) were estimated using the population of the Genoa Cancer Registry. A multivariate Poisson regression model was used to estimate relative risks (RRs) when the study groups were compared directly. By June 1999, 19 invasive breast cancers had developed in the cohort of women. The age SIR of breast cancer calculated for the whole cohort was 3.32 (95% confidence interval 2.00-5.18). The ratio was not affected by age and was only moderately increased in women with a positive family history of breast cancer and type I cysts (i.e., those with a Na+/K+ ratio <3). However, it was significantly increased in women with high EGF concentrations. Direct comparisons confirmed that age, cyst type and family history only moderately increased the RR, whereas EGF concentration was a strong predictor of risk. Our results confirm that women affected by palpable cysts have an increased risk of developing breast cancer and suggest that the risk is higher in women with high intracystic EGF concentrations.

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.

Expression and action of transforming growth factor beta (TGFbeta1, TGFbeta2, and TGFbeta3) during embryonic rat testis development.

The objective of the current study was to determine the role of transforming growth factor beta (TGFbeta) during seminiferous cord formation and embryonic testis development. The expression pattern of mRNA for TGFbeta isoforms was evaluated during testis development through a quantitative reverse transcription-polymerase chain reaction (QRT-PCR) procedure. Expression of mRNA for TGFbeta1 was highest at postnatal day 0 (P0) and P10. In contrast, TGFbeta2 was high at embryonic day 15 (E15), declined at E16, and showed a transient increase at P0 through P3 of testis development. Interestingly, expression of mRNA for TGFbeta3 was high during embryonic development and then declined after P3. Immunohistochemical localization of TGFbeta1 and TGFbeta2 demonstrated expression in Sertoli cells at E14 and in the seminiferous cords at P0. Selective interstitial cells expressed high concentrations of TGFbeta1 and TGFbeta2 in P0 testis. TGFbeta3 was expressed in selective cells at the junction of the E14 testis and mesonephros. The cells expressing TGFbeta3 in the testis appeared to be preperitubular cells that resided around the seminiferous cords. TGFbeta3 was localized to gonocytes in P0 testis. TGFbeta1 was found to have no influence on seminiferous cord formation in embryonic organ cultures of E13 testis. In contrast, growth of both E13 and E14 embryonic organ cultures was inhibited by TGFbeta1 and resulted in reduced testis size (40% of controls) with fewer cords present. A P0 testis cell culture and thymidine incorporation assay were used to directly examine the effects of recombinant TGFbeta1. TGFbeta1 alone had no influence on thymidine incorporation in P0 testis cell cultures when compared to controls. Interestingly, TGFbeta1 inhibited epidermal growth factor (EGF), and 10% calf serum stimulated P0 testis cell growth but not FSH-stimulated growth. Therefore, TGFbeta1 appears to inhibit testis growth in both the embryonic and early postnatal periods. The hormonal regulation of TGFbeta expression was measured using P0 testis cell cultures and a QRT-PCR procedure for each TGFbeta isoform. High concentrations of EGF stimulated expression of mRNA for TGFbeta1 after 24 h but suppressed expression of TGFbeta3. In contrast, there was no effect of FSH on TGFbeta isoform expression. In summary, TGFbeta regulates embryonic and P0 testis growth through inhibiting the actions of positive growth factors such as EGF. In addition, EGF but not FSH appears to regulate TGFbeta isoform expression. Combined observations from the present study demonstrate that TGFbeta isoforms are differentially expressed and appear to be regulators of testis growth during the embryonic and early postnatal periods.

Involvement of MAP Kinase-Independent Protein Kinase C Signaling Pathway in the EGF-Induced p21(WAF1/Cip1) Expression and Growth Inhibition of A431 Cells

Previous studies have revealed that the growth inhibition of A431 cells overexpressing epidermal growth factor (EGF) receptors by a high concentration of EGF is mainly due to the expression of cycline dependent kinase (CDK) inhibitor p21(WAF1/Cip1). However, the signal transduction mechanism from the activated EGF receptor to the induction of p21(WAF1/Cip1) gene is still poorly understood. We investigated which signaling pathway plays an important role in p21(WAF1/Cip1) expression and growth inhibition by using specific inhibitors of the signaling molecules. A broad PKC inhibitor, PKCδ inhibitor, but not the conventional PKC inhibitor suppressed the EGF-induced p21(WAF1/Cip1) expression and the growth inhibition of A431 cells. These inhibitors did not alter either the activation of EGF receptor or the stimulation of MAP kinase at detectable levels. Furthermore, we found that the induction of p21(WAF1/Cip1) at the early phase (within 12 hr after stimulation) by a high concentration of EGF was independent of the MAP kinase activation by using dominant negative Ras. These results suggest that PKC, especially PKCδ plays a crucial role in the EGF-induced p21(WAF1/Cip1) expression, resulting in the growth inhibition of A431 cells.