High Concentrations Of EGF Research Articles

In Vitro Differentiation of Human Amniotic Epithelial Cells into Hepatocyte-like Cells.

Human amniotic epithelial cells (hAECs) represent an interesting clinical alternative to human embryonic (hESCs) and induced pluripotent (hiPSCs) stem cells in regenerative medicine. The potential of hAECs can be enhanced ex vivo by their partial pre-differentiation. The aim of this study was to evaluate the effectiveness of 18-day differentiation of hAECs into endodermal cells, hepatic precursor cells, and cells showing functional features of hepatocytes using culture media supplemented with high (100 ng/mL) concentrations of EGF or HGF. The cells obtained after differentiation showed changes in morphology and increased expression of AFP, ALB, CYP3A4, CYP3A7, and GSTP1 genes. HGF was more effective than EGF in increasing the expression of liver-specific genes in hAECs. However, EGF stimulated the differentiation process more efficiently and yielded more hepatocyte-like cells capable of synthesizing α-fetoprotein during differentiation. Additionally, after 18 days, GST transferases, albumin, and CYP P450s, which proved their partial functionality, were expressed. In summary, HGF and EGF at a dose of 100 ng/mL can be successfully used to obtain hepatocyte-like cells between days 7 and 18 of hAEC differentiation. However, the effectiveness of this process is lower compared with hiPSC differentiation; therefore, optimization of the composition of the medium requires further research.

D-Propranolol Impairs EGFR Trafficking and Destabilizes Mutant p53 Counteracting AKT Signaling and Tumor Malignancy.

Simple SummaryCancer progression is frequently driven by altered functions of EGFR belonging to the tyrosine-kinase family of growth factor receptors and by the transcription factor p53, which is called the “genome guardian”. We report that D-Propranolol, previously used for other purposes in human patients, has antitumor effects involving a redistribution of cell surface EGFR to intracellular compartments and degradation of gain-of-function mutants of p53 (GOF-mutp53). These effects can be seen in cancer cell lines expressing EGFR and GOF-mutp53 and are reproduced in vivo, reducing tumor growth and prolonging survival of xenografted mice. D-Propranolol is proposed as a prototype drug for a new strategy against highly aggressive EGFR- and mutp53-expressing tumors.Cancer therapy may be improved by the simultaneous interference of two or more oncogenic pathways contributing to tumor progression and aggressiveness, such as EGFR and p53. Tumor cells expressing gain-of-function (GOF) mutants of p53 (mutp53) are usually resistant to EGFR inhibitors and display invasive migration and AKT-mediated survival associated with enhanced EGFR recycling. D-Propranolol (D-Prop), the non-beta blocker enantiomer of propranolol, was previously shown to induce EGFR internalization through a PKA inhibitory pathway that blocks the recycling of the receptor. Here, we first show that D-Prop decreases the levels of EGFR at the surface of GOF mutp53 cells, relocating the receptor towards recycling endosomes, both in the absence of ligand and during stimulation with high concentrations of EGF or TGF-α. D-Prop also inactivates AKT signaling and reduces the invasive migration and viability of these mutp53 cells. Unexpectedly, mutp53 protein, which is stabilized by interaction with the chaperone HSP90 and mediates cell oncogenic addiction, becomes destabilized after D-Prop treatment. HSP90 phosphorylation by PKA and its interaction with mutp53 are decreased by D-Prop, releasing mutp53 towards proteasomal degradation. Furthermore, a single daily dose of D-Prop reproduces most of these effects in xenografts of aggressive gallbladder cancerous G-415 cells expressing GOF R282W mutp53, resulting in reduced tumor growth and extended mice survival. D-Prop then emerges as an old drug endowed with a novel therapeutic potential against EGFR- and mutp53-driven tumor traits that are common to a large variety of cancers.

P2.01-33 Survival Benefit of Depleting High Serum EGF Concentration in Advanced NSCLC Patients as Switch Maintenance Therapy

P2.01-33 Survival Benefit of Depleting High Serum EGF Concentration in Advanced NSCLC Patients as Switch Maintenance Therapy

CIMAvax-EGF: A New Therapeutic Vaccine for Advanced Non-Small Cell Lung Cancer Patients.

Lung cancer is the common fatal illness with the highest incidence and mortality globally. Epidermal growth factor receptor overexpression by tumor cells is associated with uncontrolled proliferation, angiogenesis, anti-apoptotic signals, metastization, and invasiveness. CIMAvax-EGF vaccine consists of a chemical conjugate of the EGF with the P64 protein derived from the Meningitis B bacteria and Montanide ISA 51, as adjuvant. The vaccine is projected to induce antibodies against EGF that results in EGF withdrawal. CIMAvax-EGF demonstrated to be safe and immunogenic in advanced non-small cell lung cancer (NSCLC) patients. The efficacy study was an open-label, multicentric Phase III clinical trial, which enrolled 405 advanced NSCLC patients. Patients with proven stage IIIB/IV NSCLC, who had completed four to six cycles of chemotherapy (CTP) were randomized to receive CIMAvax-EGF or best supportive care. CIMAvax-EGF resulted in a significantly larger overall survival in patients receiving at least four doses. High EGF concentration at baseline was a good predictive biomarker of the vaccine activity and a poor prognostic biomarker for the non-treated population. The proportion of CD8+CD28− cells, CD4 cells, and the CD4/CD8 ratio after first-line CTP was also associated with CIMAvax-EGF clinical benefit. After completing the Phase III, a Phase IV trial was done where the vaccine was administered in primary care units. Administering the vaccine at primary care institutions granted better access and treatment compliance. Safety was confirmed. Several clinical trials are currently ongoing to validate EGF as a predictive biomarker of CIMAvax-EGF efficacy.

Blocking the PI3K pathway or the presence of high concentrations of EGF inhibits the spontaneous activation of ovine primordial follicles in vitro

Blocking the PI3K pathway or the presence of high concentrations of EGF inhibits the spontaneous activation of ovine primordial follicles in vitro

EGF-based cancer vaccine: Optimizing predictive and surrogate biomarkers.

3013 Background: EGFR is overexpressed in many epithelial tumors. EGF is one of the most important growth factors that stimulates EGFR, in a paracrine way. CIMAvax-EGF is a therapeutic cancer vaccine intended to induce antibodies against EGF. It is composed by recombinant EGF conjugated to P64 from N.Meningitides as a carrier and Montanide, as adjuvant. Methods: Two controlled trials were done in advanced NSCLC and castration-resistant prostate cancer patients (CRPC). A multicentric, randomized Phase III trial was designed to assess the efficacy, immunogenicity, and safety of CIMAVAx-EGF in advanced NSCLC patients. Patients with histology- or cytology-proven NSCLC at stage IIIB/ IV were enrolled. All subjects received 4 platinum-based cycles before entering the study. On the other hand, a multicentric, randomized Phase II trial was designed to assess the immunogenicity and safety of the vaccine in CRPC patients. The EGF cancer vaccine was administered in combination with mitoxantrone and prednisone. Results: 405 patients bearing NSCLC and 200 men with CRPC were enrolled in the 2 trials. In both studies the vaccine was immunogenic. Antibody titers against EGF increased with vaccination and EGF concentration in sera showed a fast reduction after immunization. The anti-EGF antibody response was directly correlated with overall survival. CIMAVax-EGF was safe and most prevalent adverse events were grade 1-2 injection site pain, fever, headache, nausea, vomiting, and chills. The vaccine significantly increased the survival of the NSCLC patients while it did not augment significantly the overall survival of the CRPC patients. In both studies, EGF concentration was measured at baseline and it was found to be much higher than in normal subjects. High EGF concentration predicted greater benefit after vaccination. On the contrary, NSCLC and prostate control patients with high levels of EGF had a poorer outcome. Conclusions: Antibody response against EGF is a surrogate marker of survival. High EGF concentration might be a predictive marker of vaccine efficacy and a poor prognostic biomarker for non-vaccinated NSCLC and CRPC patients. The predictive and prognostic value of the EGF concentration will be validated prospectively. Clinical trial information: IIC EC 081.

EZ spheres: A stable and expandable culture system for the generation of pre-rosette multipotent stem cells from human ESCs and iPSCs

We have developed a simple method to generate and expand multipotent, self-renewing pre-rosette neural stem cells from both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) without utilizing embryoid body formation, manual selection techniques, or complex combinations of small molecules. Human ESC and iPSC colonies were lifted and placed in a neural stem cell medium containing high concentrations of EGF and FGF-2. Cell aggregates (termed EZ spheres) could be expanded for long periods using a chopping method that maintained cell–cell contact. Early passage EZ spheres rapidly down-regulated OCT4 and up-regulated SOX2 and nestin expression. They retained the potential to form neural rosettes and consistently differentiated into a range of central and peripheral neural lineages. Thus, they represent a very early neural stem cell with greater differentiation flexibility than other previously described methods. As such, they will be useful for the rapidly expanding field of neurological development and disease modeling, high-content screening, and regenerative therapies based on pluripotent stem cell technology.

Suppression of EGFR endocytosis by dynamin depletion reveals that EGFR signaling occurs primarily at the plasma membrane

The role of endocytosis in the control of EGF receptor (EGFR) activation and cell signaling was explored by using mouse fibroblasts in which dynamin was conditionally depleted. Dynamin is a GTPase shown to play an important role in the control clathrin mediated endocytosis of EGFR and other cell surface receptors. In this report, we demonstrate that EGF binding activity and the display of high and low affinity EGFRs on the cell surface are not affected by dynamin depletion. By contrast, dynamin depletion leads to a strong inhibition of EGFR endocytosis, robust enhancement of EGFR autophosphorylation and ubiquitination, and slower kinetics of EGFR degradation. Surprisingly, MAPK stimulation induced by either low or high EGF concentrations is not affected by dynamin depletion. While a similar initial Akt response is detected in control or dynamin depleted fibroblasts, a somewhat more sustained Akt stimulation is detected in the dynamin depleted cells. These experiments demonstrate that dynamin-mediated endocytosis leads to attenuation of EGFR activation and degradation and that stimulation of the MAPK response and Akt activation are primarily mediated by activated EGFR located in the plasma membrane.

A Look at Transactivation of the EGF Receptor by Angiotensin II

A growing number of clinical and experimental studies show that the renin–angiotensin system (RAS) is involved in the progression of CKD.1 In fact, pharmacological inhibitors of the RAS such as angiotensin-converting enzyme inhibitors and angiotensin II (Ang II) type I receptor (AT1R) blockers are the most reliable and effective tools known to attenuate progression of parenchymal changes in CKD.1 However, despite the clinical evidence, understanding how RAS inhibition exerts its renoprotective function at the molecular level remains unclear.

EGFR induces expression of IRF‐1 via STAT1 and STAT3 activation leading to growth arrest of human cancer cells

Recently, we reported that epidermal growth factor receptor (EGFR) induce expression of a module of genes known to be inducible by interferons and particularly interferon-gamma. Here we show that the module is tightly regulated by EGFR in the 2 human cancer cell lines that overexpress EGFR, A431 and HN5. The module of genes included the tumor suppressor IRF-1, which was used as a prototypical member to further investigate the regulation and function of the module. Ligand-activated EGFR induce expression of IRF-1 via phosphorylation of STAT1 and STAT3. In contrast, cells expressing the constitutively active cancer specific receptor EGFRvIII are unable to mediate phosphorylation of these STATs and thereby incapable of inducing IRF-1. We also demonstrate that IRF-1 is expressed in an EGF dose-dependent manner, which correlates with inhibition of cell proliferation, and that the regulation of IRF-1 is partially dependent on intracellular Src family kinase activity. Treatment with the dual specific Abl/c-Src kinase inhibitor AZD0530 significantly reduces the growth inhibitory effect of high EGF concentrations, signifying that EGFR induced IRF-1 is responsible for the observed growth inhibition. In addition, we show that media from these EGF treated cancer cells upregulate the activation marker CD69 on both B-cells and T-cells in peripheral blood. Taken together, these results suggest that cells acquiring sustained high activity of oncogenes such as EGFR are able to activate genes, whose products mediate growth arrest and activate immune effector cells, and which potentially could be involved in alerting the immune system in vivo leading to elimination of the transformed cells.

EGF‐Induced Activation of the EGF Receptor Does Not Trigger Mobilization of Caveolae

Caveolae-dependent endocytosis has recently been proposed in the uptake of EGF receptor (EGFR) at high concentrations of ligand. Consistently, upon incubation of HEp2 and HeLa cells with methyl-beta-cyclodextrin, we observed a small inhibitory effect on endocytosis of ligated EGFR in HEp2 cells. However, immunoelectron microscopy showed the same relative amount of bound EGF localizing to caveolae on incubation with high and low concentrations of EGF, not supporting rapid recruitment of EGFR to caveolae. Live-cell microscopy furthermore demonstrated that incubating HEp2 cells with high concentrations of EGF did not increase the mobility of caveolae. By RNA-interference-mediated knockdown of clathrin heavy chain in HEp2 and HeLa cells, we found that endocytosis of EGFR was efficiently inhibited both at high and low concentrations of EGF. Our results show that caveolae are not involved in endocytosis of EGF-bound EGFR to any significant degree and that high concentrations of EGF do not further mobilize caveolae.

Farnesyltransferase Inhibitors and Human Malignant Pleural Mesothelioma: A First-Step Comparative Translational Study

It is known that the potential clinical use of farnesyltransferase inhibitors (FTI) could be expanded to include cancers harboring activated receptor tyrosine kinases. Approximately 70% of malignant pleural mesotheliomas (MPM) overexpress epidermal growth factor receptors (EGFR) and a subset express both EGFR and transforming growth factor alpha (TGF-alpha), suggesting an autocrine role for EGFR in MPM. We checked on MPM cells (10 human cell lines, 11 primary cultures obtained by human biopsies, and 7 short-term normal mesothelial cell cultures) concerning the following: (a) the relative overexpression of EGFR (Western blotting, flow cytometry, immunohistochemistry), (b) the relative expression of EGFR ligands (EGF, amphiregulin, TGF-alpha, ELISA), (c) the relative increase of the activated form of Ras (Ras-bound GTP) after EGF stimulation (Ras activation assay), (d) the efficacy of five different FTIs (HDJ2 prenylation, cell cytotoxicity, and apoptosis using ApopTag and gel ladder). EGFR was overexpressed in MPM cells compared with normal pleural mesothelial cells in equivalent levels as in non-small cell lung cancer cells A459. MPM cells constitutively expressed EGFR ligands; however, Ras activation was attenuated at high EGF concentrations (100 ng/mL). Growth of MPM cells was substantially not affected by treatment with different FTIs (SCH66336, BMS-214662, R115777, RPR-115135, and Manumycin). Among these, BMS-214662 was the only one moderately active. BMS-214662 triggered apoptosis in a small fraction of cells (not higher than 30%) that was paralleled by a slight decrease in the levels of TGF-alpha secreted by treated MPM cells. Our data highlighted the concept that the same signaling pathway can be regulated in different ways and these regulations can differ between different cells of different origin.

Overexpression of the human c-erbB (EGF receptor) protooncogene fails to alter the lifespan or promote tumourigenic growth of normal and SV40-transformed human fibroblasts

c-erbB was introduced into normal human fibroblasts, MRC-5, which expressed normal levels of EGF receptor and in a SV40-transformed cell line, MRC-5V1, derived from them, which expressed markedly reduced levels of EGF receptor mRNA. MRC-5 overexpressing c-erbB, responded mitogenically to EGF. However, addition of high EGF concentrations markedly reduced DNA synthesis and resulted in the inhibition of cellular growth. In contrast, MRC-5V1 exhibited an increase in DNA synthesis in an EGF-dependent manner which was enhanced by overexpression of c-erbB. These cells, unlike MRC-5, also produced TGF alpha, an EGF receptor ligand which is often associated with cellular transformation. Ligand-activation of EGF receptor did not alter the lifespan, induce focus formation or anchorage-independence of MRC-5 and all the cell types remained non-tumourigenic in nude mice. However, c-erbB induced the expression of tPA, c-jun and junB in both MRC-5 and MRC-5V1. The data suggest that overexpression and activation of c-erbB is unlikely to play a role in immortalisation of human diploid fibroblasts but it may contribute to cellular transformation.

P53 TUMOR-SUPPRESSOR GENE AND CYCLIC ADENOSINE-MONOPHOSPHATE IN BREAST AND ESOPHAGEAL CANCER ARE REGULATED THROUGH HIGH-CONCENTRATION OF EPIDERMAL GROWTH-FACTOR

To reveal the molecular mechanism of the growth-inhibitory effects of high concentrations of EGF, accumulations of p53 protein and p21ras expression in MX-1 and UM-1 breast cancer and ES-4 esophageal cancer transplanted into nude mice were investigated after local injections of 2 mug of EGF or 2 mug of TGF-beta. The accumulation of p53 protein and expression of p21ras were determined by using the methods of Western immunoblotting and p53 mutant selective quantitative ELISA assay. p53 mutation was investigated by using the PCR analysis and DNA sequencing. Contents of intra-cellular-cAMP of these tumors were also determined by radioimmunoassay. Results showed that the high concentration of EGF induced the accumulations not only of wild type p53 protein, but also of mutant p53 protein in these tumors growth-inhibited by EGF. In ES-4 esophageal cancer, 2 mug of EGF induced the up-regulation of p53 and the down-regulation of p21ras. On the contrary, 2 mug of TGF-beta induced the down-regulation of p53 and the up-regulation of p21ras in UM-1 human breast cancer. The point mutation of p53 gene was found at codon 181 contained C to T transversions (Amino acid switch: Arg --> Cys) in ES-4 esophageal cancer. The accumulations of p53 proteins were also associated with the down regulated intra-cellular-cAMP induced by high concentrations of EGF. These results indicate that p53 gene and ras gene are involved in the signal pathway of EGF and that the p53 gene may have some important role as a negative growth factor by suppressing ras oncogene in the mechanism of tumor growth inhibition through the high concentration of EGF.

Atypical receptor-mediated signal transduction events in the EGF-dependent growth-inhibited cell line, MDA-468.

It is now generally considered that early signalling from tyrosine kinases that induce mitogenesis is initiated through the formation of heteromeric complexes consisting of the autophosphorylated tyrosine kinase and a number of tyrosylphosphorylated proteins, including phospholipase C-gamma (PLC-gamma) and GTPase activating protein (GAP). However, since much of this work has been performed on proliferative, chimeric cell lines expressing heterologous receptor molecules, we examined the nature of the epidermal growth factor receptor (EGFR) signalling complex formation in the human breast cancer cell line, MDA-468. This cell line has an amplified, native EGFR gene, correspondingly overexpresses the EGFR, and its growth in culture is inversely related to the EGF concentration. Our results indicate that in MDA-468 cells, both the EGFR and PLC-gamma are phosphorylated on tyrosine residues and can be co-immunoprecipitated. This occurs at both high and low EGF concentrations regardless of the proliferative endpoint. The molecular association is correlated with a significant increase in total inositol phosphates formed in response to the growth factor treatment. In contrast, however, there is no evidence that GAP is either phosphorylated on tyrosine residues or forms a complex with the activated EGFR in EGF-treated MDA-468 cells. These observations suggest that as a model for growth factor action, the formation of heteromeric protein signalling complexes may demonstrate considerable diversity depending upon both cell type and physiology.

Signal transduction by the epidermal growth factor receptor is attenuated by a COOH-terminal domain serine phosphorylation site.

It has been proposed that the acute desensitization of epidermal growth factor receptor (EGF-R) function can be accounted for, in part, by the effect of EGF to increase phosphorylation of the receptor at Ser1046/7 (Countaway, J.L., Nairn, A.C., and Davis, R.J. (1992) J. Biol. Chem. 267, 1129-1140). Here, we show that the mutational removal of this phosphorylation site causes an activation of EGF-R function and a potentiation of signal transduction. The mechanism of potentiation results from 1) defective down-regulation of the EGF-R when cells are incubated with high concentrations of EGF; and 2) increased EGF-stimulated tyrosine phosphorylation. The increased EGF-stimulated phosphorylation is associated with an alteration of the apparent specificity of tyrosine phosphorylation and is independent of the down-regulation defect. Together, these data strongly support the hypothesis that Ser1046/7 is a biologically significant site of regulatory phosphorylation of the EGF-R.

Anomalous binding of epidermal growth factor to A431 cells is due to the effect of high receptor densities and a saturable endocytic system.

This study was conducted to determine how extraordinarily high numbers of epidermal growth factor receptors (EGF-R) affected the binding and internalization of EGF in the transformed cell line A431. I found that at low EGF concentrations, the kinetics of binding behaved as a nonsaturable, first-order process showing no evidence of multiple-affinity classes of receptors. However, EGF dissociation rates were strongly dependent on the degree of receptor occupancy in both intact cells and isolated membranes. This occupancy-dependent dissociation appears to be due to diffusion-limited binding. EGF-induced receptor internalization was rapid and first order when the absolute number of occupied receptors was below 4 x 10(3) min-1. However, at higher occupancies the specific internalization rate progressively declined to a final limiting value of 20% normal. The saturation of EGF-R endocytosis was specific since internalization of transferrin receptors was not affected by high concentrations of either transferrin or EGF. Saturation of EGF-R endocytosis probably involves a specific component of the endocytic pathway since fluid phase endocytosis increased coordinately with EGF-R occupancy. I conclude that there are several aspects of EGF-R dynamics on A431 cells are neither similar to the behavior of EGF-R in other cell types nor similar to the reported behavior of other hormone receptors. Although A431 cells have an extraordinary number of EGF-R, they do not seem to have corresponding levels of at least two other crucial cell surface components: one that mediates EGF-induced rapid receptor internalization and one that attenuates EGF-induced membrane responses. These factors, in addition to the presence of diffusion-limited binding at low EGF concentrations, are probably responsible for the appearance of multiple-affinity classes of receptors in this cell type.

The source of urinary epidermal growth factor in humans

To clarify the source of human urine EGF, we studied EGF renal clearance in 20 healthy, young adult subjects. Immunoreactive EGF was measured hourly in EDTA plasma, heparin plasma, serum and urine of 12 males and 8 females during a 3 h study period. Plasma and urine creatinine and creatinine clearance were measured and calculated hourly. Mean (and SEM) creatinine clearance was similar in males and females (118 +/- 12 vs 105 +/- 6 ml/min). EGF was not detectable in plasma, whereas relatively high levels were measured in serum (2.5 +/- 0.25 vs 1.5 +/- 0.18 ng/ml in males and females respectively p less than 0.05). Urine EGF excretion averaged 1641 +/- 233 ng/h in males and 1507 +/- 191 ng/h in females (p greater than 0.05). A significant correlation was observed between urine creatinine and urine EGF concentrations in both male (r = 0.98, p less than 0.01) and female (r = 0.94, p less than 0.01) subjects. EGF immunoreactivity in urine and serum eluted from G-75 sephadex columns similarly to recombinant 6000 Mr hEGF. Urine excretion of EGF approximated 1.5 micrograms/h or 25 ng/mg creatine. The high concentrations of EGF found in urine in the face of non-detectable levels of EGF in plasma favor the hypothesis that EGF in urine is derived from kidney synthesis and secretion. The significant positive correlation between urine creatinine and urine EGF suggests a functional correlation between glomerular filtration and the process of tubular EGF excretion.

Mechanism of synergistic induction of DNA synthesis by epidermal growth factor and tumor promoters.

Evidence is presented to support our previously proposed hypothesis that the hyperplastic effect of tumor promoters is related to their ability to alter existing physiological levels of growth factors in target tissues. Epidermal growth factor and phorbol ester tumor promoters acted synergistically at low (0.001-0.05 ng/ml) but not high (greater than 0.1 ng/ml) EGF concentrations to induce DNA synthesis in cultured Rat-1 fibroblast cells. The degree of synergism correlated with the tumor-promoting ability of the compound. The tumor promoters decreased 125I-EGF binding to cellular receptors in a dose-dependent manner that also correlated with the tumor-promoting ability of the compound. The inhibition of EGF binding by phorbol ester compounds resulted in a decrease in the amount of EGF degraded as compared to control cultures. At limiting EGF concentrations, the sparing of EGF degradation resulted in an increase in the amount of EGF remaining in the culture medium after 12 h of incubation and a concomitant increase in the amount of EGF bound to phorbol ester-treated cells at this time as compared to control cultures. The ability of a phorbol ester compound to alter EGF degradation and to stimulate DNA synthesis synergistically with EGF correlated with the tumor-promoting ability of the compound and occurred only a low EGF concentrations.

Internalization and processing of the EGF receptor in the induction of DNA synthesis in cultured fibroblasts: The endocytic activation hypothesis

The addition of EGF to cultured murine 3T3 cells produces a decrease in EGF binding activity with concomitant internalization and degradation of the initially bound EGF. When the EGF receptor on cultured 3T3 cells is affinity labeled with high specific activity 125I-EGF, and the fate of the affinity labeled EGF-receptor complex determined, the loss in binding activity was accounted for by receptor internalization and subsequent proteolytic processing of the EGF receptor molecules in the lysosomes. Studies of the effects of EGF concentration on EGF binding by cells, EGF-induced receptor internalization and EGF-induced stimulation of 3H-thymidine uptake into cellular DNA show that there is a direct correlation between EGF-induced receptor internalization and EGF-induced stimulation of DNA synthesis, but not between EGF binding and EGF-induced stimulation of DNA synthesis. This correlation is lost at high EGF concentrations, where stimulation of DNA synthesis is suboptimal. Optimal stimulation of DNA synthesis requires a minimum of 6 h of incubation of EGF with cells, and the suboptimal stimulation of DNA synthesis at high EGF concentration is intensified when the period of incubation of EGF with cells is less than 6 h. These data are consistent with a model of hormone signal transmission by Endocytic Activation, wherein the activation of EGF-induced processes requires constant EGF-induced internalization of receptor for a requisite 6-8 h period as an obligatory step in production of "second messenger" in the action of this hormone.