Epidermal growth factor receptor expression related to differentiation capacity in normal and transformed keratinocytes

Epidermal growth factor (EGF) and Ca2+ have been indicated to play a major role in skin development. We have used normal keratinocytes, SV40-transformed keratinocytes (SVK14) and various squamous carcinoma cell (SCC) lines as in vitro model system to study the effect of the extracellular Ca2+ concentration of EGF-receptor expression in relation to the capability of cells to differentiate. The cell lines used exhibit a decreasing capacity to differentiate in the order of keratinocytes approximately SVK14 greater than SCC-12F2 greater than SCC-15 greater than SCC-12B2 greater than SCC-4, as judged from Ca2+-ionophore-induced cornified envelope formation. Under normal Ca2+ conditions, all cell lines (except for SCC-15) exhibited two classes of EGF-binding sites. The number of low-affinity binding sites increased considerably as cells were less able to differentiate, while the apparent dissociation constant (kd) was similar in all cell lines. In contrast, the properties of high-affinity EGF binding varied in the various cell lines without a clear relationship to the degree of differentiation capacity. Lowering the extracellular Ca2+ concentration to 0.06 mM resulted in a decrease of Ca2+ ionophore-induced cornified envelope formation, demonstrating the decreased ability to differentiate under these conditions. The decreased ability to differentiate was accompanied by a marked increase in the number of EGF-binding sites, but without a change of the kd. Furthermore, no high-affinity EGF-binding sites were detectable under these conditions. Finally, addition of Ca2+ to low Ca2+-cultured cells caused a rapid decrease of EGF binding in all cell lines, most prominently in normal keratinocytes and SCC-12F2 cells. The data presented demonstrate: The combination of normal keratinocytes, SVK14 and the various SCC lines provides an attractive model system to study differentiation in vitro; EGF-receptor expression is related to the state of differentiation, both phenomena being sensitive to the external Ca2+ concentration; and EGF-receptor expression is related to the capability of cells to differentiate.     

Phorbol ester binding and protein kinase C activity in normal and transformed human keratinocytes

Normal keratinocytes, SV40-transformed keratinocytes (SVK14), and various squamous carcinoma cell (SCC) lines have been used as an in vitro model system to study the properties of phorbol ester receptor and protein kinase C expression during keratinocyte differentiation. The cell lines used exhibit a decreasing capacity to differentiate in the order of keratinocytes approximately SVK14 greater than SCC-12F2 greater than SCC-15 greater than SCC-4; moreover, all cell lines respond to a low external Ca2+ concentration by a decreased capacity to differentiate. Normal keratinocytes exhibited the highest number of phorbol ester receptors as compared to the other cell lines, while each individual cell line exhibited a higher number of phorbol ester receptors during growth under normal Ca2+ conditions as compared to cells grown under low Ca2+ conditions. The apparent dissociation constant (Kd) demonstrated only small variations in the various cell lines. In contrast, the cytoplasmic protein kinase C activity, was found to be higher in cells grown under low Ca2+ conditions than in cells grown under normal Ca2+ conditions, indicating the absence of a causal relationship between cytoplasmic protein kinase C activity and phorbol ester receptor expression. Therefore the properties of protein kinase C have been determined in more detail in normal keratinocytes and SCC-15 cells. These studies revealed differences between protein kinase C properties from the two cell lines grown under normal and low Ca2+ conditions. The differences included the effect of phorbol 12-myristate 13-acetate (PMA) on the redistribution pattern of protein kinase C between the cytoplasmic and particulate fractions as well as the activating effect of diolein in vitro on protein kinase C activity, partly purified from particulate or cytoplasmic fractions. These observations demonstrate that the functional protein kinase C activity of keratinocytes is determined by various endogenous and exogenous activators and that these activators are modulated differently in various cell lines, under various growth conditions (low Ca2+ versus normal Ca2+).     

Human esophageal carcinoma cells have fewer, but higher affinity epidermal growth factor receptors

Squamous cell carcinomas have recently been shown to contain increased numbers of epidermal growth factor (EGF) receptors. Since EGF has an important role in epithelial growth and differentiation, it is possible that modulation of its receptor may have an important role in neoplasia. In an attempt to further explore the relationship of EGF receptor expression to malignant transformation, we examined 14 squamous cell carcinoma cell lines of the esophagus for the number and affinity of EGF receptors. Seven cell lines were newly isolated by this laboratory and recently characterized. The seven additional cell lines were obtained from Japan (4 cell lines) and South Africa (3 cell lines). Surprisingly, we found that esophageal carcinomas contained lowered quantities of surface EGF receptors (2- to 100-fold) and that the affinity of the EGF receptor was increased (6- to 100-fold) when compared to normal esophageal epithelial cells. Moreover, the biologic response of esophageal carcinoma cells to EGF differed markedly from that of other squamous cell tumor cells exhibiting elevated numbers of receptors, such as A431 and SCC-15. Human esophageal carcinoma cells were maximally stimulated by the addition of 5 ng/ml of EGF, similar to normal esophageal keratinocytes, but in contrast to normal cells were not inhibited by the higher concentrations tested (up to 40 ng/ml). On the other hand, addition of any EGF to the medium (beyond that normally present in serum) was found to dramatically inhibit the growth of A431 and SCC-15 cells. Our findings indicate that squamous cell neoplasia is not dependent upon increased numbers of cell surface EGF receptors, that EGF receptor number may have a determinant role in EGF cell toxicity, and that the stimulatory response of cells to EGF may reflect a complex function of EGF receptor number, affinity, and occupancy.     

Regulation of lipid synthesis in relation to keratinocyte differentiation capacity

Cultured keratinocytes and squamous carcinoma cells provide a useful model system for studying the processes involved in the regulation of differentiation, as the differentiation capacity of the cells can be modulated experimentally by changing the extracellular calcium concentration. Furthermore, the squamous carcinoma cell lines exhibit a defect in their differentiation capacity which they express to different extents. In this paper, the effect of external lipoproteins has been studied on lipid synthesis in normal keratinocytes and three squamous carcinoma cell (SCC) lines which showed a decreasing capacity to differentiate in the order of normal keratinocytes greater than SCC-12F2 greater than SCC-15 greater than SCC-4. The ability of the cells to form cornified envelopes was taken as a measure of differentiation capacity. The rate of total lipid synthesis as well as the phospholipid-neutral lipid ratio decreased in the order SCC-4 greater than SCC-15 greater than SCC-12F2 greater than or equal to normal keratinocytes, clearly correlating with the differentiation capacity of the cells. Because of the high rate of phospholipid synthesis and the low rate of ceramide synthesis, it is concluded that, under these in vitro conditions used, the maturation of keratinocytes proceeds to a lesser extent than that seen under in vivo conditions. In proliferating cells, in which the low-density lipoprotein (LDL) receptor is operative to a high extent, the rate of lipogenesis, especially that of neutral lipids, responded dramatically to changes of extracellular lipoprotein concentration. In the presence of lipoproteins a marked decrease of cholesterol and triacylglycerol synthesis and an increase of cholesterol ester synthesis has been observed. On the other hand, in differentiating cells lipogenesis appeared to be independent of extracellular lipoproteins, due to the absence of the LDL uptake mechanism, the only exception being the synthesis of triacylglycerols, the rate of which could be modulated to a certain extent by extracellular lipoproteins. The results presented here demonstrate a close inverse relationship between the regulation of lipogenesis by extracellular lipoproteins and the ability of the cells to differentiate.     

Binding of an antagonistic monoclonal antibody to an intact and fragmented EGF-receptor polypeptide

A murine monoclonal antibody (No. 425) raised against human A431 carcinoma cells specifically immunoprecipitates the 170,000 molecular weight epidermal growth factor (EGF)-receptor from extracts of A431 cells as well as from extracts of human placenta and cultured fibroblasts, but does not recognize the murine receptor. Binding to the external domain of the human EGF-receptor was indicated by indirect immunofluorescent staining of fixed nonpermeable cells. The antibody binds to both glyco- and aglycoreceptor forms, indicating that the epitope is a part of the polypeptide chain. Binding of the antibody to the receptor is conformation dependent; i.e., denatured receptors lacking EGF-binding activity are not recognized by the antibody. The results of antibody binding studies indicate that the epitope is closely linked to the EGF binding active site, and is common to both high- and low-affinity EGF-receptors. Interaction of this epitope with the antibody inhibits EGF binding and bioactivity, and triggers receptor down-regulation, but does not generate EGFlike kinase-stimulatory or mitogenic responses either in vitro or in vivo. The antibody was tested for its ability to bind to domain-sized fragments of the 170-kDa EGF-receptor. It can recognize both the proteolytically generated 110-kDa EGF binding peptide, and a soluble 100-kDa EGF-receptor secreted by A431 cells. This indicates that the epitope recognized this antibody retains its conformation after proteolytic separation of the EGF binding domain from the rest of the receptor molecule.     

Association of EGF and LDL receptors with the cytoskeleton of cultured keratinocytes

In this paper we demonstrate that isolated cytoskeletons of normal keratinocytes cultured under differentiation inducing conditions exhibit a high level of epidermal growth factor (EGF) binding. This binding is approximately 300% higher than the binding of intact cells. In contrast, various squamous carcinoma cell lines or normal keratinocytes cultured under differentiation retarding conditions exhibit EGF binding to isolated cytoskeletons which is around 10-20% of the binding to intact cells. Incubation of normal keratinocytes in the presence of arotinoid ethyl sulfone resulted in a marked decrease of the ability of the cells to differentiate, and a decrease of EGF binding to isolated cytoskeletons. These results suggest a close relationship between the differentiation capacity of the cells and the presence of cytoskeleton-associated EGF receptors. Similar results were obtained for low density lipoprotein (LDL) binding.     

Isolation and characteristics of monoclonal antibodies to the external domain of the EGF receptor in human A431 epidermoid carcinoma

The monoclonal antibody to the epidermal growth factor (EGF) receptor was generated after fusion of PAI myeloma cells with immunized BALB/c mouse spleen cells, using intact A431 epidermoid carcinoma cells as an immunogen. The antibody, denoted 5A9, is an IgG, which recognizes a protein with molecular mass 170 kDa during immunoblot analysis, immunoprecipitates phosphoprotein with molecular mass 170 kDa from the membrane preparations of A431 cells, and, according to immunofluorescence experiments, is distributed in the cell similar to the EGF-rhodamine conjugate. It is concluded that the produced antibodies are specific to EGF-receptor. At the same time the 5A9 (50 nM) do not compete with EGF for binding with high and low affinity receptors. They fail to induce internalization of the EGF-receptor and do not exert influence on intracellular degradation of EGF-receptor. Monoclonal antibodies 5A9 are also unable to inhibit the EGF-induced protein kinase activity of the receptor and do not stimulate protein kinase activity by themselves. Thus, the prepared monoclonal antibodies can be used to register the EGF-receptor cellular localization without affecting biologic activity of the receptor.     

Proliferating, transformed keratinocytes cultured under low Ca2+ conditions exhibit high-affinity epidermal growth factor receptors

In a previously published report (Exp. Cell. Res. 161:421 (1985] we have demonstrated that cultured normal and transformed keratinocytes exhibit two classes of EGF binding sites after growth under normal Ca2+ conditions but only low-affinity binding sites after growth under low Ca2+ conditions. Here we demonstrate the presence of high-affinity binding sites in transformed keratinocytes grown under low Ca2+ conditions, using a specific monoclonal anti EGF-receptor antibody.     

Pseudomonas exotoxin A-epidermal growth factor (EGF) mutant chimeric protein as an indicator for identifying amino acid residues important in EGF-receptor interaction.

Epidermal growth factor (EGF) was fused to the carboxyl end of a modified pseudomonas exotoxin A that has its toxin binding domain deleted. This chimeric toxin designated as PE(delta Ia)-EGF kills A431 cells through the EGF receptor-mediated pathway. In this study, we used a random mutagenesis approach to make point mutations on EGF, followed by replacing the wild type EGF in PE(delta Ia)-EGF with these EGF mutants. We have constructed 14 different PE(delta Ia)-EGFmutants, and examined their EGF receptor binding activity as well as their cytotoxicity to A431 cells. Our results showed that individual mutations of Val19 to Glu and Val34 to Asp in the EGF domain of PE(delta Ia)-EGFmutants resulted in an increase in the binding affinity to EGF receptor and cytotoxicity to A431 cells. On the other hand, individual mutations of His16 to Asp and Gly18 to Ala in the EGF domain of PE(delta Ia)-EGFmutants lead to a decrease in the binding affinity to EGF receptor and cytotoxicity to A431 cells. In addition, mutations of any of the cysteine residues of EGF in PE(delta Ia)-EGFmutants resulted in the loss of their binding activity to EGF receptor and a corresponding loss of their cytotoxicity. This study indicates that the cytotoxicity of PE(delta Ia)-EGFmutant to EGF receptor-bearing cells may be used as an indicator to screen mutations of EGF important in EGF-receptor interactions.     

Functional state of the epidermal growth factor-receptor complexes during their internalization in A-431 cells.

Functional state of internalized epidermal growth factor (EGF) receptor in A-431 cells has been studied. The use of photoaffinity [125I]EGF derivative allowed us to establish that inside the cell the EGF retains its connection with the receptor. With the help of polyclonal antibodies to phosphotyrosine, it has been shown that EGF-receptor complexes maintain their phosphorylated state during internalization. The internalized EGF receptor kinase as well as that localized in the plasma membrane appeared to be able to phosphorylate synthetic peptide substrate introduced into the cell.     

Comparison of the effect of the epidermal growth factor (EGF) and its cyanogen bromide derivative on EGF receptor phosphorylation and uridine uptake in a cell culture

In contrast to the intact EGF, a cyanogen bromide derivative of EGF (EGF-CNBr) does not induce an increase in uridine phosphorylation rate in 3T3 cells, the ability of the EGF-CNBr to stimulate autophosphorylation of the EGF-receptor in A-431 cells being reserved. EGF and EGF-CNBr were used in concentrations promoting their equivalent binding with EGF receptor in both the series of experiments, which was necessary because of a decreased affinity to binding EGF-CNBr. Thus, the EGF-induced receptor autophosphorylation was not enough for uridine kinase activation. The differences between EGF and EGF-CNBr cellular processing made it possible to discuss potential ways of uridine-kinase activity regulation during the early period of stimulation of quiescent cell cultures.     

Structure and localization of genes encoding aberrant and normal epidermal growth factor receptor RNAs from A431 human carcinoma cells.

A431 cells have an amplification of the epidermal growth factor (EGF) receptor gene, the cellular homolog of the v-erb B oncogene, and overproduce an aberrant 2.9-kilobase RNA that encodes a portion of the EGF receptor. A cDNA (pE15) for the aberrant RNA was cloned, sequenced, and used to analyze genomic DNA blots from A431 and normal cells. These data indicate that the aberrant RNA is created by a gene rearrangement within chromosome 7, resulting in a fusion of the 5' portion of the EGF receptor gene to an unidentified region of genomic DNA. The unidentified sequences are amplified to about the same degree (20- to 30-fold) as the EGF receptor sequences. In situ hybridization to chromosomes from normal cells and A431 cells show that both the EGF receptor gene and the unidentified DNA are localized to the p14-p12 region of chromosome 7. By using cDNA fragments to probe DNA blots from mouse-A431 somatic cell hybrids, the rearranged receptor gene was shown to be associated with translocation chromosome M4.     

Demonstration of an intact complex of epidermal growth factor and its receptor in the endosomes of A-431 cells

The compartmentalization of the epidermal growth factor (EGF) receptors in A-431 cells was studied using centrifugation of the microsomal fraction of these cells in continuous Percoll gradient. The existence of an intact (non-degraded) EGF receptor in plasma membrane and endosome fraction was demonstrated by electrophoretic analysis of in vitro phosphorylated Percoll fractions. No phosphorylated receptor was revealed in lysosomal fraction by this method. The existence of non dissociated EGF-receptor complexes in intracellular compartments 30 minutes after the start of internalization was proven using a synthesized photoreactive labeled EGF derivative (125I-EGF-SANAH). The removing of pH gradient in organellar membranes by 10 mkM of monensin did not affect dissociation from its receptor. The data obtained proved the existence of non-dissociated and non-degraded EGF-receptor complexes in the endosomal compartment of A-431 cells.     

Analysis of morphology and receptor metabolism in clonal variant A431 cells with differing growth responses to epidermal growth factor

Human epidermoid carcinoma A431 cell clones have been obtained whose growth is inhibited, stimulated, or unaffected by epidermal growth factor (EGF). In clones exhibiting each type of growth response, EGF induced similar morphologic changes consisting of aggregation of cells into dense clusters with baring of large areas of the culture dish. The similarity of the clones' morphologic responses, despite their differing growth responses, indicates that the effects of EGF on morphology are distinct from effects on growth. Cells whose growth was inhibited by EGF contained high numbers of EGF receptors, whereas the concentration of EGF receptors was reduced in cells whose growth was stimulated or unaffected by EGF. There were, however, no consistent differences in EGF receptor concentrations between stimulated or null clones. Cells that exhibited each type of growth response displayed similar rates of EGF binding to receptors, rates of internalization of EGF, and rates and extent of EGF-induced receptor down-regulation. Changes in EGF-stimulated tyrosine-specific protein kinase activity paralleled changes in EGF receptors, both between clones and upon down-regulation. These studies indicate that a reduction in the concentration of EGF receptors in A431 cells allows escape from the growth inhibitory effects of EGF, but suggest that the pattern of growth response depends on biochemical events subsequent to EGF-receptor metabolism and activation of tyrosine-specific protein kinase.     

Effects of epidermal growth factor on transferrin receptor phosphorylation and surface expression in malignant epithelial cells

The transferrin (Tf) receptor is a major transmembrane protein which provides iron for normal and malignant cell growth. Epidermal growth factor (EGF) has been reported to rapidly and transiently alter the number of surface Tf receptors in normal and transformed epithelial cells. To investigate mechanisms of EGF-induced changes in surface Tf display, EGF effects on surface Tf receptors were compared in two cell lines which differ in their number of EGF receptors and growth responses to EGF. In cloned A431 cells with high receptor numbers which are growth-inhibited by EGF, EGF caused a 50% decrease in Tf receptor expression after 30 min. In contrast, EGF induced a rapid, transitory increase (within 5 min) in the number of surface Tf receptors on KB carcinoma cells which returned to basal levels by 15 min. The observed changes in Tf receptor display were due to altered receptor distribution and not changes in ligand affinity or total cellular transferrin receptor pools. Anti-EGF receptor monoclonal antibody blocked effects of EGF on transferrin receptor expression. Since the antibody is internalized and causes EGF receptor down-regulation, effects on transferrin receptor expression were independent of these events. EGF-induced alterations in Tf receptor display occurred even when cells were pretreated with colchicine, suggesting that changes in surface Tf binding were not mediated by cytoskeletal components. Na orthovanadate, which mimics some early cellular effects of EGF, duplicated EGF's effects on A431 Tf receptors, but had no effect on KB cells, suggesting these responses occur by differing mechanisms. To determine whether EGF caused changes in Tf receptor phosphorylation, 32P-labelled Tf receptors were immunoprecipitated after EGF treatment. After exposure to EGF, A431 cells showed no change in Tf phosphorylation, but KB cells showed a transient, 6-fold increase in transferrin receptor phosphorylation on serine residues. In both A431 and KB cells, phorbol ester (PMA) also increased phosphorylation on transferrin receptors, but had little effect on surface Tf receptor expression. In malignant cell lines, EGE induces rapid, variable changes in transferrin receptor expression and phosphorylation which differ from the effects of PMA. These early responses to EGF appear to differ with the cell type and correlate poorly with alterations in Tf receptor phosphorylation. These results suggest Tf receptor phosphorylation does not regulate Tf receptor display in all cells.     

Enhancement of calcium uptake and phosphatidylinositol turnover by epidermal growth factor in A-431 cells

Epidermal growth factor (EGF) stimulates the incorporation of 32Pi and [3H]inositol into phosphatidylinositol (5-10-fold) in A-431 cells. EGF also stimulates the incorporation of 32Pi into phosphatidic acid (up to 10-fold). These effects are attributed to an acceleration of the turnover of phosphatidylinositol as a consequence of the binding of EGF to its membrane receptor. The extent of the phosphatidylinositol response to EGF parallels the extent of hormone binding. The phosphatidylinositol response to EGF appears to be dependent on an influx of calcium since (a) external calcium is required for the enhancement of phosphatidylinositol turnover, (2) the accumulation of 45Ca by A-431 cells is stimulated by EGF, (3) blockage of calcium influx with LaCl3 inhibits stimulation of phosphatidylinositol turnover, and (4) calcium influx via ionophore A23187 is sufficient to stimulate phosphatidylinositol turnover. Since the binding, internalization, and degradation of 125I-labeled EGF in A-431 cells are unaffected by the omission of calcium from the medium, external calcium and phosphatidylinositol turnover are not necessary for the internalization and degradation of the EGF-receptor complex.     

Epidermal growth factor-induced activation and translocation of c-Src to the cytoskeleton depends on the actin binding domain of the EGF-receptor

In the epidermal growth factor (EGF)-receptor signal transduction cascade, the non-receptor tyrosine kinase c-Src has been demonstrated to become activated upon EGF stimulation. In this paper we show that c-Src associates with the cytoskeleton and co-isolates with actin filaments upon EGF treatment of NIH-3T3 cells transfected with the EGF receptor. Immunofluorescence studies using CLSM show colocalization of F-actin and endogenous c-Src predominantly around endosomes and not on stress fibers and cell–cell contacts. Stimulation of EGF receptor-transfected NIH-3T3 cells with EGF induces an activation and translocation of c-Src to the cytoskeleton. These processes depend upon the presence of the actin binding domain of the EGF-receptor since in cells that express EGF-receptors lacking this domain, EGF fails to induce an activation and translocation to the cytoskeleton of c-Src. These data suggest a role for the actin binding domain of the EGF-receptor in the translocation of c-Src.     

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.