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.     

Monoclonal antibodies against the human epidermal growth factor receptor from A431 cells. Isolation, characterization, and use in the purification of active epidermal growth factor receptor

A431 cells have been used as an immunogen for generating monoclonal antibodies against the epidermal growth factor (EGF) receptor. Two immunoglobulin M and eight immunoglobulin G3 anti-EGF receptor antibodies were cloned. All ten antibodies immunoprecipitated biosynthetically labeled mature A431 cell EGF receptor and were able to recognize the receptor in Western blotting. However, none of the antibodies immunoprecipitated precursor polypeptides of the A431 cell EGF receptor, neither did they recognize EGF receptors from human foreskin fibroblasts, human placenta, nor a human-mouse hybrid cell expressing EGF receptor. The antibodies were found to bind to glycolipids from A431 cells and it was shown that the determinant involved was the blood group A antigen. It appears that this determinant is present on both the EGF receptor and glycolipids of A431 cells but is not expressed on EGF receptors from other human cells tested. One of the monoclonal antibodies raised was used for immunoaffinity purification of the EGF receptor. The procedure took advantage of the carbohydrate nature of the antigenic determinant by employing sugar-specific elution. The mild conditions permitted the purification of A431 cell EGF receptor (70-80% pure) that possessed an intrinsic EGF-stimulated tyrosine kinase activity with a specific activity of about 20 nmol/min/mg.     

Epidermal growth factor receptor metabolism and protein kinase activity in human A431 cells infected with Snyder-Theilen feline sarcoma virus or harvey or Kirsten murine sarcoma virus.

When human A431 cells, which carry high numbers of epidermal growth factor (EGF) receptors, are exposed to EGF, the total content of phosphotyrosine in cell protein is increased, the EGF receptor becomes phosphorylated at tyrosine, and new phosphotyrosine-containing 36,000- and 81,000-dalton proteins are detected. We examined the properties of A431 cells infected with Snyder-Theilen feline sarcoma virus, whose transforming protein has associated tyrosine protein kinase activity, and Harvey and Kirsten sarcoma viruses, whose transforming proteins do not. In all cases, the infected cells were more rounded and more capable of anchorage-independent growth than the uninfected cells. EGF receptors were assayed functionally by measuring EGF binding and structurally by metabolic labeling and immunoprecipitation. In no case did infection appear to alter the rate of EGF receptor synthesis, but infection reduced EGF receptor stability by about 50% for cloned Harvey sarcoma virus-infected cells and by 80% for cloned feline sarcoma virus-infected cells. The corresponding reductions in EGF binding were 70 and 90%, respectively. The proteins of feline sarcoma virus-infected A431 cells contained an increased amount of phosphotyrosine, and the 36,000- and 81,000-dalton phosphoproteins were detected. The EGF receptor was not detectably phosphorylated at tyrosine, however, unless the cells were exposed to EGF. The Harvey and Kirsten sarcoma virus-infected cells did not exhibit elevated levels of phosphotyrosine either in the total cell proteins or in the EGF receptor, nor were the 36,000- and 81,000-dalton proteins detectable. However, these phosphoproteins were found in the infected cells after EGF treatment. Thus, all of the infected A431 cells exhibited reduced EGF binding and increased degradation of EGF receptors, yet their patterns of protein phosphorylation were distinct from those of EGF-treated A431 cells.     

Prolonged induction of p21Cip1/WAF1/CDK2/PCNA complex by epidermal growth factor receptor activation mediates ligand-induced A431 cell growth inhibition

Proliferation of some cultured human tumor cell lines bearing high numbers of epidermal growth factor (EGF) receptors is paradoxically inhibited by EGF in nanomolar concentrations. In the present study, we have investigated the biochemical mechanism of growth inhibition in A431 human squamous carcinoma cells exposed to exogenous EGF. In parallel, we studied a selected subpopulation, A431-F, which is resistant to EGF-mediated growth inhibition. We observed a marked reduction in cyclin-dependent kinase-2 (CDK2) activity when A431 and A431-F cells were cultured with 20 nM EGF for 4 h. After further continuous exposure of A431 cells to EGF, the CDK2 activity remained at a low level and was accompanied by persistent G1 arrest. In contrast, the early reduced CDK2 activity and G1 accumulation in A431-F cells was only transient. We found that, at early time points (4-8 h), EGF induces p21Cip1/WAF1 mRNA and protein expression in both EGF-sensitive A431 cells and EGF-resistant A431-F cells. But only in A431 cells, was p21Cip1/WAF1 expression sustained at a significantly increased level for up to 5 d after addition of EGF. Induction of p21Cip1/WAF1 by EGF could be inhibited by a specific EGF receptor tyrosine kinase inhibitor, tyrphostin AG1478, suggesting that p21Cip1/WAF1 induction was a consequence of receptor tyrosine kinase activation by EGF. We also demonstrated that the increased p21Cip1/WAF1 was associated with both CDK2 and proliferating cell nuclear antigen (PCNA). Taken together, our results demonstrate that p21Cip1/WAF1 is an important mediator of EGF-induced G1 arrest and growth inhibition in A431 cells.     

Expression of the human EGF receptor with ligand-stimulatable kinase activity in insect cells using a baculovirus vector.

The mechanism by which the binding of epidermal growth factor (EGF) to specific cell surface receptors induces a range of biological responses remains poorly understood. An important part of the study of signal transduction in this system involves the production of sufficient native and mutant EGF receptor species for X-ray crystallographic and spectroscopic analysis. Baculovirus vectors containing the cDNA encoding the human EGF receptor protein have here been utilized to infect insect cells. This results in expression of a 155-kb transmembrane protein which is recognized by four antibodies against different regions of the human EGF receptor. Studies with tunicamycin, monensen and endoglycosidase H show the difference in size between the recombinant and the native receptor is due to alterations in glycocsylation. Studies of [125I] EGF binding shows a Kd of 2 X 10(-9) M in intact infected insect cells which falls to 2 X 10(-7) M upon detergent solubilization. The recombinant protein exhibits an EGF-stimulated tyrosine protein kinase activity and an analysis of tryptic peptides shows that the phosphate acceptor sites are similar to those of the EGF receptor isolated from A431 cells. These observations indicate that functional EGF receptor can be expressed in insect cells, and furthermore, this system can be used for large-scale production.     

Inability of anti-epidermal growth factor receptor monoclonal antibody to block "autocrine" growth stimulation in transforming growth factor-secreting melanoma cells

Mouse monoclonal antibodies to the human epidermal growth factor (EGF) receptor were raised by immunizing with plasma membrane vesicles prepared from A431 cells. This paper describes the characterization of one of the IgG anti-receptor monoclonal antibodies generated and its use to probe the role of transforming growth factor (TGF) in the autonomous growth of a melanoma cell line in culture. This antibody blocks: 1) the binding of 125I-EGF to the A431 EGF receptor; 2) the EGF stimulation of the EGF-dependent protein kinase in vitro; and 3) human fibroblast DNA synthesis and proliferation in culture. It can precipitate the EGF receptor from metabolically labeled A431 cells and human fibroblasts and these receptors have indistinguishable peptide maps. No EGF receptor could be detected by immunoprecipitation after fibroblasts were treated with EGF or conditioned medium from the melanoma cells which secrete EGF-like TGF (alpha TGF). The antibody itself did not down-regulate the receptor but could block down-regulation caused by EGF and alpha TGF. Despite its ability to block EGF-stimulated growth and down-regulation in fibroblasts, the antibody was unable to block the growth and soft agar colony formation of alpha TGF-secreting melanoma cells, nor could the antibody detect EGF receptor in these cells under the conditions developed to prevent down-regulation and lysosomal degradation of the EGF receptor. These studies suggest that these melanoma cells do not have the intact EGF receptor and that the secretion of alpha TGF by these cells plays no role in their growth in culture. The absence of receptor cannot be explained by down-regulation by secreted alpha TGF.     

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.     

Biosynthesis of the epidermal growth factor receptor in human squamous cell carcinoma lines: secretion of the truncated receptor is not common to epidermal growth factor receptor-hyperproducing cells

The biosynthesis of the EGF receptor was examined in the epidermoid carcinoma cell line A431 and five novel cell lines from human squamous cell carcinomas possessing high numbers of EGF receptors. Newly synthesized EGF receptors were visualized by labeling with [35S]methionine and immunoprecipitation with a monoclonal anti-EGF receptor antibody. In addition, the processing of the EGF receptor and its intracellular transport was analyzed by distinguishing cell surface receptors from intracellular receptors and by treating cells with inhibitors such as tunicamycin, monensin and brefeldin A. These analyses revealed that in all the tumor cell lines the EGF receptor is synthesized as a glycosylated protein of Mr 160,000 which is converted to the receptor of Mr 170,000 through posttranslational glycosylation. The receptors of Mr 160,000 and 170,000 appeared to possess high mannose type oligosaccharide chains because endoglycosidase H treatment reduced their molecular weights by approximately 30,000. A431 was the only tumor cell line studied that secreted the truncated EGF receptor of Mr 110,000. In A431 cells, the truncated EGF receptor was generated from a protein of Mr 60,000 through tunicamycin- and monensin-sensitive glycosylation. A431 cells treated with monensin secreted the truncated receptor as a Mr 95,000 form.     

Inhibition of NF-kappaB sensitizes A431 cells to epidermal growth factor-induced apoptosis,whereas its activation by ectopic expression of RelA confers resistance

Epidermal growth factor (EGF) is a well known mitogen, but it paradoxically induces apoptosis in cells that overexpress its receptor. We demonstrate for the first time that the EGF-induced apoptosis is accelerated if NF-kappaB is inactivated. To inactivate NF-kappaB, human epidermoid carcinoma cells (A431) that overexpress EGF receptor were stably transfected with an IkappaB-alpha double mutant construct. Under the NF-kappaB-inactivated condition, A431 cells were more sensitive to EGF with decreased cell viability and increased externalization of phosphatidylserine on the cell surface, DNA fragmentation, and activation of caspases (3 and 8 but not 9), typical features of apoptosis. These results were further supported by the potentiation of the growth inhibitory effects of EGF by chemical inhibitors of NF-kappaB (curcumin and sodium salicylate) and the protective role of RelA evidenced by the resistance of A431-RelA cells (stably transfected with RelA) to EGF-induced apoptosis. EGF treatment or ectopic expression of RelA in A431 cells induced DNA binding activity of NF-kappaB (p50 and RelA) and the expression of c-IAP1, a downstream target of NF-kappaB. A431-RelA cells exhibited spontaneous phosphorylation of Akt (a downstream target of phosphatidylinositol 3-kinase and regulator of NF-kappaB) and EGF treatment stimulated it further. Blocking this basal Akt phosphorylation with LY294002, an inhibitor of phosphatidylinositol 3-kinase, did not affect their viability but blocking of EGF-induced phosphorylation of Akt sensitized the otherwise resistant A431-RelA cells to EGF-mediated growth inhibition. Our results favor an anti-apoptotic role for NF-kappaB in the regulation of EGF-induced apoptosis.     

Loss of three major auto phosphorylation sites in the EGF receptor does not block the mitogenic action of EGF

The EGF receptor cDNA has been transfected into receptor-negative Chinese hamster ovary (CHO) cells. A mutant cell line (CHO 11) was isolated that expresses a receptor of lower molecular weight than the EGF receptor from A431 cells (150,000 daltons compared to 170,000 daltons) and which appeared as a doublet on SDS-PAGE. By digestion of the receptor with endoglycosidase F it was shown that an altered pattern of glycosylation could not account for the smaller size of the protein, although it could explain the appearance of the CHO 11 receptor as a doublet protein. A deletion was located to the transfected cDNA and shown to involve the removal of coding sequences for the most C-terminal 20,000 daltons of the EGF receptor, which contains the three major autophosphorylation sites. Despite the loss of these sites the EGF receptor from CHO 11 cells binds EGF, demonstrates protein tyrosine kinase activity in response to EGF, and transduces a mitogenic signal. The CHO 11 receptor protein is still autophosphorylated on alternative tyrosine residues. We conclude that phosphorylation of the three tyrosines (P1, P2, and P3) in the C-terminal domain of the receptor is not required for signal transduction by the EGF receptor in these cells.     

Epidermal growth factor receptor. Characterization of a monoclonal antibody specific for the receptor of A431 cells

A monoclonal antibody to the epidermal growth factor (EGF) receptor of A431 cells was obtained after fusion of immunized BALB/c mouse spleen cells with NS-1 myeloma cells. Specific binding of the antibody to the plasma membrane of A431 cells was demonstrated by indirect immunofluorescence and electron microscopy. The antibody did not react with human KB cells, normal rat kidney cells, or Swiss 3T3 cells. The antibody is an IgG3K; it specifically immunoprecipitated a Mr approximately 170,000 protein from radiolabeled A431 cell extracts. This protein is phosphorylated in a EGF-dependent manner in intact A431 cells and in Triton X-100-solubilized plasma membranes. The specificity of the interaction of the antibody with the Mr = 170,000 protein was confirmed by electrophoretic transfer of A431 cell proteins to nitrocellulose followed by incubation with the antibody and 125I-protein A. When 125I-EGF was covalently cross-linked to its receptor, the 125I-EGF-receptor complex was specifically precipitated by the antibody. The monoclonal antibody did not inhibit the binding of 125I-EGF to its receptor in intact A431 cells and also failed to stimulate the phosphorylation of the Triton X-100-solubilized EGF receptor. The results indicate that the antibody and EGF bind to different sites on the EGF receptor. The antibody will be useful for isolating the EGF receptor in an unactivated form.     

Regulation of the epidermal growth factor receptor phosphorylation state by sphingosine in A431 human epidermoid carcinoma cells

The regulation of protein phosphorylation by sphingosine in A431 human epidermoid carcinoma cells was examined. Sphingosine is a competitive inhibitor of phorbol ester binding to protein kinase C (Ca2+/phospholipid-dependent enzyme) and potently inhibits phosphotransferase activity in vitro. Addition of sphingosine to intact A431 cells caused an inhibition of the phorbol ester-stimulated phosphorylation of two protein kinase C substrates, epidermal growth factor (EGF) receptor threonine 654 and transferrin receptor serine 24. We conclude that sphingosine inhibits the activity of protein kinase C in intact A431 cells. However, further experiments demonstrated that sphingosine-treatment of A431 cells resulted in the regulation of the EGF receptor by a mechanism that was independent of protein kinase C. First, sphingosine caused an increase in the threonine phosphorylation of the EGF receptor on a unique tryptic peptide. Second, sphingosine caused an increase in the affinity of the EGF receptor in A431 and in Chinese hamster ovary cells expressing wild-type (Thr654) and mutated (Ala654) EGF receptors. Sphingosine was also observed to cause an increase in the number of EGF-binding sites expressed at the surface of A431 cells. Examination of the time course of sphingosine action demonstrated that the effects on EGF binding were rapid (maximal at 2 mins) and were observed prior to the stimulation of receptor phosphorylation (maximal at 20 mins). We conclude that sphingosine is a potently bioactive molecule that modulates cellular functions by: 1) inhibiting protein kinase C; 2) stimulating a protein kinase C-independent pathway of protein phosphorylation; and 3) increasing the affinity and number of cell surface EGF receptors.     

cAMP-mediated modulation of signal transduction of epidermal growth factor (EGF) receptor systems in human epidermoid carcinoma A431 cells. Depression of EGF-dependent diacylglycerol production and EGF receptor phosphorylation.

While a cAMP-dependent protein kinase (protein kinase A) has been suggested to phosphorylate epidermal growth factor (EGF) receptor in vitro, both intrinsic and EGF- or potent phorbol tumor promoter-induced phosphorylation of EGF receptor were found to be depressed in human epidermoid carcinoma A431 cells by prior incubation of the cells with various protein kinase A activators (e.g. cholera toxin, forskolin, cAMP analogues, or a combination of prostaglandin E1 and 3-isobutyl-1-methylxanthine). Protein kinase A activators did not change significantly either the number of EGF receptors or their affinity for EGF. The tryptic phosphopeptide map of EGF receptors from cells treated with cholera toxin alone or cholera toxin followed by EGF revealed unique peptides whose serine phosphorylation was preferentially depressed. However, the catalytic subunit of protein kinase A phosphorylated no threonine and little serine in the EGF receptors in the plasma membranes of isolated A431 cells in vitro, while serine residues in an unidentified 170-kDa membrane protein(s) other than EGF receptor were heavily phosphorylated. Pretreatment of the cells with forskolin blocked 1,2-diacylglycerol induction by EGF; growth inhibition by nanomolar levels of EGF could be partially restored by the presence of forskolin. These results indicate that an increase in intracellular cAMP modulates the EGF receptor signal transduction system by reducing EGF-induced production of diacylglycerol without direct phosphorylation of EGF receptors by protein kinase A in A431 cells.     

Similarities and differences between the effects of epidermal growth factor and Rous sarcoma virus

We have derived a line of A431 human tumor cells infected with Rous sarcoma virus (RSV). The infected cells contain the RSV-transforming protein, pp60src, which has characteristic tyrosine specific protein kinase activity. As in other RSV-transformed cells, a 36,000-dalton protein is phosphorylated in RSV-infected A431 cells. Addition of epidermal growth factor (EGF) to the cells induces further phosphorylation of this protein. In contrast, this phosphoprotein is not detected in uninfected A431 cells, except when treated with EGF. Increased phosphorylation of the EGF receptor protein and of an 81,000- dalton cellular protein is dependent upon addition of EGF to the culture fluids, in both control and RSV-infected A431 cells. The results are discussed with reference to the similarities and differences between the tyrosine-specific protein kinases induced by RSV and activated by EGF.     

The competence of transformed keratinocytes to differentiate is accompanied by amplification of the LDL- and EGF-receptor genes but not of the insulin receptor gene

The possible relationship between cell surface receptor numbers, receptor gene expression for low density lipoprotein (LDL), insulin and epidermal growth factor (EGF), and differentiation capacity has been studied in normal and SV40 transformed (SVK14) keratinocytes, various squamous carcinoma cell (SCC) lines and A431 cells. Our recent studies demonstrated that an inverse relationship exists between LDL- and EGF-receptor binding and the ability to differentiate of both normal and transformed keratinocytes. In the present study cloned LDL- and EGF-receptor complementary DNAs were used as probes to identify both LDL and EGF receptor gene fragments on genomic DNA blots. The extent of hybridisation was found to be increased to the highest extent in A431 cells and decreased in other cells in the following order SCC-4 greater than SCC-15. In SCC-12F2, SVK14 and normal keratinocytes no increase has been observed. The increased hybridisation of LDL- and EGF-receptors in A431, SCC-4 and SCC-15 cells was found to be due to gene amplification and not to aneuploidy. In contrast to the LDL- and EGF-receptor binding, no correlation has been found between insulin receptor binding and ability of cells to differentiate. Furthermore, no amplification of insulin receptor gene has been observed in any of the cells under study.     

Small interfering RNAs suppress the expression of endogenous and GFP-fused epidermal growth factor receptor (erbB1) and induce apoptosis in erbB1-overexpressing cells

Deregulated and excessive expression of epidermal growth factor receptor (EGFR or erbB1), a transmembrane receptor tyrosine kinase specific for the epidermal growth factor (EGF), is a feature and/or cause of a wide range of human cancers, and thus inhibition of its expression is potentially therapeutic. In RNA interference (RNAi), duplexes of 21-nucleotide RNAs (small interfering RNA, siRNA) corresponding to mRNA sequences of particular genes are used to efficiently inhibit the expression of the target proteins in mammalian cells. Here we show that by using RNAi the expression of endogenous erbB1 can be specifically and extensively (90%) suppressed in A431 human epidermoid carcinoma cells. As a consequence, EGF-induced tyrosine phosphorylation was inhibited and cell proliferation was reduced due to induction of apoptosis. We established an inverse correlation between the level of expressed erbB1 and EGF sensitivity on a cell-by-cell basis using flow cytometry. A431 cells expressing endogenous erbB1 were transfected with erbB1 fused C-terminally to enhanced green fluorescent protein (EGFP). Selective inhibition of the expression of the fusion protein was achieved with an siRNA specific for the EGFP mRNA, whereas the erbB1-specific siRNAs inhibited the expression of both molecules. siRNA-mediated inhibition of erbB1 and other erbB tyrosine kinases may constitute a useful therapeutic approach in the treatment of human cancer.