Relation of epidermal growth factor receptor concentration to growth of human epidermoid carcinoma A431 cells

The relation between the concentration of epidermal growth factor (EGF) receptor/kinase and effects of EGF on cell proliferation has been studied using variant A431 cells and antagonist anti-EGF receptor monoclonal antibodies. Clonal A431 cell variants selected for escape from the EGF-mediated growth inhibition of parental A431 cells all have reduced concentrations of EGF receptor/kinase; Harvey sarcoma virus-transformed A431 cells, which have escaped from EGF-mediated growth inhibition, also have reduced EGF receptors. Three clonal variants which have reacquired EGF-mediated growth inhibition have 2- to 4-fold more EGF receptor than their respective parent variant. A biphasic response with stimulation at low and inhibition at high concentrations of EGF was especially evident in revertants of clone 29. Three separate antagonist monoclonal anti-EGF receptor antibodies block the growth inhibitory effects of EGF and uncover EGF-mediated growth stimulation. These studies indicate that in A431 cell variants a continuum of ligand-activated EGF receptors determines proliferative responses from low concentrations of active receptors under basal conditions to intermediate concentrations causing growth stimulation to high concentrations, causing inhibition of cell proliferation.     

Incomplete epidermal differentiation of A431 epidermoid carcinoma cells

Summary A431 malignant keratinocytes, although derived from a muco-cutaneous carcinoma of the vulva, fail to achieve terminal epidermal differentiation in culture as shown by their inability to form cornified envelopes. Even after culture in a serum-free medium (MCDB 153) containing no retinoic acid and a high (10⁻³ M) calcium concentration (conditions known to facilitate epidermal differentiation), the cells do not become competent as shown by the fact that subsequent treatment with a calcium ionophore is unable to provoke the formation of cornified envelopes. Nevertheless, A431 cells are able to synthesize the envelope precursor involucrin. The block in formation of cornified envelopes is thus not due to a lack in involucrin. The results described here suggest that the absence of cross-linking of this molecule is due to a lowered epidermal membrane-bound transglutaminase activity in A431 cells, enhances involucrin accumulation in these cells, although in normal human keratinocytes it stimulates growth and reduces involucrin synthesis. These results suggest that involucrin synthesis is triggered by the arrest of growth. EDITOR'S STATEMENT The A431 cell line has been used extensively in the study of EGF receptors and effects, and recently has been employed in studies of surface membrane receptors for other factors, as well as studies of extracellular matrix synthesis and deposition and tumor promoter activities. The expanding use of A431 cells calls for a more thorough understanding of the cell type it represents and the degree to which it represents a general in vitro model of normal or neoplastic epidermal cells. This article addresses some of these questions.     

Rapid rounding of human epidermoid carcinoma cells A-431 induced by epidermal growth factor

Epidermal growth factor (EGF) induces rapid rounding of A-431 human epidermoid carcinoma cells in Ca(++)-free medium. Cell rounding is not induced by a variety of other polypeptide hormones, antiserum to cell membranes, local anesthetics, colchicine, cytochalasin B, or cyclic nucleotides. However, trypsin, like EGF, induces rounding of A- 431 cells in the absence of Ca(++). Both trypsin- and EGF-induced rounding are temperature dependent, appear to be energy dependent, and are inhibited by cytochalasins, suggesting that the active participation of microfilaments in cell rounding. However, a medium transfer experiment suggests that EGF-induced rounding is not attributable to secretion of a protease, and a number of serine protease inhibitors have no effect on the EGF-induced rounding process. Cell rounding is not attributable to the slight stimulation by EGF of the release of Ca(++) that is observed in the Ca(++)-free medium, as stimulation of such release by the ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 neither induces cell rounding nor blocks EGF-induced rounding. Cells that have rounded up after treatment with EGF or trypsin spread out upon addition of Ca(++) to the medium, even in the continuing presence of EGF or typsin. Like the cell-rounding process, the cell-spreading process is temperature dependent, appears to be energy dependent, and is inhibited by cytochalasin B. Thus, EGF does not destroy the ability of the cell to spread; rather, in the presence of the EGF (or trypsin), cell spreading and the maintenance of the flattened state become dependent on external Ca(++). Because untreated cells remain flattened in the absence of Ca(++), the data suggest that EGF may disrupt Ca(++)-independent mechanisms of adhesion normally present in A-431 cells.     

Biosynthesis of the epidermal growth factor receptor in human epidermoid carcinoma-derived A431 cells

Using human-specific antibody reagents, we have examined the biosynthesis of the epidermal growth factor receptor in human epidermoid carcinoma-derived A431 cells. Four Mr species (Mr = 70,000, 95,000, 135,000, and 145,000) are detected when cells are subjected to a brief pulse of L-[35S]methionine; an Mr = 165,000 species is detected after 45-60 min of exposure of cells to radiolabel. In pulse-chase experiments, the four lower Mr species appear to bear a precursor relation to the Mr = 165,000 protein. The molecule acquires N-linked oligosaccharide cotranslationally, and two of the species (Mr = 95,000 and 145,000) are susceptible to digestion with endo-beta-N-acetylglucosaminidase H. The Mr = 145,000 and Mr = 165,000 proteins, which become labeled with 125I-epidermal growth factor after treatment of intact cells with a bifunctional cross-linking reagent, are phosphorylated at serine and threonine on identical tryptic peptides.     

Aspects of the metabolism of the epidermal growth factor receptor in A431 human epidermoid carcinoma cells.

The biosynthesis and posttranslational metabolism of the epidermal growth factor (EGF) receptor were examined in the A431 human epidermoid carcinoma cell line. Polyclonal antibody against the receptor specifically immunoprecipitated two [35S]methionine-labeled proteins of Mr = 160,000 and 170,000. Pulse chase experiments showed the Mr = 160,000 protein to be a precursor of the Mr = 170,000 protein. Preincubation with tunicamycin resulted in immunoprecipitation of a single band of Mr = 130,000, whereas monensin inhibited maturation to the Mr = 170,000 form. Digestion of the Mr = 160,000 and 170,000 proteins with endoglycosidase H resulted in the appearance of Mr = 130,000 and 165,000 proteins, respectively. Prolonged pulse-chase experiments indicated that the half-life of the receptor is ca. 20 h in the absence of EGF and 5 h in the presence of EGF. Approximately three- to five-fold more phosphate is incorporated into the mature receptor upon addition of EGF, due primarily to increases in levels of phosphotyrosine and phosphoserine. Phosphate was also present on the Mr = 160,000 protein and the Mr = 130,000 protein found in the presence of tunicamycin.     

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.     

Cell strains of A431 epidermoid carcinoma with altered epidermal growth factor reception

Epidermal growth factor inhibits proliferation of A431 cells when added to the cultural medium. Strains of A431 cells, resistant to EGF (800 ng/ml), were obtained by one-step selection after the treatment of these cells by 1-methyl-3-nitro-1-nitrosoguanidine (MNNG). Two of the obtained strains differ from the initial line in the EGF reception.     

Immunohistochemical localization of basic fibroblast growth factor in A431 human epidermoid carcinoma cells

Summary The intracellular location of basic fibroblast growth factor (bFGF) was determined in A431 human epidermoid carcinoma cells both on immunofluorescence and on immunoelectron microscopy using a monoclonal anti-bFGF antibody. The immunofluorescence was located in the cytoplasm in quiescent cells. Following the addition of FCS to the culture medium of quiescent sparse cells the growth factor was translocated to and accumulated in the nucleolus. Immunogold particles were dense near the ribosomes, but were not recognized in the cytoplasmic structures concerned with the usual secretory pathway such as the rough endoplasmic reticulum, the Golgi apparatus, and secretory granules. These results suggest that endogenous bFGF undergoes intracellular sorting and enters the nucleoli in A431 cells according to an extracellular growth signal.     

Adenosine modulates cell growth in human epidermoid carcinoma (A431) cells.

Adenosine mediates many physiological functions via activation of extracellular receptors. The modulation of cell growth by adenosine was found to be receptor-mediated. In A431 cells adenosine evoked a biphasic response in which a low concentration (approximately 10 microM) produced inhibition of colony formation but at higher concentrations (up to 100 microM) this inhibition was progressively reversed. Evidence for the involvement of A1 (inhibitory) and A2 (stimulatory) adenosine receptors in regulating cell growth of these tumor cells was obtained through plating efficiency studies based on the relative potency of adenosine agonists and antagonists. When both A1 and A2 receptors were blocked, colony formation or growth was not inhibited at low concentrations of adenosine but was inhibited at high adenosine concentrations.     

Recycling of epidermal growth factor-receptor complexes in A431 cells: identification of dual pathways

The intracellular sorting of EGF-receptor complexes (EGF-RC) has been studied in human epidermoid carcinoma A431 cells. Recycling of EGF was found to occur rapidly after internalization at 37 degrees C. The initial rate of EGF recycling was reduced at 18 degrees C. A significant pool of internalized EGF was incapable of recycling at 18 degrees C but began to recycle when cells were warmed to 37 degrees C. The relative rate of EGF outflow at 37 degrees C from cells exposed to an 18 degrees C temperature block was slower (t1/2 approximately 20 min) than the rate from cells not exposed to a temperature block (t1/2 approximately 5-7 min). These data suggest that there might be both short- and long-time cycles of EGF recycling in A431 cells. Examination of the intracellular EGF-RC dissociation and dynamics of short- and long-time recycling indicated that EGF recycled as EGF-RC. Moreover, EGF receptors that were covalently labeled with a photoactivatable derivative of 125I-EGF recycled via the long-time pathway at a rate similar to that of 125I-EGF. Since EGF-RC degradation was also blocked at 18 degrees C, we propose that sorting to the lysosomal and long-time recycling pathway may occur after a highly temperature-sensitive step, presumably in the late endosomes.     

Immunohistochemical localization of basic fibroblast growth factor in A431 human epidermoid carcinoma cells.

The intracellular location of basic fibroblast growth factor (bFGF) was determined in A431 human epidermoid carcinoma cells both on immunofluorescence and on immunoelectron microscopy using a monoclonal anti-bFGF antibody. The immunofluorescence was located in the cytoplasm in quiescent cells. Following the addition of FCS to the culture medium of quiescent sparse cells the growth factor was translocated to and accumulated in the nucleolus. Immunogold particles were dense near the ribosomes, but were not recognized in the cytoplasmic structures concerned with the usual secretory pathway such as the rough endoplasmic reticulum, the Golgi apparatus, and secretory granules. These results suggest that endogenous bFGF undergoes intracellular sorting and enters the nucleoli in A431 cells according to an extracellular growth signal.     

Isolation of an evolutionarily conserved epidermal growth factor receptor cDNA from human A431 carcinoma cells

Complementary DNA corresponding to total poly(A)+-RNA from the human A431 epidermoid carcinoma cell line was cloned in the phage expression vector lambda gt 11. An epidermal growth factor (EGF) receptor cDNA clone was obtained by screening of the expression library with a rabbit polyclonal antibody (IgG), raised to the purified A431 EGF receptor, in combination with [125I]protein A of S. aureus. The cloned cDNA was able to select, by hybridization, messenger RNA which was translated in Xenopus oocytes and yielded an immunoprecipitable EGF receptor protein of Mr = 160,000. The insert of this cDNA (phEGFR-1), is approximately 880 base pairs in length and encodes the carboxyterminal portion of the EGF receptor protein. Its sequence is evolutionarily conserved among vertebrates as shown by hybridization to unique chromosomal DNA sequences from human, baboon, dog, rat, mouse and frog.     

EGF induces cell cycle arrest of A431 human epidermoid carcinoma cells

The human carcinoma cell line A431 is unusual in that physiologic concentrations of epidermal growth factor (EGF) inhibit proliferation. In the presence of 5-10 nM EGF proliferation of A431 cells is abruptly and markedly decreased compared to the untreated control cultures, with little loss of cell viability over a 4-day period. This study was initiated to examine how EGF affects the progression of A431 cells through the cell cycle. Flow cytometric analysis of DNA in EGF-treated cells reveals a marked change in the cell cycle distribution. The percentage of cells in late S/G2 increases and early S phase is nearly depleted. Since addition of the mitotic inhibitor vinblastine causes accumulation of cells in mitosis and prevents reentry of cells into G1, it is possible to distinguish between slow progression through G1 and G2 and blocks in those phases. When control cells, not treated with EGF, are exposed to vinblastine, the cells accumulate mitotic figures, as expected, and show progression into S, thus diminishing the number of cells in G1. In contrast, no mitotic figures are found among the EGF-treated cells in the presence or absence of vinblastine, and progression from G1 into S is not observed, as the number of cells in G1 remains constant. These results suggest that there are two EGF-induced blocks in cell cycle transversal; one is in late S and/or G2, blocking entry into mitosis, and the other is in G1, blocking entry into S phase. After 24 hours of EGF treatment, DNA synthesis is reduced to less than 10% compared to untreated controls as measured by the incorporation of [3H]thymidine or BrdU. In contrast, protein synthesis is inhibited by about twofold. Although inhibition of protein synthesis is less extensive, it occurs 6 hours prior to an equivalent inhibition of DNA synthesis. The rapid decrease in protein synthesis may result in the subsequent cell cycle arrest which occurs several hours later.     

Ceramide stimulates epidermal growth factor receptor phosphorylation in A431 human epidermoid carcinoma cells. Evidence that ceramide may mediate sphingosine action

Recent studies suggest the existence of a signal transduction pathway involving sphingomyelin and derivatives (Kolesnick, R. N. (1989) J. Biol. Chem. 264, 7617-7623). The present studies compare effects of ceramide, sphingosine, and N,N-dimethylsphingosine on epidermal growth factor (EGF) receptor phosphorylation in A431 human epidermoid carcinoma cells. To increase ceramide solubility, a ceramide containing octanoic acid at the second position (C8-cer) was synthesized. C8-cer induced time- and concentration-dependent EGF receptor phosphorylation. This event was detectable by 2 min and maximal by 10 min. As little as 0.1 microM C8-cer was effective, and 3 microM C8-cer induced maximal phosphorylation to 1.9-fold of control. EGF (20 nM) increased phosphorylation to 2.1-fold of control. Sphingosine stimulated receptor phosphorylation over the same concentration range (0.03-3 microM) and to the same extent (1.8-fold of control) as ceramide. The effects of C8-cer and sphingosine were similar by three separate criteria, phosphoamino acid analysis, anti-phosphotyrosine antibody immunoblotting, and phosphopeptide mapping by high performance liquid chromatography. Phosphorylation occurred specifically on threonine residues. N,N-Dimethylsphingosine, a potential derivative of sphingosine, was less effective. Since sphingosine and ceramide are interconvertible, the level of each compound was measured under conditions sufficient for EGF receptor phosphorylation. C8-cer (0.1-1 microM) induced dose-responsive elevation of cellular ceramide from 132 to 232 pmol.10(6) cells-1. In contrast, cellular sphingosine levels did not rise. This suggests that C8-cer acts without conversion to sphingosine. Exogenous sphingosine (0.1-1 microM) also increased cellular ceramide levels to 227 pmol.10(6) cells-1, but did not increase its own cellular level of 12 pmol.10(6) cells-1. Higher sphingosine concentrations that induced no further increase in EGF receptor phosphorylation produced very large elevations in cellular sphingosine. Hence, at effective concentrations, both compounds elevated cellular ceramide but not sphingosine levels. Additional studies performed with [3H]sphingosine demonstrated that cells contain substantially less N,N-dimethylsphingosine than free sphingosine and, during short term incubation, convert less than 5% of added sphingosine to N,N-dimethylsphingosine. These studies provide evidence that ceramide may have bioeffector properties and suggest sphingosine may act in part by conversion to ceramide.     

Intracellular epidermal interleukin 1-like factors in the human epidermoid carcinoma cell line A431

Normal human epidermal cells produce, in primary culture, activities which stimulate the release of PGE2 and collagenase by dermal fibroblasts; this factor(s) might play an important role in epidermal-dermal interactions. Since these activities were mainly found in the cell lysates with only little being detected in the conditioned media, we investigated further the problem of cell-associated versus released activity in the model of the human epidermoid carcinoma cell line A431. The activities were consistently found in the cell lysate and in the conditioned media only when the cells were leaky. No membrane-associated activities were identified. Purification of the cytosolic activities were identified. Purification of the cytosolic activities yielded two differently charged species both with a MW of approximately 17K. The copurification of PGE2- and collagenase-stimulating activities with thymocyte comitogenic activity suggests a close physiochemical relation to IL-1. The activities described here might therefore correspond to the intracellular counterpart of epidermal IL-1 formerly described as epidermal cell-derived thymocyte activating factor (ETAF) and identified in the conditioned medium of cultured epidermal cells. These observations are of importance when studying the modulation of these activities.     

Amlodipine,a Ca2+ channel blocker,suppresses phosphorylation of epidermal growth factor receptor in human epidermoid carcinoma A431 cells

AimsAmlodipine, a dihydropyridine Ca²⁺ channel blocker, inhibits the proliferation of human epidermoid carcinoma A431 cells in vitro and in vivo. This study examined the underlying mechanism of this antiproliferative effect in relation to epidermal growth factor receptor (EGFR) signaling.Main methodsThe tyrosine phosphorylated active state of EGFR in A431 cells incubated with the test agents was evaluated by western blot with anti-phosphotyrosine antibody. EGFR phosphorylation levels in A431 xenograft tumors were assessed by immunostaining of matrigel plug sections and western blotting for phosphoEGFR in A431 xenograft tumor homogenates.Key findingsIn vitro treatment of exponentially growing A431 cells with amlodipine decreased the tyrosine phosphorylation states of EGFR. Amlodipine also suppressed the EGF-stimulated phosphorylation of EGFR and a membrane scaffolding protein, caveolin-1, in serum-starved A431 cells. Amlodipine attenuated the EGF-stimulated phosphorylation of EGFR coimmunoprecipitated with caveolin-1 without affecting the EGFR/caveolin-1 interaction. Crosslinking experiments showed that amlodipine also suppressed the EGF-stimulated phosphorylation of EGFR predimers. Addition of cholesterol abolished these inhibitory effects of amlodipine plus its inhibition of cell growth. Furthermore, treatment of mice with amlodipine (10 mg/kg/day × 7 days, i.p.) decreased the levels of phosphorylated EGFR in A431 xenograft tumors.SignificanceThe results indicated that amlodipine inhibits tyrosine phosphorylation of EGFR in vitro and in vivo, possibly via modulating cholesterol-rich, caveolin-1-containing membrane microdomains.     

Epidermal growth factor inhibits growth of A431 human epidermoid carcinoma in serum-free cell culture

A medium consisting of a rich basal nutrient mixture supplemented with bovine insulin (10 micrograms/ml), human transferrin (10 micrograms/ml), human cold-insoluble globulin (5 micrograms/ml), and ethanolamine (0.5 mM) supported the growth of the A431 human epidermoid cell line in the absence of serum with a generation time equal to that of cells in serum-containing medium. Addition of epidermal growth factor (EGF) to this culture medium at concentration mitogenic for other cell types resulted in a marked inhibition of A431 cell growth. Inhibitory effects of EGF were observed at 1 ng/ml and near-maximal effects were observed at 10 ng/ml. The inhibitory effect of EGF could be reversed by the omission of EGF in subsequent medium changes and could be prevented by the addition of anti-EGF antibody to the culture medium. Inhibition of A431 cell growth by EGF also could be demonstrated in serum-containing medium.     

Reciprocal effects of epidermal growth factor and transforming growth factor beta on the anchorage-dependent and -independent growth of A431 epidermoid carcinoma cells

The effects of epidermal growth factor (EGF) and transforming growth factor beta (TGF beta) on the growth of A431 epidermoid carcinoma cells were studied. Whereas the monolayer growth of A431 cells was inhibited by EGF, it was stimulated by TGF beta. Contrary to the effects on the monolayer growth, EGF stimulated the soft agar growth of A431 cells. The stimulatory effects of TGF beta on the anchorage-dependent growth were antagonized by EGF and those of EGF on anchorage-independent growth were antagonized by TGF beta. These results suggest that both factors not only convey the proliferative signals to A431 cells but also induce phenotypic changes, resulting in a preference for either anchorage-dependent or anchorage-independent growth. Moreover, as the stimulatory effects of EGF on the soft agar growth of A431 cells paralleled its reported stimulatory effects on their in vivo growth, it is also suggested that in vivo responses of cells to certain growth factors may correlate better with their responses in soft agar culture than with those in monolayer culture.     

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.