Epidermal growth factor induces terminal differentiation in human epidermoid carcinoma cells

Previous studies have reported that the proliferation of A431 cells, a human squamous cell carcinoma cell line, was stimulated by picomolar epidermal growth factor (EGF) but inhibited by nanomolar EGF. This biphasic dose-response phenomenon is not observed in normal human epithelial cells where nanomolar EGF is usually mitogenic. We have examined the effects of inhibitory and stimulatory concentrations of EGF on the growth and differentiation of A431 cells. In the presence of 100 pM EGF, A431 cells showed a mild increase in growth rate (129% of control) compared to cells grown in the absence of EGF. At 10 nM EGF, growth inhibition to 63% of control was observed. EGF at 10 nM stimulates a twofold increase both in cornified envelope formation and in epidermal transglutaminase activity, suggesting that high concentrations of EGF induce terminal differentiation in A431 cells. Mitogenic concentrations of EGF (100 pM) had no significant effect on these differentiation markers. Chronic exposure of A431 cells to 20 or 50 nM EGF resulted in EGF-resistant A431 variants that are neither growth arrested nor induced to terminally differentiate by 10 nM EGF. Removal of EGF from the growth medium of the EGF-resistant cells resulted in the reversion of these cells back to the wild-type A431 biphasic response pattern within 2 weeks. Our results suggest that A431 cells have the capacity to non-mutatively alter their response pattern to EGF in vitro to maintain themselves in a state of optimum proliferation and away from terminal differentiation. © 1993 Wiley-Liss, Inc.     

Activation of calcium and phospholipid-dependent protein kinase by epidermal growth factor (EGF) in A431 cells: attenuation by 12-0-tetradecanoylphorbol-13-acetate (TPA)

A calcium and phospholipid-dependent protein kinase (protein kinase C) was detected in the crude soluble extracts of A431 human epidermoid carcinoma cells. The enzyme required calcium, phosphatidylserine or phosphatidylinositol, and diacylglycerol (DG) for maximal activation. Protein kinase C phosphorylated both endogenous cytosolic proteins and various histones. Addition of epidermal growth factor (EGF) to A431 cultures resulted in a 2 to 3-fold stimulation of protein kinase activity. 12-0-tetradecanoylphorbol-13-acetate (TPA) in concert with EGF attenuated the EGF-induced enhanced phosphorylation of endogenous proteins. It is conceivable that DG, derived from phosphatidylinositol turnover, acts as a natural activator of protein kinase C activity.     

Construction of a Novel Extracellular Matrix using a New Genetically Engineered Epidermal Growth Factor Fused to IgG-Fc

The design of artificial extracellular matrices has attracted much attention in tissue engineering as well as in cell biology research. An immobilized recombinant epidermal growth factor (EGF), fused to an immunoglobulin G (IgG) Fc region (abbreviated as EGF-Fc) has been constructed. Mouse fibroblast Swiss 3T3 cells adhered both to EGF-Fc-coated and collagen-coated surfaces. Phosphorylation of EGF receptor in A431 cells was induced by immobilized EGF-Fc as well as soluble EGF. Immobilized EGF-Fc continuously activated mitogen-activated protein kinase (MAPK) in A431 cells whereas MAPK activation induced by soluble EGF decreased rapidly with time. The cytoskeleton of A431 cells adhering onto immobilized EGF-Fc was filopodia whereas that of the cells adhering onto collagen in the presence of soluble EGF was lammellipodia.     

Activation of a cytosolic serine protein kinase by epidermal growth factor

Exposure of A-431 cells to epidermal growth factor (EGF) results in a rapid enhancement (approximately 10-fold) of cytosolic serine protein kinase activity. The increase in serine kinase activity may be detected using a number of peptide and protein substrates. Enhancement of kinase activity occurs within 1 min of exposure of the cells to EGF and reaches a maximum in 5 min. Similar results were obtained with a variety of cell lines. We have partially purified the EGF-activated kinase from A-431 cells. It has an apparent molecular mass of approximately 100 kDa by gel filtration. One distinguishing property of the enzyme is its sensitivity to inhibition by micromolar quantities of polyarginine; polylysine has no effect. The EGF-activated kinase is unaffected by cyclic nucleotides, Ca2+/calmodulin, Ca2+/diolein/phosphatidylserine, or heparin. The enhancement of cytosolic serine kinase activity in A-431 cells appears to be an early event in cell "activation" by a number of biological response modifiers including EGF, bradykinin, 12-O-tetradecanoylphorbol-13-acetate, and histamine.     

Epidermal growth factor induces activation of protein kinase FA and ATP.Mg-dependent protein phosphatase in A431 cells

The cytosolic fractions from epidermal growth factor (EGF)-treated A431 cells exhibit a marked increase in activities of ATP.Mg-dependent protein phosphatase and its activating factor (protein kinase FA) when compared to controls in the absence of EGF. By contrast, the Triton X-100-solubilized membrane fractions from the same EGF-treated cells exhibit a corresponding decrease in protein kinase FA activity. The EGF-dependent activation of protein kinase FA and ATP.Mg-dependent protein phosphatase occurred within physiological concentrations of EGF (ED50 = 5 x 10(-10) M). The changes of kinase and phosphatase activities which were measured concomitantly exhibit very similar characteristics as to EGF sensitivity and time dependence. The EGF-induced kinase and phosphatase activation occurred very rapidly, reaching the maximal activity levels within 3 min. Moreover, the EGF effect is transient; both EGF-stimulated phosphatase and kinase activities returned to control levels within 30 min. Taken together, the results suggest that EGF may induce the activation of kinase FA in the membrane and thereby promotes the activation of ATP.Mg-dependent phosphatase in the cytosol. Exposure of A431 cells to exogenous phospholipase C also resulted in the activation of endogenous kinase FA and ATP.Mg-dependent phosphatase in a similar pattern produced by EGF. This further suggests that phospholipase C can mimic EGF to mediate the activation of kinase FA and ATP.Mg-dependent phosphatase in A431 cells. By its dual role as a multisubstrate protein kinase and as an activating factor of multisubstrate protein phosphatase, protein kinase FA may represent a transmembrane signal of EGF.     

Transforming growth factor beta modulates phosphorylation of the epidermal growth factor receptor and proliferation of A431 cells.

Transforming growth factor beta (TGF-beta) increased the phosphorylation of the epidermal growth factor (EGF) receptor and inhibited the growth of A431 cells. Incubation with TGF-beta induced maximal EGF receptor phosphorylation to levels 1.5-fold higher than controls. Phosphorylation increased more prominently (4-5-fold) on tyrosine residues as determined by phosphoamino acid analysis and antiphosphotyrosine antibody immunoblotting. The kinase activity of EGF receptor was also elevated 2.5-fold when cells were cultured in the presence of TGF-beta. The antiproliferative effect of TGF-beta on A431 cells was accompanied by prolongation of G0-G1 phase and by morphological changes. TGF-beta augmented the growth inhibition of A431 cells which could be induced by EGF. In parallel, the specific EGF-induced increase in total phosphorylation of the EGF receptor was also augmented in the presence of TGF-beta. In cells cultured with TGF-beta, the phosphorylation of EGF receptor tyrosines induced by 20-min exposure to EGF was further increased 2-3-fold, suggesting additive effects upon receptor phosphorylation. EGF receptor activation by TGF-beta is characterized by kinetics quite distinct from that induced by EGF and therefore appears to take place through an independent mechanism. The TGF-beta-induced elevation in the phosphorylation of the EGF receptor may have a role in the augmented growth inhibition of A431 cells observed in the presence of EGF and TGF-beta.     

Effect of photo-immobilization of epidermal growth factor on the cellular behaviors

We constructed photo-reactive epidermal growth factor (EGF) bearing p-azido phenylalanine at the C-terminal (HEGFP) by genetic engineering to investigate the possibility of immobilized EGF as a novel artificial extracellular matrix (ECM). The constructed recombinant protein was immobilized to glass surface by ultraviolet irradiation. A431 cells adhered both to HEGFP-immobilized and collagen-coated surfaces. Interaction between immobilized HEGFP and EGF receptors in the A431 cells was independent of Mg(2+) although integrin-mediated cell adhesion to natural ECMs is dependent on Mg(2+). Phosphorylation of EGF receptors in A431 cells was induced by immobilized HEGFP as same as soluble EGF. DNA uptake of hepatocytes decreased by immobilized HEGFP whereas it increased by soluble EGF. Liver-specific functions of hepatocytes were maintained for 3 days by immobilized HEGFP whereas they were not maintained by soluble EGF, indicating that immobilized HEGFP follows different signal transduction pathway from soluble EGF.     

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.     

Effects of protein kinase C activation after epidermal growth factor binding on epidermal growth factor receptor phosphorylation

The possible role of epidermal growth factor (EGF) receptor phosphorylation at threonine 654 in modulating the protein-tyrosine kinase activity of EGF-treated A431 cells has been studied. It has been suggested that EGF could indirectly activate a protein-serine/threonine kinase, protein kinase C, that can phosphorylate the EGF receptor at threonine 654. Protein kinase C is known to be activated, and threonine 654 is phosphorylated, when A431 cells are exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA). The protein-tyrosine kinase activity of EGF receptors is normally evidenced in EGF-treated cells by phosphorylation of the receptor at tyrosine. This is inhibited when TPA-treated cells are exposed to EGF. We now show that receptor phosphorylation at threonine 654 can also be detected in EGF-treated A431 cells, presumably due to indirect stimulation of protein kinase C or a similar kinase. Some receptor molecules are phosphorylated both at threonine 654 and at tyrosine. Since prior phosphorylation at threonine 654 inhibits autophosphorylation, we propose that protein kinase C can phosphorylate the threonine 654 of autophosphorylated receptors. This provides evidence for models in which protein kinase C activation, consequent upon EGF binding, could reduce the protein-tyrosine kinase activity of the EGF receptor. Indeed, we find that 12-O-tetradecanoylphorbol-13-acetate, added 10 min after EGF, further increases threonine 654 phosphorylation and induces the loss of tyrosine phosphate from A431 cell EGF receptors.     

Epidermal growth factor stimulates diacylglycerol kinase in isolated plasma membrane vesicles from A431 cells

We have examined the effect of epidermal growth factor(EGF) on three kinds of kinases activities, phosphatidylinositol(PI) kinase, phosphatidylinositol 4-phosphate[PI(4)P] kinase and diacylglycerol(DG) kinase that make important roles in the regulation of inositol phospholipids metabolism. When isolated plasma membrane vesicles from A431 cells were incubated at 30 degrees C with [gamma-32P]ATP and exogenously added DG, EGF enhanced the activity of DG kinase approximately 2-fold. This stimulation is found to be dose-dependent with a half maximal activation at 1 nM. In this case, EGF increased Vmax without changing Km Value for ATP or DG. Although this activation was observed in the absence of detergent, it was more evident when membrane vesicles were treated with 1 mM deoxycholate. Interestingly, the effect of EGF was only detected in magnesium containing medium. The use of manganese instead of magnesium diminished the stimulatory effect in either condition, presence or absence of deoxycholate. On the other hand, the stimulation of PI kinase or PI(4)P kinase activity was not caused by EGF. These results suggest that DG kinase activation by EGF makes important roles in cellular responses leading to cell growth.     

Tumor necrosis factor modulates epidermal growth factor receptor phosphorylation and kinase activity in human tumor cells. Correlation with cytotoxicity

Tumor necrosis factor (TNF) is a cytokine which induces cytotoxicity in some but not all tumor cells. Initial studies of five tumor cell lines demonstrated that TNF was able to rapidly (within 30 min) modulate tyrosine protein kinase activity of epidermal growth factor (EGF) receptors on tumor cell lines which were sensitive to the cytotoxic effects of TNF but not alter EGF receptor kinase activity in TNF-resistant tumor cells. Two tumor cell lines (ME-180 cervical carcinoma and T24 bladder carcinoma) which have been shown to express similar TNF-binding characteristics but differ in their sensitivity to the cytotoxic actions of TNF were chosen for further characterization. Treatment of TNF-sensitive ME-180 cells with 1 nM TNF resulted in a 3-fold stimulation of EGF receptor tyrosine protein kinase activity within 10 min which correlated with increased phosphorylation of EGF receptor protein itself. In addition, dose-response studies indicate that similar concentrations of TNF modulate both ME-180 cell growth and EGF receptor kinase activity. Treatment of TNF-resistant T24 cells showed that TNF had no significant effect on their growth, EGF receptor tyrosine protein kinase activity, or phosphorylation of EGF receptor protein although EGF receptor kinase activity was stimulated by EGF. Quantitation of receptors expressed on the surface of ME-180 and T24 cells demonstrated a 3-fold difference between the number of EGF-binding sites on T24 (100,000) versus ME-180 cells (300,000), suggesting the relative abundance of EGF receptor does not solely account for differential effects of TNF on EGF receptor activation in these two cell lines. Phosphoamino acid analysis of EGF receptor from 32P-equilibrated ME-180 cells demonstrated that TNF-induced phosphorylation of amino acids which was quantitatively similar to that of EGF but distinct from the effects of phorbol ester. However, unlike EGF, TNF was unable to stimulate EGF receptor kinase activity in ME-180 cell lysates. The kinetics of EGF receptor activation and the metabolic consequence of activation of EGF receptor activity by TNF appear to be distinct from those induced by EGF. These results suggest that TNF-induced modulation of EGF receptor occurs through a unique mechanism and may play a role in the cytotoxic actions of TNF.     

Inhibition of EGF-induced phospholipase C activation in A431 cells by erbstatin, a tyrosine kinase inhibitor

Erbstatin, a tyrosine kinase inhibitor, inhibited epidermal growth factor (EGF)-induced inositol phosphate production in cultured A431 cells. However, it did not inhibit ATP-induced inositol phosphate production. Cytosolic but not membrane-associated phospholipase C was activated by EGF, and erbstatin inhibited enhancement of the phospholipase C activity in EGF-treated cells. Thus, tyrosine kinase of A431 cells is suggested to be functionally involved in phospholipase C activation.     

Biosynthesis of the epidermal growth factor receptor: post-translational glycosylation-independent acquisition of tyrosine kinase autophosphorylation activity

We previously showed that the epidermal growth factor (EGF) receptor in human A431 epidermoid carcinoma cells undergoes a slow post-translational modification whereby it acquires (t1/2 = 30-40 min) EGF binding capacity (Slieker, L.J., et. al. (1986) J. Biol. Chem., 261, 15233-15241). This activation occurs in the endoplasmic reticulum and requires core N-linked glycosylation. By employing both anti-EGF receptor and anti-phosphotyrosine antibodies to immunoprecipitate receptor pulse-labeled with [35S]methionine, we demonstrate here that the EGF receptor also acquires tyrosine kinase autophosphorylation activity post-translationally (t1/2 = 10-15 min). The acquisition of tyrosine kinase activity is independent of the acquisition of EGF binding capacity, since it precedes the latter process and does not require N-linked glycosylation.     

EGF receptor degrades via the proteasome-dependent pathway in A-431 cells

It is known that a lot of cell receptors degrade by ubiquitine-proteasome pathway. Here we show that degradation of the epidermal growth factor (EGF) receptor is proteasome-dependent. Treatment of A-431 cells with lactacystine, an inhibitor of proteolytic activities of 26S proteasomes, induces accumulation of the receptor in cells. Incubation of cell lysates with isolated 26S proteasomes leads to diminishing EGF receptor in these cells. Active (tyrosine phosphorylated) EGF receptor is a target of proteolysis by proteasomes.     

Epidermal growth factor (EGF) promotes phosphorylation at threonine-654 of the EGF receptor: possible role of protein kinase C in homologous regulation of the EGF receptor

Treatment of cells with tumor-promoting phorbol diesters, which causes activation of protein kinase C, leads to phosphorylation of the epidermal growth factor (EGF) receptor at threonine-654. Addition of phorbol diesters to intact cells causes inhibition of the EGF-induced tyrosine-protein kinase activity of the EGF receptor and it has been suggested that this effect of phorbol diesters is mediated by the phosphorylation of the receptor by protein kinase C. We measured the activity of protein kinase C in A431 cells by determining the incorporation of [32P]phosphate into peptides containing threonine-654 obtained by trypsin digestion of EGF receptors. After 3 h of exposure to serum-free medium, A431 cells had no detectable protein kinase C activity. Addition of EGF to these cells resulted in [32P] incorporation into threonine-654 as well as into tyrosine residues. This indicates that EGF promotes the activation of protein kinase C in A431 cells. The phosphorylation of threonine-654 induced by EGF was maximal after only 5 min of EGF addition and the [32P] incorporation into threonine-654 reached 50% of the [32P] in a tyrosine-containing peptide. This indicates that a significant percentage of the total EGF receptors are phosphorylated by protein kinase C. A variety of external stimuli activate Na+/H+ exchange, including EGF, phorbol diesters, and hypertonicity. To ascertain whether activation of protein kinase C is an intracellular common effector of all of these systems, we measured the activity of protein kinase C after exposure of A431 cells to hyperosmotic conditions and observed no effect on phosphorylation of threonine-654, therefore, activation of Na+/H+ exchange by hypertonic medium is independent of protein kinase C activity. Since stimulation of protein kinase C by phorbol diesters results in a decrease in EGF receptor activity, the stimulation of protein kinase C activity by addition of EGF to A431 cells contributes to a feedback mechanism which results in the attenuation of EGF receptor function.     

Demonstration of epidermal growth factor-induced receptor dimerization in living cells using a chemical covalent cross-linking agent

We have used the soluble covalent cross-linking agent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDAC) to examine the capacity of epidermal growth factor (EGF) to stimulate the dimerization of purified EGF receptor, of EGF receptor in membrane preparations and in intact A431 cells. The addition of EGF either to membranes from A431 cells or to EGF receptor which was purified from A431 cells by immunoaffinity chromatography caused the appearance of a cross-linked product of Mr 340,000 which was identified using EGF receptor-specific antibodies as an EGF receptor dimer. Three independent approaches including biosynthetic labeling, surface iodination, and immunoblotting experiments were utilized to follow EGF receptor dimerization in living A431 cells. These approaches provided consistent results indicating that EGF induced rapid dimerization of EGF receptor in living cells, suggesting that this process may play a role in transmembrane signalling mediated by EGF.     

Ligand-induced activation of A431 cell epidermal growth factor receptors occurs primarily by an autocrine pathway that acts upon receptors on the surface rather than intracellularly

A431 cells express high numbers of epidermal growth factor (EGF) receptors and produce a ligand for these receptors, transforming growth factor-alpha (TGF-alpha). We have obtained evidence that the EGF receptors on these cells may be activated through an "autocrine" pathway by ligand and have investigated whether activation of phosphorylation of the receptor by the endogenously produced TGF-alpha occurs intracellularly or at the cell surface. When A431 cells were cultured under serum-free conditions, in the absence of exogenous ligand, EGF receptors were found to have a basal level of phosphorylation. When cells were labeled by culturing with 32Pi in the continuous presence of monoclonal antibodies that block binding of TGF-alpha to the EGF receptor, phosphorylation decreased to 30 +/- 10% of the basal level. This reduction could not be accounted for by the decrease in receptor content attributable to down-regulation and catabolism of EGF receptors that resulted from the binding of anti-receptor monoclonal antibodies. The reduction in receptor phosphorylation mediated by antibody was accompanied by the accumulation of increased levels of secreted TGF-alpha species in the culture medium. We also pulse-labeled A431 cells for 15 min with [35S]cysteine and immunoprecipitated the cell lysate with anti-phosphotyrosine antibody after various chase periods. Tyrosine-phosphorylated EGF receptor became detectable after 40 min of chase and reached a maximum after 4-6 h; these times are in agreement with the intervals required for EGF receptors to reach the cell surface after synthesis and then to achieve maximal expression. In addition, only the 170-kDa, mature EGF receptor species, and not the 160-kDa intracellular precursor, was immunoprecipitated with the anti-phosphotyrosine antibody. The results of these pulse-chase experiments and the finding that anti-receptor monoclonal antibody can block receptor phosphorylation suggest that activation of EGF receptors can result from the binding of an endogenous ligand (presumably TGF-alpha), which occurs at the cell surface and not during receptor biosynthesis and intracellular processing.     

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.     

Activation of 45Ca2+ influx and 22Na+/H+ exchange by epidermal growth factor and vanadate in A431 cells is independent of phosphatidylinositol turnover and is inhibited by phorbol ester and diacylglycerol

Both epidermal growth factor (EGF) and vanadate can activate 45Ca2+ influx into A431 epidermal carcinoma cells, without a detectable lag period possibly via a voltage-independent calcium channel. 22Na+/H+ exchange and 45Ca2+ uptake are mutually independent. Neither EGF nor vanadate induce any significant change in the steady-state levels of [1,3-3H]glycerol-labeled diacylglycerol, myo-[2-3H]inositol-labeled inositol trisphosphate or in 32P-labeled polyphosphoinositides or phosphatidic acid over the first 10 min of treatment, suggesting that the EGF receptor is not directly coupled to phosphatidylinositol turnover and that the two ion fluxes are not induced via a kinase C-dependent pathway. An increase in turnover of polyphosphoinositides can be detected in EGF-stimulated cells by nonequilibrium labeling with [32P]phosphate, but the increase shows a lag of about 1 min under the conditions used to detect 45Ca2+ influx. Chelation of free Ca2+ decreases but does not abolish the EGF-stimulated turnover. Preincubation with tetradecanoylphorbol acetate or 1-oleoyl-2-acetylglycerol inhibits the increase in 45Ca2+ uptake by both EGF and vanadate. Tetradecanoylphorbol acetate alone does not alter the basal rate of influx when added together with 45Ca2+. Surprisingly, the activation by vanadate and its inhibition by phorbol 12-myristate 13-acetate are unaffected by down-regulation of the EGF receptors through prior incubation with growth factor. Therefore, in A431 cells the activation of Na+/H+ exchange and Ca2+ influx appear to be independent of phosphatidylinositol turnover, and the EGF receptor does not itself function as a Ca2+ channel. Vanadate apparently activates influx through a mechanism distinct from or distal to the EGF receptor.     

Cytoskeletal changes in A-431 cells under the action of epidermal growth factor]

By the use of rhodamine-phalloidin, the distribution of actin in A-431 cells during the action of epidermal growth factor (EGF) has been studied. Changes in the pattern of staining are observed in 30-60 s after addition of the EGF. Microvilli and wrinkles are created on the cell surface. Following a 5-10 min action of EGF, rhodamine-phalloidin stained intensely ruffles and cell borders. After 60 min, the ruffling of cell surface disappeared, and actin was seen concentrating on the cell borders only. Electron microscopy of the EGF-treated A-431 cells lysed by Triton X-100 also revealed some vigorous fibrillar bunches on the cell edges.