The tumor-promoter 12-0-tetradecanoyl-phorbol-13-acetate, which shares several biological activities with EGF, was also effective in stimulating an increase in the rate of pinocytosis .

The morphological effects of epidermal growth factor (EGF) on human carcinoma cells A-431 have been examined by scanning electron microscopy . These flat polygonal cells normally exhibit only small membrane folds, but show extensive ruffling and extension of filopodia within 5 min of exposure to EGF at 37°C . This ruffling activity is transient, subsiding within another 5-15 min, but several other changes in surface morphology follow . Within the first hour of exposure to the hormone, the cell surface becomes exceedingly smooth and the nuclei seem to protrude above the plane of the otherwise thin monolayer, giving the cells a "fried egg" appearance . Cells at the edges of colonies gradually retract from the substrate, leading to reorganization, by 12 h, of the monolayer into multilayered colonies . EGF thus induces both rapid and long-term alterations in the morphology ofthese epidermoid cells .KEY WORDS scanning electron microscopy membrane ruffling -growth factor motility " cell shape changeThe regulation of the shape and motility of cultured cells is currently a subject of interest from both morphological and biochemical points of view . In several instances, hormones have been shown to influence the morphology of target cells in culture (e.g., references 27,21,16) .In our recent studies concerning the mechanism of action of epidermal growth factor (EGF), we have used the human epidermoid carcinoma cell line A-431, taking advantage of the extraordinarily high number (2-3 x 10) of EGF receptors present on these cells (9,12) . This property of A-431 cells has facilitated the visualization of hormone binding and internalization (12, 13) as well as the discoveries that EGF rapidly stimulates fluid pinocytosis in whole cells (14) and protein phosphorylation in isolated membranes (6, 7).In this report, we describe rapid and striking morphological changes which are induced in A-431 cells by treatment with EGF . MATERIALS AND METHODS Cell CultureA-431 human epithelioid carcinoma cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) . Semiconfluent cultures were prepared in 35-mm Falcon dishes containing 22-mm square cover glasses . Scanning Electron Microscopy (SEM)Samples were processed for SEM by a modification of the OTOTO method (20) . Cells were washed rapidly with Dulbecco's phosphate-buffered saline (PBS) and fixed for 1 h with 4% glutaraldehyde in PBS . After three 10-min washes with 0.1 M sodium phosphate buffer (pH 7 .1), cultures were postfixed for 2 h with l% OS04 in the same buffer . Six distilled-water washes were followed successively by treatment with 1% thiocarbohydrazide (TCH) (30 min), 1% OS0 4 (2 h), 1% TCH (30 min), and 1% OS0 4 (2 h). Between the TCH and OS0 4 treatments, and after the final incubation with Os04, cultures were washed six times for 2 .5 min each with distilled water . The cultures on coverslips were then dehydrated through a graded ethanol series and critical-point dried from C02. Coverslips were cut into quarters, which were ...

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 the active participation of microfilaments in cell rounding . However, a medium transfer experiment suggests that EGFinduced 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 Ca'-free medium, as stimulation of such release by the ionophore 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 trypsin. 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 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.Cells grown in monolayer culture may become rounded in response to a wide variety of substances . These include hormones, cyclic nucleotides (23, 28, 29, 31, 43), proteases (12, 32, 36), and antisera raised against membrane proteins (13,41,42). In addition, cells normally round up during mitosis. Cell rounding presumably occurs because of reorganization of the cytoskeleton, but the biochemical mechanisms leading to such reorganization are not yet known.Several recent studies concerned with the mechanism of action of epidermal growth factor (EGF) have employed the human epidermoid carcinoma cell line A-431. The unusually high number of receptors for EGF present on these cells has facilitated visualization of the binding of EGF and clustering and endocytosis of the EGF/receptor complex (16,18,25), as well as demonstration of the rapid stimulation of the rates of fluid pinocytosis in whole cells (19) and protein phosphorylation in isolated membranes (6, 7) by EGF. In addition, we have recently reported that A-431 cells undergo a sequence of shape 422