Rapid induction of morphological changes in human carcinoma cells A-431 by epidermal growth factors.

Abstract

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 ...

Pit formation and rapid changes in surface morphology of sympathetic neurons in response to nerve growth factor.

“…Such changes were initiated within minutes of exposure to NGF and far preceded subsequent responses such as initiation of neurite outgrowth and alterations in protein synthesis. Similar types of surface changes have also been reported (2) …”

“…Extensive surface architectural changes induced within seconds of peptide treatment are not limited to NGF . Human carcinoma A-431 cells show extensive ruffling and extension of filopodia possibly beginning as early as 30 s and subsiding within 5-15 min after treatment with epidermal growth factor (EGF) (2). Also, recent studies indicate that cultured thyroid epithelial cells display pit formation within 30 min of exposure to thyroid-stimulating hormone?…”

Simple method for quantification of fast plasma membrane movements

“…F-LD 3210. 4 and F-LD 2010.25 objectives were used in combination with a long working distance condensor (Zeiss No. 465224).…”

Growth Factor‐induced Proliferative Responses of Human and Dmh‐induced Rat Colorectal Tumour Cell Lines

“…The peptide mEGF is known to influence the in vitro growth of both normal and malignant cells (Cohen, 1965;Chinkers, McKanna and Cohen, 1979). In addition, the reports of Li et aL (1982) suggest that this peptide influences the development of rat colonic tumours in vivo.…”