Culture of A-431 human epidermoid carcinoma cells in serum-free medium: effect of culture conditions on the binding of [125I]-epidermal growth factor.

Abstract

Serum-free culture conditions that permit the continuous growth of A-431 human epidermoid carcinoma cells were developed. In Dulbecco's modified Eagle's synthetic nutritional medium (DME) supplemented with fetuin, insulin, transferrin, biotin, and oleic acid-fatty acid-free bovine serum albumin complex A-431 cells grew at a rate comparable to that observed in the presence of calf or fetal calf serum. Of the factors tested, oleic acid had the most pronounced stimulatory effect on the growth and [3H]-thymidine incorporation of A-431 cells in serum-free medium. A-431 cells have a high number of receptors for epidermal growth factor (EGF); they bind and rapidly internalize EGF. Nevertheless, EGF did not stimulate either the growth or the [3H]-thymidine incorporation of these cells. Analyses of [125I]-EGF binding data indicated that A-431 cells grown in the presence of calf serum had about 3.2-3.9 X 10(6) specific, saturable EGF receptor sites on their surface. Linear Scatchard plots indicated a single class of noninteracting receptors with an apparent equilibrium dissociation constant of about 2.8 X 10(-9) M. The average number of receptors of A-431 cells maintained in DME supplemented with only fetuin, insulin, and transferrin for several months was significantly less, 1.54 X 10(6), than that of A-431 stock cells cultured in the same medium for 2 days only (2.68 X 10(6)). The apparent dissociation constants for the same cell populations were, however, similar, 4.5 X 10(-9) M and 4.1 X 10(-9) M, respectively. Stimulation of growth by oleic acid resulted in about 20% decrease in the average number of receptor sites, with an increase in the apparent equilibrium dissociation constant.