ADAPTATION OF EGF RECEPTOR SIGNAL-TRANSDUCTION TO 3-DIMENSIONAL CULTURE CONDITIONS - CHANGES IN SURFACE-RECEPTOR EXPRESSION AND PROTEIN-TYROSINE PHOSPHORYLATION

A431 cells grown as three-dimensional spheroids show growth stimulatio n in response to nanomolar concentrations of EGF in contrast to monola yer cultures that show inhibition. In investigating the alterations in EGF signal transduction that underlie this modification of the prolif erative response, we have compared the expression of EGF receptors on A431 cells under these conditions and related our findings to tyrosine phosphorylation and the growth response. EGF receptors were measured by I-125-EGF binding to trypsin-dispersed cells. Unexpectedly, dispers ion of the monolayers caused an 80% decrease in surface EGF receptor, although, after dispersion, EGF receptor was digested by trypsin with a half-life of 69 +/- 32 min. No evidence for a comparable loss of cel lular EGF receptor was seen on trypsin dispersion of spheroids. After allowing for this effect, we found that the receptor density on nondis persed monolayers (5 x 10(6) per cell) was twentyfold greater than tha t on spheroids (0.25 x 10(6) per cell). EGF-induced tyrosine phosphory lation was confined to the outermost cells of the spheroid, although t he presence of surface-expressed EGF binding sites could be demonstrat ed throughout the structure and the number of EGF receptors/cell on di spersed spheroid cells showed a single distribution peak by flow cytom etry, with no evidence for more than one population. Using RCM-lysozym e as a substrate, tyrosine phosphatase activity in spheroids lay withi n the range observed in monolayer cultures. Autophosphorylation of the EGF receptor following EGF stimulation in monolayer cultures of A431 cells rose rapidly in the first 10 seconds and then slowly increased f or at least 3 h. In spheroids, it reached a maximum within 10 seconds and then declined over 3 h. Since the microenvironment within a tumor resembles that in a spheroid, a similar reduction in surface EGF recep tor expression may be expected in tumors relative to monolayer culture s, together with corresponding growth stimulation in response to EGF. (C) 1994 Wiley-Liss, Inc.