The NBT-II rat carcinoma cell line exhibits two mutually exclusive responses to FGF-1 and EGF, entering mitosis at cell confluency while undergoing an epithelium-to-mesenchyme transition (EMT) when cultured at subconfluency. EMT is characterized by acquisition of cell motility, modifications of cell morphology, and cell dissociation correlating with the loss of desmosomes from cellular cortex. The pleiotropic effects of EGF and FGF-1 on NBT-II cells suggest that multiple signaling pathways may be activated. We demonstrate here that growth factor activation is linked to at least two intracellular signaling pathways. One pathway leading to EMT involves an early and sustained stimulation of pp60c-src kinase activity, which is not observed during the growth factor-induced entry into the cell cycle. Overexpression of normal c-src causes a subpopulation of cells to undergo spontaneous EMT and sensitizes the rest of the population to the scattering activity of EGF and FGF-1 without affecting their mitogenic responsiveness. Addition of cholera toxin, a cAMP-elevating agent, severely perturbs growth factor induction of EMT without altering pp60c-src activation, therefore demonstrating that cAMP blockade takes place downstream or independently of pp60c-src. On the other hand, overexpression of a mutated, constitutively activated form of pp60c-src does not block cell dispersion while strongly inhibiting growth factor-induced entry into cell division. Moreover, stable transfection of a dominant negative mutant of c-src inhibits the scattering response without affecting mitogenesis induced by the growth factors. Altogether, these results suggest a role for pp60c-src in epithelial cell scattering and indicate that pp60c-src might contribute unequally to the two separate biological activities engendered by a single signal.

Changes of cell morphology and the state of differentiation are known to play important roles in embryogenesis as well as in carcinogenesis. Examples of particularly profound changes are the conversions of epithelial to mesenchymal cells; i.e., the dissociation of some or all polygonal, polar epithelial cells and their transformation into elongate, fibroblastoid cells of high motility. As an in vitro model system for such changes in cell morphology, we have used cell cultures of the rat bladder carcinoma-derived cell line NBT-II which, on exposure to inducing medium containing a commercial serum substitute (Ultroser G), show an extensive change in their organization (epithelial-mesenchymal transition): the junctions between the epithelial cells are split, the epithelial cell organization is lost, and the resulting individual cells become motile and assume a spindle-like fibroblastoid appearance. Using immunofluorescence microscopy and biochemical protein characterization techniques, we show that this change is accompanied by a redistribution of desmosomal plaque proteins (desmoplakins, desmoglein, plakoglobin) and by a reorganization of the cytokeratin and the actin-fodrin filament systems. Moreover, intermediate-sized filaments of the vimentin type are formed in the fibroblastoid cells. We demonstrate that the modulation of desmosomal proteins, specifically an increase in soluble desmoplakins, is a relatively early event in cell dissociation and in epithelial-mesenchymal transition. In this process, a latent period of 5 h upon addition of inducing medium precedes the removal of these desmosomal components from the plasma membrane. The transition, which is reversible, is dependent on continued protein synthesis and phosphorylation but not on the presence of the inducing medium beyond the initial 2-h period. We discuss the value of this experimental system as a physiologically relevant approach for studying the regulation of the assembly and disassembly of desmosomes and other intercellular adhesion structures, and as a model of the conversion of cells from one state of differentiation into another.