We have investigated the regulation of adhesion of metastatic human breast carcinoma cells to various protein substrates in the presence of the protein kinase C (PKC) activator TPA, or calcium ionophore A23187.Both TPA and A23187 dramatically enhanced MDA-MB-435 cell adhesion to type IV collagen, vitronectin, and, to some extent, fibronectin and laminin. Adhesion to bovine serum albumin and polylysine were not affected.TPA and A23187 induced substantial dose-dependent effects which were apparent after 30 min and 60 min incubations.A23187, but not TPA, induced a release of arachidonic acid (AA) from MDA-MB-435 cells.NDGA, a lipoxygenase inhibitor, prevented A23187 and exogenous AA, but not TPA, from stimulating cell adhesion to collagen IV. Increase in adhesion to vitronectin induced by A23187 and AA was only partially inhibited by NDGA treatment. Calphostin C, a PKC inhibitor, blocked the stimulation of adhesion by A23187, exogenous AA and TPA to both collagen IV and vitronectin. Results suggest that calcium mobilization activates the release of AA and its metabolism through a lipoxygenase pathway leading to a rapid increase of cell adhesion to collagen IV, whereas other mechanisms regulate adhesion to vitronectin. Finally, PKC activation, occurring downstream from calcium mobilization or the AA effects, is a key event involved in the regulation of adhesion to both proteins. The novel PKC epsilon and mu isosymes were found to translocate to the membrane fraction following treatment with arachidonic acid. In addition, specific signal transduction proteins are phosphorylated in responce to treatment with fatty acids. We are currently extending these studies to examine breast carcinoma cell spreading and migration on extracellular matrix proteins.
