Roles of calcium in aggregation of rat ascites hepatoma cells by cell surface-associated adhesive factor

Our previous studies have shown that a cell surface-associated adhesive factor (AF), separated from rat ascites hepatoma AH136B cells of a differentiated type and highly purified by chromatography, induces not only aggregation of dissociated AH136B cells or rat ascites hepatoma AH109A cells of an undifferentiated type but also adhesiveness characterized by the development of junctional complexes; the AF-induced aggregation of the cells was Ca2+-dependent. Further analysis of the roles of Ca2+ in cell aggregation was performed using AH109A cells (present as single cells in vivo). (1) AF clearly enhanced 45Ca uptake by the cells; (2) calmodulin was isolated from the cells; (3) calmodulin inhibitor, W-7 (N-(6-amino-hexyl)-5-chloro-1-naphthalenesulphonamide), strongly inhibited aggregation of the cells; (4) W-7 also inhibited the clustering or capping of AF-binding sites on the cell surface; (5) binding of 125I-labelled AF to the cells was independent of Ca2+ concentration; (6) binding of 125I-labelled AF to AF-conjugated beads was not observed, independently of the presence of Ca2+. These findings suggest that Ca2+ and Ca2+-activated calmodulin may play a key role in the process of aggregation of the cells by controlling the microfilament components and that Ca2+ may not be involved either in the interactions between AF and its cellular receptor or in linkages of AF molecules.