Enhancement of phorbol ester induced cell aggregation after alterations in asparagine-linked oligosaccharides

Abstract

Human erythroleukemia (K-562) cells grown in the presence of phorbol 12,13-dibutyrate formed aggregates of cells not seen in untreated control cultures. Furthermore, the proportion of cells in aggregates and the size of the aggregates both increased dramatically in cultures treated with both phorbol ester and kifunensine, an inhibitor of asparagine-linked oligosaccharide processing. Relative to control cells, phorbol ester treated cells exhibited a greater proportion of N-linked oligosaccharides of the complex-type. Kifunensine prevented this change and caused an accumulation of Man9GlcNAc2. The enhanced aggregation of cells treated with phorbol ester plus kifunensine depended on phorbol ester concentration and was blocked by inhibitors of protein kinase C (H7, sphinganine and sangivamycin). In flow cytometry analysis, phorbol ester treated K-562 cells showed an increase in CD44, a glycoprotein involved in cell adhesion. Moreover, monoclonal antibody to CD44 augmented reaggregation of phorbol ester treated cells. The results implicate phorbol ester induction of CD44 in aggregation of K-562 cells and demonstrate that the presence of high mannose-type asparagine-linked oligosaccharides on cell glycoproteins correlates with increased aggregation of phorbol ester treated cells.