SUSTAINED ACTIVATION OF PROTEIN-KINASE-C IS ESSENTIAL TO HL-60 CELL-DIFFERENTIATION TO MACROPHAGE

Although a single dose of phorbol 12-myristate 13-acetate (PMA) allowed HL-60 cells to differentiate to macrophages, a single dose of membrane-permeant diacylglycerol (DAG), 1,2-dioctanoylglycerol (1,2-DiC8), was normally insufficient to differentiate these cells. These cells metabolized 1,2-DiC8 very rapidly, and 1,2-DiC8 available to protein kinase C (PKC) activation was removed from the incubation medium at a rate proportional to cell density. However, increasing the duration of exposure of HL-60 cells to this DAG either by its repeated addition or by decreasing the cell density greatly enhanced their differentiation to macrophages as measured by CD11b expression. During this differentiation induced by DAG, neither measurable translocation nor depletion (down-regulation) of PKC was observed. When the cells were exposed to PMA, on the other hand, some PKC subspecies were instantaneously translocated to membranes and subsequently disappeared very quickly, whereas the alpha-subspecies was decreased to the level of almost-equal-to 60% of the resting cell, but thereafter its activity was maintained at a nearly constant level in membranes. After almost-equal-to 4 hr, the PKC subspecies, once depleted, reappeared gradually in the membrane fraction. The results suggest that sustained activation of PKC is essential to differentiation of HL-60 cells to macrophages, and depletion of the enzyme is not needed. Perhaps translocation of PKC represents an extreme state of the active form of the enzyme, which may result from PMA action, and the alpha-subspecies presumably plays a key role in HL-60 cell differentiation.