INDUCTION OF PLASMINOGEN ACTIVATOR AND PLASMINOGEN ACTIVATOR INHIBITOR ACTIVITY DURING DIFFERENTIATION OF HL-60 CELLS

Abstract

While increased production of plasminogen activator(PA) has been associated with malignant growth and differentiation, the effect of cell maturation on plasminogen activator inhibitor (PAI) production has not yet been defined. We therefore determined the effect of phorbol myristate acetate(PMA), an agent that induces monocytoid cellular maturation, on the secretion of both PA and PAI in HL-60 cells, a promyelocytic leukemia cell line. Cells were seeded at 5x105/ml and grown in a 5% CO2 incubator at 37°C in a serum-free medium in the absence or presence of 1.6 nM PMA. Experiments were performed either in polypropylene tubes to which cells did not adhere, or in petri dishes that provided surfaces for cellular adherence. Supernatants were assayed for PA activity with a functional chromogenic assay using plasminogen and S-2251 with urokinase (UK) as a standard. PAI was measured by determining the inhibition of exogenous UK (0.05 U/ml final) after incubation with the supernatants at 22°C for 30 min. In the absence of PMA, supernatants of control cells grown in tubes or petri dishes had a PA activity of 2 mU/106 cells at 72 hr. After exposure to PMA for 72 hr, PA activity in supernatants of non-adherent cells grown in tubes increased 50-fold over control, while only an 8-fold increase was measured in supernatants of cells that had been incubated in petri dishes and allowed to undergo adherence. The PA had a Mr of 56 kD as determined by fibrin zymography, and functional activity was completely inhibited by antibodies to UK. While there was no inhibition of exogenous UK in supernatants of control cells grown in tubes or petri dishes, there was a 50% inhibition of UK added to the supernatants of the PMA-treated cells grown under non-adherent conditions in tubes. Inhibition of exogenous UK was 94% by supernatants from PMA-treated cells that had undergone adherence in petri dishes. The inhibitory activity was completely abolished by incubation of the supernatants with IgG purified from antiserum to PAI-2 (courtesy Dr. Kruithof). These data indicate that PMA induces the production of UK-like PA and also an inhibitor similar to PAI-2, which has been found in monocytes. Furthermore, the regulation of both PA and PAI production appears to depend on conditions that affect cellular adherence.