EFFECT OF RADICAL SCAVENGERS ON TNF-ALPHA-MEDIATED ACTIVATION OF THE UPA IN CULTURED-CELLS

Active oxygen, produced by cultured cells following stimulation with v arious growth factors, seems to be involved in signal transduction lea ding to cellular responses such as gene expression and growth modulati on. In the present study, the intracellular oxidation state was measur ed in immortalized human endothelial cells (ECV304) after treatment wi th tumor necrosis factor (TNF)alpha, using a fluorescent dye and a las er-scanning confocal microscope. The intracellular oxidation state was increased 60 min after the addition of TNF alpha, and this increase w as abolished by a radical scavenger, N-acetylcysteine (NAC), which is also a precursor of glutathione, and by pyrrolidine dithiocarbamate (P DTC). TNF alpha increased the steady state level of urokinase-type pla sminogen activator (uPA), and NAC inhibited this increase at a dose th at also inhibited the increase in the intracellular oxidation state. P DTC, on the other hand, did not affect the induction of the uPA gene b y TNF alpha. These results suggest that intracellular glutathione leve l rather than the oxidation state is necessary for the induction of th e uPA gene by TNF alpha.