DEXAMETHASONE-INDUCED EXPRESSION OF PHOSPHATASE INHIBITS GENERATION OF REACTIVE OXYGEN SPECIES IN EHRLICH ASCITES TUMOR-CELLS

Both pre-activated and phorbol ester tetradecanoyl phorbol myristate a cetate (TPA) activated reactive oxygen species (ROS) generation were i nhibited by dexamethasone in vivo. Time kinetics on influence of dexam ethasone on cytosolic phosphoprotein phosphatase activity revealed tha t, when compared to phosphatase activity in cytosol of control Ehrlich ascites tumor (EAT) cells, a 5-fold increase in specific activity is seen in the cytosol of EAT cells treated (in vivo, 0-90 min. 1 mg/kg b ody weight) with dexamethasone. Dexamethasone induced phosphatase was partially purified by conventional ion-exchange and gel filtration col umn chromatographic techniques. Purified phosphatase had a molecular w eight of 70 KDa by SDS-PAGE. A dose-dependent inhibition of TPA activa ted ROS generation by partially purified phosphatase in permeabilized EAT cells suggested that dephosphorylation is a major regulatory mecha nism in ''switching off'' of the respiratory burst. Anti-phosphatase a ntibodies were raised, purified and were used to quantitate cytosolic phosphatase by ELISA, which revealed that dexamethasone induces 6-fold increase in expression of phosphatase in EAT cells by 120 min. The ex pression of phosphatase in EAT cell cytosol was further confirmed by i mmunostaining using anti-phosphatase antibodies, the results of which showed intense blue staining on development with BCIP/NBT.