We recently demonstrated serine/threonine phosphatase (protein phosphatase 2A, PP2A), a crucial enzyme in apoptosis control, within the plasma membrane as well as soluble fraction. This study aimed to determine whether gonadotropin-releasing hormone (GnRH) affects the membrane PP2A-associated apoptosis and the enzyme activity in ovarian cancer cells. PP2A activity was assessed by measuring the dephosphorylation of phosphopeptide highly selective for the PP2A in plasma membranes isolated from ovarian cancer SK-Ov-3 and Caov-3 cells. Apoptosis was quantified by nuclear morphology after staining with Hoechst 33342 and by loss of plasma membrane phospholipid asymmetry using Cy3-conjugated annexin-V. Treatment with doxorubicin (1 microM) for 48 h caused parallel increases in the percentage of cells undergoing apoptosis (71.3+/-5.5% vs. 2.2+/-0.8% for control, P<0.01) and decrease in the membrane-associated PP2A activity (to 38.5+/-8.2% of control, P<0.01). In cells simultaneously incubated with GnRH agonist leuprolide (1 microM) for 48 h, the maximum doxorubicine-induced apoptosis and membrane PP2A inhibition were reduced to 38.4+/-6.2% (P<0.01) and to 82.1+/-7.3% of control (P<0.01), respectively. The GnRH agonist alone caused apoptotic cell death accounting for only 6.8+/-2.1% (P<0.01) and had no effect on membrane PP2A activity. These findings demonstrate that PP2A inhibition is closely coupled to the onset of apoptosis in ovarian cancer cells exposed to doxorubicin. GnRH agonist may protect against the inhibition of membrane-associated PP2A activity and thus retard doxorubicin-induced apoptosis, suggesting an additional activity of GnRH to protect ovarian cancer cells from stimulated apoptotic cell death.