Figure 3
Cytostatic activity of IFN-γ, NG-hydroxy-L-arginine and the effect of polyamines on IFN-γ-induced cytostasis. NMP-1 cells were seeded at 104 cells/well into 96-well plates in RPMI 1640 with 10% fetal calf serum and cultured overnight. The next day, the cells were washed with phosphate buffered salines, and the medium was replaced with a low-arginine, serum-free medium (5% bovine serum albumin, 1.5% Iscove's modified Dulbecco's medium [GIBCO], 0.2% glucose, 0.01% magnesium sulfate, 0.016% calcium chloride, transferrin 5.5 μg/ml, and insulin 4 μg/ml in Hank's balanced salt medium [GIBCO], pH 7.4) alone or with rIFN-γ (500 U/ml). The arginine concentration of this medium was 6 μM, lower than plasma concentration (normal range, 50–126 μM [38]. After another 24 hours, rIFN-γ (500 U/ml), NG-hydroxy-L-arginine (250 μM), or rIFN-γ plus ornithine (1 mM), putrescine (1 mM), or spermidine (0.5 mM) was added. The cells were then pulsed with [3H] thymidine and incubated for another 24 hours. The cytostatic activity was determined as described in Materials and Methods. Similar results were observed with other EOC cell lines and when experiments were done with RPMI 1640 supplemented with 10% fetal calf serum. Data shown are representative of 3 separate experiments.