Role of endogenous prostaglandin E2 in erythropoietin production and dome formation by human renal carcinoma cells in culture

Abstract

Studies were carried out on the role of endogenous prostaglandin E2 (PGE2) in erythropoietin (Ep) production and dome formation in primary monolayer cultures of a human renal carcinoma from a patient with erythrocytosis that has been serially transplanted into BALB/c athymic nude mice. The metabolism of [14C]arachidonic acid (14C-AA) by cultured renal carcinoma cells, which were plated in 25-cm2 flasks at a density of 2 x 104 cells/cm3 and grown for 6, 12 (confluence, 13 x 104 cells/cm2), 16, 24, and 30 d in Eagle's minimum essential medium (MEM) supplemented with 10% fetal bovine serum, was examined by using radiometric thin-layer chromatography (TLC). TLC revealed PGE2 to be the major metabolite of 14C-AA produced by the cultured cells throughout the 30 d of cultivation. In addition,the cultured cells at each time period were incubated for 24 h in 5 ml of serum-free Eagle's MEM and the levels of PGE2 and Ep in the incubated media were measured via radioimmunoassay. PGE2 levels in the serum-free media incubated with the cultured cells grown for 6 d were significantly (P < 0.001) elevated (174 ± 2.5 pg/ml, n = 5), compared with the unincubated control media (1.5 ± 0.19 pg/ml, n = 5) and gradually decreased at each time period to 97.6 ± 4.4 pg/ml (n = 5) at 30 d. On the other hand, the levels of EP in the incubated media of the cells grown for 6 d were 11.5 ± 0.52 mU/ml (n = 5) compared with 7.6 ± 0.62 mU/ml (n = 5) in the control media. However, after the cultured cells became confluent, the levels of EP in the incubated media showed a marked increase to 222.9 ± 5.26 mU/ml (n = 5) at 30 d of cultivation. Multicellular hemicysts (domes) developed after the cultured cells reached confluence and their numbers increased with increasing time in confluence in parallel with the increase in Ep. Meclofenamate (MF) (3 x 10-6-3 x 10-5 M), a prostaglandin synthesis inhibitor, produced a significant dose-related decrease in PGE2, Ep, and dome formation without producing a significant effect on cell viability in the 30-d cells. This inhibitory effect of MF on Ep production and dome formation was completely abolished by the addition of 10-8 M PGE2 to the incubation medium. In conclusion, endogenous PGE2 plays an important role in supporting and/or stimulating EP production and dome formation in cultured renal carcinoma cells.