CHARACTERIZATION OF AUTOCRINE INDUCIBLE PROSTAGLANDIN-H SYNTHASE-2 (PGHS-2) IN HUMAN OSTEOSARCOMA CELLS

The human osteosarcoma 143.98.2 cell line was found to express high le vels of prostaglandin synthase-2 (PGHS-2) without detectable levels of prostaglandin synthase-1 (PGHS-1) as measured by reverse transcriptas e-polymerase chain reaction (RT-PCR) and immunoblot analysis. Maximal levels of PGHS-2 induction were attained when the cells were grown bey ond confluence. The osteosarcoma cells also secrete IL-1 alpha, IL-1 b eta and TNF alpha in the culture medium. PGHS-2 expression was inducib le by the exogenous addition of these cytokines as well as conditioned media from auto-induced cultures and inhibitable by treatment with de xamethasone. In contrast, undifferentiated U937 cells selectively expr ess PGHS-1 as analyzed by RT-PCR and Western blotting. The effects of non-steroidal anti-inflammatory drugs (NSAIDs) on the cellular PGE(2) production mediated by each isoform of human PGHS were determined usin g osteosarcoma and undifferentiated U937 cells. When cells were preinc ubated with inhibitors to allow time-dependent inhibition prior to ara chidonic acid stimulation, NS-398, CGP 28238, L-745,337, SC-58125 all behaved as potent (IC50 = 1 - 30 nM) and selective inhibitors of PGHS- 2, in contrast to indomethacin, flurbiprofen or diclofenac which are p otent inhibitors of both enzymes. DuP-697 and sulindac sulfide were al so potent inhibitors of PGHS-2 but both compounds inhibited cellular P GHS-1 activity at higher doses (IC50 = 0.2 - 0.4 mu M). Time-dependent inhibition of PGE(2) production in osteosarcoma cells was observed fo r indomethacin, diclofenac and etodolac. The synthesis of PGE(2) by U9 37 cells was strongly dependent on exogenous arachidonic acid (100-fol d stimulation) whereas confluent osteosarcoma cells also produced PGE( 2) without exogenous stimulus (7-fold stimulation by arachidonic acid) . Osteosarcoma cells grown beyond confluence released more PGE(2) from endogenous substrate than arachidonic acid stimulated undifferentiate d U937 cells. These results indicate that osteosarcoma cells selective ly express PGHS-2 with an autocrine regulation and effective utilizati on of endogenous arachidonic acid for PGE(2) synthesis.