prostaglandin-d2 and Leukemia--Megakaryoblastic--Acute

The cytosol fraction of human platelets did not convert prostaglandin (PG) H2 to PGD2. However, a homogenate of human megakaryoblastic CMK cells (precursor cells of platelets) produced PGD2 from PGH2. The PGD synthase activity was localized in the cytosol of CMK cells, and absolutely required glutathione. The catalytic properties and Western and Northern blottings indicated that the enzyme was PGD synthase of the hematopoietic type rather than the lipocalin type. When CMK cells were differentiated to megakaryocytes with phorbol ester along with induction of cyclooxygenase-1, the PGD synthase activity increased about 2-fold for 2 days and then decreased. In another human megakaryoblastic cell line, Dami, the PGD synthase increased about 10-fold by the addition of phorbol ester. Thus, the PGD synthase, which was undetectable in platelets, appeared during differentiation of megakaryoblasts to megakaryocytes.

Topics: Chromatography, Affinity; Glutathione; Humans; Intramolecular Oxidoreductases; Leukemia, Megakaryoblastic, Acute; Lipocalins; Megakaryocytes; Prostaglandin D2; Tetradecanoylphorbol Acetate; Thromboxane A2; Tumor Cells, Cultured

MEG-01s, an established human megakaryoblastic leukemia cell line, exhibited specific high-affinity binding sites for [3H]iloprost, a stable prostaglandin (PG) I2 analogue, for [3H]SQ-29548, a stable thromboxane (TX) A2 antagonist and, for [3H]PGE2/PGE1, but not for [3H]PGD2. In the MEG-01s cells, iloprost/PGI2, or PGE1 stimulated cAMP production with ED50 values practically identical to the IC50 values for the [3H] iloprost binding. STA2 and U46619, TXA2/PGH2 agonists, PGE2/PGE1, iloprost/PGI2, and thrombin elevated the intracellular concentrations of Ca2+ ([Ca2+]i), as determined by Fura-2 fluorescence signals. Elevation of [Ca2+]i by PGE2/PGE1 and iloprost, but not that by TX-agonists or thrombin, was totally dependent on the presence of extracellular Ca2+. This effect by PGE2/PGE1 was partially inhibited by prior treatment of the cells with islet-activating protein (IAP), while that by TX-agonists or by PGI2/iloprost was not affected. We tentatively conclude from these results that: (1) MEG-01s cells express (a) PGI2/PGE1 receptor(s) coupled to adenylate cyclase and Ca2+ influx, a TXA2/PGH2 receptor coupled to the phosphatidylinositol-turnover-Ca2+ system, and the PGE2/PGE1 receptor coupled to Ca2+ influx; (2) the receptors for TXA2/PGH2 and iloprost and those for PGE2/PGE1 and thrombin are coupled to IAP-insensitive and IAP-sensitive GTP-binding proteins, respectively, and function in a different manner to elevate [Ca2+]i. Thus, the MEG-01s cell line is a pertinent model for studying eicosanoid receptor-mediated signal transduction in platelet/megakaryocyte systems.

Topics: Adenylate Cyclase Toxin; Alprostadil; Blood Platelets; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Cyclic AMP; Cyclic GMP; Dinoprostone; Epoprostenol; Fatty Acids, Unsaturated; Humans; Hydrazines; Iloprost; Leukemia, Megakaryoblastic, Acute; Pertussis Toxin; Prostaglandin D2; Prostaglandins F; Receptors, Prostaglandin; Receptors, Thromboxane; Thrombin; Tumor Cells, Cultured; Virulence Factors, Bordetella