iloprost and Leukemia--Megakaryoblastic--Acute

Prostacyclin (prostaglandin I2, PGI2) is a potent vasodilator and inhibitor of platelet aggregation. Although it is well known that the specific receptor for prostacyclin (PGI2-R) is abundantly expressed on platelets, PGI2-R expression in megakaryocytes is poorly understood. In this study, we examined its expression in leukemic or normal megakaryocytes. PGI2-R mRNA was expressed in human leukemic cell lines of megakaryocytic nature as evaluated by Northern blot analysis. Phorbol 12-myristate 13-acetate (PMA), interleukin-1 (IL-1), IL-3, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), thrombopoietin (TPO), and tumor necrosis factor-alpha (TNF-alpha) enhanced PGI2-R mRNA expression. The enhancement of PGI2-R expression by PMA and TPO was associated with the upregulation of platelet factor 4 or glycoprotein IIb mRNA expression. Iloprost, an agonist of prostacyclin, induced significant cyclic (c)AMP synthesis in these leukemic cells indicating that interaction of PGI2-R and its ligand can induce postreceptor signal transduction. Furthermore, iloprost-induced cAMP synthesis was enhanced by the pretreatment with PMA or the cytokines that promoted PGI2-R expression. PMA and TPO also increased the specific binding of [3H]iloprost to these cells. Pooled normal megakaryocytic colonies from TPO-containing semisolid culture of purified human CD34+ cells expressed PGI2-R, which were increased as the megakaryocytes matured with the peak expression before proplatelet formation, as evaluated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). These results indicate that PGI2-R is expressed in human megakaryocytes and is upregulated by cytokines involved in thrombopoiesis or inflammation. Also, it was indicated that megakaryocytic maturation accompanies enhancement of PGI2-R expression.

Topics: Cell Differentiation; Cells, Cultured; Colforsin; Cyclic AMP; Gene Expression Regulation; Gene Expression Regulation, Leukemic; Granulocyte-Macrophage Colony-Stimulating Factor; Hematologic Neoplasms; Hematopoietic Stem Cells; HL-60 Cells; Humans; Iloprost; Interleukins; Leukemia, Megakaryoblastic, Acute; Megakaryocytes; Neoplasm Proteins; Platelet Factor 4; Platelet Glycoprotein GPIIb-IIIa Complex; Receptors, Epoprostenol; Receptors, Prostaglandin; Recombinant Proteins; RNA, Messenger; RNA, Neoplasm; Tetradecanoylphorbol Acetate; Thrombopoietin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

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