12-hydroxy-5-8-10-14-eicosatetraenoic-acid and Leukemia--Erythroblastic--Acute

Haploinsufficiency of RUNX1 (also known as CBFA2/AML1) is associated with familial thrombocytopenia, platelet dysfunction, and predisposition to acute leukemia. We have reported on a patient with thrombocytopenia and impaired agonist-induced aggregation, secretion, and protein phosphorylation associated with a RUNX1 mutation. Expression profiling of platelets revealed approximately 5-fold decreased expression of 12-lipoxygenase (12-LO, gene ALOX12), which catalyzes 12-hydroxyeicosatetraenoic acid production from arachidonic acid. We hypothesized that ALOX12 is a direct transcriptional target gene of RUNX1. In present studies, agonist-induced platelet 12-HETE production was decreased in the patient. Four RUNX1 consensus sites were identified in the 2-kb promoter region of ALOX12 (at -1498, -1491, -708, -526 from ATG). In luciferase reporter studies in human erythroleukemia cells, mutation of each site decreased activity; overexpression of RUNX1 up-regulated promoter activity, which was abolished by mutation of RUNX1 sites. Gel shift studies, including with recombinant protein, revealed RUNX1 binding to each site. Chromatin immunoprecipitation revealed in vivo RUNX1 binding in the region of interest. siRNA knockdown of RUNX1 decreased RUNX1 and 12-LO proteins. ALOX12 is a direct transcriptional target of RUNX1. Our studies provide further proof of principle that platelet expression profiling can elucidate novel alterations in platelets with inherited dysfunction.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate 12-Lipoxygenase; Arachidonic Acid; Base Sequence; Binding Sites; Blood Platelets; Cell Line, Tumor; Chromatin Immunoprecipitation; Consensus Sequence; Core Binding Factor Alpha 2 Subunit; Electrophoretic Mobility Shift Assay; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Genes, Reporter; Haploidy; Humans; Leukemia, Erythroblastic, Acute; Luciferases; Molecular Sequence Data; Platelet Activation; Promoter Regions, Genetic; Protein Binding; RNA, Small Interfering; Tetradecanoylphorbol Acetate; Thrombin

Cytosol and nuclei of Lewis lung carcinoma (LLC) cells contain high affinity binding sites specific for the arachidonic acid metabolite 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE). In this report we present evidence that the cytosolic 12(S)-HETE binding complex also occurs in human erythroleukemia (HEL) and promonocytic leukemia (U937) cells as well as in murine 3T3-L1 preadipocytes but not in intestinal epithelial cells (Int407). The cytosolic 650 kDa 12(S)-HETE-binding complex was found to consist of subunits; raising the ATP concentration in cytosol led to conversion of the 650 kDa complex to a 50 kDa binding component, presumably the actual 12(S)-HETE binding polypeptide. Lowering of the cytosolic concentration of ATP had the opposite effect, i.e., the amount of the 650 kDa complex increased. Another subunit of the 650 kDa complex was identified as heat shock protein 70 (hsp70) by Western blot analyses and coimmunoprecipitation. Hsp70 was present in substoichiometric amounts, in an approximate 1:6 ratio. The multimeric nature of the binding complex and the identification of hsp70 as a subunit suggest that there are similarities between the 12(S)-HETE binding protein and receptors of the steroid/thyroid hormone superfamily.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 3T3 Cells; Adenosine Triphosphate; Animals; Blotting, Western; Cell Line; Cell Nucleus; Cytosol; Epithelial Cells; HSP70 Heat-Shock Proteins; Humans; Immunosorbent Techniques; Intestinal Mucosa; Leukemia, Erythroblastic, Acute; Leukemia, Monocytic, Acute; Mice; Neoplasms; Receptors, Eicosanoid; Tumor Cells, Cultured