Eicosanoid forming enzyme mRNA in human tissues. Analysis by quantitative polymerase chain reaction.
The key enzymes in the formation of eicosanoids, including leukocyte 5-lipoxygenase (5LX), platelet 12-lipoxygenase (12LX), reticulocyte 15-lipoxygenase (15LX), prostaglandin G/H synthase cyclooxygenase, and leukotriene A4 (LTA) hydrolase have been studied extensively in recent years. Little is known, however, about the regulation of these enzymes at the gene level. We have developed a quantitative polymerase chain reaction (PCR) assay to quantify the mRNAs for these five enzymes, as well as for cytoplasmic beta-actin (bACT) mRNA. Human erythroleukemia (HEL) cells, which display megakaryocytic/erythroid characteristics, were selected as a source of RNA to characterize the assay. These cells expressed mRNA for bACT, LTA, cyclooxygenase, and 12LX (in decreasing order). mRNA for 5LX and 15LX was undetectable. Bronchoalveolar lavage fluid cells obtained from asthmatic patients, primarily alveolar macrophages, contained mRNA for bACT, LTA, 5LX, cyclooxygenase, and 15LX (in decreasing order). Treatment of HEL cells with phorbol 12-myristate 13-acetate or steroid administration to asthmatic patients apparently selectively regulated certain of these target genes. The utility of this assay in quantifying mRNA for the various target genes in blood cells, including platelets from patients with chronic myelogenous leukemia, has also been demonstrated. Studies on the regulation of genes for enzymes involved in the leukotriene and prostaglandin biosynthetic pathways, especially when only small tissue samples are available, will be facilitated with this approach.
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Division of Clinical Pharmacology, Vanderbilt University, Nashville, Tennessee 37232.
FitzGerald
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D000199:Actins / Q000032:analysis; D001092:Arachidonate 12-Lipoxygenase / Q000032:analysis; D001093:Arachidonate 15-Lipoxygenase / Q000032:analysis; D001094:Arachidonate 5-Lipoxygenase / Q000032:analysis; D001249:Asthma / Q000378:metabolism; D001483:Base Sequence; D001792:Blood Platelets / Q000201:enzymology; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry; D004851:Epoxide Hydrolases / Q000032:analysis; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism; D007962:Leukocytes / Q000201:enzymology; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011451:Prostaglandin-Endoperoxide Synthases / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis*; D012156:Reticulocytes / Q000201:enzymology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects
D000199:Actins; D012333:RNA, Messenger; D001092:Arachidonate 12-Lipoxygenase; D001093:Arachidonate 15-Lipoxygenase; D001094:Arachidonate 5-Lipoxygenase; D011451:Prostaglandin-Endoperoxide Synthases; D004851:Epoxide Hydrolases; D013755:Tetradecanoylphorbol Acetate; C043134:leukotriene A4 hydrolase