ABSTRACT
BACKGROUND: Overexpression of cyclooxygenase-2 (COX-2) has been implicated in carcinogenesis of human colorectal cancer which is one of the leading types of cancer in Western countries. MATERIALS AND METHODS: We analyzed the COX-2 expression and activity using RT-PCR, Western blot, immunocytochemistry, RP-HPLC and EIA analysis in 0% and 10% fetal bovine serum (FBS) cultured cells. RESULTS: HT 29 cl.19A cells exhibited a COX-2 expression called "constitutive" in the absence of FBS in culture media. This particular expression was not the result of a mutation of the HT29 cl.19A COX-2 gene promotor. CONCLUSION: In our study, the human colon adenocarcinoma cell line, HT29 cl.19A, expressed COX-2 abnormally. This expression appeared to be at the same time inducible by the action of classical exogenous inducers such as FBS or interleukin-1 beta and "constitutive" if none of these compounds were present.
Subject(s)
Isoenzymes/biosynthesis , Prostaglandin-Endoperoxide Synthases/biosynthesis , Animals , Arachidonic Acid/metabolism , Cattle , Cell Division , Cloning, Molecular , Colorectal Neoplasms/enzymology , Culture Media , Culture Media, Serum-Free , Cyclooxygenase 2 , DNA Primers , HT29 Cells , Humans , Immunohistochemistry , Isoenzymes/genetics , Membrane Proteins , Polymerase Chain Reaction , Promoter Regions, Genetic , Prostaglandin-Endoperoxide Synthases/genetics , Recombinant Proteins/biosynthesisABSTRACT
5-Lipoxygenase products are pro-inflammatory mediators. Their roles and cellular origin in chronic inflammatory rheumatisms such as rheumatoid arthritis (RA) are poorly understood. The expression of arachidonate 5-lipoxygenase (5-LOX, arachidonate: oxygen 5-oxydoreductase; EC 1.13.11.34) and the 5-lipoxygenase activating protein (FLAP) genes in osteoarthritis and RA synoviocytes was studied at the transcriptional level by reverse transcription-polymerase chain reaction (RT-PCR) methodology. Arachidonic acid metabolism was analyzed by reverse-phase high pressure liquid chromatography. 5-LOX and FLAP mRNA were detectable using RT-PCR in all sources of synoviocytes tested. The expression of 5-LOX and FLAP mRNA led to the synthesis of 5-LOX metabolites. 12- and 15-LOX activities were also present. These LOX products can participate in inflammatory processes leading to joint destruction in RA.
Subject(s)
Arachidonate 5-Lipoxygenase/genetics , Arachidonic Acid/metabolism , Carrier Proteins/genetics , Membrane Proteins/genetics , Synovial Membrane/metabolism , 5-Lipoxygenase-Activating Proteins , Arachidonate 5-Lipoxygenase/metabolism , Arthritis, Rheumatoid/metabolism , Base Sequence , Calcimycin/pharmacology , Carrier Proteins/metabolism , Cells, Cultured , Chromatography, High Pressure Liquid , DNA Primers , Electrophoresis, Agar Gel , Fluorescent Antibody Technique, Indirect , Humans , Hydroxyeicosatetraenoic Acids/metabolism , Leukotrienes/metabolism , Membrane Proteins/metabolism , Molecular Sequence Data , Osteoarthritis/metabolism , Polymerase Chain Reaction , RNA, Messenger/genetics , RNA, Messenger/metabolism , Synovial Membrane/enzymologyABSTRACT
The expression of arachidonate 5-lipoxygenase (5-LOX, arachidonate: oxygen 5-oxidoreductase; EC 1.13.11.34) and the 5-lipoxygenase activating protein (FLAP) genes in lymphoblastoid B and T cell lines was studied at the transcriptional level by reverse transcription-PCR analysis. Two B cell lines, CESS and SKW 6.4, were found to express both 5-LOX and FLAP mRNA. One T cell line, MOLT 4, expressed only FLAP mRNA. Upon stimulation of intact cells by calcium ionophore, B lymphoblastoid cells produced 5, 12 and 15 hydroxyeicosatetraenoic acids as well as leukotriene B4.
Subject(s)
Arachidonate 5-Lipoxygenase/biosynthesis , B-Lymphocytes/metabolism , Carrier Proteins/biosynthesis , Membrane Proteins/biosynthesis , T-Lymphocytes/metabolism , 5-Lipoxygenase-Activating Proteins , Arachidonate 5-Lipoxygenase/genetics , Arachidonic Acid/metabolism , B-Lymphocytes/enzymology , Base Sequence , Carrier Proteins/genetics , Cell Line , Gene Expression , Humans , Membrane Proteins/genetics , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Messenger/analysis , T-Lymphocytes/enzymology , Transcription, GeneticABSTRACT
A new property of ursolic acid, lipoxygenase and cyclooxygenase inhibition, has been described in an acetone-extract of heather flowers (Calluna vulgaris) which could help explain the anti-inflammatory characteristics of this plant. In mouse peritoneal macrophages, human platelets and differentiated HL60 leukemic cells, ursolic acid, at 1 microM, blocks arachidonate metabolism.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Cyclooxygenase Inhibitors/pharmacology , Lipoxygenase Inhibitors/pharmacology , Triterpenes/pharmacology , Animals , Antineoplastic Agents, Phytogenic/chemistry , Blood Platelets/drug effects , Humans , Leukemia, Experimental , Macrophages/drug effects , Mice , Triterpenes/chemistry , Tumor Cells, Cultured/drug effects , Ursolic AcidABSTRACT
The most powerful technique for eicosanoids identification is gas chromatography prior to mass spectrometry. Results are highly dependent upon the choice of appropriate gas chromatographic systems; the critical point of the system being the quality of the column. Our work is intended to provide a general overview of open tubular capillary columns which can be used for separation, identification and quantification of eicosanoids.
Subject(s)
Fatty Acids, Unsaturated/metabolism , Arachidonic Acid , Arachidonic Acids/analysis , Arachidonic Acids/metabolism , Chromatography, Gas/methods , Chromatography, High Pressure Liquid , Fatty Acids, Unsaturated/analysis , Hydroxyeicosatetraenoic Acids/analysis , Quality ControlABSTRACT
Specific binding of mouse lymphocytes for 15 HETE was examined by incubating cells with [14C]-15 HETE, 1 X 10(-8) to 1 X 10(-10)M. It was observed that the specific binding of radiolabeled 15 HETE is a function of time, of temperature and is modified by Ca2+ and dithiothreitol. When a fluorescent probe was embedded in the phospholipid core of the lymphocyte membrane and its motion analysed by fluorescence polarization, it was observed that 15 HETE increases the viscosity of the plasmatic membrane.
Subject(s)
Arachidonic Acids/blood , Hydroxyeicosatetraenoic Acids , Lymphocytes/metabolism , Membrane Fluidity , Animals , Calcium/pharmacology , Dithiothreitol/pharmacology , Fluorescence Polarization , Mice , Temperature , Time FactorsABSTRACT
We have investigated the in vivo effects of 15 HETE on C57Bl/6 (H-2b) mice injected IP daily with this product. After that the 15 HETE treated animals and the controls were challenged in vivo by DBA/2 (H-2d) cells. Splenocytes from 15 HETE injected animals were either stimulated in vitro by lectins or cocultivated with DBA/2 irradiated splenocytes. It was observed that the response of splenocytes from in vivo treated animals is weaker than the control's response. The data suggest that 15 HETE induce the generation of suppressor cells.
Subject(s)
Arachidonic Acids/pharmacology , Hydroxyeicosatetraenoic Acids , Immunosuppression Therapy , Animals , Concanavalin A/pharmacology , Flow Cytometry , Lymphocytes/drug effects , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Phytohemagglutinins/pharmacologyABSTRACT
Arachidonic acid can be transformed into a series of HPETEs by the lipoxygenase enzyme activity of mouse peritoneal macrophages. These resulting HPETEs inhibit some mouse lymphocyte responses. When mice are injected with 15-L-HPETE, their splenocytes show a decreased [3H]thymidine uptake after lectin stimulation.
