Evaluation of the activity and localization of 15-lipoxygenase-1 after introduction into human colorectal carcinoma Caco-2 cells.
In human colorectal carcinoma Caco-2 cells, sodium butyrate (NaBT) induces the expression of the reticulocyte, 15-lipoxygenase-1 (15-LO-1) and causes these cells to undergo differentiation and apoptosis. 15-LO-1 is also expressed in human colorectal epithelium with a significant higher expression observed in colorectal tumors. In this study, we have prepared stable Caco-2 cells that expressed 15-LO-1 under control of an inducible promoter. These cells provide a model system to study regulation of 15-LO-1 activity in colorectal cells without the interfering presence of NaBT and are useful to study the biological function of 15-LO-1. The expressed 15-LO-1 was highly active as measured in cell lysates, but we were unable to detect metabolism in intact cells. The addition of calcium to the media for the Caco-2 cells was required for 15-LO-1 to translocate from the cytosol to the membrane which is frequently a requirement for lipoxygenase activity. Despite the addition of calcium and translocation, little lipoxygenase activity was detected with intact cells. However, after removal of phenol red, a common constituent of cell culture media, we were able to detect 15-LO-1 activity in the transfected Caco-2 cultured cells. Thus the presence of calcium and the absence of antioxidants present in commonly used culture media are required for expressed 15-LO-1 to be catalytically active and to permit an examination of its biological effects.
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Eicosanoid Biochemistry Section, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
Kamitani
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Cornicelli
J A
JA
Eling
T E
TE
D001093:Arachidonate 15-Lipoxygenase / Q000096:biosynthesis*; D016718:Arachidonic Acid / Q000378:metabolism; D015152:Blotting, Northern; D015153:Blotting, Western; D018938:Caco-2 Cells; D002118:Calcium / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D015179:Colorectal Neoplasms / Q000201:enzymology*; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D019787:Linoleic Acid / Q000378:metabolism; D008084:Lipoxygenase / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D010637:Phenolsulfonphthalein / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D021381:Protein Transport; D013347:Subcellular Fractions; D013997:Time Factors; D014407:Tumor Cells, Cultured
D016718:Arachidonic Acid; D019787:Linoleic Acid; D008084:Lipoxygenase; D001093:Arachidonate 15-Lipoxygenase; D010637:Phenolsulfonphthalein; D002118:Calcium