Evaluation of the activity and localization of 15-Lipoxygenase-1 after introduction into human colorectal carcinoma Caco-2 cells

Abstract

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.