| Research Abstract |
theta-Toxin is a cholesterol-binding, pore-forming cytolysin of Clostridium perfringens. To detect cell surface cholerterol, we prepared a theta-toxin derivative, BCtheta by biotinylation of a protease-nicked theta-toxin, which has the same binding affinity for cholesterol as theta-toxin without cytolytic activity. Human erthyrocytes, V79 cells and human umbilical vein endothelial cells (HUVEC), were stained with BCtheta coupled with FITC-avidin, and then the cell were analyzed by either flow cytometry or laser confocal microscopy. Treatment of the cells with digitonin, a cholesterol-sequestering reagent, decreased the fluorescence intensity to the background level, indicating that BCtheta staining is specific for choresterol, The fluorescence intensity of erthrocytes pre-permeabilized with a small amount of theta-toxin increased more than ten-fold, suggesting higher cholesterol contents in the inner layer of the plasma membrane. When cells were cultured with cholestrol-depleted medium
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, the fluorescence intensity stained by BCtheta decreased remarkably in V79 cells, but did not change in HUVEC.This indicates that cell surface cholesterol may be provided in different ways with these two cell lines. These results suggest that BCtheta can be a useful probe for visualizing cell surface cholesterol and for evaluating the effects of cellular events on me topology and distribution of cholesterol. Next the membrane fluidity of the cholestrol-poor bovine carotid artery endothelial cells (BAEC) was examined. Chorestrol-poor BAEC were obtained by treating the cells with 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors under 10% low density lipoprotein (LDL)-deficient serum condition for 2 days. Simvastatin reduced the intracellular cholesterol content significantly at a concentration of 0.1 mug/ml. The reduction in the cholesterol content was accompanied by the enhancement of the cell membrane fluidity which was measured by a photobleaching technique. Additional data suggested that the reduction in cholesterol content referred to the reduction in the proliferation of BAEC.Both VEGF and bFGF activated mitogen-activated protein (MAP) kinase in BAE cells, however, EPA selectively inhibited VEGF-induced, but not bFGF-induced activation of MAP kinase. Flk-1 expression was inhibited dose-dependently in EPA-treated cells whilst Flt-1 expression was increased in EPA treated cells. This in vitro inhibitory effect by EPA on Flk-l receptor expression provides indirect evidence that one of the mechanisms of EPA for anti-tumor action in vivo may be related to its anti-angiogenic action. Less
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