Effect of docosahexaenoic acid on ras post-translational processing and localization in a transgenic mouse colonic cell line

Abstract

The acquisition of a chronically activated ras gene is a critical early step in colon cancer development, with membrane localization being a prerequisite for malignant transformation. Fish oil supplemented diets, containing docosahexaenoic acid (DHA), compared to corn oil supplemented diets, containing linoleic acid (LA), lower tumor incidence and decrease membrane ras localization in the azoxymethane-injected rat colon model. Herein, we examine the mechanism by which fish oil decreases ras membrane localization and determine its effect on ras activity. Young adult mouse colon cells over-expressing H-ras (YAMC-ras), were grown for 72 h in media containing either 50 []M LA or DHA or control. Cell lipid analysis demonstrated enrichment of DHA or LA metabolites into membrane phospholipids. Cell number was significantly reduced (p<0.005) by DHA incubation compared to LA and untreated media only (control) with no effect on viability. Immunoblotting showed that total ras expression was not significantly altered (P>0.05) by fatty acid treatment. Ras membrane to cytosol ratio, an indication of ras localization, was lower (P<0.0001) in DHA treated cells (4.87) compared to LA treatment (8.31). Ras subcellular localization was also assessed using confocal immunofluorescence. Since post-translational processing is necessary for membrane localization, key steps involving HMG CoA reductive expression, ras farnesylation, and palmitoylation were examined. No effect by fatty acid treatment (p>0.05) was found. To study the effect of decreased membrane localization on ras activity, the percentage of GTP relative to GDP bound ras was measured in subcellular fractions. Following metabolic labeling with ³²P-orthophosphate, DHA compared to LA significantly reduced the level of active (GTP bound) ras in the membrane (18.8% vs. 25.3% respectively, p<0.005). In summary, the decreased ras membrane to cytosol ratio in DHA treated colon cells is associated with decreased GTP binding in the absence of any effect on post-translational processing. Altered cell membrane composition or issuance on ras nucleotide exchange due to DHA treatment may be responsible for the decreased membrane association and GTP binding by ras.