Effect of membrane lipid alteration on the growth, phospholipase C activity and G protein of HT-29 tumor cells

The objective of the present study was to examine the effect of modifying the fatty acid composition of membranes on cell growth and phosphoinositide specific phospholipase C (PLC) activity in HT-29 colon cancer cells. Cells were seeded at a density of 12x10(3) cells/cm(2) and supplemented with 30 mu M of either 18:0, 18.2 (n6) or 18:3 (n3) complexed to bovine serum albumin (BSA) in DMEM medium. Cell growth was followed for 12 days. The 18.0 supplemented cells (control) reached maximum growth at day nine which was greater than either 18.2 (n6) or 18:3 (n3) supplemented cells. There was no difference between the latter two groups in their growth. To investigate the fatty acid incorporation of the supplemented fatty acid and how they may influence composition in the cell membrane, we examined the fatty acid composition of each phospholipid (PL) species. Both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were significantly influenced by the type of fatty acid supplemented. Supplementation with 18.0 resulted in HT-29 cell membranes having more monounsaturated fatty acids than the cells grown in the other fatty acids. Polyunsaturated fatty acid (PUFA) supplementation (both 18.2 and 18:3) resulted in the enrichment of PUFA in the PL fractions. Cells supplemented with 18:3 (n3) had the highest unsaturation index in membrane PE as compared to the other phospholipid species. PLC activity of the membranes was measured using PIP2 as a substrate in the presence of 15 mu g alamethicin and 42 mu M free calcium. The contribution of G protein to the activity of the enzyme was assessed using GTP gamma(S). PLC activity of HT-29 cells was 16% higher in the presence of GTP gamma(S) than without in all groups. The enrichment of the membranes with different fatty acids had no effect on GTPy(S) response. GTP gamma(S)-activated PLC activity of 18:3 (n3) supplemented cells was 81% of those supplemented with either 18:0 or 18:2 (n6) cells. It is concluded that the decrease in cell proliferation with supplementation with 18:3 (n3) may be mediated through its inhibitory effect on PLC, which provides the second messengers for protein kinase C (PKC) activation, PLC may be influenced by an increased unsaturation index of the PE fraction of the HT-29 tumor cell membranes.