INHIBITORS OF CHOLESTEROL BIOSYNTHESIS: EFFECTS OF 14ALPHA-ETHYL-5ALPHA-CHOLEST-7-ENE-3BETA,15ALPHA-DIOL IN CULTURED MAMMALIAN CELLS. (VOLUMES I AND II) (OXYGENATED STEROLS)
Doctor of Philosophy
14(alpha)-Ethyl-5(alpha)-cholest-7-ene-3(beta),15(alpha)-diol (0.5 uM) inhibited the synthesis of C(,27) sterols from radioactive acetate in Chinese hamster ovary (CHO-Kl) cells. This inhibition was accompanied by a striking accumulation of labeled lanosterol and 24,25-dehydrolanosterol which were characterized by chromatographic analyses on silicic acid-Super Cel columns and on alumina-Super Cel-silver nitrate columns and by the results of cocrystallization experiments. The inhibition of the metabolism of these two C(,30) sterols occurred after only 15 minutes of exposure of the cells to the 14(alpha)-ethylsteryl diol. Analyses of the labeled C(,27) sterols formed from radioactive acetate in CHO-Kl cells in the absence of the 14(alpha)-ethylsteryl diol revealed that, under the conditions employed, most of the radioactivity had the chromatographic mobility of 5(alpha)-cholesta-8,24-dien-3(beta)-ol and 5(alpha)-cholest-8-en-3(beta)-ol. The 14(alpha)-ethylsteryl diol (0.5 (mu)M) also caused the inhibition of the metabolism of lanosterol and 24,25-dihydrolanosterol in CRl, a mutant CHO line in which the levels of HMG-CoA reductase activity are not suppressed by 25-hydroxycholesterol, 14(alpha)-ethyl-5(alpha)-cholest-7-ene-3(beta),15(alpha)-diol and 5(alpha)-cholest-8(14)-en-3(beta)-ol-15-one. Incubations of CHO-Kl cells with low concentrations (0.1 (mu)M) of the 14(alpha)-ethylsteryl diol for longer periods of time (2-10 days) in lipid-deficient medium caused significant changes in sterol composition of the cells and of their plasma membranes. After two days of incubation, 67% of the total sterols of the cells consisted of lanosterol (52%) and 24,25-dihydrolanosterol (15%). After 2-3 days of the incubation with 14(alpha)-ethylsteryl diol (0.1 (mu)M) in medium containing lipid-deficient serum, the CHO-Kl cells showed a prominent morphological change (elongation). On day 4, the mean value of the ratio of the longest to the shortest cell dimension was 6.29 (+OR-) 0.34, while that of control cells was 2.92 (+OR-) 0.10 (115% increase; p < 0.001). The cell elongation was reversed by removal of the 14(alpha) ethylsteryl diol from medium or by the addition of colcemid (0.67 (mu)M) to medium containing the 14(alpha)-ethylsteryl diol. The cell elongation was prevented when the cells were incubated with the 14(alpha)-ethylsteryl diol in medium containing whole serum. Moreover, incubation of the cells with cholesterol (10 (mu)M) also prevented the cell elongation induced by the 14(alpha)-ethylsteryl diol. Miconazole nitrate (10 uM), a nonsteroidal antifungal agent, also caused an accumulation of lanosterol and cell elongation in CHO-Kl cells.