Consequences of blocked sterol synthesis in cultured cells. DNA synthesis and membrane composition

Abstract

When cell cultures were incubated with certain oxygenated derivatives of cholesterol which specifically depress 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity (i.e. 25-hydroxycholesterol, 7-ketocholesterol, or 20 alpha-hydroxycholesterol), a number of consequences followed the decline in enzyme activity. DNA synthesis, expressed in terms of (methyl-3H)thymidine incorporation/mg of cellular protein, declined progressively after the addition of an inhibitor to L-cell cultures and had essentially ceased by 80 h. A decline in the rate of cell growth, as determined by the measurement of cellular protein, became apparent about 24h after the addition of the inhibitor. These effects of the inhibitory sterols were reversed when mevalonate or cholesterol was added to the medium within 48 h after the addition of the inhibitor. Specific rates of uridine incorporation into RNA and of leucine into protein were not significantly altered during 80 h of incubation with the inhibitor. Sterol synthesis was suppressed; and the concentration of desmosterol in L-cells declined to about one-half of the control level within 36 h. Since phospholipid concentrations were not altered significantly by the treatment, the molar ratio of sterol to phospholipids declined to levels lower than any previously reported for mammalian cells. Changes in the molar ratio of sterol to phospholipids in plasma membranes isolated from L-cells that had been incubated with 25-hydroxycholesterol or cholesterol were in agreement with those found in cells under similar conditions. The inhibitory sterols caused a similar but slower decline in the concentrations of unesterified and total cholesterol in fetal mouse liver cell cultures and rat hepatoma cell cultures.