A group of oxygenated sterols has been identified as potent and specific inhibitors of sterol biosynthesis. The ability of these compounds to inhibit sterol synthesis in cultured cells and the ineffectiveness of cholesterol under the same conditions suggest that feedback regulation of sterol biosynthesis may be brought about by an oxygenated sterol rather than by cholesterol. The nature of the regulatory sterol may vary in different cells with their specific requirements for cholesterol as a structural component or as a precursor of other steroid products. The use of oxygenated sterols to block sterol synthesis in cultured cells provides new information regarding the role of sterol in cell membrane structure and function. For example, de novo sterol synthesis is required for DNA synthesis and cell division by some cultured cells. Studies with cultured cells, and with rats and mice in vivo, suggest that oxygenated sterols could be of value in the treatment of several important human diseases.

Incubation of peripheral blood or isolated lymphocytes of C57L/J mice with phytohemagglutinin stimulated the incorporation of thymidine into DNA of lymphocytes as they transformed into large lymphoblasts. DNA synthesis began after about 24 hr of incubation and reached a peak at 48 hours. The de-novo synthesis of sterols from acetate was stimulated much earlier, at 4 hr of incubation, and the rate reached a maximum at 24 hr, approximately at the time DNA synthesis began. (6)(7)(8)11) prevented both the increase in sterol synthesis and the increase in DNA synthesis that are associated with blastogenesis. DNA synthesis was not affected when the inhibitor was added after the cycle in sterol synthesis had reached its maximum. These observations of an apparent relationship between sterol synthesis and DNA synthesis are described in this report. MATERIALS AND METHODSAnalysis of Lipid, CO2 Production and 3-Hydroxy-3-methylglutaryl Coenzyme A (HAIG-CoA) Reductase Activity in Whole Blood and Isolated Lymphocytes. Female C57L/J strain mice, aged 3-4 months old, were supplied by the Production Department of the Jackson Laboratory. They were decapitated following asphyxiation in CO2 and blood was pooled from several animals in sterile test tubes that contained sodium heparin (30 USP units of heparin per ml of blood; Sigma). Aliquots (0.5 ml) of the heparinized blood were pipetted into sterile 24 ml Erlenmeyer flasks containing 5 ml of RPMI 1640 medium (Grand Island Biological Co.) with or without PHA (Difco, PHA-MI 0.5 mg/ml of final concentration). The flasks were sealed with stoppers and incubated in a shaking water bath at 370 for various lengths of time. Two hours before the end of incubation 50 jl of RPMI medium containing 25 4Ci of [1-_4C]acetate (58.5 Ci/mol, New England Nuclear) was added to the incubation mixture and the flasks were sealed with stoppers fitted with plastic cups (Kontes). The procedures for analysis of radioactive C02, fatty acids, and sterol fractions of the samples were described l)reviously (4,5).In some experiments isolated lymphocytes were used in place of whole blood. The procedure used for isolation of lymphocytes has been described (4, 9). Approximately 7 ml of heparinized blood pooled from eight animals was passed through a column containing an equal volume of 0.3 mm glass beads to remove the "sticky" population of platelets. The blood was then mixed with 7 ml of RPMLI medium containing Hepes (N-2-hydroxyetlhylp)iperazinie-N'-2-ethanesulfonic acid) 1950

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Sterol synthesis in liver in vivo is regulated at the site of the reaction catalyzed by 3-hydroxy-3-methylglutaryl-CoA reductase through a feedback system thought to involve either cholesterol or one or more of the products of its metabolism. Cholesterol feeding results in repression of the synthesis of the enzyme, but inactivation of the enzyme seems to precede repressroblasts is not inhibited by purified exogenous cholesterol. However, derivatives of cholesterol produced by the introduction of a ketone or hydroxyl function in the 7, 20, 22 or 25 positions effectively inhibit sterol synthesis by specifically depressing the level of HMG CoA reductase activity. As a result of this specific effect prolonged incubation of an inhibitory sterol with growing L cells results in depletion of cellular sterol. Growth of the culture then ceases and the cells die unless an appropriate sterol or a sterol precursor is supplied in the medium. The inhibitory sterols, 25-hydroxycholesterol and 7-ketocholesterol appear to be taken up by L cells through processes that involve their specific interactions with saturable cellular receptors. The uptake of cholesterol by L cells appears to be by a different process--possibly through physical diffusion.

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