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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The kinetics of sterol synthesis and DNA synthesis in polyclonally activated, concanavalin A-stimulated spleen cell cultures were analyzed. Inhibition of DNA synthesis by 1-f-Darabinofuranosylcytosine (Ara-C) did not abrogate the formation of cytotoxic effector cells. However, inhibition of sterol synthesis by 25-hydroxycholesterol inhibited formation ofcytotoxic effector cells as well as cellular proliferation. The inhibition ofcytotoxicity correlated well with the dose of 25-hydroxycholesterol administered and was dependent on the time ofadministration. The agent had to be present when sterol synthesis occurred normally during the time lapse before DNA synthesis began. Compactin had the same effect as 25-hydroxycholesterol. The effects of inhibition of sterol biosynthesis on cytotoxicity could be counteracted by addition of cholesterol-containing liposomes. Based on these experiments, the links between proliferation and differentiation in lymphocytes are discussed.Lymphocytes stimulated by mitogens such as concanavalin A (Con A) simulate to some degree a synchronized cell population during the first proliferative cycle after stimulation (1-3). Many metabolic phases have been demonstrated to occur during this activation, among them being a distinct cycle ofsterol synthesis (4-6), a cycle ofcAMP synthesis (7), and ultimately the S phase of DNA synthesis (4,5). Recently the important observation was made that one or more subpopulations of lectin-stimulated splenic lymphocytes differentiate into cytotoxic effector cells. This cytotoxicity appeared to be polyclonal because its detection required a ligand, such as phytohemagglutinin (PHA) (8). MacDonald and Lees (9) subsequently showed that these cells differentiated into cytotoxic lymphocytes, even if DNA synthesis was completely inhibited.In this paper we demonstrate, using specific inhibitors of sterol biosynthesis, that the polyclonal induction of naive, splenic lymphocytes into differentiated cytotoxic lymphocytes calls for the synthesis ofcholesterol as an absolute requirement, whether DNA synthesis is inhibited or not. We will use this experimental model system to discuss some aspects of the relationship of proliferation and differentiation in immunocompetent cell populations. Our experiments result in the hypothesis that proliferation is not a prerequisite for differentiation but rather appears to be controlled by the latter in normal tissues (as opposed to uncontrolled proliferation in transformed cells) and merely serves to amplify the product of differentiation.MATERIALS AND METHODS Chemicals. Bactophytohemagglutinin M (PHA-M) was purchased from Difco. The hydrochloride salt of 1-13-D-arabinofuranosylcytosine (Ara-C) was purchased from Calbiochem, Con A from Miles, and a-methyl D-mannoside from Sigma. Sterols were purchased from Steraloids (Wilton, NH), recrystallized from methanol three times (10), dissolved in absolute ethanol, and added to Dulbecco's modified Eagle's medium (DME medium) containing 5% bovine serum albumin (Pentex, Miles) (11).C...

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