Relationship between sterol synthesis and DNA synthesis in phytohemagglutinin-stimulated mouse lymphocytes.

Abstract

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

Mir-33 regulates cell proliferation and cell cycle progression

“…1 As an essential component of mammalian cell membranes, cells require cholesterol for proliferation. [2][3][4][5] In addition to its structural requirement, cholesterol is important for other cell functions, such as bile acid and hormone synthesis as well as during embryonic development. The requirement of cholesterol for cell growth and division of mammalian cells has been known for many years, [2][3][4] but whether this is just a consequence of its use for membrane formation or whether it also plays a regulatory role in this process has not been clarified.…”

mTOR signaling and transcriptional regulation in T lymphocytes

“…27 Rapid upregulation of sterol synthesis accompanies lymphocyte activation. 28 Kidani et al demonstrated that the master transcription factors for lipid biosynthesis, the sterol regulatory elementbinding proteins SREBP1 and SREBP2, activate fatty acid and cholesterol synthesis in T cells upon mitogenic stimulation. 29 T cells deficient in the SREBP chaperone molecule SCAP, which controls the processing and subsequent transcriptional activity of SREBPs, 30 fail to undergo metabolic reprograming, including lipid biosynthesis, glycolysis and ATP production.…”

Inhibition of sterol and DNA syntheses in phytohemagglutinin-stimulated human lymphocytes by 7.ALPHA.-hydroxycholesterol.

“…The sterol synthetic capacity of PHA-stimulated lymphocytes was measured by the digitonin-precipitative method (Knaus et al 1959;Chen et al 1975). PHA-stimulated lymphocytes in the culture medium were cultured in a glass test tube with a Molten cap.…”

“…Sterol and DNA synthesis during blastogenesis of lymphocytes are suppressed by the addition of 25-hydroxycholesterol and 20a-hydroxycholesterol (Chen et al 1975 ;Pratt et al 1977). Oxygenated cholesterol derivatives such as 7a-hydroxycholesterol (7a-HC), 7f-hydroxycholesterol, 7-ketocholesterol, 20a-hydroxycholesterol, and 25-hydroxycholesterol regulate sterol synthesis in normal and malignant mammalian cell lines at the site of the reaction catalyzed by 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase ; EC 1.1.1.34 ; Kandutsch and Chen 1973, ;Bell et al 1976 ; Pratt et al 1977 ; Kandutsch et al 1978).…”