concanavalin-a and 25-hydroxycholesterol

3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase is anchored to the endoplasmic reticulum (ER) membrane by a hydrophobic NH2-terminal domain that contains seven apparent membrane-spanning regions and a single N-linked carbohydrate chain. The catalytic domain, which includes the COOH-terminal two-thirds of the protein, extends into the cytoplasm. The enzyme is normally degraded with a rapid half-life (2 h), but when cells are depleted of cholesterol, its half-life is prolonged to 11 h. Addition of sterols accelerates degradation by fivefold. To explore the requirements for regulated degradation, we prepared expressible reductase cDNAs from which we either deleted two contiguous membrane-spanning regions (numbers 4 and 5) or abolished the single site for N-linked glycosylation. When expressed in hamster cells after transfection, both enzymes retained catalytic activity. The deletion-bearing enzyme continued to be degraded with a rapid half-life in the presence of sterols, but it no longer was stabilized when sterols were depleted. The glycosylation-minus enzyme was degraded at a normal rate and was stabilized normally by sterol deprivation. When cells were induced to overexpress the deletion-bearing enzyme, they did not incorporate it into neatly arranged crystalloid ER tubules, as occurred with the normal and carbohydrate-minus enzymes. Rather, the deletion-bearing enzyme was incorporated into hypertrophied but disordered sheets of ER membrane. We conclude that the carbohydrate component of HMG CoA reductase is not required for proper subcellular localization or regulated degradation. In contrast, the native structure of the transmembrane component is required to form a normal crystalloid ER and to allow the enzyme to undergo regulated degradation by sterols.

Topics: Animals; Cell Line; Cholesterol; Concanavalin A; Endoplasmic Reticulum; Glycosylation; Half-Life; Hydroxycholesterols; Hydroxymethylglutaryl CoA Reductases; Microscopy, Electron; Mutation; Transfection

Topics: Acetates; Adult; B-Lymphocytes; Cells, Cultured; Cholesterol; Concanavalin A; DNA; Female; Humans; Hydroxycholesterols; Leukemia, Hairy Cell; Lovastatin; Male; Mevalonic Acid; Middle Aged; Naphthalenes

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-beta-D-arabinofuranosylcytosine (Ara-C) did not abrogate the formation of cytotoxic effector cells. However, inhibition of sterol synthesis by 25-hydroxycholesterol inhibited formation of cytotoxic effector cells as well as cellular proliferation. The inhibition of cytotoxicity correlated well with the dose of 25-hydroxycholesterol administered and was dependent on the time of administration. 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.

Topics: Animals; Cell Differentiation; Cell Division; Cells, Cultured; Cholesterol; Concanavalin A; Cytotoxicity, Immunologic; DNA Replication; Filipin; Hydroxycholesterols; Lymphocyte Activation; Lymphocytes; Mice; Spleen; Time Factors

Mevalonic acid (5 x 10(-4)-1 x 10(-2) M) stimulates DNA synthesis, morphologic transformation and cell cycling in peripheral blood human lymphocytes. Other organic acid anions which serve as cholesterol and mevalonate precursors are devoid of such effects. Both ML-236B and 25-hydroxycholesterol, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, inhibit concanavalin A-induced lymphocyte transformation, but only the inhibition by ML-236B can be overcome by exogenous mevalonate. In contrast, only 25-hydroxycholesterol inhibits mevalonate-induced lymphocyte DNA synthesis. The effects of mevalonic acid on lymphocytes cannot be reproduced by isopentenyl adenine or isopentenyl adenosine. Unregulated endogenous cellular synthesis of mevalonic acid may contribute to uncontrolled growth in certain malignant cell lines.

Topics: Concanavalin A; DNA; Humans; Hydroxycholesterols; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lovastatin; Lymphocyte Activation; Lymphocytes; Mevalonic Acid; Naphthalenes

A concanavalin-A-resistant mutant of Chinese-hamster ovary cells has been shown to have altered cholesterol content and 3-hydroxy-3-methylglutaryl coenzyme A reductase activity compared to wild-type and spontaneous-revertant cell lines. These changes are associated with insensitivity of the mutant reductase activity to suppression by low-density lipoprotein and impaired receptor-mediated binding and uptake of 125I-labelled low-density lipoprotein.

Topics: Animals; Cell Line; Chloroquine; Concanavalin A; Cricetinae; Cricetulus; Drug Resistance; Female; Hydroxycholesterols; Hydroxymethylglutaryl CoA Reductases; Kinetics; Lipoproteins, LDL; Ovary

Topics: Animals; Cholesterol; Concanavalin A; Dactinomycin; Dexamethasone; DNA; Female; Hydroxycholesterols; In Vitro Techniques; Lipids; Mice; Mice, Inbred C57BL; Steroids; Thymus Gland