Mevalonate deprivation leads to aneuploidy in Chinese hamster ovary cells.

After exposure to compactin, the competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, 22% of CHO-K1 cells contained abnormally high numbers of chromosomes. In two populations of cells selected for compactin resistance 31 and 33% of the cells contain more than 22 chromosomes. Some cell lines isolated from these populations have the wild type chromosome number of 20-21, while others have a broad distribution of chromosome number, often with a mean around 36-40. Finally, Chinese hamster ovary cells that are mutant for 3-hydroxy-3-methylglutaryl-CoA reductase and therefore auxotrophic for mevalonate were starved for that compound. This treatment also increased the number of cells containing extra chromosomes. These results indicate that interruption of the cellular supply of mevalonate results in abnormal chromosome number.

Influence of cholestyramine feeding on mevalonate-activating enzymes.

Phosphorylation and decarboxylation of mevalonic acid have been measured in different tissues of chicks fed a cholestyramine diet from hatching until 18 days of age. Hepatic and intestinal phosphorylation and decarboxylation of mevalonate were slightly although significantly increased from 15 days of treatment. Direct measurements of 5-pyrophosphomevalonate decarboxylase activity using the specific substrate of this enzyme corroborated these data. Brain enzymatic activities remained unaltered with respect to controls. These results suggest that enzymes responsible for the conversion of mevalonate to isopentenyl pyrophosphate from neonatal chick liver and intestine alter their activities in a coordinate fashion and may play an important role in the regulation of cholesterogenesis in these tissues.

Requirement for 24(S),25-epoxycholesterol for the viability of cultured fibroblasts.

Previous studies on a somatic cell mutant auxotrophic for mevalonate (Mev-1) have shown that these cells rapidly lose viability when deprived of mevalonic acid in culture medium supplemented with serum cholesterol. Testing of all known end products of mevalonate metabolism in cultured mammalian cells has been conducted to determine the basis for this mevalonate requirement. It has been found that the recently discovered mevalonate metabolite 24(S),25-epoxycholesterol produces a partial restoration of viability of Mev-1 cells starved for mevalonate, whereas other structurally similar oxysterols do not. It appears that 24(S),25-epoxycholesterol has a specific, vital cellular function in CHO-K1 cells.

Partial prevention of compactin (ML-236B) inhibition of in vitro hematopoiesis by dolichol and dolichyl phosphate.

We have reported that the exogenous addition of dolichyl phosphate (Dol-P) enhances the colony-forming capacities of early erythroid progenitors (BFU-E), late erythroid progenitors (CFU-E), and granulocyte-macrophage progenitors (CFU-GM) in adult mouse bone marrow, and that dolichol (Dol) enhances that of only CFU-E (Int. J. Cell Cloning 3:313, 1985). Compactin (2.5-10 microM), a specific inhibitor of mevalonate biosynthesis that causes a decrease of endogenous Dol biosynthesis, inhibited colony formation of CFU-GM. Exogenous addition of Dol-P partially prevented this inhibition, but Dol and the other mevalonate metabolites, such as cholesterol, coenzyme Q10, and isopentenyladenine, could not. In addition, we have found that the colony-forming capacity of CFU-E in fetal mouse liver was not enhanced by exogenous Dol or Dol-P. But the decrease of colony formation or DNA synthesis of fetal CFU-E in the presence of compactin was prevented by the exogenous addition of Dol or Dol-P.

Regulation of acyl CoA: cholesterol acyl transferase (ACAT) activity by mevalonate and cholesterol in isolated rat hepatocytes during perinatal development.

Acyl CoA: cholesterol acyl transferase (ACAT) activity presents marked oscillations and differential sensitivity to the "in vitro" stimulation of the kinase-phosphatase modulatory system in the perinatal rat liver. The regulation of this enzyme activity by some modulators generally active in adulthood, such as cholesterol, lipoproteins and mevalonate, has been studied in hepatocytes isolated at different developmental stages. A lack of effect of mevalonate and a positive effort of lipoprotein cholesterol have been observed at the fetal and neonatal stages. A differential prevalence is suggested of one of the two modulatory mechanisms (phosphorylation-dephosphorylation system, or substrate effect) at each developmental stage.

Role of mevalonate in regulation of cholesterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase in cultured cells and their cytoplasts.

H4-II-E-C3 hepatoma cells in culture respond to lipid-depleted media and to mevinolin with increased sterol synthesis from [14C]acetate and rise of 3-hydroxy-3-methylglutaryl coenzyme A reductase levels. Mevalonate at 4 mM concentration represses sterol synthesis and the reductase, and completely abolishes the effects of mevinolin. Mevalonate has little or no effect on sterol synthesis or reductase in enucleated hepatoma cells (cytoplasts) or on reductase in cytoplasts of cultured Chinese hamster ovary (CHO) cells. The sterol-synthesizing system of hepatoma cell cytoplasts and the reductase in the cytoplasts of CHO cells were completely stable for at least 4 hr. While reductase levels and sterol synthesis from acetate followed parallel courses, the effects on sterol synthesis--both increases and decreases--exceeded those on reductase. In vitro translation of hepatoma cell poly(A)+RNAs under various culture conditions gave an immunoprecipitable polypeptide with a mass of 97,000 daltons. The poly(A)+RNA from cells exposed for 24 hr to lipid-depleted media plus mevinolin (1 microgram/ml) contained 2.8 to 3.6 times more reductase-specific mRNA than that of cells kept in full-growth medium, or cells exposed to lipid-depleted media plus mevinolin plus mevalonate. Northern blot hybridization of H4 cell poly(A)+RNAs with [32P]cDNA to the reductase of CHO cells gave two 32P-labeled bands of 4.6 and 4.2 K-bases of relative intensities 1.0, 0.61-1.1, 2.56, and 1.79 from cells kept, respectively, in full-growth medium, lipid-depleted medium plus mevinolin plus mevalonate, lipid-depleted medium plus mevinolin, and lipid-depleted medium. These values approximate the reductase levels of these cells. We conclude that mevalonate suppresses cholesterol biosynthesis in part by being a source of a product that decreases the level of reductase-specific mRNA.

Cholesterol and mevalonic acid are independent requirements for the in vitro proliferation of human bone marrow granulocyte progenitor cells: studies using ML-236B.

ML-236B is a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, the key regulatory enzyme in the sequence that catalyzes the conversion of acetate to mevalonic acid in cholesterol biosynthesis. This compound caused marked inhibition of human bone marrow granulocyte progenitor cell (CFU-C) proliferation, the 50% inhibitory concentration (IHD50) being 2.0 X 10(6)M. Inhibition of colony formation was reversed by mevalonic acid but not by cholesterol. ML-236B also inhibited DNA synthesis and acetate incorporation into cholesterol in marrow mononuclear cells (IHD50 = 5.6 x 10(6)M and 3.2 x 10(7)M, respectively). No inhibition of mevalonate incorporation into cholesterol was observed. These results differ from those observed with 25-hydroxycholesterol, another inhibitor of HMG CoA reductase. The latter compound also inhibited CFU-C proliferation and cholesterol biosynthesis from acetate; inhibition of colony formation was reversed by cholesterol but not by mevalonic acid. In addition, 25-hydroxycholesterol inhibited cholesterol synthesis from mevalonic acid precursor. We conclude that: (1) ML-236B is a potent inhibitor of CFU-C proliferation, DNA synthesis, and cholesterol biosynthesis from acetate precursor in marrow mononuclear cells; (2) the effects of ML-236B are completely reversed by mevalonic acid but not by cholesterol, suggesting that mevalonic acid per se or one or more of its nonsterol products are critical for cell growth; (3) the inhibitory effects of 25-hydroxycholesterol on CFU-C proliferation and cholesterol biosynthesis are not solely a result of its inhibition of HMG CoA reductase, but are due in part to inhibition of enzymatic steps distal to mevalonic acid in the sterol synthetic pathway; and (4) mevalonic acid and cholesterol are independent requirements for CFU-C proliferation and differentiation in vitro.

A study of the Influence of mevalonic acid and its metabolites on the morphology of swiss 3T3 cells.

We used two model systems to investigate the effect of compactin, a competitive inhibitor of beta-hydroxy beta-methylglutarylcoenzyme A reductase, on the shape of Swiss 3T3 cells. We maintained cells in a quiescent state in medium deficient in platelet-derived growth factor (PDGF), or we added PDGF to quiescent cells to initiate traverse through a single cell cycle. In both systems, the cells responded to compactin by acquiring a characteristic rounded shape. Cell rounding seemed to depend on an induced deficiency of mevalonic acid (MVA) since the response could be prevented or reversed by adding MVA to the culture medium. Compactin-induced rounding appeared in PDGF-stimulated cells concomitantly with a compactin-mediated inhibition of DNA synthesis, and both effects had similar sensitivities to exogenous compactin and MVA. However, cell rounding seemed to be unrelated to other, previously observed effects of MVA deficiency. Compactin did not influence the total content of cell cholesterol, and little cholesterol was formed when we added radioactive MVA to round cells to effect shape change reversal. Measurement of the dolichol-dependent glycosylation of cell protein revealed no evidence of dolichol deficiency. In addition, reversal of cell rounding by MVA was not prevented by concentrations of tunicamycin that effectively blocked the incorporation of radioactive mannose into cell protein or by concentrations of cycloheximide that blocked protein synthesis. Taken together, our results suggest a new role for MVA or its products in the maintenance of cell shape.