Requirement for mevalonate in cycling cells: quantitative and temporal aspects

In order to investigate a requirement for isoprenoid compounds in the cell cycle, DNA synthesis was examined in cultured Chinese hamster ovary cells in which mevalonate biosynthesis was blocked with mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Treatment of exponentially-growing cultures with mevinolin led to a decline in DNA synthesis and cell cycle arrest in G1. Synchronous DNA synthesis and cell division could be restored in the arrested cultures, in the absence of exogenous mevalonate, by removing the inhibitor from the culture thereby allowing expression of an induced level of HMG-CoA reductase. In order to quantitate the mevalonate requirement for entry into S phase, recovery of DNA synthesis was made dependent upon added mevalonate by preventing the induction of the enzyme using 25-hydroxycholesterol, a specific repressor of HMG-CoA reductase synthesis. When cultures were treated with both inhibitors, optimal recovery of DNA synthesis was obtained with 200 micrograms/ml mevalonate following an 8 h lag, whereas a progressively longer lag-time was found with lower concentrations of mevalonate. Exogenous dolichol, ubiquinone, or isopentenyladenine had no effect on the arrest or recovery of DNA synthesis. Cholesterol was required during the arrest incubation for cell viability, but was not sufficient for recovery in the absence of mevalonate. The recovery of DNA synthesis by 200 micrograms/ml mevalonate, which was maximal 14-16 h after the addition of mevalonate, only required that the mevalonate be present for the first 4 h, whereas more than an 8-h incubation was required for maximal recovery with 25 micrograms/ml mevalonate. Maximal recovery at either concentration of mevalonate was achieved after approximately 400 fmol mevalonate/micrograms protein was incorporated into non-saponifiable lipids. This quantity represents approximately 0.1% of the mevalonate required for the synthesis of total cellular isoprenoid compounds. The results indicate that production of a quantitatively minor product(s) of mevalonate metabolism is required during the first 4 h following release of the block before other cellular events necessary for entry into S phase can occur.     

Differential effect of inflammation and dexamethasone on dolichol and dolichol phosphate synthesis

Inflammation and glucocorticoids stimulate hepatic glycoprotein synthesis, resulting in an increased secretion of serum glycoproteins. We now present evidence that the synthesis of dolichol and dolichol phosphate from mevalonate is increased in hepatocytes from inflamed rats. Also, in inflamed rats, the levels of dolichol and dolichol phosphate are increased in liver homogenates and microsomes. Dexamethasone treatment of the cells, however, does not increase the synthesis of dolichol and dolichol phosphate from mevalonate. The results suggest that the inflammation-induced dolichol-linked saccharide and glycoprotein synthesis is possibly mediated through an increase in the level of dolichol and dolichol phosphate in the liver. Since dexamethasone treatment does not increase the synthesis of dolichol and dolichol phosphate, its action on glycoprotein synthesis appears to be different and to affect the induction of enzymes in mannosyl phosphoryl dolichol- and dolichol-linked oligosaccharide synthesis.     

Relationships among dolichyl phosphate, glycoprotein synthesis, and cell culture growth

Following treatment of Chinese hamster ovary cells with inhibitors of mevalonate biosynthesis in the presence of exogenous cholesterol, the cellular concentration of phosphorylated dolichol and the incorporation of [3H]mannose into dolichol-linked saccharides and N-linked glycoproteins declined coincident with a decline in DNA synthesis. Addition of mevalonate to the culture medium increased rates of mannose incorporation into lipid-linked saccharides and restored mannose incorporation into N-linked glycoproteins to control levels within 4 h. After an additional 4 h, synchronized DNA synthesis began. Inhibition of the synthesis of lipid-linked oligosaccharides and N-linked glycoproteins by tunicamycin prevented the induction of DNA synthesis by mevalonate, indicating that glycoprotein synthesis was required for cell division. The results suggest that the rate of cell culture growth may be influenced by the level of dolichyl phosphate acting to limit the synthesis of N-linked glycoproteins.     

Cell Cycle-Specific Requirement for Mevalonate, but Not for Cholesterol, for DNA Synthesis in Glial Primary Cultures

The requirement for the sterol biosynthetic pathway for the occurrence of DNA synthesis in glial cells and, in particular, the relative roles of cholesterol and of mevalonate have been studied. Primary cultures of developing glial cells were synchronized by reducing the content of fetal calf serum (FCS) in the culture medium from 10% to 0.1% (vol/vol) for 48 h between days 4 and 6 in culture. Reversal of the resulting quiescent state by the return of the cultures to 10% serum caused after 24 h a marked increase in DNA synthesis, and this increase was prevented by the simultaneous addition of mevinolin, a specific inhibitor of the sterol biosynthetic pathway at the 3-hydroxy-3-methylglutaryl coenzyme A reductase step, at the time of serum repletion. A dose-dependent reversal of the mevinolin inhibition of DNA synthesis occurred with simultaneous addition of mevalonate to the culture medium. The induction of DNA synthesis by serum repletion, its inhibition by mevinolin, and the reversal of the inhibition by mevalonate were unaffected by a 95% reduction in exogenous cholesterol produced by utilization of lipoprotein-poor serum (LPPS) rather than FCS. Similarly, return of quiescent cultures to 10% LPPS containing mevinolin and sufficient low-density lipoprotein (LDL) to raise the cholesterol concentration 80-fold failed to restore DNA synthesis. In addition, reversal of the mevinolin inhibition of DNA synthesis by mevalonate occurred despite the continuous presence of mevinolin if mevalonate was added as late as 12 h after serum repletion, but not if added after 16 h or more.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differentiation of neuroblastoma cells induced by an inhibitor of mevalonate synthesis: relation of neurite outgrowth and acetylcholinesterase activity to changes in cell proliferation and blocked isoprenoid synthesis

Mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, stimulates neurite outgrowth and acetylcholinesterase (ACE) activity in C1300 (Neuro-2A) murine neuroblastoma cells. Sprouting of neurites began within 4-8 h, before changes in cell proliferation could be detected by [3H]thymidine incorporation or flow cytometry. In contrast, the increase in ACE activity was temporally correlated with suppression of DNA synthesis, which occurred after 8 h. The activity of the membrane marker enzyme phosphodiesterase I was not stimulated by mevinolin. Suppression of protein synthesis with cycloheximide blocked the induction of ACE activity but only partially inhibited neurite outgrowth in the mevinolin-treated cultures. When mevinolin was removed from the culture medium, most of the cells retracted their neurites within 2 h, but ACE activity did not decline until DNA synthesis began to return to control levels after 10 h. Similarly, retraction of neurites in differentiated cells exposed to colchicine was not accompanied by a decrease in ACE activity. DNA histograms suggested that mevinolin arrests neuroblastoma cells in both the G1 and G2/M compartments of the cell cycle. Other cytostatic drugs that arrest cells at different stages of the cell cycle did not cause Neuro-2A cells to form neurites such as those seen in the mevinolin-treated cultures. When incorporation of [3H]acetate into isoprenoid compounds was studied in cultures containing mevinolin in concentrations ranging from 0.25 microM to 25 microM, the labeling of cholesterol, dolichol, and ubiquinone was suppressed by 90% or more at all concentrations. However, significant growth arrest and cell differentiation were observed only at the highest concentrations of mevinolin. Supplementing the medium with 100 microM mevalonate prevented the cellular response to mevinolin, but additions of cholesterol, dolichol, ubiquinone, or isopentenyl adenine were generally ineffective. The cholesterol content of neuroblastoma cells incubated with 25 microM mevinolin for 24 h was not diminished, and protein glycosylation, measured by [3H]mannose incorporation, was decreased only after 24 h at high mevinolin concentration. These studies suggest that the stimulation of neurite outgrowth and the increase in ACE activity induced by mevinolin are independent phenomena. Whereas neurite outgrowth is not related directly to the effects of mevinolin on cell cycling, the induction of ACE is correlated with the inhibition of cell proliferation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mevalonate controls cytoskeleton organization and cell morphology in thyroid epithelial cells

Blockade of mevalonate synthesis by the 3-hydroxy-3-methylglutaryl Coenzyme A reductase inhibitor mevinolin (lovastatin) causes FRTL-5 thyroid cells to undergo significant morphological changes; these include a transition from a flat, polygonal to a round shape, the development of cytoplasmic arborizations, and the loss of contact between neighboring cells. Immunofluorescence studies of cytoskeletal structures show that, at early times after administering the drug, and before the round phenotype develops, stress fibers disassemble while the peripheral actin filaments, which are adjacent to the cytoplasmic face of the plasma membrane, appear largely unaffected. Subsequently, when this cortical actin network becomes fragmented, cells start to round up and become separated from neighbors. Microtubules become disconnected from the plasma membrane and retract toward the cell center, although they do not appear depolymerized; indeed, at this stage, cytoplasmic elongations contain mostly intact microtubules. After exposure to mevinolin FRTL-5 cells also lose vinculin-related substrate contacts. Treatment of cells with either cycloheximide or colchicine abolishes morphological changes induced by mevinolin, suggesting that ongoing protein synthesis and microtubule integrity are prerequisites for the drug to be effective. Both cytoskeletal and morphological perturbations can be reversed by mevalonate, but not by cholesterol or the non-sterol derivatives of mevalonate such as dolichol, ubiquinone, and isopentenyladenine, individually or in combination. It is suggested that mevalonate deficiency may impair formation of isoprenylated proteins important for cytoskeletal organization and stability. © 1993 Wiley-Liss, Inc.     

Increased nonsterol isoprenoids, dolichol and ubiquinone, in the Smith-Lemli-Opitz syndrome: effects of dietary cholesterol

Smith-Lemli-Opitz syndrome (SLOS) is an inherited autosomal recessive cholesterol deficiency disorder. Our studies have shown that in SLOS children, urinary mevalonate excretion is normal and reflects hepatic HMG-CoA reductase activity but not ultimate sterol synthesis. Hence, we hypothesized that in SLOS there may be increased diversion of mevalonate to nonsterol isoprenoid synthesis. To test our hypothesis, we measured urinary dolichol and ubiquinone, two nonsterol isoprenoids, in 16 children with SLOS and 15 controls, all fed a low-cholesterol diet. The urinary excretion of both dolichol (P < 0.002) and ubiquinone (P < 0.02) in SLOS children was 7-fold higher than in control children, whereas mevalonate excretion was comparable. In a subset of 12 SLOS children, a high-cholesterol diet decreased urinary mevalonate excretion by 61% (P < 0.001), dolichol by 70% (P < 0.001), and ubiquinone by 67% (P < 0.03). Our hypothesis that in SLOS children, normal urinary mevalonate excretion results from increased diversion of mevalonate into the production of nonsterol isoprenoids is supported. Dietary cholesterol supplementation reduced urinary mevalonate and nonsterol isoprenoid excretion but did not change the relative ratios of their excretion. Therefore, in SLOS, a secondary peripheral regulation of isoprenoid synthesis may be stimulated.     

Cell Cycling of Astrocytes and Their Precursors in Primary Cultures: A Mevalonate Requirement Identified in Late G1, but Before the G1/S Transition, Involves Polypeptides

The relationship between mevalonate and cell cycling was investigated in developing glial cells. Primary cultures of newborn rat brains were serum-depleted (0.1%, vol/vol) for 48 h on days 4-6 in vitro, then returned to 10% calf serum (time 0). After 48 h, 70-80% of the cells were glial fibrillary acidic protein (GFAP)-negative by indirect immunofluorescence; 79 +/- 7% were GFAP-positive after an additional 3 days. Serum shift-up resulted in 12 h of quiescence, and then by 20 h (S phase) in increased proportions of cells synthesizing DNA (from 15 +/- 6% to 75 +/- 4% by bromodeoxyuridine immunofluorescence at 12 h and 20 h, respectively) and rates of DNA synthesis (42 +/- 6 versus 380 +/- 32 cpm/micrograms of protein/h of [3H]thymidine uptake). Additional mevalonate (25 mM) for 30 min at 10 h reversed the inhibition of DNA synthesis apparent with mevinolin (150 microM), an inhibitor of mevalonate synthesis, present from time 0. Cycloheximide added simultaneously with mevalonate prevented this reversal of inhibition. To cause arrest at G1/S, cultures were exposed to hydroxyurea between 10 and 22 h. By 3 h after hydroxyurea removal, bromodeoxyuridine-labeled nuclei increased from 0% to 75 +/- 9%, and DNA synthesis increased 10-fold. Mevinolin failed to inhibit these increases. Thus, primary astroglial precursors stimulated to progress through the cell cycle express a mevalonate requirement in late G1, but before the G1/S transition. The effect of mevalonate was characterized further as being brief (30 min) and as requiring polypeptides.     

Dolichol-Linked Glycoprotein Synthesis in G1 Is Necessary for DNA Synthesis in Synchronized Primary Cultures of Cerebral Glia

Primary cultures of newborn rat cerebrum, which are composed of glial cells (principally astroglia), were used for examining the relationship between dolichol-linked glycoprotein synthesis and DNA synthesis in developing cerebral glia. The cells were synchronized by reducing the content of fetal calf serum in the culture medium from 10 to 0.1% (vol/vol) for 48 h between days 4 and 6 in culture. Reversal of the quiescent state by return of the cultures to 10% serum causes a marked increase in DNA synthesis 12-24 h later. A sharp increase in glycoprotein synthesis (incorporation of [3H]mannose) occurred in the first 12 h after serum repletion, preceding the increase in DNA synthesis. Tunicamycin, an inhibitor of the dolichol-linked pathway to glycoprotein synthesis at the first committed step in oligosaccharide formation, promptly and completely prevented the increase in glycoprotein synthesis and, in addition, the subsequent increase in DNA synthesis. The effects of tunicamycin on glycoprotein and DNA syntheses were reversible, and no comparable effect on total protein synthesis was observed. When tunicamycin was added only during a temporally circumscribed period in G1, i.e., from 3 to 9 h after serum repletion, the increase in DNA synthesis between 12 and 24 h after repletion was still markedly inhibited, i.e., to approximately 45% of the value in untreated cultures. The data thus show that there is a requirement for dolichol-linked glycoprotein synthesis for the subsequent occurrence of DNA synthesis and that this requirement is expressed late in the G1 phase of the cell cycle.     

Isoprenylated proteins in cultured cells: subcellular distribution and changes related to altered morphology and growth arrest induced by mevalonate deprivation

In the presence of lovastatin (mevinolin), an inhibitor of endogenous mevalonate synthesis, C1300 murine neuroblastoma cells incorporated (2-14C)mevalonate into several discrete polypeptides that were separable by SDS-PAGE. The electrophoretic pattern of the labeled proteins did not vary substantially when cells were homogenized with Ca++, Mg++, high concentrations of NaCl or phosphatase inhibitor, or when cells were lysed immediately in trichloroacetic acid. When cells that had been prelabeled with (14C)mevalonate were incubated with lovastatin and simultaneously deprived of exogenous mevalonate, there was a 50-60% decline in the concentration of protein-bound isoprenoid label within 17 h. In contrast, there was little change in the radioactivity in the sterol, dolichol, or ubiquinone fractions. The time course of the decline in mevalonate-derived label in cellular polypeptides paralleled the onset of neurite outgrowth and preceded the decline of DNA synthesis, suggesting that a decreased intracellular concentration of protein-bound isoprenoid groups may contribute to the well-documented effects of mevalonate deprivation on cell morphology and cell cycling. Fractionation of neuroblastoma cells by differential centrifugation and sucrose density-gradient centrifugation revealed that mevalonate-labeled proteins of 53 kDA, 22-26 kDa, and 17 kDa were concentrated in the cytosol. Proteins migrating at 45 kDa were found in both the soluble and particulate fractions, including those enriched in mitochondria and plasma membrane. The isoprenylated proteins migrating at approximately 66 kDa were localized exclusively in the nuclear fraction. When chromatin was removed from the nuclei by extraction with 2 M NaCl, the 66 kDa isoprenylated proteins remained associated with the residual components of the nuclear matrix and lamina. Isoprenylated proteins with electrophoretic mobilities similar to those observed in neuroblastoma cells were detected in a variety of established cell lines. However, there was considerable variation among cell lines in the overall efficiency of protein labeling with (14C) mevalonate and in the prominence and mobilities of specific labeled proteins in the 45-70 kDa range. Comparisons of paired transformed vs. nontransformed fibroblast cell lines suggested that the profile of mevalonate-labeled proteins in a given cell line is not altered by malignant transformation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Glycoprotein synthesis in Drosophila Kc cells. Biosynthesis of dolichol-linked saccharides

The biosynthesis of dolichol and dolichol-linked saccharide intermediates in glycoprotein synthesis was studied in an embryonic Drosophila cell line (Kc) that lacks the squalene-cholesterol branch of the polyisoprenoid biosynthetic pathway. Kc cells were labeled with [5-3H]mevalonic acid and the radioactive lipids formed were analyzed. Although the major labeled product was coenzyme Q, dolichol and a variety of dolichol derivatives could be readily detected. On the basis of their chromatographic and chemical properties, these derivatives were identified as dolichyl phosphate, glucosylphosphoryldolichol, mannosylphosphoryldolichol, and oligosaccharylpyrophosphoryldolichol. Both short term (4-h) and steady state (4-day) labeling experiments with mevalonate, rather than sugars as previously used, were performed to assess the level of these intermediates. The results of these studies, using a precursor common to all the intermediates, reveal that the early intermediates, N-acetylglucosaminylpyrophosphoryldolichol and N,N'-diacetylchitobiosylpyrophosphoryldolichol, are present at very low levels (less than 5%) relative to the other intermediates on the pathway to oligosaccharylpyrophosphoryldolichol. The total amount of dolichol intermediates remained essentially constant during the chase phase of pulse-chase experiments, indicating the absence of a major catabolic pathway for the polyisoprenoid backbone. As expected, however, the sugar moiety, studied with mannosylphosphoryldolichol, underwent rapid turnover. These results are discussed in the context of our current understanding of the pathway whereby dolichol derivatives participate in glycoprotein synthesis.     

Differential interaction of branch-specific inhibitors of isoprenoid biosynthesis with cell cycle progression in tobacco BY-2 cells

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGR), which catalyzes the formation of mevalonic acid (MVA), can be specifically blocked by mevinolin. Inhibition of HMGR in vivo leads to an arrest in cell cycle progression in tobacco ( Nicotiana tabacum L. Bright Yellow 2) cells. As MVA in plants is the common precursor of a myriad of isoprenoid products synthesized in the cytosol and mitochondria, it is difficult to identify among such MVA-dependent molecules those whose lack may lead to cell cycle arrest. In an attempt to do so, branch-specific inhibitors of the cytosolic isoprenoid pathway downstream from MVA were used to study their capacity to block cell cycle progression. The effects of squalestatin (sterol biosynthesis inhibitor), chaetomellic acid A and patulin (protein prenyltransferase (PT) inhibitors) and tunicamycin (inhibitor of dolichol-dependent protein glycosyl transferase, thus mimicking the effect of an absence of dolichol) were compared to those induced by mevinolin. In this way, squalestatin and chaetomellic acid were identified as behaving like true cell cycle inhibitors, in that they led to a specific arrest in the cell cycle. However, they did not exactly mimic the mevinolin-induced effects. Patulin proved to be of high general toxicity, which suggests that it may affect other reactions besides blockage of protein isoprenylation. Finally, tunicamycin efficiently blocked growth of cell suspension cultures, but did not arrest the cells in a specific phase of the cell cycle. Results are discussed in the context of a better understanding of the essential implication of isoprenoids in plant cell cycle progression.     

Inhibition by 6-fluoromevalonate demonstrates that mevalonate or one of the mevalonate phosphates is necessary for lymphocyte proliferation

The sterol synthesis inhibitor 6-fluoromevalonate (Fmev) was used to explore the role of mevalonate products in lymphocyte proliferation. Fmev blocks the synthesis of isopentenyl pyrophosphate and all more distal products in the sterol pathway. When cells were cultured in lipoprotein-deficient medium, Fmev (200 microM) completely inhibited mitogen-stimulated human lymphocyte proliferation, quantified by measuring DNA synthesis. The addition of low density lipoprotein (LDL) restored lymphocyte responses to normal, whereas mevalonate was totally ineffective. Similar results were obtained with concentrations of Fmev up to 1 mM. These results contrast with those observed when sterol biosynthesis was blocked with lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase. When lymphocyte proliferation was blocked with lovastatin (5 microM), either high concentrations of mevalonate or LDL together with low concentrations of mevalonate was required to restore responses. In contrast, neither LDL nor low concentrations of mevalonate when alone was able to restore lymphocyte DNA synthesis in cultures blocked with 5 microM lovastatin. The effect of Fmev on the capacity of exogenous mevalonate to restore proliferation of lovastatin-blocked lymphocytes was directly examined. Fmev had no effect on the capacity of LDL plus low concentrations of mevalonate to restore DNA synthesis to lovastatin-blocked lymphocytes, indicating that the synthesis of the necessary factor from mevalonate was unaltered by Fmev. Fmev profoundly blocked lymphocyte endogenous sterol synthesis, decreasing incorporation of radiolabeled acetate into digitonin-precipitable sterols by up to 98%. LDL did not alter the capacity of Fmev to block sterol synthesis. The possibility that Fmev allowed shunting of endogenous mevalonate into essential lipid products was assessed by examining the incorporation of radiolabeled mevalonate. Fmev (200 microM) inhibited the incorporation of mevalonate into all lipids, including ubiquinone, dolichol, and other non-sterol lipids by up to 98%, and this was not altered by LDL. Furthermore, Fmev (200 microM) suppressed the incorporation of radiolabeled mevalonate into protein by up to 97%. These data confirm that a product of mevalonate is essential for cell proliferation. However, the results indicate that the required product is directly synthesized from mevalonate or mevalonate phosphates rather than from a more distal isoprenoid metabolite.     

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.     

Incorporation of mevalonate into dolichol and other isoprenoids during estrogen-induced chick oviduct differentiation

Incorporation of [14C]mevalonate into dolichol and other isoprenoid compounds by chick oviduct explants has been studied. A reliable assay of dolichol biosynthesis employing several chromatographic procedures, including two-dimentional TLC, was developed. Incorporation of [14C]mevalonate into dolichol by oviduct explants was linear for at least 6 h. The effect of estrogen-induced differentiation was studied by incubation of explants obtained from chicks treated for various periods of time with diethylstilbestrol. Mevalonate incorporation into dolichol, when expressed as cpm per g of tissue, was not affected by estrogen treatment, but since the oviduct increased about 100-fold in mass during differentiation, each oviduct synthesizes about 100-fold more dolichol. In most tissues, the major product of mevalonate incorporation is cholesterol. However, although approx. 90% of the non-saponifiable 14C-labeled compounds were in the so-called 'cholesterol fraction', oviduct explants from estrogenized chicks synthesized little, if any, cholesterol. A number of cholesterol biosynthetic intermediates were observed, with compounds comigrating with squalene and lanosterol accounting for about 50% of the total. Since the estrogenized chick has serum cholesterol levels in the range of 800-900 mg/dl, these results suggest that oviduct has secondary control points which allow it to inhibit cholesterol synthesis when mevalonate is used as the precursor. In support of this hypothesis is the observation that explants from untreated chicks can incorporate mevalonate into cholesterol.     

Effects of mevinolin treatment on tissue dolichol and ubiquinone levels in the rat.

Rats were treated with mevinolin by intraperitoneal injection (15 days) or dietary administration (30 days). The cholesterol, dolichol, dolichyl phosphate and ubiquinone contents of the liver, brain, heart, muscle and blood were then investigated. The cholesterol contents of these organs did not change significantly, with the exception of muscle. Intraperitoneal administration of the drug increases the amount of dolichol in liver, muscle and blood and decreases the dolichyl-P amount in muscle. The same treatment increases the level of ubiquinone in muscle and blood and decreases this value in liver and heart. Oral administration decreases dolichol, dolichyl-P and ubiquinone levels in heart and muscle, while in liver the dolichol level is elevated and ubiquinone level lowered. In brain the amount of dolichyl-P is increased. Intraperitoneal injection of mevinolin also modifies the liver dolichol and dolichyl-P isoprenoid pattern, with an increase in shorter chain polyisoprenes. The levels of dolichol and ubiquinone in the blood do not follow the changes observed in other tissues. Incorporation of [3H]acetate into cholesterol by liver slices prepared from mevinolin-treated rats exhibited an increase, whereas in brain no change was seen. Labeling of dolichol and ubiquinone was increased in both liver and brain, but incorporation into dolichyl phosphate remained relatively stable. The results indicate that mevinolin affects not only HMG-CoA reductase but, to some extent, also affects certain of the peripheral enzymes, resulting in considerable effects on the various mevalonate pathway lipids.     

Obligatory Relationship Between the Sterol Biosynthetic Pathway and DNA Synthesis and Cellular Proliferation in Glial Primary Cultures

Primary cultures of newborn rat brain, which are composed predominantly of astroglia, were used to examine the relationship between the sterol biosynthetic pathway and DNA synthesis and cellular proliferation. Reduction of the fetal calf serum content of the culture medium from 10 to 0.1% (vol/vol) for an interval of 48 h between days 4 and 6 in culture resulted in a quiescent state characterized by inhibition of DNA synthesis and cellular proliferation. When 10% fetal calf serum was returned to the medium for these quiescent cells, within 24 h DNA synthesis increased markedly. Preceding the rise in DNA synthesis was an increase in sterol synthesis, which occurred within 12 h of the return of the quiescent cells to the 10% fetal calf serum. Exposure of the quiescent cells to mevinolin, a specific inhibitor of sterol synthesis at the 3-hydroxy-3-methylglutaryl-CoA reductase step, completely inhibited the increase in DNA synthesis that followed serum repletion. The increase in total protein synthesis that followed serum repletion was not similarly inhibited by mevinolin. When mevinolin was removed after causing the 24-h inhibition of DNA synthesis, the cultured cells underwent active DNA synthesis and proliferation. Thus, inhibition of the sterol biosynthetic pathway resulted in a specific and reversible inhibition of DNA synthesis and glial proliferation in developing glial cells. These findings establish a valuable system for the examination of glial proliferation, i.e., primary glial cultures subjected to serum depletion and subsequent repletion. Moreover, the data establish an obligatory relationship between the sterol biosynthetic pathway and DNA synthesis and cellular proliferation in developing glia.     

Isoprenoid regulation of cell growth: Identification of mevalonate-labelled compounds inducing DNA synthesis in human breast cancer cells depleted of serum and mevalonate

Growth arrest induced by serum depletion and/or treatment with mevinolin (an inhibitor of mevalonate synthesis) in the human breast cancer cell line Hs578T was overcome by exogenous mevalonate, indicating that some product or metabolite of mevalonate may be involved in the mediation of serum-regulated growth of these cells. In the search for such compounds we first tested a variety of known end products of mevalonate with respect to their ability to counteract the inhibition of DNA synthesis caused by serum-free medium and mevinolin. Thereby high doses (10 μg/ml) of dolichol-20 were found to cause a partial counteraction. After straight-phase HPLC purification of endogenous lipids, isolated from ³H- or ¹⁴C-mevalonate-labelled Hs578T cultures, we found that non-sterol lipids co-eluting with dolichols efficiently induced DNA synthesis. After further purification with reverse-phase HPLC it was confirmed that virtually all of this effect was achieved by compound(s) (seen as a single UV and radioactive peak) co-eluting with dolichol-20. Nanogram doses, at most, of this (these) compound(s) elicited a substantial stimulation of DNA synthesis. The lipid(s) also counteracted the inhibition by mevinolin of N-linked glycosylation, indicating that it (they) also interfere(s) with this processing. Since treatment with tunicamycin (an inhibitor of N-linked glycosylation) abolished this growth-stimulative effect, N-linked glycosylation seems to be a necessary event in the processes leading to lipid-induced initiation of DNA synthesis.     

Enhanced production of dolichol,but not dolichyl phosphate,in the earliest stages of rat liver regeneration

The regenerating liver presents a changed ability to use mevalonate 16 hr after partial hepatectomy. The dolichol content and its synthesis from mevalonate is increased, while no variation of dolichyl-P and ubiquinone parameters are detectable.The greater amount ofmevalonate utilized to form dolichol, but not dolichyl-P, in this proliferating system, raises some questions about the physiological significance of these isoprenoid compounds and about their biosynthetic sequence.