Cell cycle-specific growth inhibition of human breast cancer cells induced by metabolic inhibitors

Proliferation of exponentially growing breast cancer cells (lineHs578T) was blocked specifically in G₁ by 3-hydroxy-3-methylglutarylCoenzyme A (HMG CoA) reductase inhibition, as well as by inhibitionof N-linked glycosylation. As a consequence of these inhibitoryconditions, the cells were synchronized in the G₁ stage of thecell cycle. The similarities in the kinetic responses pointto the possibility that the two different types of metabolicinhibitions block cell cycle progression by common mechanisms.One possibility is that the inhibition of HMG CoA reductaseactivity also leads to a depressed rate of N-linked glycosylation,which in turn may constitute the critical event for cell cycleprogression and cell growth. In order to investigate whetherthis relationship exists in breast cancer cells, cells synchronizedin G₁ by mevinolin (an inhibitor of HMG CoA reductase) wereused. Upon addition of mevalonate, whose endogenous synthesisis catalysed by HMG CoA reductase, the cells entered S phaseafter a 4 h pre-replicative period. Mevalonate stimulation alsoled to a rapid and substantial increase in N-linked glycosylation,measured by determining the uptake of radioactive glucosamine.This metabolic event was found to be of critical importancefor the initiation of DNA synthesis. However, as soon as thecells had entered S phase, they were independent of the levelof N-linked glycosylation. breast cancer cells glycosylation HMG CoA reductase     

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.     

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)     

Isoprenoids and astroglial cell cycling: diminished mevalonate availability and inhibition of dolichol-linked glycoprotein synthesis arrest cycling through distinct mechanisms.

Primary astroglial cultures were used to compare the relationships to cell cycling of dolichol-linked glycoprotein synthesis, and of availability of mevalonate, the precursor of dolichol and other isoprenoid lipids. With shift-up to 10% serum (time 0) after 48 h of serum depletion, the proportion of cells in S phase (bromodeoxyuridine immunofluorescence) remained under 15% for 12 h, then increased by 20 h to 72 +/- 10%; DNA synthetic rates (thymidine incorporation) increased 5-fold. S phase transition was prevented by addition at 10-12 h of tunicamycin, an inhibitor of transfer of saccharide moieties to dolichol. Mevinolin, an inhibitor of mevalonate biosynthesis, also blocked cycle progression when added at this time. However, mevinolin markedly inhibited the isoprenoid pathway, as reflected by over 90% reduction of sterol synthesis, without inhibiting net glycoprotein synthesis. Removal of mevinolin after a 24 h exposure delayed S phase until 48 h, following recovery of sterol synthesis, even though kinetics of glycoprotein synthesis were unaffected. Tunicamycin removal after 24 h spared sterol synthesis, but caused delay of S phase until 72 h, following recovery of glycoprotein synthesis. In mevinolin-treated cultures, S phase transition was restored by 1 h of exposure to mevalonate at 10 h, although cycling was thereby rendered sensitive to inhibition by cycloheximide and by tunicamycin. Cell cycle progression following hydroxyurea exposure and release was unaffected by mevinolin, tunicamycin, or cycloheximide. Thus, in these developing astroglia, mevalonate and its isoprenoid derivatives have at least two cell cycle-specific roles: dolichol-linked glycoprotein synthesis is required at or before the G1/S transition, while a distinct mevalonate requirement is apparent also in late G1.     

Mevalonate-regulated mechanisms in cell growth control: role of dolichyl phosphate in expression of the insulin-like growth factor-1 receptor (IGF-1R) in comparison to Ras prenylation and expression of c-myc

One or more mevalonate derivatives of non-sterol type have beenproposed to be of indispensable importance for cell growth.Conceivable mevalonate-dependent mechanisms involved in growthcontrol are farnesylation of Ras proteins, regulation of c-mycexpression, and N-linked glycosylation of the IGF-1 receptor.The latter mechanism might be rate-limited by dolichyl phosphate,which acts as a donor of oligosaccharides in glycoprotein synthesisin the endoplasmic reticulum. In order to study the significancefor cell proliferation of the three aforementioned mevalonate-dependentprocessings, their inhibitory response due to mevalonate deprivationwas explored and compared with the effect on DNA synthesis inthe malignant melanoma cell line SK-MEL-2. We found that mevalonatedepletion due to treatment with 3 µM lovastatin for 24h, which efficiently growth-arrested the cells, hardly at allaffected the expression of c-myc, and although Ras prenylationwas inhibited by 50%, the most pronounced effect of lovastatinwas seen on N-linked glycosylation of IGF-1 receptors, whichwas inhibited by more than 95%. The order and magnitude of thedecreased IGF-1 receptor glycosylation, which was followed bya decreased expression of IGF-1 receptors at the cell membrane,correlated well with the inhibition of DNA synthesis. We investigatedwhether dolichol, and in particular dolichyl phosphate, throughits participation in N-linked glycosylation, act as regulatorsof IGF-1 receptor expression. First, we could confirm that exogenousdolichol became phosphorylated and in this form took part inthe glycosylation processing. Secondly, we showed that dolichylphosphate, in a dose-dependent manner, could increase the numberof IGF-1 receptors at the cell membrane, simultaneously as DNAsynthesis was stimulated. Taken together, our results providedirect evidence for an important role of dolichyl phosphateas a regulator of cell growth through limiting N-linked glycosylationof the IGF-1 receptor. dolichyl phosphate IGF-1 receptor c-myc N-linked glycosylation Ras     

Profiling and comparative analysis of glycoproteins in Hs578BST and Hs578T and investigation of prolyl 4-Hydroxylase alpha polypeptide II expression and influence in breast cancer cells

To identify potential cancer related glycoproteins in breast cancer cells, we enriched N-linked glycoproteins by lentil lectin from the human breast cancer cell line Hs578T and the normal breast cell line Hs578BST for proteomic comparison. Glycoproteins were separated and compared by two-dimensional electrophoresis. Twenty-four glycoproteins were identified that expressed remarkably differently, among which nine were involved in the progress of collagen synthesis. Prolyl 4-hydroxylase alpha polypeptide II (P4HA2) expression and influence in breast cancer was further investigated. Immunohistochemistry revealed that P4HA2 was upregulated in breast tumor cells compared with its adjacent normal tissues. Moreover, overexpression and RNA interference of P4HA2 showed that P4HA2 expression suppressed cell proliferation and migration in Hs578T in vitro.     

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.     

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)     

Induction of sesquiterpenoid biosynthesis in tobacco cell suspension cultures by fungal elicitor

Large amounts of the sesquiterpenoid capsidiol accumulated in the media of tobacco (Nicotiana tabacum L. cv KY14) cell suspension cultures upon addition of fungal elicitor. Capsidiol accumulation was proportional to the amount of elicitor added. The accumulation of capsidiol was preceded by a transient increase in the capsidiol de novo synthesis rate as measured by the incorporation of exogenous [¹⁴C]acetate. Changes in 3-hydroxy-3-methylglutaryl-CoA reductase activity (HMGR; EC 1.1.1.34), an enzyme of general isoprenoid metabolism, paralleled the changes in [¹⁴C]acetate incorporation into capsidiol. Incubation of the cell cultures with mevinolin, a potent in vitro inhibitor of the tobacco HMGR enzyme activity, inhibited the elicitor-induced capsidiol accumulation in a concentration dependent manner. [¹⁴C]Acetate incorporation into capsidiol was likewise inhibited by mevinolin treatment. Unexpectedly, [³H] mevalonate incorporation into capsidiol was also partially inhibited by mevinolin, suggesting that mevinolin may effect secondary sites of sesquiterpenoid biosynthesis in vivo beyond HMGR. The data indicated the importance of the induced HMGR activity for capsidiol production in elicitor-treated tobacco cell suspension cultures.     

Isoprenoid synthesis in isolated embryonic Drosophila cells. Sterol-independent regulatory signal molecule is distal to isopentenyl 1-pyrophosphates

Embryonic Drosophila cells (Kc cells) were used to further characterize sterol-independent modulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity. 3-Methyl-3-5-dihydroxyvalerate (mevalonate), 3-fluoromethyl-3,5-dihydroxyvalerate (fluoromevalonate), and 3-ethyl-3,5-dihydroxyvalerate (homomevalonate) were tested as modulators. Although mevalonate caused a rapid, reversible suppression of reductase activity, fluoro- and homomevalonate increased activity; fluoromevalonate was more effective than homomevalonate. Mevalonate, added simultaneously with fluoromevalonate, blocked the analogue's effect on Kc cell reductase activity. However, mevalonate did not suppress an established fluoromevalonate increase in HMG-CoA reductase activity. Fluoromevalonate blocked [1-14C, 5-3H]mevalonate conversion to 14CO2- and 3H-labeled lipids and [3H] mevalonate 5-pyrophosphate accumulated. Neither protein nor RNA synthesis were required for mevalonate-mediated suppression of reductase activity. However, fluoromevalonate's effect on reductase activity required protein synthesis. Furthermore, in the absence of protein synthesis, fluoromevalonate-stabilized Kc cell HMG-CoA reductase activity. We have concluded that mevalonate, fluoromevalonate, homomevalonate, and compactin (mevinolin) modulated HMG-CoA reductase activity because they altered isoprenoid carbon flow to a post-isopentenyl 1-pyrophosphate regulatory, signal molecule.     

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.     

Inhibition by mevinolin of plant growth, sterol formation and pigment accumulation

To obtain information on the importance of a functional mevalonate synthesis for plant growth and development, we investigated the effect of mevinolin, a highly specific inhibitor of 3-hydroxy-3-methylglutaryl (HMG) coenzyme A reductase (the mevalonate-producing enzyme) on growth, sterol accumulation and pigment formation of radish seedlings (Raphanus sativus L. cv. Saxa Treib) and in part also wheat seedlings (Triticum aestivum L. cv. Kolibri). Mevinolin applied during germination inhibits root elongation and development of lateral roots in etiolated and light-grown radish seedlings. This effect cannot be overcome by exogenous GA₃, but by addition of mevalonic acid, the product of the internally inhibited reaction. This emphazises the specifity of the mevinolin effect and indicates that the biosynthesis of mevalonic acid is a mandatory requirement for root growth. In light-grown radish seedlings mevinolin also affects hypocotyl length-growth and inhibits sterol accumulation, but has little effect on the chlorophyll and carotenoid accumulation in the chloroplasts of the cotyledons. This indicates the possible presence of an independent mevalonate synthesizing pathway within the plastids and suggests a low transport rate of mevinolin from the radish roots to the cotyledons. When mevinolin is directly applied to the leaves at higher concentrations, it also reduces the light-induced chlorophyll and carotenoid accumulation as has been shown with etiolated primary leaves of wheat. This inhibition is age-dependent and proceeds to a higher extent in older than in younger etiolated leaf tissue. From our results we conclude that plastids possess an independent HMG-CoA reductase. In the cotyledons of radish, mevinolin seems to induce a senescence retardation and sun-type growth response, as has been evaluated by measuring the fast and slow chlorophyll fluorescence induction kinetics (Kautsky effect). These responses may be due to inhibitor-induced changes in the intracellular phytohormone balance.     

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.     

Inhibition of a plant sesquiterpene cyclase by mevinolin

The specificity of mevinolin as an inhibitor of sterol and sesquiterpene metabolism in tobacco cell suspension cultures was examined. Exogenous mevinolin inhibited [14C]acetate, but not [3H]mevalonate incorporation into free sterols. In contrast, mevinolin inhibited the incorporation of both [14C]acetate and [3H]mevalonate into capsidiol, an extracellular sesquiterpene. Microsomal 3-hydroxy-3-methylglutaryl Coenzyme A reductase was inhibited greater than 90% by microM mevinolin, while squalene synthetase was insensitive to even 600 microM mevinolin. Sesquiterpene cyclase, the first branch point enzyme specific for sesquiterpene biosynthesis, was inhibited in a dose-dependent manner by mevinolin with a 50% reduction in activity at 100 microM. Kinetic analysis indicated that the mechanism for inhibition was complex with mevinolin acting as both a competitive and noncompetitive inhibitor. The results suggest that the mevinolin inhibition of [3H]mevalonate incorporation into extracellular sesquiterpenes can, in part, be attributed to a secondary, but specific, site of inhibition, the sesquiterpene cyclase.     

Dolichols, ubiquinones, geranylgeraniol and farnesol as the major metabolites of mevalonate in Phytophthora cactorum

Farnesol, geranylgeraniol, dolichols and ubiquinones were the main radioactive components of the unsaponifiable lipid recovered from Phytophthora cactorum grown in aerated cultures containing [2-¹⁴C]mevalonate. The ¹⁴C recovered in each of these components was in the approximate proportion 2:4:3:5. When the culture was not aerated no radioactive ubiquinone was recovered. Most of the ¹⁴C recovered in the dolichols was found in dolichol-15 (37%), with decreasing amounts in dolichol-14 (30%) and -13 (14%) and only a little (5%) in dolichol-16, whereas the major components, by weight, of the mixture (13μg/g of damp-dry tissue) were dolichol-14, -15 and -16 in the approximate proportion of 1:3:1. Radioautography of appropriate chromatograms indicated the presence also of traces of radioactivity in dolichol-9, -10, -11, -12 and -17. Most (80%) of the ¹⁴C recovered in the ubiquinones was associated with ubiquinone-9, the rest being in ubiquinone-8. Most (80%) of the weight of ubiquinones (19μg/g of damp-dry tissue) was also ubiquinone-9. The identification of these compounds was by chromatographic methods and, for the ubiquinones and dolichols, was confirmed by mass spectrometry. In addition, the incorporation of 4R- and/or 4S-³H from [4-³H]-mevalonates showed the expected stereochemistry of biosynthesis, namely that farnesol, geranylgeraniol and ubiquinones were biogenetically all trans and the dolichols each contained three biogenetically trans isoprene residues, the remaining residues being biogenetically cis. The distribution of ¹⁴C in the components of the whole lipid of the fungus was consistent with 97% of both the farnesol and geranylgeraniol being present as the fatty acid ester. The corresponding value for dolichols was 37%. The observation by other workers, that this fungus does not form either squalene or sterol, was confirmed.     

Mechanism of Photoregulated Carotenogenesis in Rhodotorula minuta VIII. Effect of Mevinolin on Photoinduced Carotenogenesis

Mevinolin, which is a highly specific competitive inhibitorof 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase,was used in a search for photoinducible enzyme(s) other thanHMG-CoA reductase in the pathway of carotenoid biosynthesisin Rhodotorula minuta. The photoinduced production of carotenoids was competitivelyinhibited by mevinolin. The concentration of mevinolin thatis required to inhibit completely the production of carotenoidsdepends on the light dose given to the cells. However, the relationshipbetween the inhibition ratio and the concentration of mevinolinwas almost identical regardless of the light dose. These resultssuggest that the activity of enzymes involved in the formationof HMG-CoA may not be affected by light. When an adequate amount of mevalonate was added to the growthmedium that contained sufficient mevinolin for the completeinhibition of the photoinduction of the production of carotenoids,the same quantity of carotenoids was produced as in the absenceof mevinolin. Moreover, the production of carotenoids in thepresence of both mevinolin and mevalonate was inhibited by cycloheximide. It appears from these results that one or more photoinducibleenzymes, such as HMG-CoA reductase, may be present in the carotenogenicpathway beyond mevalonate. (Received April 12, 1989; Accepted January 16, 1990)     

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.     

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.     

The effect of protein synthesis inhibitors on the glycosylation site occupancy of recombinant human prolactin

The relationship between synthesis and N-liked glycosylation site occupancy of recombinant human prolactin produced from C127 cells was studied with the aid of a battery of protein synthesis inhibitors. Non-lethal concentrations of sodium fluoride, gougerotin, puromycin, anisomycin, and emetine did not alter site occupancy, but low concentrations (<10g ml^–1) of cycloheximide increased the fraction of secreted prolactin bearing oligosaccharide from 20% to 80% of the total. Cycloheximide is an inhibitor of the elongation step of protein synthesis. The observed increase in glycosylation site occupancy upon addition of cycloheximide is consistent with the current opinion that the initial glycosylation event occurs cotranslationally during a limited time period. Cycloheximide may extend this time period by reducing elongation rate. However, the absence of any effect from treatment with other inhibitors of elongation suggests that cycloheximide is unique in its behavior on this system.Abbreviations clp-PRL clipped form of prolactin - DMEM/F12 11 Dulbecco's Modified Eagle's Medium/Ham's nutrient mixture F12 - G-PRL glycosylated (N-linked) fraction of prolaction - NG-PRL prolactin fraction without N-linked glycosylation - PMSF phenylmethylsulfonylfluoride     

The biosynthesis of C55 polyprenols by a cell-free preparation of Lactobacillus plantarum

A cell-free supernatant of lysates of Lactobacillus plantarum catalyses the synthesis of lipids from [2-¹⁴C]mevalonate. Of the added mevalonate, 7.5% is incorporated into lipids, which were fractionated into five components. About 4% of the radioactivity in these lipids co-chromatographs with compounds shown by mass spectrometry, n.m.r. and i.r. spectroscopy to be C₅₅ polyprenols, and about 2% co-chromatographs with a hexamer. The rest of the radioactivity is in more complex fractions. Analysis by mass spectrometry, n.m.r. and i.r. spectroscopy shows that the major C₅₅ polyprenol is undecaprenol, accompanied by an isomer containing one reduced isoprene unit. A Kuhn–Roth degradation of [¹⁴C]polyprenols indicates that the supernatant catalyses synthesis of these compounds de novo.