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.     

Interrelationships between mevalonate metabolism and the mitogenic signaling pathway in T lymphocyte proliferation.

Upon stimulation with antigen or antibodies directed at the CD3.T cell receptor complex, T lymphocytes undergo a series of biochemical events that result in DNA synthesis and cellular proliferation. The purpose of the current study was to explore the role of mevalonic acid and its metabolites in this process. Stimulation of freshly isolated human T cells with immobilized anti-CD3 monoclonal antibody (mAb) results in the induction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase message, with maximum induction occurring at 24 h of culture, approximately 12 h before the onset of DNA synthesis. Protein kinase C (PKC) probably mediates this induction, as H7, which inhibits PKC and cyclic nucleotide-dependent protein kinases, but not HA1004, which inhibits all of these protein kinases except PKC, completely abrogates the appearance of HMG-CoA reductase message. The importance of HMG-CoA reductase induction and mevalonate production in cell cycle progression was demonstrated by the observation that either 25-hydroxycholesterol, which inhibits this induction, or lovastatin, a competitive inhibitor of HMG-CoA reductase, inhibited anti-CD3-induced T cell mitogenesis in a dose-dependent manner. The presence of lovastatin during the first 24-36 h of culture results in a progressive delay of cell cycle progression, whereas this agent, when present only for the first 12 h of culture, had no effect on T cell proliferation. These results suggest that mevalonate is required for cell cycle progression from mid-G1 into late G1. Exogenous mevalonate overcomes the antiproliferative effect of lovastatin but not of 25-hydroxycholesterol. Since 25-hydroxycholesterol suppresses the metabolism of mevalonic acid at multiple points, this result suggests that one or more metabolites of mevalonate, rather than mevalonate itself, plays an essential role in cell cycle progression. One metabolite of mevalonate, farnesol pyrophosphate, may play such a role, since free farnesol suppresses anti-CD3 mAb-induced T cell proliferation in a concentration-dependent manner. In mAb is associated with PKC-dependent induction of HMG-CoA reductase which, in turn, leads to the generation of mevalonic acid and its metabolites, one or more of which play a requisite role in cell cycle progression.     

Inhibition of farnesylation blocks growth but not differentiation in FRTL-5 thyroid cells.

The cholesterol lowering drug lovastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, blocks DNA synthesis and proliferation of thyrotropin (TSH) primed FRTL-5 rat thyroid cells. The blockade can be completely prevented and/or reversed by mevalonate and largely prevented and/or reversed by farnesol whereas cholesterol and/or other non-sterol mevalonate derivatives such as ubiquinone, dolichol or isopentenyladenosine are ineffective. TSH-dependent augmentation of cyclic-AMP and cAMP dependent differentiated functions, such as iodide uptake, are unaffected by lovastatin. 3H-Thymidine incorporation into DNA is also decreased by alpha-hydroxyfarnesyl-phosphonic acid, an inhibitor of protein farnesylation which mimicks the effect of lovastatin since it also leaves unaffected TSH stimulated iodide uptake. It is suggested that the HMG-CoA reductase inhibitor lovastatin affects cell proliferation mainly through inhibition of protein farnesylation which results in altered function proteins relevant for proliferation control, notably p21ras and/or other small GTPases.     

Post-translational isoprenylation of cellular proteins is altered in response to mevalonate availability

Cells incorporate isoprenoid products derived from mevalonate (MVA) into several unique proteins. The aim of this study was to delineate the effects of blocking MVA synthesis on the covalent isoprenylation of these proteins in murine erythroleukemia cells. Inhibition of protein synthesis with cycloheximide prevented the incorporation of [3H]MVA into proteins, suggesting that isoprenylation normally occurs immediately after synthesis of the polypeptides. However, incubation of cells with lovastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, for as little as 1 h prior to addition of cycloheximide rendered the isoprenylation step insensitive to cycloheximide. Lovastatin had no apparent effect on the stability of the isoprenylated proteins, but the development of cycloheximide insensitivity during the lovastatin preincubation was dependent on synthesis of new protein during that period. Addition of 50-200 microM MVA to the culture medium eliminated the effects of preincubation with lovastatin. Preincubation of cells with 25-hydroxycholesterol, which suppresses the synthesis and enhances the degradation of HMG-CoA reductase but is not a competitive enzyme inhibitor, did not induce cycloheximide-insensitivity of the isoprenylation reaction. The results suggest that blocking MVA synthesis with lovastatin causes a rapid depletion of isoprenoid groups available for protein modification. Consequently, there is an accumulation of non-isoprenylated substrate proteins. Shifts in the ratio of modified vs. unmodified proteins in response to MVA availability may have implications for the changes in cell morphology, cell proliferation and HMG-CoA reductase gene expression that occur when cells are subjected to MVA deprivation.     

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.     

Effect of inhibition of cholesterol synthetic pathway on the activation of Ras and MAP kinase in mesangial cells

Intermediary metabolites of cholesterol synthetic pathway are involved in cell proliferation. Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, blocks mevalonate synthesis, and has been shown to inhibit mesangial cell proliferation associated with diverse glomerular diseases. Since inhibition of farnesylation and plasma membrane anchorage of the Ras proteins is one suggested mechanism by which lovastatin prevents cellular proliferation, we investigated the effect of lovastatin and key mevalonate metabolites on the activation of mitogen-activated protein kinase (MAP kinase) and Ras in murine glomerular mesangial cells. The preincubation of mesangial cells with lovastatin inhibited the activation of MAP kinase stimulated by either FBS, PDGF, or EGF. Mevalonic acid and farnesyl-pyrophosphate, but not cholesterol or LDL, significantly prevented lovastatin-induced inhibition of agonist-stimulated MAP kinase. Lovastatin inhibited agonist-induced activation of Ras, and mevalonic acid and farnesylpyrophosphate antagonized this effect. Parallel to the MAP kinase and Ras data, lovastatin suppressed cell growth stimulated by serum, and mevalonic acid and farnesylpyrophosphate prevented lovastatin-mediated inhibition of cellular growth. These results suggest that lovastatin, by inhibiting the synthesis of farnesol, a key isoprenoid metabolite of mevalonate, modulates Ras-mediated cell signaling events associated with mesangial cell proliferation.     

Effects of compactin, mevalonate and low-density lipoprotein on 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and low-density-lipoprotein-receptor activity in the human hepatoma cell line Hep G2.

Compactin, an inhibitor of HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase, decreased cholesterol synthesis in intact Hep G2 cells. However, after the inhibitor was washed away, the HMG-CoA-reductase activity determined in the cell homogenate was found to be increased. Also the high-affinity association of LDL (low-density lipoprotein) to Hep G2 cells was elevated after incubation with compactin. Lipoprotein-depleted serum, present in the incubation medium, potentiated the compactin effect compared with incubation in the presence of human serum albumin. Addition of either mevalonate or LDL prevented the compactin-induced rise in activities of both HMG-CoA reductase and LDL receptor in a comparable manner. It is concluded that in this human hepatoma cell line, as in non-transformed cells, both endogenous mevalonate or mevalonate-derived products and exogenous cholesterol are able to modulate the HMG-CoA reductase activity as well as the LDL-receptor activity.     

The cytotoxicity of mevalonate, lovastatin and carminomycin with respect to MOLT-4 human malignant T-lymphoblasts in vitro]

It was shown in vitro that high concentrations of lovastatin, a competitive inhibitor of hydroxymethyl glutaryl coenzyme A (HMG-CoA) reductase inhibited human malignant cells MOLT-4. The activity of lovastatin in doses of 50-250 microM was dose-dependent. Addition of mevalonate in a concentration of 3 mM to the growth medium completely prevented the cytotoxic effect of 100 microM of lovastatin. At the same time, exogenous mevalonate did not decrease the cytotoxicity of the anthracycline antibiotic carminomycin. Moreover, in a high concentration (7 mM) mevalonate slowly but significantly inhibited the growth of the malignant target cells and the effect was added to the cytotoxic effect of carminomycin low concentrations (0.08 to 0.175 microgram/ml). The results and the literature data suggested that combination of mevalonate, HMG-CoA reductase inhibitors and anthracyclines could be useful in tumor chemotherapy. The suggestion needs further investigation.     

Effect of geraniol on fatty-acid and mevalonate metabolism in the human hepatoma cell line Hep G2.

Monoterpenes have multiple pharmacological effects on the metabolism of mevalonate. Geraniol, a dietary monoterpene, has in vitro and in vivo anti-tumor activity against several cell lines. We have studied the effects of geraniol on growth, fatty-acid metabolism, and mevalonate metabolism in the human hepatocarcinoma cell line Hep G2. Up to 100 micromol geraniol/L inhibited the growth rate and 3-hydroxymethylglutaryl coenzyme A reductase (HMG-CoA) reductase activity of these cells. At the same concentrations, it increased the incorporation of cholesterol from the medium in a dose-dependent manner. Geraniol-treated cells incorporated less 14C-acetate into nonsaponifiable lipids, inhibiting its incorporation into cholesterol but not into squalene and lanosterol. This is indicative of an inhibition in cholesterol synthesis at a step between lanosterol and cholesterol, a fact confirmed when cells were incubated with 3H-mevalonate. The incorporation of 3H-mevalonate into protein was also inhibited, whereas its incorporation into fatty acid increased. An inhibition of delta5 desaturase activity was demonstrated by the inhibition of the conversion of 14C-dihomo-gamma-linolenic acid into arachidonic acid. Geraniol has multiple effects on mevalonate and lipid metabolism in Hep G2 cells, affecting cell proliferation. Although mevalonate depletion is not responsible for cellular growth, it affects cholesterogenesis, protein prenylation, and fatty-acid metabolism.     

Hep-G2 cells and primary rat hepatocytes differ in their response to inhibitors of HMG-CoA reductase

CI-981, a novel synthetic inhibitor of HMG-CoA reductase, was previously reported to be highly liver-selective using an ex vivo approach. In order to determine liver-selectivity at the cellular level, CI-981 was evaluated in cell culture and compared to lovastatin, pravastatin, fluvastatin and BMY-21950. Using human cell lines, none of the compounds tested showed liver-selectivity, i.e. strong inhibition of cholesterol synthesis in Hep-G2 cells (liver model) but weak inhibition in human fibroblasts (peripheral cell model). In contrast, all drugs tested produced equal and potent inhibition of sterol synthesis in primary cultures of rat hepatocytes, and CI-981, pravastatin and BMY-21950 were more than 100-fold more potent in rat hepatocytes compared to human fibroblasts. Since all compounds were also equally potent at inhibiting sterol synthesis in a rat subcellular system and in vivo, the data suggest that the use of Hep-G2 cells may not be the cell system of choice in which to study inhibition of hepatic cholesterogenesis or to demonstrate liver selectivity of inhibitors of HMG-CoA reductase.     

Lovastatin induces apoptosis in a metastatic ovarian tumour cell line

Lovastatin is a very specific and potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which regulates a rate-limiting step in the cellular synthesis of isoprenoid and cholesterol. In this study, we demonstrate that treatment of rat ovarian metastatic OV1N cells with lovastatin induces apoptosis. Furthermore, apoptotic death of lovastatin-treated OV1N cells can be prevented by the addition of either mevalonic acid (an immediate metabolite of HMG-CoA) or farnesyl pyrophosphate (one of the downstream products of mevalonic acid metabolism). However, metabolic derivatives of farnesyl pyrophosphate failed to prevent the apoptotic effect of lovastatin on cells. Therefore farnesyl pyrophosphate appears to be important for cell survival and the relationship of this compound to protein farnesylation and apoptosis induction is discussed.     

Sterol-independent regulation of 3-hydroxy-3-methylglutaryl-CoA reductase by mevalonate in Chinese hamster ovary cells. Magnitude and specificity

In this paper, we assess the relative degree of regulation of the rate-limiting enzyme of isoprenoid biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, by sterol and nonsterol products of mevalonate by utilizing cultured Chinese hamster ovary cells blocked in sterol synthesis. We also examine the two other enzymes of mevalonate biosynthesis, acetoacetyl-CoA thiolase and HMG-CoA synthase, for regulation by mevalonate supplements. These studies indicate that in proliferating fibroblasts, treatment with mevalonic acid can produce a suppression of HMG-CoA reductase activity similar to magnitude to that caused by oxygenated sterols. In contrast, HMG-CoA synthase and acetoacetyl-CoA thiolase are only weakly regulated by mevalonate when compared with 25-hydroxycholesterol. Furthermore, neither HMG-CoA synthase nor acetoacetyl-CoA thiolase exhibits the multivalent control response by sterol and mevalonate supplements in the absence of endogenous mevalonate synthesis which is characteristic of nonsterol regulation of HMG-CoA reductase. These observations suggest that nonsterol regulation of HMG-CoA reductase is specific to that enzyme in contrast to the pleiotropic regulation of enzymes of sterol biosynthesis observed with oxygenated sterols. In Chinese hamster ovary cells supplemented with mevalonate at concentrations that are inhibitory to reductase activity, at least 80% of the inhibition appears to be mediated by nonsterol products of mevalonate. In addition, feed-back regulation of HMG-CoA reductase by endogenously synthesized nonsterol isoprenoids in the absence of exogenous sterol or mevalonate supplements also produces a 70% inhibition of the enzyme activity.     

HMG-CoA reductase inhibitors induce apoptosis of lymphoma cells by promoting ROS generation and regulating Akt,Erk and p38 signals via suppression of mevalonate pathway

Statins, the inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, are widely used cholesterol-lowering drugs. Convincing evidence indicates that statins stimulate apoptotic cell death in several types of proliferating tumor cells in a cholesterol-lowering-independent manner. The objective here was to elucidate the molecular mechanism by which statins induce lymphoma cells death. Statins (atorvastatin, fluvastatin and simvastatin) treatment enhanced the DNA fragmentation and the activation of proapoptotic members such as caspase-3, PARP and Bax, but suppressed the activation of anti-apoptotic molecule Bcl-2 in lymphoma cells including A20 and EL4 cells, which was accompanied by inhibition of cell survival. Both increase in levels of reactive oxygen species (ROS) and activation of p38 MAPK and decrease in mitochondrial membrane potential and activation of Akt and Erk pathways were observed in statin-treated lymphoma cells. Statin-induced cytotoxic effects, DNA fragmentation and changes of activation of caspase-3, Akt, Erk and p38 were blocked by antioxidant (N-acetylcysteine) and metabolic products of the HMG-CoA reductase reaction, such as mevalonate, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). These results suggests that HMG-CoA reductase inhibitors induce lymphoma cells apoptosis by increasing intracellular ROS generation and p38 activation and suppressing activation of Akt and Erk pathways, through inhibition of metabolic products of the HMG-CoA reductase reaction including mevalonate, FPP and GGPP.     

Arrest of 3T3 cells in G1 phase by low density lipoprotein

Low density lipoprotein (LDL) and high density lipoprotein (HDL) were purified from normal human serum by KBr density gradient centrifugation and gel filtration through Sepharose 4B. LDL reversibly inhibited proliferation of Swiss/3T3 cells, whereas HDL had no inhibitory effect on cell growth. The LDL-induced inhibition was LDL-dose dependent and was reversed by the addition of mevalonate, a product of the reaction of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34). These data suggest that a specific reduction in the activity of HMG-CoA reductase produced by the addition of LDL is the main cause of the inhibition of cell proliferation. Studies of the effect of LDL on the cell cycle showed that it inhibited the entry of cells arrested in G0/G1 into the S phase but that it did not affect the transition of cells at the G1/S boundary into the M phase. The cell cycle of 3T3 is arrested solely in G1 by LDL.     

Increase of latent HMG-CoA reductase activity with increasing density of cell cultures

The total (active latent) activity of HMG-CoA reductase declined linearly with increasing cell density in cultures of three lines of mammalian cells. The active form disappeared almost entirely under this condition, while the latent (presumably phosphorylated) form increased to some extent. The disappearance of active HMG-CoA reductase with concomitant increase in the proportion of latent HMG-CoA reductase was correlated with the decline in cellular multiplication and sterol synthesis. These results suggest that interconversion of HMG-CoA reductase between active and inactive forms through phosphorylation-dephosphorylation can be associated with changes in the rate of cellular proliferation in cell cultures. However, the decreased rate of sterol synthesis followed more closely the slower disappearance of the total HMG-CoA reductase activity than the rapid decrease of the active form of the reductase alone. Therefore, changes in the rate of cellular proliferation can affect the interconversion of HMG-CoA reductase between active and inactive forms through reversible phosphorylation. However, phosphorylation of the enzyme to the inactive form appears not to be the mechanism by which the sterol synthetic rate is regulated in confluent cell cultures. Rather, the amount of total HMG-CoA reductase determines the rate of sterol synthesis.     

Lovastatin-induced RhoA modulation and its effect on senescence in prostate cancer cells

Lovastatin inhibits a 3-hydroxy 3-methylglutaryl coenzyme A reductase and prevents the synthesis of cholesterol precursors, such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), responsible for important cell signaling in cell proliferation and migration. Recently, the anti-cancer effect of lovastatin has been suggested in various tumor types. In this study, we showed that a low dose lovastatin induced senescence and G1 cell cycle arrest in human prostate cancer cells. Addition of GGPP or mevalonate, but not FPP, prevented the lovastatin-induced G1 phase cell cycle arrest and cell senescence. We found that constitutively active RhoA (caRhoA) reversed lovastatin-induced senescence in caRhoA-transfected PC-3 cells. Thus, we postulate that modulation of RhoA may be critical in lovastatin-induced senescence in PC-3 cells.     

Inhibition of protein geranylgeranylation and RhoA/RhoA kinase pathway induces apoptosis in human endothelial cells

Geranylgeranylation of RhoA small G-protein is essential for its localization to cell membranes and for its biological functions. Many RhoA effects are mediated by its downstream effector RhoA kinase. The role of protein geranylgeranylation and the RhoA pathway in the regulation of endothelial cell survival has not been elucidated. The hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor lovastatin depletes cellular pools of geranylgeranyl pyrophosphate and farnesol pyrophosphate and thereby inhibits both geranylgeranylation and farnesylation. Human umbilical vein endothelial cells (HUVECs) were exposed to lovastatin (3 microm-30 microm) for 48 h, and cell death was quantitatively determined by cytoplasmic histone-associated DNA fragments as well as caspase-3 activity. The assays showed that lovastatin caused a dose-dependent endothelial cell death. The addition of geranylgeraniol, which restores geranylgeranylation, rescued HUVEC from apoptosis. The geranylgeranyltransferase inhibitor GGTI-298, but not the farnesyltransferase inhibitor FTI-277, induced apoptosis in HUVEC. Cell death was also induced by a blockade of RhoA function by exoenzyme C3. In addition, treatment of HUVEC with the RhoA kinase inhibitors Y-27632 and HA-1077 caused dose-dependent cell death. Y-27632 did not inhibit other well known survival pathways, such as NF-kappa B, ERK, and phosphatidylinositol 3-kinase/Akt. However, there was an increase in p53 protein level concomitant with Y-27632-induced cell death. Unlike the apoptosis induced by TNF-alpha, which occurs only with inhibition of new protein synthesis, apoptosis induced by inhibitors of HMG-CoA reductase, geranylgeranyltransferase, or RhoA kinase was blocked by cycloheximide. Our data indicate that inhibition of protein geranylgeranylation and RhoA pathways induce apoptosis in HUVEC and that induction of p53 or other proapoptotic proteins is required for this process.     

Dose-dependent effects of lovastatin on cell cycle progression. Distinct requirement of cholesterol and non-sterol mevalonate derivatives

The mevalonate pathway is tightly linked to cell proliferation. The aim of the present study is to determine the relationship between the inhibition of this pathway by lovastatin and the cell cycle. HL-60 and MOLT-4 human cell lines were cultured in a cholesterol-free medium and treated with increasing concentrations of lovastatin, and their effects on cell proliferation and the cell cycle were analyzed. Lovastatin was much more efficient in inhibiting cholesterol biosynthesis than protein prenylation. As a result of this, lovastatin blocked cell proliferation at any concentration used, but its effects on cell cycle distribution varied. At relatively low lovastatin concentrations (less than 10 microM), cells accumulated preferentially in G(2) phase, an effect which was both prevented and reversed by low-density lipoprotein cholesterol. At higher concentrations (50 microM), the cell cycle was also arrested at G(1) phase. In cells treated with lovastatin, those arrested at G(1) progressed through S upon mevalonate provision, whereas cholesterol supply allowed cells arrested at G(2) to traverse M phase. These results demonstrate the distinct roles of mevalonate, or its non-sterol derivatives, and cholesterol in cell cycle progression, both being required for normal cell cycling.     

Balancing a heterologous mevalonate pathway for improved isoprenoid production in Escherichia coli

Engineering biosynthetic pathways in microbes for the production of complex chemicals and pharmaceuticals is an attractive alternative to chemical synthesis. However, in transferring large pathways to alternate hosts and manipulating expression levels, the native regulation of carbon flux through the pathway may be lost leading to imbalances in the pathways. Previously, Escherichia coli was engineered to produce large quantities of isoprenoids by creating a mevalonate-based isopentenyl pyrophosphate biosynthetic pathway [Martin, V.J., Pitera, D.J., Withers, S.T., Newman, J.D., Keasling, J.D., 2003. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat. Biotechnol. 21, 796-802]. The strain produces high levels of isoprenoids, but upon further investigation we discovered that the accumulation of pathway intermediates limited flux and that high-level expression of the mevalonate pathway enzymes inhibited cell growth. Gene titration studies and metabolite profiling using liquid chromatography-mass spectrometry linked the growth inhibition phenotype with the accumulation of the pathway intermediate 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA). Such an accumulation implies that the activity of HMG-CoA reductase was insufficient to balance flux in the engineered pathway. By modulating HMG-CoA reductase production, we eliminated the pathway bottleneck and increased mevalonate production. These results demonstrate that balancing carbon flux through the heterologous pathway is a key determinant in optimizing isoprenoid biosynthesis in microbial hosts.     

Effect of lovastatin on acyl-CoA: cholesterol O-acyltransferase (ACAT) activity and the basolateral-membrane secretion of newly synthesized lipids by CaCo-2 cells.

Lovastatin, a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity, was used to study the regulation of cholesterol metabolism and the basolateral-membrane secretion of triacylglycerol and cholesterol in the human intestinal cell line CaCo-2. At 0.1 microgram/ml, lovastatin decreased 3H2O incorporation into cholesterol by 71%. In membranes prepared from cells incubated with lovastatin for 18 h, HMG-CoA reductase activity was induced 4-8-fold. Mevalonolactone prevented this induction. In intact cells, lovastatin (10 micrograms/ml) decreased cholesterol esterification by 50%. The reductase inhibitor decreased membrane acyl-CoA:cholesterol O-acyltransferase (ACAT) activity by 50% at 5 micrograms/ml. ACAT inhibition by lavastatin was not reversed by adding excess of cholesterol or fatty acyl-CoA to the assay. Lovastatin, in the presence or absence of mevalonolactone, decreased the basolateral secretion of newly synthesized cholesteryl esters and triacylglycerols. Lovastatin also inhibited the esterification of absorbed cholesterol and the secretion of this newly synthesized cholesteryl ester. Lovastatin is a potent inhibitor of cholesterol synthesis in CaCo-2 cells. Moreover, it is a direct inhibitor of ACAT activity, independently of its effect on HMG-CoA reductase and cholesterol synthesis.