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.     

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.     

Negative regulation of cell proliferation by mevalonate or one of the mevalonate phosphates

The role of mevalonate and its products in the regulation of cellular proliferation was examined using 6-fluoromevalonate (Fmev), a compound that blocks the conversion of mevalonate pyrophosphate to isopentenyl pyrophosphate. Fmev suppressed DNA synthesis by a variety of transformed and malignant T cell, B cell, and myeloid cell lines. In contrast to results previously reported with mitogen-stimulated human peripheral blood T cell DNA synthesis, low concentrations of low density lipoprotein (LDL) alone could not restore proliferation to these cell lines. The same concentrations of LDL were able to provide sufficient cholesterol and support the growth of all cell lines when mevalonate synthesis was blocked with a specific inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, lovastatin. Fmev-mediated inhibition was totally prevented in some but not all cell lines when the concentration of exogenous LDL was increased 5-10-fold above that required to permit proliferation of lovastatin-blocked cells. Residual HMG-CoA reductase activity of cells cultured with LDL inversely correlated with the restoration of growth to Fmev-blocked cultures. Confirmation of the critical role of HMG-CoA reductase activity and mevalonate synthesis in the inhibition of cellular proliferation by Fmev was obtained by demonstrating that the specific inhibitor of this enzyme, lovastatin, restored proliferation of Fmev-blocked cells. Furthermore, supplementation of cultures with mevalonate, the product of HMG-CoA reductase activity, markedly inhibited proliferation of Fmev-blocked cells. These findings indicate that mevalonate or one of the mevalonate phosphates, which accumulates in Fmev-blocked cells, is a critical negative regulator of cellular proliferation.     

The mevalonate/isoprenoid pathway inhibitor apomine (SR-45023A) is antiproliferative and induces apoptosis similar to farnesol

Apomine (SR-45023A) is a new antineoplastic compound which is currently in clinical trials and representative of the family of cholesterol synthesis inhibitors 1,1-bisphosphonate esters. Apomine inhibits growth of a wide variety of tumor cell lines with IC(50) values ranging from 5 to 14 microM. The antiproliferative activity of apomine was studied in comparison with that of other inhibitors of the mevalonate/isoprenoid pathway of cholesterol synthesis, simvastatin, farnesol, and 25-hydroxycholesterol. All these compounds inhibit 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity. Apomine (IC(50) = 14 microM), simvastatin (IC(50) = 3 microM), farnesol (IC(50) = 60 microM), and 25-hydroxycholesterol (IC(50) = 2 microM) inhibited HL60 cell growth. Growth inhibition due to simvastatin was reverted by mevalonate, whereas the antiproliferative activity of apomine, farnesol, and 25-hydroxycholesterol was not. Apomine triggered apoptosis in HL60 cells in less than 2 h. Apomine and farnesol induced caspase-3 activity at concentrations similar to their IC(50) values for cell proliferation, whereas a 10-fold excess of simvastatin was necessary to trigger apoptosis compared to its potency on proliferation. Caspase-3 activity was not induced by 25-hydroxycholesterol. The overall similar profile on mevalonate synthesis inhibition, cell growth inhibition, and apoptosis suggests that apomine acts as a synthetic mimetic of farnesol.     

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.     

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.     

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.     

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase synthesis by a non-sterol mevalonate-derived product in Mev-1 cells. Apparent translational control

A somatic cell mutant (Mev-1) auxotrophic for mevalonate by virtue of a complete lack of detectable 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase activity has been shown to demonstrate a requirement for a non-sterol mevalonate-derived product for regulation of synthesis of HMG-CoA reductase. A comparison of the effects of 25-hydroxycholesterol and the combination of 25-hydroxycholesterol and mevalonate on HMG-CoA reductase activity, synthesis, and mRNA levels in Mev-1 is presented in this report. The results show a close correlation between activity, rate of synthesis, and mRNA levels for Mev-1 cells treated with 25-hydroxycholesterol alone. Under the conditions of these experiments these effects are relatively small (approximately a 4-fold decrease). A much larger inhibition of HMG-CoA reductase activity and rate of synthesis (approximately 50-fold) is observed upon treatment of Mev-1 cells with a combination of 25-hydroxycholesterol and mevalonate. Yet, under these conditions mRNA levels are still reduced by only a factor of 4. These results are interpreted to suggest that the non-sterol mevalonate-derived regulatory product of HMG-CoA reductase acts by a translational control mechanism.     

Accumulation of regulatory oxysterols, 32-oxolanosterol and 32-hydroxylanosterol in mevalonate-treated cell cultures

In a previous publication (Saucier, S.E., A.A., Taylor, F.R., Spencer, T.A., Phirwa, S., and Gayen, A.K., J. Biol. Chem. (1985) 260, 14571-14579), we demonstrated that cultured Chinese hamster lung (Dede) cells contain 24(S),25-epoxycholesterol and 25-hydroxycholesterol in cellular concentrations within the range required to repress 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. In this paper, we show that the addition to the culture medium of a concentration of mevalonate high enough to repress the reductase by 90% resulted in the appearance of two new regulatory oxysterols. The two new sterols are believed to be 32-oxolanosterol and 32-hydroxylanosterol on the basis of high performance liquid chromatography (HPLC) retention times and mass spectrometric and nuclear magnetic resonance spectroscopic data and by NaBH4 reduction of the putative aldehyde to material which had the HPLC retention time of the putative alcohol. The cellular concentrations of 24(S),25-epoxycholesterol and 25-hydroxycholesterol were not significantly changed by the presence of mevalonate. However, there was approximately a 30% increase in total HMG-CoA reductase repressor units which can be ascribed to the 32-oxolanosterol and 32-hydroxylanosterol, where 1 unit equals the repressor activity of 1 ng of 25-hydroxycholesterol. These results support the idea that the level of HMG-CoA reductase activity in growing cell cultures is determined by intracellular oxysterol metabolites and that relatively small changes in their numbers or concentrations can alter the level of HMG-CoA reductase activity.     

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.     

Regulation of osteoclast differentiation by statins

HMG-CoA reductase inhibitor (statin) treatment is frontline therapy for lowering plasma cholesterol levels in patients with hyperlipidemia. In a few case studies, analysis of clinical data has revealed a decreased risk of fracture in patients on statin therapy. However, this reduction in the incidence of fracture is not always observed nor is it supported by an increase in bone density, which further complicates our understanding of the role of statins in bone metabolism. Thus, the precise role of statins in bone metabolism remains poorly understood. In this study, we examined the effect of statin treatment on osteoclastogenesis. Treatment with lovastatin resulted in a significant, dose-dependent decrease in the numbers of differentiated osteoclasts and decreased cholesterol biosynthesis activity with an EC(50) similar to that observed in freshly isolated rat or cultured human liver cells. Studies assessing the role of mevalonate metabolites in the development of the osteoclasts demonstrated that geranylgeraniol, but not squalene or farnesol was important for the development and differentiation of osteoclasts, implicating protein geranylgeranylation rather than protein farnesylation as a key factor in the osteoclast differentiation process. In conclusion, our data indicate that lovastatin inhibits osteoclast development through inhibition of geranylgeranylation of key prenylated proteins and that the bone effects of statins are at least partially due to their effects on osteoclast numbers.     

Lack of correlation between 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and lovastatin resistance in nerve growth factor treated PC-12 cells

Summary 1. The relationships among the mevalonic acid (MVA) forming enzyme, 3-hydroxy-3-methylglutaryl coenzyme A (CoA) reductase, cell growth and differentiation, and the cytotoxic effects of the reductase inhibitor lovastatin were studied in PC-12 cells, exposed to growth factors.2. When added individually, nerve growth factor (NGF), basic fibroblast growth factor, and epidermal growth factor induce an increase in HMG-CoA reductase activity in cells grown in serum-containing medium. In the presence of serum, the effect of NGF on HMG-CoA reductase is persistent.3. Short-term serum starvation and long-term NGF treatment, in combination, have an additive effect, resulting in a high reductase activity.4. Unlike serum and MVA, which downregulate levels of HMG-CoA reductase by accelerating its degradation, NGF upregulates reductase by slowing the rate of its degradation. This mechanism, however, appears to operate only in the presence of serum, as after prolonged growth with NGF in serum-free medium, cells have a low reductase activity.5. PC-12 cells grown in the absence of NGF are highly sensitive to lovastatin (25 µM) and more than 70% of the cells die after 48 hr. NGF confers lovastatin resistance on cells grown in the presence or in the absence of serum (only 30–40% cell death after 48 hr with lovastatin).6. NGF-induced resistance on lovastatin develops with time and is apparent only in the well-differentiated PC-12 cells whether or not the cells express a high reductase activity.7. Thus, levels of HMG-CoA reductase activity and lovastatin resistance in PC-12 cells are not directly correlated, though clearly inversed lovastatin cytotoxicity and elevated reductase activities are expressed during the period of cell proliferation.8. These data suggest that fully differentiated neuronal cells may not be affected by prolonged high doses of lovastatin.     

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.     

Reversible inactivation of 3-hydroxy-3-methylglutaryl coenzyme A reductase: reductase kinase and mevalonate kinase are separate enzymes

Reductase kinase and mevalonate kinase are separated by: a) ammonium sulfate fractionation; b) chromatography on agarose-Procion Red HE3B; and c) chromatography on DEAE-Sephacel. Fractions containing only reductase kinase reversibly inactivated microsomal or homogeneous HMG-CoA reductase. Fractions containing only mevalonate kinase revealed artifactual reductase kinase activity in the absence of EDTA or mevalonic acid; however, addition of EDTA or mevalonate before reductase assay completely blocked any apparent decline in HMG-CoA reductase activity. Under these conditions no dephosphorylation (reactivation) was observed by phosphatase. The combined results demonstrate unequivocally that reductase kinase and mevalonate kinase are two different enzymes and inactivation of HMG-CoA reductase is catalyzed by ATP-Mg-dependent reductase kinase.     

An overview on the biological activity and anti-cancer mechanism of lovastatin

Lovastatin, a secondary metabolite isolated from fungi, is often used as a representative drug to reduce blood lipid concentration and treat hypercholesterolemia. Its structure is similar to that of HMG-CoA. Lovastatin inhibits the binding of the substrate to HMG-CoA reductase, and strongly competes with HMG-CoA reductase (HMGR), thereby exerting a hypolipidemic effect. Further, its safety has been confirmed in vivo and in vitro. Lovastatin also has anti-inflammatory, anti-cancer, and neuroprotective effects. Therefore, the biological activity of lovastatin, especially its anti-cancer effect, has garnered research attention. Several in vitro studies have confirmed that lovastatin has a significant inhibitory effect on cancer cell viability in a variety of cancers (such as breast, liver, cervical, lung, and colon cancer). At the same time, lovastatin can also increase the sensitivity of some types of cancer cells to chemotherapeutic drugs and strengthen their therapeutic effect. Lovastatin inhibits cell proliferation and regulates cancer cell signaling pathways, thereby inducing apoptosis and cell cycle arrest. This article reviews the structure, biosynthetic pathways, and applications of lovastatin, focusing on the anti-cancer effects and mechanisms of action.     

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.     

Studies on the relationships between 7α-hydroxylation and the ability of 25- and 27-hydroxycholesterol to suppress the activity of HMG-CoA reductase

The metabolism of 25-hydroxycholesterol in different cell types was studied and the role of 7α-hydroxylation for the effect of 25-hydroxycholesterol on the activity of HMG-CoA reductase was determined. Human diploid fibroblasts (HDF) and the human melanoma cell line SK-MEL-2 converted 25-hydroxycholesterol into 7α,25-dihydroxycholesterol and 7α,25-dihydroxy-4-cholesten-3-one while the virus-transformed fibroblast line 90VA-VI, the colon carcinoma cell line WiDr and the breast cancer cell line MDA-231 did not express 7α-hydroxylase activity. The 7α-hydroxylation of 25-hydroxycholesterol in HDF could be stimulated by dexamethasone and cortisol and inhibited by metyrapone. An unidentified, possibly 4-hydroxylated, metabolite was formed by 90VA-VI cells and a polar, probably conjugated, metabolite was formed by WiDr cells. The 7α-hydroxylated metabolites of 25-hydroxycholesterol suppressed the activity of HMG-CoA reductase to a similar extent as 25-hydroxycholesterol in HDF but not in 90VA-VI cells, while the 7α-hydroxylated metabolites of 27-hydroxycholesterol suppressed the activity of HMG-CoA reductase also in 90VA-VI cells. The suppression of HMG-CoA reductase activity by 25- and 27-hydroxycholesterol was decreased or abolished by dehydroepiandrosterone or pregnenolone which have little or no effect on the 7α-hydroxylation. The results indicate that 7α-hydroxylation is not directly involved, positively or negatively, in the action of 25- or 27-hydroxycholesterol as suppressors of HMG-CoA reductase activity.     

Lipid requirement for cell cycling : The effect of selective inhibition of lipid synthesis

Tissue culture cells require lipid which must be provided exogenously or synthesized via endogenous pathways. The exogenous supplies can be largely removed by growing cells in medium containing delipidized serum. Pathways for synthesis of lipid can then be blocked at three steps: (1) fatty acids by removal of biotin, an essential coenzyme; (2) phosphatidylcholine and sphingomyelin by deleting choline from the growth medium; and (3) cholesterol by inhibiting HMG-CoA reductase with 25-hydroxycholesterol. Sustained proliferation is prevented when lipid synthesis is blocked at any one of these steps. Cell proliferation resumes upon restoring synthesis with biotin, choline, or mevalonate (the product of the HMG-CoA reductase reaction) or by providing the lipid end products oleic acid or cholesterol. Using a combined cytophotometric-autoradiographic analysis to determine cell cycle distributions we have demonstrated that prereplicative (G1) cell cycle arrests develop in parallel with the proliferative inhibition. Each of the G1 arrests can be reversed by restoring the synthetic pathways or their lipid products. These observations suggest a causal relationship between the supply of lipids and passage through G1.     

Geranylgeraniol Overcomes the Block of Cell Proliferation by Lovastatin in C6 Glioma Cells

Abstract: It is well documented that 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors prevent cultured mammalian cells from progressing through the cell cycle, suggesting a critical role for a mevalonate-derived product. Recently, it has been shown that free geranylgeraniol (GG-OH) and farnesol (F-OH) can be utilized by C6 glioma cells for protein isoprenylation. The ability of GG-OH and F-OH to restore protein geranylgeranylation or farnesylation selectively has enabled us to examine the possibility that mevalonate is essential for cell proliferation because it is a precursor of farnesyl pyrophosphate or geranylgeranyl pyrophosphate, the isoprenyl donors involved in the post-translational modification of key regulatory proteins. In this study we report that GG-OH, as well as mevalonate, overcomes the arrest of cell proliferation of C6 glioma cells treated with lovastatin, as assessed by increased cell numbers and a stimulation in [³H]thymidine incorporation. The increase in cell number and [³H]thymidine incorporation were significantly lower when F-OH was added. Under these conditions [³H]mevalonate and [³H]GG-OH are actively incorporated into a set of isoprenylated proteins in the size range of small, GTP-binding proteins (19–27 kDa) and a polypeptide with the molecular size (46 kDa) of the smaller isoform of 2′,3′-cyclic nucleotide 3′-phosphodiesterase. Analysis of the proteins metabolically labeled by [³H]mevalonate and [³H]GG-OH reveals the presence of labeled proteins containing geranylgeranylated cysteinyl residues. Consistent with geranylgeranylated proteins playing a critical role in the entry of C6 cells into the cell cycle, a (phosphonoacetamido)oxy derivative of GG-OH, a drug previously shown to interfere with protein geranylgeranylation, prevented the increase in cell number when mevalonate or GG-OH was added to lovastatin-treated cells. These results strongly suggest that geranylgeranylated proteins are essential for progression of C6 cells into the S phase of the cell cycle and provide the first evidence that the “salvage” pathway for the utilization of the free isoprenols is physiologically significant in the CNS.