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ICANCER RESEARCH57. 3498â€”3505.AugustIS. 9971 Regulation of Proliferation and Ras Localization in Transformed Cells by Products of Mevalonate Metabolism'

Jennifer A. Cuthbert2 and Peter E. Lipsky

Department of Internal Medicine. The Unit'ersitv of Texas Southwestern Medical (â€˜enterat Dallas. Dallas. Texas 75235-9151

ABSTRACT position 186, the removal of the three COOH-terminal amino acids at positions 187â€”189, and carboxymethylation of the new COOH-termi Lovastatin, an inhibitor of 3-hydroxy.3-methylglutaryl (HMG) CoA nab cysteine. In addition, either palmitybation of other cysteine resi reductase, and 6-fluoromevalonate (Fmev), an inhibitor of diphospho dues in the COOH terminus (H-Ras, N-Ras, and K-RasA) or a mevalonate decarboxylase, blocked the synthesis of downstream meval. onate products, including prenyl-derived lipids, and prevented membrane pobybasic domain (K-RasB) is important in enhancing membrane localization of Ras in the myeloid cell line U.937. In contrast to lovastatin, association (7). These processes occur stepwise, and the first step, that which induced cytosol localization of Ras in U-937 cells, Fmev failed to of farnesybation of the full-length polypeptide, is thereby essential for increase cytosolic Ras and also completely prevented the proliferation of plasma membrane localization (12â€”14).Thus, compounds and muta U.937 cells. Growth of U-937 cells was restored by the addition of lovas tions that block the process of farnesylation interfere with the trans tatin to Fmev-blocked cells. These results implied that a product of formation and proliferation that are dependent upon mutationally mevalonate metabolism proximal to isopentenyl diphosphate was respon. activated Ras. sible for the suppression of proliferation. To delineate the action of this Lovastatin, a specific inhibitor of HMG3 CoA reductase, blocks endogenous inhibitor of cell proliferation and determine the relationship between its impact on Ras localization and cell proliferation, the effect of farnesylation of Ras (12, 13) because HMG CoA reductase catalyzes Fmev on a variety of leukemia- and lymphoma-denved cells was exam the synthesis of mevabonate, the precursor of the farnesyl lipid moiety med. Whereas Fmev blocked the growth of these cell lines, there were (Ref. 15; see Fig. 7 for biochemical pathways). Unfarnesylated Ras more than 50-fold differences in the concentrations required to inhibit the remains in the cytosob of bovastatin-blocked cells. In addition, inhib growth of individual cell lines by 90%. Regardless of its effect on cell itors of the enzyme farnesyltransferase prevent posuranslational mod proliferation, the biochemical effect of Fmev was similar. Thus, Fmev ification and membrane association of Ras (8, 9). Farnesyltransferase uniformly prevented the conversion of radiolabeled mevalonate to isopen. inhibitors prevent cell growth that is dependent on mutationally acti tenyl diphosphate and other downstream products, including synthesis of vated Ras (8â€”11).We have demonstrated that Fmev, a fluorinated sterol and nonsterol lipids and prenylation of proteins. A correlation was mevabonate analogue that blocks the conversion of mevabonate noted between higher intrinsic rates of mevalonate synthesis by a cell and susceptibility to inhibition by Fmev. Thus, sensitivity of a cell line to diphosphate to isopentenyl diphosphate (16, 17), also inhibits fame inhibition by Fmev was associated with markedly increased rates of HMG sybation of Ras (18, 19). Like bovastatin, Fmev prevents membrane CoA reductase activity that were further increased by incubation with localization of Ras (19). Unlike lovastatin, however, Fmev does not Fmev. Whereas Fmev depleted cellular levels of the prenylated protein induce the accumulation of Ras in the cytosob (19). Moreover, Fmev Ras in the sensitive cell line U-937, there was no depletion of cellular Ras also induces the activity of an endogenous inhibitor of cellular pro levels in the resistant cell line EL-4, but rather, there was a shift of Ras liferation (19, 20). This inhibitor is a product of mevabonate proximal from membrane to cytosol, as expected for inhibition of prenylation. to isopentenyb diphosphate (19, 20). The current studies were under These results suggest that leukemic cells with increased HMG CoA re taken to examine the potential dysregubation of this inhibitor of ductase activity produce increased levels of an endogenous mevalonate. cellular growth in various transformed cell lines and determine the derived inhibitor that leads to Ras depletion and suppression of cell growth. As a result, inhibition of the growth of these transformed cells relationship of its impact on Ras localization and cellular growth. might be specifically accomplished by Fmev.

MATERIALS AND METHODS INTRODUCTION Cell Culture and Measurement of DNA, Sterol, and Mevalonate Syn Ras, a Mr 21,000 guanine nucleotide-binding protein, is activated thesis. The following leukemia- and lymphoma-derived cell lines were ob by signal transduction pathways involved in growth and differentia tamed from the American Type Culture Collection: the B-cell line Daudi tion (I). Mutations that constitutively activate Ras are found in â€”20% (Burkitt lymphoma ATCC CCL 213); the lymphoma cell line EL-4 (ATCC of all human tumors ( 1, 2) and are particularly common in pancreatic TIB 39); the acute monocytic leukemia cell line THP-l (ATCC TIB 202); the (3) and colon (4) cancer. Cellular transformation by mutationally acute promyelocytic leukemia line HL-60 (ATCC CCL 240); and the histio activated Ras requires plasma membrane association of the oncogenic cytic lymphoma-derived myeloid cell line, U-937 (ATCC CRL 1593). The normal human B-lymphoblastoid cell line, MarB, that had been generated by protein (5â€”7).This observation has been exploited in the development transformation of peripheral blood mononuclear cells with Epstein-Barr virus of potential cancer chemotherapeutic agents that block membrane using standard techniques (21) was obtained from Dr. S. Zwillich (University association of Ras (8â€”bI). of Texas Southwestern Medical School). For some experiments, the Epstein Initially, newly synthesized Ras is localized in the cytosol (12â€”14). Barr virus-transformed human B-lymphoblastoid cell line 526.1! (22) was @â€˜seriesof posttranslational modifications of the COOH terminus used. The Jurkat T-cell line was obtained from Dr. L. Davis (University of â€˜esults in plasma membrane targeting (12â€”14). These include the Texas Southwestern Medical School). All cells were maintained in RPMI-l640 ddition of a 15-carbon farnesyl lipid to the cysteine at amino acid (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% (v/v) iron-supplemented BCS (HyClone Laboratories, Inc., Logan, UT). For some Received11/18/96;accepted6/17/97. experiments, cells were adapted to medium supplemented with 10%(v/v) LPP, The costs of publication of this article were defrayed in part by the payment of page prepared as detailed (18). Fmev was synthesized by CPM Laboratory (Car arges. This article must therefore be hereby marked advertisement in accordance with rollton, TX) using the procedure of Quistadt et a!. (23). Lovastatin (Merck, U.S.C. Section 1734 solely to indicate this fact. I This work was supported by Grant CN-l08 from the American Cancer Society. 2 To whom requests for reprints should be addressed. Phone: (214) 648-2666: Fax: 3 The abbreviations used are: HMG, 3-hydroxy-3-methylglutaryl; Fmev, 6-fluorom @ 4) 648-5678; E-mail: [email protected]. evalonate; BCS, bovine calf serum; LPP, lipoprotein-poor plasma. 3498

Sharp and Dohme, Rahway, NJ) and the sodium salt of mevalonate were n-propanol:ammonium hydroxide:water (6:3: 1) as the solvent system. Radio prepared as reported previously (24) and added where indicated. labeled standards that were cochromatographed were 15-3Hlmevalonolactone, For measurements of DNA synthesis, cells were cultured (@5 X lO@cells [5-'4Clmevalonate phosphate (Amersham Corp., Arlington Heights, IL), and per well) in triplicate in microtiter plates, with additions as indicated in the 15- 4Clmevalonate diphosphate (Amersham). After treatment with individual experiments; in preliminary experiments, this concentration of cells EN3HANCE, the chromatograms were subjected to fluorography. The fluoro allowed maximal proliferation during the ensuing 1â€”4-dayincubation.DNA graphically identified spots were scraped and counted by liquid scintillation synthesis was measured by the incorporation of [3H]thymidine as described spectroscopy. (24). For measurements of sterol synthesis, 1 X 106cells/mI were cultured in Detection of Ras by Immunoblotting. Cells were incubatedfor varying 17- X 100-mm polypropylene tubes, with additions as indicated in the mdi lengths of time in complete medium with or without Fmev and lovastatin, as vidual experiments. Incorporation of [I- â€˜4C]acetateintodigitonin-precipitable indicated in the individual experiments. After harvesting, the number of cells sterols was measured in triplicate incubations as detailed previously (24). recovered was quantitated. Soluble (cytosolic) and particulate (membrane) For measurements of mevalonate synthesis (HMG CoA reductase activity), fractions were separated by 100,000 X g centrifugation after homogenization cells cultured in T25 tissue culture flasks in medium supplemented with 10% in hypotonic buffer and removal of nuclei by low-speed centrifugation (28). (v/v) BCS were harvested, washed by centrifugation to remove serum proteins, Proteins in the soluble fractions were concentrated by centrifugation using and counted, and the cell pellet was frozen at â€”90Â°Cuntilthe activity was Microcon 10 microconcentrators (Amicon, Inc., Beverly, MA). Both cytosol assayed. HMG CoA reductase activity was measured by a modification of the and membrane fractions were solubilized in SDS buffer, and protein content procedures of Brown et a!. (25) and Panini er a!. (26) as described (19). was assayed (DC protein assay; Bio-Rad). Equal amounts of protein were Briefly, cell pellets were resuspended in activation buffer containing 200 msi loaded in each lane after the addition of 2-mercaptoethanol and bromphenol KCI, 50 mMpotassium phosphate (pH 7.4), 5 mMDY)', 5 mr@iEDTA,5 mrvi blue. Individual proteins were separated by SDS-PAGE and transferred to EGTA, 25 mMimidazole, 100 @Mleupeptin,and 0.25% (v/v) Tergitol nonionic polyvinylidene fluoride membranes (Millipore). Ras proteins were identified detergent (Union Carbide Chemicals, Danbury, CT), sonicated, and incubated by immunoblotting with mouse monoclonal anti-Ras antibody LA045 against for 30 mm at 37Â°C.AfterI mm ofcentrifugation at 16,000 X g, aliquots of the amino acid residues 96-1 18 of H-, K-, and N-Ras (29, 30). Bound antibody was supernatant were harvested for measurements of HMG CoA reductase activity detected with affinity-purified horseradish peroxidase-conjugated goat anti (50 pA)and protein. To each reaction tube, an equal volume of 2X mix 1200 mouse IgG and enhanced chemiluminescence (Amersham International, UK) mM potassium phosphate (pH 7.4), 12.5 mM DTF, and 5 mt@iEGTAI, 175 @xg as previously detailed (19). of NADPH, and 3.75 nmol of [â€˜4C]HMGCoA (Dupont NEN Research Products, Boston, MA) was added. Following incubation in a shaking water bath for 1 h at 37Â°C,thereaction was stopped with 5 N HCI, followed by RESULTS incubation for 30 mm at 37Â°Cafteraddition of 2 @.tmolof[3Hjmevalonolactone Ras Localization Is Affected Differently by Fmev and Lovasta (Dupont) as an internal recovery standard. After a 20-mm centrifugation at 16,000 x g, 50 @.dofsupernatant were spotted onto plastic-backed silica gel G tin in a Human Transformed Cell Line. The initial experiments plates and chromatographed using a 1:1 mixture of benzene:acetone as the examined the effects of Fmev and lovastatin on membrane bocaliza solvent system. Chromatograms were sprayed with EN3HANCE (Dupont), tion of Ras in the human histiocytic bymphoma-denved cell line mevalonate was visualized by fluorography at â€”90Â°Cfor 2â€”3 days, and U-937. In untreated U-937 cells, the majority of immunoreactive Ras [t4Cjmevalonate was quantitated by liquid scintillation spectroscopy. Rates of was detected in the 100,000 X g pellet or crude membrane fraction HMG CoA reductase activity were calculated using [3H]mevalonatefor cor (Fig. la, Lane 1). A small amount of immunodetectable Ras was rection and are expressed as pmol/h/l06 cells and pmollmin/mg of cell protein. observed in the 100,000 X g supernatant or cytosol fraction (Fig. bb, Incorporation of Mevalonate into Cellular Proteins and Lipids. Cells Lane 1). The addition of Fmev was accompanied by a concentration were cultured in complete medium supplemented with or without 5 MM dependent decrease in membrane-associated Ras (Fig. ba, Lanes 2 and bovastatinand varying concentrations of Fmev, as indicated in the individual 3). There was no concomitant increase in cytosol-locabized Ras (Fig. experiments. Cells were incubated with [5-3Hlmevalonolactone (specific ac lb. Lanes 2 and 3). In contrast, incubation with 5 MM lovastatin tivity, 27.8 Ci/mmol; Dupont) and harvested after a 24-h incubation. Following extensive washing to remove unincorporated radioactivity, cells were lysed resulted in a decrease in membrane-localized Ras (Fig. la, Lane 4), after resuspension in water and fractionated by a modified Bligh and Dyer together with a substantial increase in cytosolic Ras (Fig. lb. Lane 4). method as described previously (18). Cell proteins partitioned to the interphase When cells were incubated with both Fmev and lovastatin, the results and were precipitated with 10% (v/v) trichioracetic acid after removal of the were similar to those obtained with bovastatin alone. The effects of upper methanol:water phase and lower chloroform phase. The precipitates Fmev and bovastatin on endogenous cholesterol synthesis were simi were washed sequentially with 10% trichloracetic acid, ice-cold acetone, and lar. Each caused a concentration-dependent inhibition of lipid synthe ice-cold ethanol and solubilized by heating in sample buffer containing 2% sis. Thus, 50 MMFmev decreased incorporation of radiolabeled me (wlv) SDS, 50 mM 2-mercaptoethanol, and 25 mM NaOH, and the radioactivity valonate into lipid by 80 Â±5% (mean Â±SE; n 3), and this incorporated into protein was quantified in an aliquot by liquid scintillation increased to 93 Â±1% inhibition with 500 ,tM Fmev. Lovastatin (0.5 spectroscopy. The results are presented as fmol incorporated into protein per MM) decreased incorporation of radiolabeled acetate into sterols from million cells cultured. One-dimensional SDS-PAGE was performed according to Laemmli (27), using a 4% polyacrylamide stacking gel and a 12.5% 236.3 Â±6.6 pmol/h/l06 cells (n = 3) to 24.1 Â±0.7 pmollh/106 cells polyacrylamide separating gel. Gels were stained with Coomassie blue, (89.8% inhibition) and to 3.0 Â±0.4 pmol/h/l06 cells (98.7% inhibi destained, permeated with EN3HANCE, dried, and exposed to KOdak XAR-5 tion) with 5 MM lovastatin. film at â€”90Â°Cfor7â€”14dayswith intensifying screens. Fmev-inhibited Proliferation of U-937 Cells Is Restored by Incorporation of radiolabeled mevalonolactone into lipid products was Lovastatin. The effects of Fmev and lovastatin on the proliferation of determined as detailed previously (18). Before fractionation, unlabeled mass U-937 cells were examined next. As illustrated in the representative quantities of sterols and intermediates, together with [â€˜4C]cholesterylester experiment depicted in Fig. 2, the addition of increasing concentra (Dupont), were added as internal control standards, and lipids were extracted tions of Fmev resulted in a dose-dependent inhibition of proliferation. into the chloroform phase by centrifugation. Radioactivity incorporated into Concentrations of Fmev that decreased membrane-associated Ras total lipids was quantified in an aliquot by liquid scintillation spectroscopy. (Fig. Ia, Lane 2) completely prevented the continued growth of U-937 Individual lipids were separated by TLC, identified by iodine visualization and comparisonwith authenticstandards,and quantitatedby liquid scintillation cells (Fig. 2). Previous studies had indicated that Fmev-mediated spectroscopy as described previously (18). inhibition of cell proliferation was related to the action of an inhibitor The methanol:water (aqueous) phase was concentrated by evaporation to that was a product of mevalonate proximal to isopentenyl diphosphate dryness, resuspended in methanol:water (1:0.9, v:v), spotted onto LK6D chan (19, 20). To address this possibility, the effects of lovastatin, an neled silica gel plates (Whatman Inc., Clifton, NJ) and chromatographed using inhibitor of HMG CoA reductase and therefore mevalonate synthesis, 3499

Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1997 American Association for Cancer Research. Ras AND MEVALONATES a. MEMBRANE When EL-4 cells were cultured in lipoprotein-deficient medium and, therefore, became dependent on endogenously synthesized cho Lovastatin besterol for ongoing membrane synthesis, the antiproliferative effect Control 5@iM of Fmev was greatly enhanced (IC50, 4 @LM;IC@,14 MM),as would be expected if Fmev was effectively inhibiting the production of choles II I terol under these conditions. These results imply that Fmev inhibited Fmev 0 50500 0 50500 mevabonate metabolism effectively in the resistant lines when endog (@.tM) enous sterol synthesis was required. However, when endogenous sterol synthesis was not required because the cells were cultured in sterol-containing medium, there was a 50-fold disparity in the con centration needed for comparable inhibition of proliferation in resist ant cell lines compared to sensitive ones. Fmev Suppresses MevabonateMetabolism Equivalently in Sen sitive and Resistant Cell Lines. The simplest explanation for the 123456 differences in the effectiveness of Fmev was that Fmev suppressed mevalonate metabolism less efficiently in the relatively resistant cell b. CYTOSOL lines. This possibility was directly addressed by measuring the effect Lovastatin of Fmev on the metabolism of radiolabeled mevabonate in U-937 and Control [email protected] EL-4 cells. For these experiments, cells were incubated with trace quantities of [3H]mevalonate in the presence or absence of varying II I concentrations of Fmev for 24 h, and then cell bysates were separated Fmev 0 50500 0 50500 into lipid, protein, and small molecular weight aqueous fractions. In (MM) both U-937 and EL-4 cells, Fmev inhibited the incorporation of [3H]mevalonate into lipid and protein fractions (Fig. 4a) in a concen tration-dependent manner (data not shown). There was a reciprocal increase in labeled mevabonate in the aqueous phase in Fmev-blocked U-937 and EL-4 cells (Fig. â€˜la).This increase represents mevabonate, mevalonate phosphate, and mevabonate diphosphate (data not shown). The addition of 500 MMFmev suppressed mevabonate incorporation 123456 into lipid by 93 Â± 1% and 95 Â±1% (n â€”3) and into protein by Fig. I. Both Fmev and lovastatin deplete membrane-associated Ras. whereas only 43 Â±7% and 83 Â±5% (n 3) in U-937 and EL-4 cells, respectively. lovastatin increases cytosolic Ras. U-937 cells were cultured in medium supplemented The concentrations of Fmev required to inhibit incorporation of me with 10% BCS for 48 h and with or without Fmev (50 or 500 @sM)andlovastatin (5 @sM) vabonate into lipid by 50% in U-937 and EL-4 cells were similar (6 as indicated, after which they were separated into membrane (particulate; a) and cytosol (soluble: b) fractions, and Ras was identified by immunobloning as detailed in â€œMaterials and 4 MMfor U-937 and EL-4 cells, respectively, calculated from cells and Methods.â€• incubated with varying concentrations of Fmev). Mevabonate modi fication of proteins was more readily inhibited in EL-4 cells (IC50, 18 .LM) than in U-937 cells (inhibited <50% with 500 p.M Fmev). on Fmev-mediated suppression were examined. As shown in Fig. 2, It was possible that the presence of unlabeled, endogenously syn lovastatin (0.5 and 5 MM)had no effect on DNA synthesis in control thesized mevabonate was confounding the analysis of the inhibitory U-937 cells. When added to Fmev-bbocked cells, however, lovastatin effect of Fmev. To examine this possibility, endogenous mevalonate restored proliferation. These data indicate that the inhibition of U-937 synthesis was prevented by blocking the activity of HMG CoA cell proliferation by Fmev was associated with the accumulation of a reductase with 5 MMlovastatin. This concentration of bovastatin de mevabonate-derived inhibitor. creased incorporation of radiolabeled acetate into digitonin-precipita Cell Lines Differ in the Fmev Concentration Required to In ble sterols by 98% (n = 2). The effect of Fmev on the metabolism hibit Proliferation. The effectiveness of Fmev at inhibiting the of radiolabeled mevalonate was qualitatively unchanged by the pres growth of different transformed cell lines was then examined to ence of bovastatin. Thus, Fmev blocked incorporation of labeled determine whether other leukemia- and bymphoma-derived cell lines would exhibit the same profound suppression of cell growth observed with U-937 cells. All cells were cultured under conditions imparting (I) Z5U Cl) optimal proliferation in the absence of inhibitors. Cell lines differed .c 200 substantially in the impact of a specific concentration of Fmev. Proliferation of some cell lines (illustrated in Fig. 3), was markedly c@ 150 suppressed by very low concentrations of Fmev. Thus, the calculated @Ltati@! @4100 concentration of Fmev that inhibited proliferation by 50% (IC50) I, Onhl ranged between 4 MM(526. 11) and 11 @.LM(Mar-B),and the concen \ . 0.5jiM @ tration causing 90% inhibition (IC@) ranged between 12 MM(526. 11) 50 â€˜@\ â€¢5iiM @ and 31 ,.LM(Daudi) for cells that were classified as â€œsensitiveâ€•to 0 -@//--@-o@-o@ Fmev. In contrast, other cell lines were relatively â€œresistantâ€•toFmev 0 10 100 500 mediated suppression (Fig. 3, bottom). For these cell lines, the IC50 of Fluoromevalonate(liM) Fmev ranged between 114.1 (Jurkat) and 304.1 MM(HL-60). Similar Fig. 2. Lovastatin restores growth of Fmev-blocked U-937 cells. U-937 cells were results were obtained repeatedly in sensitive U-937 cells (IC50, 7 Â±3 cultured in medium supplemented with 10% BCS and incubated with varying concentra tions of Fmev (5â€”500@xM)alone(0) or with 0.5 @sMlovastatin(â€¢)or5 @sMlovastatin(U) MM, mean Â±SE, n = 3; IC@, 14 Â± 3 @.tM;see also Fig. 2) and resistant for 4 days before quantification of DNA synthesis by the incorporation of [3Hlthymidine. EL-4 cells (IC50, 225 Â±49 @M,n = 3). Data points, means of triplicate determinations; bars, SE. 3500

U-937cells â€œSensitiveâ€•CellLines 50 50 @, 200 200 S 1C50 @ 40 AIC9O 40 @. 150 150 30 A 30 A 100 100 o@ I 20 20 Fig. 3. Cell lines differ in the concentration of @ @l50 50 I A â€¢ 00 @ Fmev required for inhibition. Cell lines were cultured 10 in medium supplemented with 10% BCS (solid sym I. . @ bols) or 10% LPP (open symbols) and incubated with 0 ..@...â€¢,.....0 0 0 @ varying concentrations of Fmev (0â€”500gsM)for 4 0@__ 100 500 days before quantification of DNA synthesis. The concentrations of Fmev required for 50% inhibition Fluoromevalonate(jiM) (IC50; â€¢and 0) and 90% inhibition (IC@; A and @s) â€œI were calculated. In some cell lines, only 50% inhibi tion was calculated because 90% inhibition was not EL-4cells â€œResistant'CellLines achieved with the addition of 500 p@ Fmev. Left, 400 400 @uu A 500 representative DNA synthesis of a sensitive cell line â€˜1 (U-937) and a resistant cell line (EL-4). Right, IC.@ lC5Oâ€¢0 @ and IC,@determined for cell lines that are sensitive or 400 lC9O A L@ 400 resistant to Fmev. Data points, means of triplicate E 300 300 experiments; bars, SE. 0. 0 300 300 200 200 . .i+: 200 100 100

@ â€”â€˜-#...@0 wo'@@Ã³@Â° Is 0 0 Fluoromevalonate(jiM)

mevabonate into lipid and protein in a similar, concentration-depen Cell Lines Also Differ in the Effects of Fmev on Ras Localiza. dent manner (Fig. 4b). There were quantitative differences in the lion and Depletion. Similar to the findings in U-937 cells (Fig. 1), distribution of labeled mevalonate within cells with and without Fmev (500 MM) alone decreased membrane-localized Ras in EL-4 suppression of endogenous mevalonate synthesis (compare Fig. 4, a cells (Fig. 5, Lane 2). Low concentrations of bovastatin (0.5 MM)also and b), but there was no effect on the differential capacity of Fmev to modestly decreased membrane-localized Ras in EL-4 cells (Lane 3). suppress mevalonate metabolism in U-937 and EL-4 cells. The major In contrast to U-937 cells, however, Fmev increased cytosolic Ras in impact of bovastatin was that the incorporation of radiobabeled meva EL-4 cells (Lane 6). Thus, the effect of Fmev on localization of Ras bonate into protein was increased by â€”3-fold (EL-4) to > 10-fold in EL-4 cells was similar to the effect of lovastatin (Lane 7). (U-937). The aqueous (small molecular weight) fraction was Un Lovastatin Inhibits Proliferation of EL-4 Cells and Does Not changed by bovastatin, whereas less radiobabeled mevalonate was Restore Fmev-blocked Growth. Low concentrations of bovastatin incorporated into lipid. In Fmev-bbocked cells (Fig. 4b), the inclusion (0.5 ELM)did not alter growth of unblocked EL-4 cells (Fig. 6). of bovastatin marginally increased the residual incorporation of me However, the addition of higher concentrations (5 MM lovastatin) valonate into protein, somewhat more in U-937 cells than in EL-4 inhibitedproliferationsubstantially.Whereasthe low concentrationof cells (P > 0.05). However, the accumulation of radiolabeled meval bovastatin (0.5 ELM)restored the growth of U-937 cells that was onate in the aqueous (small molecular weight) fraction of Fmev suppressed by Fmev (Fig. 2), there was no effect on the proliferation blocked cells was unaltered by bovastatin. These results clearly dem of Fmev-blocked EL-4 cells, despite less inhibition by Fmev (Fig. 6). onstrate the efficacy of Fmev at blocking the conversion of Furthermore, the addition of a higher concentration of lovastatin (5 mevalonate to downstream products in both U-937 and EL-4 cells but @.LM)to EL-4 cells resulted in increased inhibition of cell growth and do not provide an explanation for the differential resistance of these a complete blockade of proliferation with concentrations of Fmev transformed cells to the antiproliferative effect of Fmev. Differences in HMG CoA Reductase Activity and Induction Table I HMG CoA reductaseinEL-4 activity is significant/v greater in V.937 cells than Correlate with the Degree of Fmev Inhibition. The possibility that FmevU-937 cells and is further induced by sensitive and resistant cells might differ in the accumulation of a wereincubatedand EL-4 cells were cultured in medium supplemented with 10% BCS and mevalonate-derived inhibitor was addressed by determining HMG cellswere with or without varying concentrations of Fmev, as indicated. After 24 h, harvested, and 11MGCoA reductase activity was determined as detailed in â€œMate CoA reductase activity in the cell lines in the presence or absence of rials and Methods.â€•Resultsare means Â±SE of triplicatedeterminations.HMG Fmev. In control U-937 cells, bevels of HMG CoA reductase activity activity(pmol/h/mgCoA reductase were almost 6-fold higher than in comparably cultured EL-4 cells protein)Addition of (Table 1). This difference was additionally amplified by incubation cellsControl Concentration (g.tM) U-937 cells EL-4 with Fmev. Thus, Fmev increased the already elevated bevels of HMG IFmev 418 Â±28 73 Â± CoA reductase activity in U-937 cells by an additional >2-fold. In 99Â±7Fmev 5 456Â±34 contrast, Fmev caused only a 0.4-fold increase in HMG CoA reduc 2Fmev 50 1040 Â±21 104 Â± 500 764Â±31 87Â±6 tase activity in EL-4 cells. 3501

0 200 400 600 800 0 100 200 300 AQUEOUS Nil __1H

500pM

@ 0 100 200 300 0 50 100 150 200 PROTEIN Nil I

500pM Fig. 4. Mevalonate metabolism in sensitive and resistant cell lines is comparably inhibited by Fmev. a, U-937 and EL-4 cells were 0 5 10 15 0 10 20 30 cultured in 10%BCS, with or without Fmev (500 pM)as indicated and incubated with [5-3Hlmevalonolactone for 24 h. Cells were separated @H]MevalonateIncorporation(fmoisI10@cells) into lipid. protein, and aqueous fractions, and then the individual components in the various fractions were separated by TLC or SDS PAGE, identified by comparison with authentic standards, visualized b by fluorography. and quantitated by liquid scintillation spectroscopy. Fmev U-937cells EL-4cells b, cellswereculwredandprocessedasin a, exceptthat5 @silovastatin LIPID was added to all cultures. Columns, means of three separate experi menu; bars, SE. NilI

0 200 400 600 0 25 50 75 100 AQUEOUS NilI H H

500pM

0 100 200 300 0 50 100 150 200 PROTEIN Nil I I-

500pM

0 50 100 150 0 40 80 120

@H]MevalonateIncorporation(fmolsI10@cells)

greater than 100 MM.These data suggest that, in EL-4 cells, unlike phate. Rather, sensitivity to Fmev-mediated inhibition was associated U-937 cells, the inhibitory effect of bovastatin on cell growth is with an elevated rate of HMG CoA reductase activity in the highly dominant. sensitive cell line, U-937, with a further induction of activity follow ing incubation with Fmev. In contrast, the Fmev-resistant cell line, DISCUSSION EL-4, had a significantly lower rate of HMG CoA reductase activity The current studies explored the relationship of Ras expression and that was not substantially altered by Fmev. In addition, U-937 cells inhibition of proliferation by Fmev, a fluorinated mevalonate ana manifested substantial depletion ofceblular Ras as a result ofthe Fmev bogue. The results demonstrate that there is a range of effects of Fmev block, whereas Ras was not diminished but rather relocated from the on different cell types, with the concentration required for 90% membrane to the cytosol, as would be expected from a decrease in inhibition (IC@) varying over a >-50-fobd range. This variability prenybation in the Fmev-resistant cell line, EL-4. Thus, as shown in could not be explained by differences in the capacity of Fmev to block Fig. 7, the findings are consistent with the conclusion that Fmev the conversion of mevalonate phosphate(s) to isopentenyl diphos blocked U-937 cells accumulate a mevalonate-derived inhibitor that 3502

of proliferation. Furthermore, when the intracellular pool of mevab Membrane Cytosol â€¢1 onate was depleted with bovastatin, the capacity of Fmev to inhibit the I @l F Fmev - + - + â€” + â€” + incorporation of radiobabebed mevalonate was unchanged or enhanced in both cell lines, making it unlikely that fluctuations in the endoge Lovastatin â€”â€”+ + â€”â€”+ + nouspoo1sizeconfoundedtheanalysis. In contrast to the similarity of Fmev-blocked mevalonate metabo lism in sensitive and resistant cells, mevalonate synthesis was dispar ate between the cell lines. Not only was mevalonate synthesis (HMG CoA reductase activity) substantially greater in U-937 cells, but it was also further induced by Fmev. The induction of HMG CoA reductase

@@@ ____ _ - . @-- . activity was observed with low concentrations of Fmev, consistent with the pattern of inhibition of proliferation by bow concentrations of Fmev in these cells. The additional increase in HMG CoA reductase activity in Fmev-blocked cells is compatible with the activity of the 2 3 4 5 6 7 8 proposednonsterolmevalonateproduct(s),distaltoisopentenyl diphosphate (31), that regulate(s) HMG CoA reductase by altering Fig. 5. Effect of Fmev and lovastatin on immunodetectable Ras in EL-4 cells. EL-4 degradation and/or translation (1 5). The diminished impact of Fmev cells were cultured for 48 h in medium supplemented with 10% BCS, with or without .. .. Fmev(500ELM)andlovastatin(0.5 @,asindicated,afterwhichtheywereseparatedinto on HMG CoA reductase activlty of EL-4 cells 15lfl accordance wlth cytosol (soluble) and membrane (particulate) fractions, and Ras was identified by immu- the diminished flux of mevalonate metabolites in this cell line due to noblotting as detailed in â€œMaterialsandMethods.â€• the decreased HMG CoA reductase activity. Labeled mevalonate and mevalonate phosphates accumulated in the Fmev-bbocked cells. Although the accumulation of radioactivity was 400 similar in EL-4 cells and U-937 cells, the actual intracellular concen Cl) tration will vary with the relative HMG CoA reductase activity of the Cl, 0 300 cell line. Lovastatin increased proliferation of Fmev-blocked U-937 â€” cells, rather than additionally decreasing proliferation, as would be predicted if a compound downstream of mevalonate diphosphate were U) @<200 essential for cell growth. The most reasonable explanation for this Zn. observation is that bovastatin decreased mevalonate synthesis and thereby decreased the accumulation of an inhibitor derived from â€˜j'lOO -I mevalonate or the mevalonate phosphates. The data presented herein w therefore demonstrate that intracellular levels of mevalonate and the 0 mevalonate phosphate(s) detennine the effectiveness of Fmev in 0 10 100 blocking proliferation of many cell lines. Fluoromevalonate (jtM) The precise molecular nature of the mevalonate-derived inhibitor

Fig. 6. Lovastatin fails to restore growth of Fmev-blocked EL-4 cells. EL-4 cells were and the actual process whereby cellular proliferation is blocked re culturedinmediumsupplementedwith10%BCSandincubatedwithvaryingconcentra main unknown. However, one intriguing possibility, suggested by the tions of Fmev (5â€”500pM)alone (0) or with 0.5 @5Mlovastatin(@)or 5 psi lovastatin (U) studies presented herein, is that the amount of total cellular Ras in for 4 days before quantification of DNA synthesis by the incorporation of [3H]thymidine. Data points, means of triplicate determinations; bars, SE. transformed cells is an important determinant of growth. In contrast, the specific intracellular localization of Ras may be less critical. The effect of cytosolic localization of Ras may be dependent on whether depletes Ras. These results suggest that inhibitors of diphosphomeva Ras is oncogenically activated or normal. Thus, recent reports indicate bonate decarboxylase, such as Fmev, may be effective as chemother that complexes of oncogenic Ras with Raf accumulate in the cyto apeutic agents for cancer cells exhibiting elevated levels of HMG plasm when farnesylation is blocked, for example, by farnesyltrans CoA reductase activity. The findings also imply that mevalonate, a mevalonate phosphate, or a product derived from these metabolic intermediates is an important regulator of Ras metabolism. Acetate The proliferation of many cell lines was suppressed in a concen tration-dependent manner by Fmev, even though cells were cultured HMG-COA in medium containing sufficient plasma lipoproteins to provide cho ,____ @3Lovastafln â€”iâ€”.HMG.COAreductase e besterol for membrane synthesis. However, the concentration of Fmev that decreased proliferation by 90% varied greatly (>50-fold) be I lati Mevalonate- Mev&onati tween cell lines of both human and murine origin (this report and Ref. on @_ dsdvâ€¢d Mevalonate Phosphate dsrlvsd 19). The trivial explanation that Fmev was not blocking mevalonate 0 InhibItor Growth Promoter @ metabolism in resistant cell lines was clearly eliminated. However, Mevalonate Diphosphate J synthesis of cholesterol from mevalonate represents a low-affinity, Â® Diphosphomevalonate? Fm.v Decarboxylase high-capacity pathway (15). Therefore, it remained possible that a IsopentenylpiphosPhate high-affinity, low-capacity pathway might not have been as com pletely suppressed. This possibility was addressed directly by meas Famesyl Dlphosphate urements of the capacity of Fmev to prevent incorporation of radio Prenylated @__- -@â€˜â€œâ€˜@-- _ Cholesterol labeled mevalonate into lipids and proteins. These experiments Proteins PrenylUpids

demonstrated that mevalonate metabolism in EL-4 cells was very Fig. 7. Schematic representation of the steps in mevalonate metabolism and the effectively blocked by Fmev, although these cells resisted inhibition putativeregulatorymetabolite(s)generatedafterinhibitionbyFmev. 3503

Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1997 American Association for Cancer Research. Ras AND MEVALONATES ferase inhibitors (32). The complexes cannot signal to the mitogen oncogenic Ras. HMG CoA reductase activity is tightly regulated in activated protein kinase cascade downstream of Ras and are thus normal cells, and this control would protect nonmalignant cells from inactive (32). Similarly, inactive cytosolic Ras-Raf complexes were accumulating inhibitors of proliferation. Criteria that predict Fmev detected when oncogenic Ras was mutated to prevent famesylation mediated inhibition of proliferation in vitro may also predict in vivo (cysteine I86 â€”@serine;Ref. 33). In contrast, Ras-Raf complexes were responsiveness. Thus, the ability to determine HMG CoA reductase not observed when normal Ras was mutated to block farnesylation, activity in circulating leukemic cells might permit selection of those and signaling downstream of Raf was not blocked (33). These obser patients with a greater likelihood of responding to Fmev as an anti vations suggest that cytosolic localization of oncogenic Ras may canceragent. affect proliferation by complexing the effector Raf and preventing downstream signaling via the mitogen-activated protein kinase cas ACKNOWLEDGMENTS cade. Cytosolic localization of normal Ras, as in U-937 and EL-4 cells, is predicted to be without effect on proliferation because sig The authors thank Angela Diem and Sol Kim for excellent technical nabing downstream of Raf is not blocked if Ras is normal (33). assistance. The depletionof total Rasin Fmev-blockedU-937 cellsimpliesthat mevabonate, a mevabonate phosphate, or a derivative of these meta REFERENCES bolic intermediates is involved in Ras metabolism, either directly or I. Barbacid, M. ras genes. Annu. Rev. Biochem., 56: 779â€”827, 1987. indirectly. Because the effects of Fmev and bovastatin are disparate, 2. Bos, J. L. The ras gene family and human carcinogenesis. Mutat. 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Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1997 American Association for Cancer Research. Regulation of Proliferation and Ras Localization in Transformed Cells by Products of Mevalonate Metabolism

Jennifer A. Cuthbert and Peter E. Lipsky

Cancer Res 1997;57:3498-3505.

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