©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Differential Effects of Monoterpenes and Lovastatin on RAS Processing (*)

(Received for publication, January 4, 1995; and in revised form, April 26, 1995)

Raymond J. Hohl (§) , Kriste Lewis

From the Departments of Internal Medicine and Pharmacology, University of Iowa, Iowa City, Iowa 52242

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Limonene and related monoterpenes have been shown to impair the incorporation of mevalonic acid-derived isoprene compounds, that is farnesyl pyrophosphate, into RAS and RAS-related proteins. As farnesylation is critical for RAS's membrane localization and function, the isoprenylation pathways have received attention as potential targets of anti-RAS pharmacologic maneuvers. We have expanded on these prior studies and demonstrate that one of limonene's metabolic derivatives, perillyl alcohol, decreases the levels of antigenic RAS in the human-derived myeloid THP-1 and lymphoid RPMI-8402 cell lines. Both limonene and perillyl alcohol decrease levels of [S]methionine-labeled RAS proteins in cells that have been pulsed with radiolabeled methionine for 4 h. In contrast, lovastatin, which inhibits hydroxymethylglutaryl coenzyme A reductase and thus depletes cells of farnesyl pyrophosphate, does not diminish levels of total antigenic RAS but rather results in a shift in the RAS protein; levels of farnesylated RAS decrease whereas levels of unmodified/unfarnesylated RAS increase. As limonene and perillyl alcohol do not induce such a shift, we conclude that these monoterpenes decrease farnesylated RAS protein levels by a mechanism that is clearly distinct from that of either depleting cells of farnesyl pyrophosphate or inhibiting the enzyme farnesyl protein transferase that catalyzes the post-translational farnesylation of RAS. Perillyl alcohol decreases antigenic RAS levels but does not decrease levels of another membrane-tethered protein, the subunit of the heterotrimeric G protein. Furthermore, perillyl alcohol decreases the levels of radiolabeled methionine incorporated into immunoprecipitable RAS to a greater extent than it decreases radiolabeled methionine incorporated into total cellular protein. Thus there is some degree of specificity for the activity of perillyl alcohol to depress RAS levels.


INTRODUCTION

Limonene, and many of its monoterpene metabolites, displays both chemopreventive and chemotherapeutic activity against chemically induced tumors in rodents(1-6). The mechanism(s) that underlie such anti-cancer activity is/are uncertain but have been postulated to be related to the observation that limonene can decrease levels of isoprenylated RAS and RAS-related proteins in cells maintained in tissue culture(7) . Post-translational RAS farnesylation is necessary for RAS to be localized to the cytoplasmic membrane; such membrane localization is critical for RAS's growth promoting and transforming activities(8, 9, 10) . Pharmacological strategies to block RAS farnesylation are shown in Fig. 1and minimally include: 1) depletion of farnesyl pyrophosphate necessary for RAS farnesylation and 2) inhibition of the farnesyl protein transferase that catalyzes RAS modification. The former strategy is accomplished with high concentrations of the hydroxymethylglutaryl coenzyme A (HMG-CoA)()reductase inhibitors(8) , such as lovastatin(11) , and the latter strategy is accomplished through use of a number of novel RAS and farnesyl pyrophosphate analogs(12, 13, 14, 15, 16, 17, 18) . Limonene is believed to fit in this scheme by interfering with the incorporation of mevalonic acid-derived products into RAS proteins, possibly at the level of impairing farnesyl protein transferase activity(7) .


Figure 1: Isoprene biosynthesis and RAS processing.



Investigations revealing the effects of limonene and its structural analogs on RAS isoprenylation are generally based upon experiments that have followed the incorporation of radiolabeled mevalonic acid into protein fractions and immunoprecipitable RAS in HMG-CoA reductase-inhibited cells(8, 18, 19) . In such cells that are depleted of mevalonic acid by HMG-CoA reductase inhibition, there is a diminution of exogenously supplied radiolabeled mevalonic acid into immunoprecipitable RAS with increasing concentrations of limonene and related monoterpenes(8, 18, 19) . This experimental design is based on the studies that originally demonstrated that mevalonic acid, or one or more of its metabolic derivatives, was incorporated into the protein fraction of eukaryotic cells(20) . Expanding on prior investigations with limonene we utilized an alternate experimental system to study the effects of limonene and one of its more potent metabolic derivatives, perillyl alcohol, on RAS expression in two human-derived leukemia cell lines. Specifically, we have compared and contrasted the effects of limonene and perillyl alcohol to those of lovastatin on RAS processing.


EXPERIMENTAL PROCEDURES

Cell Culture

THP-1 cells (American Type Culture Collection) and RPMI-8402 cells (M. Diaz, Loyola University) were grown in RPMI-1640 media supplemented with 10% heat-inactivated fetal calf serum. Cells were maintained in TI-75 culture flasks at 37 °C in humidified 5% CO atmosphere. Trypan blue exclusion was measured by incubating cells for 5 min with a 1:1 0.4 mg/dl trypan blue solution and counting clear viable and blue nonviable cells with a hemocytometer. All studies with limonene contained a final media concentration of 0.1% ethanol which does not alter the parameters measured in these studies (data not shown).

Measurement of RAS

Cells (1 10 cells at 1 10 cells/ml) in logarithmic phase growth were incubated in RPMI-1640 media with 10% fetal calf serum supplemented with (R)-limonene, (R)-perillyl alcohol, or lovastatin for 24-72 h. Cells were pelleted by centrifugation at 800 g for 10 min and washed twice with cold PBS (140 mM NaCl, 2.7 mM KCl, 8.0 mM NaHPO, and 1.5 mM KHPO). Cells were resuspended in 0.1 ml of lysing buffer (20 mM Tris, pH 7.5, 0.1 M NaCl, 1.0% Nonidet P-40, 0.05 g/dl sodium deoxycholate) and vortexed intermittently for 30 min. The cell pellets were dounced 10 times and centrifuged at 10,000 g for 30 min. Protein content of the supernatant was assayed according to the method of Lowry et al.(21) .

Cell lysate containing 100 µg of protein was subjected to electrophoretic fractionation on denaturing 15% polyacrylamide gels (SDS-polyacrylamide gel electrophoresis). H-ras standard was loaded to one lane on each gel. The gel slab was transblotted to Immobilon-Polyvinylidene difluoride (Millipore, Bedford, MA) prior to blocking for 2 h with blocking buffer (5 mg/dl skim milk, 1 mg/dl bovine serum albumin (BSA), 0.01% antifoam A in PBS). The membrane was then sequentially incubated with NCC-004 anti-RAS antibody (22) in hybridoma supernatant for 2 h, biotinylated rabbit anti-mouse IgG for 2 h, and avidin-linked horseradish peroxidase (Vectastain ABC, Vector Laboratories) for 30 min. The membrane was washed five times with 0.1% Tween 20 in PBS between each incubation. The RAS bands were visualized by incubation of the membrane with 0.7 mM 3,3-diaminobenzidine and 0.006% HO in 10 mM Tris, pH 7.5.

Immunoprecipitation of Radiolabeled RAS

Cells were washed and incubated at 3 10 cells/ml in methione-free RPMI-1640 media for 1.5 h with or without limonene, perillyl alcohol, or lovastatin. [S]Methionine/cysteine (1180 Ci/mM) was added to a final media concentration of 0.045 mCi/ml. After incubation for 4 h, the cells were harvested and lysed as described above in the Measurement of RAS section with the exception that 1 g/dl BSA was added to the lysing buffer.

Radiolabeled cell lysate containing 200 µg of protein was precleared by incubation with Protein A-Sepharose CL4B complexed with rabbit-anti-rat IgG. After centrifugal separation of the lysate from the preclearing sepharose, the RAS was immunoprecipitated from the lysate by the addition of 150 µl of a 10 g/dl mixture of a complex of Protein A-Sepharose CL4B, rabbit-anti-rat IgG, and Y13-259 rat-anti-RAS IgG. The Sepharose was washed five times with 1 g/dl BSA in RIPA (0.15 M NaCl, 1 g/dl sodium deoxycholate, 0.1 g/dl SDS, 1% Triton X-100, 1 mM phenylmethanesulfonyl fluoride, and 0.05 M Tris, pH 7.4) buffer and five times with 1 g/dl BSA in PBS prior to boiling for 5 min in 40 µl of 0.05 M Tris, pH 6.8, 30% glycerol, 10% 2-mercaptoethanol, 2 g/dl SDS, 0.025 g/dl bromphenol blue, and 0.001 g/dl methyl green. The supernatant was loaded to 5% glycerol, 15% polyacrylamide gels and subjected to electrophoretic fractionation. The gels were fixed in 7% acetic acid and 5% methanol for 1 h, washed with HO, and enhanced with a 1 M sodium salicylate solution for 30 min. Gels were then dried and exposed to Kodak XAR5 (X-OMAT) film for 48 h. With development, the density of the bands corresponded to the amount of radiolabeled protein.

The amount of radiolabel incorporated into the protein fraction of the cell lysate was measured as tricarboxylic acid-precipitated protein. After incubation with [S]methionine/cysteine and cell lysis, 100 µl of 1 g/dl BSA in 40 mM methionine was added to 5 µl of cell lysate. The volume was increased to 180 µl by adding HO prior to the addition of 20 µl of 100 g/dl tricarboxylic acid. After precipitation of the protein fraction, the supernatant was discarded and the precipitated protein washed twice with 0.4 ml of 10 g/dl tricarboxylic acid. The precipitant was resuspended in TS-2 tissue solubilizer (RPI, Mount Prospect, IL) and, after complete dissolution, the radiolabel was quantified by scintillation spectroscopy using Neutralizer (RPI) scintillation mixture.

Measurement of Subunit of the Heterotrimeric G Protein

Cells were incubated and lysed, cell lysate was subjected to electrophoretic fractionation, the gel slab was transblotted, and the Immobilon polyvinylidene difluoride membrane blocked with blocking buffer as described under Measurement of RAS section. The membrane was then sequentially incubated with 1:1000 dilution of anti-G protein, -Subunit Internal in blocking buffer for 2 h and then biotinylated goat anti-rabbit IgG for 2 h. The membrane was then processed beginning with incubation to aviden-linked horseradish peroxidase as described under Measurement of RAS. The densities of the subunit bands were quantified by analyzing the image acquired with a Hewlett Packard ScanJet IIcx (Palo Alto, CA) with Mocha Image Analysis software from Jandel Scientific (San Rafael, CA).

Materials

(R)-Limonene (>99% pure) was obtained from Aldrich, and stock solutions were freshly prepared before each experiment by diluting limonene in 10% ethanol-tissue culture media solution. (R)-Perillyl alcohol was obtained from Fluka (Ronkonkoma, NY), and stock solutions were prepared before each experiment by mixing perillyl alcohol with tissue culture media. RPMI-1640 media and fetal calf serum were obtained from the Hybridoma Facility at the University of Iowa. Methionine and cysteine-free media and TranS-label (methionine/cysteine) were from ICN Biochemicals (Costa Mesa, CA). Lovastatin was kindly provided by Alfred Alberts of Merck, Sharpe & Dohme and was converted to the open acid prior to use. Protein A-Sepharose CL4B was from Sigma. NCC-004 anti-RAS IgG was kindly provided by Dr. Setsuo Hirohashi (National Cancer Center, Tokyo), H-ras standard was kindly provided by Dr. Robert Deschenes (University of Iowa), Y13-259 anti-RAS IgG was from Oncogene Science (Uniondale, NY), rabbit anti-mouse IgG, goat anti-rabbit IgG, and Vectastain ABC kits were from Vector Laboratories (Burlingame, CA), anti-apolipoprotein B antibody was from Biodesign International (Kennebunkport, MA), rabbit-anti-rat IgG was from Cappel Laboratories (Durham, NC), and rabbit anti-G-Protein -Subunit Internal (40-54) and G-protein, Bovine Brain were from Calbiochem.


RESULTS

Lovastatin's Effects on RAS Protein Levels

To determine the effects of lovastatin on total antigenic RAS protein levels, THP-1 cells were incubated with 0, 10, 25, and 50 µM lovastatin for 24 h prior to harvesting and lysing. Mevalonic acid to a final concentration of 10 mM was added to control cultures that contained 0 and 50 µM lovastatin. Fig. 2is a Western blot of cell lysate that demonstrates that in control cells without lovastatin there is only one dominant RAS band that corresponds to farnesylated RAS protein. The density seen streaking below the RAS band is a consequence of some degradation of the RAS protein into smaller molecular weight fragments. Increasing concentrations of lovastatin from 10 to 50 µM results in the accumulation of increasing amounts of unmodified RAS protein that migrates with an apparent higher molecular weight than does the farnesylated RAS protein. Mevalonic acid has no effect on the levels of antigenic RAS protein and completely reverses the effects of lovastatin in that only the farnesylated RAS protein is seen. The same experiment using RPMI-8402 cells yielded the same results; selected conditions from this experiment are displayed in Fig. 2.


Figure 2: Effects of lovastatin on RAS isoprenylation in THP-1 and RPMI-8402 cells. This Western blot was developed as described under ``Experimental Procedures.'' Above each lane is listed the cell type and treatment with lovastatin (Lov) and/or mevalonic acid (Mev). Each lane contains 100 µg of protein from cell lysate.



Limonene and Perillyl Alcohol's Effect on Antigenic RAS Levels

To contrast the effects of (R)-limonene with lovastatin on total antigenic RAS protein levels, THP-1 and RPMI-8402 cells were incubated with 0.1 mM lovastatin and 0 to 5 mM limonene for 24 h prior to harvesting and lysing. Fig. 3demonstrates that limonene, unlike lovastatin, does not result in accumulation of unmodified RAS protein. There is a suggestion that total RAS levels decrease slightly at the highest concentrations (5 mM) of limonene employed although at 5 mM limonene cell viability was reduced to 78% in THP-1 cells and 60% in RPMI-8402 cells.


Figure 3: Effects of lovastatin and limonene on RAS isoprenylation in THP-1 and RPMI-8402 cells. These Western blots were developed as described under ``Experimental Procedures.'' The top panel displays the lysates from the THP-1 cells while the bottom panel displays the lysates from the RPMI-8402 cells. Each lane contains 100 µg of protein from cell lysate and is labeled with the concentration of lovastatin or limonene used in the incubations. Control cells were incubated without drug. H-ras standard (RAS STD) was also loaded on a separate lane. This standard migrates as a single band with an apparent molecular weight that approximates that of unmodified RAS. Higher concentrations of lovastatin were used than in Fig. 2so as to nearly completely block RAS farnesylation as evidenced by the presence of only one RAS band that corresponds to the unmodified protein.



Perillyl alcohol is an in vivo oxidative metabolite of limonene that has been demonstrated to be more potent than limonene at depressing RAS levels(18) . THP-1 and RPMI-8402 cells were incubated with 0.1 mM lovastatin or 0-5 mM perillyl alcohol for 24 h prior to harvesting and lysing. Fig. 4demonstrates that perillyl alcohol, unlike lovastatin, does not result in accumulation of unmodified RAS protein. It is apparent that perillyl alcohol depresses levels of RAS in a more potent manner than does limonene ( Fig. 4compared to Fig. 3) although perillyl alcohol proved more toxic to cells than did limonene. At 5 mM perillyl alcohol cell viability was severely reduced to <25%, thus allowing the conclusion that even at toxic levels of perillyl alcohol there is still no accumulation of unmodified RAS protein.


Figure 4: Effects of lovastatin and perillyl alcohol on RAS isoprenylation in THP-1 and RPMI-8402 cells. These Western blots were developed as described under ``Experimental Procedures.'' The top panel displays the lysates from the THP-1 cells while the bottom panel displays the lysates from the RPMI-8402 cells. Each lane contains 100 µg of protein from cell lysate and is labeled with the concentration of lovastatin or perillyl alcohol used in the incubations. Control cells were incubated without drug. H-ras standard (RAS STD) was loaded on a separate lane.



Lovastatin, Limonene, and Perillyl Alcohol's Effect on Newly Synthesized RAS

To verify the integrity of the immunoprecipitation methods, THP-1 cell lysate was treated and immunoprecipitated with Protein A-Sepharose complexed with or without primary Y13-259 anti-RAS antibody. Fig. 5demonstrates that the RAS band does not precipitate unless the Y13-259 antibody is included in the reaction mixture. To further assess this specificity, cells were pulsed with radiolabeled methionine and lysed. Immunoprecipitation of cell lysate with the Y13-259 antibody resulted in two electrophoretically separable radiolabeled RAS bands as shown in the control lanes for both RPMI-8402 and THP-1 cells in Fig. 6. The addition of excess amounts of H-ras standard competes with radiolabeled RAS for binding to the Y13-259-Sepharose complex and markedly diminishes the intensity of the radiolabeled RAS bands (data not shown).


Figure 5: Specificity of the immunoprecipitation for RAS. THP-1 cells were lysed, precleared, and 200 µg of protein from cell lysate immunoprecipitated as described under ``Experimental Procedures.'' The immunoprecipitate using the Protein A-Sepharose CL4b/rabbit-anti-rat IgG/Y13-259 rat-anti-RAS IgG complex is refered to as +Ab whereas the immunoprecipitate using the Protein A-Sepharose CL4b/rabbit-anti-rat IgG complex (without the Y13-259 rat anti-RAS IgG) is refered to as -Ab. After fractionation by SDS-polyacrylamide gel electrophoresis, this Western blot was developed as described under ``Experimental Procedures.'' The RAS protein is only immunoprecipitated and visualized when the anti-RAS antibody is complexed to the Sepharose.




Figure 6: Comparative effects lovastatin, limonene, and perillyl alcohol on the farnesylation of newly synthesized RAS. RPMI-8402 and THP-1 cells were radiolabeled and 200 µg of protein from cell lysate immunoprecipitated as described under ``Experimental Procedures.'' For this autoradiogram, each lane is labeled according to cell type and whether there was exposure to lovastatin, limonene, or perillyl alcohol. The lane labeled as control + Apo B MAB refers to immunoprecipitant obtained when anti-apolipoprotein B antibody is used in place of Y13-259 anti-RAS antibody. As expected, no radiolabeled bands that migrate with the anticipated molecular weight of the RAS proteins are visualized in this lane.



To assess whether limonene and perillyl alcohol would diminish the synthesis of new radiolabeled RAS protein, RPMI-8402 and THP-1 cells were pulsed with radiolabeled methionine after incubation without added agents or with lovastatin, limonene, or perillyl alcohol. Fig. 6is the autoradiogram from this experiment. Control cells demonstrate that radiolabel is incorporated into bands that migrate as would farnesylated and unmodified/unfarnesylated RAS protein. Lovastatin at 0.05 mM concentrations completely blocks incorporation of radiolabel into farnesylated RAS protein as evidenced by the lack of a faster moving protein in these treated cells. In contrast, limonene and perillyl alcohol do not result in a selective decrease of radiolabel into farnesylated RAS protein but rather decrease overall levels of radiolabeled RAS proteins. For the RPMI-8402 cells there are sufficient concentrations of limonene and perillyl alcohol to demonstrate a concentration dependence of this phenomenon. As in the Western blots displayed in Fig. 3and Fig. 4, this effect is more evident in the cells treated with perillyl alcohol as compared to those treated with limonene. The treatment of cells with limonene or perillyl alcohol did not alter the ratio of radiolabeled farnesylated to radiolabeled unmodified RAS protein. For control purposes it is important to note that the RPMI-8402 and THP-1 cells maintained viability, as assessed by trypan blue exclusion, to greater than 75% with this short exposure to these concentrations of limonene or perillyl alcohol.

Specificity of Perillyl Alcohol's Effects on RAS

To clarify the specificity for perillyl alcohol to depress levels of antigenic RAS, THP-1 and RPMI-8402 cells were incubated with perillyl alcohol ranging from 0 to 1.0 mM for 16 h prior to harvesting and lysing. Fig. 7displays a Western blot of an acrylamide gel that contains 50 µg of THP-1 cell lysate in each lane; RPMI-8402 cells yielded similar results (not shown). The blot was developed using the anti-G protein (-subunit) as primary antibody. Lovastatin does not alter the migration of the G protein consistent with understanding that the -subunit of the heterotrimeric G protein is not isoprenylated(23, 24) . Visualization of the intensity of the bands reveals that the cellular levels of the G protein are relatively constant when cells are incubated with incrementally increasing concentrations of perillyl alcohol. This is verified by the densitometric analysis of the G bands which is shown in the tabular portion of Fig. 7. Also shown in this figure is that RAS levels are decreased in a concentration-dependent fashion in perillyl alcohol-treated cells. Cell viability is maintained to greater than 80% with these concentrations of perillyl alcohol. Thus, perillyl alcohol decreases antigenic levels of RAS without decreasing G protein (-subunit) levels and cell viabilities.


Figure 7: Effects of perillyl alcohol on G protein and RAS levels, cell viability, and newly synthesized RAS. THP-1 cells were incubated with lovastatin or perillyl alcohol prior to harvesting. The Western blot displays the G protein levels detected in cell lysate as described under ``Experimental Procedures.'' A separate lane contains an aliquot of G protein standard. In the tabular form of this figure, G(% control) refers to the relative densities of G protein bands expressed as percent density of the G band in treated as compared to control cells. RAS (% control) refers to the levels of RAS proteins in the lysates of these cells, as determined by Western blot (not shown) and densitometric analysis, and is expressed as percent density of the RAS band in treated as compared to control cells. Cell viability (%) refers to percent viable cells assessed by trypan blue exclusion. S-RAS/total protein (% control) is the amount of radiolabeled methionine incorporated into RAS in 200 µg of cellular protein and is expressed as percent of control cells.



To determine whether the decreased incorporation of radiolabeled methionine to immunoprecipitable RAS by perillyl alcohol is specific to RAS or is a consequence of a general decrease of protein synthesis, THP-1 and RPMI-8402 cells were incubated with [S]methionine for the last 4 h of a 16-h incubation with 0-1.0 mM perillyl alcohol. Cells were lysed and RAS protein was immunoprecipitated from 200 µg of cellular protein as described earlier. As is shown in the tabular portion of Fig. 7, the amounts of radiolabel incorporated into RAS are decreased in a concentration-dependent fashion in perillyl alcohol-treated THP-1 cells; the radiolabeled methionine incorporated into RAS in cells treated with 1.0 mM perillyl alcohol is decreased to 32% that of control cells. In contrast, the amount of radiolabeled methionine incorporated into 200 µg of cellular protein, assessed as tricarboxylic acid precipitable counts in 200 µg of protein and expressed as the percent tricarboxylic acid precipitable counts in 200 µg of protein in treated as compared to control cells was only minimally decreased. Cells treated with 1.0 mM perillyl alcohol had a reduction of radiolabeled methionine incorporated into RAS to 87% that of control cells. Thus, perillyl alcohol decreased radiolabeled methionine into immunoprecipitable RAS in a more selective fashion than by simply decreasing total protein synthesis. Studies with RPMI-8402 cells demonstrated this same selectivity of perillyl alcohol to decrease RAS synthesis (not shown).


DISCUSSION

These experiments demonstrate that limonene and perillyl alcohol act differently than lovastatin with respect to their activity to suppress levels of farnesylated RAS protein. In these two different human-derived cell lines, limonene and, more potently, perillyl alcohol decrease farnesylated RAS without increasing unmodified/unfarnesylated RAS levels. This result contrasts with that observed with lovastatin which decreases farnesylated RAS and increases unmodified/unfarnesylated RAS levels. Lovastatin's activity is entirely consistent with HMG-CoA reductase inhibition and farnesyl pyrophosphate depletion (Fig. 1)(9, 25) . In contrast the data displayed in Fig. 3and Fig. 4suggest that the monoterpenes either decrease RAS transcription/translation or increase RAS degradation. The high concentrations of monoterpenes used in the experiments shown in Fig. 3and Fig. 4markedly diminish cell viability and thus reinforce the finding that even excessively toxic levels of monoterpenes do not result in the accumulation of unmodified/unfarnesylated RAS in these cell lines. Furthermore, the activity of lovastatin to shift RAS from a predominantly farnesylated to an unmodified/unfarnesylated form is also associated with the newly developed farnesyl protein transferase inhibitors (Fig. 1)(13, 15, 16) . Because this shift in RAS species is not observed with the monoterpenes then we can conclude that it is highly unlikely, in contrast to previous suggestions, that limonene and perillyl alcohol significantly block RAS processing at the level of farnesyl protein transferase(7, 18, 19) .

The differences in the conclusions derived from our experiments and those reported by others (7, 18, 19) lie in the techniques used to measure RAS levels. These previous reports used radiolabeled mevalonic acid to label the RAS proteins and therefore could not distinguish between agents that decrease levels of the farnesylated RAS proteins by direct inhibition of the enzyme farnesyl protein transferase, by depletion of farnesyl pyrophosphate, by decrease of RAS synthesis at the transcriptional or translational level, or by increase in degradation rates of farnesylated RAS. Additionally, to achieve sufficiently high mevalonic acid specific activity to radiolabel the isoprenylated proteins to the degree needed to detect the proteins by autoradiogram analysis these studies co-incubated cells with radiolabeled mevalonic acid, lovastatin, and the monoterpene to be tested; lovastatin diminishes endogenous mevalonic acid synthesis and thus maintains the high specific activity of radiolabeled mevalonic acid(20) . Thus, these investigations could not conclusively distinguish the effects of lovastatin from that of the monoterpenes on RAS processing. The studies reported herein allowed us to assess the effects of treating cells with the monoterpenes alone.

That perillyl alcohol was effective at decreasing antigenic RAS levels to a markedly greater degree than limonene is consistent with the known in vivo oxidation of limonene to perillyl alcohol(18) . Our in vitro studies support the hypothesis that the chemopreventive and chemotherapeutic activity of limonene may likely be a consequence of metabolism to the more active perillyl alcohol. The THP-1 and RPMI-8402 cells used in our studies are incapable of metabolically activating sufficiently large quantities of limonene to generate enough perillyl alcohol to decrease RAS levels.

We observed the presence of only a single farnesylated RAS band in the Western blot analysis of cell lysate from unlabeled control cells (Fig. 2-4) and the presence of both the farnesylated and a slower migrating band in immunoprecipitated RAS from radiolabeled cells (Fig. 6). This finding is likely due to the relatively short duration of the pulse of [S]methionine as compared to the longer half-life of the RAS protein in these cells. Although there are no data available that describes the RAS half-life in the human-derived leukocyte cells used in these present studies, there are reports that the half-life of the H-RAS protein in NIH 3T3 cells that have been transfected with normal human H-RAS cDNA is approximately 20 h(26) . Were the half-life of the RAS proteins studied in these present experiments to be similarly long then it is not unexpected that the intensity of the radiolabel in the unmodified/unfarnesylated RAS bands would approximate the intensity of the radiolabel in the farnesylated bands (Fig. 6), especially given the high ratio of farnesylated to unfarnesylated RAS proteins in untreated cells (Figs. 2-4).

A concern with these studies is that the high concentrations of perillyl alcohol used for the experiment in Fig. 4may have decreased RAS levels because of nonspecific cytotoxicity. The experiment shown in Fig. 4is important because it unambiguously shows that even at toxic levels of perillyl alcohol there is no accumulation of unmodified RAS protein. The concentrations of perillyl alcohol used in the study shown in Fig. 7still decreased RAS protein levels but had only minimal effects on cell viability. Furthermore, we have shown in Fig. 7that the levels of another membrane-tethered protein, the subunit of a heterotrimeric G protein (23, 24) that is expressed in the leukemia cell lines in this study, remain relatively constant while the RAS levels in these cells are decreased. Thus, it seems unlikely that perillyl alcohol's effects are exclusively the result of nonspecific toxicity. To further assess the specificity issue, we have compared the amount of radiolabeled methionine incorporation into immunoprecipitable RAS to the amount of radiolabeled methionine incorporated into total protein fractions. Fig. 7shows that the radiolabeled methionine incorporated into RAS is diminished more than that incorporated into total cell protein. This suggests that the mechanism that underlies perillyl alcohol's effect to decrease antigenic RAS may be a consequence of decreased RAS synthesis, particularly at and below 0.5 mM perillyl alcohol. At 0.75 and 1.0 mM perillyl alcohol, there is continued low new RAS synthesis but even lower RAS levels suggesting that enhanced RAS degradation may partially explain the effect of these higher concentrations of perillyl alcohol to depress RAS levels. Further studies will be necessary to clarify these findings; such studies will also improve understanding of the regulation of RAS synthesis and degradation.

The finding that the monoterpenes decrease farnesylated RAS levels by a different mechanism than does either lovastatin or the newly developed farnesyl protein transferase inhibitors is important because it supports the possibility that combinations of monoterpenes, lovastatin, and the farnesyl protein transferase inhibitors might be more effective at decreasing farnesylated RAS levels than each agent alone. In fact, there is a recent report that the combination of limonene and pravastatin, another competitive inhibitor of HMG CoA reductase, is more effective at decreasing the levels of isoprenylated RAS in human-derived Hep G2 cells than either compound alone(19) . Finally, further dissection of mechanisms that underlie the activity of the isoprene compounds to decrease RAS levels will likely include detailed analysis of the effects of these compounds on the rates of synthesis and degradation of the RAS proteins. Such studies will improve understanding of the mechanisms that regulate RAS expression and may suggest new approaches that can be used to pharmacologically manipulate RAS levels in eukaryotic cells.


FOOTNOTES

*
This work was supported by grants 92SG20 and 93B46 from the American Institute for Cancer Research and Carver Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of an American Society of Clinical Oncology Young Investigator Award and a Pharmaceutical Research and Manufacturers of America Foundation Faculty Development Award in Clinical Pharmacology. To whom correspondence should be addressed: Depts. of Internal Medicine & Pharmacology, C32-I GH, University of Iowa, Iowa City, Iowa 52242. Tel.: 319-356-8110; Fax: 319-353-8383.

The abbreviations used are: HMG-CoA, hydroxymethylglutaryl coenzyme A; PBS, phosphate-buffered saline; BSA, bovine serum albumin.


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