(Received for publication, January 4, 1995; and in revised form, April 26, 1995)
From the
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
[
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)
Figure 1:
Isoprene
biosynthesis and RAS processing.
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%
H
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
H
The amount of
radiolabel incorporated into the protein fraction of the cell lysate
was measured as tricarboxylic acid-precipitated protein. After
incubation with [
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.
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.
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.
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.
Figure 7:
Effects of perillyl alcohol on G
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
[
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
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.
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.
(
)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) .
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.
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 Na
HPO
, and 1.5 mM KH
PO
). 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) .
O
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.
O, 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.
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 H
O 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
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 of the Heterotrimeric 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.
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.
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.
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.
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).
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.
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).
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).
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.