(Received for publication, January 3, 1995; and in revised form, June 5, 1995)
From the
S-trans,trans-Farnesylthiosalicylic
acid (FTS) is a novel farnesylated rigid carboxylic acid derivative. In
cell-free systems, it acts as a potent competitive inhibitor (K = 2.6 µM) of the
enzyme prenylated protein methyltransferase (PPMTase), which methylates
the carboxyl-terminal S-prenylcysteine in a large number of
prenylated proteins including Ras. In such systems, FTS inhibits Ras
methylation but not Ras farnesylation. Inhibition of the PPMTase by FTS
in homogenates or membranes of a variety of tissues and cell lines is
inferred from a block in the methylation of exogenously added
substrates such as N-acetyl-S-trans,trans-farnesyl-L-cysteine
and of endogenous substrates including small GTP-binding proteins. FTS
can also inhibit methylation of these proteins in intact cells (e.g. in Rat-1 fibroblasts, Ras-transformed Rat-1, and B16
melanoma cells). Unlike in cell-free systems, however, relatively high
concentrations of FTS (50-100 µM) are required for
partial blocking (10-40%) of protein methylation in the intact
cells. Thus, FTS is a weak inhibitor of methylation in intact cells.
Because methylation is the last step in the processing of Ras and
related proteins, FTS is not likely to affect steps that precede it, e.g. protein prenylation. This may explain why the growth and
gross morphology of a variety of cultured cell types (including Chinese
hamster ovary, NIH3T3, Rat1, B16 melanoma, and PC12) is not affected by
up to 25 µM FTS and is consistent with the observed lack
of FTS-induced cytotoxicity. Nevertheless, FTS reduces the levels of
Ras in cell membranes and can inhibit Ras-dependent cell growth in
vitro, independently of methylation. It inhibits the growth of
human Ha-ras-transformed cells (EJ cells) and reverses their
transformed morphology in a dose-dependent manner (0.1-10
µM). The drug does not interfere with the growth of cells
transformed by v-Raf or T-antigen but inhibits the growth of
ErbB2-transformed cells and blocks the mitogenic effects of epidermal
and basic fibroblast growth factors, thus implying its selectivity
toward Ras growth signaling, possibly via modulation of Ras-Raf
communication. Taken together, the results raise the possibility that
FTS may specifically interfere with the interaction of Ras with a
farnesylcysteine recognition domain in the cell membrane. This drug,
and perhaps other farnesylated rigid carboxylic acid analogs, may be
used for in vitro characterization of the PPMTase and for the
identification of a putative Ras farnesylcysteine recognition domain in
cell membranes.
The free carboxyl group of the terminal farnesylcysteine or
geranylgeranylcysteine in a large number of prenylated proteins is
methylated by the prenylated protein methyltransferase (PPMTase), ()a unique enzyme among methyltransferases(1) . This
enzyme associates tightly with the particulate fractions in eukaryotic
cells (2, 3, 4, 5) and has an
absolute requirement that its substrates contain the free carboxyl
group and S-farnesyl or S-geranylgeranyl
moieties(4, 5, 6) . The sequence of the yeast
PPMTase gene (STE14) indeed predicts a hydrophobic
membrane-associated protein(7, 8) . Many of its
natural substrates are key regulatory elements in receptor signal
transduction
pathways(9, 10, 11, 12, 13, 14, 15) ,
for example Ras and the heterotrimeric G-proteins. Methylation of these
proteins would increase the hydrophobicity of their carboxyl terminus,
already rendered hydrophobic by
prenylation(16, 17, 18, 19) .
Because methylation is the last and only possible reversible step in
the processing of such prenylated proteins, it is likely that the
PPMTase modulates their interactions with membranes and other proteins.
It is also reasonable to assume that the methyl-acceptor recognition
site in the PPMTase shares some similarities with prenylcysteine
recognition domains found in target sites for prenylated proteins. Such
sites are not necessarily located within the lipid
bilayer(20) .
The view that protein prenylation would mediate protein-protein interaction(20) , as well as the possibility of separate targets for the methylated and unmethylated forms of prenylated proteins, has recently been discussed(5) . Very little is known about the nature of prenylcysteine recognition domains. They are likely to exist, however, in at least three other proteins besides the PPMTase: the GDP-dissociation inhibitory protein, which binds only to the fully processed rab3A(21) , and the yeast a-factor transporter (8) and receptor(8, 22) , which require the fully processed a-factor. Also, the existence of farnesylcysteine recognition domains for Ras proteins may be predicted from the absolute requirement of the farnesylcysteine for Ras oncoprotein membrane anchorage and transforming activity(10, 19, 23) .
An approach that has provided valuable information on the prenylcysteine recognition domain of the PPMTase is the use of synthetic farnesyl derivatives. To be recognized by this domain, a PPMTase substrate need not be a peptide; N-acetyl-S-trans,trans-farnesyl-L-cysteine (AFC) is an excellent substrate for the enzyme(5, 6) . Other N-acetylated farnesylcysteine analogs can also act as substrates provided that they do not have bulky moieties(24) . Whereas such moieties can prevent association of farnesyl derivatives with the methyl-acceptor recognition domain(24) , the total absence of an amino group is tolerated, as shown by the fact that S-farnesylthiopropionic acid does serve as a PPMTase substrate(6, 24, 25) . Moreover, S-farnesylthioacetic acid is recognized by the PPMTase, though it does not serve as substrate but rather as a competitive inhibitor with respect to AFC(6, 25) .
On the basis of such observations, we have now prepared a novel rigid S-farnesyl carboxylic acid derivative, which, like S-farnesylthioacetic acid, is not an amino acid. We show here that it acts as a pure competitive inhibitor of the PPMTase in cell-free systems. Most interestingly, the results presented here together with those presented in a related study (26) suggest that rigid farnesylated carboxylic acid derivatives can inhibit Ras-dependent cell growth in a mechanism unrelated to their ability to inhibit the PPMTase.
A reconstituted
system for cell-free Ras processing was obtained by a two-step
procedure. In the first step(30) , bacterially expressed human
Ha-Ras (2.5 µg) and partially purified rat brain
farnesyltransferase (2 µg) were incubated (total volume 10 µl)
for 1 h at 37 °C with 15 pmol
[H]farnesylpyrophosphate (15 Ci/mmol, ARC), 50
mM Tris-HCl, pH 7.4, 50 µM ZnCl
, 5
mM MgCl
, 20 mM KCl, 1 mM dithiothreitol, 4% Me
SO (control), or 50 µM FTS in 4% Me
SO. Proteins were separated by SDS-PAGE,
and
H-farnesylated Ras was determined by direct counting of
2-mm gel sections in a Lipoluma/Lumasolve scintillation mixture.
Neither Ras alone nor farnesyltransferase alone yielded a signal above
background. In the second step, samples incubated as described above
each received 5 µl of rat brain microsomal membranes (5 µg of
protein), which served as a source of protease and methyltransferase,
and 10 µl (110 pmol) of S-[methyl-
H]adenosyl-L-methionine
(85 Ci/mmol). Me
SO and FTS concentrations in the test
samples were adjusted to 4% and 50 µM, respectively, and
the samples were then incubated for 30 min at 37 °C. Proteins were
separated by SDS-PAGE, and
H-labeled methylesters were
determined by the base hydrolysis vapor phase diffusion
assay(2, 26, 27) . Assay background counts
(no Ras added) were 10% of signals. Control experiments showed that by
itself the
H-farnesylated Ras, which was formed in the
first step, did not yield base-labile diffusible counts. This enabled
us to perform the double tritium labeling procedure described above.
In an attempt to determine whether FTS (Fig. 1a, inset) can interact with PPMTase, we
used a rat cerebellum-derived preparation of synaptosomal membranes,
which are highly enriched in the enzyme(25) . With AFC as a
substrate, V in this preparation is 60
pmol/min/mg protein. Thus, together with a sensitive methylation assay (25, 26) , it should be possible to detect substrate
or inhibitor activity. Incubation of synaptosomal membranes with 25
µM [methyl-
H]AdoMet and 200
µM FTS under conditions that promote efficient methylation
of either AFC or S-farnesylthiopropionic acid (25, 26) yielded no methylated products in the
standard methylation assay. If FTS were a substrate, the formation of
H-methylated FTS should have been detected in this assay,
in which total lipophilic compounds are extracted by
chloroform/methanol. We conclude that FTS is not a substrate for rat
cerebellum PPMTase. In contrast, FTS inhibited the methylation of AFC
in a dose-dependent (Fig. 1a) and competitive manner (Fig. 1b). The estimated K
value
for FTS in this preparation is 2.6 ± 1.5 µM (n = 4), comparable to the values recorded for S-farnesylthioacetic acid in the same preparation (25) or in rod outer segments(6) . As shown in Fig. 1c, in the synaptosomal membrane preparation FTS
could also inhibit methylation of endogenous 21-26-kDa proteins,
which represent mostly GTP-binding proteins(26) . Thus, in this
cell-free system, FTS can compete with natural substrates for the
prenylcysteine recognition domain in the PPMTase. To examine whether
FTS can also inhibit the PPMTase in other cell-free systems, we
repeated the experiments with homogenates of a variety of tissues and
cell types. These include mouse brain, kidney, pancreas, heart and
testis, human endometrium and endometrial carcinomas, Rat1 and NIH3T3
fibroblasts, pheochromocytoma PC12 cells, B16 melanoma cells, human
Ha-Ras-transformed rat1 (EJ) cells, and human endometrial carcinoma
HEC1A cells. In all cases, FTS caused a dose-dependent inhibition of
AFC methylation. Typical results obtained with homogenates of EJ cells
are shown in Fig. 1a. We conclude that FTS is likely to
inhibit PPMTase in any cell-free system, even though its inhibitory
potency may vary among cell types and tissues (see Fig. 1a). Such variations were also observed with the
PPMTase inhibitor S-farnesylthioacetic acid(24) .
Figure 1:
Inhibition of
PPMTase by FTS. The chemical structure of FTS is shown in the boxedinset. a, dose-dependent inhibition of PPMTase
in rat brain synaptosomes () and in homogenates of EJ cells
(
) by FTS. Enzyme assays were performed in the presence of 25
µM [methyl-
H]AdoMet, 50
µM AFC as a methyl acceptor, and the indicated
concentrations of FTS. b, competitive inhibition of AFC
methylation by FTS. Enzyme assays were performed in the absence
(
) and in the presence (
) of 10 µM FTS and
various concentrations of AFC. Data are presented in the form of
double-reciprocal plots. c, FTS inhibits methylation of
21-26-kDa GTP-binding proteins in rat cerebellum synaptosomes.
Synaptosomal membranes were incubated for 60 min with 2.4 µM [methyl-
H]AdoMet (15 Ci/mmol) in
the absence (
) and in the presence (
) of 100 µM FTS. Proteins were then separated by SDS-PAGE, and carboxyl
methylesters were determined in gel
slices(25, 26) .
Further studies were performed with intact cells. We first examined
whether FTS can inhibit protein carboxyl methylation in the cells.
Cells were metabolically labeled with
[methyl-H]methionine in the absence and
in the presence of 100 µM FTS.
H-Labeled
methylesters were then determined (2, 26) in the group
of 21-26-kDa proteins following their separation by SDS-PAGE.
Typical results obtained with EJ, Rat1, and B16 cells (Fig. 2, a-c) indicated that FTS inhibits methylation of the
21-26-kDa proteins. The degree of inhibition was, however,
relatively low (30-40%), and even lower (10-20%) when the
cells were grown for 3 days in the presence of 50 µM FTS
and then labeled with
[methyl-
H]methionine (not shown).
Figure 2:
Inhibition of methylation in intact cells
by FTS. Methylation was determined following metabolic labeling for 2 h
with [methyl-H]methionine in the absence
(
) and in the presence (
) of 100 µM FTS (see
``Experimental Procedures''). Cell homogenates were separated
by SDS-PAGE, and carboxylmethyl esters were determined in gel slices
corresponding to gel migration of 20-30 kDa. a, Rat1
cells; b, EJ cells; and c, B16
cells.
The
ability of FTS to inhibit carboxyl methylation of Ras in intact cells
was examined in
[methyl-H]methionine-labeled human
Ha-ras-transformed Rat1 (EJ) cells. As shown in Fig. 3a, FTS (100 µM) inhibited Ras
methylation by 40%. Labeling of EJ cells exposed to 50 µM FTS for 3 days gave similar results, except that Ras methylation
was inhibited by only 20-30%. Thus, FTS appears to be a weak
inhibitor of carboxyl methylation in intact cells. It therefore
probably does not affect processing steps that precede methylation of
prenylated proteins, such as
farnesylation(16, 19, 20, 23) .
Indeed, FTS (50 µM) did not inhibit farnesylation of
Ha-Ras in a reconstituted cell-free processing system (Fig. 3b). It did, however, partially inhibit Ras
methylation (Fig. 3c), a finding consistent with
competitive inhibition of the methyltransferase in cell-free systems (Fig. 1).
Figure 3:
Inhibition of Ras carboxyl methylation but
not of Ras farnesylation by FTS. a, effect of 100 µM FTS on Ras methylation in intact human Ha-Ras-transformed Rat1
cells. Ras proteins were immunoprecipitated from the cell lysates by
Y13-259 anti-Ras antibodies, following metabolic labeling with
[methyl-H]methionine as detailed in Fig. 2(see ``Experimental Procedures''). The
immunoprecipitated proteins were separated by SDS-PAGE, and
carboxylmethyl esters were determined in gel slices corresponding to
gel migration of 20-30 kDa. b, farnesylation of
bacterially expressed human Ha-Ras in a cell-free Ras processing
system. Partially purified farnesyltransferase (2 µg) and Ha-Ras
(2.5 µg) were incubated with 15 pmol of
[
H]farnesylpyrophosphate for 1 h at 37 °C in
the absence (
) and in the presence (
) of 50 µM FTS (see ``Experimental Procedures''). Proteins were
separated by SDS-PAGE, and gel slices were directly counted. c, inhibition of human Ha-Ras methylation in the cell-free Ras
processing system. Rat brain microsomal membranes (5 µg of protein)
and 110 pmol of [methyl-
H]AdoMet were
added to the reaction mixture that contained the farnesylated Ras.
Following 30 min of incubation at 37 °C in the absence (
) or
in the presence (
) of 50 µM FTS, the proteins were
separated by SDS-PAGE, and carboxylmethyl esters were determined in gel
slices (see ``Experimental
Procedures'').
In view of the fact that several prenylated
proteins apparently play important roles in cell growth and in
cytoskeleton-membrane
interactions(16, 17, 18, 19, 20) ,
it was reasonable to examine whether FTS can affect the growth and/or
morphology of cultured cells. Accordingly, a variety of cell types ( Table 1and Table 2) were grown under normal serum and
medium conditions for 5 days in the presence of the solvent (0.1%
MeSO) or of 25 µM FTS in the solvent (see
``Experimental Procedures''). Cell morphology was examined
under the light microscope, with cell counting and trypan blue
exclusion staining to detect dead cells and MTT staining to detect live
cells. A typical example of an experiment in which Rat1 cells were
stained with MTT is shown in Fig. 4, a-c, and the
estimated numbers of cells in the various cell types tested are
summarized in Table 1and Table 2. The MTT data indicated
that under the specified conditions, FTS was not toxic and did not
affect the growth of Rat1 (Fig. 4, a-c) or of the
other tested cell types, namely NIH3T3, CHE, Chinese hamster ovary,
PC12, and B16 cells, bovine capillary endothelial cells, mouse
cerebellum granular cells, and rat brain astrocytes. This was confirmed
by direct cell counting ( Table 1and Table 2). No gross
effects of FTS on cell morphology were detected in these cell types,
and trypan blue staining indicated that cell death in the presence of
25 µM FTS (5-10%) was similar to that observed in
controls.
Figure 4:
FTS is not cytotoxic in Rat 1 or EJ cells.
Rat1 (a-c) or EJ (d-f) cells were plated
at a density of 2 10
/well in the absence and in the
presence of the indicated concentrations of FTS. The cells were either
stained with MTT on the day of plating (day 0 in c and f) or grown for 1, 3, and 5 days and then stained with MTT.
Typical photomicrographs of 5-day cultures of Rat1 (a, b) and of EJ (d, e) are shown (magnification
100
). Quadruplicate samples were used for the spectrophotometric
determination (A
, A
) of
MTT-stained cells, in control wells (openbars), and
drug-treated wells (hatchedbars). Their mean values
(±S.D.) as determined on days 0, 1, and 3 in cultures of Rat1 (c) and EJ (f) are
presented.
We therefore conclude that FTS is not cytotoxic and would probably have no effect in vitro on the unstimulated growth and morphology of a variety of non-transformed cells or of some types of transformed cells such as the pheochromocytoma PC12 and melanoma B16 cells. In contrast, FTS had a profound effect on the growth of Rat1 cells transformed by the human Ha-ras oncogene (EJ). At concentrations lower than required for inhibition of Ras methylation, FTS caused a dose-dependent (0.1-10 µM) and time-dependent (3-10 days) inhibition of cell growth in EJ cells (Fig. 5a). Cultures grown for 5 days in the presence of 5 µM FTS had fewer and smaller foci than control cultures, and most cells had the flat morphology reminiscent of non-transformed Rat1 cells (Fig. 5b).
Figure 5:
Inhibition of EJ cell growth by FTS. a. Ha-Ras-transformed Rat1 (EJ) cells () or
untransformed Rat1 cells (
) were plated at a density of 2
10
cells/well in 24-well plates and grown in the presence
of the solvent (0.1% Me
SO) or the indicated FTS
concentration. Quadruplicate cultures of solvent or drug-treated cells
were maintained in culture for 3, 7, or 10 days and then collected from
the plates and counted. Data are expressed as the ratio between the
number of cells in the drug-treated cultures and the number in the
solvent-treated cultures. The actual cell numbers
(
10
) on days 3, 7, and 10 were 1.7, 29, and 55 for
the solvent-treated Rat1 cultures and 2.5, 42, and 140 for the
solvent-treated Ras-transformed Rat1 cultures. Results shown are from
one of five experiments with similar results. Within each experiment,
the means varied by 15% or less. Variations between experiments yielded
estimates varying between 55 and 78% (maximal inhibition of cell
growth). b, typical photomicrographs (magnification
100
) of Rat1 and Ha-Ras-transformed Rat1 cells grown for 5 days
in the presence of solvent or 50 µM FTS or 5 µM FTS.
The change in EJ cell morphology developed rapidly and was apparent already 24 h to 48 h after drug treatment (Fig. 6). Reversibility of the effect of FTS on EJ cell growth was evident in experiments in which the cells were exposed to 25 µM FTS for 48 h, then washed and replated in a drug-free medium. The number of cells in the replated cultures (determined 5 days after replating) presented 90 ± 7% (n = 4) of controls. These results are also consistent with lack of FTS-induced cytotoxicity in EJ cells.
Figure 6:
FTS alters the morphology of EJ cells. EJ
cells were plated at a density of 2 10
cells/well
in 24-well plates and 24 h later received either 0.1% Me
SO
(control) or 25 µM FTS. The cells were then
photomicrographed either 24 h (a and b) or 48 h (c and d) following the
treatments.
Fig. 4, d and e, shows MTT-stained EJ cells that were grown
in the absence and in the presence of 10 µM FTS. Despite
the reduction in cell number, most of the EJ cells were stained by MTT.
Trypan blue staining confirmed that FTS did not cause death of the EJ
cells. Thus, the observed reduction in the spectrophotometrically
determined MTT staining of the FTS-treated EJ cultures (Fig. 4f) probably reflects the reduced number of cells
and not cell death. In separate experiments, it could be demonstrated
that [H]thymidine incorporation was reduced by
55% when EJ cells were grown for 24 h in the presence of 25 µM FTS.
While FTS (up to 50 µM) had no effect on the growth or morphology of non-transformed Rat1 cells ( Fig. 4and Fig. 5), it did block their mitogenic response to basic fibroblast and epidermal growth factors (Table 2). We conclude that FTS can affect growth signaling either when Ras is activated transiently through a tyrosine-kinase growth factor-receptor pathway (39) or when it is activated constitutively. This conclusion was supported by experiments showing that the growth of NIH3T3 cells transformed by the Ras-activating tyrosine-kinase ErbB2 oncogene(40) , and of human endometrial carcinoma HEC1A cells that express activated K-Ras(34) , was inhibited by FTS (Table 2). In contrast, growth of cells transformed by the v-Raf oncogene, whose normal cellular form c-Raf1 is recruited by Ras (41, 42) to the plasma membrane where it is activated (43) , or of cells transformed by the nuclear T-antigen oncogene(31) , was not affected by up to 25 µM FTS (Table 2).
Taken together, the results suggest that FTS may
directly affect the membrane anchorage of Ras and thereby interfere
with Ras-Raf communication. This implies that FTS alters the
distribution of cellular Ras. To test this possibility, EJ cells were
exposed to 25 µM FTS for 12 h, and the amounts of Ras
present in the particulate (P) and in the cytosolic
(S
) fractions of the cells were determined by
immunoprecipitation combined with immunoblotting (see
``Experimental Procedures''). In agreement with earlier
studies(19) , Ras was found to be localized predominantly to
the P
fraction of the untreated
Ha-ras-transformed cells (Fig. 7). In contrast, most of
the Ras in the FTS-treated cells was localized to the S
fraction (Fig. 7).
Figure 7:
Cellular distribution of Ras proteins in
FTS-treated EJ cells. EJ cells were plated at a density of 2
10
cells/75-cm
flask and received 24 h later
either 0.1% Me
SO (control) or 25 µM FTS. 12 h
later, Ras proteins were immunoprecipitated by antibody Y13-259
from 150 µg of protein of the P
fractions and from
the equivalent amount of protein of the S
fractions of
the cells. The immunoprecipitated proteins were then subjected to
SDS-PAGE followed by immunoblotting and ECL as detailed under
``Experimental Procedures.'' The film was exposed 5
min.
The results of this study indicate that the farnesylated
rigid carboxylic acid derivative FTS is a potent competitive inhibitor
of the PPMTase (K = 2.6 µM) in
cell-free systems. In such systems, FTS can inhibit methylation of
added synthetic substrates, such as AFC and added Ras, and methylation
of endogenous proteins.
Unlike in cell-free systems, in intact cells FTS is a relatively weak inhibitor of methylation. It is possible that FTS crosses the plasma membrane with low efficacy, perhaps due to its carboxyl moiety. Alternatively, the PPMTase in intact cells, unlike the enzyme in broken cells, may be partially protected from interactions with exogenously added farnesyl derivatives. Other possibilities, such as cell-derived formation of inactive FTS metabolites or induction of PPMTase, cannot be ruled out. Whatever the cause, it is clear that FTS may be a useful tool for studies of PPMTase in cell-free systems but not for studies of methylation of prenylated proteins in intact cells. To achieve significant inhibition of methylation of such proteins in the cells, concentrations higher than 100 µM would be required. This, however, could be an advantage for studying interactions at relatively low FTS concentrations with farnesylcysteine recognition domains other than the one known in the PPMTase.
Such recognition domains for farnesylcysteine are likely to be critical for the activity of Ras oncoproteins, since their carboxyl-terminal farnesylcysteine, but not in its carboxymethylated form(44) , is essential for membrane anchorage and transforming activity(10, 19, 23) . Similarities between farnesylysteine domains in Ras targets and in the PPMTase may therefore allow farnesylcysteine mimetics independently to inhibit both carboxylmethylation and Ras-dependent cell growth. This is demonstrated here with FTS, which inhibited the growth of Ha-ras-transformed Rat1 cells and reversed their transformed morphology at concentrations (0.1-10 µM) lower than those required to inhibit protein methylation. FTS is therefore one example of a farnesyl derivative that may actually bind to a distinctive farnesylcysteine recognition domain with a higher affinity than its affinity for the PPMTase. This possibility is strengthened by the demonstrated effect of FTS on Ras localization (Fig. 7) and by recent results obtained in a related study with human platelets(24) . In that case, it was demonstrated that the PPMTase substrate, AFC, and farnesylcysteine derivatives that do not interact with the enzyme can all inhibit platelet aggregation(24) .
The possibility that aside from inhibiting Ras membrane association, FTS may also affect the interaction of proteins other than Ras with their own targets cannot be ruled out. In such a case, parameters other than Ras signaling would be affected by FTS. Additional experiments are required to test this possibility and to examine whether, for example, the effects of FTS on cell morphology and on cell growth are separable, as in the case of the farnesyltransferase inhibitor L-739,749(45) . The latter was found to induce morphological reversion of ras-transformed Rat1 cells and to inhibit their anchorage-independent growth by a mechanism that is unrelated to inhibition of Ras processing yet involves regulation of the actin cytoskeleton(45) . Inhibition of the anchorage-dependent growth by L-739,749 appears, however, to correlate with the inhibition of Ras processing(45) . Because the effects of FTS on EJ cell morphology and on Ras localization are both relatively fast (apparent within 24 h or less) and because the inhibition of DNA synthesis by FTS is also detected after 24 h of treatment, we cannot tell whether or not the effects of FTS on cell morphology and on cell growth are separable.
The lack of FTS toxicity at concentrations that effectively inhibit Ras-dependent cell growth may be attributable to its low efficacy in inhibiting the processing of prenylated proteins in intact cells. In addition to Ras, many of the proteins involved in regulatory mechanisms in mammalian cells are prenylated. In those that undergo both prenylation and methylation, prenylation occurs first(16, 17, 18, 19, 20, 23, 44) . Therefore, despite its ability to inhibit methyltransferase in vitro, FTS probably does not affect processing steps that precede methylation in intact cells, and as shown here, even Ras methylation is only weakly affected. Our results then suggest that the consequence of FTS action is a diminution in the levels of Ras in the membranes with a concomitant accumulation of fully processed Ras in the cytosol.
In this respect, the action of FTS is in sharp contrast to the actions of farnesyltransferase inhibitors(29, 46, 47) , which inevitably prevent all of the covalent modifications in the Ras CAAX box (C = cysteine, A = aliphatic and, X = any amino acid). It is not unlikely that FTS, and perhaps other farnesyl derivatives of rigid carboxylic acids(26) , interact specifically with Ras farnesylcysteine anchorage sites. High-affinity binding of FTS to such sites would probably affect proper anchoring of Ras in the plasma membrane and thereby disrupt the Ras-dependent activation of c-Raf1. Indeed, our results suggest that FTS affects membrane-anchorage of Ras and inhibits the growth signaling of tyrosine kinases (epidermal and basic fibroblast growth factor receptors and Erb-B2), which use Ras-Raf1 to recruit the mitogen-activated protein kinase cascade(39, 40, 43) . This and the lack of an effect on cells transformed by the constitutively activated v-Raf strongly suggest that FTS indeed interferes with tyrosine kinase-activated Ras-Raf communication. In this sense, the consequences of FTS actions would be similar to those of CAAX farnesyltransferase inhibitors. Indeed, neither farnesyltransferase inhibitors (29, 46) nor FTS affected the growth of untransformed fibroblasts grown in the absence of tyrosine kinase-receptor ligands. We report here that the unstimulated growth of cell types other than fibroblasts is also not affected by FTS. Together, our results suggest that FTS can selectively affect Ras-dependent cell growth but not Ras-independent cell division.
It is important to note, however, that the CAAX farnesyltransferase inhibitor BZA-5B does not block the epidermal growth factor-induced activation of the mitogen-activated protein kinase cascade in Rat1 cells(47) . This would imply that BZA-5B, unlike FTS, does not block the growth factor's mitogenic effect. Two alternative explanations have been proposed for the observed resistance of Rat1 cells to BZA-5B(47) . The one assumes that this drug does not affect farnesylation of endogenous K- or N-Ras and the other that the cells may use alternative, Ras-independent signaling pathways. FTS is not expected to be selective toward one or other types of Ras, as it only mimics the common structure of their farnesylcysteine. Therefore, usage of Ras-independent pathways is probably the more likely explanation for the resistance of unstimulated Rat1 cells to FTS. Because of the different actions of FTS and CAAX farnesyltransferase inhibitors, it will be interesting to examine whether these two types of inhibitors of Ras oncoprotein-dependent cell growth can act synergistically. Our results suggest potential uses for FTS and its analogs (26) in the characterization of farnesylcysteine recognition domains and in the design of treatments of Ras-dependent cancers.