The inhibition of Ras farnesylation leads to an increase
in p27
Kip1
and G1 cell cycle arrest
Hadas Reuveni*, Shoshana Klein and Alexander Levitzki
Department of Biological Chemistry, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel
HR12 is a novel farnesyltransferase inhibitor (FTI). We have
shown previously that HR12 induces phenotypic reversion
of H-ras
V12
-transformed Rat1 (Rat1/ras) fibroblasts. This
reversion was characterized by formation of cell–cell con-
tacts, focal adhesions and stress fibers. Here we show that
HR12 inhibits anchorage independent and dependent
growth of Rat1/ras cells. HR12 also suppresses motility and
proliferation of Rat1/ras cells, in a wound healing assay.
Rat1 fibroblasts transformed with myristoylated H-ras
V12
(Rat1/myr-ras) were resistant to HR12. Thus, the effects of
HR12 are due to the inhibition of farnesylation of Ras. Cell
growth of Rat1/ras cells was arrested at the G1 phase of
the cell cycle. Analysis of cell cycle components showed that
HR12 treatment of Rat1/ras cells led to elevated cellular
levels of the cyclin-dependent kinase inhibitor p27
Kip1
and
inhibition of the kinase activity of the cyclin E/Cdk2
complex. This is the first time an FTI has been shown to lead
to a rise in p27
Kip1
levels in ras-transformed cells. The data

ability to completely reverse the transformed phenotype of
oncogenic H-Ras-transformed Rat1 (Rat1/ras) fibroblasts.
This reversion entailed the assembly of adheren junctions,
concomitant with induction of cadherin and b-catenin.
Focal adhesions and actin stress fibers were formed, and the
overall cell morphology was indistinguishable from that of
nontransformed Rat1 cells.
Cell adhesion affects cell growth and invasion. Cadherin,
the primary cell–cell adhesion molecule, acts as a suppressor
of cancer cell invasion [7,8], and the loss of cadherin function
is required for tumor progression in vivo [9,10]. Moreover,
the activation or overexpression of cadherin has been shown
to arrest cell growth at the G1 phase, following an increase in
the p27
Kip1
level and dephosphorylation of the retino-
blastoma protein (pRb) [11,12]. The present report shows
that HR12 inhibits anchorage dependent and independent
growth of Rat1/ras cells, and suppresses motility and
proliferation in an in vitro ‘wound healing’ assay. We further
show that HR12 arrests Rat1/ras cells at the G1 phase of the
cell cycle, following up-regulation of the cell cycle inhibitor
p27
Kip1
and down-regulation of the kinase activity of the
cyclin E/cyclin-dependent kinase-2 (Cdk2) complex.
Progression of mammalian cell division through the cell
cycle is governed by the sequential formation, activation
and subsequent inactivation of Cdk complexes [13]. The
activation of Cdks depends upon multiple levels of regula-

p27
Kip1
and p57
Kip2
) with broader specificity [15]. Over-
expression of the CKIs causes G1 arrest [15,17–21].
Ras plays a central role in integrating mitogenic signals
and cell cycle progression. Interference with normal Ras
function by injection of anti-Ras Igs or by the expression of
the dominant negative (DN) mutant, Ras
N17
,blocksthe
proliferation of NIH3T3 cells [22–24]. In particular, Ras
was shown to control cell cycle progression at the early G1
stage by induction of cyclin D1, and to control the
progression and passage through the restriction point at
late G1, by down-regulation of the Cdk inhibitor p27
Kip1
[25–27]. Expression of DN-Ras
N17
in fibroblasts caused
p27
Kip1
accumulation, resulting in suppression of Cdk
activities and G1 arrest [26,28]. Oncogenic Ras-transformed
epithelial and fibroblast cells were shown to express reduced
levels of p27
Kip1
protein [29]. p27
Kip1

Cip1
levels was mediated by the inhibition
of non-Ras farnesylated proteins [32,34,35]. For the first
time, we report here on an FTI that causes p27
Kip1
levels
in Rat1/ras cells to be elevated in a Ras-dependent
manner, resulting in inhibition of the kinase activity of the
cyclin E/Cdk2 complex. We suggest that this is the
mechanism by which HR12 suppresses proliferation and
motility and arrests Rat1/ras cell growth at the G1 phase
of the cell cycle.
Experimental procedures
Materials and cell cultures
All cell lines were maintained and treated in growth medium
[Dulbecco’s Modified Eagles Medium (DMEM) containing
10% fetal bovine serum (Biological Industries Bet-Haemek
Ltd, Israel)]. Rat1/myr-ras cells were maintained under
G418 selection. Rat1 and Rat1/ras cells were described
previously [6]. Rat1/myr-ras cells [36] were kindly provided
by Yoel Kloog (Tel-Aviv University, Israel). HR12 was
synthesized and purified as described before [5,6].
Anchorage-dependent and independent cell growth
assays
Colony formation in soft agar was performed essentially
as described previously [37]. A suspension of separated
Rat1/ras or Rat1/myr-ras cells was plated in agar at a
density of 5 000 cells per well in a 96-well plate in growth
medium containing 0.3% agar (50 lL per well), on top of
a layer of growth medium containing 1% agar (100 lL

wound width was measured at various time points.
Cell cycle analysis
Rat1/ras and Rat1/myr-ras were grown to subconfluence in
growth medium in the presence or absence of 20 l
M
HR12
for 48 h. In the last 30 min of treatment, the cells were
exposed to 10 l
M
bromodeoxyuridine (BrdU; Amersham),
followed by harvesting and fixation in 70% ethanol. The
cells were stained with fluorescein isothiocyanate (FITC)-
conjugated anti-BrdU Ig (Dako, Denmark) and propidium
iodide (PI, Sigma) as described before [38]. A total of 10 000
stained cells were analysed in a fluorescence-activated cell
sorter.
Immunostaining
Immunostaining was conducted as described previously [6].
Cells were plated on coverslips in DMEM containing 10%
FBS and maintained at 37 °Cwith5%CO
2
.Afterseeding
(24 h), the medium was replaced with medium containing
20 l
M
HR12. Twenty four hours later, the medium was
again replaced with fresh medium containing 20 l
M
HR12.
Following 48 h of exposure to HR12, cells were fixed at

Cells were treated with HR12 at various concentrations
in growth medium for 48 h, and lysed in Laemmli sample
buffer (50 m
M
Tris/HCl, pH 6.8, 5% 2-mercaptoethanol,
3% SDS and 0.5 mgÆmL
)1
bromophenol-blue). Aliquots
of cell extracts containing equal amounts of protein were
resolved by SDS/PAGE and electroblotted onto nitrocellu-
lose filters. The membranes were blocked with lowfat milk
diluted 1 : 20 in NaCl/Tris containing 0.2% Tween-20
(blocking solution), incubated with primary Igs overnight at
4 °C, and then with horseradish peroxidase-conjugated
secondary antibodies for 75 min at room temperature.
Immunoreactive bands were visualized using enhanced
chemiluminescence, and quantified using
NIH
-
IMAGE
1.61
program ( Each experi-
ment was repeated at least two times. Each figure shows a
representative blot, and its corresponding
NIH
-
IMAGE
ana-
lysis. Arbitrary values are shown, except where otherwise
stated.

orthovanadate, 20 m
M
2-glycerophosphate, 50 m
M
sodium
fluoride, 5 m
M
sodium pyrophosphate, 10 lgÆmL
)1
soy-
bean trypsin inhibitor, 10 lgÆmL
)1
leupeptin, 1 lgÆmL
)1
aprotonin, 313 lgÆmL
)1
benzamidine, 0.2 m
M
4-(2-amino-
ethyl)-benzenesulfonylfluoride (AEBSF) and 0.1% 2-merca-
ptoethanol. Lysates were centrifuged at 19 000 g for 10 min
and supernatants were subjected to immunoprecipitation.
For each sample, 75 lL of 10% protein A–Sepharose were
incubated for 1 h at 4 °Cwith2lg of either anti-(cyclin E)
(M-20), anti-Cdk2 (M-2), anti-(cyclin D1) (72–13G) or anti-
Cdk6 (C-21) Igs. Antibodies used for immunoprecipitation
were purchased from Santa Cruz Biotechnology. Super-
natants (500 lg of each) were incubated with the Ig-coupled
protein A for 1 h at 4 °C. As negative controls, Igs were
‘blocked’ by the inclusion of 2 lg of blocking peptide during

PhosphorImager screen to measure intensity of the
32
P-
labelled substrates, and then blocked with blocking
solution and immunoblotted with antibodies against the
immunocomplex components (as described in Immuno-
blotting).
Metabolic labeling
Rat1/ras cells were cultured in 60 mm Petri dishes
(120 000 cells per dish). The medium was replaced with
fresh medium every 24 h. HR12 (20 l
M
) was added to
the relevant samples 24 h after the cells were plated.
Following 48 h exposure to HR12, the plates were
washed three times with NaCl/P
i
. Starvation medium
[dialysed FBS (10%) in medium lacking both methionine
and cysteine; Biological Industries Beth HaEmek], with
HR12 in the relevant samples, was added for 1 h.
35
S-Met/Cys Promix (200 lCiÆmL
)1
; Amersham-Pharma-
cia) was then added. N-acetyl-leucyl-leucyl-norleucynal
(LLnL) (50 l
M
) was added to the appropriate samples.
After 3 h exposure to

M
). This effect was selective, as the
growth of the parental nontransformed Rat1 cells was not
Ó FEBS 2003 FTI-induced p27Kip1 increase and G1 arrest (Eur. J. Biochem. 270) 2761
affected at all by HR12 up to a concentration of 25 l
M
,
and only a minor effect of 50 l
M
HR12 was observed
(Fig. 1B).
HR12 treatment of Rat1/ras cells suppresses
in vitro
monolayer ‘wound healing’
We then tested whether HR12 treatment of Rat1/ras cells
suppresses the ability of cells present at the edges of a
‘wounded’ Rat1/ras monolayer to move out of the layer and
‘repair’ the wound. This assay characterizes the proliferative
and motility potentials of the cells, both of which are
suppressed by cell–cell contacts. Figure 2 shows that while
Rat1/ras cells rapidly repaired the wound, HR12 treatment
dramatically suppressed this process.
HR12 induces arrest of Rat1/ras cells at the G1 phase
of the cell cycle
We next examined the effect of HR12 on the distribution
of Rat1/ras cells in the cell cycle. To resolve the G1, S and
G2/M phases, we double-labelled the cells with BrdU and
propidium-iodide, as described in Experimental procedures.
Figure 3 shows that HR12 treatment of Rat1/ras cells
induced G1 arrest, concomitant with a 25% reduction in

Kip1
levels
and to a decrease in pRb phosphorylation
in a dose-dependent manner
To analyse the cell cycle components affected by HR12
treatment, we prepared whole cell lysates of Rat1/ras cells
that had been exposed to HR12 at various concentrations
for 48 h. Immunoblotting with Igs against cell cycle
components led to several interesting findings. First, the
levels of the Cdk-inhibitor p27
Kip1
increased upon HR12
treatment in a dose-dependent manner (Fig. 5). Second, the
levels of the Cdk-inhibitor p21
Cip1
dropped (Fig. 5). The
levels of the Cdk-inhibitor p16
INK4A
were also examined,
Fig. 1. Inhibition of anchorage independent and dependent growth of
Rat1/ras cells by HR12. (A) Rat1/ras cells were grown in a layer of
0.3% soft agar in a 96-well plate, and exposed to HR12 at the indicated
concentrations, in triplicate. After 7 days, the colonies were stained
with MTT and photographed. Quantification was performed by
extraction of the color and measurement of the absorbance at 570 nm.
(B) HR12 selectively inhibited the growth of Rat1/ras cells, without
affecting the growth of nontransformed Rat1 cells. Rat1 and Rat1/ras
cells were grown in monolayers in 96-well plates, and exposed to HR12
at the indicated concentrations. Three days later the cells were har-
vested and counted.

replaced every 24 h. Quantification of the wound width vs. time is
presented.
DNA incorporation (BrdU)
DNA content (PI)
no treatment
HR12
S
S
G1
G2/M
G1
G2/M
% of Rat1/ras population
G1
S
G2/M
0
10
20
30
40
50
60
70
no treatment
HR12
Fig. 3. HR12 induces G1 arrest of Rat1/ras cells. Rat1/ras cells were
treated with 20 l
M
HR12 for 48 h, exposed to BrdU for 30 min,

cells treated with HR12 is much longer (> 240 min) than the
t
1/2
of p27
Kip1
in untreated Rat1/ras cells (< 100 min). Thus,
HR12 leads to stabilization of the p27
Kip1
protein.
To examine whether HR12 also affects the rate of
expression and/or synthesis of p27
Kip1
, we blocked protea-
some-mediated proteolysis by using LLnL, an inhibitor of
the chymotryptic site on the proteasome [42,43]. Rat1/ras
cells were treated with 20 l
M
HR12 for 48 h and 50 l
M
LLnL was added to the cell medium for the last 3 h of
treatment. Figure 6B shows that p27
Kip1
levels in the cell
lysate increased 2.5-fold as a result of LLnL treatment. This
result confirms the essential role of the proteasome in p27
Kip1
down-regulation in Rat1/ras cells. There was no significant
difference between the amount of p27 synthesized after
addition of LLnL when HR12 was absent (D1inFig.6B)
and the amount of p27 synthesized after addition of LLnL

We next examined whether G1 phase cyclin-dependent
kinase activity is affected by the elevation in cellular p27
Kip1
levels. Rat1/ras cells were treated with 20 l
M
HR12 for 24
and 48 h, and lysates immunoprecipitated with anti-
(cyclin E) (Fig. 7A) or anti-Cdk2 (Fig. 7B). Kinase activity
of the cyclin E/Cdk2 complex was measured using his-
tone H1 and [c
32
P]ATP as substrates for the anti-(cyclin E)
immunoprecipitates. The mixtures were separated using
SDS/PAGE, blotted onto a nitrocellulose filter and exposed
to a PhosphorImager screen to quantify the levels of phos-
phorylated histone H1. The levels of the components of the
immunocomplex were probed by immunoblotting the same
blot with the relevant antibodies, as described in Experimen-
tal procedures. The kinase activity of the cyclin E/Cdk2
complex was significantly inhibited in Rat1/ras cells treated
with HR12. Furthermore, the levels of Cdk-inhibitor p27
Kip1
bound to the cyclin E/Cdk2 immunocomplexes in HR12-
treated cells were at least three- to fourfold higher than those
of p27
Kip1
bound to the cyclin E/Cdk2 immunocomplexes in
untreated cells (Fig. 7A). Correspondingly, Fig. 8B shows
that the p27
Kip1

0
20
40
60
80
100
1 3 5 152448wash
hours of exposure to HR12
Fig. 4. The time course of the inhibition of Ras processing by HR12
correlates with the hypophosphorylation of pRb. Rat1/ras cells grown in
medium containing 10% FBS were treated with 20 l
M
HR12 for the
indicated time periods, or exposed to 20 l
M
HR12 for 48 h, washed,
and incubated without the inhibitor for 24 h longer, before lysis
(wash). Lysates were immunoblotted with anti-Ras and anti-phospho-
Ser795-pRb (p-pRb) Igs. (up) Unprocessed Ras, (p) processed Ras.
The upper graph shows the levels of processed Ras, as a percentage of
total Ras, over the course of HR12 treatment. The lower graph shows
levels of pRb phosphorylation, compared to the untreated sample at
thesametimepoint.
2764 H. Reuveni et al. (Eur. J. Biochem. 270) Ó FEBS 2003
complexes, immunoprecipitated by anti-Cdk6 Ig, from
lysates of untreated and HR12-treated Rat1/ras cells, were
assayed using GST-pRb and [c
32
P]ATP as substrates. The
immunocomplex components were visualized by immuno-

no effect. It did not change the cell cycle distribution
(Fig. 9A). It had a minor effect on cell-growth in soft agar at
concentrations up to 25 l
M
(Fig. 9B). The IC
50
of growth
inhibition in soft agar was about sevenfold higher for Rat1/
myr-ras cells than for Rat1/ras cells. We have shown
previously that HR12 induces the assembly of adheren
junctions labelled with b-catenin and complete morpho-
logical reversion of Rat1/ras cells ([6] and Fig. 9C). In the
Rat1/myr-ras cells, HR12 had no effect on b-catenin
distribution within the cells, as measured by immunostain-
ing (Fig. 9C). Moreover, no morphological change of Rat1/
myr-ras cells was induced by HR12 treatment (Fig. 9C).
HR12 did not affect the rate of ‘wound healing’ of Rat1/
myr-ras cells (Fig. 9D), in contrast to its suppressive effect
on Rat1/ras cells (Fig. 2). Finally, HR12 was not found to
affect p27
Kip1
levels, pRb phosphorylation or cyclin D1
levels in Rat1/myr-ras cells (Fig. 9E).
Discussion
HR12 effects are mediated by Ras inhibition
The inhibition of farnesyltransferase was developed origin-
ally as a strategy to block oncogenic Ras function.
Nonetheless, the actual target of FTIs is a matter of
controversy [44]. We have reported recently on the devel-
opment of a novel FTI, HR12 [5]. We have shown that

Cyclin E
0
50
100
150
0 0.5 1.5 4.5 13 40
Cyclin E
0
50
100
150
p27
Kip1
0 0.5 1.5 4.5 13 40
0
50
100
p-pRb/pRb
0 0.5 1.5 4.5 13 40
pS795-pRb
pRb
M HR12
0
0.5
1.5
4.5
13
40
p27
Kip1

levels or pRb phosphorylation in Rat1/
myr-ras cells (Fig. 9E). Lastly, Rat1/myr-ras cells were
much less sensitive to HR12 than Rat1/ras cells in a soft
agar assay (Fig. 9B). Resistance to HR12 was also seen with
NIH3T3 fibroblasts transformed by myr-ras, unlike
NIH3T3 cells transformed by farnesylation-dependent
oncogenic ras (data not shown). Thus, the effects of
HR12 on the proliferation, motility, cytoskeletal rearrange-
ment and morphology of Rat1/ras cells are mediated
through the inhibition of Ras farnesylation.
P27
Kip1
inhibition of Cdk2 mediates HR12-induced G1
arrest
We show that HR12 treatment leads to accumulation of
Rat1/ras cells in G1, with a corresponding reduction in the
number of S phase cells (Fig. 3). It has been shown that Ras
controls progression through the late G1 phase of the cell
cycle by controlling the levels of p27
Kip1
[25–27]. Treating
Rat1/ras cells with HR12, we saw a strong correlation
HR12-treated Rat1/ras cells
Untreated Rat1/ras cells
A
p27
Kip1
actin
0
50

LLnL: - - + +
HR12: - + - +
0
2
4
6
8
∆2
∆1
Fig. 6. HR12 enhances the half-life of p27
Kip1
protein, with no effect on
its synthesis rate. (A) HR12 leads to stabilization of the p27
Kip1
protein.
Rat1/ras cells were treated with 20 l
M
HR12 for 48 h, followed by the
addition of 100 l
M
cycloheximide (chx) to the cell medium. Lysates
were prepared at the indicated time periods after chx addition, and
immunoblotted with anti-p27
Kip1
Ig and with anti-actin Ig as a control.
The diagram shows quantification of the intensity of the p27
Kip1
bands, calibrated to the intensity of the actin bands, where the zero
time value was designated 100%. (B, C) HR12 does not affect the
synthesis rate of p27

Cdk4 and Cdk6, but their kinase activities were not
inhibited (Fig. 8). This result is not surprising, for while
p27
Kip1
functions as an inhibitor of cyclin E/Cdk2, it also
plays a role in the assembly and activation of the cyclin D/
Cdk4 and cyclin D/Cdk6 complexes [46–48].
One of the best-characterized substrates of the Cdk
enzymes is the retinoblastoma protein (pRb). Hypophos-
phorylated pRb binds target proteins and arrests cells in the
G1 phase of the cell cycle. This arrest is relieved by Cdk-
mediated hyperphosphorylation of pRb, which in turn
promotes the expression of factors that are essential for cell
cycle progression. Treatment of Rat1/ras cells with HR12
led to a decrease in pRb phosphorylation (Fig. 5). There
was a good correlation between the inhibition of Ras-
processing and of pRb dephosphorylation, in terms of both
kinetics and dose-responsiveness (Figs 4 and 5).
Our data contrast with those of Du et al. [32,49], who
reported that their FTI led to an increase in p21
CIP1
levels, in
the same Rat1/ras model we used. These authors, who did
not report any effect on p27
Kip1
, attribute the increase in
p21
CIP1
to the increase in geranylgeranylated RhoB caused
by inhibition of RhoB farnesylation (‘FTI-RhoB Hypothe-

pathway [29], the PI3K pathway [26] or the Rho pathway
[52] and (c) repression of p27
Kip1
transcription through the
activation of the PI3K/PKB pathway, which prevents
the forkhead transcription factors from translocating to
the nucleus [41].
The PI3K/PKB pathway is unlikely to be responsible for
the observed increase in p27
Kip1
levels, as treatment of Rat1/
ras cells with HR12 for 48 h led to activation (rather than
Fig. 7. HR12 treatment of Rat1/ras cells leads to an increase in the level
of p27
Kip1
in the Cyclin E/Cdk2 complex and inhibition of cyclin E/Cdk2
kinase activity. (A) Rat1/ras cells were treated with 20 l
M
HR12 for 24
and 48 h. Cell lysates were prepared and immunoprecipitated with
polyclonal anti-(cyclin E) Ig. As a negative control, the anti-(cyclin E)
Ig was preincubated with a blocking peptide (BP). The immunopre-
cipitates were tested for kinase activity with histone-H1 as a substrate,
as described in Experimental procedures, followed by separation on
SDS/PAGE and blotting. The blot was exposed to a PhosphorImager
screen or to X-ray film to quantify kinase activity ([
32
P]-H1). To
visualize the levels of the individual proteins in the immunoprecipitates
the same blot was immunoreacted with monoclonal anti-(cyclin E),

proteasome pathway plays an essential role in p27
Kip1
degradation, and indeed the specific proteasome inhibitor,
LLnL, induced accumulation of p27
Kip1
protein in Rat1/ras
cells (Fig. 6B). Ras positively regulates RhoA [53], and
RhoA leads to cyclin E/Cdk2 activation [54]. The cyclin E/
Cdk2 complex phosphorylates p27
Kip1
at Thr187 and leads
it to degradation through the ubiquitin/proteasome path-
way [27,55,56]. There is a positive loop between p27
Kip1
protein and cyclin E/Cdk2 in which p27
Kip1
serves both as a
substrate and as an inhibitor of Cdk2. In summary, HR12
inhibits the degradation of the p27
Kip1
protein in Rat1/ras
cells, possibly via the Ras-to-RhoA pathway.
Is the increase in p27
Kip1
mediated by the induction
of cell–cell contacts?
p27
Kip1
levels are controlled by cadherin mediated cell–cell
contacts that are themselves regulated by Ras [6,57].

blots were probed with polyclonal anti-cyclin D1 or polyclonal anti-
Cdk6 and with monoclonal anti-p27
Kip1
antibodies.
Fig. 9. Resistance of Rat1/myr-ras cells to HR12. After 48 h treatment
with 20 l
M
HR12 in medium containing 10% FBS, Rat1/myr-ras cells
were (A) analysed for cell cycle distribution, (C) fixed and stained with
anti-(b-catenin), (D) subjected to a wound healing assay, or (E) lysed
and immunoblotted with antibodies against Ras, phospho-pRb (p-
pRb), pRb, p27
Kip1
and cyclin D1. The growth of Rat1/myr-ras cells in
soft agar was examined also (B). Rat1/myr-ras were resistant to HR12
effects, including suppression of ‘wound healing’, morphology rever-
sion, assembly of adherens junctions, G1 arrest, up-regulation of
p27
Kip1
and hypophosphorylation of pRb.
2768 H. Reuveni et al. (Eur. J. Biochem. 270) Ó FEBS 2003
inactivation of cyclinE/cdk2, dephosphorylation of pRb,
and G1 arrest of Rat1/ras cells [[6] and this study). This
raises the possibility that the HR12-induced increase in
p27
Kip1
levels might be the consequence of the induction
of cell–cell contacts, rather than the outcome of a signal
transduction pathway leading from Ras to p27
Kip1

pathway by Ras should lead to cyclin D1 stabilization.
Nonetheless, we detected no change in cyclin D1 levels
upon treatment of Rat1/ras cells with HR12 (Fig. 5). This
might be due to the opposite effects of the Mek/Erk and
the PI3K/PKB pathways: the former is strongly inhibited
by HR12, whereas the latter is induced (possibly as a
consequence of the assembly of adhesion sites) [6]. We
conclude that the inhibition of Ras processing by HR12
leads to cell cycle arrest through a p27
Kip1
-mediated
mechanism, with no obvious involvement of cyclin D1.
HR12 inhibits ‘wound healing’ of Rat1/ras cells
The in vitro monolayer ‘wound healing’ assay combines
aspects of cell proliferation and migration. Treatment of
Rat1/ras cells with HR12 leads to an increase in p27
Kip1
levels, whereas the induction of cell proliferation during
wound healing is accompanied by a decrease in p27
Kip1
levels
[62]. This may be one reason for the suppression of wound
healing by HR12. Furthermore, HR12 may also affect
wound healing by inhibiting cell migration. First, HR12
leads to the formation of cell–cell contacts that may interfere
with the freedom of the cells to move. Second, induction of
stress fibers may also reduce motility in fibroblasts [63]. The
sustained activation of the Mek/Erk pathway in Ras-
transformed fibroblasts leads to inhibition of ROCK and
Rho kinase, consequent loss of stress fibers, and enhanced

and found that it suppresses invasive growth and prolifer-
ation of Rat1/ras cells. Our data show conclusively that the
effects of HR12 are due to inhibition of Ras farnesylation.
HR12 appears to arrest ras-transformed fibroblasts speci-
fically, with minimal effects on nontransformed cells,
encouraging us to anticipate minimal toxic side-effects of
HR12. In light of the connection between loss of p27
Kip1
protein and metastasis and proliferation, agents such as
HR12 that lead to stabilization of p27
Kip1
protein could
serve as anticancer drugs with high antimetastatic and
antiproliferative potentials.
Acknowledgements
We would like to thank Sharon Simmer and Shirlee Lichtman for their
help with the manuscript and Prof. Benjamin Geiger for generous help
and advice.
References
1. Zhang, F.L. & Casey, P.J. (1996) Protein prenylation: molecular
mechanisms and functional consequences. Annu. Rev. Biochem.
65, 241–269.
2. Hancock, J.F., Magee, A.I., Childs, J.E. & Marshall, C.J. (1989)
All ras proteins are polyisoprenylated but only some are palmi-
toylated. Cell 57, 1167–1177.
3. Jackson, J.H., Cochrane, C.G., Bourne, J.R., Solski, P.A., Der
Buss,J.E.,Cox,A.D.,Hisaka,M.M.,Graham,S.M.,DerBuss,
J.E.&., C.J. (1992) Isoprenoid addition to Ras protein is the cri-
tical modification for its membrane association and transforming
activity. Proc. Natl Acad. Sci. USA 89, 6403–6407.

suppression is mediated by the cyclin-dependent kinase inhibitor
p27 (KIP1). J. Cell Biol. 142, 557–571.
13. Sherr, C.J. (1993) Mammalian G1 cyclins. Cell 73, 1059–1065.
14. Draetta, G.F. (1994) Mammalian G1 cyclins. Curr. Opin. Cell
Biol. 6, 842–846.
15. Sherr, C.J. & Roberts, J.M. (1995) Inhibitors of mammalian G1
cyclin-dependent kinases. Genes Dev. 9, 1149–1163.
16. Massague, J. & Roberts, J.M. (1995) Cell cycle 1995: constructing
cell physiology with molecular building blocks. Curr. Opin. Cell
Biol. 7, 769–772.
17. Guan, K.L., Jenkins, C.W., Li, Y., Nichols, M.A., Wu, X.,
O’Keefe, C.L., Matera, A.G. & Xiong, Y. (1994) Growth sup-
pression by p18, a p16INK4/MTS1- and p14INK4B/MTS2-rela-
ted CDK6 inhibitor, correlates with wild-type pRb function.
Genes Dev. 8, 2939–2952.
18. Coats, S., Flanagan, W.M., Nourse, J. & Roberts, J.M. (1996)
Requirement of p27Kip1 for restriction point control of the
fibroblast cell cycle. Science 272, 877–880.
19. Harper, J.W., Adami, G.R., Wei, N., Keyomarsi, K. & Elledge,
S.J. (1993) The p21 Cdk-interacting protein Cip1 is a potent
inhibitor of G1 cyclin-dependent kinases. Cell 75, 805–816.
20. Polyak,K.,Lee,M.H.,Erdjument-Bromage,H.,Koff,A.,Rob-
erts, J.M., Tempst, P. & Massague, J. (1994) Cloning of p27Kip1,
a cyclin-dependent kinase inhibitor and a potential mediator of
extracellular antimitogenic signals. Cell 78, 59–66.
21. Rivard, N., L’Allemain, G., Bartek, J. & Pouyssegur, J. (1996)
Abrogation of p27Kip1 by cDNA antisense suppresses quiescence
(G0 state) in fibroblasts. J. Biol. Chem. 271, 18337–18341.
22. Cai, H., Szeberenyi, J. & Cooper, G.M. (1990) Effect of a domi-
nant inhibitory Ha-ras mutation on mitogenic signal transduction

31. Sgambato, A., Cittadini, A., Faraglia, B. & Weinstein, I.B. (2000)
Multiple functions of p27 (Kip1) and its alterations in tumor cells:
areview.J. Cell Physiol. 183, 18–27.
32. Du, W., Lebowitz, P.F. & Prendergast, G.C. (1999) Cell growth
inhibition by farnesyltransferase inhibitors is mediated by gain of
geranylgeranylated RhoB. Mol. Cell Biol. 19, 1831–1840.
33. Ashar,H.,James,L.,Gray,K.,Carr,D.,McGuirk,M.,Maxwell,
E., Black, S., Armstrong, L., Doll, R., Taveras, A., Bishop, W. &
Kirschmeier, P. (2001) The farnesyl transferase inhibitor SCH
66336 induces a G (2) fi M or G (1) pause in sensitive human
tumor cell lines. Exp. Cell Res. 262, 17–27.
34. Lebowitz, P. & Prendergast, G. (1998) Non-Ras targets of
farnesyltransferase inhibitors: focus on Rho. Oncogene 17, 1439–
1445.
35. Prendergast, G. & Oliff, A. (2000) Farnesyltransferase inhibitors:
antineoplastic properties, mechanisms of action, and clinical
prospects. Cancer Biol. 10, 443–452.
36. Haklai, R., Weisz, M.G., Elad, G., Paz, A., Marciano, D., Egozi,
Y., Ben-Baruch, G. & Kloog, Y. (1998) Dislodgment and
accelerated degradation of Ras. Biochemistry 37, 1306–1314.
37. Horowitz, D. & King, A.G. (2000) Colorimetric determination
of inhibition of hematopoietic progenitor cells in soft agar.
J. Immunol. Methods 244, 49–58.
38. Resnitzky, D., Hengst, L. & Reed, S.I. (1995) Cyclin A-associated
kinase activity is rate limiting for entrance into S phase and
is negatively regulated in G1 by p27Kip1. Mol. Cell Biol. 15,
4347–4352.
39. Agrawal,D.,Hauser,P.,McPherson,F.,Dong,F.,Garcia,A.&
Pledger, W.J. (1996) Repression of p27kip1 synthesis by platelet-
derived growth factor in BALB/c 3T3 cells. Mol. Cell Biol. 16,

47. LaBaer, J., Garrett, M.D., Stevenson, L.F., Slingerland, J.M.,
Sandhu, C., Chou, H.S., Fattaey, A. & Harlow, E. (1997) New
functional activities for the p21 family of CDK inhibitors. Genes
Dev. 11, 847–862.
48. Cheng, M., Olivier, P., Diehl, J.A., Fero, M., Roussel, M.F.,
Roberts, J.M. & Sherr, C.J. (1999) The p21 (Cip1) and p27 (Kip1)
CDK ‘inhibitors’ are essential activators of cyclin
D
-dependent
kinases in murine fibroblasts. EMBO J. 18, 1571–1583.
Ó FEBS 2003 FTI-induced p27Kip1 increase and G1 arrest (Eur. J. Biochem. 270) 2771
49. Du, W. & Prendergast, G. (1999) Geranylgeranylated RhoB
mediates suppression of human tumor cell growth by farnesyl-
transferase inhibitors. Cancer Res. 59, 5492–5496.
50. Hengst, L. & Reed, S.I. (1996) Translational control of p27Kip1
accumulation during the cell cycle. Science 271, 1861–1864.
51. Hirai, A., Nakamura, S., Noguchi, Y., Yasuda, T., Kitagawa, M.,
Tatsuno, I., Oeda, T., Tahara, K., Terano, T., Narumiya, S.,
Kohn, L.D. & Saito, Y. (1997) Geranylgeranylated rho small
GTPase (s) are essential for the degradation of p27Kip1 and
facilitate the progression from G1 to S phase in growth-stimulated
rat FRTL-5 cells. J. Biol. Chem. 272, 13–16.
52. Vidal, A., Millard, S.S., Miller, J.P. & Koff, A. (2002) Rho activity
can alter the translation of p27 mRNA and is important for
RasV12-induced transformation in a manner dependent on p27
status. J. Biol. Chem. 277, 16433–16440.
53. Zohn, I.M., Campbell, S.L., Khosravi-Far, R., Der Rossman,
K.L.&.,.Bellone, C.J. & Baldassare, J.J. (1999) RhoA stimulates
p27 (Kip) degradation through its regulation of cyclin E/CDK2
activity. J. Biol. Chem. 274, 3396–3401.

63. Sahai, E., Olson, M.F. & Marshall, C.J. (2001) Cross-talk
between Ras and Rho signalling pathways in transformation
favours proliferation and increased motility. EMBO J. 20,
755–766.
2772 H. Reuveni et al. (Eur. J. Biochem. 270) Ó FEBS 2003