Báo cáo khoa học: The inhibition of Ras farnesylation leads to an increase in p27Kip1 and G1 cell cycle arrest pdf

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

suggest a new mechanism for FTI action, whereby in ras-
transformed cells, the FTI causes an increase in p27
Kip1
levels, which in turn inhibit cyclin E/Cdk2 activity, leading
to G1 arrest.
Keywords: farnesyl transferase inhibitor (FTI); p27
Kip1
;Ras;
cell cycle.
Localization of Ras proteins in the plasma membrane
follows a series of post-translational modifications [1] and is
crucial to the functioning of these proteins [2,3]. The first
and essential step in this process is farnesylation, whereby a
farnesyl group (C
15
-isoprenoid) is covalently attached to the
cysteine residue of the C-terminal CAAX sequence of Ras
[4]. Farnesylation is mediated by the enzyme farnesyltrans-
ferase (FT). The three C-terminal residues, AAX, are then
proteolytically cleaved and the new carboxy-terminus is
methylated. H-Ras, N-Ras and K-Ras4A are also palmi-
toylated on one or more upstream cysteine residues.
Mutationally activated ras genes are found in  30% of
all human cancers. As farnesylation is required for the
oncogenic activity of activated Ras, there has been much
interest in the development of FT inhibitors (FTIs) for
anticancer treatment.
We have developed an FTI, cysteine-N-methyl-valine-
N-cyclohexyl-glycine-methionine-methyl ester, called HR12
[5]. We have demonstrated recently [6] the compound’s

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-

tion: the synthesis of the cyclins and their assembly into
cyclin/Cdk complexes [14], the phosphorylation of the
Cdks, and the inhibitory action of the Cdk inhibitors (CKIs)
in these complexes [15,16]. CKIs identified in mammalian
cells are classified into two main categories: the INK4
Correspondence to A. Levitzki, Department of Biological Chemistry,
Institute of Life Sciences, The Hebrew University of Jerusalem,
Jerusalem, Israel 91904.
Fax: + 972 2 6512958, Tel.: + 972 2 6585404,
E-mail:
Abbreviations: Cdk, cyclin dependent kinase; CKI, Cdk inhibitor;
Erk, extracellular-signal regulated kinase; FT, farnesyltransferase;
FTI, farnesyltransferase inhibitor; LLnL, N-acetyl-leucyl-leucyl-
norleucynal; mAb, monoclonal antibody; MAPK, mitogen activated
protein kinase; Mek, MAPK kinase; PI3K, phosphatidylinositol-
3¢OH-kinase; PKB, protein kinase B; pRb, retinoblastoma protein;
Rat1/ras, H-ras
V12
-transformed Rat1 cell line; Rat1/myr-ras,
myristoylated H-ras
V12
-transformed Rat1 cell line.
*Present address: Keryx Biopharmaceuticals, PO Box 23706,
Jerusalem, Israel.
(Received 11 March 2003, revised 29 April 2003, accepted 1 May 2003)
Eur. J. Biochem. 270, 2759–2772 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03647.x
proteins, which bind to and specifically inhibit Cdk4 and
Cdk6 complexes [17], and the Kip/Cip inhibitors (p21
Cip1
,

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

is thus a key factor in
Ras regulation of progression through the late G1 phase
and through the restriction point, the latter being a
prerequisite for entry into the S phase.
Reduced expression of the p27
Kip1
protein has been
observed in a variety of human malignancies, and in
particular, the progressive loss of p27
Kip1
is commonly
observed during the progression from normal cells to
benign and malignant tumors. p27
Kip1
appears to play a
role in the switch from cell proliferation to differentiation,
and loss of p27
Kip1
is associated with a poorly differen-
tiated phenotype in several human malignancies, suggest-
ing that potentiation of p27
Kip1
might be a useful strategy
in cancer treatment (reviewed in [30,31]). FTIs, which
were designed as inhibitors of Ras localization in the
membrane, have been reported to elevate p21
Cip1
levels in
Rat1/ras cells [32,33]. It has been claimed that the
elevationinp21

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

per well). Growth medium (50 lL) supplemented with
HR12 at four times the indicated concentration was
added on top. Seven to nine days after plating, the cells
were stained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT; Sigma) and photographed.
The color developed by viable colonies was extracted by
the addition of 100 lL per well of solubilization buffer
containing 20% (w/v) SDS, 50% (v/v) dimethylforma-
mide, 2% (v/v) acetic acid and 0.25
M
HCl. Following
incubation of the plate at 37 °C overnight, absorbance at
570 nm was read in an ELISA Reader. The assays were
performed in triplicates.
For anchorage-dependent growth curves, Rat1 (1500
cells/well) and Rat1/ras (3300 cells/well) cells were plated on
96-well plates. One day after seeding, cultures were treated
with HR12 at various concentrations (triplicate samples
were made for each concentration) in growth medium.
Medium with and without HR12 was replaced after two
days. Cells were counted 96 h after seeding.
In vitro
monolayer ‘wound healing’ assay
Rat1/ras and Rat1/myr-ras were grown to confluence in
60 mm plates under growth medium, in the presence or
absence of 20 l
M
HR12. Monolayers were wounded using a
rubber policeman or a micropipette tip, and visualized using
a phase-contrast microscope. Pictures were taken and the

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

37 °C, as follows. Cells were washed once with NaCl/P
i
,
fixed and permeabilized in a solution containing 3%
paraformaldehyde, 50 m
M
Mes buffer pH 6, 0.5% Triton
X-100and5m
M
CaCl
2
, for 30 s, followed by 1 h incuba-
tion in the same solution without Triton. The fixed cells
were incubated with anti-(b-catenin) Ig (Transduction
Laboratories, dilution 1 : 20 in NaCl/P
i
)for30minat
2760 H. Reuveni et al. (Eur. J. Biochem. 270) Ó FEBS 2003
room temperature, washed three times with NaCl/P
i
and
incubated with the secondary antibody, Cy3-conjugated
goat anti-(mouse IgG) (Jackson ImmunoResearch Labor-
atories USA, dilution 1 : 80 in NaCl/P
i
). Following stain-
ing, coverslips were mounted in Elvanol. Fluorescent images
were recorded with a Zeiss Axiophot microscope equipped
for fluorescence using · 66/1.4 or · 100/1.3 objectives.
Immunoblotting

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.

Anti-p27
Kip1
monoclonal antibodies (mAb; cat#K25020)
and anti-(Rap1A/K-rev) mAb (cat#R22020) were provided
by Transduction Laboratories (KY). Polyclonal Igs against
cyclin D1 (M20), p21
Cip1
(C-19), Cdk2 (M-2) and Cdk6
(C-21) came from Santa-Cruz Biotechnologies. Anti-Ras Ig
was produced from hybridoma Y13-259. Polyclonal anti-
phospho-pRb(Ser795) Ig came from New England BioLabs
(MA). Monoclonal anti-pRb (G3-245) came from Pharmi-
gen (San Diego, CA, USA). Monoclonal anti-cyclin E
(HE12) came from Upstate Biotechnology.
Immunoprecipitation
Rat1/ras cells were treated with 20 l
M
HR12 in growth
medium for 24 and 48 h, and lysed at 4 °C in lysis buffer
containing 50 m
M
Tris/HCl pH 7.5, 1 m
M
EGTA, 1 m
M
EDTA, 1% Triton X-100, 0.27
M
sucrose, 1 m
M
sodium

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

coupling. The immunoprecipitates were washed twice with
lysis buffer and once with kinase buffer containing 50 m
M
Hepes pH 7.4, 10 m
M
magnesium acetate, 1 m
M
dithio-
threitol and 1 l
M
ATP.
Cyclin-dependent kinase (Cdk) assays
The assay for Cdk2 activity was performed by adding
40 lL of kinase buffer containing 10 lCi [c
32
P]ATP and
2.5 lg histone H1 (freshly prepared) to the anti-(cyclin E)
immunoprecipitates. The activities of Cdk4 and Cdk6
were measured as follows: 40 lL of kinase buffer
containing 5 lCi [c
32
P]ATP and 1 lgofGST-pRb
(C-terminal fragment of pRb; Santa Cruz Biotechnology)
were added to the anti-(cyclin D1) and anti-Cdk6 immuno-
precipitates. The mixtures were agitated for 20 min (for
Cdk2) or 30 min (for Cdk4/6) at 30 °C, and the reactions
were halted by the addition of 15 lLperassayof4·
Laemmli sample buffer. Samples were separated on 12%
SDS/PAGE and electroblotted onto nitrocellulose filters.
The blots were exposed either to X-ray film or to a

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

35
S-Met/Cys Promix, with or
without LLnL, the plates were washed with NaCl/P
i
and
the cells lysed. Anti-p27
Kip1
mAb (# K25020) was
coupled to protein G-sepharose (Amersham-Pharmacia)
and served for immunoprecipitation. Following SDS/
PAGE and blotting, the membrane was exposed to X-ray
film.
Results
HR12 treatment of Rat1/ras cells inhibits anchorage-
dependent and independent cell-growth
We first characterized the effect of HR12 on anchorage-
independent growth of Rat1/ras cells, using the assay for
colony growth in soft agar. HR12 treatment inhibited the
growth of Rat1/ras cells in soft agar in a dose-dependent
manner, with an IC
50
value of 5 l
M
(Fig. 1A). The
inhibition led to a decrease in both colony size and the
number of colonies. The growth rate of Rat1/ras cells in a
monolayer was also inhibited by HR12 in a dose dependent
manner (IC
50
¼ 12 l

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

the number of Rat1/ras cells in the S phase.
The time course of HR12-induced inhibition of Ras
processing correlates with the decrease in pRb
phosphorylation
As phosphorylation of pRb is one of the key events required
for G1/S transition, we examined whether HR12 affects
pRb phosphorylation in Rat1/ras cells, and whether the
timing of Ras-processing and pRb phosphorylation are
correlated. We treated Rat1/ras cells with 20 l
M
HR12 in
growth medium and lysed the treated and untreated cells at
the indicated times (Fig. 4). Immunoblots of the lysates
were probed with anti-Ras Ig and with anti-phospho-
pRb(Ser795) Ig. Unprocessed-Ras was separated from proc-
essed Ras in 15% SDS/PAGE. As we had shown previously,
during the course of HR12 treatment, unprocessed Ras
accumulated, whereas processed-Ras disappeared ([6] and
Fig. 4, upper panel). We found there to be a corresponding
decrease in phosphorylation of pRb. This dephosphoryla-
tion followed the same kinetics as the inhibition of Ras
processing (Fig. 4, lower panel). When HR12 was removed
following 48 h treatment with 20 l
M
HR12, processed-Ras
accumulated and pRb was phosphorylated simultaneously
(Fig. 4 ‘wash’). Thus, the inhibition of Ras processing caused
by HR12 was reversible, and relief of this inhibition
correlated with the return of pRb phosphorylation.
HR12 leads to an increase in p27

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.

2762 H. Reuveni et al. (Eur. J. Biochem. 270) Ó FEBS 2003
but no increase was detected (data not shown). The levels of
cyclin D1 and cyclin E were not affected by HR12
treatmentupto40l
M
(Fig. 5); furthermore, their levels
remained unchanged over the course of HR12 treatment
(data not shown). Finally, a dose-dependent decrease in
the hyper-phosphorylated form of pRb (Fig. 5, pRb, upper
band) was evident using an Ig against both the hyper- and
the hypo-phosphorylated forms of pRb. We note, how-
ever, that in Rat1/ras cells the proportion of hyper-
phosphorylated pRb was lower than in other cell lines
(data not shown). Therefore, we also used an Ig specific for
phospho-Ser795 of pRb: a dose-dependent reduction in
phosphorylation was evident in both the hyper- and the
hypo-phosphorylated bands (Fig. 5, pS795-pRb). In sum-
mary, we observed that treatment of Rat1/ras cells with
increasing concentrations of HR12 led to a dose-dependent
increase in p27
Kip1
levels accompanied by a corresponding,
dose-dependent decrease in pRb-phosphorylation.
Fig. 2. HR12 suppresses in vitro monolayer ‘wound healing’ of Rat1/ras
cells. Rat1/ras cells were grown to confluence in the presence (right
column)ortheabsence(leftcolumn)of20l
M
HR12. At time 0, the
monolayer was wounded and phase-contrast photomicrographs were
taken at the indicated time points. Medium with or without HR12 was

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,

harvested and fixed in 70% ethanol. The cells were double-stained with
FITC-labelled anti-BrdU and propidium-iodide (PI), and analysed by
flow cytometry.
Ó FEBS 2003 FTI-induced p27Kip1 increase and G1 arrest (Eur. J. Biochem. 270) 2763
HR12 inhibits the degradation of p27
Kip1
protein
in Rat1/ras cells
Three different mechanisms have recently been implicated in
the regulation of p27
Kip1
levels: (a) variations in the rate of
synthesis of the protein [25,29,39]; (b) variations in the rate
of degradation [40] and (c) transcriptional control [41]. To
evaluate the contribution of HR12 to the stability of the
p27
Kip1
protein, we blocked the expression of new p27
Kip1
protein by cycloheximide treatment of the cells, and observed
p27
Kip1
levels in whole cell lysates by immunoblotting with
anti-p27
Kip1
Igs. Figure 6A shows that the t
1/2
of p27
Kip1
in

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

when HR12 was present (D2 in Fig. 6B), suggesting that
HR12 does not influence the rate of synthesis of p27
Kip1
.
To confirm this finding, we labelled newly synthesized
proteins with
35
S-Met/Cys Promix during the last 3 h of
HR12 treatment, as described in the Experimental proce-
dures section. The amount of label incorporated into
immunoprecipitated p27
Kip1
in samples that were treated
with HR12 was equivalent to or lower than the amount in
untreated samples, whether or not LLnL was present during
the metabolic labelling (Fig. 6C). These findings confirm
that HR12 does not enhance the rate of p27
Kip1
synthesis,
indicating that the increase in amounts of p27
Kip1
in the
presence of HR12 reflects a longer p27
Kip1
half-life.
HR12 treatment of Rat1/ras cells leads to the
accumulation of p27
Kip1
in the cyclin E/Cdk2 complex
and to the inhibition of its kinase activity

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

levels present in anti-Cdk2 immunoprecip-
itates were significantly higher in the HR12-treated Rat1/ras
cell complexes than in untreated cell complexes.
HR12 treatment of Rat1/ras cells induces an increase in
p27
Kip1
levels in the cyclin D1/Cdk6 and cyclin D1/Cdk4
complexes, with no inhibitory effect on their kinase
activities
We evaluated the ability of HR12 to affect p27
Kip1
content
in the cyclin D1/Cdk6 and cyclin D1/Cdk4 G1 phase
complexes and also evaluated its effect on the kinase activity
of these complexes. The kinase activities of cyclin D1/Cdk6
p-pRb
0
40
80
120
pS795-pRb
HR12 - + - + - + - + - + - +
time (hr) 1 3 5 15 24 48
wash
Ras
Processed Ras
(% of total Ras)
1 3 5 15 24 48 wash
up
p

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-

blotting as described above. Figure 8A shows that cyclin
D1/Cdk6 complexes bound much higher levels of p27
Kip1
in
cells treated with HR12 than in untreated cells. However, no
change in kinase activity was detected. Immunoprecipita-
tion of cyclin D1/Cdk4 and cyclin D1/Cdk6 complexes
using anti-(cyclin D1) Igs revealed an HR12-induced
increase of p27
Kip1
levels in the complexes. This was
accompanied by increased kinase activity of these immuno-
precipitates (Fig. 8B).
Fibroblasts transformed by farnesylation-independent
myristoylated-Ha-Ras are resistant to HR12-induced
G1 arrest, suppression of
in vitro
monolayer ‘wound
healing’, cytoskeletal recovery and p27
Kip1
increase
To examine whether the effects of HR12 on the cell cycle,
cell motility and cell cycle components are mediated
exclusively by its effect on Ras, rather than on the
farnesylation of other protein(s), we examined the effects
of HR12 on Rat1 cells transformed by myr-Ras (Rat1/myr-
ras). Myr-Ras is an oncogenic Ha-Ras engineered to bind
the membrane constitutively through N-myristoylation with
no dependence on FT for its function. Figure 9 shows that
treatment of Rat1/myr-ras with HR12 for 48 h had almost

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

Fig. 5. HR12 treatment of Rat1/ras cells induces hypophosphorylation
of pRb and elevation of p27
Kip1
levels in a dose-dependent manner. Rat1/
ras cells were exposed to HR12 at the indicated concentrations for
48 h, lysed and immunoblotted with Igs against phospho-Ser795-pRb
(p-pRb), pRb (pRb), p27
Kip1
,p21
Cip1
, cyclin D1 and cyclin E. In the
case of pRb, the level of the phosphorylated protein was normalized to
the level of the total protein.
Cyclin D1
0
50
100
150
Cyclin D1
0 0.5 1.5 4.5 13 40
p21
Cip1
0
60
80
40
p21
Cip1
20
0 0.5 1.5 4.5 13 40

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

Ó FEBS 2003 FTI-induced p27Kip1 increase and G1 arrest (Eur. J. Biochem. 270) 2765
Rat1/ras cells treated with HR12 undergo complete mor-
phological reversion and dramatic assembly of adheren
junctions, concomitant with an increase in cadherin and
b-catenin levels. These effects are mediated via Ras [6].
In the current paper, we report upon the effects of HR12 on
growth and on the cell cycle. We find that HR12 suppresses
anchorage-dependent and independent growth and motility
of Rat1/ras (Figs 1 and 2). Furthermore, treatment with
HR12 leads to arrest of cell growth at the G1 phase of the
cell cycle (Fig. 3). It has been argued recently that FTIs
inhibit the growth of Rat1/ras cells [32] and induce
morphological reversion [45] through an inhibitory mech-
anism that is Ras-independent and depends on the farnesy-
lation of RhoB (the ‘FTI-RhoB hypothesis’, reviewed in
[34,35,44]). In contrast, our results show clearly that the
effects of HR12 are mediated via Ras. In Rat1/myr-ras cells,
Ras function is no longer dependent on farnesylation. If the
effects of HR12 were due to inhibition of a farnesylated
protein other than Ras, the myristoylated-Ras transformed
cells would have been affected by HR12. In Fig. 9 we show
that HR12 had no effect on cell cycle distribution (Fig. 9A)
or the rate of ‘wound healing’ (Fig. 9D) of Rat1/myr-ras
cells. Moreover, no cytoskeletal or morphological changes
were observed in HR12-treated Rat1/myr-ras cells, while
Rat1/ras cells were driven toward complete morphological
and cytoskeletal reversion following HR12 treatment
(Fig. 9C). In accordance with the above data, HR12 had
no effect on 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

100
0 20 60 100 120 240
p27
Kip1
/actin
p27
Kip1
actin
0
50
100
0 20 60 100 120 240
exposure to chx (min)
p27
Kip1
/actin
C
B
p27
Kip1
actin
LLnL: - - + +
HR12: - + - +
p27
Kip1
/actin
[
35
S]p27
Kip1

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

Kip1
. (B) Rat1/ras cells were treated with 20 l
M
HR12 for 48 h, and 50 l
M
LLnL was added to the medium 3 h before
lysis. Immunoblotting and quantification were performed as described
above. (C) Rat1/ras cells were treated with HR12 for 48 h, starved for
1 h, and labelled with
35
S-Met/Cys Promix in the presence or absence
of 50 l
M
LLnL for 3 h. The lysates were immunoprecipitated with
anti-p27
Kip1
, immunoblotted and exposed to X-ray film.
2766 H. Reuveni et al. (Eur. J. Biochem. 270) Ó FEBS 2003
between the inhibition of Ras processing and the accumu-
lation of p27
Kip1
([6]andFig. 5).Weobservedanincreasein
p27
Kip1
levels in the cyclin E/Cdk2 complex, and a
corresponding reduction in the kinase activity of the
complex (Fig. 7). Treatment of Rat1/ras cells with HR12
also led to an increase in the level of p27
Kip1
complexed with

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-

sis’). We report here on a striking increase in p27
Kip1
levels
following HR12 treatment. Moreover, we have shown that
this increase is correlated with increased amounts of p27
Kip1
in complex with Cdk2 and with reduced Cdk2 kinase
activity. Our data provide a plausible mechanism for the G1
arrest of Rat1/ras cells caused by HR12. We did not observe
an increase in p21
CIP1
levels, under the same conditions of
HR12 treatment (Fig. 5).
HR12 leads to stabilization of p27
Kip1
The amounts of p27
Kip1
are regulated at the levels of
transcription [41], translation [39,50] and post-translational
degradation by the ubiquitin-proteasome pathway [40]. Ras
has been reported to down-regulate p27
Kip1
by all three
mechanisms: (a) control of p27
Kip1
degradation, by regula-
tion of the RhoA pathway [27,29,51]; (b) repression of
p27
Kip1
synthesis, mediated either by the Raf/Mek/Erk

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),

polyclonal anti-Cdk2 and monoclonal anti-p27
Kip1
Igs. (B) Rat1/ras
cells were treated as in A, and immunoprecipitated with polyclonal
anti-Cdk2 Ig. Immunoprecipitates were immunoblotted with anti-
Cdk2 and anti-p27
Kip1
Ig.
Ó FEBS 2003 FTI-induced p27Kip1 increase and G1 arrest (Eur. J. Biochem. 270) 2767
repression) of PKB. This activation of PKB was probably a
secondary event that arose as a consequence of the assembly
and activation of focal adhesions and cell–cell contacts [6].
The Raf/Mek/Erk pathway was strongly inhibited in Rat1/
ras cells treated with HR12 [6]. However, we do not believe
this to be the regulatory pathway that leads to reduced levels
of p27
Kip1
because HR12 treatment had no effect on the rate
of p27
Kip1
synthesis and expression (Fig. 6B) and inhibition
of Mek by PD98059 had no effect on p27
Kip1
levels in Rat1/
ras cells (data not shown). The half-life of the p27
Kip1
protein was much longer in the presence of HR12 (Fig. 6A),
showing that HR12 stabilizes p27
Kip1
. The ubiquitin-

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].

Levenberg et al.andSt.Croixet al. recently showed that
overexpression or activation of cadherin leads to depho-
sphorylation of pRb, increased levels of p27
Kip1
and a
reduction in cyclinE/Cdk2 levels, resulting in arrest of cell
growth [11,12]. Moreover, the levels of p27
Kip1
mRNA
remained constant in contact-inhibited cells [50] and the
half-life of p27
Kip1
protein was much longer in contact-
inhibited cells than in cells growing exponentially [40].
These phenomena are strikingly similar to the conse-
quences of HR12 treatment: increased levels of cadherin,
assembly of cell–cell contacts, stabilization of p27
Kip1
,
Fig. 8. HR12 treatment of Rat1/ras cells does not induce inhibition of
the kinase activity of cyclin D1/Cdk6 or cyclin D1/Cdk4 complexes.
Rat1/ras cells were treated as in Fig. 7 and immunoprecipitated with
anti-Cdk6 (A) or anti-(cyclin D1) (B) Igs. The immunoprecipitates
were tested for kinase activity with GST-pRb as a substrate, as des-
cribed in Experimental procedures, followed by separation on SDS/
PAGE and blotting. The blots were exposed to a PhosphorImager
screen or to X-ray film to quantify kinase activity, [
32
P]pRb. To
visualize the levels of the proteins in the immunoprecipitates the same

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

.
Although we cannot exclude this interpretation, we and
others saw no effect of Mek/Erk inhibition on p27
Kip1
Ó FEBS 2003 FTI-induced p27Kip1 increase and G1 arrest (Eur. J. Biochem. 270) 2769
levels ([27,52] and our unpublished data), whereas the
Mek inhibitor, PD98059 led to complete recovery of cell–
cell contacts [6]. We therefore postulate that the stabiliza-
tion of p27
Kip1
by HR12 is not a consequence of the
assembly of cell–cell contacts, but rather reflects Ras to
p27
Kip1
signalling.
No role for cyclin D1 in HR12-mediated G1 arrest
It has been reported that the central effect of Ras on
progression through the G1 phase of the cell cycle is not
mediated solely by down-regulation of p27
Kip1
but also by
the induction of cyclin D1 [25,27]. Cyclin D1 is controlled
by two distinct signalling pathways downstream of Ras:
the Raf/Mek/Erk pathway, which induces transcription of
cyclin D1 (reviewed in [26]), and the PI3K pathway,
which leads to increased translation of cyclin D1 [58] and
to its stabilization [59–61]. Ras activates the PI3K/PKB
pathway and PKB in turn phosphorylates and deactivates
GSK3. Active GSK3 phosphorylates cyclin D1, triggering
its degradation. Hence, activation of the PI3K/PKB

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

cell motility [63]. In our studies, HR12 treatment of Rat1/ras
cells led to Mek/Erk inhibition, accompanied by induction of
stress fibers [6], and suppression of ‘wound healing’.
HR12 as a potential anticancer drug
Progressive loss of p27
Kip1
protein is commonly observed
during progression from normal cells to benign and
malignant tumors and has been associated with the meta-
static potential of carcinomas (reviewed in [30,31]). Ectopic
expression of p27
Kip1
is associated with decreased malignant
behaviour. The p27
Kip1
gene is only rarely mutated in cancer
cells. The reduced p27
Kip1
expression observed in a variety
of human malignancies is regulated primarily at a post-
translational level [30,31]. As HR12 leads to stabilization
and accumulation of p27
Kip1
in ras-transformed cells, it is an
attractive candidate for treatment of cancers involving
oncogenic Ras. Furthermore, HR12 is the first FTI that has
beenshowntoleadtoelevatedp27
Kip1
levels.
We developed HR12 as an inhibitor of Ras-farnesylation,

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.

4. Kato,K.,Cox,A.D.,Hisaka,M.M.,Graham,S.M.,Buss,J.E.
and Der, C.J. (1992) Isoprenoid addition to Ras protein is the
critical modification for its membrane association and trans-
forming activity. Proc. Natl Acad. Sci. USA, 89, 6403–6407.
5. Reuveni, H., Gitler, A., Poradosu, E., Gilon, C. & Levitzki, A.
(1997) Synthesis and biological activity of semipeptoid farnesyl-
transferase inhibitors. Bioorg.Med.Chem.5, 85–92.
6. Reuveni, H., Geiger, T., Geiger, B. & Levitzki, A. (2000) Reversal
of the ras-induced transformed phenotype by HR12, a novel ras
farnesylation inhibitor, is mediated by the Mek/Erk pathway.
J. Cell Biol. 151, 1179–1192.
7. Frixen, U.H., Behrens, J., Sachs, M., Eberle, G., Voss, B., Warda,
A., Lochner, D. & Birchmeier, W. (1991) E-cadherin-mediated
cell-cell adhesion prevents invasiveness of human carcinoma cells.
J. Cell Biol. 113, 173–185.
8. Vleminckx, K., Vakaet, L. Jr, Mareel, M., Fiers, W. & van Roy, F.
(1991) Genetic manipulation of E-cadherin expression by epithelial
tumor cells reveals an invasion suppressor role. Cell 66, 107–119.
9. Perl, A.K., Wilgenbus, P., Dahl, U., Semb, H. & Christofori, G.
(1998) A causal role for E-cadherin in the transition from ade-
noma to carcinoma. Nature 392, 190–193.
10. Birchmeier, W. & Behrens, J. (1994) Cadherin expression in car-
cinomas: role in the formation of cell junctions and the prevention
of invasiveness. Biochim. Biophys. Acta 1198, 11–26.
11. Levenberg, S., Yarden, A., Kam, Z. & Geiger, B. (1999) p27 is
involved in N-cadherin-mediated contact inhibition of cell growth
and S-phase entry. Oncogene 18, 869–876.
2770 H. Reuveni et al. (Eur. J. Biochem. 270) Ó FEBS 2003
12. St. Croix, B., Sheehan, C., Rak, J.W., Florenes, V.A., Slingerland,
J.M. & Kerbel, R.S. (1998) E-Cadherin-dependent growth

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

in NIH 3T3 cells. Mol. Cell Biol. 10, 5314–5323.
23. Feig,L.A.&Cooper,G.M.(1988)InhibitionofNIH3T3cell
proliferation by a mutant ras protein with preferential affinity for
GDP. Mol. Cell Biol. 8, 3235–3243.
24. Mulcahy,L.S.,Smith,M.R.&Stacey,D.W.(1985)Requirement
for ras proto-oncogene function during serum-stimulated growth
of NIH 3T3 cells. Nature 313, 241–243.
25. Aktas, H., Cai, H. & Cooper, G.M. (1997) Ras links growth
factor signaling to the cell cycle machinery via regulation of
cyclin D1 and the Cdk inhibitor p27KIP1. Mol. Cell Biol. 17,
3850–3857.
26. Takuwa, N. & Takuwa, Y. (1997) Ras activity late in G1 phase
required for p27kip1 downregulation, passage through the
restriction point, and entry into S phase in growth factor-stimu-
lated NIH 3T3 fibroblasts. Mol. Cell Biol. 17, 5348–5358.
27. Weber,J.D.,Hu,W.,Jefcoat,S.C.Jr,Raben,D.M.&Baldassare,
J.J. (1997) Ras-stimulated extracellular signal-related kinase 1 and
RhoA activities coordinate platelet-derived growth factor-induced
G1 progression through the independent regulation of cyclin D1
and p27. J. Biol. Chem. 272, 32966–32971.
28. Kawada, M., Yamagoe, S., Murakami, Y., Suzuki, K., Mizuno, S.
& Uehara, Y. (1997) Induction of p27Kip1 degradation and
anchorage independence by Ras through the MAP kinase sig-
naling pathway. Oncogene 15, 629–637.
29. Rivard,N.,Boucher,M.J.,Asselin,C.&L’Allemain,G.(1999)
MAP kinase cascade is required for p27 downregulation and S
phase entry in fibroblasts and epithelial cells. Am.J.Physiol.277,
C652–C664.
30. Slingerland, J. & Pagano, M. (2000) Regulation of the cdk inhibitor
p27 and its deregulation in cancer. J. Cell Physiol. 183, 10–17.

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,

4327–4336.
40. Pagano, M., Tam, S.W., Theodoras, A.M., Beer-Romero, P., Del
Sal, G., Chau, V., Yew, P.R., Draetta, G.F. & Rolfe, M. (1995)
Role of the ubiquitin-proteasome pathway in regulating abun-
dance of the cyclin-dependent kinase inhibitor p27. Science 269,
682–685.
41. Medema, R.H., Kops, G.J., Bos, J.L. & Burgering, B.M. (2000)
AFX-like Forkhead transcription factors mediate cell-cycle
regulation by Ras and PKB through p27kip1. Nature 404,
782–787.
42. Rock,K.L.,Gramm,C.,Rothstein,L.,Clark,K.,Stein,R.,Dick,
L.,Hwang,D.&Goldberg,A.L.(1994)Inhibitorsofthepro-
teasome block the degradation of most cell proteins and the gen-
eration of peptides presented on MHC class I molecules. Cell 78,
761–771.
43. Palombella, V.J., Rando, O.J., Goldberg, A.L. & Maniatis, T.
(1994) The ubiquitin-proteasome pathway is required for proces-
sing the NF-kappa B1 precursor protein and the activation of
NF-kappa B. Cell 78, 773–785.
44. Prendergast, G.C. (2000) Farnesyltransferase inhibitors: antineo-
plastic mechanism and clinical prospects. Curr. Opin. Cell Biol. 12,
166–173.
45. Prendergast, G.C., Davide, J.P., deSolms, S.J., Giuliani, E.A.,
Graham, S.L., Gibbs, J.B., Oliff, A. & Kohl, N.E. (1994) Farne-
syltransferase inhibition causes morphological reversion of ras-
transformed cells by a complex mechanism that involves regulation
of the actin cytoskeleton. Mol. Cell Biol. 14, 4193–4202.
46. Zhang, H., Hannon, G.J. & Beach, D. (1994) p21-containing
cyclin kinases exist in both active and inactive states. Genes Dev
8, 1750–1758.

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.

54. Hu, W., 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.
55. Sheaff, R.J., Groudine, M., Gordon, M., Roberts, J.M. & Clur-
man, B.E. (1997) Cyclin E-CDK2 is a regulator of p27Kip1. Genes
Dev. 11, 1464–1478.
56. Vlach, J., Hennecke, S. & Amati, B. (1997) Phosphorylation-
dependent degradation of the cyclin-dependent kinase inhibitor
p27. EMBO J. 16, 5334–5344.
57. Potempa, S. & Ridley, A.J. (1998) Activation of both MAP kinase
and phosphatidylinositide 3-kinase by Ras is required for hepa-
tocyte growth factor/scatter factor-induced adherens junction
disassembly. Mol. Biol. Cell 9, 2185–2200.
58. Muise-Helmericks, R.C., Grimes, H.L., Bellacosa, A.,
Malstrom, S.E., Tsichlis, P.N. & Rosen, N. (1998) Cyclin D
expression is controlled post-transcriptionally via a phosphatidyl-
inositol 3-kinase/Akt-dependent pathway. J. Biol. Chem. 273,
29864–29872.
59. Diehl, J.A., Cheng, M., Roussel, M.F. & Sherr, C.J. (1998) Gly-
cogen synthase kinase-3beta regulates cyclin D1 proteolysis and
subcellular localization. Genes Dev. 12, 3499–3511.
60. Diehl, J.A., Zindy, F. & Sherr, C.J. (1997) Inhibition of cyclin D1
phosphorylation on threonine-286 prevents its rapid degradation
via the ubiquitin-proteasome pathway. Genes Dev. 11, 957–972.
61. Frame, S. & Balmain, A. (2000) Integration of positive and
negative growth signals during ras pathway activation in vivo.
Curr. Opin. Genet. Dev. 10, 106–113.
62. Chen, D., Walsh, K. & Wang, J. (2000) Regulation of cdk2
activity in endothelial cells that are inhibited from growth by cell
contact. Arterioscler. Thromb. Vasc. Biol. 20, 629–635.

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