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: levitzki@vms.huji.ac.il. 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 (http://rsb.info.nih.gov/nih-image/). 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 [...]... FTI-induced p27Kip1 increase and G1 arrest (Eur J Biochem 270) 2769 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 p27Kip1 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 p27Kip1. .. [30,31] As HR12 leads to stabilization and accumulation of p27Kip1 in ras- transformed cells, it is an attractive candidate for treatment of cancers involving oncogenic Ras Furthermore, HR12 is the first FTI that has been shown to lead to elevated p27Kip1 levels We developed HR12 as an inhibitor of Ras- farnesylation, and found that it suppresses invasive growth and proliferation of Rat1 /ras cells Our data... pathway in Rastransformed 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 p27Kip1 protein is commonly observed during progression... cell cycle arrest through a p27Kip1- 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 p27Kip1 levels, whereas the induction of cell proliferation during wound healing is accompanied by... that the effects of HR12 are due to inhibition of Ras farnesylation HR12 appears to arrest ras- transformed fibroblasts specifically, with minimal effects on nontransformed cells, encouraging us to anticipate minimal toxic side-effects of HR12 In light of the connection between loss of p27Kip1 protein and metastasis and proliferation, agents such as HR12 that lead to stabilization of p27Kip1 protein could... 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. .. & 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-stimulated NIH 3T3 fibroblasts Mol Cell Biol 17, 5348–5358... contacts, but rather reflects Ras to p27Kip1 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 p27Kip1 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... decrease in p27Kip1 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 . 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. 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. 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