the farnesyl transferase inhibitor sch 66336 induces a g2 → m or g1 pause in sensitive human tumor...
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Experimental Cell Research 262, 17–27 (2001)doi:10.1006/excr.2000.5076, available online at http://www.idealibrary.com on
The Farnesyl Transferase Inhibitor SCH 66336 Induces a G2 3 Mor G1 Pause in Sensitive Human Tumor Cell Lines
Hena R. Ashar, Linda James, Kimberly Gray, Donna Carr, Marnie McGuirk, Eugene Maxwell, Stuart Black,Lydia Armstrong, Ronald J. Doll,* Arthur G. Taveras,* W. Robert Bishop, and Paul Kirschmeier1
Department of Tumor Biology and *Chemistry, Schering-Plough Research Institute, 2015 Galloping Hill Road,
Kenilworth, New Jersey 07033SCH 66336 is a potent farnesyl transferase inhibitor(FTI) in clinical development. It efficiently preventsthe membrane association of H-ras, but not K- or N-ras.Yet, in soft agar, it reverts the anchorage-independentgrowth of human tumor cell lines (hTCLs) harboringH-ras, K-ras, and N-ras mutations, implying that block-ing farnesylation of proteins besides ras may be re-sponsible for this effect. Experiments show that SCH66336 altered the cell cycle distribution of sensitivehuman tumor cells in two distinct ways. Most sensitivehTCLs accumulated in the G23M phase after the FTIreatment, but those with an activated H-ras accumu-ated in G1 phase, suggesting that the biological effects
induced by FTIs in cells with an activated H-ras aredistinct from other sensitive cells. A careful genotypiccomparison of the hTCLs revealed that those cellswith wild-type p53 are especially sensitive to the FTIs.In these cells p53 and its downstream target genep21Cip1 are induced after treatment with SCH 66336 for24 h. These data suggest that cell cycle effects, eitherG1 or G23M accumulation, and p53 status are impor-ant for mediating the effects of FTIs on tumor cells.
© 2001 Academic Press
Key Words: FTI; p53; cell cycle; mitotic pause; ras.
INTRODUCTION
Ras is an essential component in the transduction ofextracellular signals that induce proliferation and dif-ferentiation. For mutationally activated ras to betransforming, it must be posttranslationally preny-lated [1–3]. Normally, this reaction involves the en-zyme, farnesyl transferase (FPT),2 which catalyzes thetransfer of a farnesyl group to a conserved cysteine
1 To whom all correspondence and reprint requests should beaddressed at Mail stop 4600, SPRI, 2015 Galloping Hill Rd.,Kenilworth, NJ 07033. Fax: (908) 740-7115. E-mail:[email protected].
2 Abbreviations used: FTI, farnesyl protein transferase inhibitor;FPT, farnesyl protein transferase; GGPT-1, geranylgeranyl protein
transferase type I; and hTCLs human tumor cell lines.17
residue in the C-terminal tetrapeptide, CAAX (where Ais generally an aliphatic amino acid and X is a methi-onine, glutamine, serine or threonine) [2]. The additionof the hydrophobic isoprenoid moiety is necessary forthe membrane association of ras, for subsequent trans-duction of cellular signals, and for transforming activ-ity [1, 3].
Farnesyl transferase inhibitors (FTIs) were devel-oped initially to inhibit the growth of tumors that con-tained constitutively active ras proteins [2, 4, 5]. FTIsefficiently restore contact inhibition and suppress theanchorage independence of H-ras transformed cells invitro [6–8]. Additional efficacy studies using trans-genic mouse models expressing activated H-ras showedthat FTIs induced a complete regression of large well-established tumor masses [9]. Because of these char-acteristics, the FTIs show promise as cancer chemo-therapeutic agents.
In human tumor cells H-ras mutations are not asprevalent as K or N-ras mutations [10]. Previous ex-periments characterizing the response of hTCLs toFTIs demonstrate that the sensitivity to FTIs does notcorrelate with the presence of activated ras proteins[11, 12]. A number of hTCLs that express activatedK-ras proteins are relatively sensitive to FTI treat-ment, while others expressing an activated K-ras pro-teins are resistant. One potential explanation for thelack of sensitivity of some mutant K-ras- or N-ras-expressing cells is the observation that N-ras and K-ras are alternatively prenylated by geranylgeranyl pro-tein transferase (GGPT-I) in cells treated with FTIs[13–16]. Thus the membrane localization of N- or K-rasmay persist in the presence of FTIs. Furthermore,some cell lines that lack ras mutations were found to behighly sensitive to FTI treatment [11, 12]. Taken to-gether, these results suggest that FTIs do suppress thetransformed phenotype of a number of hTCLs and thatexcept for cell lines with an activated H-ras, the rasgenotype of a cell cannot be used to predict the sensi-tivity of tumor cells to FTI treatment.
Clearly, the biological basis for FTI action in all
tumor types is not well understood. To elucidate the0014-4827/01 $35.00Copyright © 2001 by Academic Press
All rights of reproduction in any form reserved.
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18 ASHAR ET AL.
mechanism(s) of growth inhibition of tumor cells byFTIs, hTCLs sensitive to the action of FTIs were com-pared with those hTCLs that are resistant to FTIs.Results presented show that FTI treatment affects thecell cycle distribution in sensitive cell lines. However,the cell cycle responses to FTI treatment with theK-ras-transformed pancreatic cancer cell lines, Ca-pan-2 and MIA Pa Ca-2; the colon cancer cell line,HCT116; and the lung cancer cell carcinoma, NCI-H460, differed from those seen in the H-ras-trans-formed cell lines, the human bladder carcinoma, T24and H-ras-transformed NIH 3T3. Furthermore, hTCLsthat are wild type for p53 appear to be more sensitiveto FTI treatment, and both p53 and p21Cip1 are inducedby FTIs in several hTCLs with wild-type p53 alleles, aspreviously observed [18]. The combined results suggesta possible explanation for understanding the sensitiv-ity of human tumor cells to FTIs. The presence of anactivated H-ras in a hTCL is predictive of sensitivity tothe FTI. However, with either K-ras or non-ras muta-tions the sensitivity of hTCLs to FTIs is enhanced bythe presence of a wild-type p53 and depends on acombination of other factors (or genes) that influencecell cycle regulation.
MATERIALS AND METHODS
Cell culture and growth assays. All the hTCLs, which includecells derived from pancreatic carcinomas (AsPC-I, MIA Pa Ca-2, andPANC-I), colon carcinomas (HCT116, SW620, SW480, DLD-I, HCT-15, and LoVo), lung carcinomas (NCI-H460, HTB-183, HTB-175,A549), bladder carcinoma (T24), breast carcinomas (MCF7, T-47D,MDA-MB-453, BT-474, BT-549, BT-20, and Hs 578T), leukemias(MOLT-4, and K-562), and melanomas (SK-MEL-5, and SK-MEL-28), were obtained from American Type Culture Collection (Rock-ville, MD) and maintained according to their recommendation. Thehuman melanoma line, LOX, was established by O. Fodstad from theInstitute for Cancer Research, Norwegian Radium Hospital, Monte-bello, Oslo [19]. The p53 status of the human tumor cells was ob-tained from the summary of the anticancer drug screen from thedevelopmental therapeutics program, NCI [20, http://epnws1.ncifcrf.gov:2345/dis3d/dtp.html]. The construction, cloning,and selection of NIH 3T3 cells transfected with an activated H-ras orK-ras have been described previously [9].
Soft agar assays were performed in 6-well dishes by seeding 10,000cells in 0.35% agar with a 0.6% agar feed layer. The drug tested wasincluded in both layers at five different concentrations. Cells weregrown for 14 days and colonies were stained with 1% MTT in PBS.The colony counts were used to draw growth curves to determine theIC50 of the drug (the concentration of the drug required for 50%eduction in the colony formation).
Farnesyl transferase inhibitors. The farnesyl transferase inhibitors,CH 66336 and SCH 66177, were previously described (Table 1) [15].Cell cycle analysis. For FACs analysis 0.5–1 3 106 cells were
rown in 10-cm tissue culture plates with or without 1 mM SCH6336. After 36–84 h, cells were collected from the culture mediumnd mixed with cells that were trypsinized. All cells were washedith PBS, fixed with 70% methanol for 30 min at 4°C, and stainedirectly with propidium iodide solution (0.05 mg/ml propidium io-
ide, 0.1% Triton X-100, 0.1 mM EDTA, 0.05 mg/mL RNAse A).0,000 stained cells were analyzed by flow cytometry in a Bectonickinson flow cytometer and analyzed using MODFIT LT (Verityoftware Inc.).Cell fractionation and Western blot analysis. The indicated
TCLs or H-ras-transformed NIH 3T3 cells were incubated in theresence of the specified concentrations of SCH 66177 for 3–4 days.ells were harvested in 2 ml of PBS, centrifuged at 1200 rpm, andesuspended in 500 ml of homogenization buffer (20 mM Hepes, pH.5, 1 mM EDTA, 1 mM EGTA, 2 mg/ml) containing a protease
inhibitor (PIC: aprotinin, 2 mg/ml soybean trypsin inhibitor, 4 mMenzamidine, 0.7 mg/ml pepstatin, 2 mM EDTA, 0.4 mM Tris, pH
7.8). The cells were then lyzed in a glass teflon homogenizer andnuclei were removed by centrifugation for 5 s at 13,000 rpm. Thesupernatant was centrifuged cold at 45,000 rpm. The pelleted mem-brane fraction was resuspended in membrane solubilization buffer(50 mM Hepes, pH 7.8, 10% glycerol, 100 mM NaCl, 1 mM EDTA, 1mM EGTA, 0.5% Triton X-100, and PIC). The soluble and nonsolubleprotein fractions were then separated by SDS-PAGE on 12% gels.The proteins were blotted onto nitrocellulose and probed with eitherthe anti-H-ras polyclonal, H-ras (C-20), anti K-ras Mab, F234, or theanti N-ras Mab, F155 (Santa Cruz Laboratories). Proteins werevisualized using an HRP-linked secondary antibody and enhancedchemiluminesence (Amersham).
Detection of p53 and p21Cip1. To detect p53 induction, 1–2 3 106
cells were grown for 24–72 h with or without the indicated concen-trations of FTI or doxorubicin and then scraped in PBS. A finalconcentration of 1% Nonidet P-40 was added to the extracts beforeincubating the cells on ice for 30 min. Samples were sonicated threetimes for 5–10 s each and insoluble material was removed by cen-trifugation at 13000 rpm for 5 min. The protein concentration inthese extracts was determined and 100–150 mg of total cellular
rotein was separated on 4–20% acrylamide gels for Western anal-sis. The separated proteins were transferred onto nitrocellulose androbed for p53 with the specific antibody PAB1801 (Zymed Labs) or21Cip1 with the antibody Cip1/Waf1 (clone 70) from Transduction
Laboratories. Proteins were detected by chemiluminescence (ECL,Amersham).
RESULTS
The Farnesyl Transferase Inhibitors, SCH 66336 andSCH 66177
SCH 66336 and SCH 66177 have previously beendescribed as farnesyl transferase inhibitors (FPT IC50
1.9 and 1.3 nM, respectively) with no inhibitory actionon GGPT-1 at concentrations as high as 50 mM [21].
he structures of the two compounds are very similarnd are shown in Table 1.
TI Mediate in a G1 Pause in Cells with an ActivatedH-ras
Cell lines with activated H-ras mutations are ex-tremely sensitive to antitransformation effects of FTIs[6–8]. To evaluate the ability of the FTI SCH 66336 toalter the cell cycle distribution and restore cellularcheckpoints in cells with an activated H-ras, flow cy-tometry was performed. H-ras-transformed NIH 3T3cells accumulate in the G1 phase on treatment with 1mM SCH 66336. After 72 h, the population of cells in
the G1 phase increase from 59.9 to 79.6%. This is ac-
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19CELL CYCLE CHANGES RESULTING FROM FTI TREATMENT
companied by a corresponding decrease of cells in the Sphase population [34.6 to 14.7%]. The population in theG23M phase remains unaffected (Fig. 1a and Table 2).
he accumulation of cells in the G1 phase was alsoobserved in T24 cells, a human bladder carcinoma cellwith an activated H-ras and mutant p53. (Fig. 1b andTable 2). For both cell lines the G1 pause was consis-tently observed in FTI-treated cells when the experi-ments were repeated. Since mutant p53 does not affectthe G1 pause observed in the T24 after FTI treatment,p53 is not required for the cell cycle changes or thetumor regression that occurs in H-ras mutant cellsafter FTI treatment [22].
Several Sensitive Cells Accumulate in the G23MPhase on FTI Treatment
Cell cycle analysis was extended to hTCLs with avarying ras genotype. Flow cytometry was performedon sensitive hTCLs with (A) K-ras mutations: the lungtumor carcinoma cell line, NCI-H460; the colon cancerline HCT116; and the pancreatic tumor line MIA PaCa-2; (B) wild-type ras: the breast tumor cell linesMCF7 (reviewed in Table 2). In contrast to cells withH-ras mutations, these cells do not accumulate in G1
TABLE 1
Farnesyl Protein Transferase Inhibitors
Compound Structure
IC50 for enzymeFPTa to H-ras
(nM)
CH 66177 1.3
SCH 66336 1.9
a Ability of the compound to inhibit the transfer of [3H]farnesylpyrophosphate to activated H-ras in vitro [21].
upon FTI treatment. Rather, HCT116, NCI-H460,
MCF7, and MIA Pa Ca-2 pause in the G23M phase(accumulation of more than 25% cells in G23M) upontreatment with SCH 66336 for 24–72 h (Figs. 1c, d, e,f, and Table 2). The cells in G23M more than doubledin HCT116, NCI-H460, and MIA Pa Ca-2 (12.4 to36.4% in HCT116, 11.3 to 25.0% in NCI-H460, and 13.3to 34.3% in MIA Pa Ca-2) and there was also an in-crease in the G23M population in MCF-7 cells (25.5 to36.2% in MCF-7). A similar accumulation of the G23M
opulation was consistently observed in repeated ex-eriments.Furthermore, flow cytometric analysis was per-
ormed with a hTCL that is resistant to the FTI treat-ent [11, 12]. No significant cell cycle changes were
een with FTI SCH 66336 treatment in the tumor celline T47-D, which has mutant p53 and no known ras
utations (Fig. 1g and Table 2).
arnesyl Transferase Inhibitors Prevent theMembrane Association of H-ras but NotK-ras or N-ras
In NIH 3T3 cells transformed with an H-ras, 1 mMSCH 66177 prevents the attachment of H-ras to theplasma membrane, and ras is associated with the sol-uble protein fraction (Fig. 2a). Lerner et al. have re-ported that preventing the recruitment of H-ras to theplasma membrane blocks the constitutive activation ofthe MAPK cascade and results in the accumulation ofinactive Ras/Raf complexes in the cytoplasm [23]. How-ever, with K-ras and N-ras, which are substrates forthe protein geranylgeranyltransferase I, their prenyla-tion and subsequent membrane attachment could notbe prevented by FTIs in any of the hTCLs studied (Fig.2b) [14]. It has previously been observed that the gera-nylgeranylated forms of ras proteins retain transform-ing activity [23–26]. Thus, the transforming signalstransduced through the activated, membrane-associ-ated K-ras and N-ras remain active.
To establish that the disruption of ras processingcorrelated in vivo with the reversion of transformedcells to their nontransformed state, the ability of theFTI to alter the anchorage-independent growth of NIH3T3 cells transformed with an H-ras or a K-ras wastested in soft agar. The IC50 obtained is similar for bothFTIs, SCH 66336 and SCH 66177 (Table 3). As ex-pected, SCH 66336 effectively suppresses the anchor-age independence of H-ras-transformed NIH 3T3 cells(IC50 5 50 nM) but K-ras-transformed cells were lesssensitive (IC50 5 700 nM). Similar effects are observedwith SCH 66177. These results indicate that SCH66336 and SCH 66177 effectively enter the cells wherethey disrupt the farnesylation of cellular proteins with-out affecting their geranylgeranylation, and in H-ras-transformed NIH 3T3 cells, result in a visible pheno-
typic alteration.
20 ASHAR ET AL.
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21CELL CYCLE CHANGES RESULTING FROM FTI TREATMENT
Human Tumor Cell Lines That Are Wild Type for p53Show Enhanced Sensitivity to FTI Treatment
Human tumor cell lines were grouped according totheir p53 or ras genotype and screened by soft agaranalysis to determine their sensitivity to SCH 66336and SCH 66177 (Table 3). When the cell lines aregrouped by the ras status, our data corroborate withpreviously reported work [8, 11, 12] (Fig. 3a). Severalinvestigators have previously shown that cell linestransformed with H-ras mutations, like EJ-1 and T24,are reverted by FTIs [11]. Similarly, the H-ras-trans-formed NIH 3T3 are also sensitive to SCH 66336.Though N-ras and K-ras are geranylgeranylated in thepresence of FTIs, cell lines with N-ras mutations likethe hematopoietic tumor line, Molt-4 (IC50 5 25 nMwith SCH 66336), and K-ras mutations, HCT116 andNCI-H460 (Table 2), are very sensitive to FTIs. Celllines with wild-type ras, like MCF7 and LOX (IC50 5 40nM with SCH 66336) are also sensitive to treatmentwith SCH 66336, whereas others like K-562, T47-D,and BT-474 are relatively resistant [11, 12].
A clear correlation was observed between the pres-ence of wild-type p53 and the sensitivity to the FTI,SCH 66336. Most hTCLs that are wild type for p53have an IC50 , 100 nM to the FTI SCH 66336 (Fig. 3b).
ost cells with a mutant p53 have an IC50 . 200 nM tothe FTI SCH 66336. However, it must be noted that atleast one human tumor cell line with mutant p53,
FIG. 1. Cell cycle analysis of hTCLs treated with FTI 66336. Humand compared with untreated cells that were grown for the same timeunder Materials and Methods, and the data were analyzed and plotted uis plotted on the abcissa and the number of cells on the ordinate. TFTI-treated cells are shown on the right. The percentage of cells in G1 p
TABLE 2
Effects of SCH 66336 on the Cell Cycle Distribution ofHuman Tumor Cell Lines
Cell line G1 G23M S
NIH 3T3 H-ras Untreated 59.9 5.4 34.61 mM SCH 66336 79.6 5.5 14.7
T24 Untreated 74.8 6.8 19.21 mM SCH 66336 89.5 2.4 8.0
HCT116 Untreated 49.1 12.4 38.41 mM SCH 66336 29.0 36.4 37.4
NCI-H460 Untreated 49.0 11.3 39.51 mM SCH 66336 43.3 25.0 31.6
MCF-7 Untreated 36.2 25.5 38.21 mM SCH 66336 32.3 36.2 31.4
MIA Pa Ca-2 Untreated 52.1 13.3 34.51 mM SCH 66336 34.6 34.3 31.0
T47-D Untreated 46.6 16.0 37.31 mM SCH 66336 49.2 17.0 33.7
t 72 h. (b) T24 at 72 h. (c) HCT116 at 72 h. (d) NCI-H460 at 72 h. (e) MC
BT-20, is as sensitive to FTIs as cell lines with wild-type p53, suggesting that there may exist other celllines with mutant p53 that are sensitive to FTI treat-ment. This also indicates that though the presence of awild-type p53 contributes to the sensitivity of manyhTCLs to FTIs, other hTCLs may harbor distinct de-terminant(s) of sensitivity.
FTI Treatment Induces p53 and p21Cip1 Only inTumor Cells with Wild-Type p53
hTCLs that are p53 wild type are sensitive to FTItreatment. The induction of p53 and one of its down-stream target gene p21Cip1 has been observed by Sepp-Lorenzino and Rosen with the FTI L-744,832 [18]. Todetermine if the p53 gene is active and induced in cellstreated with SCH 66336, Western blot analysis wasperformed on proteins from tumor cells with wild-typeor mutant p53 alleles. p53 was induced by SCH 66336in the tumor cells with wild-type p53 alleles, HCT116and NCI-H460 (Fig. 4a). Though MIA Pa Ca-2 andSW480 both express the mutant form of p53, no induc-tion of p53 was seen in these cells. A doublet was seenin HCT116, MIA Pa Ca-2, and SW480, which mayrepresent a proteolytically cleaved form of p53 [27].The lack of p53 induction in MIA Pa Ca-2 cells, whichare sensitive to FTI treatment, strengthens the obser-vation that FTI sensitivity could be conferred throughp53-independent pathways. In order to determinewhether a p53-independent induction of p21Cip1 con-tributed to the G1 or a G23M pause following FTIreatment, Western blot analysis with the p21Cip1 anti-
body was performed on hTCLs with mutant and wild-type p53. The expression of p21Cip1 varied in cells witha mutant p53. Whereas no expression of p21Cip1 wasdetected by Western analysis in MIA Pa Ca-2 cells,p21Cip1 was detected in SW480 cells (Fig. 4b), but noadditional induction occurred in the presence of FTI.Importantly, as an increase in expression levels ofp21Cip1 was not observed in either MIA Pa Ca-2 cells orSW480 cells, the induction of p21Cip1 was not necessaryfor the accumulation of cells in G23M.
The kinetics of p53 and p21Cip1 induction in FTI treatedtumor cells were compared with those of doxorubicin, anantitumor agent that is known to induce p53 and p21Cip1.In HCT116 cells, both p53 and p21Cip1 were induced
ithin 2 h of treatment with 0.5 mM doxorubicin (Fig. 4c),whereas, in cells treated with 0.5 mM SCH 66336, theinduction of p53 and p21Cip1 was observed between 24 and
tumor cells were treated with 1 mM SCH 66336 for the indicated timeiod. Cells were collected and examined by flow cytometry as discussedg MODFIT (Verity Software Inc.). For every cell line, the DNA content
data from the control/untreated cells are plotted on the left and thee, G23M phase, and S phase are shown in Table 3. (a) H-ras NIH 3T3
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22 ASHAR ET AL.
48 h (Fig. 4d). Also, the induction of p53 and p21Cip1 withdoxorubicin was much stronger than that seen with SCH66336, when the same amount of total cellular protein
FIG. 2. (a) H-ras remains in the soluble fraction of the cell in theresence of SCH 66177. H-ras-transformed NIH 3T3 cells were in-ubated in the presence of the indicated concentrations of SCH 66177s described under Materials and Methods. The soluble (S) andarticulate (P) phases were separated on 12% SDS-PAGE gels. Theroteins were transferred to nitrocellulose filters and probed with anntibody specific for H-ras. Proteins were visualized using an HRP-inked secondary antibody and enhanced chemiluminescence (ECL).b) K and N-ras are membrane-bound in presence of the FTIs. Thendicated cells were incubated without (2) or with (1) 1 mM SCH6177 and soluble (S) and particulate (P) fractions were prepared asescribed under Materials and Methods. Forty micrograms of pro-ein from each fraction was separated by SDS-PAGE and blottednto nitrocellulose. The filters were probed with either F-234 (anti--ras) or F-155 (anti-N-ras) and visualized using a HRP-linked sec-ndary antibody and enhanced chemiluminescence (ECL).
was compared.
b Did not grow in soft agar.
Kinetics of the G23M Pause and p53/p21Cip1
Induction in Cells with Wild-Type p53
Both flow cytometry and Western blot analysis wereperformed in parallel on A549 cells (wild type for p53),that were treated with 1 mM SCH 66336 for 24, 48, and72 h. By 24 h, 25.8% of the cells had accumulated inG23M in response to treatment with 1 mM SCH 66336(compared to 11.74% cells G23M in untreated A549cells) (Fig. 5a). The response after 24 h was near max-imal. Between 24 and 72 h, only a marginal increase ofthe G23M fraction in FTI treated cells was observedFig. 5a).
As with the other two p53 wild-type cell lines,CT116 and NCI-H460, FTI treatment induced both53 and p21Cip1. In A549 cells, a weak induction of p53
was observed following 24 h exposure to 1 mM SCH66336, and the maximal induction was observed be-tween 24 and 48 h. The induction of p21Cip1 occurredbetween 24 and 48 h and followed the accumulation ofp53. Our data show that the maximal G23M accumu-ation occurred before maximum induction of p53 andrior to the induction of p21Cip1 (Fig. 5b). This suggests
the existence of a temporal relationship between theG23M pause and the p53 and p21Cip1 induction inhTCLs with wild-type p53.
DISCUSSION
This study describes a broad analysis of hTCLs ge-netically characterized at the p53 and ras loci for theirsensitivity to the farnesyl transferase inhibitor, SCH66336, a compound currently undergoing clinical tri-
als. Since the mechanism of action of the FTIs is notTABLE 3
Effect of the Farnesyl Transferase Inhibitors on Cell Lines
Cell line Source p53 Ras mut FTI IC50 (nM)a
NIH 3T3 H-ras H-ras SCH66336 50SCH66177 40
NIH 3T3 K-ras K-ras SCH66336 700SCH66177 600
T24 Bladder carcinoma mut H-ras b
MIA Pa Ca-2 Pancreatic carcinoma mut K-ras SCH66336 300SCH66177 295
NCI-H460Large cell lungcarcinoma wt K-ras SCH66336 40–80
SCH66177 40HCT116 Colon carcinoma wt K-ras SCH66336 74
SCH66177 87MCF7 Breast carcinoma wt none SCH66336 50T-47D Breast carcinoma mut none SCH66336 2700
a The effect of FTIs on and human tumor cell lines and H-ras-transformed NIH 3T3 cells was analyzed by performing soft agar assays asescribed under Materials and Methods.
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23CELL CYCLE CHANGES RESULTING FROM FTI TREATMENT
completely understood, a set of experiments examiningFTI effects on the cell cycle was performed. The cellcycle distribution of hTCLs that are sensitive to FTIswas correlated with the genetic background of the
FIG. 3. Effect of SCH 66336 on transformed cells. The indicateMethods. The IC50 of each cell line for SCH 66336 was determinedplotted according to their IC50 in nM (ordinate). Cells were groupedmutations, ■ shows cells with K-ras mutations, Œ shows cells with N(b) where cells with wild-type (wt) p53 are indicated by F and with
hTCLs and the ability of the FTI to induce p53 and
p21Cip1. The major findings reported in this study arethat a G1 arrest due to treatment with FTI is onlyobserved in cells with H-ras mutations, namely T24human tumor cells and H-ras-transformed NIH 3T3
ll lines were grown in soft agar as described under Materials andells were grouped according to their genomic status (abscissa) andpending on their ras status in (a), where F shows cells with H-rass mutations, and are cells with wild-type ras; and p53 status intant (mut) p53 are indicated by ■.
d ce. Cde-ra
cells. In other sensitive cell lines, whether p53 wild
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24 ASHAR ET AL.
type or mutant, FTI treatment resulted in a G23Mccumulation.Cell cycle analysis was performed on hTCLs in the
resence and absence of the FTI. If the primary effectf the FTIs was through inhibition of ras signaling, a
13S transition pause would be predicted. Flow cyto-etric analysis of lines containing an activating H-rasutation, namely H-ras-NIH 3T3 and T24, demon-
trated that FTI treatment resulted in the expected G1
arrest. The observation was consistent with resultsshowing that FTIs revert the morphological trans-formed phenotype and restore contact inhibition intransformed cells [6–8] and tumors [9, 22] with anactivated H-ras. The G1 block can be explained by theact that FTIs completely abrogate the proliferationignals that emanate in the cell from membrane-bound
FIG. 4. FTIs induce the expression of p53 and p21Cip1 (a) Westernlot analysis was performed with 150 mg of total protein from the
indicated hTCLs grown in the absence (2) or presence (1) of 1 mMCH 66336 for 48 h and probed with anti-p53 (PAB1801) as de-cribed under Materials and Methods. (b) Western blot analysis waserformed with 150 mg of total protein from the indicated hTCLsrown in the absence (2) or presence (1) of 1 mM SCH 66336 for 48 h
and probed with anti-p21Cip1 (Cip1/Waf1, clone 70) as described un-der Materials and Methods. (c) HCT116 cells were grown in presenceof 500 nM doxorubicin and total protein extracts from the cellscollected at the indicated time points were used for Western blotanalysis for the detection of p53 and p21Cip1. Total protein from
CT116 cells without doxorubicin (0) was also analyzed (d). Westernnalysis of total protein extracts from HCT116 cells treated with 0.5
mM SCH 66336 for 24, 48, and 72 h. Total protein from untreatedcells was also analyzed (0). All blots were probed with anti p53 andanti p21Cip1 antibodies and detected using ECL.
ctivated H-ras [23]. FTI-mediated cycle effects on cells
containing a mutant H-ras are independent of p53status, as T24, the bladder carcinoma cell line, pausesin G1 even though this line has a mutant p53 allele.Experiments using transgenic MMTV-v-Ha-ras miceinterbred with p53(2/2) mice also demonstrated G1
accumulation and tumor regression which was as effi-cient as tumor regression from MMTV-v-Ha-ras trans-genic mice treated with the FTI L-744,832 [22]. Thissupports the view that the FTI-mediated cycle effectson cells containing a mutant H-ras are not dependenton the presence of a functional wild-type p53.
In contrast to cells with an activated H-ras, sensitivecells with wild-type ras or K-ras mutations accumu-lated only in G23M in response to FTI treatment. Asimilar result was also reported in one other cell line,the lung carcinoma A549 [28, 29]. Importantly, it wasrecently demonstrated that the cell cycle arrest duringG23M could be attributed to p53 since at least twodifferent p53-inducible proteins, p21Cip1 or GADD45, orboth, appear to be involved in augmenting the tran-sient pause in late G2 [30, 31]. In another study, Sepp-Lorenzino et al. report a G1 (or a G1 and G23M) pausewith both MCF-7 and HCT116 cells on treatment witha structurally distinct FTI [18]. Experiments describedabove demonstrated that while both tumor cell linesremain very sensitive to the FTI SCH 66336, theyaccumulated in G23M in response to this compound.
FIG. 5. Kinetics of the G23M pause and p53/p21Cip1 induction inA549 cells. A549 cells were grown in the absence and presence of 1mM SCH 66336 for 24, 48, and 72 h. Cells from each time point wereharvested for flow cytometry and Western blot analysis as describedunder Materials and Methods. (a) Number of cells in G0-G1, S, and
23M phases obtained by ModFit analysis in A549 cells for eachtime point (24, 48, 72 h) was plotted for untreated (U) and FTI-treated cells (FTI). (b) Western blot analysis for the induction of p53
and p21Cip1 in A549 cells treated with 1 mM SCH 66336.
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25CELL CYCLE CHANGES RESULTING FROM FTI TREATMENT
No further evidence was seen for the accumulation ofcells in G1 following treatment with 1 mM SCH 66336for 36 to 72 h.
The cell cycle analysis was extended to cell lines thatwere mutant for p53. Those hTCLs with mutant p53that were moderately sensitive to SCH 66336, like MIAPa Ca-2, did not respond to FTI by inducing p21Cip1.Treatment of MIA Pa Ca-2 cells with the FTI did,however, result in accumulation of the cell populationin G23M. Since the G23M pause is observed in cells
ith mutant p53, functional wild-type p53 is not abso-utely required for the FTI-mediated G23M pause.he G23M pause was observed as early as 24 h after
FTI treatment A549 cells (Fig. 5). This supports theidea that a primary response to FTI treatment is dis-regulation of the cell cycle at G23M. This effect alonen hTCLs may lead to moderate sensitivity as observedith the MIA Pa Ca-2 cells (Fig. 3a). The presence ofild-type p53 could augment this effect by mediatingdditional cellular responses and greater sensitivity toTI treatment, as observed with HCT116 and NCI-460 (Fig. 3a).In this study T47-D, a breast carcinoma cell line
esistant to structurally distinct FTIs, SCH 66336 and-744,832, was also examined [12]. This cell line doesot show any significant G1 or G23M accumulation in
response to FTI treatment. What makes these cellsresistant to FTI? It has been demonstrated that theenzyme farnesyl transferase is completely inhibited inthese cells [P. Dayananth and J. English, unpublishedobservations]. Perhaps these cells have inherent defi-ciencies in regulating cellular checkpoints that resultin unresponsiveness to FTI treatment.
The mechanism of action of FTIs can be explained incells with activating H-ras mutations, since FTIs in-hibit the prenylation of H-ras completely, but theiraction on sensitive cell lines with other genotypes ispoorly defined. The presence of several hTCLs withK-ras and N-ras mutations that are sensitive to FTIssuggest that the biological effects of FTIs must stemfrom inhibition of prenylation of other proteins besidesras. This is supported by the observation that FTIs donot prevent the membrane attachment of K-ras or N-ras and therefore signals transduced through the Raseffector pathways like the Raf-MAPK pathway re-mains active in cells that contain an activated K-ras orN-ras. The fact that FTIs impel cellular events thatresult in a G23M pause may indicate that in thesecells, a potential target for FTIs is a farnesylated pro-tein(s) that contributes to progression of the mitoticphase of the cell cycle. This protein(s) may not exclu-sively mediate FTI sensitivity but may contribute toFTI sensitivity.
In agreement with other studies a comparison of thegenotypes of the sensitive cells showed no association
between the presence of a mutationally activated rasallele and sensitivity to the FTI, SCH 66336 [11, 12,31]. However, this study revealed a clear correlationbetween the presence of wild-type p53 and an in-creased sensitivity to the FTI. It is important to notethat cell lines that were sensitive to SCH 66336 werealso sensitive to structurally distinct FTIs. In general,hTCLs that were sensitive to SCH 66336, namelyMCF-7, LoVo, Molt-4, and NCI-H460, were also sensi-tive to L-744,832; whereas, T47-D, Panc-1, and AsPC-1were resistant to the effects of both FTIs [12]. Simi-larly, MIA Pa Ca-2 and HCT116 were sensitive to bothSCH 66336 and B956 [11], and A549 was sensitive andPANC-1 was resistant to both SCH 66336 and FTI-277[28]. These results suggest that the sensitivity of agiven cell line results from the inhibition of the enzymefarnesyl transferase.
The relationship of FTI sensitivity to p53 genotypewas explored further by examining FTI-treated cellsfor the induction of p53 and one of the genes it regu-lates, p21Cip1 [33]. In cells that are wild type for p53,treatment with SCH 66336 led to an accumulation ofp53 and consequently p21Cip1. Both the accumulation ofp53 and the induction of p21Cip1 were also observedwith a structurally distinct FTI in cells with wild-typep53 [18]. This suggests very strongly that inhibition offarnesyl transferase leads to the induction of p53 in acompound structure-independent way. The level of ac-cumulation of p53 and p21Cip1 proteins was markedlydifferent from their accumulation after treatment ofcells with doxorubicin. In presence of the FTI, p53expression was less, and also occurred much slower,reaching a maximal level of accumulation after 24–48h. This could result from the accumulation of fewer p53molecules per cell, or the induction of p53 in a specificsubpopulation of the cells. In either case, our resultsindicate that induction of p53 by FTI treatment con-tributes significantly to the sensitivity of cell lines thatare wild type for p53.
The molecular mechanism by which SCH 66336 in-duces p53 is at present unknown. Because the kineticsand extent of p53 induction are distinct from thoseobserved following doxorubicin treatment, the molecu-lar mechanism leading to induction is also likely to bedistinct. Notably, neither p53 expression nor sensitiv-ity of cells with wild-type p53 to FTI treatment isinfluenced by the ras status of the cell. For example,NCI-H460 cells with a K-ras mutation and MCF-7 cellswith wild type ras are equally sensitive to FTI treat-ment. Since FTIs also induce a G23M pause in thesehuman tumor cell lines, it was tempting to speculatethat the p53 induction is a consequence of the cell cyclepause, and that FTI treatment must alter protein(s)affecting mitosis by interfering with DNA replicationor stabilization. The identification of potential candi-date proteins is the subject of current research. Two
centromere-associated proteins CENP-E and CENP-F,
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1
1
1
1
1
1
1
2
26 ASHAR ET AL.
expressed during G23M, were recently shown to bearnesylated [28] and may mediate some of the cellycle effects in response to FTIs.
How does wild-type p53 sensitize cells to FTI treat-ent? One possibility is that an apoptotic program is
nitiated in tumor cells as a result of p53 induction34–36]. In addition, p53 may activate cellular check-oints via its transcriptional properties or repress pro-eins that are required for the maintenance of a trans-ormed state. It must also be noted that, though theresence of wild-type p53 does contribute significantlyo FTI sensitivity, some human tumor cells with mu-ant p53 alleles, like BT-20, may also be as sensitive toTIs as NIH 3T3 cells transformed with H-ras.Our study clearly connects FTI sensitivity to a spe-
ific genotype, the presence of functional wild-type p53r an activated H-ras. Based on FTI sensitivity, mostells could be placed in three different groups, the veryensitive (IC50 , 100 nM to SCH 66336) with wild-type53 and/or an activated H-ras mutation, moderatelyensitive (IC50 . 200 and , 1000 nM) with mutant p53,
and the resistant cells. The existence of two sensitivitygroups implies that in a tumor cell, FTIs may elicit atleast two distinct cellular responses, only one of whichis p53 dependent. This is consistent with the view thatthe inhibition of farnesylation of more than one proteinmay be required to suppress the malignant phenotype[6–9, 37–39] and that the ultimate sensitivity of thecells to FTI depends on the genetic alterations withinthe tumor cell.
We thank Dr. Suxing Liu for her help with the data analysis of thep53 sensitivity of hTCLs to FTIs.
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