From the Program in Cell Biology and Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10021
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ABSTRACT |
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Farnesylation is required for the membrane partition and function of several proteins, including Ras. Farnesyl-protein transferase inhibitors (FTIs) were developed to prevent Ras processing and thus to be effective agents for the treatment of cancers harboring mutated ras. However, FTIs inhibit the growth of most tumor cells and several xenograft models, irrespective of whether they possess mutated ras. Furthermore, the antiproliferative effect is not correlated with inhibition of Ki-Ras processing; tumors with wild type ras are inhibited, and FTIs are not particularly toxic. These data suggest that the mechanism of FTI action is complex and may involve other targets besides Ras. To begin to understand how FTIs work, we investigated the mechanism of growth inhibition. FTI causes G1 arrest in a subset of sensitive lines. This is accomplished by transcriptional induction of p21, which mediates the inhibition of cyclin E-associated protein kinase activity, pRb hypophosphorylation and inhibition of DNA replication. Induction of p21 is p53-dependent; it does not occur in cells with mutant p53 or in cells expressing human papillomavirus E6. However, neither p53 nor p21 are required for inhibition of cell proliferation. FTI still blocks the growth of cells deficient in these proteins. In the absence of p21, G1 block is relaxed, DNA replication is not affected, and cells become polyploid and undergo apoptosis. These results suggest that farnesylated protein(s) may be involved in regulating p53 function and in coordinating entrance into S, and that the consequences of FTI treatment are a function of the other mutations found in the tumor cell.
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INTRODUCTION |
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The biologic activity of some proteins is dependent upon their isoprenylation. For example, farnesylation of Ras and geranylgeranylation of small GTP-binding proteins of the Rab and Rho families are necessary for proper subcellular localization and for efficient interaction with regulators and effectors (1, 2). This led to the idea that specific inhibitors of farnesyl-protein transferase would be useful anticancer agents by virtue of their ability to prevent Ras processing (1, 3-5). Several natural products with anti farnesyl-protein transferase activity have been identified, but farnesyl-protein transferase inhibitors (FTIs)1 with little activity against related geranylgeranyl-protein transferase I, were also rationally designed (reviewed in Coleman et al. (3)). These selective agents, including the peptidomimetic FTI L-744,832 we used in our studies (6), inhibit protein farnesylation generally and Ha-Ras processing specifically. Accordingly, FTIs effectively revert the phenotype of Ha-ras-transformed fibroblasts and have remarkable effects on animal tumors that are Ha-ras-dependent (7, 8). They prevent the development of breast tumors when administered to murine mammary tumor virus-Ha-ras transgenic mice and when these tumors are allowed to develop, cause their complete remission (7, 8).
However, it is not clear that the effects of the drug are due predominantly to inhibition of Ras processing. First, the kinetics for inhibition of cell growth and initiation and maintenance of morphological reversion of v-ras-transformed fibroblasts by FTI does not correlate with the kinetics for inhibition of Ras processing (9). Second, a majority of human tumor cell lines are sensitive to FTI whether or not they contain mutated ras genes (6). Furthermore, although the processing of Ki- and N-Ras is much less sensitive to FTI than is that of Ha-Ras (10-14), tumor cell lines with the former mutations can be very sensitive to the drug (6). Finally, although FTI inhibits the processing of wild type Ha-Ras, the drug is selective for tumor cells and is remarkably nontoxic in vivo (6-8).
These observations suggest that the phenotype induced by FTI might be due to effects on other farnesylated target proteins (3-5). These include lamins, nuclear matrix proteins, Rho B, among others. In fact, Prendergast et al. (9, 15) have shown that in transformed fibroblasts the growth and morphological changes induced by FTI correlate more closely with inhibition of RhoB processing than with changes in Ras. This is unlikely to be true in epithelial lineages, however, in which inhibition of growth by FTI is unassociated with characteristic changes in morphology and cytoskeletal architecture.
The mechanism underlying FTI action is of interest for several reasons. The action of the drug is likely to be secondary to specific inhibition of farnesyl-protein transferase. This suggests that one or several farnesylated protein(s) yet to be identified (or to be assigned such a function), play a critical role in the biology of transformation. Second, several FTIs are currently entering phase I trials as anticancer agents in humans. Rational clinical development will depend on an understanding of the biochemical basis for their inhibition of cell growth. We undertook to approach these questions by examining the mechanism(s) by which FTI arrested the growth of cancer cells. We found that in a subset of sensitive lines, FTI arrests cells in either G1 or G1 and G2. In these cell lines, FTI caused a p53-dependent induction of p21waf1/cip1/sdi, which led to inhibition of cyclin E- and A-associated protein kinase activities, accumulation of hypophosphorylated retinoblastoma protein (pRb), inhibition of DNA synthesis, and cell cycle arrest. However, whereas p21 induction was necessary for G1 block, it was not necessary for the cytotoxic effects of the drug. In cells in which p53 or p21 were knocked out, FTI caused endoreduplication of DNA and increased apoptosis. These data show that FTI affects cells in a complex manner that is, as it might be expected, dependent upon the complement of mutations in cell cycle regulatory genes in the tumor cell. They also suggest that farnesylated protein(s) may be involved in regulating cellular activity of p53.
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EXPERIMENTAL PROCEDURES |
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Materials--
FTI L-744,832 was a generous gift
from Drs. N. Kohl and A. Oliff at Merck & Co., West Point, PA. The drug
was dissolved in phosphate-buffered saline (PBS) to a final
concentration of 50 mM. Stocks were fractionated and stored
at 80 °C.
Cell Lines-- The human tumor cell lines MCF7, MDA MB-468, Colo 205, DU145, and PC3 were from the American Tissue Culture Collection. HCT116 p21 knockouts and parentals (16) were kindly provided by Drs. T. Waldman and B. Vogelstein (John Hopkins University, Baltimore, MD). All cells were grown in Dulbecco's modified Eagle's medium:Ham's F-12 (1:1) supplemented with 10% fetal bovine serum, 2 mM glutamine, and 50 units/ml each of streptomycin and penicillin at 37 °C in a humidified incubator with 5% CO2, air.
Cell Proliferation and Soft Agar Growth-- To investigate the effect of the FTI L-744,832 on the proliferation of MCF7 and HCT116 cells, growth curves and soft agar colony formation assays were performed as described in Sepp-Lorenzino et al. (6). In order to collect cells for cell cycle analysis and apoptosis and to obtain protein samples, cultures were seeded at a density of 2,500 cells/cm2 and incubated with or without 20 µM FTI for increasing time periods. Cells floating in the medium were collected by centrifugation, and attached cells were trypsinized. These populations were combined. The cell number was determined with a Coulter counter, and aliquots were prepared for protein and kinase analysis and determination of cell cycle parameters and apoptosis.
Flow Cytometry and Determination of Apoptosis-- Nuclei were prepared according to Giaretti and Nusse (17), stained with ethidium iodide, and analyzed with a Becton Dickinson fluorescence-activated cell sorter. Conversely, samples for whole cell flow analysis were prepared according to Nusse et al. (18). Dual label FACS analysis was employed to determine the effect of the drug on DNA replication. Thirty minutes before harvest, cells were pulsed-labeled with 10 µM bromodeoxyuridine (BrdUrd). Samples were fixed and stained with anti-BrdUrd monoclonal antibodies conjugated with fluorescein (Becton Dickinson) and with propidium iodide prior to FACS analysis.
Apoptosis was assessed with quantitative fluorescence microscopy and terminal deoxynucleotidyl transferase assays combined with flow cytometry. Terminal deoxynucleotidyl transferase assays were performed with the apo-BrdUrd kit from Pharmingen according to the manufacturer's instructions. For quantitative fluorescence microscopy, 105 cells were fixed in 3% paraformaldehyde in PBS and stored at 4 °C until further analysis. Cells were then washed with PBS and resuspended in 25 µl of PBS containing 3 µg/ml bisbenzimide. Over 300 cells were counted with a fluorescence microscope, and the percentage of apoptosis was determined.RNA Isolation and Northern Blots-- Cells were extracted with RNAzol (Quality Biotech). Thirty µg of total RNA was analyzed by Northern blotting using a HindIII-XbaI 0.7-kilobase pair fragment of p21 cDNA as a probe (kindly provided by Dr. J. Massagué, Memorial Sloan-Kettering). The probe was labeled by random priming. Blots were also probed for a housekeeping gene (glyceraldehyde-3-phosphate dehydrogenase) as a control for RNA loading. p21 and glyceraldehyde-3-phosphate dehydrogenase mRNA levels were quantitated with a PhosphorImager and p21 mRNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA.
Protein Analysis--
Extracts for protein analysis were
prepared from PBS-washed cell pellets that had been stored at
20 °C. Pellets were lysed with 2% SDS, for pRb detection or with
1% Nonidet P-40 for Cdk activity assays. For total cell lysates,
pellets were dissolved in 2% SDS in 50 mM Tris, pH 8, boiled 10 min, and sonicated three times for 30 s each time on
ice. Nonidet P-40 lysates were obtained by incubating the cell pellets
on ice for 15 min in 1% Nonidet P-40, 50 mM Tris, pH 7.4, 150 mM NaCl, 40 mM NaF, 1 mM
V04Na3, 10 µg/ml each of soybean trysin
inhibitor, aprotinin, and leupeptin, and 1 mM
phenylmethylsulfonyl fluoride. Insoluble material was removed by
centrifugation at 15,000 × g for 5 min. Protein
concentration was measured with the bicinchoninic acid protein assay
(Pierce). Ten percent polyacrylamide gels were used to detect cyclins,
Cdks, and p21, whereas 7% gels were used for analysis of pRb. Gels
were transferred onto nitrocellulose and probed with specific
antibodies. All the antibodies for immunoblotting were from Santa Cruz.
Protein detection was by chemiluminescence (ECL, Amersham Corp.).
In Vitro Kinase Reactions--
Cyclin-associated Cdk 2 and Cdc 2 protein kinase activity were assayed in vitro with histone
H1 as the substrate. One-hundred µg of Nonidet P-40 lysates were
immunoprecipitated with 2 µg of anti-cyclin antibodies (sc-751 for
cyclin A, sc-198 for cyclin E, and sc-181 for cyclin B, Santa Cruz
Biotechnology). Immune complexes were washed four times with Nonidet
P-40 buffer and twice with 20 mM Tris-HCl, pH 7.4, 7.5 mM MgCl2, and 1 mM dithiothreitol. Kinase reactions were carried out for 15 min at 37 °C in 40 µl of
kinase buffer containing 20 mM Tris HCl pH 7.4, 7.5 mM MgCl2, 1 mM dithiothreitol, 30 µM ATP, 10 µCi of [-32P]ATP, and 2 µg of histone H1. Cdk 4 activity was analyzed in anti-Cdk 4 immunoprecipitates with a glutathione S-transferase-pRb fragment (Promega) as a substrate. Reactions were terminated by addition of 5× SDS-polyacrylamide gel electrophoreiss sample buffer and then boiled for 5 min. Proteins were separated on 10% gels and
transferred onto nitrocellulose, and blots were exposed to x-ray film
or to a PhosphorImager screen. Kinase activity was quantitated with a
FUJIX PhosphorImager. Blots were also probed for cyclins, kinases, and
p21 using specific antibodies and detection by chemiluminescence (ECL,
Amersham).
Transfections--
Transient and stable transfections were
carried out using Lipofectin (Life Technologies, Inc.) according to the
manufacturers' instructions. Transient transfections were employed to
study the effect of FTI on the induction of the p21 promoter.
Luciferase reporter constructs under control of the p21 promoter,
either inact (WWP) or lacking the major p53 regulatory site (DM) (19), were cotransfected with SV40--galactosidase into MCF7 cells. After
16 h, cultures were split into 6-well plates and treated for
either 24 or 48 h with or without 20 µM FTI in
triplicate. Cells were harvested, and aliquots were employed for
determination of
-galactosidase and luciferase activity. Luciferase
activity was normalized to
-galactosidase, which was unaffected by
the FTI.
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RESULTS |
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We have previously shown that FTI has a profound inhibitory effect on the anchorage-dependent and -independent growth of a majority of human tumor cell lines (6). In order to begin to identify the target(s) responsible for the antiproliferative effect of the FTI, we investigated the mechanism of growth inhibition in several of the sensitive lines identified in our previous screen (6). FTI inhibited the growth of various cell lines without apparent changes in cell cycle parameters. On the other hand, in another subset of sensitive lines, inhibition of growth was accompanied by cell cycle arrest. For example, the human breast carcinoma cell line MCF7 is sensitive to the FTI (6). These cells are estrogen-dependent human breast cancer cells, harboring wild type p53 and ras, and functional pRb; they posses low levels of epidermal growth factor receptor and ErbB2 and abundant levels of insulin-like growth factor I receptor (20). As shown in Fig. 1A, FTI inhibited MCF7 cell proliferation and resulted in an increase in the proportion of G1 cells at the expense of cells in S phase, whereas the proportion of cells in G2 and mitosis remained largely unchanged (Fig. 1B). G1 arrest was accompanied by hypophosphorylation of pRb as seen in Fig. 1C. Control cells maintained hyperphosphorylated pRb while in a logarithmic growth state; hypophosphorylation of pRb was detected at days 5 and 6 when cultures began to reach confluence.
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The mechanism by which FTI induced the hypophosphorylation of pRb and G1 block was investigated. FTI treatment led to a time-dependent inhibition of cyclin E- and A-associated kinase activities, whereas Cdk 4 kinase activity remained unchanged (Fig. 2A and not shown). The kinase activity of cyclin E·Cdk 2 complexes declined earlier than that associated with cyclin A (24 h versus 48-72 h). FTI induced a decline in the steady-state levels of cyclin A (2-3-fold) and a slight decrease in Cdk 2, which might explain the effect on kinase activity of this complex (Fig. 2B). On the other hand, the levels of Cdk 4 and cyclins E and D3 remained largely unchanged, whereas FTI increased cyclin D1 levels by 2-3-fold (Fig. 2B).
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As cyclin E kinase levels fell in the absence of major changes in cyclin E and Cdk 2 expression, the effects of FTI on the expression of Cdk inhibitors (Cdi) were determined. No changes were observed in the levels of p27, p15, p16, p18, and p19 (not shown); however, p21waf1/cip1/sdi was induced by treatment of MCF7 cells with 20 µM FTI (Fig. 3A). We also found that p21 levels rose with increased cell density in control cultures, yet the levels induced by FTI treatment are 2.5-5-fold higher than those of controls. It is also important to point out that the cell density in FTI-treated cultures remained very low compared with control cultures (Fig. 1A), thus if a direct comparison could be made under equal density conditions the induction of p21 by FTI would be of a greater magnitude.
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The kinetics of induction of p21 by FTI were compared with those of doxorubicin, an agent known to induce DNA damage and p21. Cells were treated for increasing time periods with either 20 µM FTI or 350 nM doxorubicin and p21 levels were compared with those of untreated controls (Fig. 3B). Doxorubin induced a rapid and marked increase in the steady-state levels of p21, detected after 3 h of drug treatment. In contrast, FTI induction took at least 16 h to become evident. A maximun p21 level was attained after 24 h in FTI when p21 was 5-fold higher than that in untreated controls. The induction of p21 by the FTI was accompanied by an increase in the steady-state levels of p21 mRNA (Fig. 3C). Increased p21 mRNA was also detected in control cultures as cell density increased; however, FTI induction was 4-fold greater (484 versus 2012 arbitrary units).
We observed that the cells in which FTI induced p21 and caused G1 arrest had wild type p53. MCF7 and human colon carcinoma HCT116 are both p53 wild type cells, and in both, FTI treatment leads to increased p21, Cdk inhibition, Rb hypophosphorylation, and cell cycle block (Figs. 1, 2, and 6). On the contrary, FTI did not induce p21 in cell lines with mutated p53 (such as Colo 205, DU145, PC3, and MDA MB-468) that are sensitive to its effects on cell growth (Fig. 4A). In view of these results we sought to determine whether the induction of p21 in response to the FTI was dependent on p53.
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The effect of the FTI on the transcriptional activation of the p21
promoter and its p53 dependence were investigated using a set of
reporter constructs in which the luciferase gene was driven either by
the intact p21 promoter (WWP-luc) or by a truncated p21 promoter, in
which the major p53-binding element at 2.4 kilobase pairs is missing
(DM-luc) (19). Transcription from the wild type p21 promoter was
induced by the FTI in p53 wild type cells, but not in cells with mutant
p53 (Fig. 4B). In addition, the p53 binding site was
necessary for transcriptional activation from the p21 promoter by FTI,
as no significant changes in transcription were detected from the
truncated promoter in p53 wild type cells treated with FTI (Fig.
4B).
The requirement for p53 was further investigated by assessing the effects of FTI on cells engineered to express the E6 gene of the human papilloma virus 16 (HPV16). HPV16 E6 specifically targets p53 for degradation via the ubiquitin-dependent pathway (21). E6 was introduced into HCT116 cells and stable transfectants were isolated 2 weeks later as G418-resistant clones. Expression of E6 was confirmed by immunodetection of the AU1-tagged protein. These clones contained decreased basal levels of p53, and the up-regulation of p53 following DNA damage was attenuated (Fig. 5A). The induction of p21 by doxorubicin was decreased in the E6-expressing clones compared with neomycin-resistant controls, and no increase in p21 occurred in response to the FTI (Fig. 5A). Moreover, the small increase in p53 levels detected in the neomycin controls treated with FTI was absent from the p53 deficient cells. These results support the conclusion that the FTI induces the transcriptional activation of the p21 gene by a p53-dependent mechanism.
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Are p21 and p53 required for the antiproliferative effect of the FTI?
For this purpose we tested the effect of the FTI on the
anchorage-dependent and -independent growth of HCT116 cells expressing HPV16 E6 or lacking the p21 gene (16). As seen in Fig.
5B, inability of the FTI to induce p21 due to absence of the
gene or to loss of wild type p53 function did not alter the capacity of
the drug to inhibit cell growth. This result is in agreement with our
previous finding that FTI effectively inhibited the proliferation of
cells with mutated p53 (6). Thus, it seems that p21 induction
constitutes only one of the pathways through which FTI inhibits cell
growth. If that is the case, what is the mechanism of growth inhibition
in the absence of p21? We compared the effects of the FTI on HCT116
parental cells and on their counterparts lacking p21. As expected, only
the parental cells could up-regulate p21 in response to FTI treatment
and induce the hypophosphorylation of Rb (Fig.
6A). Increased cellular levels
of p21 led to enhanced association of the Cdi with cyclin-Cdk complexes
and consequential inhibition of cyclin E- and A-associated kinase
activity (Fig. 6B). In contrast, in p21/
cells the FTI
neither caused decreased Cdk activity or pRb hypophosphorylation (Fig.
6, A and B). These results suggest that the
inhibition of cyclin E-associated protein kinase activity and induction
of Rb hypophosphorylation by FTI require the p53-dependent
induction of p21.
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Induction of cell cycle block by FTI also required p21. FTI blocked DNA
replication in parental cells; there were 4-fold fewer BrdUrd-positive
cells in FTI-treated cultures compared with untreated controls (Fig.
6C). Conversely, p21/
cells in which the FTI did not
inhibit Cdk activity, continued to synthesize DNA in the presence of
the drug (Fig. 6C). FACS analysis corroborated these results
and revealed that the lack of Cdk inhibition and consequential unrestricted DNA replication was associated with the formation of
polyploid cells (Fig. 7A).
While the FTI caused parental cells to arrest in G1 and
G2 at the expense of cells in S, the response of p21
knockout cells was different; S phase remained prominent, the
G1 block was relaxed, and cells accumulated with greater
than 4n DNA content. Ungated FACS analysis revealed the
appearance of 8n and 16n peaks in the p21
/
cells treated with the drug (Fig. 7A). FTI also caused the
development of polyploid cells in cells expressing HPV16 E6 (Fig.
7B).
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Bisbenzimide staining showed that parental cells exposed to the FTI
were predominantly in interphase, whereas p21 knockout cultures showed
an increased proportion of cells in mitosis (3.7% versus
9.25%) (Fig. 8, A-D).
Double-staining with anti-tubulin antibodies provided evidence for
endoreduplication in the FTI-treated p21/
cells, as mitotic forms
with four centrioles were common (Fig. 8E). However, some of
these mitosis were abnormal and seemed to lead into apoptosis (Fig.
8D). Cell cycle analysis in combination with terminal
deoxynucleotide transferase assays corroborated these results and
suggested that inhibition of protein farnesylation in the absence of a
p21-mediated cell cycle block, translates into increased vulnerability
for cell death.
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DISCUSSION |
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FTIs were developed as drugs that would cause the specific inhibition of Ras processing and would thus be effective agents for the treatment of tumors with mutations in the ras proto-oncogene (1, 3, 4). Potent and highly specific FTIs are now available, and they have proven in preclinical studies to be promising drugs. However, contrary to expectations, FTIs inhibit the growth of tumor cells with wild type ras and inhibit the processing of this protein while causing only minor toxicity to normal tissue (6-8). Moreover, FTIs inhibit the growth of tumor cells containing mutations in the Ki-ras gene (6) at doses that inhibit the processing of Ha-Ras but not Ki-Ras (10-14).2 These data suggest both that FTI may exert its effects on cancer cells by inhibiting other targets (for example, Rho B (9, 15, 22)) and that the role played by activated Ras in human epithelial tumors is poorly understood. We undertook studies to determine the mechanism of induction of growth inhibition by FTI in order to develop a context in which to investigate these issues.
Most tumor cell lines, which were sensitive to the FTI in terms of inhibition of growth, did not undergo a cell cycle specific growth arrest in response to the drug. However, a subset of sensitive lines did block in G1 or in G1 and G2. These results are in agreement with the recent observations of Sebti and collaborators (23, 24). They compared the cell cycle effects of the farnesyl-protein transferase inhibitor FTI-277 and of a geranylgeranyl-protein transferase I inhibitor in human tumor cell lines. Unlike inhibition of protein geranylgeranylation, which caused a G0/G1 arrest in all cell lines examined, inhibition of farnesylation resulted in either no effect, or in G1 or G2/M blockade, in a cell line-specific fashion.
We concentrated our efforts in trying to understand whether there was a pattern that would explain the cell cycle-specific effects of the FTI. We found that cell lines that arrested in G1 (or in G1 and G2/M), were wild type for p53. The three p53-wild type human tumor cell lines we studied, MCF7 (breast), HCT116 (colon), and LNCaP (prostate), arrested in G1 in response to FTI treatment (Figs. 1B, 6B, and 7).3 The only other cell line in our inventory that is p53 wild type is the immortalized human breast epithelial line MCF10A. However, we have previously shown (6) that untransformed cells are severalfold more resistant for growth inhibition than cancer lines, thus, at 20 µM FTI no cell cycle effects were observed (not shown).
As shown in this study for MCF7 and HCT116, growth arrest was associated with activation of p53, inhibition of cyclin E-dependent protein kinase, and hypophosphorylation of the Rb protein. Inhibition of cyclin E·Cdk 2 occurred prior to cell cycle arrest and in the absence of significant changes in cyclin E·Cdk 2 or increase in p27 expression (Figs. 2 and 6, and not shown). We investigated the mechanism of cyclin E-dependent protein kinase inhibition and found that it was mediated by a p53-dependent induction of the Cdi p21waf1/cip1/sdi.
p21 plays a crucial role in controlling G1/S progression and in coordinating the G1 and G2 checkpoints (16, 19, 26-28). Besides inhibiting Cdk activity, p21 also affects DNA replication (but not DNA repair) directly, by binding to and interfering with the role of proliferating cell nuclear antigen in replication (29-33). Moreover, p21 was also shown to regulate the activity of stress-activated protein kinases (34). We found that increased p21 was responsible for the FTI-induced inhibition of cyclin E·Cdk 2 kinase activity, which in turn resulted in decreased Rb phosphorylation, inhibition of G1/S progression and cell cycle arrest. HCT116 cells in which p21 had been knocked out no longer inhibited cyclin E-dependent protein kinase activity in response to the FTI, nor did they arrest in G1 (Figs. 6 and 7). The results strongly suggest that the primary basis for induction of G1 arrest by FTI is induction of p21.
The increase in p21 levels by the FTI was detected after 16 h of drug treatment and reached a maximum after 48 h (Fig. 3, A and B) when nuclear accumulation was evident (data not shown). Nuclear translocation of p21 following up-regulation was also detected in other cell systems (35, 36). The increase in p21 protein levels was explained, in part, by a concomitant elevation in the levels of p21 mRNA (Fig. 3C). We cannot exclude at the moment the participation of other types of posttranslational regulation as it was reported that p21 levels can be controlled by changes in protein and mRNA stability (37-39).
We also detected elevated levels of p21 in untreated cells as their growth rates decreased as a function of cell density (Fig. 3, A and B). Given that the cell density of the FTI-treated cultures is severalfold lower that that of untreated controls (Fig. 1A), the mechanism for p21 up-regulation due to FTI treatment is density-independent. Another observation from our kinetic studies is that the acumulation of p21 due to treatment with FTI is delayed compared with the increase caused by direct DNA damage inflicted with doxorubicin (Fig. 3B). It is possible that this variation reflects the two different mechanism of actions for these drugs. While the effect of doxorubicin is direct and p21 is rapidly induced, the lag time observed in FTI-treated cultures might reflect the half-life of a farnesylated protein.
FTI caused a p21-dependent G1 arrest in only a
few epithelial tumor cell lines, and these all contained wild type p53.
Reduction of p53 expression by introduction of E6 protein prevented FTI induction of p21 (Fig. 5A); cells expressing mutated p53 did
not up-regulate p21 in response to the FTI, whereas wild type cells did
(Fig. 4A), and translocated p21 into the nucleus (data not shown), an indicator of wild type p53 function (36). Second, FTI
induced transcription from the p21 promoter (Fig. 4B) only in p53 wild type cells, and this required the major p53 binding site in
the promoter (Fig. 4B). Deletion of the p53 binding sequence at 1.3 kilobase pairs (19) decreased the magnitude of the FTI induction (Fig. 4B). The truncated promoter might still
contain a weak p53-responsive site at
75 which could account for the residual activity seen by us and others (19, 39). Third, we investigated whether FTI altered p53 levels. In MCF7 cells, p53 levels
were unchanged by FTI (data not shown). However, MCF7 cells express
high basal levels of wild type p53 and p53 activation is not associated
with further increases in its steady-state levels (40, 41). On the
contrary, HCT116 cells have low basal levels of p53, which become
elevated upon DNA damage. In these cells, the FTI led to a modest
induction of p53 (1.5-2-fold increase) as detected by immunoblotting
(Figs. 5A and 6A). Finally, it was reported that
activation of p53 is correlated with up-regulation of cyclin D1
(42-44). We did observe an increase in the steady-state levels of
cyclin D1 (2-3-fold) in response to FTI treatment (Fig. 6B). The weight of the evidence is, then, that FTI induces
p21 via a p53-dependent pathway. The mechanism whereby FTI
activates p53 transcriptional activity is unknown. The p53 effect could result from induction of low level DNA damage, but we have no evidence
for this. It is also possible that a farnesylated protein plays a role
in maintaining DNA stability or functions in the p53 sensor pathway, or
that the FTI suppresses a pathway that down-regulates both p53 function
and DNA replication.
The majority of tumor cell lines with mutated p53 were growth inhibited
by FTI, but did not arrest in G1 (6) (data not shown). It
was not surprising, then, to find that when p21 was knocked out from or
E6 was expressed in tumor cells with wild type p53, FTI still caused
cell growth arrest and death, although it no longer caused
G1 block or inhibition of DNA synthesis (Figs. 6 and 7).
Instead, unscheduled endoreduplication of DNA occurred and polyploid
cells underwent apoptosis (Figs. 6-8). Polyploidy was also induced in
HCT116 p21/
cells by doxorubicin and other chemotherapeutic agents
(28). Although many parallels can be drawn between the response of
p21
/
cells to DNA damaging agents and to the FTI, there are also
differences, as FTI-treated cells entered mitosis (Fig. 8, D
and E), whereas other treatments led to arrest in a
"G2-like state" (16, 28). It is possible that the
mismatch repair deficiency of HCT116 cells (45) may contribute to
polyploidy; however, rereplication was also observed in human and mouse
cells lacking p53, Rb, or p21 when treated with drugs that interfere
with mitotic spindle assembly (25, 46). In addition to allowing cells
to continue to cycle, loss of checkpoint control under these conditions
is associated with bulk replication of DNA and apoptosis (25). The
mechanism underlying this phenomenon is obscure.
In MCF7 and HCT116 cells, the effects of the FTI vary as a function of p53 and p21 status. This reinforces the point that the effects of perturbing particular regulatory pathways are dependent upon the cellular context. HCT116 contains mutated Ki-ras; MCF7 contains wild type ras. The effects of FTI may vary as a function of ras genotype, but in these cells, the FTI effect on cell cycle was primarily dependent on p53 status. These data suggest that the consequences of administering FTI to patients with tumors with or without mutated p53 will be different. Clinical protocols will have to stratify patients for p53 as well as ras status. Tumors with mutant p53 and wild type ras, such as some breast, prostate, and small cell lung cancers, may be especially sensitive to treatment with DNA-damaging agents in combination with FTI.
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ACKNOWLEDGEMENTS |
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The excellent technical assistance of Zhengping Penny Ma and Merna Timaul-Holmes is acknowledged. The authors thank Drs. Nancy Kohl, Jay Gibbs and Allen Oliff (Merck Research Laboratories) for providing the FTI and critical comments, Drs. Todd Waldmann and Bert Vogelstein (John Hopkins University) for the HCT 116 p21 knockout cells, Diane Domingo (Sloan-Kettering Institute) for FACS analysis, Dr. Katia Manova from the Molecular Cytology Core Facility (Sloan-Kettering Institute) for invaluable help with fluorescence microscopy, Susan Krueger from the Center for Biomedical Imaging Technology, University of Connecticut Health Center, for confocal microscopy, and Dr. Gabrielle Tjaden and other members of the Rosen laboratory for helpful discussions.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health NCI Breast SPORE program grant (to N. R.) and Career Development Award P50CA68425-02 (to L. S. L.), and by Merck & Co.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
L. S.-L. dedicates this paper to the memory of Gabriel Lorenzino Sepp.
To whom correspondence should be addressed: Memorial
Sloan-Kettering Cancer Center, 1275 York Ave., Box 333, New York, NY 10021. Tel.: 212-639-2824; Fax: 212-717-3627; E-mail:
l-sepp{at}ski.mskcc.org.
The abbreviations used are: FTI, farnesyl-protein transferase inhibitor; PBS, phosphate-buffered saline; BrdUrd, bromodeoxyrudine; FACS, fluorescein-activated cell sorter.
2 L. Sepp-Lorenzino and N. Rosen, unpublished observations.
3 L. Sepp-Lorenzino, G. Tjaden, M. M. Moasser, M. Timaul-Holmes, Z. Ma, N. E. Kohl, J. B. Gibbs, A. Oliff, H. I. Scher, and N. Rosen, manuscript submittted for publication.
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