1 McArdle Laboratory for Cancer Research, Department of Oncology, Room 506A, 1400 University Ave, Madison, WI 53706, USA
2 Present address: Howard Hughes Medical Institute, Department of Cellular and Molecular Medicine, HHMI/CMM-West, Room 219, University of California-San Diego School of Medicine, Mail Code 0686, 9500 Gilman Drive, La Jolla, CA 92093-0686, USA
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Abstract |
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Abbreviations: POH; monoterpene perillyl alcohol; TGFß; transforming growth factor ß; Thr-160; threonine-160
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Introduction |
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The monoterpenes have been shown to have diverse cellular and molecular effects both in vivo and in vitro. The induction of hepatic phase I and phase II detoxifying enzymes by monoterpenes may underlie their blocking action at the initiation stage of chemical carcinogenesis (2,6,7). In addition, monoterpenes can modulate the mevalonate pathway at several points distal to HMG-CoA reductase, including inhibition of the isoprenylation of certain small G proteins (810), and inhibition of both the synthesis of ubiquinone and the conversion of lathosterol to cholesterol (11). Dietary monoterpene-induced tumor regression is associated with changes in the cellular level of several gene products, especially those involved in the transforming growth factor ß (TGFß) signaling pathway (12,13). For example, in regressing mammary carcinomas, monoterpene induced the expression of mannose 6-phosphate (M6P)/insulin-like growth factor II (IGF-II) receptor, which binds a variety of extracellular ligands, including the mammary mitogen IGF-II, and mediates their endocytosis and degradation (14). In contrast, this receptor also participates in the activation of the secreted latent TGFß (15). In vivo, the anticancer effects of monoterpenes on liver, colon and mammary tumorigenesis are associated with inhibition of cellular proliferation and induction of apoptosis in regressing tumors (9,10,13,16,17). In vitro, limonene and perillyl alcohol inhibit cell proliferation (10) and induce apoptosis (K.Kim and M.N.Gould, unpublished data) in a variety of cell lines at doses within a pharmacologically achievable range. In this study, we focus on mechanisms underlying the cytostatic effect induced by monoterpene perillyl alcohol (POH) in the transformed murine mammary epithelial TM6 culture model.
Progression through the cell cycle is governed by a family of serine/threonine protein kinase complexes, which are composed of both a regulatory subunit (the cyclin) and a catalytic subunit (the cyclin-dependent kinase or Cdk) (18). The oscillation of specific Cdk activities dictates the temporal order of cell-cycle events. For example, one of the important molecular events required for passage through a rate-limiting G1 phase preceding DNA synthesis, or the restriction point, is the inactivation of the retinoblastoma susceptibility gene product pRB by hyperphosphorylation by Cdks (19). pRB phosphorylation prevents pRB from binding with and regulating E2F transcription factors and thus allows the expression of E2F-responsive genes necessary for DNA replication and cell-cycle progression (20,21).
The activation of Cdks is regulated by various mechanisms including proteinprotein interactions (with cyclins, Cdk inhibitors and assembly factors), protein degradation, transcriptional control, subcellular localization and multiple phosphorylation (22,23). D-type cyclins are induced as part of the delayed early responses to mitogenic stimuli. They form active holoenzymes with their catalytic partners Cdk4 and Cdk6 by mid-G1, and are required for the initiation of G1S phase (19,24). Cyclin D-associated Cdks are responsible for the initial phosphorylation of pRB family proteins (18). Expression of cyclin E and the onset of its associated Cdk2 kinase activities is delayed relative to D-type cyclins, and is also rate-limiting for the G1S progression of the cell cycle (18). The cyclin E- and cyclin A-associated Cdk2 complete and/or maintain phosphorylation of pRB (18). One group of important negative regulators for Cdks are the Cdk inhibitors (CKIs), which fall into two distinct families based on their sequence homology. One family of CKIs includes p16INK4a, p15INK4b, p18INK4c and p19INK4d. They act specifically on cyclin DCdk4/6 complexes by preventing cyclin D from binding (23,25). The second family of Cdk inhibitors includes p21WAF1, p27Kip1 and p57Kip2. They were thought to be potent inhibitors of a wide range of cyclinCdk complexes (23,25). However, recent data suggest that Cip/Kip family inhibitors selectively inhibit cyclinCdk2 activities and are required for the activation for the cyclin DCdk4/6 in vivo (2628). p21 is known to accumulate in response to DNA damage in a p53-dependent manner and can be induced in a p53-independent fashion (23).
Furthermore, the activities of Cdks can be both negatively or positively regulated by phosphorylation of critical residues in Cdks (22,29). Phosphorylation of a highly conserved threonine (Thr)-160 residue by a Cdk-activating kinase (CAK) is required for the Cdk activity (22). Interestingly, p21WAF1 or p27Kip1 in the Cip family or p18INK4c in the INK4 family can prevent CAK from phosphorylating the Cdk subunit by making the Thr-160-like site inaccessible to the CAK enzyme (30,31).
In this study, we have chosen the murine mammary epithelial TM6 cells as a model to study the effect of POH on cell proliferation and cell-cycle progression. We have found that POH treatment inhibited TM6 proliferation in a dose-dependent manner and caused cell-cycle arrest in the early G1 phase and slowed the cell-cycle progression at the G2M phase. The early G1 arrest was associated with the reduction in both cyclin D1-associated and cyclin E-associated kinase activities. POH treatment led to a reduction of cyclin D1 levels in asynchronous cells and prevented the formation of active cyclin D1-associated kinase complexes in synchronous cells during the exit of G0. The reduction of cyclin E-associated kinase activity is consistent with an increased p21 association with cyclin E-associated kinase and a reduction of the activating phosphorylation of Cdk2 at the Thr-160 site.
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Materials and methods |
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Cell culture
The murine transformed mammary epithelial TM6 cell line (32,33) was a generous gift from Dr Daniel Medina (Baylor College of Medicine, Houston, TX). Cells were maintained in Dulbecco's modified Eagle medium (DMEM)/Ham's F12 (1:1) medium (Sigma), pH 7.5, supplemented with HEPES buffer, heat-inactivated bovine serum (2%, HyClone Laboratories, Logan, UT), insulin (10 µg/ml, Collaborative Biomedical Products, Bedford, MA), epidermal growth factor (5 ng/ml, EGF, Collaborative Biomedical Products) and gentamycin (5 µg/ml, Gibco BRL, Gaithersburg, MD) at 37°C in 95% humidified air and 5% CO2. The cells were routinely passaged using 0.1% trypsinEDTA. To achieve synchronization at the G0 phase of the cell cycle, TM6 cells were serum- and growth factor-starved for 48 h prior to the replacement with complete medium. To achieve a late G1early S phase synchronization, TM6 cells were treated with 200 mM mimosine (Sigma, stock solution prepared in 1 M NH4OH) for 16 h, then washed and fed with complete medium.
Proliferation assay
One day before treatment, TM6 cells were plated into 6 well (35 mm diameter) culture plates in triplicate. Cells were either untreated or treated with various concentrations of POH. The cells were trypsinized and collected at the start of treatment (0 h), 9 and 19.5 h afterwards. The viable cells determined by trypan blue (Gibco BRL) exclusion were counted using a hemacytometer.
Cell-cycle analysis
TM6 cells were labeled with 20 mM bromodeoxyuridine (BrdU; Sigma) for 20 min, then trypsinized, harvested and fixed in 70% ethanol0.34% Tween-20 at 4°C for at least 1 h. After digestion with pepsin, the resultant nuclei were incubated with a mouse anti-BrdU antibody (Becton Dickinson, San Jose, CA), followed by a fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary antibody (Sigma). The nuclear DNA was then stained with propidium iodide (PI; Boehringer Mannheim, Indianapolis, IN) overnight. Both DNA content and BrdU incorporation data were acquired and analyzed on a FACScan flow cytometry system (Becton Dickinson). Cell-cycle distribution was analyzed using ModFit LT 2.0 (Verity Software House, Topsham, ME) or CellQuest (Becton Dickinson Immunocytometry Systems, San Jose, CA) software. In all cases, proper gating was used to exclude doublets and other cell aggregates for analysis.
To follow cell-cycle progression through the S and G2M phases, actively proliferating TM6 cells were pulse-labeled with 1 mM BrdU (Sigma) for 30 min, and then the medium was replaced by fresh medium either with or without 0.5 mM POH. The cells were recovered at the start of treatment (0 h), 2, 5, 10 and 15 h afterwards, and flow cytometric assays were performed (20 000 events/determination). The BrdU-positive cells were gated for DNA content analysis using CellQuest software (Becton Dickinson).
Western blot analysis
Cell monolayers were washed twice with ice-cold phosphate-buffered saline (PBS) and cells were then scraped into ice-cold PBS and pelleted by centrifugation. The cell pellets were stored at 80°C before analyses. Cell lysates were prepared in ice-cold lysis buffer [50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol, 1 mM dithiothreitol (DTT), 0.1% Tween-20, 10 mM ß-glycerophosphate, 1 mM NaF, 0.1 mM sodium orthovanadate, 0.1 mM PMSF, 1 mg/ml aprotinin, 20 mM leupeptin] followed by sonication 20 s in an ice-bath. The lysates were then clarified by centrifugation at 10 000 g for 15 min, and protein concentrations were determined by the bicinchonine acid (BCA) protein assay kit (Pierce, Rockford, IL). Thirty micrograms of protein per lane were resolved on either 12 or 15% SDSPAGE gels for most analyses, or on 7.5% SDSPAGE gels for pRB phosphorylation, and then transferred to 0.45 mm PVDF filter membranes (Millipore Corporation, Bedford, MA). The membranes were incubated with a specific antibody followed by a second antibody (anti-mouse IgG or anti-rabbit IgG) conjugated with alkaline phosphatase (Sigma) and proteins of interest were visualized using the Vistra Fluorescence Western blotting system (Amersham Life Science, Arlington Heights, IL) and a FluorImager (Molecular Dynamics, Sunnyvale, CA). Protein abundance was quantified using ImageQuant Software (Molecular Dynamics). The data displayed are representative of three or more independent experiments.
In vitro immune complex kinase assay and immunoprecipitation
The cell lysates were prepared as described as above. Equivalent amounts of protein were pre-cleared by incubating with normal serum and protein A/Gagarose (Cytosignal, San Diego, CA or Santa Cruz Biotechnology). For analysis of cyclin D1- or cyclin E-associated kinase activity, the kinase complexes were immunoprecipitated with 10 µl of mouse monoclonal anti-cyclin D1 (72-13G, Santa Cruz Biotechnology) or rabbit polyclonal anti-cyclin E (M-20, Santa Cruz Biotechnology) antibody, respectively, for 4 h followed by incubation with 20 µl of protein A/Gagarose (CytoSignal) for an additional 2 h. Immunoprecipitates were recovered by centrifugation, washed twice with lysis buffer, three times with base buffer (50 mM HEPES, pH 8.0, 10 mM MgCl2 and 1 mM DTT), and once with kinase buffer (50 mM HEPES, pH 8.0, 10 mM MgCl2, 2.5 mM EGTA, 1 mM DTT, 20 µmM ATP, 10 µM ß-glycerophosphate, 0.1 mM sodium orthovanadate, 1 mM NaF). The kinase reaction was carried out at 30°C for 30 min by resuspending the beads in 40 µl of kinase buffer containing 10 mCi [-32P]ATP (Dupont NEN) and 0.8 µg glutathione S-transferase (GST)C-terminal pRB (QED Bioscience, San Diego, CA) to determine cyclin D1-associated kinase activity (250 µg protein/reaction), or that containing 2 µg histone H1 (Sigma) and 2 mCi [
-32P]ATP to determine cyclin E-associated kinase activity (100 µg protein/reaction) before it was terminated by addition of 5x SDS Laemmli's sample buffer. Unlabelled ATP (1 µl of 10 mM stock solution) was also added to each kinase reaction mixture following termination to reduce background. The reaction mixture was boiled for 5 min and resolved on 10% SDSPAGE gels, which were later dried. Incorporation of 32P into GSTpRB or histone H1 was quantitated using a PhosphorImager with ImageQuant software (Molecular Dynamics). The data displayed are representative of three independent experiments.
For examining the physical proteinprotein interaction, the above pre-cleared cell lysates were adjusted by adding Nonidet-40 lysis buffer [50 mM TrisHCl, pH 7.5, 150 mM NaCl, 0.5% Nonidet-40, 50 mM NaF, 1 mM DTT, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF), 25 µg/ml leupeptin, 25 µg/ml aprotinin and 10 µg/ml trypsin inhibitor] as described by Jenkins and Xiong (34). The proteins of interest were immunoprecipitated with 10 µl of a specific antibody for 4 h followed by incubation with 20 µl of protein A/Gagarose (Santa Cruz biotechnology, or CytoSignal) for an additional 2 h. The agarose beads were recovered by centrifugation, and washed four times with Nonidet P-40 lysis buffer. After the final aspiration of lysis buffer, equal volume of 2x SDS Laemmli's sample buffer was added to the agarose and the precipitated proteins were denatured by boiling for 3 min, and separated in a 15% SDSPAGE gel. The proteins of interest were detected by western blot analysis as described above.
Northern blot analysis
Total cellular RNA was extracted using RNeasy Mini kit (Qiagen, Chatsworth, CA). Ten micrograms of total RNA per lane were separated by formaldehyde1% agarose gel electrophoresis, transferred to Zeta-Probe® nylon blotting membranes (Bio-Rad, Hercules, CA) using PosiBlot® Pressure Blotter (Stratagene, La Jolla, CA), and crosslinked using UV Stratalinker 2400 (Stratagene). A DNA probe used for murine p21 mRNA detection was generated by PCR (1 min at 94°C, 1 min at 55°C, 1 min at 72°C) from cDNAs synthesized from mRNA isolated from murine Neuro-2A cells using SuperScript cDNA synthesis kit (Gibco BRL) using the primer pair: 5'-CACAGCGATATCCAGACATTC-3' and 5'-CCTCCTGACCCACAGC-3'. Human cyclin-D1 cDNA was kindly provided by Dr David Boothman (Case Western Reserve University, Cleveland, OH). All DNA probes were labeled with [-32P]dATP or [
-32P]dCTP using DECAprime II kit (Ambion, Austin, TX) and unincorporated nucleotides were removed by NICK® columns (Pharmacia, Piscataway, NJ). The hybridization was performed using QuickHyb (Clontech, Palo Alto, CA). Bands of interest were analyzed using a PhosphorImager with ImageQuant software (Molecular Dynamics). Equivalent loading of RNA samples was confirmed by hybridization with a DNA probe complementary to ribosomal 36B4.
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Results |
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Effects of POH on cell-cycle progression in synchronous TM6 cells
We next focused on examining the effects of POH on the G1S progression of cells synchronized either by release from quiescence (G0) or from late G1. TM6 cells were synchronized in G0 phase by serum and growth factor starvation for 48 h and followed by re-feeding with complete growth medium. Cell-cycle progression after release from serum starvation was evaluated by flow cytometric analysis for both BrdU incorporation and DNA content (Figure 2C). Between 70 and 80% of the cells were arrested in the G0 phase after serum starvation for 48 h. Ten hours after release by serum addition, 30% of TM6 cells progressed into early S phase. After 15 h, 60% of cells entered the S phase, while after 21 h the majority of cells passed through the G2M phase and entered the subsequent G1 phase. In contrast, the majority (6580%) of cells exposed to 0.5 mM POH concurrently with serum addition remained in the G0G1 phase.
We then examined the cell-cycle progression in TM6 cells synchronized by the plant non-protein amino acid mimosine. Mimosine has been shown to reversibly arrest cells in the late G1 or early S phase (3537). The cells were synchronized by release from mimosine incubation. Cell-cycle progression in the presence of or absence of POH was evaluated by flow cytometric analysis of BrdU incorporation and DNA content. After release from the mimosine block, both untreated cells and cells treated with POH entered the early S phase in ~2 h (Figure 2D). Within 4.5 h, similar percentages of cells entered the S phase. POH treatment did not prevent the onset of S phase and the S to G2M cell-cycle progression up to this point. Twelve hours after release, the control cells completed mitosis and progressed through the S phase, while the cells treated with POH were blocked at the G2M phase and/or the subsequent G0G1 phase. Thus, the G1 cell-cycle blockage point of POH appears to be located at early G1 to mid-G1. Once the cells pass into the late G1, the completion of S phase seems to be less sensitive to POH treatment.
Inhibition of pRB phosphorylation and cyclin D1- and cyclin E-associated Cdk activities by POH treatment in asynchronous cells
As pRB phosphorylation has been associated with the G1S transition, we investigated the phosphorylation status of pRB in response to POH treatment in asynchronous cells. Phosphorylated pRB is indicated by a retarded electrophoretic mobility in denaturing polyacrylamide gels in comparison to unphosphorylated or hypophosphorylated pRB. In asynchronously growing TM6 cells, the majority of pRB was hyperphosphorylated (the slower migrating forms). In contrast, in cells exposed to 0.5 mM POH for 5 and 15 h, the percentage of cells in the G1, S, G2M phases were 62.9, 6.0 and 15.6% for the POH-treated cells versus 36.1, 39.5 and 14.4%, respectively, for the untreated cells. The hyperphosphorylated pRB forms (slower migrating) were reduced, as the hypophosphorylated forms (faster migrating) increased (Figure 3A). Therefore, POH treatment resulted in the inhibition of pRB phosphorylation.
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Effects of POH on levels of cyclins Cdks, and the Cdk inhibitor p21WAF1 in asynchronous cells
To address how the cyclin-associated kinase activities are affected by POH treatment, we examined the expression of cyclins and Cdks involving the G1S transition in asynchronous TM6 cells. POH treatment reduced the cyclin D1 protein levels by 14% (P = 0.053, n = 3) after 5 h and by 47% (P = 0.017, n = 3) after 15 h, respectively (Figure 4A). Such a reduction in cyclin D1 protein levels might be responsible for the reduction in the cyclin D1-associated kinase activity after 15 h (Figure 3B
). Meanwhile, no difference in the levels of cyclin E and Cdk4 proteins were observed in these cells (Figure 4B
), whereas cyclin A protein levels were slightly but reproducibly reduced, followed by a reduction of the percentage of cells in the S phase after 15 h treatment of POH (Figure 4B
).
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We also examined the effect of POH treatment on the phosphorylation of Cdk2 at Thr-160 site, which is required for the full activation of Cdk2 (22). The detection of the Thr-160 phosphorylated Cdk2 benefited from its faster electrophoretic mobility on denaturing polyacrylamide gels compared with that of the unphosphorylated form (38). In asynchronous TM6 cells, POH treatment for 15 h caused a decrease in the abundance of the faster migrating form of Cdk2, which represents the active Thr-160 phosphorylated form (Figure 4E). Such a decrease of the active form of Cdk2 occurs later compared with the increased binding of p21WAF1/Cip1 with cyclin ECdk2 complexes (Figure 4D
) and correlated well with the reduction of cyclin ECdk2 kinase activity (Figure 3C
).
Inhibition of pRB phosphorylation and G1 Cdk activities by POH treatment in synchronous cells
We also followed up the changes induced by POH treatment in synchronous TM6 cells released from serum starvation. In cells under serum and growth factor starvation (in the G0 phase), the majority of pRB was unphosphorylated (the faster migrating band, Figure 5A). Starting from 5 h after release from the serum starvation, the pRB became phosphorylated (the slower migrating forms) as the cells underwent the G1 to S transition and entered the S phase subsequently. However, pRB in cells under POH remained as the slower migrating forms, presumably unphosphorylated or hypophosphorylated, as the cells were arrested at the G0G1 phase during the period of POH treatment.
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Effects on the levels of cyclins, Cdks, p21 and the Cdk2 phosphorylation by POH treatment in synchronous cells
To understand how cyclin D1-associated kinase activities were affected by POH treatment in the synchronous cells, we examined the cyclin D1 protein levels. As predicted, cyclin D1 protein levels increased as cells exited quiescence and progressed through the G1 and S phase. Whereas over 70% cells did not progress through the G1 phase under POH treatment, surprisingly similar levels of cyclin D1 were present in the untreated and the 0.5 mM POH-treated cells (Figure 6A) despite an attenuated induction of cyclin D1 mRNA levels (Figure 6C
) and lack of cyclin D1-associated kinase activities in the POH-treated cells (Figure 5B
). Only POH at a dose of 1 mM blocked the induction of cyclin D1 protein totally along with the induction of the cyclin D1-associated kinase activity (data not shown). POH treatment did not change the protein levels of Cdk4 either (Figure 6A
). Furthermore, when the association of Cdk4 and cyclin D1 was examined by western blot analysis in cyclin D1 immunoprecipitate, comparable levels of D1 were associated with Cdk4 in the POH-treated cells and in the untreated cells (Figure 6A
). Similar levels of Cdk6 were associated with cyclin D1 in the untreated and in the POH-treated cells (Figure 6B
). The results suggest that some as yet unknown mechanisms, other than prevention of cyclin D1 and Cdk4/6 interaction but still forming functional cyclin D1Cdk4/6 complexes, were responsible for the POH inhibition of cyclin D1-associated kinase activities during the exit of G0.
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In synchronized TM6 cells, the p21 message and protein levels increased as the cells exited quiescence (Figure 6C and D). The total cellular p21 protein levels in the POH-treated cells were modestly but consistently higher (by 1030%) than those in the control cells (Figure 6D
). Furthermore, the association of p21 with the cyclin ECdk2 complex was increased by 2- and 5-fold at 10 and 15 h, respectively, by POH treatment (Figure 6D
) in contrast to the minimal p21 association with cyclin E in untreated cells. POH treatment further increased p21 transcript levels compared with untreated cells (Figure 6C
). Meanwhile, the levels of p21 associated with the cyclin D1Cdk complexes were generally comparable in both the POH-treated cells and the control cells (Figure 6B
). Therefore, the increase of p21 in cyclin ECdk2 complexes may also result from a specific interaction with in the cyclin ECdk2 complexes independently of the increase in total cellular p21 protein levels.
In addition, Cdk2 protein levels remained constant under 0.5 mM POH while increased 2-fold in the control (Figure 6A). Using 15% SDSPAGE gel, the different phosphorylated forms of Cdk2 at Thr-160 site can be separated. The relative abundance of the active form of Cdk2 (faster migrating) increased at 15 h as the untreated cells progress through the G1 and S phase. Such an increase was modestly inhibited in the POH-treated cells (Figure 6E
). A comparison of the effects of POH on cell-cycle related molecules is given in Table I
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Discussion |
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POH treatment affects the cell cycle by causing a G1 arrest and a transient G2M arrest. Extended POH exposure results in an accumulation of cells predominantly in the G1 phase (data not shown). Examination of the cell-cycle progression of the BrdU pulse-labeled cells further suggested that the G2M arrest caused by POH treatment is incomplete and transient. The underlying mechanism of the G2M arrest induced by POH has yet to be identified. POH treatment blocked the S-phase entry in synchronous cells released from serum starvation; however, it did not affect the S phase progression in cells pre-treated with and released from mimosine. Therefore, the POH-induced G1 arrest occurs at early to mid-G1. Such an arrest position appears to reside earlier than the late G1 proximal to G1S border or at early S phase, the acting point of mimosine induced cell-cycle arrest (35).
We have thus focused on studying the molecular factors involved in the regulation of G1S transition, as they are less likely the consequence of cell-cycle arrest. In contrast, the expression of factors associated with other cell-cycle phases, such as the reduced expression of S phase cyclin A, may be due to lack of cell-cycle progression. To distinguish the molecular changes associated with the G1 block and the cause of G1 arrest, we have examined these changes in both asynchronous cells and synchronous cells released from serum starvation. We found that the early G1 arrest may be in part related to the effect of POH treatment on pRB phosphorylation. In addition, POH treatment reduced cyclin D1-associated kinase activities. Furthermore, POH treatment induced an increase in the association of Cdk inhibitor p21 with cyclin ECdk2 complexes and inhibited the activating phosphorylation of Cdk2.
Among all changes associated with the G1 arrest, we found that an increase of p21 association with cyclin ECdk2 complexes might be important in causing G1 arrest. Such an increase of p21 binding with cyclin Ecdk2 complexes happens in asynchronous cells after 5 h exposure to POH, whereas there is no major change in cell-cycle distribution. The paradox is that at this 5 h timepoint we did not observe a major inhibition of the cyclin E-associated kinase activities. It is possible that the inhibition of cyclin E-associated kinase activity may also require an inhibition of activating phosphorylation of Cdk2 at Thr-160 site. Hitomi et al. has reported that elevated p21 association with cyclin ECdk2 induced by prostaglandin A2 could not completely account for the reduction in kinase activity and the activating phosphorylation of Cdk2 at the Thr-160 site which correlates most closely with the inhibition of kinase activities (41). However, here the reduction in active phosphorylation of Cdk2 appears later than the induction of p21WAF1. Although we could not exclude the possibility that POH treatment might affect CAK, we suggest that POH treatment induced p21 association with cyclin ECdk2 complexes. However, the consequence of this interaction, the major inhibition of cyclin E-associated kinase activity, may occur later when the Cdk2-activating phosphorylation is inhibited as a result of a blockage of CAK from phosphorylating Cdk2 by p21.
The increased p21 association with cyclin ECdk2 complexes thus may have resulted partly from the increase in total cellular p21 protein levels and partly from a specific proteinprotein interaction. In TM6 cells, POH exposure (at 0.5 mM) resulted in a 2-fold increase in the p21 mRNA levels after 5 h treatment. The increase in p21 mRNA levels caused by POH treatment may be due to increased p21 gene transcription, as such an increase is sensitive to the RNA synthesis inhibitor actinomycin D (data not shown). p21 was initially found under the transcriptional control of wild-type p53, especially following DNA damage (23). However, p21 can also be induced by p53-independent mechanisms. In the TM6 cell line, both alleles of the p53 gene are mutated with a Trp138 substitution and a D123129 allele in p53 gene (33). Therefore, the upregulation of p21 mRNA by POH is independent of functional p53 protein. Based on our biochemical studies of these monoterpenes and findings by others on p21-inducing agents, we speculate a possible linkage of the POH-induced inhibition of protein isoprenylation with p21 induction. POH treatment inhibits the mevalonate pathway downstream of HMG-CoA reductase (8,10,11). This inhibition includes inhibiting geranylgeranyl protein transferase (GGPT) activity (42). Interestingly, the HMG-CoA reductase inhibitor lovastatin and specific GGPT inhibitors induces p21 mRNA independent of p53 (43,44). Furthermore, the geranylgeranylated small G protein Rho has been linked to a repression of p21 upregulation in response to Raf activation (45,46). The induction of p21 by POH treatment is potentially mediated through the disruption of isoprenylated protein functions.
Compared with the POH-induced increase in the p21 message RNA levels, the increase in total cellular p21 protein levels ranged from 0.5- to 2-fold in POH treated asynchronous cells and modestly increased by 1030% in synchronous cells when compared with the control cells. Nevertheless, POH treatment caused a more dramatic increase in the association of p21 with cyclin ECdk2 complexes. In contrast, the association of p21 with cyclin D1Cdk complexes did not increase. It is yet unclear how such a specific increase of p21 association with cyclin E complex was achieved by POH treatment. It has been proposed that an important function of D-type cyclins is acting to sequester p21 from binding with cyclin E complexes (25). It is possible that the reduction of D-type cyclins by POH treatment could lead to more p21 available for interacting with cyclin E complexes.
In addition, in synchronous cells released from serum starvation (during the cell-cycle entry from quiescence), POH treatment prevented the induction of cyclin D1-associated kinase activity. In contrast, the inhibition of cyclin D1-associated kinase activities by POH treatment in asynchronous cells was less dramatic. This could be due to differences between cells that move from a quiescent state to a proliferative state and actively proliferating cells traversing G1. As the former cells exit from the G0 phase, cyclin D1 is synthesized in the cytoplasm, assembled with its kinase partners Cdk4 and Cdk6 into complexes, and translocated into the nuclei in response to serum-mediated signaling (47). POH treatment may block the induction of cyclin D1-associated kinases by preventing the synthesis or activation of unidentified rate-limiting activating regulator(s), which are responsible for the formation or proper localization of active cyclin DCdk complexes. In contrast, in actively cycling cells, the activating factor(s) for cyclin DCdk complexes are not rate-limiting. POH-induced inhibition of cyclin D1-associated kinase activity reflects the downregulation of cyclin D1 proteins.
Furthermore, surprisingly equivalent levels of cyclin D1Cdk6 and cyclin D1Cdk4 complexes exist in control and 0.5 mM POH-treated synchronized TM6 cells released from serum starvation despite the strong inhibition of cyclin D1-associated kinase activity caused by POH treatment. Therefore, some mechanisms other than those preventing cyclin D1Cdk6 complex formation could be involved to inhibit cyclin D1Cdk activities. Among a number of possibilities, it is unlikely due to currently known CKIs. The inhibitors of the INK4 family would disrupt the association of Cdk4/6 with cyclin D (23,25). In addition, the members of the Kip family, p21 and p27, were found to be associated with cyclin D1; however, their association may play activating roles in cyclin DCdk4/6 complex formation (25). In our case, no correlation between the p21 or p27 association and the inhibition of cyclin DCdk4/6 activity was found following POH treatment (Figures 4D, 6B and data not shown). Alternatively, POH treatment might affect the post-translational modifications of cyclin D1Cdk4/6 complexes, such as the phosphorylation state of Cdk4 and Cdk6. For example, the tyrosine phosphorylation regulation of Cdk4 has been shown to be associated with the cell-cycle exit into the G0 phase, contact inhibition (48), and G1 arrest induced by UV irradiation and TGFß treatment (49,50). Finally, POH treatment could prevent the formation of active cyclin D1Cdk4/6 holoenzymes by affecting additional factors in cyclin D1Cdk4/6 complexes and subcellular localization of the complexes. It has been shown by Mahony et al. that only the nuclear localized cyclin DCdk6 complexes with estimated molecular mass of 150170 kDa are catalytically active in T cells (51). Similar complexes were also observed in mammary epithelial cells (52). The identity and nature of the components of the active holoenzymes are as yet unknown.
In summary, POH can cause both apoptosis and cell cycle arrest both in vivo and in vitro (13). Interestingly, in certain in vitro systems POH preferentially induces either apoptosis or cell-cycle arrest. In the cell line studied here, the predominant effect of POH is cell-cycle arrest in G1. This inhibition was associated with a reduction in both cyclin D- and E-associated kinase activities. The data may provide insight in exploring which tissues and tumor types are likely to respond to POH treatment in both cancer prevention and treatment applications. This could help direct future clinical trials.
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Notes |
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Acknowledgments |
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References |
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