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The Journal of Immunology, 2004, 172: 7272-7281.
Copyright © 2004 by The American Association of Immunologists

IL-1{beta} Suppresses Prolonged Akt Activation and Expression of E2F-1 and Cyclin A in Breast Cancer Cells1

Wen Hong Shen*, Steve T. Jackson*, Suzanne R. Broussard*, Robert H. McCusker*, Klemen Strle*, Gregory G. Freund{ddagger}, Rodney W. Johnson{dagger}, Robert Dantzer§ and Keith W. Kelley2,*

* Laboratory of Immunophysiology, {dagger} Laboratory of Integrative Biology, Department of Animal Sciences, and {ddagger} Department of Pathology, College of Medicine, University of Illinois, Urbana, IL 61801; and § Integrative Neurobiology, Formation de Recherche en Evolution, Centre National de la Recherche Scientifique, Unité Mixte de Recherche, Institut National de la Recherche Agronomique, Institut François Magendie des Neurosciences, Bordeaux, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell cycle aberrations occurring at the G1/S checkpoint often lead to uncontrolled cell proliferation and tumor growth. We recently demonstrated that IL-1{beta} inhibits insulin-like growth factor (IGF)-I-induced cell proliferation by preventing cells from entering the S phase of the cell cycle, leading to G0/G1 arrest. Notably, IL-1{beta} suppresses the ability of the IGF-I receptor tyrosine kinase to phosphorylate its major docking protein, insulin receptor substrate-1, in MCF-7 breast carcinoma cells. In this study, we extend this juxtamembrane cross-talk between cytokine and growth factor receptors to downstream cell cycle machinery. IL-1{beta} reduces the ability of IGF-I to activate Cdk2 and to induce E2F-1, cyclin A, and cyclin A-dependent phosphorylation of a retinoblastoma tumor suppressor substrate. Long-term activation of the phosphatidylinositol 3-kinase/Akt signaling pathway, but not the mammalian target of rapamycin or mitogen-activated protein kinase pathways, is required for IGF-I to hyperphosphorylate retinoblastoma and to cause accumulation of E2F-1 and cyclin A. In the absence of IGF-I to induce Akt activation and cell cycle progression, IL-1{beta} has no effect. IL-1{beta} induces p21Cip1/Waf1, which may contribute to its inhibition of IGF-I-activated Cdk2. Collectively, these data establish a novel mechanism by which prolonged Akt phosphorylation serves as a convergent target for both IGF-I and IL-1{beta}; stimulation by growth factors such as IGF-I promotes G1-S phase progression, whereas IL-1{beta} antagonizes IGF-I-induced Akt phosphorylation to induce cytostasis. In this manner, Akt serves as a critical bridge that links proximal receptor signaling events to more distal cell cycle machinery.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The G1/S phase checkpoint is the major cell cycle transition point in which cells are susceptible to extracellular mitotic and antiproliferative signals (1). Progression through the G1/S checkpoint is driven by activation of G1 cyclin-dependent kinases (CDKs), 3 and the enzymatic activity of these G1 CDKs requires heterodimer formation with their cyclin partners (2). G1 CDK activity and G1 cyclin availability define a final common pathway for growth factors (3) and cytostatic cytokines (4). IL-1{beta} is a prototypical proinflammatory cytokine that exerts a plethora of biological activities, including tumor regression (5). The tumor-suppressing property of IL-1{beta} has been attributed mostly to its ability to prime antitumor immunity (6), but the mechanism for its direct cytostatic actions in suppressing cell cycle progression is largely unknown. We recently reported that the antiproliferative action of IL-1{beta} on human breast cancer cells is exhibited not by killing the cells but rather by preventing the ability of the late G1 progression factor, insulin-like growth factor (IGF)-I, to promote progression from late G1 into the S phase of the cell cycle (7). This cross-talk between proinflammatory cytokine and growth factor receptors is similar in principle to that between the B cell receptor and the {beta}2-adrenergic receptor for the neurotransmitter norepinephrine (8) and that between the IGF-I receptor and integrin-associated protein for thrombospondin-1 (9).

Three sets of G1 cyclin-CDK complexes are sequentially assembled to drive G1-S progression in mammalian cells: D-type cyclins plus Cdk4 and Cdk6, cyclin E plus Cdk2, and cyclin A plus Cdk2 (2). Among these complexes, cyclin A-associated CDKs play a critical role in initiation of DNA replication and subsequent S phase entry by phosphorylating components of the DNA replication machinery (10). Activated CDKs hyperphosphorylate and inactivate the retinoblastoma (RB) tumor suppressor protein. This releases E2F transcription factors, allowing them to activate genes required for S phase entry (2). Constitutively elevated expression of cyclin A in breast cancer patients is positively related to early cancer relapse and death (11). Preneoplastic changes, such as nuclear abnormalities and hyperplasia, have been found in mammary tissue-specific cyclin A transgenic mice (12). These oncogenic properties can be mimicked in vitro by ectopic expression of cyclin A, which accelerates entry of G1 cells into the S phase (13).

Activation of phosphatidylinositol 3-kinase (PI3K) or its downstream effectors, primarily Akt, leads to transformation and tumorigenesis of human mammary epithelial cells (14). This oncogenic property of the PI3K/Akt signaling pathway may be directly related to its role in promoting G1 to S phase progression via elevated G1 CDK enzymatic activity (15) and induction of E2F transcriptional activity (16). We recently established that IGF-I causes accumulation of cyclin A and hyperphosphorylation of RB in human MCF-7 breast carcinoma cells by inducing de novo synthesis of the E2F-1 transcription factor (17). More importantly, all these IGF-I-dependent mitotic effects are inhibited by TNF-{alpha}. In addition to cyclin A, IGF-I can also elevate other G1 cyclins, such as cyclin D1 (18) and cyclin E (19). Although important intracellular pathways, such as PI3K/Akt and mitogen-activated protein kinase, have been reported to mediate IGF-I-responsive expression of cyclin D1 (20), little direct evidence is yet available to demonstrate involvement of these two pathways in IGF-I-induced accumulation of cyclin A or cyclin E and the key G1 transcription factor, E2F-1. Moreover, it is unknown whether another important proinflammatory cytokine, IL-1{beta}, causes cytostasis by suppressing these key G1 and S phase regulators.

We recently reported that the cytostatic property of IL-1{beta} is associated with its ability to impair IGF-I-promoted juxtamembrane receptor signaling, specifically, tyrosine phosphorylation of insulin receptor substrate (IRS)-1 (7). In addition, IL-1{beta}-induced G1 arrest reduces the number of IGF-I-induced cycling cells (S plus G2/M), suggesting that components at the G1/S transition point are targeted and regulated oppositely by IL-1{beta} and IGF-I. In this study, we provide direct evidence to confirm this hypothesis by demonstrating that IGF-I increases abundance of cyclin A and E2F-1, the activity of Cdk2 and cyclin A, and both the expression and hyperphosphorylation of RB through a LY294002- and wortmannin-sensitive pathway. IL-1{beta} suppresses all of these IGF-I-induced activities. Moreover, long-term Akt phosphorylation is identified not only as a critical protein that mediates this intracellular communication between IL-1{beta} and IGF-I, but also as an intermediate link between receptor proximal signaling events and distal cell cycle machinery.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and reagents

Human MCF-7 breast carcinoma cells were obtained from the American Type Culture Collection (Manassas, VA) and maintained in MEM supplemented with 10% heat-inactivated FBS (HyClone Laboratories, Logan, UT). For experimentation, cells were plated at 1 x 106 cells/ml in a volume of 2 ml of maintenance medium in six-well Costar plates (Corning, Corning, NY). The next day, cells were washed with 0.15 M PBS followed by a 24 h incubation in culture medium consisting of phenol red-free MEM supplemented with 5 µg/ml human transferrin and 30 nM sodium selenite (Sigma-Aldrich, St. Louis, MO). Cells were treated with different concentrations of IL-1{beta} with or without IGF-I (100 ng/ml, both from Intergen, Purchase, NY) for 24 h. Inhibitors LY294002 (50 µM), PD98059 (50 µM), rapamycin (50 nM), or wortmannin (100 nM) (all from Sigma-Aldrich) were added for 30 min before treatments with IGF-I (100 ng/ml) for either 15 min or 24 h. Abs used for immunoprecipitation and immunoblotting included rabbit anti-human cyclin A (H-432, sc-751; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-human cyclin E (C-19, sc-198; Santa Cruz Biotechnology), rabbit anti-human Cdk2 (M2, sc-163; Santa Cruz Biotechnology), mouse anti-human cyclin A (BF683, sc-239; Santa Cruz Biotechnology), mouse anti-human Cdk2 (D-12, sc-6248; Santa Cruz Biotechnology), mouse anti-human E2F-1 (KH95, sc-251; Santa Cruz Biotechnology), goat anti-human actin (I-19, sc-1616; Santa Cruz Biotechnology), mouse anti-human RB (554136, clone G3-245; BD PharMingen, San Diego, CA), rabbit anti-human phospho-RB (Ser807/811, no. 9308; Cell Signaling, Beverly, MA), rabbit anti-human phospho-RB (Thr356, no. 21-578; BioSource International, Camarillo, CA), rabbit anti-mouse phospho-Akt (Ser473, no. 9271; Cell Signaling), rabbit anti-mouse Akt (no. 9272; Cell Signaling), rabbit anti-human phospho-p70S6K (Thr389, no. 9205; Cell Signaling), rabbit anti-human p70S6K (no. 9202; Cell Signaling), mouse anti-human phospho-ERK (Tyr204, E-4, sc-7383; Santa Cruz Biotechnology), rabbit anti-rat ERK (K-23, sc-94; Santa Cruz Biotechnology), rabbit anti-human p21 (C-19, sc-397; Santa Cruz Biotechnology), HRP-linked donkey anti-goat IgG (Santa Cruz Biotechnology) and HRP-linked donkey anti-rabbit and sheep anti-mouse IgG (Amersham, Arlington Heights, IL).

DNA synthesis

Following incubation for 24 h in serum-free MEM to synchronize cells in G0, MCF-7 cells were washed three times (400 x g), adjusted to 5 x 104 cells/ml in culture medium and plated into 96-well plates (Costar 3596, Corning) in a volume 200 µl per well. Cells were then treated in triplicate with wortmannin (100 nM; Sigma-Aldrich), LY294002 (50 µM), PD98059 (50 µM), or rapamycin (50 µM) for 30 min before IGF-I (100 ng/ml) treatment for an additional 24 h. Cells were pulsed with [3H]TdR (1 µCi per well; 1 Ci = 37 GBq; ICN Pharmaceuticals, Costa Mesa, CA) and harvested onto fiberglass filters with a PHD cell harvester (Cambridge Technology, Cambridge, MA), as we previously described (7). The filters were dried and then submerged in 3 ml of scintillation fluid, and [3H]-radioactivity was determined on a Beckman Coulter LS 6000 IC scintillation counter (Fullerton, CA).

Immunoblotting

Whole cell lysates were prepared from MCF-7 cells in whole cell lysis buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, containing freshly added 1 mM PMSF, 1 mM NaF, 48 trypsin inhibitory units of aprotinin, 40 nM leupeptin, and 2 µg/ml pepstatin; Sigma-Aldrich). Following centrifugation at 16,000 x g at 4°C for 15 min, protein concentration in the supernatant was determined with a protein assay kit (Bio-Rad, Hercules, CA). Equal amounts of protein in whole cell lysates (20–50 µg) were mixed with reducing sample buffer (0.92 M Tris-HCl, pH 8.8, 1.5% SDS, 4% glycerol, and 280 mM 2-ME) and separated in discontinuous SDS-PAGE products. Proteins were transferred with a Bio-Rad Trans-Blot electrophoretic transfer device to Immune-Blot polyvinylidene difluoride membranes. Membranes were blocked for 1 h at room temperature with 1% BSA or 5% skim milk dissolved in TBS (20 mM Tris-HCl, pH 7.4, 150 mM NaCl) supplemented with 0.1% Tween 20. The membranes were then incubated in the same blocking buffer with the indicated Abs for 1 h at room temperature. Blots were extensively rinsed and then incubated with an HRP-labeled species-matched secondary Ab for another 1 h. Immunoreactive bands were visualized using ECL detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ) and exposure to B-PLUS X-ray film (Central Illinois X-ray, Bloomington, IL). Reprobing was performed following incubation of membranes in heated (>55°C) stripping buffer (100 mM 2-ME, 2% SDS, 62.5 mM Tris-Cl, pH 6.7) for 30 min. Intensity of immunoreactive protein bands on autoradiograms was quantified by scanning with an Agfa Duoscan T1200 scanner followed by analysis using GelExpert 3.5 software (NucleoTech, San Mateo, CA). To account for minor variations in protein loading into the gels, data were calculated as a ratio of the densitometric intensity of the protein of interest relative to its loading control.

Immunoprecipitation, in vitro kinase assay, and in vivo association of Cdk2 and cyclin A

Following incubation with different concentrations of IL-1{beta} with or without IGF-I (100 ng/ml) for 24 h, MCF-7 cells were lysed with ice-cold CDK lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 0.1% Triton X-100, 10 mM 0.1% Nonidet P-40, 1 mM EDTA, 2.5 mM EGTA, 10 mM glycerophosphate, with freshly added 50 mM NaF and 1 mM DTT, 1 mM PMSF, 1 mM NaF, 48 trypsin inhibitory units of aprotinin, 40 nM leupeptin, and 2 µg/ml pepstatin). Cdk2 or cyclin A was immunoprecipitated from equal amounts of protein (~200 µg) from whole cell lysates. Immunoprecipitates were washed three times with CDK lysis buffer and twice with CDK buffer (50 mM HEPES, pH 7.5, 10 mM MgCl2, 10 mM MnCl2, and 1 mM DTT). Kinase reactions were conducted by incubation of immune complexes in 50 µl reaction buffer (20 µM cold ATP, 5 µCi [{gamma}-32P]ATP, and ~0.5–1 µg CDK substrate) at 37°C for 30 min. Both a truncated RB protein (p56RB, amino acids 379–928; QED, San Diego, CA) and histone H1 (Roche Diagnostics, Indianapolis, IN) were used as CDK substrates. Reactions were terminated by addition of 20 µl SDS sample buffer, and the samples were subsequently subjected to 10% SDS-PAGE. Following fixation in 20% methanol and 10% acetic acid for 30 min, gels were dried in a slab gel drier (SGD4050; Furma Scientific, Marietta, OH) and phosphorylation of p56RB peptide or histone H1 was detected with either a PhosphorImager (Typhoon 8600; Molecular Dynamics, Piscataway, NJ) or with B-PLUS X-ray film (Central Illinois X-ray).

Aliquots of Cdk2 immunoprecipitates that were used for the in vitro kinase activities were used for measurement of in vivo association of cyclin A with Cdk2. Briefly, the Cdk2 immunoprecipitates were mixed with a nonreducing sample buffer (0.92 M Tris-HCl, pH 8.8, 1.5% SDS, 4% glycerol) and maintained at room temperature for 1 h before electrophoresis on 10% SDS-PAGE gels. Electrophoresis under nonreducing conditions prevented reduced H chain and L chain of the rabbit immunoprecipitating Ab from masking appearance of both cyclin A (~50 kDa) and Cdk2 (~30 kDa), respectively. As an additional control for these experiments, immunoblotting was conducted using a monoclonal mouse cyclin A Ab, which was subsequently detected with an HRP-conjugated sheep anti-mouse Ab. Equal amounts of Cdk2 in each immunoprecipitate sample were confirmed by reprobing the same membrane with a monoclonal Cdk2 Ab.

Statistical analysis

Statistical analyses were performed using the Statistical Analysis System for Microsoft Windows (22). All data, including standardized densitometric intensities from replicate autoradiograms, were analyzed as a completely randomized design using standard ANOVA procedures. Treatment differences were assessed by Duncan’s multiple range tests. All experiments were independently replicated at least three times and data were summarized as a mean ± SEM. Two-sided values of p < 0.05 or p < 0.01 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IGF-I-induced elevation of both E2F-1 and cyclin A and hyperphosphorylation of RB occurs by a LY294002-sensitive, but PD98059- and rapamycin-insensitive pathway

IGF-I has long been known as a late G1 progression factor (23), which occurs by regulating expression of proteins at the G1/S checkpoint (24). Our recent experiments confirmed this idea by showing that IGF-I causes quiescent cells to exit G0, nearly doubling the proportion of cells in S phase from 8% to 15% (7). Cell cycle progression can be regulated by three major signaling pathways: PI3K (25), mammalian target of rapamycin (mTOR) (26), and extracellular signal-regulated kinase (ERK)1 and ERK2 (27). Akt and p70S6K are downstream signaling effectors of PI3K and mTOR, respectively, and phosphorylation of Akt (Ser473) and p70S6K (Thr389) is required for full activation of Akt (28, 29) and p70S6K (28, 29). We first conducted experiments to determine whether IGF-I activates these proteins and whether blocking concentrations of LY294002 (30), rapamycin (31), and PD98059 (32) would specifically block them.

MCF-7 cells were treated for 30 min with each inhibitor before IGF-I stimulation. As shown in Fig. 1A, IGF-I (100 ng/ml for 15 min) induced site-specific phosphorylation of Akt, p70S6K, and ERK1/2. Moreover, the inhibitors potently and specifically blocked IGF-I-stimulated activation of each pathway. For example, PD98059 and rapamycin independently prevented IGF-I from phosphorylating ERK1/2 and p70S6K, respectively, whereas LY294002 blocked the ability of IGF-I to phosphorylate both Akt and p70S6K. LY294002 inhibits PI3K by competing with ATP for binding to the p110 catalytic subunit (30), whereas another commonly used selective PI3K inhibitor, wortmannin, covalently binds and inhibits PI3K (33). This mechanistic difference did not distinguish their specificity in blocking IGF-I early signaling, as shown by similar inhibition of short-term IGF-I-induced activation of both Akt and p70S6K by wortmannin (data not shown). These data are consistent with our earlier findings with murine myeloid progenitor cells (34). Interestingly, we found that both Akt and p70S6K, but not ERK1/2 (data not shown), remained phosphorylated as long as 24 h following addition of IGF-I (Fig. 1B). As expected, we observed a similar specificity of inhibition in prolonged IGF-I-induced activation of Akt and p70S6K by LY294002 and rapamycin as was observed with the shorter, 15 min exposure to IGF-I. In contrast, wortmannin specifically blocked IGF-I-induced prolonged Akt phosphorylation at Ser473 without affecting sustained activation of p70S6K.



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  		FIGURE 1. DNA synthesis and expression of cell cycle proteins are induced by IGF-I via a PI3K/Akt-sensitive pathway. A, Inhibitors of PI3K (LY294002, 50 µM), mTOR (rapamycin, 50 nM), and ERK1/2 (PD98059, 50 µM) were added to human MCF-7 breast carcinoma cells 30 min before stimulation with IGF-I (100 ng/ml) for another 15 min. Activation of the three different pathways were analyzed by Western blot analysis with phospho-specific Abs, and total amount of Akt, p70S6K, and ERK were used as loading controls. B, Akt and p70S6K were activated by long-term exposure to IGF-I.The PI3K inhibitors, LY294002 and wortmannin (100 nM), both blocked Akt phosphorylation at Ser473. However, phosphorylation of p70S6K was blocked by LY294002, but not by wortmannin. C, MCF-7 cells were washed and arrested in G0 by culturing for 24 h in serum-free medium. The PI3K inhibitor LY294002 (50 µM), the ERK1/2 inhibitor PD98059 (50 µM) or the mTOR inhibitor rapamycin (50 nM) were added 30 min before IGF-I (100 ng/ml), and the treatments continued for an additional 18 h. After cell labeling with [3H]TdR for an additional 6 h, DNA synthesis was measured by [3H]TdR incorporation. Data represent the mean ± SEM of three independent experiments. Although all three inhibitors significantly reduced the ability of IGF-I to induce DNA synthesis, the inhibition caused by LY294002 was significantly greater than that caused by either PD98059 (p < 0.01) or by rapamycin (p < 0.05). D, MCF-7 cells were incubated with IGF-I (100 ng/ml) for 24 h following a 30 min preincubation with LY294002 (50 µM), PD98059 (50 µM), or rapamycin (50 nM). Western blot analysis was performed with specific Abs for E2F-1, cyclin A, and RB in whole cell lysates. {beta}-actin was used as a loading control. A representative Western blot is shown, followed by graphs that represent the means ± SEM of densitometric ratios of each protein relative to the loading control in three independent experiments. **, p < 0.01. E, Wortmannin (100 nM) was added to MCF-7 cells 30 min before IGF-I treatment for 24 h. Western blotting was performed as described in D.

 
We then tested the ability of these pharmacologic compounds at the same concentrations to inhibit IGF-I-induced DNA synthesis. As we previously reported (7), IGF-I significantly (p < 0.01) increased DNA synthesis in MCF-7 cells (Fig. 1C). In the presence of the PI3K inhibitor, LY294002, the ability of IGF-I to promote DNA synthesis was completely blocked. Similar experiments with wortmannin (100 nM) showed that it inhibited IGF-I-induced DNA synthesis by 76 ± 2% (p < 0.01, data not shown). Rapamycin was not as potent as LY294002 in reducing IGF-I-induced DNA synthesis. The ERK1/2 inhibitor, PD98059, was the least efficacious in reducing the ability of IGF-I to promote DNA synthesis. Indeed, the inhibition caused by LY294002 was significantly greater than that caused by either PD98059 (p < 0.01) or rapamycin (p < 0.05). This inhibition was not due to cytotoxicity because in the absence of IGF-I, none of the inhibitors reduced basal [3H]TdR incorporation into DNA (Fig. 1C) or cell viability (data not shown). These results confirm those of others (35, 36) and point to the importance of the PI3K-related pathway in mediating IGF-I-induced DNA synthesis in MCF-7 cells.

As an important G1 cyclin, cyclin A is synthesized at onset of the S phase (10). Expression of cyclin A is mediated by the E2F-1 transcription factor (37) and activation of E2F-1 is directly related to hyperphosphorylation of the RB protein (38). To investigate the role of IGF-I in regulating these critical G1/S checkpoint proteins, we analyzed expression of G1 cyclins and E2F-1, as well as RB phosphorylation in cells treated with IGF-I for 24 h. IGF-I caused a dramatic accumulation in cyclin A (Fig. 1, D and E), although it did not affect expression of either cyclin E or cyclin D1 in these cells (17). IGF-I also increased expression of E2F-1 and caused RB phosphorylation at both Ser807/811 and Thr356 (Fig. 1E). IGF-I increased (p < 0.01) the amount of RB (as determined by relative densities of both the hypo- and hyperphosphorylated forms) compared with that of MCF-7 cells in medium only. LY294002 completely blocked IGF-I-induced E2F-1 and expression of both cyclin A and RB (Fig. 1D). Similar inhibition was observed with wortmannin treatment for 24 h (Fig. 1E). More specifically, IGF-I-induced site-specific phosphorylation of RB at both Ser807/811 and Thr356 was inhibited by wortmannin. In contrast, neither rapamycin nor PD98059 affected the ability of IGF-I to increase expression of any of these G1 regulators or to phosphorylate RB. Although activation of p70S6K was blocked by both LY294002 and rapamycin (Fig. 1, A and B), it is important to note that the IGF-I-induced increase in the amount of cyclin A, E2F-1, and RB was inhibited by LY294002 (Fig. 1D) and wortmannin (Fig. 1E) in a rapamycin-independent manner. Collectively, these experiments indicate that the PI3K/Akt pathway plays a major role in the ability of IGF-I to increase expression of both E2F-1 and cyclin A and to cause hyperphosphorylation of RB.

IL-1{beta} suppresses IGF-I-stimulated Cdk2 activity and formation of cyclin A-Cdk2 complexes

Growth factor-stimulated G1-S progression can often be inhibited by cytostatic cytokines (4, 39). We recently demonstrated that IL-1{beta} reduces the ability of IGF-I to increase the proportion of cycling MCF-7 cells (S plus G2/M) by 73% (7). Importantly, IL-1{beta} did not inhibit G1 progression in the absence of IGF-I. Because Cdk2 is a critical CDK that drives the G1-S transition (40), we immunoprecipitated Cdk2 from cells treated with IL-1{beta}, IGF-I, or both and measured Cdk2 enzymatic activity in vitro. IGF-I significantly (p < 0.01) activated Cdk2 activity, as shown by its ability to phosphorylate both a truncated RB and histone H1 substrates (Fig. 2A). More importantly, IL-1{beta} dose dependently inhibited IGF-I-induced Cdk2 activity. IL-1{beta} did not affect Cdk2 activity in the absence of IGF-I.



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  		FIGURE 2. IL-1{beta} dose dependently blocks IGF-I-induced Cdk2 activity and prevents formation of cyclin A-Cdk2 complexes. Cdk2 was immunoprecipitated from MCF-7 cells treated with different concentrations of IL-1{beta} in the absence and presence of IGF-I (100 ng/ml) for 24 h. A, Cdk2 enzymatic activity was measured with both truncated RB (p56) and histone H1 as substrates. A representative autoradiogram is shown, followed by densitometric summaries of three independent experiments. B, Cyclin A association with Cdk2 was detected by Western blotting of the Cdk2 immunoprecipitates (IP) with a specific Ab to cyclin A. A representative Western blot is shown, followed by a densitometric analysis (mean ± SEM) of three independent experiments. **, p < 0.01 as compared with the cells treated with IGF-I (100 ng/ml) alone.

 
Enzymatic activity of Cdk2 largely depends on its association with G1 cyclins (41). In this study we focused upon cyclin A because the amount of this protein (Fig. 1C), but not that of cyclin E or cyclin D1 (data not shown), was increased by 24 h treatment with IGF-I in MCF-7 cells. To investigate the physical association between cyclin A and Cdk2, amount of cyclin A was measured in Cdk2 immunoprecipitates derived from cells treated with IGF-I, IL-1{beta}, or both. These coimmunoprecipitation experiments showed that IGF-I greatly increased the amount of cyclin A that associates with Cdk2 (Fig. 2B). This induction was not due to an increase in Cdk2 protein, as shown by equal amounts of Cdk2 in each immunoprecipitate. Cotreatment with IL-1{beta} dose dependently reduced IGF-I-induced formation of the cyclin A-Cdk2 complexes. IL-1{beta} alone did not alter the association of Cdk2 with cyclin A. These data establish that IL-1{beta} prevents formation of cyclin A-Cdk2 complexes only in the presence of IGF-I.

IL-1{beta} targets IGF-I-stimulated, cyclin A-dependent CDK activity to inhibit RB phosphorylation

Because IL-1{beta} impairs formation of IGF-I-induced cyclin A-Cdk2 complexes, cyclin A itself may serve as a target for the antagonism between IL-1{beta} and IGF-I. To test this idea, we precipitated cyclin A instead of Cdk2 with a specific Ab from cells treated with different concentrations of IL-1{beta} with or without IGF-I (100 ng/ml) and measured the ability of cyclin A-associated CDKs to phosphorylate their substrates in vitro. As expected, IGF-I increased enzymatic activity ofcyclin A-associated CDKs, leading to phosphorylation of both histone H1 and a truncated RB peptide (Fig. 3). IL-1{beta} inhibited this IGF-I-stimulated cyclin A-dependent CDK activity in a dose-dependent manner. In contrast, IL-1{beta} itself did not affect cyclin A-dependent phosphorylation of either truncated RB or histone H1. These results show that IL-1{beta} suppresses IGF-I-triggered cyclin A expression and enzymatic activity that leads to RB phosphorylation and inactivation.



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  		FIGURE 3. IGF-I-stimulated cyclin A-associated CDK enzymatic activity is blocked by IL-1{beta}. Cyclin A was immunoprecipitated from cells treated as described in Fig. 2. Cyclin A-associated CDK activity was measured using in vitro kinase assays with both truncated RB peptide (p56) and histone H1 as CDK substrates. This experiment was independently repeated three times. Means ± SEM are shown in graphs representing the densitometric summary of each treatment. *, p < 0.05; **, p < 0.01 as compared with the cells treated with IGF-I (100 ng/ml) alone.

 
IL-1{beta} dose- and time-dependent inhibition of IGF-I up-regulated expression of cyclin A and E2F-1 and hyperphosphorylation of RB

The ability of IL-1{beta} to inhibit IGF-I-stimulated cyclin A activity and RB phosphorylation (Fig. 3) may directly result from a reduction in IGF-I-induced accumulation of cyclin A (Fig. 1D). Because IGF-I induces synthesis of new E2F-1 protein, which is required for cyclin A accumulation (17), IL-1{beta} may also suppress IGF-I-induced E2F-1 expression. To test this hypothesis, we analyzed expression of both cyclin A and E2F-1, as well as different phosphorylation forms of RB, in MCF-7 cells treated with different concentrations of IL-1{beta} in the absence and presence of IGF-I (100 ng/ml). A concentration-dependent reduction in IGF-I activity by IL-1{beta} was detected in the amount of cyclin A and E2F-1 (Fig. 4A).



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  		FIGURE 4. IL-1{beta} dose dependently inhibits IGF-I-stimulated accumulation of E2F-1 and cyclin A as well as hyperphosphorylation and amount of RB. A, Western blot analysis was performed with specific Abs to cyclin A or E2F-1 on whole cell lysates of MCF-7 cells treated with different concentrations of IL-1{beta} in the absence and presence of IGF-I (100 ng/ml) for 24 h. B, The same whole cell lysates from all treatments were blotted with the G3-245 mouse anti-human RB Ab. Density of the hypo- and hyperphosphorylated forms of RB was determined, and the results presented as both a ratio of hyperphosphorylated to hypophosphorylated RB (ppRB/pRB) and total amount of RB (ppRB + pRB). Graphs (A and B, bottom) represent the mean ± SEM of densitometric ratios of the appropriate protein relative to the loading control from three independent experiments (n = 3). C, Independent experiments, including the same treatments, were then conducted with site-specific RB Abs in 8% polyacrylamide gels. IGF-I increased phosphorylation of residues pSer807/811 (pS807/811) and pThr356 (pT356) of RB, and these events were inhibited by 1 ng/ml IL-1{beta}. These changes in RB site-specific phosphorylation were much more prominent than the changes of total RB amount, as shown by analyzing the same samples in 10% polyacrylamide gels and blotting with Ab G3-245. The loading control, {beta}-actin, varied <10% in all experiments. *, p < 0.05; **, p < 0.01. D, The kinetics of IL-1{beta} inhibition on IGF-I-induced cyclin A, E2F-1, and RB are represented. MCF-7 cells were treated with IL-1{beta} (20 ng/ml), IGF-I (100 ng/ml), or both for the indicated times. Expression of cyclin A, E2F-1, and RB protein was detected by Western blotting.

 
Several experiments were then conducted to more fully characterize the role of IL-1{beta} in inhibiting IGF-I-induced expression and phosphorylation of RB. Intensity of both the hypo- and hyperphosphorylated forms of RB was expressed as both a ratio (ppRB:pRB) and sum (ppRB plus pRB). This summary revealed that IL-1{beta} significantly impaired IGF-I-induced hyperphosphorylation of RB as well as the total amount of RB, although the former occurred at a five-fold lower concentration. We then used site-specific RB Abs to more specifically begin to determine the phosphorylation sites on RB that are regulated by both IGF-I and IL-1{beta} (Fig. 4C). These experiments demonstrated that as little as 1 ng/ml IL-1{beta} potently impaired both IGF-I induced phosphorylation of residues pSer807/811and pThr356 of RB. To determine the time course in which IL-1{beta} acts to inhibit these G1-S regulators that are induced by IGF-I releasing the cells from G0, we treated MCF-7 cells simultaneously with IL-1{beta} and IGF-I for different periods of time and analyzed the expression of E2F-1, cyclin A and RB, as well as site-specific phosphorylation of RB at either Ser807/811 or Thr356. Consistent with our recent report (17), we found IGF-I induces all three G1 regulators in a time-dependent manner (Fig. 4D). A dramatic inhibition by IL-1{beta} occurred at 12 h, and some inhibition was observed at earlier time points. Because MCF-7 cells require at least 12 h to exit G1 to begin synthesizing DNA, this finding is consistent with the idea that IL-1{beta}-induced G1 arrest (7) results from the inhibitory effects of IL-1{beta} on IGF-I-induced G1-S regulators. Importantly, these reductions caused by IL-1{beta} were manifested only in the presence of IGF-I. To determine whether cyclin A is the only G1 cyclin that is regulated by the combination of IL-1{beta} and IGF-I, we also analyzed the amount of cyclin E and cyclin D1. We found no effect of IL-1{beta} on the amount of these two proteins, either in the absence or presence of IGF-I (data not shown). These data indicate that IL-1{beta} impairs both the expression and phosphorylation status of the RB protein, but the phosphorylation state of RB is reduced at lower concentrations of IL-1{beta} than concentrations required to reduce the amount of RB. Collectively, these results establish that IL-1{beta} inhibits the ability of IGF-I to up-regulate key proteins during the G1-S transition.

IL-1{beta} induces expression of p21Cip1/Waf1

Mitogen-dependent progression through G1-S transition is regulated by an integrated interplay of G1 CDKs, cyclins, and CDK inhibitors. As the first recognized CDK inhibitors, p21Cip1/Waf1 can induce G1 arrest and block S phase entry by inactivating Cdks (42). A defect in p21Cip1/Waf1 increases susceptibility to chemically induced skin carcinoma formation (43), and over-expression of p21Cip1/Waf1 effectively suppresses tumor growth (44). IL-1{beta} has been shown to induce p21Cip1/Waf1 in human melanoma cells, leading to G1 arrest (45). IL-1{beta} may also stimulate expression of p21Cip1/Waf1 in our system, which may contribute to the impairment of IGF-I-induced Cdk2 (Fig. 2A) activity. To test this idea, p21Cip1/Waf1 was analyzed in MCF-7 cells treated with increasing concentrations of IL-1{beta}. As shown in Fig. 5, IL-1{beta} dose dependently induces p21Cip1/Waf1. Significant induction occurred when cells were treated with as little as 0.1 ng/ml IL-1{beta}, suggesting that the induction of p21Cip1/Waf1 may contribute to IL-1{beta} inhibition of IGF-I-stimulated Cdk2 activation.



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  		FIGURE 5. Induction of p21Cip1/Waf1 by IL-1{beta}. Following 24 h serum deprivation, human MCF-7 cells were treated with different doses of IL-1{beta} for 24 h. Expression of p21Cip1/Waf1 was analyzed by immunoblotting, and {beta}-actin was used as a loading control. A densitometric analysis (mean ± SEM) of three independent experiments is shown in the graph. *, p < 0.05; **, p < 0.01 as compared with the medium control.

 
IL-1{beta} inhibits IGF-I-induced sustained phosphorylation of Akt, but not p70S6K or ERK

IGF-I up-regulates both cyclin A and E2F-1 through a LY294002-sensitive pathway (Fig. 1C), indicating that a PI3K/Akt-related pathway may be responsible for this regulation. A logical question is whether the IGF-I-induced activation of Akt is also a target for IL-1{beta}. To test this possibility, we determined whether IL-1{beta} inhibits not only the PI3K/Akt pathway but also the mTOR/p70S6K and ERK1/2 pathways, which are activated by short-term treatment (15 min) with IGF-I in MCF-7 cells (Fig. 1B). As shown in Fig. 6, activation of both the PI3K/Akt and mTOR/p70S6K pathways remained detectable even following a 24 h continuous exposure to IGF-I. Cotreatment with IL-1{beta} reduced IGF-I-induced Akt phosphorylation by 75%. IL-1{beta} pretreatment for up to 24 h did not inhibit the immediate (15 min) IGF-I-induced activation of PI3K/Akt, mTOR/p70S6K, or ERK1/2 pathways (data not shown).



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  		FIGURE 6. IL-1{beta} targets prolonged Akt activation, but not activation of mTOR, to suppress IGF-I receptor signaling. Site-specific Ser/Thr phosphorylation of Akt and p70S6K was analyzed by Western blot lysates from MCF-7 cells treated with IGF-I (100 ng/ml), IL-1{beta} (10 ng/ml), or their combination for 24 h. Amount of Akt and p70S6K was verified by stripping and reprobing the same membrane with Abs directed to these proteins. The two graphs represent the mean ± SEM of the densitometric ratio of phospho-proteins relative to total amount of the proteins from three independent experiments; **, p < 0.01.

 
The ability of IGF-I to induce sustained p70S6K phosphorylation was unaffected by IL-1{beta}, suggesting that IL-1{beta} targets activation of Akt, but not mTOR/p70S6K, to inhibit IGF-I receptor signaling. The IGF-I-activated ERK1/2 pathway was transient, remaining detectable when cells were exposed to IGF-I for 15 min (Fig. 1B) but not for 24 h (data not shown). We did not detect any reduction in ERK phosphorylation when MCF-7 cells were coincubated with both IL-1{beta} and compared with IGF-I alone for 10, 20, or 40 min, which were all times when IGF-I was able to induce ERK1/2 phosphorylation (data not shown). Similar results were observed at 24 h, indicating that the ERK1/2 pathway is unlikely to be targeted by IL-1{beta}. These experiments point to PI3K/Akt signaling as the critical pathway that is responsible for IL-1{beta} inhibition of the ability of IGF-I to promote cell proliferation and increase expression of critical G1/S checkpoint proteins.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Acting as a prototypical proinflammatory cytokine, IL-1{beta} is recognized as one of the most pleiotropic cytokines involved in a variety of different biological events. IL-1{beta} inhibits IGF-I-induced tyrosine phosphorylation of IRS-1 and prevents IGF-I from causing cells to enter the S phase of the cell cycle, leading to G1 arrest (7). In this report, we dissect the cell cycle machinery at the G1-S boundary to reveal Akt as a key convergent protein that is targeted by both IGF-I and IL-1{beta} to regulate cell cycle progression. We recently established that IGF-I induces synthesis of E2F-1 transcription factor (Fig. 1D), which is responsible for promoting expression of downstream genes like cyclin A (17). IGF-I promotes transition through the G1 restriction point by inducing cyclin A accumulation and RB hyperphosphorylation (Fig. 1D). A LY294002- and wortmannin-sensitive, but rapamycin- and PD98059-insensitive, pathway mediates IGF-I-induced DNA synthesis (Fig. 1C), hyperphosphorylation of RB, and accumulation of both cyclin A and E2F-1 (Fig. 1D). IL-1{beta} suppresses IGF-I-induced activation of Akt (Fig. 6). As a consequence, IL-1{beta} antagonizes the ability of IGF-I to trigger activation of Cdk2 (Fig. 2), accumulation of both cyclin A and E2F-1 (Fig. 4A), expression and hyperphosphorylation of RB (Fig. 4, B and C), and ultimately, G1-S progression. In the absence of IGF-I, IL-1{beta} does not affect any of these molecular events, which is consistent with our previously established model that the major inhibitory properties of proinflammatory cytokines on growth of breast cancer cells are manifested prominently in the presence of growth factors (7).

IGF-I receptor signaling occurs via binding to its transmembrane receptor and activation of the type I IGF receptor’s intrinsic tyrosine kinase activity. This tyrosine kinase activity leads to recruitment and activation of the IRS docking proteins and cytoplasmic signaling enzymes. These early membrane signal transduction events have been widely investigated and elucidated. Similarly, the major downstream consequences of these early signaling events, in this case cell cycle progression, have also been studied and clarified to a large extent. However, understanding the links between upstream signaling events and downstream cell cycle progression is rather limited. How do cells select from a variety of signaling cascades that are initiated by growth factors to cause them to pass through the restriction point of the cell cycle? Data in this report provide evidence that IGF-I causes changes directed toward S phase entry by increasing the amount of cyclin A and E2F-1 and the subsequent hyperphosphorylation and inactivation of RB. These events and passage of cells through the cell cycle are completely blocked by PI3K inhibitors, but ERK1/2 or mTOR inhibitors do not affect them.

The ERK1/2 (46, 47) and mTOR (48, 49, 50) pathways can be involved in the G1-S transition by regulating cyclin D1 expression. However, our results demonstrate that IGF-I induces G1-S key regulators by activating the PI3K/Akt signaling cascade or related kinases in a manner independent of ERK1/2 and mTOR. The PI3K-specific inhibitor, wortmannin, blocks IGF-I-responsive prolonged activation of Akt, but not that of p70S6K (Fig. 1B). Blockage of prolonged PI3K/Akt activation with wortmannin also suppresses the ability of IGF-I to elevate both E2F-1 and cyclin A and to hyperphosphorylate RB (Fig. 1E). Interestingly, IL-1{beta} acts just like wortmannin by inhibiting IGF-I-induced Akt phosphorylation at Ser473 (Fig. 6), up-regulation of both E2F-1 and cyclin A (Fig. 4A) and phosphorylation of RB (Fig. 4, B and C). LY294002 blocks phosphorylation of both Akt and p70S6K (Fig. 1B). However, blockage of the mTOR pathway with rapamycin does not affect the ability of IGF-I to stimulate accumulation of cyclin A, activation of E2F-1 or phosphorylation of RB (Fig. 1D), the key molecular indicators for passage into the S phase in response to IGF-I. Wortmannin blocks the activation of both Akt and p70S6K evoked by short pulse of IGF-I (data not shown). However, the inhibition remains at 24 h for only IGF-I-stimulated phosphorylation of Akt, but not that of p70S6K (Fig. 1B). These data suggest that the PI3K/Akt pathway may not be responsible for IGF-I-induced prolonged activation of p70S6K. Although it is well accepted that mTOR/p70S6K resides downstream of PI3K/Akt (51), the mTOR pathway can also be regulated independently of PI3K (52, 53). Other signaling molecules, such as newly synthesized amino acids (52, 53), may mediate prolonged IGF-I-induced signals associated with activation of mTOR/p70S6K. These data not only provide direct evidence that identifies an important role for PI3K/Akt in mediating the engagement of IGF-I-stimulated early signaling events to subsequent cell cycle progression, but also suggest that IL-1{beta} specifically targets the PI3K/Akt pathway to suppress IGF-I-induced key G1-S regulators. Our results suggest that other signaling events downstream of PI3K/Akt mediate IGF-I-induced key G1-S regulators in an mTOR/p70S6K-independent manner.

IGF-I increases DNA synthesis (Fig. 1C), an indicator of cells entering the S phase, by 40-fold in MCF-7 cells. Consistent with elimination of IGF-I-induced increase in cyclin A, E2F-1 and RB phosphorylation (Fig. 1D), LY294002 completely abrogates the ability of IGF-I to induce DNA synthesis (Fig. 1C). Another PI3K-specific inhibitor, wortmannin, also significantly suppresses IGF-I-induced DNA synthesis. The failure of IGF-I to promote DNA synthesis in the presence of these inhibitors confirms an essential role of PI3K, or other PI3K-related kinases (54), in mediating IGF-I-induced cell proliferation. Structural and mechanistic differences between wortmannin and LY294002 may be responsible for their different levels of inhibition. Wortmannin inhibits PI3K by covalently binding to the p110 catalytic subunit (33), whereas LY294002 competes with ATP for binding to p110 (30). In addition, the shorter half-life (~90 min) of wortmannin (55) may also explain its reduced potency in inhibiting the relative long process of induction of DNA synthesis.

Growth factors often trigger a rapid burst of signaling, such as phosphorylation of the growth factor receptor and receptor adaptor proteins, recruitment and activation of PI3K and phosphorylation of Akt, all of which occur within seconds and subside within an hour, even in the continuous presence of growth factors (55). In comparison, over 12 h are required for a G0 fibroblast to progress to the S phase, of which 8–10 h are required for cells to reach the G1-S restriction point (56). It has been difficult to explain why a transient signal, lasting <60 min, is sufficient to drive cells through the G1 restriction point, which begins 8 h after exposure to growth factors. Recent experiments have revealed that there is an additional wave of signaling in response to growth factor stimulation. Although the first burst of signaling is dispensable, the second wave, which occurs 3–7 h poststimulation, is required for DNA synthesis and S phase entry (55, 57). In our experimental setting, activation of the PI3K/Akt pathway, shown by phosphorylation of Akt at Ser473, remains detectable following constant IGF-I stimulation for 24 h (Figs. 1B and 6) at a level that is comparable to 15 min of IGF-I simulation (Fig. 1A). In agreement with the two-wave concept, inhibition of Akt activation with LY294002 (Fig. 1D) and wortmannin (Fig. 1E) eliminates the increase in E2F-1/cyclin A and RB hyperphosphorylation following a 24-h continuous exposure to IGF-I. Interestingly, prolonged activation of both Akt and p70S6K is blocked by LY294002, whereas wortmannin only specifically blocks Akt phosphorylation (Fig. 1B). More importantly, the sustained, but not the immediate, IGF-I-responsive activation of Akt is largely inhibited when cells are simultaneously exposed to IL-1{beta} (Fig. 6), in a manner similar to wortmannin. In contrast, although activation of p70S6K is also detectable after a 24-h exposure to IGF-I, IL-1{beta} does not affect the ability of IGF-I to phosphorylate p70S6K at Thr389 (Fig. 6). Because a PI3K-related pathway and Akt activation mediate IGF-I-promoted G1-S transition and cell proliferation in an mTOR/p70S6K-independent manner (Fig. 1D), IL-1{beta} inhibits IGF-I-induced signaling (Figs. 2, 3, and 4) by targeting the pathway that is essential for IGF-I to promote cell cycle transition (Fig. 6). As for ERK1/2 pathway, IGF-I-induced ERK1/2 phosphorylation can be detected only with a 15-min treatment (Fig. 1A), but the signal is not detectable at 24 h (data not shown). These results exclude a possible role for the ERK1/2 pathway in mediating either IGF-I stimulation or IL-1{beta} inhibition of cyclin A, phosphorylation of RB or E2F-1.

Cells are responsive to extracellular growth factors only during an interval between the early to mid-G1 phase and the restriction point of the cell cycle (58). Phosphorylation of RB marks the end of this interval and enables cells to transit through the restriction point, leading to completion of the cell cycle even in the absence of growth factors. RB phosphorylation is directly related to the activity of G1 CDKs, accumulation of G1 cyclins and activation of E2F transcription factors. In this report, we demonstrate in MCF-7 breast cancer cells that IGF-I activates Cdk2, the vital G1 CDK that is necessary for cells to pass through the G1/S restriction checkpoint. This event occurs concurrently with accumulation of cyclin A and its association with Cdk2, hyperphosphorylation of RB and elevation and activation of E2F-1. Significantly, all these cell cycle-accelerating properties of IGF-I are eliminated when MCF-7 cells are simultaneously exposed to the proinflammatory cytokine, IL-1{beta}. These data also establish an essential role for PI3K/Akt pathway, through mTOR/p70S6K and ERK-independent mechanisms, as a convergent target in mediating IGF-I-induced G1/S transition and IL-1{beta}-induced antagonism. IL-1{beta} impairs IGF-I-induced Akt activation. In the absence of Akt activation by IGF-I, IL-1{beta} does not affect any of the G1-S regulators.

As a surrogate risk factor for development of various cancers, elevated concentrations of IGF-I are associated with uncontrolled cell proliferation by activating key signaling enzymes, such as PI3K/Akt, and promoting cell cycle progression by triggering critical G1-S regulators, such as RB, E2F, and G1 cyclin. The present results establish an intracellular mechanism of cross-talk between the immune (IL-1{beta}) and neuroendocrine (IGF-I) systems, both of which are expressed in the microenvironment of tumors, in regulating the growth of breast cancer cells. In this scenario, leukocyte-derived IL-1{beta} manifests its cytostatic G1 arresting property only when cells are driven into a cycling state by IGF-I. Identification of prolonged PI3K/Akt activation as an intracellular target for this IL-1{beta}-IGF-I interaction in regulating breast cancer cell cycle progression not only confirms the requirement of sustained IGF-I signaling for the transition through the restriction point, but also identify cytoplasmic proteins that provide a link between juxtamembrane early signaling events and the nuclear cell cycle machinery.


   Footnotes
 
1 This work was supported by Grant AI50442 from the National Institutes of Health (to K.W.K.). Back

2 Address correspondence and reprint requests to Dr. Keith W. Kelley, Laboratory of Immunophysiology, Department of Animal Sciences, University of Illinois, 207 Edward R. Madigan Laboratory, 1201 West Gregory Drive, Urbana, IL 61801. E-mail address: kwkelley{at}uiuc.edu Back

3 Abbreviations used in this paper: CDK, cyclin-dependent kinase; IGF, insulin-like growth factor; PI3K, phosphatidylinositol 3-kinase; mTOR, mammalian target of rapamycin; IRS, insulin receptor substrate; RB, retinoblastoma; S6K, ribosomal protein S6 kinase; ERK, extracellular signal-regulated kinase. Back

Received for publication January 27, 2004. Accepted for publication April 5, 2004.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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