The Cyclin-Dependent Kinase 2 Inhibitor Down-regulates Interleukin

The Cyclin-Dependent Kinase 2 Inhibitor Down-regulates

Interleukin-1B-Mediated Induction of Cyclooxygenase-2

Expression in Human Lung Carcinoma Cells

Partha Mukhopadhyay, M. Aktar Ali, Animesh Nandi, Peter Carreon,Hak Choy, and Debabrata Saha

Department of Radiation Oncology, Division of Molecular Radiation Biology, University of TexasSouthwestern Medical Center, Dallas, Texas

Abstract

Overexpression of cyclooxygenase-2 (COX-2) is frequentlyobserved in several human cancers, including lung, colon,and head and neck. Malignancies are also associated with thedysregulation of cell cycle events and concomitant elevatedactivity of cyclin-dependent kinases (CDK). CDK2 is a key cellcycle regulatory protein that controls the transition ofcells from G1 to S phase. In this study, we furnish severallines of evidence that show a functional role for the CDK2in interleukin-1B (IL-1B)–induced COX-2 expression in H358human non–small cell lung carcinoma cell line by blockingCDK2 activity. First, we show that BMS-387032, a potent CDK2inhibitor, blocks IL-1B-induced expression as well as steady-state mRNA levels of COX-2. Second, we show that smallinterfering RNA that abrogates CDK2 expression also blocksIL-1B-induced COX-2 expression. Third, results from in vitrokinase assays clearly show that IL-1B induces CDK2 activityin H358 cells and this activity is significantly inhibited byBMS-387032. Moreover, CDK2 inhibition blocks IL-1B-inducedbinding to the NF-IL6 element of the COX-2 promoter andinhibits transcription of the COX-2 gene. We also observedthat BMS-387032 does not inhibit endogenous expression ofCOX-2 or prostaglandin synthesis in lung carcinoma cells.Finally, we provide evidence showing that IL-1B-inducedsignaling events, such as p38 mitogen-activated proteinkinase, phosphorylated stress-activated protein kinase/c-JunNH2-terminal kinase, phosphorylated AKT, and phosphory-lated extracellular signal-regulated kinase 1/2, are notinhibited by CDK2 inhibitor. Taken together, the data suggestthat CDK2 activity may play an important event in the IL-1B-induced COX-2 expression and prostaglandin E2 synthesis andmight represent a novel target for BMS-387032. (Cancer Res2006; 66(3): 1758-66)

Introduction

The cell cycle is an exquisitely controlled and ordered sequenceof events, culminating in cell growth and division. Perturbation ofcell cycle regulation is a key factor in most human neoplasias (1).The regulatory proteins that play key roles in controlling cell cycleprogression are the cyclins, cyclin-dependent kinases (CDK), their

substrate proteins, the CDK inhibitors, and the tumor suppressorgene products, p53 and pRb. Several CDK inhibitors, such asflavopiridol, UCN-01, CYC202, and BMS-387032, are undergoingclinical evaluation and showing promising results in early clinicaltrials (2, 3). Flavopiridol inhibits multiple CDK activities, includingCDK2, CDK4, CDK6, and CDK7. A new class of CDK inhibitorN-acyl-2-aminothiazoles with nonaromatic acyl side chains (BMS-387032) is reported to be very selective and acts as a competitivesmall-molecule inhibitor of CDK2/cyclin E complex by interactingwith the ATP-binding site of CDK2, blocking cell cycle progressionand tumor cell proliferation and inducing tumor cell apoptosis (4).The IC50 of BMS-387032 toward CDK2/cyclin E is 48 nmol/L,whereas the IC50 of BMS-387032 toward CDK4/cyclin D and CDK1/cyclin B is 925 and 480 nmol/L, respectively (4). In A2780 ovariancarcinoma cell line, BMS-387032 inhibits CDK2 phosphorylationand is shown to inhibit the phosphorylation of the downstreamtargets of CDK2, including pRb, histone H1, and DNA polymerasea (1, 4).Prostaglandins, the primary metabolites of cyclooxygenase-2

(COX-2) catalyzed oxygenation of arachidonic acid, are a diversegroup of autocrine and paracrine hormones. They mediate manycellular and physiologic processes, such as cell proliferation,inflammatory and immune responses, bone development, woundhealing, hemostasis, reproductive function, glomerular filtration,and production of extracellular matrix proteins (5–9). There is alsoa strong positive correlation between overexpression of COX-2and tumorigenesis. COX-2 is overexpressed in a variety of differenttumors, including colon, lung, pancreatic, prostate, and head andneck cancers, and correlates with poor prognosis and shortenedsurvival (10–13). This increased expression of COX-2 andproduction of prostaglandins seem to provide a survival advantageto transformed cells through the inhibition of apoptosis, increasedattachment to extracellular matrix, increased invasiveness, andstimulation of angiogenesis. Given the multifarious roles of COX-2in normal physiology, it is critically important to identify agentsthat inhibit COX-2 expression in tumor cells without affectingCOX-2 expression and activity in normal cells. Because activelyproliferating tumor cells have elevated CDK activities and severaltumors also express COX-2, we investigated whether cell cycleregulatory kinases may have any role in the induction of COX-2gene expression.Chronic inflammation is linked to the development of cancer

in several organs (14). Interleukin-1h (IL-1h), an inflammatorycytokine, induces COX-2 gene expression in several cancer cell linesthrough mechanisms that involve p38 and p44/p42 mitogen-activated protein kinase (MAPK) signaling pathways (15, 16).Induction of COX-2 expression by IL-1h operates at the trans-criptional level involving transcription factors, such as nuclear

Requests for reprints: Debabrata Saha, Department of Radiation Oncology,Division of Molecular Radiation Biology, University of Texas Southwestern MedicalCenter, 2201 Inwood Road, Dallas, TX 75390-9187. Phone: 214-648-7750; Fax: 214-648-5995; E-mail: [email protected].

I2006 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-05-3317

Cancer Res 2006; 66: (3). February 1, 2006 1758 www.aacrjournals.org

Research Article

Research. on January 2, 2019. © 2006 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

factor-nB (NF-nB) and CCAAT/enhancer-binding protein (C/EBP),as well as at the level of mRNA stabilization in a extracellularsignal-regulated kinase 1/2 (ERK1/2)–dependent process (17, 18).Interestingly, IL-1h also enhances the activity of the CDK2 (19).We therefore investigated whether IL-1h-induced COX-2 expressioninvolves CDK2.Here, we report for the first time that a potent CDK2 inhibitor,

BMS-387032, down-regulates IL-1h-induced expression of COX-2in non–small cell lung carcinoma (NSCLC) cell lines (H358 andA549) without inhibiting the basal COX-2 level. We show that IL-1hinduces CDK2 activity in H358 cells and that IL-1h-induced CDK2activity may be critical for induction of COX-2 through a pathwaythat involves the C/EBP group of transcription factors. Our findingssuggest a nexus between cell cycle regulatory events and COX-2induction that could potentially represent an attractive target fortumor-specific blockage of COX-2.

Materials and Methods

Materials. CDK inhibitor BMS-387032 (molecular weight 416.99) was

obtained from Bristol-Myers Squibb (New York, NY) and dissolved indouble-distilled water (10 mmol/L) and the stock solution was stored at

�20jC. Penicillin, streptomycin, RPMI 1640, fetal bovine serum (FBS),

L-glutamine, LipofectAMINE, and Oligofectamine were obtained from

Invitrogen (Grand Island, NY). COX-2 monoclonal and COX-1 polyclonalantibodies were purchased from Cayman Chemical (Ann Arbor, MI). Anti-

actin antibodies were purchased from Sigma-Aldrich Co. (St. Louis, MO).

CDK2 monoclonal antibodies were purchased from Cell SignalingBiotechnology (Beverly, MA). Antibodies against C/EBP subunits were

obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). RNase

protection assay kit II (RPA II) was obtained from Ambion (Austin, TX).

Oligodeoxynucleotides for electrophoretic mobility shift assay (EMSA)were obtained from Invitrogen. CDK2 small interfering RNA (siRNA) was

obtained from Dharmacon (Lafayette, CO). [a-32P]UTP and [g-32P]ATP

were purchased from ICN Biochemicals (Irvine, CA).

Cell lines. H358, A549, and HCC3255 NSCLC cells (kindly provided byDr. John D. Minna, University of Texas Southwestern Medical Center, Dallas,

TX) were maintained in RPMI 1640 supplemented with 5% FBS, 50 units/mL

penicillin, 50 Ag/mL streptomycin, 2 mmol/L L-glutamine, and 25 mmol/L

HEPES. For all experiments, cells were grown to 75% confluency and thentreated as indicated.

Immunoblot analysis. Cells were preincubated with the specific kinase

inhibitors for 1 hour and then exposed to IL-1h for 8 hours. Cells wereharvested, lysed, and subjected to SDS-PAGE as mentioned earlier (20).

Membranes were incubated overnight with COX-1, COX-2, and CDK2

antibodies. The same membrane was probed with anti-h-actin antibodies

and provided as loading control. Membranes were developed by theenhanced chemiluminescence system (Amersham Pharmacia Biotech,

Piscataway, NJ) and exposed to Hyperfilm (Amersham Pharmacia Biotech).

In some experiments, the effect of specific kinase inhibitors, such as PD98059

[MAPK/ERK kinase (MEK) inhibitor], SB203580 (p38 MAPK inhibitor), andLY294002 [phosphatidylinositol 3-kinase (PI3K inhibitor)], in comparison

with BMS-387032 was quantitatively assessed by densitometric scanning of

immunoblots. Blots were digitally scanned using Adobe Photoshop version7.0 and intensity of bands corresponding to COX-2 was measured in the

green channel from inverted images. Background values were subtracted

from values obtained from the COX-2 band. Values from samples with

increasing concentration of the inhibitor were converted to percent COX-2expression relative to the value obtained from the COX-2 band from samples

with no inhibitor. Percent COX-2 expression was plotted as a function of log

inhibitor concentration (mol/L) and EC50s were calculated from sigmoid

dose curves using GraphPad Prism version 4 software.Immunoprecipitation and immune complex kinase assays. H358

cells were preincubated with BMS-387032 for 1 hour followed by the

addition of IL-1h for 2 hours. Cell lysates were prepared with ice-cold lysis

buffer [50 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl, 10% glycerol,

1% Triton X-100, 1 mmol/L EDTA, 2.5 mmol/L EGTA, 1 mmol/L DTT, 25

mmol/L h-glycerophosphate, 1 mmol/L NaF, 1 mmol/L Na3VO4, 10 Ag/mL

protease inhibitor mixture] and sonicated at 4jC and lysates were clarified

by centrifugation at 10,000 � g for 5 minutes. Whole-cell lysates (500 Ag)were preincubated with 1.0 Ag anti-CDK2 antibodies (Upstate Biotech-

nology, Lake Placid, NY) for 2 hours and the immune complex was

precipitated with protein A-Sepharose beads at 4jC overnight. The

immunoprecipitated proteins on the beads were washed thrice with 1 mL

lysis buffer and twice with a kinase buffer (containing 20 mmol/L HEPES,

2 mmol/L MgCl2, 2 mmol/L MnCl2, 10 mmol/L h-glycerophosphate, 10mmol/L NaF, 0.3 mmol/L sodium orthovanadate, 1 mmol/L DTT). The final

pellet was resuspended in 25 AL kinase buffer containing 5 Ag histone H1

(Sigma, St. Louis, MO), 20 Amol/L ATP, and 5 ACi [g-32P]ATP (4,500 ACi/mmol) and incubated for 30 minutes at 30jC with occasional mixing. The

reaction was terminated by the addition of 25 AL of 2� concentrated

Laemmli sample buffer and separated on a 12.5% SDS-polyacrylamide gel.

The gel was dried under vacuum and exposed to Kodak BioMax MS film

(Kodak, Rochester, NY) at �80jC.Prostaglandin E2 assay. Prostaglandin E2 (PGE2) was measured using

the PGE2 EIA kit (Cayman Chemical). A total of 1 � 106 cells were plated intriplicates for each experiment and serum starved for 24 hours. The serum-

free cells were treated as indicated with IL-1h (1.0 ng/mL) and BMS-387032

(300 nmol/L). After 8 hours, medium was collected and subjected to PGE2assay in a 96-well plate. The product of this enzymatic reaction has adistinct yellow color that absorbs strongly at 412 nm. The intensity of the

color is inversely proportional to the amount of free PGE2 present in the

well during the incubation.

RNA analysis. H358 cells were treated as indicated and total RNAs wereisolated by using TRIzol (Life Technologies, Grand Island, NY). RPA was

done using the RPA II kit. COX-2 open reading frame was subcloned into a

pCMV-SPORT6 obtained from Open Biosystem (Huntsville, AL) andlinearize with Tth111I enzyme (65jC, overnight). Radiolabeled riboprobes

were synthesized from 1 Ag of the linearized plasmids using T7 enzyme mix

MAXIscript kit (Ambion) for 1 hour at 37jC in a total volume of 20 AL. Thereaction buffer contained 10 mmol/L DTT, 0.5 mmol/L ATP, CTP, and GTP,2.5 mmol/L UTP, and 5 AL of 800 Ci/mmol [a-32P]UTP at 10 mCi/mL. At the

same time, actin riboprobe was also synthesized for loading control. Free

nucleotides were removed using the NucAway spin columns (Ambion).

Total RNA (10 Ag) from H358 cells were incubated at 45jC for 12 hoursin hybridization buffer with 5 � 104 cpm COX-2-labeled and 1 � 104 cpm

actin-labeled riboprobes. After hybridization, RNase digestion was carried

out at 37jC for 30 minutes. The protected fragments were precipitated and

then separated on a 5% denaturing polyacrylamide gel at 200 V for 4 hours.The gel was dried under vacuum and exposed to Kodak BioMax MS film

overnight at �80jC with intensifying screens.

COX-2 promoter activity. H358 cells were transiently transfected withCOX-2 promoter-luciferase reporter plasmids (1.0 Ag) along with pRSV-

h-gal (0.25 Ag) using LipofectAMINE. An equal amount of total DNA was

used in transfection for each experiment. Sixteen hours after transfection,

cells were serum starved for 24 hours and treated with or without IL-1h(1 ng/mL) in the presence or absence of BMS-387032 for further 8 hours.

Luciferase activity was measured by Promega (Madison, WI) luciferase

assay system using Monolight 2010 luminometer (Analytical Lumines-

cence Laboratory, San Diego, CA). Luciferase activity was normalized toh-galactosidase activity for differences in the transfection efficiency.

Oligonucleotides. Oligonucleotides containing different COX-2 pro-

moter sites were synthesized by Invitrogen as follows: NF-IL6, 5V-CCCACC-GGGCTTACGCAATT-3V (sense) and 5V-AATTGCGTAAGCCCGGTGGG-3V(antisense); mutant NF-IL6, 5V-CCCACCGGGCTTACgcttTT-3V (sense)

and 5V-AAaagcGTAAGCCCGGTGGG-3V (antisense); cyclic AMP–responsive

element (CRE), 5V-AAACAGTCATTTCGTCACATGGGCTTG-3V (sense) and5V-CAAGCCCATGTGACGAAATGACTGTTT-3V (antisense); mutant CRE,

5V-AAACAGTCActgattcaCATGGGCTTG-3V (sense) and 5V-CAAGCCCATG-tgaatcagTGACTGTTT-3V (antisense); NF-nB, 5V-AGGAGAGTGGGGAC-TACCCCCTCTG-3V (sense) and 5V-AGAGGGGGTAGTCCCCACTCTCCT-3V(antisense); and mutant NF-nB, 5V-AGGAGAGTGggtgtgtatcCCTCTG-3V

Regulation of COX-2 Expression by CDK2 Inhibitor

www.aacrjournals.org 1759 Cancer Res 2006; 66: (3). February 1, 2006



(sense) and 5V-AGAGGgatacacaccCACTCTCCT-3V(antisense). The comple-mentary oligonucleotides were annealed and purified following the

manufacturer’s protocol.

Electrophoretic mobility shift assays. Nuclear extracts were prepared

as described previously (21). Probes (double-stranded oligonucleotides)containing the NF-IL6, NF-nB, or CRE-binding site were purchased from

Invitrogen and radiolabeled using T4 polynucleotide kinase and [g-32P]ATP

(22). A typical binding reaction involved a 15-minute preincubation with

5 Ag nuclear extract, 1.5 Ag poly(deoxyinosinic-deoxycytidylic acid), 200 ngsingle-stranded oligonucleotide, 20 mmol/L HEPES-NaOH (pH 7.6),

100 mmol/L NaCl, 1 mmol/L DTT, and 2% glycerol followed by a 20-

minute incubation with 20 fmol radiolabeled probe. To assess the specificity

of DNA protein binding, radiolabeled mutant oligonucleotide was addedinstead of wild-type radiolabeled oligonucleotide to the binding reaction

mixture. In the supershift analysis, C/EBPh-specific antibodies (1 Ag) wereadded to the mixture and incubated for an additional 10 minutes at room

temperature before electrophoresis. Complexes were then resolved byelectrophoresis for 90 minutes at 200 V on a 6% native polyacrylamide gel,

dried, and processed for autoradiography.

siRNA transfection. H358 cells (1 � 105 per well) were transiently

transfected with different concentrations of CDK2 siRNA in six-well platesusing Oligofectamine. Cells were rinsed 5 hours after transfection with

1� PBS, and complete medium was added and further incubated for

40 hours. Cells were serum starved for 24 hours and treated with IL-1hfor another 8 hours in the serum-free medium. Cells were lysed andsubjected to immunoblot analysis for CDK2, COX-2, and COX-1 expression.

Determination of cell death. Cells were plated, serum starved for

24 hours, and treated with specific kinase inhibitors at increasingconcentrations for 1 hour before the addition of IL-1h. Cells were further

incubated for 8 hours. Cells were trypsinized (keeping all the floating cells)

and the cell suspension was diluted 1:1 with 0.4% trypan blue and counted

using a hemacytometer. Cells scoring positive for uptake of the dye wasconsidered dead and the % cell death was calculated using the following

formula: [(number of trypan blue positive cells) / (number of trypan blue–

positive cells + number of trypan blue negative cells)] � 100.

Results

CDK2 inhibitor BMS-387032 inhibits IL-1B-mediated induc-tion of COX-2 expression. Proinflammatory cytokines arereported to induce COX-2 expression in a variety of cell types(23–25). We tested the ability of several cytokines to induce COX-2expression in the H358 NSCLC cells. These cells were serumstarved for 24 hours and incubated with IL-1h, IL-6, IL-8, IFN-g,and tumor necrosis factor-a (TNF-a). Results in Fig. 1A show thatonly the addition of IL-1h significantly increased COX-2 expression(Fig. 1A, lane 2) after 8 hours. Enhancement of the activity ofCDK2 by IL-1h has been reported before (19). We thereforeexplored the possibility that IL-1h-induced COX-2 expressionmay involve CDK2. In Fig. 1B , H358 cells were pretreated withincreasing concentrations of the CDK2 inhibitor, BMS-387032, for1 hour and then treated with IL-1h (1 ng/mL) for further 8 hours.We observed that IL-1h strongly induced COX-2 expression(Fig. 1B, lane 2) and BMS-387032 produced a dose-dependentdecrease in IL-1h-induced COX-2 expression (Fig. 1B, lanes 3-7).Significant inhibition started at 150 nmol/L BMS-387032 where>85% COX-2 expression was blocked (Fig. 1B, lane 5), whereascomplete inhibition was achieved at 300 nmol/L (Fig. 1B, lane 7).BMS-387032 also blocked IL-1h-induced COX-2 expression in a

Figure 1. Effect of BMS-387032 on COX-1 and COX-2 expression in humanlung cancer cells. A, IL-1h induces COX-2 expression in H358 cells. Cells wereserum starved for 24 hours. Proinflammatory cytokines IL-1h, IL-6, IL-8, IFN-g,and TNF-a (1 ng/mL) were added in the fresh medium and further incubatedfor 8 hours (lanes 2-6). The cells were harvested and lysed and total cell lysates(50 Ag) were loaded onto a 10% SDS-polyacrylamide gel separately forimmunoblot analysis with antibodies specific for COX-1 and COX-2. The samemembrane was reprobed with anti-h-actin antibodies and provided as a loadingcontrol. B and C, BMS-387032 inhibits IL-1h-induced COX-2 expression inH358 and A549 cells. Serum-starved H358 and A549 cells were treated with0 to 300 nmol/L BMS-387032 for 1 hour (lanes 2-7) and stimulated with IL-1h(1 ng/mL) for further 8 hours. Cells were harvested and total cell lysates (50 Ag)were then subjected to immunoblot analysis for COX-1 and COX-2 expression.D, BMS-387032 inhibits IL-1h-induced CDK2 activity. Serum-starved H358cells were pretreated with BMS-387032 (300 nmol/L) for 1 hour before theaddition of IL-1h. Cells were harvested after 2 hours. Total cell lysates (500 Ag)were then immunoprecipitated with anti-CDK2 antibody. The kinase reaction wasdone using histone H1 as a substrate. Lane 1, control; lane 2, IL-1h; lane 3,IL-1h + BMS-387032.

Cancer Research




second NSCLC cell line, A549 (Fig. 1C, lanes 3-7). However, CDK2inhibition in both cell lines had no effect on COX-1 expression(Fig. 1B and C, middle). Data from these experiments clearlyindicate that BMS-387032 inhibits IL-1h-induced COX-2 expressionin these lung carcinoma cells in a dose-dependent manner, whereasendogenous COX-1 expression is not affected by either IL-1h or thecombination of IL-1h and BMS-387032.BMS-387032 inhibits IL-1B-induced CDK2 activity. To show

that IL-1h in fact induces CDK2 activity in H358 cells and BMS-387032 blocks this activity, we did in vitro kinase assays. CDK2 wasimmunoprecipitated from cells treated with IL-1h in the presenceor absence of BMS-387032. The immune complex was thenanalyzed for CDK2 activity using histone H1 as the substrate. Weobserve that the treatment with IL-1h strongly induced CDK2activity at 2 hours (Fig. 1D , lane 2), which is evidenced by thephosphorylation of its downstream target histone H1. Pretreatmentwith BMS-387032 significantly inhibited IL-1h-induced histone H1phosphorylation (Fig. 1D , lane 3). Immunoprecipitation of equalamounts of CDK2 protein in each kinase reaction was verified byWestern blotting with anti-CDK2 antibody.CDK2 siRNA abrogates IL-1B-induced COX-2 expression. Our

data indicate that BMS-387032 blocks the IL-1h-mediatedinduction of COX-2 in H358 cells by inhibiting CDK2. Next, wetested the possibility that ablation of the CDK2 protein has asimilar effect on IL-1h-induced COX-2 expression. Data show thatCDK2 expression is abrogated by the transfection of CDK2 siRNAat increasing concentrations (Fig. 2A). Maximum inhibition ofCDK2 expression was observed at 100 nmol/L (Fig. 2A, lane 5),whereas levels of CDK2 remained unchanged when the cells weretransfected with a similar concentration of acyl protein thio-esterase 1 (APT1) siRNA (Fig. 2A, lane 2) and serve as a negativecontrol. Silencing of CDK2 expression resulted in a strikinginhibition of IL-1h-induced COX-2 expression (Fig. 2B). We alsonoticed that transfection with CDK2 siRNA had no effect onCOX-1 expression (Fig. 2B). These data correlate well with theeffect of the small-molecule inhibitor BMS-387032 and clearlyshow that the activity as well as expression of the CDK2 is criticalfor IL-1h-induced expression of COX-2.BMS-387032 inhibits IL-1B-mediated induction of COX-2

mRNA levels. Our data show a critical requirement of CDK2during IL-1h-mediated induction of COX-2 expression. We nextanalyzed whether CDK2 inhibition by BMS-387032 had any effecton steady-state mRNA levels of COX-2. Serum-starved cells aretreated with BMS-387032 or IL-1h plus BMS-387032 for 4 hours.Total RNA was isolated and subjected to RPA as described inMaterials and Methods. The results show that a substantialinduction of COX-2 mRNA is observed in response to IL-1h(Fig. 3A , lane 2). Addition of BMS-387032 alone had no effect onCOX-2 mRNA (Fig. 3A , lane 4). However, BMS-387032 at 300nmol/L resulted in a significant (>95% inhibition) inhibition ofIL-1h-mediated induction of COX-2 mRNA (Fig. 3A , lane 3). TheRPA result is consistent with our observations indicating thatCDK2 inhibition blocks IL-1h-induced levels of the COX-2 enzyme(Fig. 1B and C).BMS-387032 inhibits IL-1B-mediated induction of PGE2

synthesis. To determine whether CDK2 inhibition blocks PGE2production through the inhibition of COX-2 gene expression,we measured IL-1h-induced PGE2 release in the presence orabsence of the BMS-387032. Results presented in Fig. 3B indicate asignificant enhancement of PGE2 release from 2 to 85 pg/mL(42.5-fold) in 8 hours in the presence of IL-1h. Addition of BMS-

387032 alone had no observable effect on the basal level of PGE2(4.7 pg/mL). However, >90% of the IL-1h-induced PGE2 synthesiswas inhibited (15 pg/mL) by the addition of BMS-387032. Thisresult is consistent with the observation that CDK2 inhibition leadsto a blockade of IL-1h-induced COX-2 mRNA and protein.BMS-387032 inhibits IL-1B-mediated induction of COX-2

promoter activity. COX-2 expression is tightly regulated atmultiple levels, including transcription and mRNA stabilization.The COX-2 gene is transcriptionally regulated by a 1,432-bpminimal promoter and is responsive to IL-1h stimulation (26).The COX-2 promoter harbors sequences for the specific bindingof transcription factors, such as NF-nB, NF-IL6-C/EBP, PEA3, CRE,and activator protein-1. We asked whether transcriptional activ-ation of the COX-2 promoter by IL-1h can be inhibited by thisCDK2 inhibitor. To address this question, we examined the effectof BMS-387032 on the transcription of a luciferase reporter

Figure 2. CDK2 siRNA inhibits IL-1h-induced COX-2 expression. A, H358 cellswere transfected using Oligofectamine with increasing doses of CDK2 siRNA(25-100 nmol/L; lanes 3-5). APT1 siRNA at 100 nmol/L concentration was alsoused for negative control (lane 2 ). After 72 hours of transfection, total cellularextracts were prepared and processed for immunoblot analysis for CDK2expression. Actin protein serves as a loading control. B, H358 cells weretransfected using Oligofectamine with CDK2 siRNA (100 nmol/L). After 40 hours,cells were serum starved for 24 hours and then treated with IL-1h (1 ng/mL)for further 8 hours. Cells were harvested and extracts were then prepared andsubjected to Western blot analysis for COX-2 (a), CDK2 (b), actin (c and e), andCOX-1 (d ). Lane 1, control; lane 2, CDK2 siRNA; lane 3, IL-1h alone;lane 4, IL-1h + CDK2 siRNA.





construct driven by the �1432/+59 and �327/+59 minimal COX-2promoter containing NF-nB, NF-IL6/C/EBP, and CRE sites. Thedata indicate that treatment with IL-1h produces a 4.4-fold(�1432/+59) and 4.8-fold (�327/+59) increase in COX-2 promoteractivity (Fig. 3C). Pretreatment with BMS-387032 resulted in asignificant inhibition of IL-1h-induced COX-2 promoter activity inboth cases. The results clearly show that the inhibitory effect ofBMS-387032 manifests at the level of transcription and accountsfor inhibition of IL-1h-induced COX-2 protein expression and PGE2levels.Effect of BMS-387032 on transcription factors that regulate

COX-2 transcription in response to IL-1B. To further elucidatethe effect of CDK2 inhibitor in the IL-1h-stimulated binding to

COX-2 regulatory sequences, we did EMSAs with radiolabeledoligonucleotides containing the binding sites for NF-nB, CRE, orNF-IL6 elements present in the COX-2 promoter. The data indicatethat IL-1h increased binding of nuclear proteins to the NF-IL6(Fig. 4A , lane 3) and NF-nB (Fig. 4B, lane 3) sites of COX-2promoter. However, no significant increase in binding wasobserved with the CRE site (Fig. 4C, lane 3). The binding to allthe three key cis-acting elements in the COX-2 promoter wasspecific because oligonucleotides containing mutations in any ofthe sequences failed to bind nuclear proteins (Fig. 4A-C, lane 5).Interestingly, preincubation with BMS-387032 followed by IL-1htreatment showed significantly reduced binding (>90%) to theNF-IL6 site (Fig. 4A, lane 4). A <15% inhibition of binding to theNF-nB site is also observed in the presence of BMS-387032 (Fig. 4B,lane 4). On the other hand, binding to the CRE sites (Fig. 4C, lane 4)of the COX-2 promoter remained unaffected. Moreover, supershiftanalysis with antibodies against C/EBPh indicated the presenceof C/EBPh in complex with the NF-IL-6 element (Fig. 4A, lane 7).We also did the supershift analysis using the other subunits ofC/EBP (a, g, and y); however, we did not observe any significantsupershifting in the NF-IL6-binding complex (data not shown).These findings strongly suggest the nuclear factor C/EBPh is thedownstream target of CDK2 because BMS-387032 prevented theIL-1h-induced binding of C/EBPh to NF-IL6 sequence of the COX-2promoter.BMS-387032 does not inhibit the endogenous expression of

COX-2. The NSCLC cell line HCC3255 exhibits high constitutiveexpression of COX-2. Therefore, we wanted to examine whetherCOX-2 expression in this cell line can be prevented by BMS-387032.Our results show that BMS-387032 does not down-regulate thebasal COX-2 as well as COX-1 expression in HCC3255 cells evenafter 48 hours of treatment (Fig. 5A, lane 4, 8). We then investigatethe effect of IL-1h on the induction of COX-2 expression in this cellline in the presence of BMS-387032. The data in Fig. 5B show thatneither IL-1h nor BMS-387032 has any effect on COX-2 expressionin HCC3255 cells (Fig. 5A and B).Effect of BMS-387032 on signaling pathways regulating

COX-2 expression. IL-1h induces the activity of several keysignaling molecules, such as p38 MAPK, phosphorylated stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK),phosphorylated AKT, and phosphorylated ERK1/2. Moreover, thesekinases are also involved in the regulation of COX-2 expression in

Figure 3. A, BMS-387032 inhibits COX-2 transcription. RPA of COX-2 mRNAwas done. Cells were treated with (+) or without (�) IL-1h (1 ng/mL) for 2 hoursand pretreated with BMS-387032 for 1 hour before the addition of IL-1h foranother 2 hours. Total RNA was isolated from H358 cells and radiolabeledriboprobes of COX-2 and human actin were synthesized as described inMaterials and Methods and the protected fragments were then separated on a5% denaturing polyacrylamide gel. Actin served as a control for equal loading ofsamples. B, BMS-387032 inhibits IL-1h-induced PGE2 synthesis. A total of1 � 106 cells were plated in triplicates for each experiment and serum starved for24 hours. The serum-free cells were treated as indicated. After 8 hours, mediumwas collected and subjected to PGE2 assay using the PGE2 EIA kit. Cellswere also harvested at the same time and total cellular extracts were preparedand subjected to immunoblot analysis for COX-2 expression (inset ).C, BMS-387032 suppresses IL-1h-mediated induction of COX-2 promoteractivity. H358 cells were cotransfected with 1.0 Ag full-length (�1,432/+52) andtruncated (�327/+52) human COX-2 promoter constructs ligated to luciferaseand 0.25 mg pRSV-h-gal using LipofectAMINE. Six hours after transfection,cells were rinsed with PBS and fresh medium was added for recovery. Cells weretreated as described in Materials and Methods. Reporter activities were thenmeasured using the Promega luciferase assay system. Luciferase activityrepresents data that were normalized to h-galactosidase activity. *, P < 0.002,compared with IL-1h (�1,432/+59) alone; **, P < 0.0005, compared with IL-1h(�327/+59) alone.

Cancer Research




carcinoma cells (27, 28). We examined whether p38 MAPK,phosphorylated SAPK/JNK, phosphorylated AKT, or phosphorylat-ed ERK1/2 might lie downstream to CDK2 in the IL-1h-inducedsignaling pathway leading to COX-2 expression. Our results

indicate that, in all cases, BMS-387032 fails to prevent the IL-1h-induced phosphorylation of p38 MAPK, phosphorylated SAPK/JNK,phosphorylated AKT, and phosphorylated ERK1/2 (Fig. 6A). Wethen examined the relative contribution of the different signaling

Figure 4. BMS-387032 inhibited IL-1h-mediated enhanced binding of C/EBPh to the NF-IL6 site of the COX-2 promoter. A to C, H358 cells were treated for 2 hourswith IL-1h and with or without 1 hour prior addition of BMS-387032 (300 nmol/L). A, nuclear extracts were incubated with labeled COX-2 NF-IL6 oligonucleotide.B, nuclear extracts were incubated with labeled COX-2 NF-nB oligonucleotide. C, nuclear extracts were incubated with labeled COX-2 CRE oligonucleotide.

Figure 5. A, BMS-387032 does not prevent endogenous COX-2expression. HCC3255 cells were treated with increasing doses(100-300 nmol/L) of BMS-387032. Cells were harvested at 24 and48 hours and subjected to immunoblot analysis for COX-1 andCOX-2 expression. B, HCC3255 cells were serum starved for24 hours and then treated with IL-1h (1 ng/mL) in the presence orabsence of BMS-387032. After 8 hours, cells were harvested andimmunoblot analysis for COX-2 expression was done.





pathways that lead to IL-1h-induced COX-2 expression. Towardthis end, we compared the effects of BMS-387032 with the effectsof specific kinase inhibitors, such as PD98059, SB203580, andLY294002, which are also known to inhibit COX-2 expression underinduced conditions (27, 28). H358 cells are treated with theseinhibitors at various concentrations (5 nmol/L to 50 Amol/L) for1 hour before the addition of IL-1h. Although p38 MAPK(SB203580), PI3K (LY290004), and MEK (PD98059) inhibitorssignificantly inhibited IL-1h-induced COX-2 expression in H358cells (Fig. 6B), the effective concentrations of these inhibitorsrequired to exert this effect were much higher when comparedwith a similar concentration range of BMS-387032 (Fig. 6C). Wemeasured the approximate EC50s for all the inhibitors used in thisstudy and found 14.5, 10.7, and 2.5 Amol/L for SB203580, LY290004,and PD98059, respectively, whereas the EC50 of BMS-387032 is0.18 Amol/L. Therefore, data in Fig. 6C show that among all theinhibitors tested BMS-387032 is the most effective in preventingCOX-2 induction by IL-1h in these lung cancer cell lines. We also

measured the % cell death in response to the treatment witheach of these inhibitors under similar experimental conditions.We observed no appreciable cell death (7-10%) after 8 hours oftreatment with these inhibitors (Fig. 6C).

Discussion

Dysregulation of the cell cycle is a critical event in the develop-ment of cancer. CDK2 is a key modulator of cell cycle and cellproliferation. The role of COX-2 in the modulation of cell cycleprogression and its overexpression in several tumor models hasalso been documented (29, 30). In this study, we have examinedthe effect of a potent inhibitor of CDK2 on cytokine-inducedCOX-2 expression, and our findings showing that CDK2 expressionand activity is critical in IL-1h-induced COX-2 expression in theNSCLC cell lines gain particular significance.COX-2 expression is predominantly controlled at two levels:

activation of transcription and stabilization of COX-2 mRNA levels.

Figure 6. A, effect of BMS-387032 on IL-1h-induced MAPK, ERK1/2, AKT, and JNK phosphorylation. Serum-starved H358 cells were treated with (+) or without (�)BMS-387032 for 1 hour followed by the addition of IL-1h (1 ng/mL) for 30 minutes. Cells were harvested after 30 minutes, and lysates were subjected to immunoblotanalysis with several phosphospecific kinase antibodies. Lane 1, control; lane 2, IL-1h alone; lane 3, IL-1h + BMS-387032. B, BMS-387032 is a potent inhibitor ofCOX-2 expression. Serum-starved H358 cells were treated with several kinase-specific inhibitors at different concentrations as indicated for 1 hour before the addition ofIL-1h (1 ng/mL). Cells were additionally incubated for 8 hours and cellular lysates (50 Ag) were subjected to immunoblot analysis using COX-2-specific antibodies.The same membranes were additionally probed with anti-actin antibodies and provided as a loading control (data not shown). C, EC50 determination. Blots were digitallyscanned and the EC50s of each inhibitor were determined as described in Materials and Methods. % Cell death in response to each inhibitor was determined bytrypan blue exclusion method.

Cancer Research




We showed that IL-1h-induced binding of C/EBPh subunit toNF-IL6 sites of the COX-2 promoter is inhibited by BMS-387032.C/EBP family of proteins has emerged as a key transactivator forCOX-2 expression induced by proinflammatory mediators. Severalreports indicated that h and y isoforms of C/EBP are involved inCOX-2 transcriptional activation by proinflammatory mediatorsin human cells (17). Saunders et al. reported that in human foreskinfibroblast cells C/EBPh activation is required for the IL-1h-inducedCOX-2 expression and is inhibited by the addition of eitheraspirin or salicylate (17). The mechanism by which these drugsinhibit the binding of C/EBPh to the COX-2 promoter site is stillunknown (31).COX-2 mRNA contains an AU-rich element that regulates COX-2

expression at the post-transcriptional level. In this study, we didnot observe any effect of either IL-1h or BMS-387032 on theexpression of a cytomegalovirus promoter-driven luciferase report-er under the control of the COX-2 3Vuntranslated region (data notshown). It also reported that activation of p38 MAPK signalingcascades are important for the increase in COX-2 stability (26, 32).In this study, we noticed that BMS-387032 did not preventp38 MAPK activation by IL-1h (Fig. 6A) and also observed thatEC50 of SB203580, a specific inhibitor of p38 MAPK, was 80-foldhigher (14.5 Amol/L) when compared with the EC50 of BMS-387032(0.181 Amol/L), indicating that IL-1h-mediated COX-2 expression ismore sensitive to CDK2 inhibition in this H358 lung carcinomacells (Fig. 6B and C).There is considerable evidence indicating that C/EBP is a

downstream target of CDK2. Gorgoni et al. reported that COX-2mRNA induction and promoter activity was blocked in C/EBPh�/�

macrophages but was rescued when C/EBPh was overexpressed(33). The transcriptional activity of C/EBPh is regulated byphosphorylation (34) and several phosphorylation sites in C/EBPhare required for its activity (34, 35). In a recent report, Shumanet al. reported that in mouse embryo fibroblast cells (NIH3T3)Ser64 and Thr189 residues within the transactivation domain ofC/EBPh were phosphorylated by CDK2 and phosphorylation onboth sites was blocked by CDK inhibitor roscovitin (35). It was alsoreported that Ser64 residue of C/EBPh is specifically phosphorylatedby CDK2 not by either CDK4 or CDK6 (35). However, the precise roleof CDK2 in the IL-h-mediated activation of human C/EBPh iscurrently unknown and this mechanism remains to be elucidated.

CDK2 was originally thought to be critically required for G1-Stransition along with cyclin E. However, recent findings indicatedthat blocking of CDK2 expression in human tumor cell lines hadlittle or no effect on their ability to proceed through the cell cycleand CDK2 knockout mice are viable and that their cells are ableto proliferate in culture in vitro (36). Previous studies reportedthat the phosphorylation of Ser64 and Thr189 by CDKs is criticalfor C/EBPh to enhance Ras-induced neoplastic transformation inmouse fibroblast cells (35, 37). These observations clearly indicatedthat the function of CDK2 is not limited to cell cycle regulation andmay be indicative of a different role in tumor cell lines. In thisstudy, we reported CDK2 expression is necessary for IL-1h-inducedCOX-2 expression (Fig. 2).COX-2 expression in various cellular systems is controlled by

multiple signaling pathways. For example, in HCC3255, NSCLCcells that exhibit a high constitutive expression of COX-2 and BMS-387032 did not inhibit the high levels of COX-2 expression in thiscell line. Our results indicate that CDK2 activity is critical in IL-1h-induced expression of COX-2 in H358 cells and may not be involvedin the high constitutive expression of COX-2 observed in HCC3255cells. This information suggests the possibility that CDK2 inhibitormight target tumors in which COX-2 expression is cytokinemediated and is less likely to be effective against tumors thatexhibit cytokine-independent COX-2 expression. Clinically, anti-COX-2 therapies have been targeted at the level of COX-2 enzymeactivity. Recent reports suggest that classic COX-2 inhibitors donot effectively discriminate between tumor COX-2 activity andother physioprotective functions of the COX-2 enzyme. Therefore,our results present an opportunity for tumor control through theselective inhibition of cytokine- and CDK2-driven COX-2 expres-sion in certain subsets of tumors.

Acknowledgments

Received 9/15/2005; revised 11/15/2005; accepted 12/1/2005.Grant support: NIH grants CA90949, CA82117, and CA21661 (H. Choy) and

American Cancer Society institutional funding and Flight Attendant Medical ResearchInstitute Young Clinical Scientist Award (D. Saha).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Hiroshi Inoue (Nara Women’s University, Nara, Japan) for the COX-2promoter constructs, Dr. John D. Minna for NSCLC cell lines, and Dr. ChaitanyaS. Nirodi (University of Texas Southwestern Medical Center) for advice and discussion.



References1. Benson C, Kaye S, Workman P, et al. Clinicalanticancer drug development: targeting the cyclin-dependent kinases. Br J Cancer 2005;92:7–12.

2. Swanton C. Cell-cycle targeted therapies. Lancet Oncol2004;5:27–36.

3. Senderowicz AM. Novel direct and indirect cyclin-dependent kinase modulators for the prevention andtreatment of human neoplasms. Cancer ChemotherPharmacol 2003;52 Suppl 1:S61–73.

4. Misra RN, Xiao HY, Kim KS, et al. N -(cycloalkylami-no)acyl-2-aminothiazole inhibitors of cyclin-dependentkinase 2. N -[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]me-thyl]thio]-2-thiazolyl]-4-piperidinecarboxamide (BMS-387032), a highly efficacious and selective antitumoragent. J Med Chem 2004;47:1719–28.

5. Marnett LJ. Aspirin and the potential role of prosta-glandins in colon cancer. Cancer Res 1992;52:5575–89.

6. Langenbach R, Morham SG, Tiano HF, et al.Prostaglandin synthase 1 gene disruption in micereduces arachidonic acid-induced inflammation and

indomethacin-induced gastric ulceration. Cell 1995;83:483–92.

7. Morham SG, Langenbach R, Loftin CD, et al.Prostaglandin synthase 2 gene disruption causes severerenal pathology in the mouse. Cell 1995;83:473–82.

8. Smith WL, Dewitt DL. Prostaglandin endoperoxide Hsynthases-1 and -2. Adv Immunol 1996;62:167–215.

9. Tsujii M, DuBois RN. Alterations in cellular adhesionand apoptosis in epithelial cells overexpressingprostaglandin endoperoxide synthase 2. Cell 1995;83:493–501.

10. Achiwa H, Yatabe Y, Hida T, et al. Prognosticsignificance of elevated cyclooxygenase 2 expression inprimary, resected lung adenocarcinomas. Clin CancerRes 1999;5:1001–5.

11. Hida T, Yatabe Y, Achiwa H, et al. Increasedexpression of cyclooxygenase 2 occurs frequently inhuman lung cancers, specifically in adenocarcinomas.Cancer Res 1998;58:3761–4.

12. Soslow RA, Dannenberg AJ, Rush D, et al. COX-2 isexpressed in human pulmonary, colonic, and mammarytumors. Cancer 2000;89:2637–45.

13. Wolff H, Saukkonen K, Anttila S, et al. Expression ofcyclooxygenase-2 in human lung carcinoma. Cancer Res1998;58:4997–5001.

14. Weitzman S, Gordon L. Inflammation and cancer:role of phagocyte-generated oxidants in carcinogenesis.Blood 1990;76:655–63.

15. Fiebich BL, Mueksch B, Boehringer M, Hull M.Interleukin-1h induces cyclooxygenase-2 and prosta-glandin E(2) synthesis in human neuroblastomacells: involvement of p38 mitogen-activated proteinkinase and nuclear factor-nB. J Neurochem 2000;75:2020–8.

16. Fan XM, Wong BC, Lin MC, et al. Interleukin-1hinduces cyclo-oxygenase-2 expression in gastric cancercells by the p38 and p44/42 mitogen-activated proteinkinase signaling pathways. J Gastroenterol Hepatol 2001;16:1098–104.

17. Saunders MA, Sansores-Garcia L, Gilroy DW, Wu KK.Selective suppression of CCAAT/enhancer-binding pro-tein h binding and cyclooxygenase-2 promoter activityby sodium salicylate in quiescent human fibroblasts.J Biol Chem 2001;276:18897–904.



Cancer Research


18. Tamura M, Sebastian S, Yang S, et al. Interleukin-1helevates cyclooxygenase-2 protein level and enzymeactivity via increasing its mRNA stability in humanendometrial stromal cells: an effect mediated byextracellularly regulated kinases 1 and 2. J ClinEndocrinol Metab 2002;87:3263–73.

19. Nathe TJ, Deou J, Walsh B, et al. Interleukin-1hinhibits expression of p21(WAF1/CIP1) and p27(KIP1)and enhances proliferation in response to platelet-derived growth factor-BB in smooth muscle cells.Arterioscler Thromb Vasc Biol 2002;22:1293–8.

20. Saha D, Sekhar KR, Cao C, et al. The antiangiogenicagent SU5416 down-regulates phorbol ester-mediatedinduction of cyclooxygenase 2 expression by inhibitingnicotinamide adenine dinucleotide phosphate oxidaseactivity. Cancer Res 2003;63:6920–7.

21. Dignam JD, Lebovitz RM, Roeder RG. Accuratetranscription initiation by RNA polymerase II in asoluble extract from isolated mammalian nuclei. NucleicAcids Res 1983;11:1475–89.

22. Sambrook J, Fritsch EF, Maniatis T. Molecularcloning: a laboratory manual. 2nd ed. Cold SpringHarbor, NY: Cold Spring Harbor Laboratory; 1989.

23. Vane JR, Bakhle YS, Botting RM. Cyclooxygenases 1and 2. Annu Rev Pharmacol Toxicol 1998;38:97–120.

24. Seibert K, Zhang Y, Leahy K, et al. Pharmacologicaland biochemical demonstration of the role of cyclo-

oxygenase 2 in inflammation and pain. Proc Natl AcadSci U S A 1994;91:12013–7.

25. Minghetti L, Polazzi E, Nicolini A, Creminon C, Levi G.Up-regulation of cyclooxygenase-2 expression in cul-tured microglia by prostaglandin E2, cyclic AMP andnon-steroidal anti-inflammatory drugs. Eur J Neurosci1997;9:934–40.

26. Tessner TG, Muhale F, Schloemann S, et al. Ionizingradiation up-regulates cyclooxygenase-2 in I407 cellsthrough p38 mitogen-activated protein kinase. Carcino-genesis 2004;25:37–45.

27. Sheng H, Williams CS, Shao J, et al. Induction ofcyclooxygenase-2 by activated Ha-ras oncogene in Rat-1fibroblasts and the role of mitogen-activated proteinkinase pathway. J Biol Chem 1998;273:22120–7.

28. Shao J, Sheng H, Inoue H, Morrow JD, DuBois RN.Regulation of constitutive cyclooxygenase-2 expressionin colon carcinoma cells. J Biol Chem 2000;275:33951–6.

29. Detjen KM, Welzel M, Wiedenmann B, Rosewicz S.Nonsteroidal anti-inflammatory drugs inhibit growth ofhuman neuroendocrine tumor cells via G1 cell-cyclearrest. Int J Cancer 2003;107:844–53.

30. Basu GD, Pathangey LB, Tinder TL, et al. Cyclo-oxygenase-2 inhibitor induces apoptosis in breastcancer cells in an in vivo model of spontaneousmetastatic breast cancer. Mol Cancer Res 2004;2:632–42.

31. Wu KK, Liou JY, Cieslik K. Transcriptional control of

COX-2 via C/EBPh. Arterioscler Thromb Vasc Biol 2005;25:679–85.

32. Subbaramaiah K, Marmo TP, Dixon DA, DannenbergAJ. Regulation of cyclooxgenase-2 mRNA stability bytaxanes: evidence for involvement of p38, MAPKAPK-2,and HuR. J Biol Chem 2003;278:37637–47.

33. Gorgoni B, Maritano D, Marthyn P, Righi M, Poli V.C/EBPh gene inactivation causes both impaired andenhanced gene expression and inverse regulation of IL-12 p40 and p35 mRNAs in macrophages. J Immunol2002;168:4055–62.

34. Nakajima T, Kinoshita S, Sasagawa T, et al. Phos-phorylation at threonine-235 by a ras-dependentmitogen-activated protein kinase cascade is essentialfor transcription factor NF-IL6. Proc Natl Acad Sci U S A1993;90:2207–11.

35. Shuman JD, Sebastian T, Kaldis P, et al. Cell cycle-dependent phosphorylation of C/EBPh mediates onco-genic cooperativity between C/EBPh and H-RasV12. MolCell Biol 2004;24:7380–91.

36. Tetsu O, McCormick F. Proliferation of cancer cellsdespite CDK2 inhibition. Cancer Cell 2003;3:233–45.

37. Zhu S, Yoon K, Sterneck E, Johnson PF, Smart RC.CCAAT/enhancer binding protein-h is a mediator ofkeratinocyte survival and skin tumorigenesis involvingoncogenic Ras signaling. Proc Natl Acad Sci U S A 2002;99:207–12.



2006;66:1758-1766. Cancer Res Partha Mukhopadhyay, M. Aktar Ali, Animesh Nandi, et al. Expression in Human Lung Carcinoma Cells

-Mediated Induction of Cyclooxygenase-2βInterleukin-1The Cyclin-Dependent Kinase 2 Inhibitor Down-regulates

Updated version

http://cancerres.aacrjournals.org/content/66/3/1758

Access the most recent version of this article at:

Cited articles

http://cancerres.aacrjournals.org/content/66/3/1758.full#ref-list-1

This article cites 35 articles, 18 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/66/3/1758.full#related-urls

This article has been cited by 4 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://cancerres.aacrjournals.org/content/66/3/1758To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/content/66/3/1758.full#ref-list-1

http://cancerres.aacrjournals.org/content/66/3/1758.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



Documents

The Cyclin-Dependent Kinase 2 Inhibitor Down-regulates Interleukin