The aryl hydrocarbon receptor-dependent deregulation of cell cycle control induced by polycyclic aromatic hydrocarbons in rat liver epithelial cells

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Mutation Research 615 (2007) 87–97

The aryl hydrocarbon receptor-dependent deregulation of cellcycle control induced by polycyclic aromatic hydrocarbons

in rat liver epithelial cells

Zdenek Andrysık a,b, Jan Vondracek a,b,∗, Miroslav Machala b, Pavel Krcmar b,Lenka Svihalkova-Sindlerova a, Anne Kranz c, Carsten Weiss c, Dagmar Faust c,

Alois Kozubık a, Cornelia Dietrich c

a Laboratory of Cytokinetics, Institute of Biophysics, 612 65 Brno, Czech Republicb Department of Chemistry and Toxicology, Veterinary Research Institute, 621 32 Brno, Czech Republic

c Institute of Toxicology, Johannes Gutenberg-University, 55131 Mainz, Germany

Received 24 July 2006; received in revised form 19 October 2006; accepted 27 October 2006Available online 1 December 2006

Abstract

Disruption of cell proliferation control by polycyclic aromatic hydrocarbons (PAHs) may contribute to their carcinogenicity. Weinvestigated role of the aryl hydrocarbon receptor (AhR) in disruption of contact inhibition in rat liver epithelial WB-F344 ‘stem-like’ cells, induced by the weakly mutagenic benz[a]anthracene (BaA), benzo[b]fluoranthene (BbF) and by the strongly mutagenicbenzo[a]pyrene (BaP). There were significant differences between the effects of BaA and BbF, and those of the strongly genotoxicBaP. Both BaA and BbF increased percentage of cells entering S-phase and cell numbers, associated with an increased expression ofCyclin A and Cyclin A/cdk2 complex activity. Their effects were significantly reduced in cells expressing a dominant-negative AhRmutant (dnAhR). Roscovitine, a chemical inhibitor of cdk2, abolished the induction of cell proliferation by BbF. However, neitherBaA nor BbF modulated expression of the principal cdk inhibitor involved in maintenance of contact inhibition, p27Kip1, or pRbphosphorylation. The strongly mutagenic BaP induced apoptosis, a decrease in total cell numbers and significantly higher percentageof cells entering S-phase than either BaA or BbF. Given that BaP induced high levels of Cyclin A/cdk2 activity, downregulationof p27Kip1 and hyperphosphorylation of pRb, the accumulation of cells in S-phase was probably due to cell proliferation, althoughS-phase arrest due to blocked replication forks can not be excluded. Both types of effects of BaP were significantly attenuated indnAhR cells. Transfection of WB-F344 cells with siRNA targeted against AhR decreased induction of Cyclin A induced by BbF or

BaP, further supporting the role of AhR in proliferative effects of PAHs. This suggest that activation of AhR plays a significant roleboth in disruption of contact inhibition by weakly mutagenic PAHs and in genotoxic effects of BaP possibly leading to enhancedcell proliferation. Thus, PAHs may increase proliferative rate and the likelihood of fixation of mutations.© 2006 Elsevier B.V. All rights reserved.

Keywords: Aryl hydrocarbon receptor; Polycyclic aromatic hydrocarbons; Contact inhibition; Cell proliferation; Rat liver ‘stem-like’ cells

∗ Corresponding author at: Institute of Biophysics, Kralovopolska 135, 612 65 Brno, Czech Republic. Tel.: +420 5415 17168;fax: +420 5412 11293.

E-mail address: [email protected] (J. Vondracek).

0027-5107/$ – see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.mrfmmm.2006.10.004

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lic). Diamidino-2-phenylindole dihydrochloride (DAPI) was

88 Z. Andrysık et al. / Mutat

1. Introduction

Polycyclic aromatic hydrocarbons (PAHs) are a largegroup of environmental organic pollutants that are ofconcern for humans, as many of them are known orsuspected carcinogens [1]. Apart from being potenttumor initiating compounds, PAHs are known to act ascomplete carcinogens producing tumors following theirrepeated application. This suggests that they may act alsoas tumor promoters, either directly or through formationof further active metabolites [2,3]. Although the mech-anisms responsible for the tumor promoting effects ofPAHs remain largely unknown, PAHs have been foundto activate several signaling pathways involved in con-trol of cell proliferation, differentiation or apoptosis[2,4,5].

A principle characteristic of many PAHs is their abil-ity to activate the aryl hydrocarbon receptor (AhR), aligand activated transcription factor, which, after dimer-ization with AhR nuclear translocator (ARNT), controlsexpression of a battery of genes involved in the con-trol of xenobiotic metabolism, including the cytochromeP450 (CYP) genes Cyp1a1, Cyp1a2, Cyp1b1, as well asa number of phase II metabolizing enzymes [6]. TheCYP enzymes play a major role in the production of theultimate DNA-binding metabolites of genotoxic PAHs[7] and they are also significantly involved in detoxifica-tion of PAHs and their removal from the organism [6].However, stimulation of transcription of genes codingfor detoxification enzymes does not explain the wholerange of toxic outcomes associated with exposure toAhR ligands. It has been proposed that the AhR affin-ity also reflects the promoting effect of PAHs in rodentcarcinogenicity tests [8]. This is in line with a mountingevidence that AhR is involved in regulation of cellulargrowth (reviewed in [9,10]).

Several PAHs and/or their metabolites have beenfound to stimulate cell proliferation in various types ofcells [2,11–13]. However, there is currently no evidencethat the AhR plays any direct role in cell prolifera-tion modulated by PAHs. Therefore, it is important tostudy these regulatory pathways, especially in the lightof the fact that stimulation of cell proliferation mightsubstantially contribute to mutagenicity of polyaromaticcompounds [14,15].

Contact inhibition is a mechanism through whichnon-transformed cells enter a reversible G1 phasearrest, when receiving anti-proliferative signals medi-

ated through cell–cell contacts [16]. Loss of contactinhibition is associated with abnormal growth and theappearance of multilayered foci in culture occurringupon cell malignant transformation [16,17]. Importantly,

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disruption of contact inhibition has been also found tobe induced by various tumor promoters [18,19]. Wehave previously observed that some PAHs, inducing 7-ethoxyresorufin deethylase activity, can stimulate cellproliferation in contact-inhibited rat liver epithelial cells[20]. This suggested that the AhR activation mightparticipate in the PAH-induced release from contact inhi-bition. However, there is so far no direct evidence thatAhR has a functional role in the proliferative effects ofPAHs either in this or in other cellular models. On theother hand, as the expression of CYP1 enzymes is undercontrol of the AhR, this may also significantly modifythe proliferative effects through formation of ultimategenotoxic metabolites of PAHs, inducing DNA damage[6,7]. This is illustrated by the fact that weak mutagensamong PAHs may induce cell proliferation, while stronggenotoxins may actually decrease cell numbers due toinduction of programmed cell death [11,20].

Taken together, the role of AhR in deregulation of cellproliferation by PAHs is unclear. In the present study,we used both expression of a dominant-negative AhRmutant and small interfering RNA (siRNA) targetingAhR, in order to determine the role of AhR activationin the PAH-induced modulation of cell cycle control.The AhR may play a dual role in regulation of cellproliferation in cells affected with PAHs that are eitherweak or strong mutagens. Therefore, we investigatedmodulation of G1/S-phase transition, cell proliferation,apoptosis and expression/activity of proteins involved inmaintenance of contact inhibition, in cells treated withboth weakly and strongly mutagenic PAHs, in order todescribe principal differences between the effects of bothgroups of compounds.

2. Materials and methods

2.1. Chemicals

Fluoranthene (CAS No. 206-44-0, purity 99.9%),benz[a]anthracene (CAS No. 86-73-7, purity 99.9%),benzo[b]fluoranthene (CAS No. 205-99-2, purity 99.9%)and benzo[a]pyrene (CAS No. 50-32-8, purity 99.9%)were provided by Ehrenstorfer (Augsburg, Germany).2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was fromCambridge Isotope Laboratories (Andover, MA, USA).Roscovitine was kindly provided by Dr. Miroslav Strnad(Institute of Experimental Botany, Olomouc, Czech Repub-

purchased from Fluka (Buchs, Switzerland). Polyvinylidenedifluoride (PVDF) membrane Hybond-P and chemilumines-cence detection reagents (ECL + Plus) were obtained fromAmersham (Amersham, Aylesbury, UK). All other chemicalswere provided by Sigma–Aldrich.

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.2. Cells

WB-F344 rat liver epithelial cells [21] were grown in mod-fied Eagle’s Minimum Essential Medium (Sigma–Aldrich,rague, Czech Republic) with 50% increased concentrationsf essential and nonessential amino acids, and supplementedith sodium pyruvate (110 mg/l), 10 mM HEPES, and 5%eat-inactivated fetal bovine serum (Sigma–Aldrich). Onlyhe cells at passage levels 15–22 were used throughout thetudy.

.3. Cell transfection and selection of stable transfectants

Plasmid pAHR�495-805, derived from pcDNAI/B6AHRy deleting the C-terminal 311 amino acids [22], was kindlyrovided by Dr. Alvaro Puga (University of Cincinnati Medi-al Centre, Cincinnati, OH). Stable transfection was performedy co-transfecting 1.75 × 106 WB-F344 cells with dnAhR ormpty vector (each 12 �g) and pPUR (1.2 �g). Six hours afterransfection, the cells were fed with fresh medium contain-ng 10% FCS. After 24 h, the cells were passaged and selectedith puromycin (1.25 �g/ml). Medium was changed every twoays. Functional expression of dn-AhR was detected by deter-ination of TCDD-dependent induction of Cyp1A1 whichas assessed by Western blotting and real-time quantitativeT-PCR.

.4. RNA interference

For transient transfection of siRNA directed against AhR orontrol siRNA, 8 × 104 cells/well were seeded in 24-well platend cultured for 24 h to reach 80–90% confluency. Transfectionas performed in a total volume of 600 �l containing 20 pmol

iRNA and 1 �l of Lipofectamine 2000 (Invitrogene, Carls-ad, CA), according to the manufacturer’s instructions. After8 h, cells were exposed for 24 h to 100 nM BbF or 1 �M BaP.otal cell extracts were prepared and subjected to Western blotnalysis as described above. AhR siRNA (directed against rathR mRNA sequence [gi: 6978474]: CGUUAGAUGUUC-UCUGUGtt (sense) and CACAGAGGAACAUCUAACGtt

antisense); control siRNA (directed against mRNA encodinghe red fluorescence protein DsRed from the coral Discosoma):GUUCCAGUACGGCUCCAAtt (sense) and UUGGAGC-GUACUGGAACUtt [23].

.5. Assessment of cell proliferation and cell cycleistribution

The proliferative effects of PAHs on confluent WB-F344ells were determined as described previously [20]. Briefly,onfluent cells were exposed to tested compounds dissolved

n DMSO for 72 h. Following the exposure the medium wasemoved, cells were harvested with trypsin and counted with

Coulter Counter (Model ZM, Coulter Electronics, Luton,K). For cell cycle analysis, harvested cells were fixed in0% ethanol, stained with propidium iodide and analyzed by

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flow cytometry as described previously [20]. A minimum of15,000 events were collected per sample. Data were analyzedusing ModFit LT version 2.0 software (Verity Software House,Topsham, ME).

2.6. Apoptosis detection

Apoptosis induced by tested compounds was assessed usingmorphological criteria (nuclear fragmentation). Cells (includ-ing floating cells) were harvested and fixed in 70% ethanol,as described above. The fixed cells were centrifuged (200 × g,5 min) and resuspended in 200 �l of supernatant. Five micro-liters of the suspension was mixed with Mowiol-DAPI solution(10% Mowiol solution was prepared in 25% glycerol, 100 mMTris–HCl, pH 8.5 and DAPI 1 �g/ml) and mounted for obser-vation with a fluorescence microscope Olympus IX70. Aminimum of 200 cells was counted per sample.

2.7. Real-time RT-PCR for quantification of CYP1A1mRNA

Total RNA was isolated from cells using the NucleoSpinRNA II kit (Macherey-Nagel, Duren, Germany). The ampli-fications of the samples were carried out in a final volumeof 20 �l in a reaction mixture containing 10 �l of QuantiTectProbe RT-PCR Master Mix, 0.2 �l of QuantiTect RT Mix (Qia-gen, Valencia, CA), 2 �l of solution of primers and probe, 5.8 �lof water and 2 �l of sample. The final concentration of eachprimer was 1.0 �M and probe was 0.2 �M. All probes werelabeled with the fluorescent reporter dye 6-carboxyfluorescein(6-FAM) on the 5′-end and with the Black Hole 1 (BH 1) flu-orescent quencher dye on the 3′-end. The amplifications wererun on the LightCycler (Roche Diagnostics, Mannheim, Ger-many) using the following program: reverse transcription at50 ◦C for 20 min and initial activation step at 95 ◦C for 15 min,followed by 35 cycles at 95 ◦C for 0 s and 60 ◦C for 60 s. Theprimer and probe sequences used for rat CYP1A1 and ref-erence gene porphobilinogen deaminase have been publishedpreviously [24].

2.8. Western blotting

Forty-eight hours treated cells were lysed in SDS samplebuffer (1% SDS, 10% glycerol, 100 mM Tris, pH 7.4). Proteinconcentration was estimated using Bio-Rad DC Protein Assay(Bio-Rad Laboratories, Inc., Hercules, CA). Following a com-mon SDS-PAGE in 7.5–12% gels proteins were transferredon a PVDF membrane using a semi-dry blotter. The pro-teins were detected with following antibodies: anti-Cyclin A(sc-751), anti-cdk2 (sc-163 M-2) from Santa Cruz Biotechnol-ogy (Santa Cruz, CA), anti-pRB (#14001A) from Pharmingen

(BD Biosciences, San Jose, CA), anti-AhR (SA-210-0025)from Biomol (Butler Pike, PA) and anti-p27 (#610242) fromTransduction Laboratories (BD Biosciences). For chemilumi-nescence detection ECLPlus reagent was used according tomanufacturer’s instructions.

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Fig. 1. Induction of CYP1A1 mRNA following 24-h treatmentwith 1 �M fluoranthene (Fla), 1 �M benzo[a]pyrene (BaP), 1 �Mbenz[a]anthracene (BaA) and 100 nM benzo[b]fluoranthene (BbF) inwild type WB-F344 cells, and in cells transfected either with dnAhRconstruct or with vector control, expressed relative to control (DMSO0.1%). Total RNA was isolated and quantitative real-time RT-PCR wasperformed. The results are expressed as mean ± S.D. of three indepen-

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2.9. Cyclin-dependent kinase 2 (cdk2) assay

For in vitro kinase assay, cells were lysed in kinase lysisbuffer (50 mM HEPES–KOH pH 7.5, 150 mM NaCl, 1 mMEDTA, 2.5 mM EGTA, 0.1% Tween 20, 1 mM DTT, 10 mg/mlleupeptin, 10 mg/ml aprotinin, 10 mM �-glycerophosphate,0.1 mM Na3VO4 and 1 mM NaF). Protein complex of CyclinA/cdk2 was isolated by immunoprecipitation with anti-CyclinA antibody. Antibody bound to target proteins was extractedusing A-protein Sepharose beads (Sigma–Aldrich). Beadswere washed three times in kinase lysis buffer and once inkinase buffer (50 mM HEPES–KOH pH 7.5, 10 mM MgCl2,2.5 mM EGTA, 1 mM DTT, 10 mM �-glycerophosphate,0.1 mM Na3VO4 and 1 mM NaF), before incubation with32P-labeled �-ATP (5 mCi per sample) and histon H1(Calbiochem-Novabiochem Corp., La Jolla, CA) for 30 minat 30 ◦C. Kinase reaction was terminated by addition of aSDS sample buffer (3% SDS, 300 mM Tris pH 7.4, 30%glycerol). Protein samples were separated on SDS-PAGE,and the phosphorylated histon H1 was visualized by autora-diography. Densitometry was performed using AIDA ImageAnalyzer software (raytest Isotopenmeßgerate, Starubenhardt,Germany).

2.10. Statistical analysis

Data were expressed as means ± S.D. and analyzed byStudent t-test (cell numbers), or by the nonparametricMann–Whitney U-test (% of cells in S-phase, % of apoptoticcells, CYP1A1 mRNA levels) and Kruskal–Wallis analysis ofvariance (ANOVA) (cdk2 activity). A P-value of less than 0.05was considered significant.

3. Results

3.1. Expression of a dominant-negative mutant formof the AhR prevents induction of CYP1A1 expressionby PAHs in WB-F344 cells

Based on our previous study [20], we selected fourmodel PAHs in order to study the role of the AhR intheir effects on cell proliferation and apoptosis in rat liverepithelial cells. We selected 3 model PAHs: BaA andBbF, both shown to induce cell proliferation in contact-inhibited WB-F344 cells; and BaP, a strong genotoxinand AhR ligand that has been previously observed toinduce cell death in this cell line [20]. As a negative con-trol we used fluoranthene (Fla), a poor AhR ligand, whichdoes not affect significantly either cell proliferation orapoptosis.

As shown in Fig. 1, Fla was a poor inducer ofCYP1A1 mRNA expression in WB-F344 cells. In con-trast both BaP and BbF, and to a lesser extent also BaA,induced significant increase in CYP1A1 in WB-F344

dent experiments. Symbols ‘*’ and ‘**’ denote a significant differencebetween wild-type cells treated with tested compound and cells trabs-fected with dnAhR construct (P < 0.05 and <0.01, respectively).

cells. In order to test the efficiency of dnAhR expres-sion to block induction of CYP1A1 mRNA, a modelAhR-regulated gene, we examined effects of PAHs onCYP1A1 expression in cells stably transfected withtruncated AHR�495-805 (dnAhR), which lacks transac-tivation domain, and in cells transfected with the emptyvector. As summarized in Fig. 1, induction of CYP1A1mRNA was strongly reduced in cells transfected with thednAhR construct, while the levels of CYP1A1 inducedby PAHs in mock-transfected cells were comparable towild type cells.

3.2. Expression of a dominant-negative mutant formof the AhR prevents induction of cell proliferation byPAHs in contact-inhibited WB-F344 cells

As the data presented above showed that stableexpression of dnAhR successfully prevents induction ofAhR-dependent gene expression by PAHs, we furtherinvestigated the impact of dnAhR on cell proliferationinduced by PAHs in contact-inhibited WB-F344 cells.As outlined in Fig. 2, BaA and BbF induced increaseof cell numbers in wild type WB-F344 cells, but not indnAhR cells. This was similar to the effects of the model

AhR ligand TCDD suggesting that, like persistent AhRligands, PAHs modulate cell proliferation in WB-F344in an AhR-dependent manner. The genotoxic AhR lig-and BaP reduced cell numbers significantly in wild type,

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Fig. 2. Effects of selected PAHs on cell proliferation in wild typeWB-F344 cells and in cells stably transfected with either dnAhR orwith empty vector. Confluent WB-F344 cells were treated with DMSO(0.1%, v/v) as a vehicle; TCDD (1 nM) as positive control; Fla (1 �M)as a negative control; BbF (100 nM), BaA (1 �M), BaP (1 �M) orTCDD 1 nM for 72 h. Following exposition, trypsinized cells werecounted on a Coulter Counter. Data represent mean values ± S.D.obtained from three independent experiments and are expressed rel-ads

batfpos

tive to group treated with DMSO. Symbol ‘*’ denotes a significantifference between sample treated with studied compound and controlample treated with DMSO (P < 0.05).

ut not in the dnAhR cells. The induction of cell prolifer-tion was also observed in vector control cells, showinghat this effect is specific and it is not related to trans-

ection with vector itself. As expected, Fla, which is aoor AhR ligand, had no effect on cell proliferation. Allf the above data were also independently verified in aeparate clone of dnAhR cells (data not shown).

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3.3. BaP-induced apoptosis is attenuated in dnAhRcells

Formation of genotoxic metabolites of BaP plays acrucial role in BaP-induced cytotoxicity and apopto-sis [25]. As the expression of several enzymes, suchas CYP1A1, involved in formation of ultimate geno-toxic metabolites is under control of the AhR [6], wenext investigated the impact of dnAhR on induction ofapoptosis by BaP, as the expression of dnAhR shouldpresumably inhibit metabolic activation of BaP andresulting DNA damage leading to cell death. Induc-tion of apoptosis, determined as fragmentation of cellnuclei, was significantly lower in dnAhR cells as com-pared to both wild type cells and cells transfected withvector control (Fig. 3). Similar data have been alsoobtained by using annexin V staining of externalizedphosphatidylserine as a measure of apoptosis induction(data not shown).

3.4. Effects of dnAhR on cell cycle proteins in cellstreated with PAHs

Downregulation of Cyclin A and upregulation ofp27Kip1 cdk inhibitor, as well as decreased cdk2 activ-ity have been previously shown to play a crucial rolein maintenance of contact inhibition [17,26–28]. There-fore, we next investigated the impact of PAHs onexpression of these proteins, as well as on phospho-rylation pattern of the pRb tumor suppressor, whichreflects progression of cells through the cell cycle, inboth wild type and dnAhR cells. We found that bothBaA and BbF increased expression of Cyclin A in wildtype, but not in dnAhR cells (Fig. 4). Interestingly,BaP induced a significantly higher increase in CyclinA than both weak mutagens, and this effect was par-tially attenuated in dnAhR cells. None of the tested PAHsaffected cdk2 levels in either wild type or in dnAhRcells. We further observed that neither BaA nor BbFwere able to reduce p27Kip1 expression (Fig. 4). In con-trast, the genotoxic BaP induced a decrease in p27Kip1

in wild type cells, while this effect was lost in dnAhRcells. Only BaP was found to induce significant pRbhyperphosphorylation. The BaP-induced hyperphospho-rylation of pRb was significantly reduced in dnAhR cells(Fig. 4).

3.5. Effects of PAHs on activity of Cyclin A/cdk2

complex

We observed that BaA and BbF, in a manner similarto TCDD, increased the percentage of cells entering S-

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Fig. 3. Effects of BaP on apoptosis in wild type WB-F344 cells and in cells stably transfected with either dnAhR or with vector control. Confluentlei staince persignific

cells were treated with 1 �M BaP for 48 h, and fragmentation of nucfluorescence microscopy. A minimum of 200 cells were counted twimean ± S.D. of three independent experiments. Symbol ‘*’ denotes awith BaP (P < 0.05).

phase in wild type cells, but not in dnAhR cells (Fig. 5A).In contrast, BaP induced significantly higher accumula-tion of cells in S-phase, which was even comparableto proliferating population of WB-F344 cells. Never-theless, a strong decrease in cells in S-phase was alsodetected in dnAhR cells treated with BaP as comparedto wild type cells, although their percentage was still

higher than in dnAhR cells treated with DMSO. Thismight be related to the fact that despite a strong reduc-tion of CYP1A1 induction, its inhibition is not complete,or that other enzymes that are able to metabolize BaP to

Fig. 4. Changes in expression of cell cycle regulatory proteins induced by PA(0.1%, v/v), TCDD (1 nM), Fla (1 �M), BbF (100 nM), BaA (1 �M) or Baperformed. In order to document equal loading of samples, expression of �-acthree independent experiments.

ed with DAPI, characteristic for apoptotic cells, was determined byeach sample. The number reflects percentage of apoptotic cells as aant difference between wild type and dnAhR cell populations treated

its proximate genotoxic pro-apoptotic metabolites arepresent in WB-F344 cells [24].

We found that BaA and BbF induced a signifi-cant increase in Cyclin A/cdk2 complex activity, whichwas similar to effects of TCDD (Fig. 5B). In contrast,BaP induced a significantly higher level of cdk2 activ-ity, which corresponded with its effects on Cyclin A

and p27Kip1 expression. The BaP-induced cdk2 activ-ity was significantly attenuated in dnAhR cells and theBbF-induced increase in cdk2 activity was completelyprevented in dnAhR WB-F344 cell variant (Fig. 5B).

Hs. Confluent population of WB-F344 cells was exposed to DMSOP (1 �M) for 48 h. Cell lysates were prepared and Western blottingtin was determined in each sample. The results are representative from

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Fig. 5. Cell cycle analyses and kinase assays performed in contact-inhibited wild type and dnAhR WB-F344 cells. Confluent cell weretreated with DMSO (0.1%, v/v), TCDD (1 nM), Fla (1 �M), BbF(100 nM), BaA (1 �M) or BaP (1 �M) for 48 h. The results were com-pared with exponentially growing cells (exp. growing). (A) Percentageof cells in S-phase of cell cycle. The data represent mean values ± S.D.obtained from three independent experiments. Symbols ‘*’ denotes asignificant difference between sample treated with studied compoundand control sample treated with DMSO (P < 0.05). (B) Activity ofthe Cyclin A/cdk2 complex in wild-type and dnAhR WB-F344 cellstreated with test compounds. Immunoprecipitation was performedusing anti-Cyclin A antibody and kinase activity of the immunoprecip-itates was measured in the presence of [�-32P]ATP using histone H1 as

Fig. 6. Induction of Cyclin A expression by BbF and BaP is AhR-dependent. Cells were transfected with siRNA targeted against AhRor against Dichostoma DsRed protein, grown for 48 h to reach conflu-ence and then treated for 24 h with DMSO (0.1%), BbF (100 nM) orBaP (1 �M) for 24 h. (A) siRNA designed against rat AhR efficientlyreduces amount of AhR protein as compared to untreated control andcells incubated with siRNA against DsRed protein. The cells weretreated as described above and the level of AhR was verified in eachsample by Western blotting. The result shown here is representative

of three independent experiments. (B) Effects of transient transfectionwith siRNA on induction of CYP1A1 and Cyclin A proteins by BbFand BaP. For a loading control, the membrane was incubated with anti-ERK2 antibody. One representative experiment out of three is shown.

In order to determine whether an increased cdk2 activ-ity plays a significant role in the observed PAH-inducedrelease from contact inhibition, we used roscovitine, asynthetic inhibitor of cdk2 [29]. We found that bothTCDD- and BbF-induced proliferative responses weresignificantly inhibited by addition of 10 �M roscovitine(Fig. 5C). This again supports the hypothesis that upreg-ulation of cdk2 activity is crucial for the proliferativeeffects of PAHs on contact-inhibited WB-F344 cells.

3.6. Small inhibitory RNA directed against AhRsuppressed both CYP1A1 and Cyclin A upregulationin BbF-treated WB-F344 cells

In order to provide further evidence that the AhRis essential for the PAH-induced cell proliferation incontact-inhibited WB-F344 cells, we transiently trans-fected WB-F344 cells with siRNA duplexes directed

a substrate. One representative result of three independent experimentsis presented. The results of densitometry shown in parallel representmeans ± S.D. of three independent experiments. Symbol ‘*’ denotes asignificant difference between sample treated with studied compoundand control sample treated with DMSO (P < 0.05). (C) Roscovitine,cdk2 inhibitor, attenuates cell numbers increase induced either by 1 nMTCDD (positive control) or 100 nM BbF. Symbol ‘*’ denotes a signifi-cant difference between group treated with tested compound and grouptreated with tested compound in combination with inhibitor (P < 0.05).

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against rat AhR [23]. As shown in Fig. 6, protein lev-els of the AhR were significantly decreased in cellstransfected with siRNA for AhR, whereas its levelswere unchanged in cells transfected with non-specificcontrol siRNA. Knock-down of the AhR by siRNAresulted in a significantly reduced CYP1A1 inductionby BbF (100 nM) or BaP (1 �M), as compared to con-trol cells or to cells transfected with control siRNA. Thisindicated that knock-down of the AhR successfully pre-vented transcriptional activation of AhR-dependent geneupon BbF and BaP treatment. As further outlined inFig. 6, knock-down of the AhR inhibited BbF-inducedincrease in Cyclin A expression, as compared to con-trols. It confirmed that elevated Cyclin A expression,which was found to be associated with increased CyclinA/cdk2 activity and proliferative effect of BbF, was AhR-dependent. Similarly, knock-down of the AhR reducedlevels of Cyclin A induced by BaP, further supportingthe results obtained with dnAhR cells.

4. Discussion

The stem cell hypothesis of cancer suggests that stemor ‘stem-like’ cells are likely to play an important role incarcinogenesis [30,31]. However, a majority of studieson carcinogenicity of PAHs have concentrated in paston prevalent mature differentiated cells in given organs,such as keratinocytes in skin or hepatocytes in the liver,despite the fact that there might be large differences, e.g.in accumulation of genotoxic PAH metabolites amongdifferent cell types [3]. The existence of stem cells inliver has been a matter of long debate, nevertheless it isnow generally agreed that oval cells in the liver are ‘stem-like’ cells descending from a population of progenitorcells involved in liver regeneration [32]. The liver con-tains a population of progenitor cells, which in case ofserious liver injury and inhibition of hepatocyte regen-erative capacity can give rise to oval cells [33]. The ovalcells, which can develop into various liver cell types,have been suggested to play a significant role in hepa-tocarcinogenesis [34–36]. Using a large series of PAHs,we have demonstrated in our previous study that PAHsmay induce either cell proliferation or a massive pertur-bation of cell cycle and apoptosis in contact-inhibitedrat liver epithelial WB-F344 cells [20], which are con-sidered to represent an in vitro model of oval cells [21].Importantly, another recent report has shown that AhRligands induce cholangiocarcinomas in rat liver [37],

thus suggesting that oval cells, being precursors of bothhepatocytes and biliary epithelial cells, might be a directtarget for carcinogenic effects of AhR ligands. There-fore, in the present study, we attempted to verify the

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hypothesis that the AhR is actively involved not onlyin regulation of PAH-metabolizing enzymes, but alsoin disruption of contact inhibition induced by PAHs.Elucidation of such mechanisms should contribute toour understanding of cellular events associated with thetumor promoting activity of PAHs and activation of theAhR [8].

There is an increasing body of evidence that theAhR participates in various pathophysiological pro-cesses involving the immune, cardiovascular and liversystem. The AhR-negative Hepa1c1c7 cells or mouseAhR−/− embryonic fibroblasts grow more slowlythan their AhR-positive counterparts [38,39]. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a prototypicalAhR ligand, can inhibit DNA synthesis in rat hepato-cytes both in vitro and in vivo [9]. TCDD also inhibitscell cycle progression in rat hepatoma cells [40]. This hasbeen attributed both to AhR-dependent transcriptionalactivation of genes such as p27Kip1 cdk inhibitor andto transcriptional co-repression of E2F activity throughassociation of AhR with pRb protein, resulting in pre-vention of p300/CBP access to E2F [40–42]. However,all of these studies have been performed using prolif-erating cells as a principal model. In contact-inhibitedrat liver epithelial cells, various types of AhR ligandsinduce a loss of contact inhibition leading to increasedcell proliferation [19,20,43,44]. This suggests that dif-ferent mechanisms may regulate cell proliferation uponAhR activation in quiescent cells that are in G1 phase ofcell cycle than in those that actively proliferate, and thatvarious subpopulations of liver cells might differentiallyrespond to AhR ligands.

We showed in the present study that expression ofa dnAhR mutant suppresses both induction of a modeltarget gene of AhR, CYP1A1, and induction of cell pro-liferation in contact-inhibited WB-F344 cells elicited byBbF and BaA (Figs. 1 and 2), two PAHs with relativelyhigh AhR-binding affinity [45]. The upregulation of cellproliferation in wild type WB-F344 cells was associatedwith increased expression of Cyclin A and signifi-cantly higher Cyclin A/cdk2 activity than in control cells(Figs. 4 and 5). Both upregulation of Cyclin A expressionand increased cdk2 activity were significantly attenu-ated in dnAhR cells. Further support for the hypothesissuggesting active involvement of the AhR in disruptionof contact inhibition induced by PAHs came from theexperiment showing that transient transfection of WB-F344 cells with siRNA against AhR attenuated both

induction of CYP1A1 and Cyclin A. Downregulation ofCyclin A gene expression during contact inhibition hasbeen shown to contribute to inhibition of cdk2 and cellproliferation by cell–cell contact [28,46]. Importantly,

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verexpression of Cyclin A may overcome the G1/Slock [47], suggesting that the observed upregulationf Cyclin A/cdk2 complex activity may play an impor-ant role in the release from contact inhibition inducedy PAHs. This is supported by the fact that roscovitine,chemical inhibitor of cdk2 [29], prevented increased

ell proliferation induced by both TCDD and BbF.Upregulation of p27Kip1 has been reported to playcrucial role in maintenance of contact inhibition

17,26,27,46]. Nevertheless, high density growth arrestay occur also in absence of p27Kip1 induction [48].e found that expression of p27Kip1, which expres-

ion is high in contact-inhibited WB-F344 cells, wasot attenuated in cells treated with either BaA or BbF,hich was again similar to the model persistent AhR

igand TCDD. In the present study, there was also noignificant increase of pRb phosphorylation followinghe treatment with either BaA or BbF. This supports theypothesis that Cyclin A functions mainly downstreamf pRb [19] during the release from contact inhibitionnduced by PAHs. It has been proposed that reduceddk2 activity in contact-inhibited cells depends both oneduction of Cyclin A and upregulation of p27Kip1, whichinds and inactivates cyclin E/cdk2 complex [46]. Ourresent results may suggest that upregulation of Cyclin Anduced by PAHs is sufficient to overcome growth arrestnduced by cell–cell contact in WB-F344 cells.

In contrast to BbF and BaA, the genotoxic AhR ligandaP induced significant level of apoptosis and mas-

ive accumulation in S-phase in wild type WB-F344ells. It has been proposed that PAHs fail to induce G1rrest, which has been suggested to increase the like-ihood of fixation of mutations [49]. Accumulation ofells in S-phase has been attributed to the activationf S-phase checkpoints, as an integral part of cellu-ar response to formation of bulky DNA adducts byAH dihydrodiol epoxides [50]. Elimination of poten-ial DNA lesions by removing damaged cells throughpoptosis is a further option for maintenance of geneticntegrity of multicellular organisms [51]. However, whenxamining more closely the effects of BaP on WB-344 cells, we observed that accumulation of cells in-phase is more likely due to an intensive cell prolif-ration than a cell cycle arrest. First, there has beenstrong increase in Cyclin A expression and Cyclin/cdk2 activity, which was even comparable to levelsbserved in exponentially-growing cells. This is in aarked contrast with previous report, where cell cycle

rrest observed in fibroblasts treated with BaP was asso-iated with post-trascriptional downregulation of Cyclin

[52]. Second, BaP induced both decreased expres-ion of p27Kip1 and hyperphosphorylation of pRb, which

earch 615 (2007) 87–97 95

again is indicative of increased cell proliferation. Takentogether, these results seem to imply that BaP induces adual response in WB-F344 cells, both programmed celldeath and compensatory cell proliferation. On the otherhand, in cannot be excluded that accumulation of cells inS-phase was associated with cell cycle block due to DNAdamages blocking the replication forks. The expressionof Cyclin A, cdk2 activity and pRb phosphorylationwere lower in dnAhR cells than in wild type cells,while p27Kip1 expression was not attenuated in dnAhRcells upon BaP treatment. A transient transfection ofWB-F344 cells with siRNA targeted against AhR alsoattenuated Cyclin A induction. Expression of dnAhRsignificantly reduced expression of CYP1A1, one of theprincipal enzymes involved in metabolic activation ofBaP [7], and it attenuated both apoptotic and proliferativeeffects of BaP. Nevertheless, the BaP-induced prolifer-ation was not fully inhibited in dnAhR cells. This maybe either due to the fact that induction of CYP1A1 wasnot fully suppressed in dnAhR WB-F344 cells, or due topresence of other enzymes involved in metabolic activa-tion of PAHs in WB-F344 cells, such as CYP1B1 [24].Dibenzo[a,l]pyrene (DBalP), a more potent genotoxinthan BaP has been reported to induce either G1- or S-phase arrest accompanied with cell death in various cellmodels, including WB-F344 cells [11,20,24,53]. How-ever, DBalP is a poor AhR ligand [54], which does notinduce CYP1A1 expression in WB-F344 cells (unpub-lished data), suggesting that there is present in WB-F344cell a sufficient pool of enzymes capable to metabolicallyactivate mutagenic PAHs, thus supporting the secondpossibility.

In conclusion, the present data suggest that activa-tion of AhR plays a significant role both in disruptionof contact inhibition by weakly mutagenic PAHs, suchas BbF or BaP, and in genotoxic effects of BaP lead-ing to enhanced cell proliferation. Both types of effectsmay increase the proliferative rate and the likelihoodof fixation of persistent DNA adducts into mutations.Given the fact that AhR ligands among PAHs belongalso among the most potent carcinogens in this group ofcompounds [1], more attention should be paid to AhR-activating properties of PAHs and related environmentalcontaminants. Their AhR-activating capacity may notonly contribute to their metabolic activation, but alsoto other processes involved in carcinogenesis, such astumor promotion.

Acknowledgments

Authors thank Dr. A. Puga for providing plasmidpAHR�495-805, and Dr. M. Strnad for kindly provid-

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ing roscovitine. This study was supported by grant No.B6004407 from the Grant Agency of the Academy ofSciences of the Czech Republic, by the Research Planof the Academy of Sciences of the Czech Republic,No. AVOZ50040507 and by the Ministry of Agricultureof the Czech Republic (grant No. 00002716201). Thiswork was partly supported by ECNIS, European Union6th Framework Program, Priority 5: “Food Quality andSafety” (Contract No. 513943).

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