Lucia Corich

Alejandro Aranda

Laura Carrassa

Cristina Bellarosa

J Donald Ostrow

MD Claudio Tiribelli

The cytotoxic effect of unconjugated bilirubin in human neuroblastoma SH-SY5Y cells is modulated by the expression level of MRP1 but not MDR1

Keywords: neurotoxicity, bilirubin, MRP1, MDR1, kernicterus

In vitro and in vivo studies have demonstrated that unconjugated bilirubin (UCB) is neurotoxic. Although previous studies suggested that both MRP1 and MDR1 proteins may protect cells against accumulation of UCB, direct comparison of their role in UCB transport was never performed. To this end, we used an inducible siRNA expression system to silence the expression of MRP1 and MDR1 in human neuroblastoma SH-SY5Y cells.. The effects of in vitro exposure to clinically-relevant levels of unbound UCB were compared between unsilenced (control) cells and cells with similar reductions in the expression of MRP1 or MDR1, documented by RT-PCR (mRNA), immunoblotting (protein), and for MDR1, the enhanced net uptake of a specific fluorescent substrate. Cytotoxicity was assessed by the MTT test. MRP1deficient cells accumulated significantly more UCB and suffered greater cytotoxicity than controls. By contrast, MDR1-deficient cells exhibited UCB uptake and cytotoxicity comparable to controls. At intermediate levels of silencing, the increased susceptibility to UCB toxicity closely correlated with the decrease of the expression of MRP1, but not of MDR1. These data support the concept that limitation of cellular UCB accumulation, due to UCB export mediated by MRP1, but not MDR1, plays an important role in preventing bilirubin encephalopathy in the newborn.

INTRODUCTION

In humans and rodents, unconjugated bilirubin (UCB) may be neurotoxic. In some neonates with severe hyperbilirubinemia, UCB accumulates in neurons and astroglial cells in specific brain regions, causing encephalopathy and kernicterus [START_REF] Wennberg | Toward understanding kernicterus: a challenge to improve the management of jaundiced newborns[END_REF]. Initial in vitro studies of the effects of UCB on mouse neuroblastoma cell lines revealed a higher sensitivity to UCB toxicity of mitotically active N2AB-1 cells than of "mature" neurons [START_REF] Notter | Differential sensitivity of neural cells to bilirubin toxicity[END_REF], and a progressive and irreversible toxicity in N-115 cells exposed to UCB for one or more hours [START_REF] Amit | Bilirubin toxicity in a neuroblastoma cell line N-115: II. Delayed effects and recovery[END_REF].

Toxic effects of UCB are greatly decreased by tight binding to plasma albumin, since only the unbound diacid species of UCB, which is dominant at physiological pH [4;5], can diffuse rapidly across cell membranes [START_REF] Zucker | Unconjugated bilirubin exhibits spontaneous diffusion through model lipid bilayers and native hepatocyte membranes[END_REF]. In vitro exposure of neurons or astrocytes to UCB has shown neuroprotection at low, physiological concentrations of unbound UCB (B f ), but neurotoxicity at modestly higher B f values [5;7]. Others reported that UCB activated the mitochondrial pathway of apoptosis in rat brain neurons, inducing mitochondrial depolarization and Bax translocation via physical interaction with membranes [START_REF] Rodrigues | Bilirubin induces apoptosis via the mitochondrial pathway in developing rat brain neurons[END_REF]. Studies performed on human NT2-N neurons showed that UCB induces rapid necrosis at high and moderate concentrations and delayed apoptosis at low and moderate concentrations [START_REF] Hanko | Bilirubin induces apoptosis and necrosis in human NT2-N neurons[END_REF]. In human neuroblastoma SH-SY5Y cells, clinically relevant UCB concentrations cause early disruption of the mitochondrial membrane potential and subsequently induce apoptosis [START_REF] Han | Bilirubin induced apoptosis of human neuroblastoma cell line SH-SY5Y and affected the mitochondrial membrane potential[END_REF].

Collectively these findings indicate that intracellular accumulation of UCB may cause serious damage to nerve cells. This prompted us to explore the mechanisms that may limit toxic intracellular accumulation of UCB. The multidrug resistance-associated protein, MRP1 (ABCC1), and the multidrug resistance protein, MDR1 (P-Glycoprotein, ABCB1), belong to the superfamily of the ATP-binding cassette (ABC) transporters [START_REF] Borst | Mammalian ABC transporters in health and disease[END_REF]. MRP1 transports amphipathic anionic, glucuronide and sulphate conjugates of steroid hormones and bile salts. In the presence of physiological concentrations of reduced glutathione, MRP1 also transports unconjugated xenobiotics and uncharged drugs [START_REF] Borst | Mammalian ABC transporters in health and disease[END_REF][START_REF] Loe | Multidrug resistance protein (MRP)-mediated transport of leukotriene C4 and chemotherapeutic agents in membrane vesicles. Demonstration of glutathione-dependent vincristine transport[END_REF][START_REF] Renes | ATP-and glutathione-dependent transport of chemotherapeutic drugs by the multidrug resistance protein MRP1[END_REF]. MDR1 transports hydrophobic, either uncharged or weakly positively charged compounds in their unmodified forms and, in particular conditions, even highly charged, anionic compounds [START_REF] Borst | Mammalian ABC transporters in health and disease[END_REF]. Both MRP1 and MDR1 protect nerve cells from toxic substances by promoting their active export from the cells [START_REF] Decleves | Functional expression of P-glycoprotein and multidrug resistanceassociated protein (Mrp1) in primary cultures of rat astrocytes[END_REF][START_REF] Falcao | Role of multidrug resistance-associated protein 1 expression in the in vitro susceptibility of rat nerve cell to unconjugated bilirubin[END_REF][START_REF] Pardridge | Brain microvascular and astrocyte localization of P-glycoprotein[END_REF][START_REF] Regina | Mrp1 multidrug resistance-associated protein and P-glycoprotein expression in rat brain microvessel endothelial cells[END_REF][START_REF] Zhang | Induction of P-glycoprotein expression in astrocytes following intracerebroventricular kainate injections[END_REF].

Studies using various experimental models demonstrated that MRP1 is directly involved in the transport of UCB [START_REF] Calligaris | Multidrug resistance associated protein 1 protects against bilirubin-induced cytotoxicity[END_REF][START_REF] Pascolo | Mechanisms for the transport of unconjugated bilirubin in human trophoblastic BeWo cells[END_REF][START_REF] Petrovic | The products of YCF1 and YLL015w (BPT1) cooperate for the ATPdependent vacuolar transport of unconjugated bilirubin in Saccharomyces cerevisiae[END_REF]. In plasma-membrane vesicles from MDCKII cells (Madin-Darby canine kidney II cells), the stable expression of human MRP1 increased significantly the export of UCB with high affinity, K m =10±3 nM (B f ) [START_REF] Rigato | The human multidrugresistance-associated protein MRP1 mediates ATP-dependent transport of unconjugated bilirubin[END_REF]. In cultured mouse astrocytes and primary cultures of rat neurons and astrocytes, functional inhibition of Mrp1 with MK571 engendered an increase in UCB-induced toxicity [START_REF] Falcao | Role of multidrug resistance-associated protein 1 expression in the in vitro susceptibility of rat nerve cell to unconjugated bilirubin[END_REF], even at a normally nontoxic B f of 40 nM [START_REF] Gennuso | Bilirubin protects astrocytes from its own toxicity by inducing up-regulation and translocation of multidrug resistance-associated protein 1 (Mrp1)[END_REF]. Collectively, these findings show that MRP1 exports UCB at relatively low, physiologically-relevant B f , and thus may protect the cell from accumulation of toxic levels of UCB.

Other studies have suggested that Mdr1/MDR1 might also transport UCB. UCB may be a weak substrate for Mdr1a [START_REF] Gosland | Bilirubin: a physiological substrate for the multidrug transporter[END_REF] and competitively inhibits the labelling of brain capillary MDR1 with a photoaffinity substrate [START_REF] Jetté | Interaction of drugs with Pglycoprotein in brain capillaries[END_REF]. A greater proportion of UCB administered intravenously is taken up by the brains of mdr1a (-/-) knockout mice compared with their mdr1a (+/+) controls [START_REF] Watchko | Brain bilirubin content is increased in P-glycoprotein-deficient transgenic null mutant mice[END_REF]. Although these studies suggest that MDR1 may be involved in the transport of UCB, they were all performed with high doses of UCB that yielded bilirubin concentrations vastly higher than clinically-relevant levels [27;28]. However, drugs known to inhibit MDR1 function may increase the risk of bilirubin encephalopathy in jaundiced neonates [START_REF] Hanko | Administration of drugs known to inhibit P-glycoprotein increases brain bilirubin and alters the regional distribution of bilirubin in rat brain[END_REF].

The aim of the present work was to compare directly the role of MRP1 and MDR1 in the prevention of UCB related cytotoxicity in the human neuroblastoma SH-SY5Y cell line, which expresses both proteins [30;31] and is highly susceptible to UCB toxicity [START_REF] Han | Bilirubin induced apoptosis of human neuroblastoma cell line SH-SY5Y and affected the mitochondrial membrane potential[END_REF]. The expression of either MRP1 or MDR1 in these cells was modulated by using small interference RNAs (siRNAs) produced by an inducible vector, and net accumulation and cytotoxicity of UCB were assessed.

Our experiments are the first to directly compare the protective role of MRP1 and MDR1 against UCB toxicity in a single cell line under identical experimental conditions. We demonstrate that knockdown of MRP1, but not MDR1: a) decreases the cellular export of UCB and increases the cytotoxicity of UCB over a range of clinically-relevant B f levels (40-140 nM); b) renders the cells sensitive to UCB toxicity at low B f levels that are non-toxic to control cells. purchased from Boehringer Mannheim, (Indianapolis, Ind., USA). iScript TM cDNA Synthesis Kit and iQ TM SYBR Green Supermix were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Cell Lysis Buffer was purchased from Cell Signaling (Beverly, MA, USA). ECL-Plus Western Blotting detection system solutions were purchased from Amersham-Pharmacia Biotech (Buckinghamshire, UK). UCB was purchased from Sigma (St. Louis, MO, USA) and purified as described by McDonagh and Assisi [START_REF] Mcdonagh | The ready isomerization of bilirubin-IX in aqueous solution[END_REF]. [ 3 H]-bilirubin (29.25 mCi/mmol) was biosynthetically labelled in vivo and then highly purified from the bilirubin conjugates in bile as described [START_REF] Bayón | Pitfalls in preparation of (3)H-unconjugated bilirubin by biosynthetic labeling from precursor (3)H-5aminolevulinic acid in the dog[END_REF]. Liquid scintillation cocktail for radioassay (Filter counter Nº 6013149) was purchased from Packard Bioscience (Groningen, The Netherlands). MRP1-A23 rabbit antibody was prepared at our laboratory [START_REF] Fernetti | Preparation of an antibody recognizing both human and rodent MRP1[END_REF], anti-Mouse IgG (Fc specific)-Peroxidase antibody produced in goat affinity isolated antibody, rabbit anti-actin affinity isolated antibody and peroxidase conjugated-goat anti-rabbit IgG-whole molecule affinity isolated antigen specific antibody were purchased from Sigma (St. Louis, MO, USA). Anti-C219 (MDR1) Monoclonal Antibody was purchased from Signet Laboratories (Dedham, MA, USA).

EXPERIMENTAL

Chemicals

Construction of pSUPERIOR-MRP1 and pSUPERIOR-MDR1 clones

We used pSUPERIOR.puro vector (Oligoengine, Seattle, WA, USA), a tetracycline (or doxycycline)-regulated vector for inducible siRNA expression. The siRNA sequence against MDR1 was obtained by Wu et al. [START_REF] Wu | Small interfering RNA-induced suppression of MDR1 (P-glycoprotein) restores sensitivity to multidrug-resistant cancer cells[END_REF] while that against MRP1 was the Silencer Validated MRP1-siRNA (#51321-1651 Ambion, Austin TX, USA). The pSUPERIOR.puro vector was digested by BglII and HindIII and the annealed oligonucleotides ligated and cloned into the vector. The constructs were confirmed by sequencing. SH-SY5Y cells were transfected (Lipofectamine 2000) with the pcDNA6/TR vector (Invitrogen, Carlsbad, CA, USA) expressing high levels of the Tetracycline repressor (TetR) gene and cells grown in medium containing 5 µg/mL blasticidin. After transient transfection of the pcDNA4/TO/lacZ vector (Invitrogen, Carlsbad, CA, USA), single colonies were isolated and expanded. They were screened for expression of the highest levels of Tet repressor from pcDNA6/TR, by assaying -galactosidase expression upon induction with 3.9 µM doxycycline, used as an alternative inducing agent to tetracycline (half-life: 48 vs. 24 hours, respectively) according to the pSUPERIOR manufacturer's suggestions. The clone showing the highest ratio of -galactosidase activity between induced and non-induced samples was selected for transfecting the pSUPERIOR vector containing the siRNA-expressing sequence targeting MRP1 or MDR1. The empty pSUPERIOR.puro vector was also transfected and used as control. Puromycin (0.8 µg/mL)resistant colonies were expanded for two additional months. The clones showing the highest knockdown of MRP1 (pSUPERIOR-MRP1) or MDR1 (pSUPERIOR-MDR1) upon induction with 3.9 µM doxycycline were selected by gene, protein and functional analysis.

Cell culture

SH-SY5Y cells were kindly supplied from Pharmacological Research Institute "Mario Negri" (Laboratory of Molecular Pharmacology) in Milan. Stable SH-SY5Y clones expressed constitutively the Tet repressor from pcDNA6/TR vector and inducibly the siRNAs from pSUPERIOR vector containing the siRNA-expressing sequence targeting MRP1 (pSUPERIOR-MRP1 clone) or MDR1 (pSUPERIOR-MDR1 clone). The cells were grown in DMEM medium supplemented with 2 mM L-Glutamine, 10% (v/v) fetal bovine serum Tetracycline-free, 1% (v/v) MEM Non Essential Amino Acids, 1% (v/v) MEM Vitamin Solution and 1% (v/v) Penicillin/Streptomycin (100 units/ml Penicillin and 100 µg/ml Streptomycin). Blasticidin (5 µg/mL) and puromycin (0.8 µg/mL) were added as selection antibiotics for pcDNA6/TR and pSUPERIOR.puro, respectively. Clones were maintained at 37° C in humidified air with 5% CO 2 . On the day prior to the experiment, confluent cells were trypsinized and plated in: 1) 35 mm plates at a density of 2.5 x 10 6 cells/well for RT-Real Time PCR and Western Blot analyses;

2) in 12-well plates at a density of 1 x 10 6 cells/well for the [ 3 H]UCB-uptake assay; and 3) in 96well plates at a density of 0.2 x 10 6 cells/well for MTT assay and for Hoechst 33342 Dye Accumulation assay. Doxycycline (3.9 µM) was used to induce the siRNA expression from pSUPERIOR.puro vector. Before the harvest or treatments, clones were incubated with and without doxycycline (induced and non-induced samples, respectively) at 37°C for various time intervals.

RNA isolation, reverse transcription and RT-Real Time PCR

pSUPERIOR-MRP1 clone was incubated with doxycycline (3.9 µM) for 24, 48 and 72 hours or for 48 hours followed by additional 48 hours without doxycycline (induced samples). pSUPERIOR-MDR1 was incubated with doxycycline (3.9 µM) for 24 and 48 hours or for 24 hours followed by additional 24 hours without doxycycline (induced samples). The control clone, stably transfected with pcDNA6/TR vector and with an empty pSUPERIOR.puro vector, was treated with doxycycline (3.9 µM) as described for pSUPERIOR-MRP1 and pSUPERIOR-MDR1 clones. For each induced sample, a non-induced sample was tested. Total RNA was isolated from each clone and from its control using TRI REAGENT TM , according to the manufacturer's recommendations. One µg of total RNA from cell culture was reverse transcribed using the iScript cDNA Synthesis Kit, according to manufacturer's instructions, on a thermal cycler (Gene Amp PCR System 2400, Perkin-Elmer, Boston, MA, USA). The relative levels of MRP1 mRNA with sense (GCCAAGAAGGAGGAGACC) and anti-sense (AGGAAGATGCTGAGGAAGG) primers and the MDR1 mRNA with sense (TGCTCAGACAGGATGTGAGTTG) and anti-sense (AATTACAGCAAGCCTGGAACC) primers were assayed by RT-Real Time PCR using iQ SYBR Green Supermix, as described previously [START_REF] Falcao | Role of multidrug resistance-associated protein 1 expression in the in vitro susceptibility of rat nerve cell to unconjugated bilirubin[END_REF]. Amplification, data acquisition and data analysis were carried out using the iCycler IQ (Bio-Rad Laboratories, Hercules, CA). Each sample was analyzed in triplicate. For each amplificate, a blank (nuclease free water) was added in place of cDNA. The thermal cycler conditions were: 95°C for 3 min; 40 cycles at 95°C for 20 minutes, 60°C for 20 minutes and 72°C for 30 minutes. In each reaction, a standard curve was generated by serial dilution of isolated and quantified cDNA (chosen among the cDNA samples) and a melting curve was generated to verify the specificity of the amplification. The results were normalized to each housekeeping gene and the initial amount of the template of each sample was determined as relative expression versus the non-induced control sample chosen as reference. -ACTIN with sense (CGCCGCCAGCTCACCATG) and anti-sense (CACGATGGAGGGGAAGACGG) primers and GAPDH with sense (CCCATGTTCGTCATGGGTGT) and anti-sense (TGGTCATGAGTCCTTCCACGATA) primers were used as endogenous controls to normalize the expression level of target genes.

Western Blotting

pSUPERIOR-MRP1 clone was incubated with doxycycline (3.9 µM) for 24, 48 and 72 hours or for 72 hours followed by 72 hours without doxycycline (induced samples). pSUPERIOR-MDR1 clone was incubated with doxycycline (3.9 µM) for 24 and 48 hours or for 48 hours followed by 48 hours without doxycycline (induced samples). The control clone, stably transfected with pcDNA6/TR vector and with an empty pSUPERIOR.puro vector, was treated with doxycycline as described for pSUPERIOR-MRP1 and pSUPERIOR-MDR1 clones. For each induced sample, a non-induced sample was tested. Proteins of each sample were subjected to SDS-PAGE in 10% acrylamide and the resolved protein bands transferred onto a nitrocellulose membrane (0.2 lm Protran BA 83, Schleicher and Schuell, Dassel, Germany).

[ 3 H]-UCB uptake in cultured cells

pSUPERIOR-MRP1 clone was treated with UCB 72 hours after induction, by doxycycline (3.9 µM), of the siRNA-expressing sequence transcription. pSUPERIOR-MDR1 clone was treated with UCB 48 hours after removing the doxycycline from cells previously incubated with doxycycline for 48 hours. These incubation conditions were predetermined to be the best for observing the silencing of MRP1 and MDR1, respectively (see Results). [ 3 H]-UCB (29.25 mCi/mmol) was dissolved in 0.3% (v/v) DMSO and diluted in culture medium supplemented with 15% (v/v) Fetal Bovine Serum containing 54 µM albumin. Cells were incubated for 30 minutes in the presence of [ 3 H]-UCB (15 µM) at B =40 nM, determined as described previously [START_REF] Roca | Factors affecting the binding of bilirubin to serum albumins: validation and application of the peroxidase method[END_REF]. f The cells were then carefully washed with ice-cold PBS to arrest UCB uptake and remove loosely-bound [ 3 H]-UCB. The quantity of [ 3 H]-UCB retained by cells, normalized for protein concentration, was measured as previously described [START_REF] Calligaris | Multidrug resistance associated protein 1 protects against bilirubin-induced cytotoxicity[END_REF].

MTT assay and Hoechst 33342 Dye Accumulation Assay

MTT assay is used for measuring the activity of enzymes that reduce MTT to formazan. quadruplicate. After the appropriate doxycycline incubation time, cells were treated for 4 hours with three different concentrations of UCB (15 µM or 20 µM or 30 µM). UCB was dissolved in 0.3%, 0.4% or 0.6% v/v of DMSO respectively, and diluted in 100 l of complete medium supplemented with 15% Fetal Bovine Serum containing 54 µM albumin. B f concentrations were 40 nM, 70 nM and 140 nM respectively, measured as described previously [START_REF] Roca | Factors affecting the binding of bilirubin to serum albumins: validation and application of the peroxidase method[END_REF]. For each induced and non-induced sample treated with UCB, an induced and a non-induced sample treated with the same volume of DMSO, but not UCB, were included as controls (CTRL).

Induced and non-induced samples, not treated with either UCB or DMSO, were tested also. MTT assay was performed as described previously [START_REF] Von | Rescue from death but not from functional impairment: caspase inhibition protects dopaminergic cells against 6-hydroxydopamine-induced apoptosis but not against the loss of their terminals[END_REF], with absorbance measured at 562 nm, using a microtiter plate reader (Beckman Coulter). MTT assay was also performed on pSUPERIOR-MDR1 clone exposed to ceftriaxone, a cephalosporin antibiotic transported by MDR1 but not MRP1 [29;38;39]. Cell viability, assessed by MTT reduction ability, of induced and non-induced samples treated with UCB+DMSO was expressed as a percentage of the respective samples treated with DMSO alone.

To assess MDR1 export function, accumulation of the Hoechst 33342 dye, a substrate for MDR1 [START_REF] Shapiro | P-glycoprotein-mediated Hoechst 33342 transport out of the lipid bilayer[END_REF], was measured in a microtiter plate fluorescence reader (Beckman Coulter) at 350 nm (excitation)/460 nm (emission), using 0.2 x 10 6 cells/well, in 96-well black plates. After the appropriate incubation time with doxycycline, cells were washed twice with Hank's solution and exposed for 6 minutes to three different concentrations of Hoechst 33342 (1.0, 2.5 and 5.0 µM), dissolved in Hank's solution. Cellular dye uptake was assessed by the increase in fluorescence.

At the end of each experiment cell viability was assessed by the MTT test, as described above.

Statistical analysis

Data are given as mean ± SD of at least three separate experiments. Statistical analysis was performed by Student "t" test and a p value less than 0.05 was considered statistically significant.

RESULTS

Gene and protein expression changes according to the incubation time with doxycycline

MRP1/MRP1 expression of pSUPERIOR-MRP1 clone. In the samples induced with doxycycline for 72 hours, MRP1 mRNA expression (Fig. 1 A) decreased by about 45% as compared to the non-induced samples (p<0.02); no decrease was observed with a shorter incubation time (48 hours). A 40% decrease (p<0.03) was also observed in samples incubated with doxycycline for 48 hours and then without doxycycline for 48 additional hours.

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Protein analysis confirmed the variations observed in gene expression. As shown in Fig. 1 B, no change in the MRP1 level was observed in both induced and non-induced samples after 48 hours of incubation with doxycycline. On the contrary, a 60% reduction (p<0.03) was found after 72 hours of incubation. A similar decrease in MRP1 content (about 50%) occurred in samples induced by doxycycline for 72 hours and then incubated without doxycycline for 72 additional hours (p<0.04). The control clone, stably transfected with pcDNA6/TR vector and with an empty pSUPERIOR.puro vector, showed a 10% up-regulation of MRP1/MRP1 after 72 hours of incubation with doxycycline (data not shown).

MDR1/MDR1 expression of pSUPERIOR-MDR1 clone. After a 24 hour incubation with doxycycline, the expression of MDR1 mRNA (Fig. 1 C) decreased by 30% (p<0.05), but partially recovered by 48 hours. The reduction in gene expression was greater than 50% (p<0.02) when the clone was incubated for 24 hours in presence of doxycycline followed by additional 24 hours in its absence. The expression of MDR1 protein (Fig. 1 D) did not show any decrease after 24 or 48 hours of incubation with doxycycline, but was reduced by about 60% (p<0.02) 48 hours after the removal of doxycycline from cells previously incubated with doxycycline for 48 hours.

Taking into account the limited specificity of the anti-C219 antibody, these results are compatible with the observation that the silencing by siRNA was hidden by the induction of MDR1/MDR1 expression caused by doxycycline, as previously reported [START_REF] Mealey | Doxycycline induces expression of P glycoprotein in MCF-7 breast carcinoma cells[END_REF]. Doxycyclinerelated induction of 25% was observed also in the control clone stably transfected with pcDNA6/TR vector and with an empty pSUPERIOR.puro vector, after a 24 (for gene) or 48 hour (for protein) incubation in presence of the antibiotic followed by a 24 (for gene) or 48 hour (for protein) incubation in its absence (data not shown). This finding explains why the silencing effect could be observed mainly only after removing the doxycycline. The small reduction in the gene but not in the protein expression observed after a 24 hour incubation with doxycycline (Fig. 1 C,D) as well as the different incubation times needed to achieve maximum silencing in gene or protein expression is related to the shorter half life of mRNA than the protein [START_REF] Aleman | P-glycoprotein, expressed in multidrug resistant cells, is not responsible for alterations in membrane fluidity or membrane potential[END_REF].

These findings defined the optimal incubation times to achieve comparable decreases in the expression levels of MRP1 or MDR1 to correlate with UCB accumulation and cytotoxicity. A c c e p t e d M a n u s c r i p t

UCB accumulates in MRP1-deficient cells, not in MDR1-deficient cells

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Since the decreased expression of both proteins was comparable in the two clones (see Fig. 1 B,D), these data confirm that MRP1, but not MDR1, can regulate the cellular content of UCB, at the low B f of 40 nM.

Impairment of transport function and viability in MDR1-deficient cells

As shown in Figure 3, compared to non-induced cells, induced pSUPERIOR-MDR1 samples showed a dramatic increase in the cellular accumulation of three different concentrations of the fluorescent dye Hoechst 33324. These results demonstrate that the knockdown of MDR1 expression resulted in functional impairment of the export of the dye, a known substrate for MDR1 [START_REF] Shapiro | P-glycoprotein-mediated Hoechst 33342 transport out of the lipid bilayer[END_REF].

When the induced clone with its MDR1 level decreased by 60% was exposed for 24 hours to 15 mM ceftriaxone, cell viability was decreased by two-thirds compared to the noninduced clone (35 ± 6% vs. 100 ± 4%, p<0.002) (data not shown). This confirmed that the decreased export function of MDR1 in the induced cells engendered greater susceptibility to cytotoxicity from a known MDR1 substrate.

UCB-induced cytotoxicity occurs in cells deficient in MRP1 but not MDR1

Figure 4 shows the cytotoxic effects of 4 hours of exposure to UCB at B f = 40, 70 and 140 nM, on pSUPERIOR-MRP1 and pSUPERIOR-MDR1 clones with comparable silencing of MRP1 and MDR1, respectively. Cell viability was assessed by MTT assay. All pSUPERIOR-MRP1 samples, whether or not induced with doxycycline for 72 hours, showed progressive reduction in cell viability by increasing B f . However, the decrease in viability was significantly greater in the induced cells with a 60% reduction of MRP1 (see Fig. 1B), at each B f level (Fig. 4A), and this effect was UCB dose-dependent (Fig. 4C).

In contrast to the pSUPERIOR-MRP1, no difference in cytotoxicity was found between induced and non-induced samples of the pSUPERIOR-MDR1 (Fig. 4 B,C), even at the highest B f (140 nM) although the induced clone reached a 60% reduction of MDR1 expression by incubation with doxycycline for 48 hours followed by additional 48 hours in its absence (see Fig. 1 D). In spite of the substantial reduction in MDR1 expression, the susceptibility of the induced clone to UCB was comparable to the one of the non-induced clone, in keeping with the lack of difference in UCB accumulation (Fig. 2). This is in sharp contrast to the clear increase, in induced pSUPERIOR-MDR1 cells, of accumulation of Hoechst 33342 (Figure 3), and cytotoxicity of ceftriaxone, two known MDR1 substrates, which confirmed the decreased export function of MDR1 in the induced cells.

In summary, greater accumulation of UCB markedly affected the cell viability of MRP1deficient cells and this effect was dose-dependent. On the contrary, the MDR1-deficient cells showed UCB accumulation and cell viability identical to their non-induced control, in spite of a demonstrated impairment in transport activity of the protein for a known substrate (Fig. 3).

Collectively these data strongly point to a role of MRP1 but not of MDR1 in the protection from UCB-induced cytotoxicity. This conclusion is further supported by the strong linear correlation between the relative expression of MRP1 and the cytotoxic effect of UCB at all B f tested (Fig. 5).

DISCUSSION

Previous studies have indicated that nerve cells are particularly sensitive to UCB toxicity [2;3;7-10;27]. MRP1 and MDR1 have each been proposed to protect cells against UCB toxicity, by limiting the intracellular accumulation of the pigment. We had previously demonstrated that MRP1 transports UCB with high affinity [START_REF] Rigato | The human multidrugresistance-associated protein MRP1 mediates ATP-dependent transport of unconjugated bilirubin[END_REF] and protects MEF cells from UCB toxicity by exporting the pigment from the cells [START_REF] Calligaris | Multidrug resistance associated protein 1 protects against bilirubin-induced cytotoxicity[END_REF]. Both Gennuso et al., using differentiated mouse astrocytes [START_REF] Gennuso | Bilirubin protects astrocytes from its own toxicity by inducing up-regulation and translocation of multidrug resistance-associated protein 1 (Mrp1)[END_REF] and, more recently Falcão et al. using mature rat astrocytes and neurons [START_REF] Falcao | Role of multidrug resistance-associated protein 1 expression in the in vitro susceptibility of rat nerve cell to unconjugated bilirubin[END_REF],

have shown dramatic increases in UCB-induced cytotoxicity (impaired MTT reduction) following blockade of Mrp1 by MK571, an inhibitor of Mrp1 function [START_REF] Leier | ATP-dependent glutathione disulphide transport mediated by the MRP gene-encoded conjugate export pump[END_REF]. Gennuso et al. [START_REF] Gennuso | Bilirubin protects astrocytes from its own toxicity by inducing up-regulation and translocation of multidrug resistance-associated protein 1 (Mrp1)[END_REF] showed further that MK571 increased vulnerability of astrocytes to UCB-induced membrane, mitochondrial and nuclear damage, even at a normally non toxic B f of 40 nM. Others have suggested that UCB is also a substrate for MDR1 [START_REF] Watchko | Brain bilirubin content is increased in P-glycoprotein-deficient transgenic null mutant mice[END_REF] and that this transporter may protects neural cells from toxicity by limiting accumulation of UCB [25;26;29]. However, those studies were performed at vastly high and clinically irrelevant concentrations of UCB.

The present study is the first to directly compare the functional roles of MRP1 and MDR1 in protecting against UCB toxicity in identical cells exposed to identical, increasing concentrations of UCB at clinically-relevant B f levels. The comparable reduction we obtained in the expression levels of MRP1 and MDR1, utilizing RNA interference technology, also allowed a direct comparison of their relative role in the transport of UCB and in the protection against UCB neurotoxicity in cells differing only in the reduced expression of either transporter.

Page 12 of 19

Our experiments demonstrated that, at clinically-relevant B f levels, protection from UCB cytotoxicity was correlated with the level of expression of MRP1 but not MDR1. No significant UCB-induced impairment in cell viability was observed even when maximal MDR1 silencing was achieved and its impaired transport function confirmed by the significantly greater cellular accumulation, in the cells with induced siRNA expression, of Hoechst 33342 dye, a substrate for MDR1 [START_REF] Shapiro | P-glycoprotein-mediated Hoechst 33342 transport out of the lipid bilayer[END_REF]. In contrast, the cytotoxic effect caused by ceftriaxone, another known substrate for The [ 3 H]-UCB uptake data confirm further the involvement of MRP1 in UCB export, previously demonstrated by using MEF cells isolated from Mrp1 knockout mice [START_REF] Calligaris | Multidrug resistance associated protein 1 protects against bilirubin-induced cytotoxicity[END_REF], and exclude participation of MDR1 in the transport of UCB at low B f levels. They indicate also that the protective effects of MRP1 against UCB cytotoxicity are largely related to the ability of this transporter to export UCB from the cell, thus limiting intracellular accumulation of the pigment [19;20;22]. This is in agreement with the chemical characteristics of the substrates of the two proteins; MDR1 "prefers" uncharged or slightly positively charged compounds, while MRP1 primarily transports amphipathic, anionic conjugates, as well as unconjugated xenobiotics and uncharged drugs [START_REF] Borst | Mammalian ABC transporters in health and disease[END_REF][START_REF] Loe | Multidrug resistance protein (MRP)-mediated transport of leukotriene C4 and chemotherapeutic agents in membrane vesicles. Demonstration of glutathione-dependent vincristine transport[END_REF][START_REF] Renes | ATP-and glutathione-dependent transport of chemotherapeutic drugs by the multidrug resistance protein MRP1[END_REF]. While our results show no role for MDR1 in the transport of UCB at B f =40 nM, they do not exclude the possibility, suggested by the work of others [24-26;29], that MDR1 transports UCB at much higher B f levels. Many compounds are substrates for both high affinity (low K m ) transporters that operate at low concentrations, and low affinity (high K m ) transporters that are operative at much higher concentrations of substrate [15;44;45]. In the case of UCB, MRP1 clearly is the high-affinity transporter, whereas MDR1 may become operative only at B f levels that are far above clinically-relevant concentrations.

Our MRP1-knockdown model validly mimics the increases in mRNA and protein expression of Mrp1 [15;46], and the decreased susceptibility to UCB toxicity [START_REF] Falcao | Bilirubin-induced immunostimulant effects and toxicity vary with neural cell type and maturation state[END_REF], that occur during maturation of rodent neurons and astrocytes. Similar changes in the expression of MRP1 might explain the higher vulnerability of premature than term infants to UCB-induced neurological dysfunction during moderate to severe neonatal jaundice [START_REF] Gourley | Bilirubin metabolism and kernicterus[END_REF][START_REF] Dennery | Neonatal hyperbilirubinemia[END_REF][START_REF] Kaplan | Understanding and preventing severe neonatal hyperbilirubinemia: is bilirubin neurotoxity really a concern in the developed world?[END_REF].

ACKNOWLEDGMENTS

This study was supported in part by a grant from Telethon (GGP05062). AA is a PhD A c c e p t e d M a n u s c r i p t

Dulbecco' s

 s Modified Eagle's Medium (DMEM), L-Glutamine, Penicillin Streptomycin and Trypsin 0.05%/EDTA 0.02% in PBS, without Phenol Red, Ca 2+ /Mg 2+ were purchased from Euroclone (Pero, Milano, Italy). Fetal Bovine Serum Tetracycline-free (Tet System Approved FBS, US-Sourced) was purchased from Clontech (Mountain View, CA, USA). MEM Non Essential Amino Acids, MEM Vitamin Solution, Opti-MEM I Reduced Serum Medium and Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA, USA). Blasticidin S hydrochloride, puromycin dihydrochloride, doxycycline hyclate, PBS (Dulbecco's Phosphate Page 4 of 19 Biochemical Journal Immediate Publication. Published on 19 Aug 2008 as manuscript BJ20080918 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080918 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society Buffered Saline) without Ca 2+ /Mg 2+ , TRI REAGENT TM , bicinchoninc acid protein assay kit, Ponceau S-solution (0.1% w/v) in 5% acetic acid (v/v), MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide], dimethyl sulfoxide (DMSO) and Hoechst 33342 were purchased from Sigma (St. Louis, MO, USA). Chlorophenol red--D-galactopyranoside (CPRG) was

  Membranes were blocked for 1 hour at room temperature in 4% milk-TTBS (0.2% Tween 20, Page 7 of 19 Biochemical Journal Immediate Publication. Published on 19 Aug 2008 as manuscript BJ20080918 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080918 A c c e p t e d M a n u s c r i p t 20mM Tris and 500mM NaCl; pH 7.5) and then incubated overnight at 4°C with a 1:600 dilution of the MRP1-A23 rabbit antibody or 1:50 dilution of the anti-C219 (MDR1) mouse antibody or 1:1500 dilution of rabbit anti-actin antibody. After washing three times with 4% milk-TTBS, the membranes were incubated in blocking solution for 1 hour at room temperature with secondary antibodies: peroxidase conjugated-goat anti-rabbit (1:6000) or anti-Mouse IgG (Fc specific)-Peroxidase (1:2000). Protein bands were detected by ECL-Plus, transferred to Kodak film and visualized by Kodak EDAS 260 (Kodak Instruments, New Haven, CT, USA) using Kodak 1D image software. The intensities of protein bands were quantified by Scion Image (Frederick, MD, USA). To quantify the relative expression of the proteins (MRP1/ACTIN or MDR1/ACTIN), serial dilutions of a reference sample were loaded on each gel, optical density recorded and a standard curve was generated from the serial dilutions by non linear regression analysis (Curver Expert 1.3 software, Starkville, MS, USA). The correlation coefficient of the calibration curve was never lower than 0.995.

  Yellow MTT is reduced to purple formazan in the mitochondria of living cells by active mitochondrial reductase enzymes; therefore conversion is used as a measure of viable cells. pSUPERIOR-MRP1 and pSUPERIOR-MDR1 clones were incubated with doxycycline (3.9 µM) for the same durations selected to analyze protein content (see above). For each induced sample, a non-induced sample was included, and each treatment was performed in Page 8 of 19 Biochemical Journal Immediate Publication. Published on 19 Aug 2008 as manuscript BJ20080918 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080918 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Fig. 2

 2 Fig. 2 shows the effects of decreased expression of MRP1 or MDR1 on the intracellular accumulation of UCB after incubation for 30 minutes with [ 3 H]UCB (B f =40 nM). Accumulation of [ 3 H]-UCB was markedly higher (2.6-fold) in the doxycycline-induced samples of the pSUPERIOR-MRP1 than in the non-induced ones (p<0.0006). By contrast, the intracellular accumulation of [ 3 H]-UCB was similar in the induced and non-induced pSUPERIOR-MDR1. Page 10 of 19

  student sponsored by a fellowship from the Ministry of Foreign Affairs, Rome, Italy. Thanks are due to Drs. Sebastian D. Calligaris and Pablo Giraudi for their help in performing the bilirubin uptake experiments. Page 13 of 19 Biochemical Journal Immediate Publication. Published on 19 Aug 2008 as manuscript BJ20080918 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080918

Figure 1 .

 1 Figure 1. Time course of the relative gene and protein expression of MRP1/MRP1 and MDR1/MDR1 in SH-SY5Y clones. Panel A, Relative MRP1 mRNA expression in pSUPERIOR-MRP1 clone at 48, 72 hours after induction by doxycycline (DOX, 3.9 µM) and 48 hours after the DOX removal from cells previously incubated with DOX for 48 hours (48/48 h). RT-Real Time PCR was performed normalizing MRP1 expression values to housekeeping genes GAPDH and -ACTIN. Each bar represents the mean ± SD of three separate experiments. *p<0.02 and § p<0.03 for Induced vs. Non-induced samples. Panel B, Western Blot analysis of MRP1 and ACTIN proteins in the same clones under the same treatments as in Panel A except DOX was removed for 72 hours from cells previously incubated with DOX for 72 hours (72/72 h). Bands were visualized by Kodak 1D image software and quantified by Scion Image software. The relative expression values were obtained by the Curver Expert software as described in Methods. Each bar represents the mean ± SD of three separate experiments. #p<0.03 and *p<0.04 for Induced vs. Non-induced samples. Panel C, Relative MDR1 mRNA expression in pSUPERIOR-MDR1 clone at 24, 48 hours after induction by doxycycline (DOX, 3.9 µM) and 24 hours after DOX removal from cells previously incubated with DOX for 24 hours (24/24h). RT-Real Time PCR was performed normalizing MDR1 expression values to housekeeping genes GAPDH and -ACTIN. Each bar represents the mean ± SD of three separate experiments. *p<0.05 and § p<0.02 for Induced vs. Non-induced samples. Panel D, Western Blot analysis of MDR1 and ACTIN proteins in the same clones under the same treatments as in Panel C, except DOX removed for 48 hours from cells previously incubated with DOX for 48 hours (48/48 h). Band visualization and quantification as in Panel B. Each bar represents the mean ± SD of three separate experiments. § p<0.02 for Induced vs. Non-induced samples.

Figure 2 . 3 [

 23 Figure 2. 3 [H]-UCB accumulation by SH-SY5Y clones MRP1-or MDR1-deficient. Before exposure to 3 [H]-UCB, the induced samples of pSUPERIOR-MRP1 clone were incubated with DOX (3.9 µM) for 72 hours while the induced samples of pSUPERIOR-MDR1 clone were incubated with DOX (3.9 µM) for 48 hours followed by additional 48 hours in medium without DOX (48/48 h). At these incubation times, the levels of MRP1 and MDR1 proteins were comparably decreased (see Fig.1 B and D). Both induced and non-induced samples were incubated with 3 [H]-UCB at B f = 40 nM for 30 minutes. The 3 [H]-UCB contents of the induced and non-induced samples were measured and normalized for the respective cell protein content. Data are reported as the mean ± SD of experiments performed in triplicate. Induced samples of pSUPERIOR-MRP1 clone accumulated 2.6 times as much 3 [H]-UCB (#p<0.0006) as non-Page 18 of 19

Figure 3 .

 3 Figure 3. Cellular accumulation of a fluorescent MDR1 substrate by SH-SY5Y clone MDR1-deficient. Before exposure to the fluorescent dye Hoechst 33342, the induced samples of pSUPERIOR-MDR1 clone were treated with DOX as described in Fig. 2. Cell content of Hoechst 33342 was measured in 96-well, black microtiter plates by a fluorescence reader at 350 nm (excitation)/ 460 nm (emission), using 0.2 x 10 6 cells/well. Panel A: Time dependent increase in cell fluorescence due to the uptake of 5 µM Hoechst in pSUPERIOR-MDR1 clone induced (--) or non-induced (--). Panel B: Cell fluorescence determined after 5 minutes of incubation with three different concentrations of Hoechst dye (1, 2.5 and 5 µM). Data are expressed as the mean ± SD of three independent measurements. #p<0.001 for Induced vs. Noninduced samples. Dramatic increases in dye accumulation by induced cells are observed at all dye concentrations.

Figure 4 .Fig. 2 .

 42 Figure 4. Cell viability of SH-SY5Y MRP1-or MDR1-deficient clones after exposure to UCB. Before exposure to UCB, the induced samples of pSUPERIOR-MRP1 clone (Panel A and C) and of pSUPERIOR-MDR1 clone (Panel B and C) were treated with DOX as described in Fig. 2. Clones were then exposed for 4 hours to increasing concentrations of unbound UCB (B f = 40, 70, and 140 nM), and viability then assessed by their ability to reduce MTT. Both induced and non-induced samples were incubated in the presence of DMSO 0.3, 0.4 or 0.6% v/v (used to dissolve the UCB) and 15% Fetal Bovine Serum (albumin concentration 54 µM). Cell viability of induced and non-induced clones is expressed as a percentage of the corresponding control (CTRL) treated with DMSO only. Each bar represents the mean ± SD of three separate experiments. #p<0.001 and *p <0.0002 for Induced vs. Non-induced samples. §p<0.02 for B f = 40 nM vs. B f = 70 nM and ~p <0.01 for B f = 70 nM vs. B f = 140 nM.

Figure 5 .

 5 Figure 5. Correlation between UCB-induced cytotoxicity and MPR1 protein expression in SH-SY5Y clone. pSUPERIOR-MRP1 clone was incubated with DOX as reported in Figure 1B and UCB treatment performed as described in Figure 4. At each free bilirubin concentration (B f ), there is a significant linear correlation between the MRP1 protein content (normalized for actin) of induced cells relative to non-induced (control) cells, and UCB cytotoxicity, expressed as cell viability assessed by MTT assay. The slope of the correlation increases with increasing B f . Symbols: = Bf 40 nM; = Bf 70 nM; = Bf 140 nM.

  Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society MDR1 [29;38;39], was significantly greater in the induced cells than in non-induced cells. These findings clearly indicate that decreased expression and function of MDR1 is not associated with greater UCB cytotoxicity at low B f values.

	Biochemical Journal Immediate Publication. Published on 19 Aug 2008 as manuscript BJ20080918
	A c c e p t e d M a n u s c r i p t	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080918

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society


Martina Takacova

Tereza Holotnakova

Jan Vondracek

Miroslav Machala

Katerina Pencikova

Katarina Gradin

Lorenz Poellinger

Jaromir Pastorek

Silvia Pastorekova

Juraj Kopacek

Role of aryl hydrocarbon receptor in modulation of the expression of the hypoxia marker carbonic anhydrase IX

Keywords: carbonic anhydrase IX, hypoxia, Ah receptor, dioxin, transcriptional regulation, nuclear translocator of AhR CA IX, carbonic anhydrase IX protein, CA9, carbonic anhydrase IX gene, HIF, hypoxia inducible factor, AhR, aryl hydrocarbon receptor, ARNT, nuclear translocator of AhR

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Carbonic anhydrase IX (CA IX) belongs to the family of Zn 2+ binding metalloenzymes (carbonic anhydrases) which catalyze the reversible conversion of carbon dioxide to bicarbonate and proton and thus are involved in gas exchange, ion transport and the acid-base balance across cell membranes and between different intracellular compartments [START_REF] Pastorekova | Carbonic anhydrases: current state of the art, therapeutic applications and future prospects[END_REF]. Fifteen human carbonic anhydrases identified to date participate in a variety of physiological processes like respiration, bone resorption, gluconeogenesis, renal acidification, formation of cerebrospinal fluid and gastric acid [START_REF] Fleming | Carbonic anhydrase IV expression in rat and human gastrointestinal tract regional, cellular, and subcellular localization[END_REF][START_REF] Hewett-Emmett | Functional diversity, conservation, and convergence in the evolution of the alpha-, beta-, and gamma-carbonic anhydrase gene families[END_REF][START_REF] Sly | Human carbonic anhydrases and carbonic anhydrase deficiencies[END_REF].

Besides physiological expression, several carbonic anhydrase isoforms have been described to be deregulated in various types of malignances [START_REF] Ivanov | Down-regulation of transmembrane carbonic anhydrases in renal cell carcinoma cell lines by wild-type von Hippel-Lindau transgenes[END_REF][START_REF] Kivela | Differential expression of cytoplasmic carbonic anhydrases, CA I and II, and membrane-associated isozymes, CA IX and XII, in normal mucosa of large intestine and in colorectal tumors[END_REF][START_REF] Parkkila | Immunohistochemical demonstration of human carbonic anhydrase isoenzyme II in brain tumours[END_REF][START_REF] Tureci | Human carbonic anhydrase XII: cDNA cloning, expression, and chromosomal localization of a carbonic anhydrase gene that is overexpressed in some renal cell cancers[END_REF]. So far, the most studied carbonic anhydrase related to oncogenesis is CA IX [START_REF] Liao | Identification of the MN antigen as a diagnostic biomarker of cervical intraepithelial squamous and glandular neoplasia and cervical carcinomas[END_REF]10]. The strong association of CA IX expression with tumor development is mainly related to diminished oxygen supply, hypoxia [START_REF] Kaluz | Expression of the hypoxia marker carbonic anhydrase IX is critically dependent on SP1 activity. Identification of a novel type of hypoxia-responsive enhancer[END_REF][START_REF] Wykoff | Hypoxia-inducible expression of tumor-associated carbonic anhydrases[END_REF]. In general, the primary cellular response to hypoxia is the stabilization of HIF-1 transcriptional complex composed of a constitutively expressed ARNT (aryl hydrocarbon receptor nuclear translocator, also known as HIF-1β) and the rate-limiting subunit HIF-1α, which is regulated by oxygen availability [START_REF] Wang | Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension[END_REF]. The resulting HIF-1 complex activates transcription of a variety of genes the promoters of which contain hypoxia response elements (HRE, 5'-RCGTG-3'). CA9 belongs to genes with a very strong hypoxia response mainly due to the unique localization of its HRE element just -3/-10 nucleotides from the transcription start site [START_REF] Wykoff | Hypoxia-inducible expression of tumor-associated carbonic anhydrases[END_REF]. Moreover, efficient hypoxia response is achieved through HIF-1 and SP1 cooperation on CA9 promoter [START_REF] Kaluz | Expression of the hypoxia marker carbonic anhydrase IX is critically dependent on SP1 activity. Identification of a novel type of hypoxia-responsive enhancer[END_REF]. The basal promoter of the CA9 gene is localized within a sequence of approximately 200 nucleotides upstream of the transcription start site.

In addition to hypoxia, expression of CA IX is also enhanced by high cell density in cell culture models [START_REF] Kaluz | Lowered oxygen tension induces expression of the hypoxia marker MN/carbonic anhydrase IX in the absence of hypoxia-inducible factor 1 alpha stabilization: a role for phosphatidylinositol 3'-kinase[END_REF] and modulated by the phosphatidylinositol-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways [START_REF] Kaluz | Lowered oxygen tension induces expression of the hypoxia marker MN/carbonic anhydrase IX in the absence of hypoxia-inducible factor 1 alpha stabilization: a role for phosphatidylinositol 3'-kinase[END_REF][START_REF] Kaluz | The role of extracellular signalregulated protein kinase in transcriptional regulation of the hypoxia marker carbonic anhydrase IX[END_REF][START_REF] Kopacek | MAPK pathway contributes to density-and hypoxia-induced expression of the tumor-associated carbonic anhydrase IX[END_REF]. Moreover, extracellular acidosis, which is characteristic for most solid tumors, increases CA IX at both protein and mRNA levels in certain cell types [17]. However, it is clear that CA IX expression could be modulated by an interplay between several stimuli and pathways with HIF-1 and SP1 representing the crucial targets of these pathways.

During the past decades, an increasing attention has started to be paid to the role of various environmental contaminants in the process of cancer development. Direct cellular response to the action of these toxic compounds is the induction of xenobiotic-metabolizing enzymes among which cytochrome P450 (CYP1A1) is best characterized (reviewed in [START_REF] Shimada | Metabolic activation of polycyclic aromatic hydrocarbons to carcinogens by cytochromes P450 1A1 and 1B1[END_REF]). The environmental toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces a broad spectrum of biochemical and toxic effects such as teratogenesis, immunosupression and tumor development [19]. Its effects are at the molecular level primarily mediated through activation of the aryl hydrocarbon receptor (AhR) [START_REF] Marlowe | Aryl hydrocarbon receptor, cell cycle regulation, toxicity, and tumorigenesis[END_REF]. Under conditions when physiological ligand or TCDD are absent, AhR is present in the cytoplasm and forms a complex stably associated with the heat shock protein 90 (Hsp90) and additional co-chaperone proteins [START_REF] Wilhelmsson | The specific DNA binding activity of the dioxin receptor is modulated by the 90 kd heat shock protein[END_REF]. After ligand binding, the activated AhR translocates into the nucleus [22], where it dimerizes with ARNT allowing both proteins to recognize the asymmetrical Ebox motif known as xenobiotic response element (XRE, 5'-GCGTG-3'), which is involved in regulation of dioxin-responsive genes [START_REF] Sun | Comparative analysis of dioxin response elements in human, mouse and rat genomic sequences[END_REF].

AhR, HIF-1α and ARNT belong to a group of structurally-related transcription factors known as basic helix/loop/helix (bHLH) -Per/ARNT/Sim (PAS) family of proteins [START_REF] Kewley | The mammalian basic helixloop-helix/PAS family of transcriptional regulators[END_REF]. The main characteristic of PAS proteins is their ability to form various homo-and heterodimeric complexes, which regulate expression of target genes. The ability of ARNT to act as a heterodimeric partner for both HIF-1α and AhR raises the possibility of cross-talk between the hypoxia and dioxin-signaling cascades. The studies published to date seem to indicate that the degree of interaction among ARNT-dependent pathways might be cell-, tissue-or species-specific [START_REF] Chan | Cross-talk between the aryl hydrocarbon receptor and hypoxia inducible factor signaling pathways. Demonstration of competition and compensation[END_REF][START_REF] Gassmann | Oxygen-and dioxinregulated gene expression in mouse hepatoma cells[END_REF] and it may depend on cellular abundance of ARNT [START_REF] Nie | Interactions between aryl hydrocarbon receptor (AhR) and hypoxia signaling pathways[END_REF]. Consequently, a possible competition for ARNT may lead to regulation of target gene expression in a positive or negative manner. Therefore, the aim of our study was to elucidate whether dioxin activation of the AhR pathway could interfere with the expression of the hypoxia marker CA IX.

Materials and methods

Cell culture and dioxin treatment

Mouse hepatoma cell lines, wild-type Hepa-1c1c7 and the ARNT-deficient subline Hepa-1c4, as well as HeLa cells derived from human cervical carcinoma, were cultured in DMEM supplemented with 10% FCS (BioWhittaker, Verviers, Belgium) and 40 µg/ml gentamicin (Lek, Ljubljana, Slovenia) in a humidified atmosphere with 5% CO 2 at 37°C. Hypoxic treatments were performed in an anaerobic workstation (Ruskin Technologies, Bridgend, UK) in 2% O 2 , 5% CO 2 , 10% H 2 and 83% N 2 at 37°C. TCDD was diluted to working concentrations of 1 nM or 10 nM. The final concentration of DMSO was less than 0.1% including controls. Cells were pre-treated with TCDD or DMSO for 1 h prior to being placed into hypoxia (2% O 2 ), for additional 24 h. Parallel control dishes were pretreated and maintained in normoxia (21% O 2 ) for the same time period.

Antibodies and plasmids

Mouse monoclonal antibody M75, specific for the human MN/CA IX protein, was characterized earlier [28]. Anti-actin goat polyclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary anti-mouse peroxidase-conjugated antibody was from Sevapharma (Prague, Czech Republic) and anti-goat peroxidase-conjugated antibody was from Dako (Glostrup, Denmark). For chromatin immunoprecipitation, rabbit polyclonal antibody against ARNT was used as described in [START_REF] Mason | Purification of the DNA binding form of dioxin receptor. Role of the Arnt cofactor in regulation of dioxin receptor function[END_REF]. Anti-human IgG antibodies (rabbit polyclonal, Abcam, Cambridge, UK) were used as a negative control and for chromatin preclearing.

Human promoter constructs were generated by an insertion of PCR-amplified -50/+37 and -174/+37 CA9 genomic fragments upstream of the firefly luciferase gene in pGL3-Basic luciferase reporter vector (Promega, Madison, WI) [START_REF] Kopacek | MAPK pathway contributes to density-and hypoxia-induced expression of the tumor-associated carbonic anhydrase IX[END_REF]. pRL-TK renilla vector (Promega) served as a transfection efficiency control. Plasmids containing full-length ARNT (pARNT/GEM7), XRE-driven reporter gene construct pTX.DIR, and dioxin receptor deletion mutant lacking the minimal PAS B motif (DR∆PASB) that functions as a constitutively active AhR (CAAhR) have been described previously [30][START_REF] Lindebro | Protein-protein interaction via PAS domains: role of the PAS domain in positive and negative regulation of the bHLH/PAS dioxin receptor-Arnt transcription factor complex[END_REF][START_REF] Mcguire | Definition of a dioxin receptor mutant that is a constitutive activator of transcription: delineation of overlapping repression and ligand binding functions within the PAS domain[END_REF]. HIF-1α cDNA in pcDNA1 expression plasmid [START_REF] Wood | Selection and analysis of a mutant cell line defective in the hypoxia-inducible factor-1 alpha-subunit (HIF-1alpha). Characterization of hif-1alpha-dependent and -independent hypoxia-inducible gene expression[END_REF] was kindly provided by Prof. Patrick Maxwell (Imperial College, London, UK). Plasmid containing SP1 cDNA was generated by Dr. Robert Tjian and purchased from Addgene (plasmid no.12097, Cambridge, MA). CBP/p300 expression plasmid was kindly provided by Dr. Jiri Vachtenheim (Third Faculty of Medicine, Prague, Czech Republic).

Transfection and luciferase assay

Cells were plated into 35-mm Petri dishes to reach ~70% density on the following day. Transient transfection was performed with 1 µg of promoter-containing luciferase construct and 50 ng of pRL-TK plasmid DNA using GenePorterII reagent (Genlantis, San Diego, CA) according to the manufacturer's recommendations. To analyze the effect of CAAhR either with or without transcription factors (ARNT, HIF-1α, SP1 or p300), the plasmids containing corresponding cDNAs were co-transfected. Cells transfected with control empty vectors (pCMV) served as negative controls. The day after transfection, cells were trypsinized and plated in triplicates into 24-well plates. Cells were allowed to attach for 24 h, and then they were either transferred to hypoxia or maintained in normoxia for additional 24 h. Reporter gene expression was assessed 72 h after transfection using Dual-Luciferase Reporter Assay System (Promega), and luciferase activity was normalized against renilla activity. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Western blotting

HeLa cells were plated in 35-mm Petri dishes to reach ~90% density on the following day. Pretreatment with TCDD or DMSO was done 1 h before the induction of hypoxia in the anaerobic workstation. Parallel control dishes were pre-treated and maintained in normoxia for the same time period. 24 h later, the cells were lysed in ice-cold RIPA buffer (1% Triton X-100, 1% deoxycholate in PBS) containing inhibitors of proteases (Roche Applied Science, Mannheim, Germany) for 30 min on ice. Cell lysates were scraped off and centrifuged at 10000 g for 10 min at 4˚C. Protein concentration was determined using the BCA protein assay reagent (Pierce, Rockford, IL).

Total protein extracts (50 µg/lane) were separated in 10% SDS-PAGE and transferred onto PVDF membrane (Immobilon TM -P, Milliopore, Billerica, MA). For CA IX detection, the membrane was incubated with M75 primary antibody diluted 1:2 in blocking buffer for 1 h. Secondary antimouse peroxidase-conjugated antibody (Sevapharma) was diluted 1:7500 in blocking buffer. For loading control, the membrane was probed with anti-actin antibody (Santa Cruz) and the polyclonal rabbit anti-goat IgG-HRP (Dako). The membrane was developed by enhanced chemiluminescence using ECL kit (Amersham Pharmacia Biotech, Buckinghamshire, UK).

The intensity of bands corresponding to individual proteins was evaluated with ImageJ 1.38x software (Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/, 1997-2007). ImageJ can create density histograms and calculate area and pixel value statistics of user-defined selections. Amount of proteins was expressed as the ratio of the intensity of each band to the intensity of the related actin internal standard.

Reverse transcription PCR

Total RNA was isolated from cells using InstaPure reagent (Eurogentec, Seraing, Belgium) according to the manufacturer's instructions. Reverse transcription was performed with M-MuLV reverse transcriptase (Finnzymes, Espoo, Finland) using random heptameric primers. The mixture of 3 µg of total RNA and random primers (400 ng/µl) was heated for 10 min at 70°C, cooled quickly on ice and supplemented with 0.5 mM dNTPs (Finnzymes), M-MuLV reverse transcriptase buffer, and 200 U of reverse transcriptase M-MuLV, incubated for 1 h at 42°C, heated for 15 min at 70°C and stored at -80°C until further use.

Real-time PCR

Real-time PCR was performed with POWER SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA) with gene-specific primers (CA9 and CYP1A1) and primers for β-actin that served as internal standards. The primers were as follows (S=sense, A=antisense): CA9 S: 5´-CCGAGCGACGCAGCCTTTGA-3´ and CA9 A: 5´-GGCTCCAGTCTCGGCTACCT-3´ (252 bp product); CYP1A1 S: 5´-TCTTTGGAGCTGGGTTTGACA-3´ and CYP1A1 A: 5´-GCTGTGGGGGATGGTGAA-3´ (227 bp product); β-actin S: 5´-CCAACCGCGAGAAGATGACC-3´ and β-actin A: 5´-GATCTTCATGAGGTAGTCAGT-3´ (236 bp product).

The amplification of the samples was carried out in a final volume of 20 µl on StepOne TM Real-Time PCR System (Applied Biosystems, Foster City, CA), using the following program: initial denaturation at 95°C for 10 min followed by 40 cycl es of: denaturation at 95°C for 15 s and annealing at 60°C for 1min. All PCR reactions were performed in triplicates and repeated three times.

Chromatin immunoprecipitation (ChIP) assay

HeLa cells were plated into 500 cm 2 plates at a ~90% density, left to attach overnight and incubated in hypoxia in absence or presence of 10 nM TCDD for 2 h. The cells were fixed in 1% formaldehyde in PBS for 10 min at room temperature. Chromatin isolation, shearing to size of ~600-800 bp and its immunoprecipitation with rabbit polyclonal antibodies against ARNT [START_REF] Mason | Purification of the DNA binding form of dioxin receptor. Role of the Arnt cofactor in regulation of dioxin receptor function[END_REF] was performed as described in [START_REF] Lofstedt | Induction of ID2 expression by hypoxia-inducible factor-1: a role in dedifferentiation of hypoxic neuroblastoma cells[END_REF]. Anti-human IgG antibodies (rabbit polyclonal, Abcam) were used 5´-CACCCTTCGACAGTTCCTCTCCCT-3´ and hCYP1A1-XRE-A: 5´-CTCCCGGGGTGGCTAGTGCTTTGA-3´; 391 bp product) were used as a positive control.

Statistical analysis

Results were analysed by two-tailed unpaired t test (Student`s test), and P < 0.05 was considered significant.

Results and discussion

TCDD inhibits hypoxia-induced CA IX expression

Our initial aim was to determine whether TCDD treatment influences expression of CA IX. For this purpose, HeLa cells grown in sparse culture were treated under both normoxic and hypoxic conditions with TCDD and DMSO as a control, and CA9 mRNA expression was analyzed using real-time PCR (Fig. 1A). As expected, the highest level of CA9 expression was observed in control HeLa cells exposed to hypoxia for 24 h, whereas TCDD treatment significantly decreased CA9 mRNA levels under hypoxic conditions. Due to sparse culture conditions of the treated cells, the expression of CA9 in normoxia was very low and statistically insignificant. Subsequently, results obtained by real-time PCR were confirmed by Western blotting of corresponding protein level (Fig. 1B). Treatment of HeLa cells with both concentrations of TCDD under hypoxic conditions resulted in a decrease in CA IX protein.

Expression of CYP1A1 was also determined by real-time PCR as a control for induction of the dioxin-responsive pathway (Fig. 1A). In accord with previous observation [30], hypoxia decreased expression of TCDD-induced CYP1A1 suggesting reciprocal influence between the hypoxia and dioxin signaling pathways in the cell culture model used in our experiments.

These experiments suggested that TCDD treatment inhibited CA9 expression at the transcriptional level. Therefore, in the next step, we decided to analyze this mode of inhibition utilizing CA9 promoter constructs in reporter gene experiments.

HIF-1 binding site within CA9 promoter is critical for dioxin mediated inhibition

Previous studies have determined the region -174 nt upstream from the transcription start site as the basal CA9 promoter. Moreover, the -50 nt proximal region of the CA9 promoter containing SP1 binding site and HRE was found as crucial for responses to cell density and hypoxia [START_REF] Kaluz | Expression of the hypoxia marker carbonic anhydrase IX is critically dependent on SP1 activity. Identification of a novel type of hypoxia-responsive enhancer[END_REF][START_REF] Kaluz | Transcriptional regulation of the MN/CA 9 gene coding for the tumor-associated carbonic anhydrase IX. Identification and characterization of a proximal silencer element[END_REF].

In order to determine the region critical for CA9 promoter inhibition caused by the dioxininduced AhR pathway, we used HeLa cells transiently transfected with the -174/+37 luciferase reporter construct [START_REF] Kopacek | MAPK pathway contributes to density-and hypoxia-induced expression of the tumor-associated carbonic anhydrase IX[END_REF] and treated with TCDD for 24 h in normoxia or hypoxia. Activation of AhR by TCDD treatment led to reduced activity of the -174/+37 luciferase reporter construct. Simultaneously, in order to examine direct effects of AhR on CA9 transcription, HeLa cells were cotransfected with 1 µg of constitutively active AhR (CAAhR), which is known to activate transcription in a ligand-independent manner. This construct is not subject to repression by the ligand binding domain in the absence of dioxin and may, in fact, under certain conditions show a more potent effect as transactivator than the ligand-occupied wild-type receptor [START_REF] Mcguire | Definition of a dioxin receptor mutant that is a constitutive activator of transcription: delineation of overlapping repression and ligand binding functions within the PAS domain[END_REF]. As shown in Fig. 2A, both TCDD treatment and CAAhR co-transfection significantly reduced promoter activities, with CAAhR being a more potent inhibitor.

To confirm functionality of the CAAhR in our assay, CAAhR-expressing HeLa cells were also co-transfected with the XRE-containing reporter plasmid pTX.DIR ([30], Fig. 2B). As expected, CAAhR dramatically elevated pTX.DIR reporter activity under normoxia and in the absence of AhR ligand. On the other hand, this activity was markedly diminished under hypoxic conditions to approximately one half of the CAAhR-induced value, thus providing additional evidence of a reciprocal impact between the hypoxia and dioxin pathways under these assay conditions.

CAAhR was also transiently expressed in HeLa cells in the presence of the minimal CA9 promoter (-50/+37) (Fig. 2A). In a manner similar to above, overexpression of CAAhR resulted in significant inhibition of the activity of the minimal reporter construct. The minimal CA9 promoter only contains SP1 and HIF-1 binding sites, therefore it seems plausible that the TCDD-and CAAhR-mediated CA9 repression may affect these transcription factors.

In the case of the CA9 promoter, both HIF-1α and SP1 are indispensable in responsiveness to hypoxic-and cell density-dependent induction. To determine the significance of these factors in CA9 repression, HeLa cells were co-transfected with equal amounts of CAAhR and expression vectors of either HIF-1α, SP1 or co-factors, and with the minimal CA9 reporter plasmid. Co-transfection with the empty vector did not have any effect on CAAhR mediated inhibition of reporter gene activity. In contrast, the expression of HIF-1α completely abrogated CAAhR-mediated inhibition of promoter activity. On the other hand, introduction of SP1 did not have this effect of counteracting CAAhR-mediated repression of CA9 expression (Fig. 2C). Moreover, transient coexpression of p300 and CAAhR did not have any effect on CAAhR-mediated inhibition of the hypoxic induction of the CA9 promoter (Fig. 2C). It is well known that the co-activator CBP/p300 functionally and physically interacts with HIF-1α/ARNT as well as with the AhR/ARNT complex and supports their functional activity in a conditionally regulated manner [START_REF] Kobayashi | CBP/p300 functions as a possible transcriptional coactivator of Ah receptor nuclear translocator (Arnt)[END_REF].

We have to keep in mind, that AhR could disrupt a HIF-1α-SP1 complex and by this mechanism reduce CA9 promoter activity. In this situation HIF-1α, due to its lower abundance comparing to SP1, could be a limiting factor.

TCCD mediated inhibition of CA9 expression is strictly dependent on the presence of ARNT

In order to form a transcriptionaly functional complex, both HIF-1α and AhR must dimerize with their common partner ARNT. It has been reported that these pathways can mutually interfere with one another by competition for recruitment of ARNT [START_REF] Chan | Cross-talk between the aryl hydrocarbon receptor and hypoxia inducible factor signaling pathways. Demonstration of competition and compensation[END_REF][START_REF] Nie | Interactions between aryl hydrocarbon receptor (AhR) and hypoxia signaling pathways[END_REF]30,[START_REF] Kim | Inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)stimulated Cyp1a1 promoter activity by hypoxic agents[END_REF]. To elucidate whether AhRmediated inhibition of CA9 expression is dependent on ARNT, we performed transfection experiments using both wild-type and ARNT-deficient mouse hepatoma cell lines. As shown in Fig. 3A, hypoxic induction of CA9 reporter activity was observed in wild-type Hepa-1c1c7 cells, but was not detected in Hepa-1c4 cells expressing a functionally deficient form of ARNT [START_REF] Hoffman | Cloning of a factor required for activity of the Ah (dioxin) receptor[END_REF]. In the presence of TCDD, hypoxic induction of CA9 promoter activity was decreased in a dioxin dosedependent manner (Fig. 3B).

Transient expression of CAAhR did not have any effect on the CA9 promoter activity in Hepa-1c4 cells either under normoxic or hypoxic conditions. On the other hand, the inhibitory effect of CAAhR was restored following co-expression of wild-type ARNT (Fig. 3C). Most notably, this inhibition became significant only after hypoxic treatment of Hepa-1c4 cells. The data obtained from these experiments demonstrate that AhR/CAAhR mediated inhibition of CA9 expression is critically dependent on a functional ARNT.

AhR activation directly affects binding of ARNT to CA9 promoter

The fact that both HIF-1α and AhR require dimerization with ARNT to form functional transcription complexes prompted us to consider the possible rivalry between both transcription factors for ARNT binding. To get a more direct proof about the possibility of cross-talk between hypoxia and dioxin pathways in the regulation of CA9 expression, we performed chromatin immunoprecipitation assay (ChIP). In order to activate CA9 expression, HeLa cells were grown in hypoxia and subsequently treated with TCDD to ensure the activation of AhR. DMSO treated cells were used as a negative control. The cells were then fixed with formaldehyde and shared chromatin was immunoprecipitated with either ARNT specific or IgG control antibodies. Finally, ARNT-specific immunoprecipitation was investigated by PCR reaction. As shown in Fig. 4, PCR performed with the primers flanking HRE within the CA9 promoter clearly demonstrates a weaker signal detected after ARNT-specific DNA precipititation in the TCDD treated cells when compared to control cells. The proper functionality and activation of AhR in our assay conditions was confirmed by PCR performed on the same precipitated DNA utilizing primers flanking XRE within the CYP1A1 promoter (Fig. 4). This result directly implicates possible inhibition of CA9 expression by decreasing number of ARNT molecules accessible for binding to HRE element within the CA9 promoter.

It has already been postulated by several research groups that activation of one pathway would inhibit the other due to a competition for ARNT or other limiting cellular factors. In accordance with our results obtained from chromatin immunoprecipitation, Chan and colleagues observed in gel mobility shift experiments that AhR inhibited formation of the HIF-1α/ARNT complex with HREs, and, conversely, that HIF-1α inhibited formation of the AhR/ARNT complex with an XRE motif [START_REF] Chan | Cross-talk between the aryl hydrocarbon receptor and hypoxia inducible factor signaling pathways. Demonstration of competition and compensation[END_REF]. In an earlier report, it was demonstrated in vitro that HIF-1α exhibits a very high affinity for ARNT resulting in a competition with the activated AhR for the recruitment of ARNT [30]. A similar observation was made following overexpression of HIF-1α, resulting in an apparent inhibition of the DNA-binding activity of dioxin-activated AhR [30]. In those experiments, however, activation of both hypoxia-and dioxin-signaling pathways was not mutually exclusive because activation of the AhR pathway did not result in inhibition of hypoxia-dependent expression of target genes. On the other hand, there is a conflicting report stating that the DNA-binding activity of AhR/ARNT heterodimers was only slightly reduced under hypoxic conditions [START_REF] Gassmann | Oxygen-and dioxinregulated gene expression in mouse hepatoma cells[END_REF]. Regardless of the identification of a crosstalk between hypoxia and dioxin pathways, even Pollenz and colleagues failed to detect competition between HIF-1α and AhR for ARNT binding [START_REF] Pollenz | Analysis of aryl hydrocarbon receptor-mediated signaling during physiological hypoxia reveals lack of competition for the aryl hydrocarbon nuclear translocator transcription factor[END_REF].

Taken together these seemingly conflicting reports would argue that the interplay between both pathways would obviously be dependent on the architecture of the target promoter/enhancer and the battery of cognate transcription factors and co-factors in close connection with the extent of chromatin remodeling but, importantly, competition for ARNT might be a valid scenario for negative regulation. In particular, this model is supported by the limiting ARNT concentrations observed in most cell types.

Very recently it has been reported that the environmentally abundant polycyclic aromatic hydrocarbon benzo[a]pyrene downregulates vascular endothelial growth factor (VEGF) expression through disruption of HIF-1α and Hsp90 association, which promotes ubiquitination and subsequent degradation of HIF-1α by the proteasome [START_REF] Li | Benzo[a]pyrene-3,6-dione inhibited VEGF expression through inducing HIF-1alpha degradation[END_REF]. This is most probably not the mechanism underlying CAAhR-mediated downregulation of CA9 expression, because, in contrast to the wild-type dioxin receptor, CAAhR lacks the minimal ligand binding domain (PASB domain) and is therefore active in both a ligand-and Hsp90-independent manner [START_REF] Mcguire | Definition of a dioxin receptor mutant that is a constitutive activator of transcription: delineation of overlapping repression and ligand binding functions within the PAS domain[END_REF].

A large scale microarray analysis has recently been employed to determine the extent of crosstalk between the hypoxia-and dioxin-signaling pathways. Interestingly, CA IX was identified among 33 unique transcripts, the expression of which was affected by both pathways [START_REF] Lee | Identification and characterization of genes susceptible to transcriptional cross-talk between the hypoxia and dioxin signaling cascades[END_REF]. Computational analysis of these genes identified some common DNA motifs present in their regulatory regions. This observation might help to distinguish between results obtained from different experiments done on different cell culture models and with distinct target genes and their regulatory regions. Nevertheless, these results should be interpreted with caution as Lee and colleagues [START_REF] Lee | Identification and characterization of genes susceptible to transcriptional cross-talk between the hypoxia and dioxin signaling cascades[END_REF] used cobalt chloride as a mimic of hypoxic conditions.

In our work we have demonstrated that the activated AhR can impair hypoxic induction of both CA IX mRNA and protein expression. We have thus observed a cross-talk between hypoxia and AhR pathways in regulation of CA9 expression. This mode of regulation seems to occur primarily at the transcriptional level. We have further provided evidence for competition between HIF-1α and AhR for limited intracellular levels of ARNT. This mode of competition for recruitment of ARNT may play a role in CA IX regulation in tumors that are exposed to xenobiotics.

From the pathophysiological point of view it is quite conceivable that exposure to dioxin could have a negative influence on normal cells and tissues that rely on protective mechanisms activated by HIF-regulated pathways. However, situation might be more complicated in tumors due to a high plasticity of malignant cells which could react to acute exposure to dioxin or other xenobiotics by a transiently reduced hypoxic response. On the other hand, chronic exposure might induce either a switch to alternative pathways, or to selection of those tumor cells, which are able to return back to the hypoxic phenotype that provides a significant survival advantage in hostile tumor microenvironment. These issues were not addressed in any of the studies published so far and remain to be resolved in different experimental settings that are currently underway.

In conclusion, our observations support the model that the existence of limiting concentrations of bHLH/PAS proteins is involved in the cross-talk among members of this protein family. The expression of CA IX is strongly induced by hypoxia and its distribution in tumor tissues significantly correlates with poor prognosis of many tumor types. Our results extend data about factors and signaling pathways involved in the regulation of CA IX expression. (A) HeLa cells transfected with 1 µg of the promoter-containing luciferase construct (full-length -174/+37 or minimal -50/+37) were either pre-treated with TCDD or co-transfected with 1 µg of CAAhR expressing plasmid or empty vector (pCMV). Transfected cells were maintained in normoxia or hypoxia for 24 h. CA9 promoter activity was measured 72 h after transfection and calculated as the ratio of luciferase to renilla activity. Results are expressed as the mean percentage of activity of the -174/+37 promoter construct observed in hypoxia ± SE of 3 independent experiments. (B) Co-transfection of 1 µg of XRE-driven reporter construct pTX.DIR confirmed the functionality of CAAhR in our assay. Results are expressed as the mean percentage of activity obtained in normoxic culture co-transfected with CAAhR ± SE of 3 independent experiments. (C) Effect of co-transfection of the same amount of HIF-1α, SP1 or p300 together with 1 µg of CAAhR on the activity of the -174/+37 luciferase construct. Co-transfection of an empty vector served as a control. Results are expressed as the mean percentage of activity of the -174/+37 promoter construct observed in hypoxia ± SE of three independent experiments. Fig. 3. Inhibition of CA9 expression mediated by TCDD or CAAhR depends on the presence of ARNT. (A) Wild-type mouse hepatoma cells Hepa-1c1c7 and the ARNT-deficient subline Hepa-1c4 cells were transfected with the -174/+37 luciferase construct and exposed to normoxia or hypoxia for 24 h. CA9 promoter activity was determined as described in Fig. 2. Results are expressed as the mean percentage of activity obtained from the wild-type cells exposed to hypoxia ± SE of 3 independent experiments. (B) Pre-treatment with TCDD (1 nM or 10 nM) in normoxia or hypoxia of Hepa-1c1c7 cells transfected with 1 µg of the -174/+37 promoter construct. Results are expressed as the mean percentage of activity obtained from the DMSO-treated cells exposed to hypoxia ± SE of 3 independent experiments. (C) Effect of 1 µg of CAAhR co-transfected in Hepa-1c4 cells with or without functional ARNT maintained for 24 h in normoxia and hypoxia. Results are expressed as the mean percentage of activity obtained from the cells with restored ARNT that were co-transfected with an empty vector ± SE of three independent experiments. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

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Biochemical

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  as a negative control and for chromatin preclearing.Purified DNA was subjected to 35 cycles of PCR with primers flanking the putative hypoxia response element (HRE) (hCA9-HRE-S: 5´-TCCTAGCTTTGGTATGGGGGAGAG-3´ and hCA9-HRE-A: 5´-AGTGACAGCAGCAGTTGCACAGT-3´; 277 bp product) within the CA9 promoter. Primers flanking the xenobiotic response element (XRE) of the CYP1A1 promoter (hCYP1A1-XRE-S:

  Biochemical Journal Immediate Publication. Published on 20 Jan 2009 as manuscript BJ20080952 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080952A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

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Biochemical

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Figure legends Fig. 1 .

 legends1 Figure legends

Fig. 2 .

 2 Fig.2. Transcriptional activity of CA9 promoter constructs after TCDD treatment or cotransfection with CAAhR. (A) HeLa cells transfected with 1 µg of the promoter-containing luciferase construct (full-length -174/+37 or minimal -50/+37) were either pre-treated with TCDD or co-transfected with 1 µg of CAAhR expressing plasmid or empty vector (pCMV). Transfected cells were maintained in normoxia or hypoxia for 24 h. CA9 promoter activity was measured 72 h after transfection and calculated as the ratio of luciferase to renilla activity. Results are expressed as the mean percentage of activity of the -174/+37 promoter construct observed in hypoxia ± SE of 3 independent experiments. (B) Co-transfection of 1 µg of XRE-driven reporter construct pTX.DIR confirmed the functionality of CAAhR in our assay. Results are expressed as the mean percentage of activity obtained in normoxic culture co-transfected with CAAhR ± SE of 3 independent experiments. (C) Effect of co-transfection of the same amount of HIF-1α, SP1 or p300 together with 1 µg of CAAhR on the activity of the -174/+37 luciferase construct. Co-transfection of an empty vector served as a control. Results are expressed as the mean percentage of activity of the -174/+37 promoter construct observed in hypoxia ± SE of three independent experiments.

Fig. 4 .

 4 Fig.4. Chromatin immunoprecipitation assay. HeLa cells were treated with 10 nM TCDD in hypoxia. DMSO treated cells were used as a negative control. DNA was immunoprecipitated with antibody specific for ARNT or anti-human IgG antibodies to visualise background signal. Recovered DNA was used in PCR with primers flanking ARNT-binding sites within CA9 (HRE) and CYP1A1 (XRE) promoters. DNA samples from non-immunoprecipitated extracts were amplified as input. Numbers given below the figures represent relative levels of PCR products (ND-not determined). A representative of three experiments is shown.

Figure 1 .

 1 Figure 1.

Figure 2 .

 2 Figure 2.

Figure 3 .

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Figure 4 .

 4 Figure 4.

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Acknowledgements

This work was supported by research grants from Slovak Research and Development Support Agency (Contract APVV-51-024905), from EU (6FP Integrated project EUROXY, LSCH-CT-2003-502932) and from the Czech Ministry of Agriculture (MZE 0002716201). M.T. and T.H. contributed equally to this work. Biochemical Journal Immediate Publication. Published on 20 Jan 2009 as manuscript BJ20080952 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080952

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.