Aromatic Hydrocarbon Receptor(AhR)⅐AhR NuclearTranslocator-and p53-mediated Induction of theMurine Multidrug Resistance mdr1Gene by3-Methylcholanthrene and Benzo(a)pyrene in Hepatoma Cells*Received for publication,September18,2000,and in revised form,November10,2000Published,JBC Papers in Press,November28,2000,DOI10.1074/jbc.M008495200Marie-Claude Mathieu,Isabelle Lapierre,Karine Brault‡,and Martine Raymond§From the Institut de Recherches Cliniques de Montre´al,Montre´al,Que´bec H2W1R7,CanadaThe mouse multidrug resistance gene family consists of three genes(mdr1,mdr2,and mdr3)encoding P-gly-coprotein.We show that the expression of mdr1is in-creased at the transcriptional level upon treatment of the hepatoma cell line Hepa-1c1c7with the polycyclic aromatic hydrocarbon3-methylcholanthrene(3-MC). This increase is not observed in the aromatic hydrocar-bon receptor(AhR)-defective TAOc1BP r c1and the AhR nuclear translocator(Arnt)-defective BP r c1variants, demonstrating that the induction of mdr1by3-MC re-quires AhR⅐Arnt.We show that the mdr1promoter (؊1165to؉84)is able to activate the expression of a reporter gene in response to3-MC in Hepa-1c1c7but not in BP r c1cells.Deletion analysis indicated that the re-gion from؊245to؊141contains cis-acting sequences mediating the induction,including a potential p53bind-ing sequence.3-MC treatment of the cells increased the levels of p53and induced p53binding to the mdr1pro-moter in an AhR⅐Arnt-dependent manner.Mutations in the p53binding site abrogated induction of mdr1by 3-MC,indicating that p53binding to the mdr1promoter is essential for the induction.Benzo(a)pyrene,a polycy-clic aromatic hydrocarbon and AhR ligand,which,like 3-MC,is oxidized by metabolizing enzymes regulated by AhR⅐Arnt,also activated p53and induced mdr1tran-scription.2,3,7,8-Tetrachlorodibenzo-p-dioxin,an AhR ligand resistant to metabolic breakdown,had no effect. These results indicate that the transcriptional induc-tion of mdr1by3-MC and benzo(a)pyrene is directly mediated by p53but that the metabolic activation of these compounds into reactive species is necessary to trigger p53activation.The ability of the anticancer drug and potent genotoxic agent daunorubicin to induce mdr1independently of AhR⅐Arnt further supports the proposition that mdr1is transcriptionally up-regulated by p53in response to DNA damage.Multidrug resistance(MDR)1is characterized by cross-resis-tance of the cells to a large number of structurally and func-tionally unrelated cytotoxic agents used in chemotherapy.In cultured cells,MDR is frequently caused by the overexpression of P-glycoprotein(Pgp),an integral membrane protein belong-ing to the ATP-binding cassette superfamily of transporters and which functions as an energy-dependent efflux pump of cytotoxic drugs(1,2).Pgp is encoded by a small family of genes with two members in humans(MDR1and MDR2/MDR3)and three in rodents(mdr1/mdr1b,mdr2,and mdr3/mdr1a)(1,2). Only one human gene(MDR1)and two rodent genes(mdr1/ mdr1b and mdr3/mdr1a)can confer MDR upon overexpression in drug-sensitive cells(1,2).The different mdr genes and Pgp isoforms are expressed in a tissue-specific manner(1,2).In the mouse,mdr1is expressed mostly in the adrenal cortex,kidney,and pregnant uterus, mdr2in the liver at the canalicular face,and mdr3in the intestine and to a lesser extent in the heart,liver,lung,and capillaries of the brain(3).Pgps are localized on the apical membrane of epithelial cells lining luminal spaces,suggesting that they function in normal tissues as transporters of toxic substances and/or specific endogenous cellular products(4). Knockout mice experiments have demonstrated a role for the mdr3gene in the maintenance of the blood-brain barrier and drug elimination and for the mdr2gene in the transport of phospholipids in the bile(5,6).No physiological function has been attributed to the mouse mdr1gene so far,since knockout mdr1(Ϫ/Ϫ)mice display no obvious physiological abnormali-ties(7).However,different experimental evidence indicates that Pgp encoded by mdr1can serve in the transport of steroids(8).A number of factors have been found to modulate the level of mdr gene expression in the liver.For example,high levels of MDR1RNA have been found in human hepatocarcinomas,and overexpression of the mdr1isoforms has also been observed in rodent liver during cholestasis,during regeneration following partial hepatectomy,during chemically induced hepatocarcino-genesis,and following administration of various natural and synthetic xenobiotics(1,2).In particular,it has been shown that expression of the rat mdr1b gene is increased in liver cells in response to treatment with various polycyclic aromatic hy-*This work was supported by a research grant from the Cancer Research Society Inc.(to M.R).The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked“advertisement”in accordance with18 U.S.C.Section1734solely to indicate this fact.‡Supported by a studentship from the Medical Research Council ofCanada.Present address:Dept.of Biological Sciences,Bio-Mega Re-search Division,Boehringer Ingelheim(Canada)Ltd.,Laval,Que´bec H7S2G5,Canada.§Supported by a scholarship from Le Fonds de la recherche en sante´du Que´bec.To whom correspondence should be addressed:Institut de recherches cliniques de Montre´al,110Pine Ave.W.,Montre´al,Que´bec H2W1R7,Canada.Tel.:514-987-5770;Fax:514-987-5764;E-mail: raymonm@ircm.qc.ca.1The abbreviations used are:MDR,multidrug resistance;Pgp,P-glycoprotein;3-MC,3-methylcholanthrene;B(a)P,benzo(a)pyrene; TCDD,2,3,7,8-tetrachlorodibenzo-p-dioxin;DN,daunorubicin;CAT, chloramphenicol acetyl transferase;AhR,aromatic hydrocarbon recep-tor;Arnt,AhR nuclear translocator;EMSA,electrophoretic mobility shift assay;DME,drug metabolizing enzymes;PAH polycyclic aromatic hydrocarbon;XRE,xenobiotic response element;bp,base pair(s);kb, kilobase pair(s).T HE J OURNAL OF B IOLOGICAL C HEMISTRY Vol.276,No.7,Issue of February16,pp.4819–4827,2001©2001by The American Society for Biochemistry and Molecular Biology,Inc.Printed in U.S.A.This paper is available on line at 4819 at ZHEJIANG UNIVERSITY, on November 21, Downloaded fromdrocarbon(PAH)compounds,including3-methylcholanthrene (3-MC),and that this increased expression occurs at the tran-scriptional level(9–11).However,the precise molecular mech-anisms involved in mdr1b regulation in response to3-MC are still unknown.PAHs are carcinogenic compounds arising from the incom-plete combustion of organic matter and are widespread in the environment,including tobacco smoke and tar.PAHs such as 3-MC and benzo(a)pyrene(B(a)P)as well as halogenated aro-matic hydrocarbons such as2,3,7,8-tetrachlorodibenzo-p-di-oxin(TCDD)are specific inducers of genes coding for drug-metabolizing enzymes(DME),including cyp1a1and cyp1a2, that code for cytochromes P450involved in metabolic oxidation (12).PAHs and TCDD bind in the cytoplasm to the aromatic hydrocarbon receptor(AhR),a member of the bHLH-PAS(basic helix-loop-helix Per-Arnt-Sim)family of transcription factors (12,13).The ligand-bound AhR translocates to the nucleus, where it binds as a heterodimer with the AhR nuclear trans-locator(Arnt;another bHLH-PAS protein)to specific cis-acting regulatory DNA sequences located in the promoter of its tar-gets(known as AH-,dioxin-,or xenobiotic-responsive elements (or AHRE,DRE,or XRE,respectively))to enhance their tran-scription(12,13).Given that mdr1b expression is increased in liver cells in response to treatment with various PAHs,it was postulated that mdr1b may be under the control of the AhR(9). However,studies failing to show mdr1induction in the liver of mice treated with TCDD,one of the most potent agonists of the AhR,suggested that mdr1expression was not regulated by AhR(14).The involvement of AhR in the regulation of mdr1 has so far remained controversial.The mouse hepatoma cell lines Hepa-1c1c7(wild type), TAOc1BP r c1(AhR-defective),and BP r c1(Arnt-defective)con-stitute a powerful experimental system to investigate the tran-scriptional regulation of different AhR⅐Arnt targets in response to xenobiotics(12).The two mutant cell lines were derived as B(a)P-resistant variants of Hepa-1c1c7and were identified based on their inability to induce aryl hydrocarbon hydroxylase activity in response to TCDD treatment(15).TAOc1BP r c1cells have a decreased level of AhR(ϳ10%of wild-type cells)and therefore decreased induction of the cyp1a1promoter and lower aryl hydrocarbon hydroxylase activity in response to TCDD and other AhR ligands(15–18).BP r c1cells have a nor-mal cytosolic AhR,which fails to accumulate in the nucleus because of a defective Arnt(15).They have virtually no basal or inducible levels of cyp1a1expression and aryl hydrocarbon hydroxylase activity(15–17).In the present report,we have used this panel of cell lines to investigate the transcriptional regulation of the murine mdr1 gene by3-MC and other xenobiotic compounds.Our results demonstrate that mdr1is transcriptionally induced by3-MC and B(a)P and that this induction is mediated by p53but also requires AhR⅐Arnt.A model for the AhR⅐Arnt-and p53-medi-ated transactivation of mdr1in response to genotoxic stress is proposed.EXPERIMENTAL PROCEDURESCell Culture—Wild-type Hepa-1c1c7and Hepa1–6,AhR-defective TAOc1BP r c1,and Arnt-defective BP r c1cells were obtained from the American Type Culture Collection(ATCC;Manassas,VA)and main-tained in culture under the conditions recommended by the ATCC. Chinese hamster ovary LR73cell lines stably transfected with plasmid constructs carrying full-length cDNAs for the mouse mdr1,mdr2,or mdr3genes(LR73mdr1,LR73mdr2,and LR73mdr3,respectively;a gift from Dr.Philippe Gros,McGill University,Montre´al,Canada)were grown as described elsewhere(19,20).For inductions,cells atϳ50% confluence were exposed to different concentrations of xenobiotics for various periods of time(the exact conditions for each experiment are indicated in the figure legends).3-MC,B(a)P,and daunorubicin were obtained from Sigma,and TCDD was obtained from the Centre d’expertise en analyze environnementale du Que´bec(Laval,Canada).Stock solutions of3-MC(5m M)and B(a)P(25m M)were prepared in Me2SO,and the stock solutions of daunorubicin(1mg/ml)were pre-pared in water.TCDD was obtained in n-nonane at a concentration of 50g/ml and was stored at room temperature.Stock solutions of3-MC, B(a)P,and daunorubicin were stored atϪ80°C.RNA Preparation—Total RNA was prepared from3-MC-treated and untreated hepatocytes as well as from the LR73mdr1,LR73mdr2,and LR73mdr3cell lines by homogenizing the cells in a solution containing guanidium hydrochloride(6M)followed by sequential ethanol precipi-tation,as described previously(21).RNase Protection Assay—The plasmid constructed to detect the mdr1 RNA consisted of a165-bp Bam HI fragment isolated from the mdr1 cDNA(positions1926–2090relative to the ATG initiation codon(22)), blunt-ended with T4DNA polymerase,and cloned into plasmid pGEM-7Z(Promega,Madison,WI)at the Sma I site,giving plasmid pmdr1-G7.This plasmid was linearized with Eco RI and used as a template to synthesize an antisense mdr1probe using SP6RNA polym-erase(Amersham Pharmacia Biotech).The pKX10–3Z plasmid consist-ing of an Xba I–Kpn I mouse-actin cDNA fragment(positions724–969 in the-actin cDNA)cloned into pGEM-3Z at the Xba I and Kpn I sites (kindly provided by Dr.Rashmi Kothary,Institut du cancer de Mon-tre´al,Montre´al,Canada)was used to generate a control actin probe. pKX10–3Z was linearized with Xba I and used to synthesize an anti-sense actin RNA probe with T7RNA polymerase.The riboprobes were synthesized in the presence of[␣-32P]UTP,and the RNase protection assay was performed according to standard protocols(23).Nuclear Run-on Transcription Assay—The run-on experiment was performed essentially as described by Fisher et al.(24).Nuclei wereisolated from Hepa-1c1c7cells treated with Me2SO or with3-MC(5M) for48h and were used to label nascent RNAs with[␣-32P]UTP.Plas-mids pVT101-U/mdr1,carrying the full-length mouse mdr1cDNA(25); pmP1450–3Ј,carrying a1.2-kb Pst I cDNA fragment overlapping part of the mouse cyp1a1cDNA(26)(obtained from the ATCC);and pKX10–3Z were linearized with Stu I,Bam HI,and Xba I,respectively.The linear-ized plasmids were denatured,immobilized in duplicate onto a nylon membrane,and hybridized with the[␣-32P]UTP-labeled RNAs for48h at65°C.The membranes were washed and exposed for7days with two intensifying screens.Slot Blot Analyses—Slot blotting was performed as previously de-scribed(21).RNA samples(10g)were denatured in7ϫSSC-7.5% formaldehyde for15min at65°C and applied to a nylon membrane (Zeta-Probe).Detection of specific RNAs was performed by hybridiza-tion at65°C in0.5M NaPO4,pH7.2,1m M EDTA,7%SDS,1%bovine serum albumin,and100g/ml salmon sperm DNA with32P-labeled DNA probes.The mdr1probe was a4.2-kb Sph I–Eco RI fragment over-lapping the full-length mouse mdr1cDNA,isolated from plasmid pGEM7/mdr1(a gift from Dr.Philippe Gros,McGill University,Mon-tre´al);the cyp1a1probe was a 1.2-kb Pst I fragment isolated from plasmid pmP1450–3Ј;and the actin probe was a245-bp Xba I–Kpn I fragment isolated from pKX10–3Z.The membranes were washed twiceat65°C with a solution containing40m M NaPO4,pH7.2,5%SDS,1 m M EDTA,0.5%bovine serum albumin and twice with a solutioncontaining40m M NaPO4,pH7.2,5%SDS,and1m M EDTA before autoradiography.Chloramphenicol Acetyl Transferase(CAT)Expression Plasmids—Plasmid pMcat5.9consists of a482-bp DNA fragment containing the dioxin-responsive elements of the cyp1a1gene cloned upstream of the mouse mammary tumor virus promoter and the CAT gene(24)(kindly provided by Dr.Allan Okey,University of Toronto).Plasmids pmdr1, p-452,p-245,p-141,and p-93(previously referred to as pSacICAT, pExo6CAT,pExo2CAT,pExo1CAT,and pAluCAT,respectively)have been described elsewhere(27).The mdr1promoter sequence in these constructs ends at positionϩ84with respect to the transcription start site(27).To produce the p53mutant constructs,pM1and pM2,plasmid pSBM13was used.This plasmid consists of a1.2-kb Sac I–Hin dIII mdr1 promoter fragment(positionsϪ1165toϩ84)cloned into M13mp18. Single-stranded DNA was prepared from pSBM13and used as a tem-plate to perform site-directed mutagenesis of the p53binding site,using the mutant oligonucleotides M15Ј-TACCTGAA T AC A TAAAGACA and M25Ј-CGTAAAGA T AA A TCTATGTA(the base changes are shown in boldface type).The resulting M1and M2mdr1promoter fragments were then excised from pSBM13with Sac I and Hin dIII,blunt-ended with T4DNA polymerase,and cloned into plasmid pCAT at the Hin dIII site also blunt-ended with T4DNA polymerase,yielding plasmids pM1 and pM2.The presence of the mutations in the resulting constructs was confirmed by DNA sequencing.Transient Transfections and CAT Assays—Cells were plated at aInduction of the Mouse mdr1Gene by PAHs4820at ZHEJIANG UNIVERSITY, on November 21, Downloaded fromdensity of 8ϫ105/60-mm plate and transfected on the following day with 10g of plasmid DNA,using a standard calcium phosphate pre-cipitation method (28).After incubation with the DNA precipitate for 16h,the cells were washed twice with phosphate-buffered saline and supplied with fresh medium containing the different xenobiotics.After 48h,the cells were collected.Cell extracts were prepared,and protein concentrations were determined by the Bradford method (29).CAT activities were assayed by standard protocols as described previously,using 2g of proteins (27).Preparation of Nuclear Extracts—Nuclear extracts were prepared according to Schreiber et al .(30),with some modifications.Cells were harvested in cold phosphate-buffered saline,0.6m M EDTA and col-lected by centrifugation.The cell pellets were resuspended in 400l of ice-cold buffer A (10m M Tris,pH 8.0,10m M KCl,0.1m M EDTA,0.1m M EGTA,1m M dithiothreitol)containing 0.5m M phenylmethylsulfonyl fluoride,10g/ml aprotinin,1g/ml pepstatin,and 5g/ml leupeptin and swelled on ice for 15min.Subsequently,25l of 10%Nonidet P-40were added,and the tubes were vortexed vigorously.The nuclear pellets were collected by centrifugation and resuspended in 100l of cold buffer C (20m M Tris,pH 8.0,400m M NaCl,1m M EDTA,1m M EGTA,1m M dithiothreitol)in the presence of protease inhibitors.The suspen-sions were shaken vigorously at 4°C for 1h and centrifuged for 15min at 4°C,and the supernatants were frozen in aliquots at Ϫ80°C.Proteinconcentrations were determined by the Bradford method (29).ElectrophoreticMobility Shift Assay—Oligonucleotides overlapping the potential p53binding site in the mdr1promoter (5Ј-GAACACGTA-AAGACAAGTCTAT)and the p53consensus sequence in the p21waf1/cip1promoter (5Ј-GAACATGTCCCAACATGTTGAG)(31)were end-labeled with ␥-32P using T4polynucleotide kinase and annealed to their respec-tive in a M M 2.5m M dithiothreitol,4%Ficoll,1g of poly(dI-dC),and 20,000cpm of radiolabeled probe.The binding reactions were carried out at room temperature for 15min.Where needed,1g of the monoclonal anti-p53antibody pAb421(32)(Calbiochem)or of the polyclonal anti-Jun or anti-Skn-1antibodies (Santa Cruz Biotechnology,Inc.,Santa Cruz,CA)was added,and the incubation was continued for an additional 15min.The complexes were separated on 5%nondenaturing polyacrylamide gels in 1ϫTBE (90m M Tris,65m M boric acid,2.5m M EDTA,pH 8.0)at 200V.The gels were exposed to XAR films (Eastman Kodak Co.)for 16h with two intensifying screens at Ϫ80°C.Western Blotting—Total proteins from 3-MC-or Me 2SO-treated Hepa-1c1c7and BP r c1cells were extracted in ice-cold buffer (10m M Tris-HCl,pH 8.0,150m M NaCl,1m M EDTA,1%Nonidet P-40,and 1%sodium deoxycholate)containing 10g/ml leupeptin,10g/ml aproti-nin,1M sodium orthovanadate,and 1m M phenylmethylsulfonyl flu-oride.Total proteins (75g/sample)or nuclear extracts (30g/sample)were separated by SDS-polyacrylamide gel electrophoresis on a 10%acrylamide gel,transferred to a nitrocellulose membrane,and analyzed with the monoclonal anti-p53antibody pAb421(32)(Calbiochem)at a concentration of 5g/ml.Immune complexes were revealed by incuba-tion with a goat anti-mouse IgG antibody coupled to alkaline phospha-tase (Bio-Rad)and developed with 5-bromo-4-chloro-3-indolylphosphate p -toluidine salt and nitro blue tetrazolium chloride substrates as rec-ommended by the manufacturer (Life Technologies,Inc.).RESULTSTranscriptional Induction of the Mouse mdr1Gene by 3-MC in Hepatoma Cells—We have used an RNase protection assay to study the expression of mdr1in the hepatoma cell line Hepa-1c1c7upon exposure to 3-MC (Fig.1).An mdr1-specific riboprobe was prepared by cloning into pGEM7-Zf a mouse mdr1cDNA fragment overlapping the linker region of the protein,this domain displaying the lowest sequence homology among the three mouse mdr cDNAs (21).When tested with RNA prepared from LR73stable transfectants expressing each of the three mouse mdr cDNAs,the mdr1riboprobe was found to recognize the mdr1RNA but not the mdr2or mdr3RNA,thus confirming its specificity (Fig.1,top right ).The mdr1probe was then used with RNA from Hepa-1c1c7cells treated or not with 3-MC (Fig.1,top left ).This experiment showed that the amount of mdr1RNA detected is very low in untreated cells but is strongly increased in 3-MC-treated cells,demonstrating that expression of the mouse mdr1gene is induced by 3-MCtreatment.The use of an actin probe confirmed that equal quantities of RNA were used in the assay (Fig.1,bottom ).A similar experiment performed with mdr2-and mdr3-specific riboprobes showed that the expression of these genes is not induced under such conditions,demonstrating that the induc-tion of mdr1expression by 3-MC is isoform-specific (data not shown).A nuclear run-on experiment was performed to determine whether mdr1induction by 3-MC occurs at the transcriptional level (Fig.2).In addition to the mouse mdr1cDNA,cDNAs for the mouse cyp1a1gene (known to be transcriptionally regu-lated by 3-MC (12))and for the actin gene were also included as positive and negative controls,respectively.The data in Fig.2show that 3-MC induces an increase in the rate of mdr1mRNA synthesis,indicating that 3-MC acts at the transcriptional level to induce mdr1gene expression in Hepa-1c1c7cells.AhR ⅐Arnt-dependent Induction of mdr1Expression by 3-MC—To determine whether the increase in mdr1expression in response to 3-MC exposure is AhR ⅐Arnt-mediated,we ana-lyzed the mdr1RNA levels upon 3-MC treatment in two wild-type hepatoma cell lines Hepa-1c1c7and Hepa 1–6and in two variant cell lines derived from Hepa-1c1c7,TAOc1BP r c1(AhR-defective)and BP r c1(Arnt-defective)(15)(Fig.3).As controls,we also analyzed the level of cyp1a1and actin expression under the same conditions (Fig.3,middle and right ,respectively).This experiment showed that mdr1is expressed at low levels in the four cell lines in the absence of 3-MC induction (Fig.3,left panel ).Upon 3-MC treatment,the expression of mdr1is in-duced in the two wild-type hepatoma cell lines (by ϳ5-fold),this induction being completely abrogated in the AhR-defective or in the Arnt-defective variants (Fig.3,left panel ).The actin control probe confirmed that equal amounts of RNA had been applied to the membrane (Fig.3,right panel ).These data clearly demonstrate that the induction of mdr1in response to 3-MC requires an intact AhR ⅐Arnt complex,like cyp1a1(Fig.3,middle )(12).The Mouse mdr1Promoter Confers 3-MC-regulated Expres-sion in an AhR ⅐Arnt-dependent Manner—To determine if reg-ulatory sequences responsible for mdr1induction by 3-MC are present in the promoter region of the gene,plasmid pmdr1,consisting of a 1.2-kb Sac I–Hin dIII DNA fragment overlapping the mdr1promoter region (positions Ϫ1165to ϩ84with respect to the transcription start site (27))fused to the CAT reporter gene,was analyzed in transient transfection experiments.Plasmid pMcat5.9,which consists of a 482-bp fragment derived from the cyp1a1promoter fused to the mouse mammary tumorF IG .1.Increased mdr1expression in Hepa-1c1c7upon 3-MC treatment.The expression of mdr1was analyzed by RNase protection assay.Total RNAs (45g)from Hepa-1c1c7cells treated with 5M 3-MC (ϩMC )or with Me 2SO (ϪMC )for 56h and from the control cell lines LR73/mdr1,LR73/mdr2,and LR73/mdr3were analyzed with an mdr1riboprobe,which protects a 169-nt fragment within the mdr1transcript,or with a -actin riboprobe,which protects a 245-nt actin transcript fragment.Autoradiography was for 15h with two intensify-ing screens (mdr1)or for 5h without intensifying screens (actin ).Induction of the Mouse mdr1Gene by PAHs4821at ZHEJIANG UNIVERSITY, on November 21, 2012 Downloaded fromvirus promoter and to the CAT gene (24),as well as the empty pCAT vector were also included as positive and negative con-trols,respectively.The three plasmids were transiently trans-fected into Hepa-1c1c7and BP r c1cells.The cells were treated with 3-MC or with Me 2SO for 48h,and the cellular extracts were prepared and assayed for CAT activity.This experiment showed that the mdr1promoter is transcriptionally active in Hepa-1c1c7cells and BP r c1cells,since it can drive the expres-sion of the CAT gene in both cell lines,albeit at low levels (Fig.4).This result is consistent with the basal level of expression of mdr1detected by slot blot analysis in these cells (Fig.3).3-MC treatment of the Hepa-1c1c7cells transfected with pmdr1re-sulted in a 10-fold induction in CAT activity as compared with untreated cells,reaching levels of CAT activity similar to those detected in the Hepa-1c1c7pMcat5.9transfectants upon 3-MC treatment.However,this induction was completely abrogated in BP r c1cells (Fig.4),consistent with the lack of mdr1induc-tion at the RNA level observed in the slot blot assay (Fig.3).Similar results were obtained upon transfection in TAOc1BP r c1cells (data not shown).These results,showing that the mdr1promoter is able to activate the expression of the reporter gene in response to 3-MC in Hepa-1c1c7but not in BP r c1and TAOc1BP r c1cells,demonstrate that (i)the mdr1promoter is able to confer 3-MC-mediated transcriptional acti-vation;(ii)this activation requires a functional AhR ⅐Arnt com-plex;and (iii)the sequences mediating this induction are lo-cated between positions Ϫ1165and ϩ84in the mdr1promoter.Two Putative XREs Located in the mdr1Promoter Are Dis-pensable for the Induction of mdr1by 3-MC—The AhR ⅐Arnt transcriptional complex binds to a specific DNA sequence,5Ј-(A/T)NGCGTG,known as an XRE to activate transcription (12).XREs render heterologous promoters responsive to xeno-biotics and function in a position-and orientation-independent manner (33,34).Examination of the mdr1promoter sequence indicated the presence of two potential XREs in an inverted orientation in the distal portion of the promoter at positionsϪ1129and Ϫ620(5Ј-CACGCAT and 5Ј-CACGCAA,respective-ly).To identify the cis -acting sequences responsible for the induction of mdr1by 3-MC and to investigate the possible involvement of these putative XREs,we analyzed the tran-scriptional activity of a series of mdr1promoter 5Ј-deletion CAT constructs after transient transfection into Hepa-1c1c7and treatment of the resulting transfectants with 3-MC (Fig.5A ).3-MC treatment of Hepa-1c1c7cells transfected with plas-mids p-452or p-245resulted in a level of CAT induction similar to that observed in cells transfected with plasmid pmdr1car-rying the full-length promoter,indicating that sequences lo-cated within positions Ϫ1165to Ϫ245are dispensable for the induction of mdr1by 3-MC,including the two putative XREs as well as a potential AP-1binding site (5Ј-TGACTCA;positions Ϫ265to Ϫ255(35))(Fig.5,B and C ).However,further deletion of a 104-bp region down to position Ϫ141(p Ϫ141)was found to greatly diminish the induction of CAT activity by 3-MC (Fig.5,B and C ),demonstrating that sequences important for the induction are located between positions Ϫ245and Ϫ141.CAT activity in the absence of 3-MC was reduced in the p Ϫ141transfectants when compared with the p Ϫ245transfectants,indicating that sequences between positions Ϫ245and Ϫ141are also involved in the basal transcriptional activity of the mdr1promoter in hepatoma cells.Finally,we found that alowF IG .2.Nuclear run-on experiment.Nuclei were isolated from Hepa-1c1c7cells treated with 5M 3-MC (ϩMC )or with Me 2SO (ϪMC )for 48h.Nascent RNAs were radiolabeled with [␣-32P]UTP and used to probe duplicate nylon membranes on which denatured cDNAs for mdr1,cyp1a1,and actin had been immobilized.The membranes were washed and exposed for 7days with two intensifyingscreens.F IG .3.AhR ⅐Arnt-dependent induction of mdr1expression by 3-MC.Total RNAs (10g)from wild-type Hepa-1c1c7and Hepa 1–6,AhR-defective TAOc1BP r c1,and Arnt-defective BP r c1cells treated (ϩMC )or not treated (ϪMC )with 3-MC at 5M for 56h were applied onto a nylon membrane.The membrane was hybridized sequentially with an mdr1(left ),a cyp1a1(middle ),and a -actin (right )probe.Autoradiography was for 18h (mdr1and cyp1a1)or for 2h (actin)with two intensifyingscreens.F IG .4.AhR ⅐Arnt-dependent induction of the mdr1promoter by 3-MC.Plasmids pCAT (no promoter),pmdr1(mdr1promoter from position Ϫ1165to ϩ84),and pMcat5.9(pMcat;482-bp fragment from the cyp1a1promoter fused to the mouse mammary tumor virus pro-moter)were transiently transfected into Hepa-1c1c7and BP r c1cells by the calcium phosphate method.The cells were then treated with 3-MC (5M )or Me 2SO for 48h.Total cellular extracts were prepared,and equal quantities of proteins (2g)were assayed for CAT activity.A ,autoradiogram of a representative CAT assay,showing the activity of plasmids pCAT,pmdr1and pMcat in Hepa-1c1c7and BP r c1cells treated (ϩ)or not treated (Ϫ)with 3-MC (MC ).The position of the [14C]chloramphenicol (CM )and of its acetylated products (AcCM )is indicated on the left .B ,quantitative analysis of CAT activities.The percentage of conversion of [14C]chloramphenicol to its acetylated de-rivatives was quantitated by liquid scintillation counting.Open bars ,ϪMC ;filled bars ,ϩMC .The results presented are the averages of three independent transfections performed in duplicate.S.D.values are rep-resented by the bars .Induction of the Mouse mdr1Gene by PAHs4822 at ZHEJIANG UNIVERSITY, on November 21, 2012 Downloaded from。
