Aromatic Hydrocarbon Receptor (AhR)·AhR Nuclear Translocator- and p53-mediated Induction of the Murine Multidrug Resistance mdr1 Gene by 3-Methylcholanthrene and Benzo(a)pyrene in Hepatoma Cells*

The mouse multidrug resistance gene family consists of three genes (mdr1, mdr2, andmdr3) encoding P-glycoprotein. We show that the expression of mdr1 is increased at the transcriptional level upon treatment of the hepatoma cell line Hepa-1c1c7 with the polycyclic aromatic hydrocarbon 3-methylcholanthrene (3-MC). This increase is not observed in the aromatic hydrocarbon receptor (AhR)-defective TAOc1BPrc1 and the AhR nuclear translocator (Arnt)-defective BPrc1 variants, demonstrating that the induction of mdr1 by 3-MC requires AhR·Arnt. We show that the mdr1 promoter (−1165 to +84) is able to activate the expression of a reporter gene in response to 3-MC in Hepa-1c1c7 but not in BPrc1 cells. Deletion analysis indicated that the region from −245 to −141 containscis-acting sequences mediating the induction, including a potential p53 binding sequence. 3-MC treatment of the cells increased the levels of p53 and induced p53 binding to the mdr1promoter in an AhR·Arnt-dependent manner. Mutations in the p53 binding site abrogated induction of mdr1 by 3-MC, indicating that p53 binding to the mdr1 promoter is essential for the induction. Benzo(a)pyrene, a polycyclic aromatic hydrocarbon and AhR ligand, which, like 3-MC, is oxidized by metabolizing enzymes regulated by AhR·Arnt, also activated p53 and induced mdr1 transcription. 2,3,7,8-Tetrachlorodibenzo-p-dioxin, an AhR ligand resistant to metabolic breakdown, had no effect. These results indicate that the transcriptional induction of mdr1 by 3-MC and benzo(a)pyrene is directly mediated by p53 but that the metabolic activation of these compounds into reactive species is necessary to trigger p53 activation. The ability of the anticancer drug and potent genotoxic agent daunorubicin to induce mdr1independently of AhR·Arnt further supports the proposition thatmdr1 is transcriptionally up-regulated by p53 in response to DNA damage.

Multidrug resistance (MDR) 1 is characterized by cross-resistance of the cells to a large number of structurally and functionally unrelated cytotoxic agents used in chemotherapy. In cultured cells, MDR is frequently caused by the overexpression of P-glycoprotein (Pgp), an integral membrane protein belonging 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 (MDR1 and 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, mdr1 is expressed mostly in the adrenal cortex, kidney, and pregnant uterus, mdr2 in the liver at the canalicular face, and mdr3 in 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 mdr3 gene in the maintenance of the blood-brain barrier and drug elimination and for the mdr2 gene in the transport of phospholipids in the bile (5,6). No physiological function has been attributed to the mouse mdr1 gene so far, since knockout mdr1 (Ϫ/Ϫ) mice display no obvious physiological abnormalities (7). However, different experimental evidence indicates that Pgp encoded by mdr1 can 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 MDR1 RNA have been found in human hepatocarcinomas, and overexpression of the mdr1 isoforms has also been observed in rodent liver during cholestasis, during regeneration following partial hepatectomy, during chemically induced hepatocarcinogenesis, 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-drocarbon (PAH) compounds, including 3-methylcholanthrene , and that this increased expression occurs at the transcriptional level (9 -11). However, the precise molecular mechanisms involved in mdr1b regulation in response to 3-MC are still unknown.
PAHs are carcinogenic compounds arising from the incomplete 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 aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are specific inducers of genes coding for drugmetabolizing enzymes (DME), including cyp1a1 and cyp1a2, that code for cytochromes P450 involved 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 translocator (Arnt; another bHLH-PAS protein) to specific cis-acting regulatory DNA sequences located in the promoter of its targets (known as AH-, dioxin-, or xenobiotic-responsive elements (or AHRE, DRE, or XRE, respectively)) to enhance their transcription (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 mdr1 induction in the liver of mice treated with TCDD, one of the most potent agonists of the AhR, suggested that mdr1 expression 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) constitute a powerful experimental system to investigate the transcriptional 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-1c1c7 and were identified based on their inability to induce aryl hydrocarbon hydroxylase activity in response to TCDD treatment (15). TAOc1BP r c1 cells have a decreased level of AhR (ϳ10% of wild-type cells) and therefore decreased induction of the cyp1a1 promoter and lower aryl hydrocarbon hydroxylase activity in response to TCDD and other AhR ligands (15)(16)(17)(18). BP r c1 cells have a normal cytosolic AhR, which fails to accumulate in the nucleus because of a defective Arnt (15). They have virtually no basal or inducible levels of cyp1a1 expression and aryl hydrocarbon hydroxylase activity (15)(16)(17).
In the present report, we have used this panel of cell lines to investigate the transcriptional regulation of the murine mdr1 gene by 3-MC and other xenobiotic compounds. Our results demonstrate that mdr1 is transcriptionally induced by 3-MC and B(a)P and that this induction is mediated by p53 but also requires AhR⅐Arnt. A model for the AhR⅐Arnt-and p53-mediated transactivation of mdr1 in response to genotoxic stress is proposed.

EXPERIMENTAL PROCEDURES
Cell Culture-Wild-type Hepa-1c1c7 and Hepa 1-6, AhR-defective TAOc1BP r c1, and Arnt-defective BP r c1 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA) and maintained in culture under the conditions recommended by the ATCC. Chinese hamster ovary LR73 cell lines stably transfected with plasmid constructs carrying full-length cDNAs for the mouse mdr1, mdr2, or mdr3 genes (LR73 mdr1, LR73 mdr2, and LR73 mdr3, respectively; a gift from Dr. Philippe Gros, McGill University, Montréal, Canada) were grown as described elsewhere (19,20). For inductions, cells at ϳ50% 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 Québec (Laval, Canada). Stock solutions of 3-MC (5 mM) and B(a)P (25 mM) were prepared in Me 2 SO, and the stock solutions of daunorubicin (1 mg/ml) were prepared in water. TCDD was obtained in n-nonane at a concentration of 50 g/ml and was stored at room temperature. Stock solutions of 3-MC, B(a)P, and daunorubicin were stored at Ϫ80°C.
RNA Preparation-Total RNA was prepared from 3-MC-treated and untreated hepatocytes as well as from the LR73 mdr1, LR73 mdr2, and LR73 mdr3 cell lines by homogenizing the cells in a solution containing guanidium hydrochloride (6 M) followed by sequential ethanol precipitation, as described previously (21).
RNase Protection Assay-The plasmid constructed to detect the mdr1 RNA consisted of a 165-bp BamHI fragment isolated from the mdr1 cDNA (positions 1926 -2090 relative to the ATG initiation codon (22)), blunt-ended with T4 DNA polymerase, and cloned into plasmid pGEM-7Z (Promega, Madison, WI) at the SmaI site, giving plasmid pmdr1-G7. This plasmid was linearized with EcoRI and used as a template to synthesize an antisense mdr1 probe using SP6 RNA polymerase (Amersham Pharmacia Biotech). The pKX10 -3Z plasmid consisting of an XbaI-KpnI mouse ␤-actin cDNA fragment (positions 724 -969 in the ␤-actin cDNA) cloned into pGEM-3Z at the XbaI and KpnI sites (kindly provided by Dr. Rashmi Kothary, Institut du cancer de Montréal, Montréal, Canada) was used to generate a control actin probe. pKX10 -3Z was linearized with XbaI and used to synthesize an antisense actin RNA probe with T7 RNA polymerase. The riboprobes were synthesized in the presence of [␣-32 P]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 were isolated from Hepa-1c1c7 cells treated with Me 2 SO or with 3-MC (5 M) for 48 h and were used to label nascent RNAs with [␣-32 P]UTP. Plasmids pVT101-U/mdr1, carrying the full-length mouse mdr1 cDNA (25); pmP1450 -3Ј, carrying a 1.2-kb PstI cDNA fragment overlapping part of the mouse cyp1a1 cDNA (26) (obtained from the ATCC); and pKX10 -3Z were linearized with StuI, BamHI, and XbaI, respectively. The linearized plasmids were denatured, immobilized in duplicate onto a nylon membrane, and hybridized with the [␣-32 P]UTP-labeled RNAs for 48 h at 65°C. The membranes were washed and exposed for 7 days with two intensifying screens.
Chloramphenicol Acetyl Transferase (CAT) Expression Plasmids-Plasmid pMcat5.9 consists of a 482-bp DNA fragment containing the dioxin-responsive elements of the cyp1a1 gene 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 mdr1 promoter sequence in these constructs ends at position ϩ84 with respect to the transcription start site (27). To produce the p53 mutant constructs, pM1 and pM2, plasmid pSBM13 was used. This plasmid consists of a 1.2-kb SacI-HindIII mdr1 promoter fragment (positions Ϫ1165 to ϩ84) cloned into M13mp18. Single-stranded DNA was prepared from pSBM13 and used as a template to perform site-directed mutagenesis of the p53 binding site, using the mutant oligonucleotides M1 5Ј-TACCTGAATACATAAAGACA and M2 5Ј-CGTAAAGATAAATCTATGTA (the base changes are shown in boldface type). The resulting M1 and M2 mdr1 promoter fragments were then excised from pSBM13 with SacI and HindIII, blunt-ended with T4 DNA polymerase, and cloned into plasmid pCAT at the HindIII site also blunt-ended with T4 DNA 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 a density of 8 ϫ 10 5 /60-mm plate and transfected on the following day with 10 g of plasmid DNA, using a standard calcium phosphate precipitation method (28). After incubation with the DNA precipitate for 16 h, the cells were washed twice with phosphate-buffered saline and supplied with fresh medium containing the different xenobiotics. After 48 h, 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 2 g 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, 0.6 mM EDTA and collected by centrifugation. The cell pellets were resuspended in 400 l of ice-cold buffer A (10 mM Tris, pH 8.0, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol) containing 0.5 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 1 g/ml pepstatin, and 5 g/ml leupeptin and swelled on ice for 15 min. Subsequently, 25 l of 10% Nonidet P-40 were added, and the tubes were vortexed vigorously. The nuclear pellets were collected by centrifugation and resuspended in 100 l of cold buffer C (20 mM Tris, pH 8.0, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol) in the presence of protease inhibitors. The suspensions were shaken vigorously at 4°C for 1 h and centrifuged for 15 min at 4°C, and the supernatants were frozen in aliquots at Ϫ80°C. Protein concentrations were determined by the Bradford method (29).
Electrophoretic Mobility Shift Assay-Oligonucleotides overlapping the potential p53 binding site in the mdr1 promoter (5Ј-GAACACGTA-AAGACAAGTCTAT) and the p53 consensus sequence in the p21 waf1/cip1 promoter (5Ј-GAACATGTCCCAACATGTTGAG) (31) were end-labeled with ␥-32 P using T4 polynucleotide kinase and annealed to their respective complementary oligonucleotides to generate double-stranded probes. The EMSA was performed with 5 g of nuclear extracts in a binding mixture containing 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 2.5 mM dithiothreitol, 4% Ficoll, 1 g of poly(dI-dC), and 20,000 cpm of radiolabeled probe. The binding reactions were carried out at room temperature for 15 min. Where needed, 1 g of the monoclonal anti-p53 antibody pAb421 (32) (Calbiochem) or of the polyclonal anti-Jun or anti-Skn-1 antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added, and the incubation was continued for an additional 15 min. The complexes were separated on 5% nondenaturing polyacrylamide gels in 1ϫ TBE (90 mM Tris, 65 mM boric acid, 2.5 mM EDTA, pH 8.0) at 200 V. The gels were exposed to XAR films (Eastman Kodak Co.) for 16 h with two intensifying screens at Ϫ80°C.

Transcriptional Induction of the Mouse mdr1 Gene by 3-MC
in Hepatoma Cells-We have used an RNase protection assay to study the expression of mdr1 in the hepatoma cell line Hepa-1c1c7 upon exposure to 3-MC (Fig. 1). An mdr1-specific riboprobe was prepared by cloning into pGEM7-Zf a mouse mdr1 cDNA 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 LR73 stable transfectants expressing each of the three mouse mdr cDNAs, the mdr1 riboprobe was found to recognize the mdr1 RNA but not the mdr2 or mdr3 RNA, thus confirming its specificity (Fig. 1, top right). The mdr1 probe was then used with RNA from Hepa-1c1c7 cells treated or not with 3-MC ( Fig. 1, top left). This experiment showed that the amount of mdr1 RNA detected is very low in untreated cells but is strongly increased in 3-MC-treated cells, demonstrating that expression of the mouse mdr1 gene is induced by 3-MC treatment. 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 induction of mdr1 expression by 3-MC is isoform-specific (data not shown).
A nuclear run-on experiment was performed to determine whether mdr1 induction by 3-MC occurs at the transcriptional level (Fig. 2). In addition to the mouse mdr1 cDNA, cDNAs for the mouse cyp1a1 gene (known to be transcriptionally regulated by 3-MC (12)) and for the actin gene were also included as positive and negative controls, respectively. The data in Fig. 2 show that 3-MC induces an increase in the rate of mdr1 mRNA synthesis, indicating that 3-MC acts at the transcriptional level to induce mdr1 gene expression in Hepa-1c1c7 cells.
AhR⅐Arnt-dependent Induction of mdr1 Expression by 3-MC-To determine whether the increase in mdr1 expression in response to 3-MC exposure is AhR⅐Arnt-mediated, we analyzed the mdr1 RNA levels upon 3-MC treatment in two wildtype hepatoma cell lines Hepa-1c1c7 and Hepa 1-6 and in two variant cell lines derived from Hepa-1c1c7, TAOc1BP r c1 (AhRdefective) and BP r c1 (Arnt-defective) (15) (Fig. 3). As controls, we also analyzed the level of cyp1a1 and actin expression under the same conditions (Fig. 3, middle and right, respectively). This experiment showed that mdr1 is 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 mdr1 is induced 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 mdr1 in response to 3-MC requires an intact AhR⅐Arnt complex, like cyp1a1 (Fig. 3, middle) (12).
The Mouse mdr1 Promoter Confers 3-MC-regulated Expression in an AhR⅐Arnt-dependent Manner-To determine if regulatory sequences responsible for mdr1 induction by 3-MC are present in the promoter region of the gene, plasmid pmdr1, consisting of a 1.2-kb SacI-HindIII DNA fragment overlapping the mdr1 promoter region (positions Ϫ1165 to ϩ84 with respect to the transcription start site (27) virus promoter and to the CAT gene (24), as well as the empty pCAT vector were also included as positive and negative controls, respectively. The three plasmids were transiently transfected into Hepa-1c1c7 and BP r c1 cells. The cells were treated with 3-MC or with Me 2 SO for 48 h, and the cellular extracts were prepared and assayed for CAT activity. This experiment showed that the mdr1 promoter is transcriptionally active in Hepa-1c1c7 cells and BP r c1 cells, since it can drive the expression 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 mdr1 detected by slot blot analysis in these cells (Fig. 3). 3-MC treatment of the Hepa-1c1c7 cells transfected with pmdr1 resulted 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-1c1c7 pMcat5.9 transfectants upon 3-MC treatment. However, this induction was completely abrogated in BP r c1 cells (Fig. 4), consistent with the lack of mdr1 induction at the RNA level observed in the slot blot assay (Fig. 3). Similar results were obtained upon transfection in TAOc1BP r c1 cells (data not shown). These results, showing that the mdr1 promoter is able to activate the expression of the reporter gene in response to 3-MC in Hepa-1c1c7 but not in BP r c1 and TAOc1BP r c1 cells, demonstrate that (i) the mdr1 promoter is able to confer 3-MC-mediated transcriptional activation; (ii) this activation requires a functional AhR⅐Arnt complex; and (iii) the sequences mediating this induction are located between positions Ϫ1165 and ϩ84 in the mdr1 promoter.
Two Putative XREs Located in the mdr1 Promoter Are Dispensable for the Induction of mdr1 by 3-MC-The AhR⅐Arnt 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 xenobiotics and function in a position-and orientation-independent manner (33,34). Examination of the mdr1 promoter sequence indicated the presence of two potential XREs in an inverted orientation in the distal portion of the promoter at positions Ϫ1129 and Ϫ620 (5Ј-CACGCAT and 5Ј-CACGCAA, respectively). To identify the cis-acting sequences responsible for the induction of mdr1 by 3-MC and to investigate the possible involvement of these putative XREs, we analyzed the transcriptional activity of a series of mdr1 promoter 5Ј-deletion CAT constructs after transient transfection into Hepa-1c1c7 and treatment of the resulting transfectants with 3-MC (Fig.  5A). 3-MC treatment of Hepa-1c1c7 cells transfected with plasmids p-452 or p-245 resulted in a level of CAT induction similar to that observed in cells transfected with plasmid pmdr1 carrying the full-length promoter, indicating that sequences located within positions Ϫ1165 to Ϫ245 are dispensable for the induction of mdr1 by 3-MC, including the two putative XREs as well as a potential AP-1 binding site (5Ј-TGACTCA; positions Ϫ265 to Ϫ255 (35)) (Fig. 5, B and C). However, further deletion of a 104-bp region down to position Ϫ141 (pϪ141) 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 Ϫ245 and Ϫ141. CAT activity in the absence of 3-MC was reduced in the pϪ141 transfectants when compared with the pϪ245 transfectants, indicating that sequences between positions Ϫ245 and Ϫ141 are also involved in the basal transcriptional activity of the mdr1 promoter in hepatoma cells. Finally, we found that a low FIG. 2. Nuclear run-on experiment. Nuclei were isolated from Hepa-1c1c7 cells treated with 5 M 3-MC (ϩMC) or with Me 2 SO (ϪMC) for 48 h. Nascent RNAs were radiolabeled with [␣-32 P]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 7 days with two intensifying screens. level of induction was still detectable in the pϪ141 but not in the pϪ93 transfectants, suggesting that sequences located between positions Ϫ141 and Ϫ93 may be required for maximal induction of mdr1. However, this residual induction was not seen in Hepa 1-6 cells in which deletion of sequences down to position Ϫ141 completely abolished the induction of CAT activity by 3-MC (data not shown). Taken together, these results indicate that (i) the two potential XREs present in the mdr1 promoter are not required for its 3-MC-regulated expression, thus suggesting an indirect role for the AhR⅐Arnt complex in the induction process, and that (ii) the region from Ϫ245 to Ϫ141 contains cis-acting sequences mediating the 3-MC-inducible mdr1 expression in hepatoma cells.
AhR⅐Arnt-dependent Induction of a p53⅐mdr1 Promoter Complex by 3-MC-Sequence comparison of the mouse mdr1 promoter with that of the rat mdr1b promoter revealed the presence of a potential p53 binding site in the DNA region from Ϫ245 to Ϫ141 of the mouse promoter (5Ј-GAACAcGTaaAGA-CAAGTCT; positions Ϫ197 to Ϫ178). The p53 binding site in the rat mdr1b promoter has been shown to mediate the induction of mdr1b by the anticancer drug daunorubicin (DN) (36). The p53 binding site in the mouse promoter contains three mismatched nucleotides (lowercase letters) when compared with the p53 consensus sequence, which consists of two copies of the 10-bp motif 5Ј-RRRC(A/T)(T/A)GYYY (37).
To assess p53 binding to the mdr1 promoter, we performed an electrophoretic mobility shift assay (EMSA), using a 32 Plabeled double-stranded oligonucleotide corresponding to the p53 binding site of the mdr1 promoter and nuclear extracts prepared from Hepa-1c1c7 and BP r c1 cells treated or not with 3-MC. Nuclear extracts from both Hepa-1c1c7 and BP r c1 cells gave rise to a complex (C1) (Fig. 6A, lanes 1, 3, and 4) that disappears only in Hepa-1c1c7 cells upon 3-MC treatment (Fig.  6A, lane 2). This result suggests that 3-MC treatment abolishes the binding of an unknown protein to the mdr1 promoter and that this effect requires the presence of an intact AhR⅐Arnt complex. It has been shown that the monoclonal antibody pAb421, which binds within the C-terminal domain of p53, activates the DNA binding activity of the protein (38). The addition of pAb421 to the binding reactions gave rise to the formation of a p53⅐pAb421⅐mdr1 complex (C2), which was detected only in Hepa-1c1c7 cells treated with 3-MC (Fig. 6A, lane  6). This complex was not formed when antibodies against the transcription factors c-Jun and Skn-1 were used as a control, confirming that C2 is specific for pAb421 and thus for p53 (Fig.  6B, lanes 6 and 8). Taken together, these results demonstrate that 3-MC treatment of Hepa-1c1c7 cells induces binding of p53 to the mdr1 promoter and that this induction requires AhR⅐Arnt.
An EMSA was also performed using the same nuclear extracts but this time with a probe overlapping the high affinity p53 binding sites in the p21 waf1/cip1 promoter (5Ј-GAACAT-GTCCcAACATGTTg) (31). As observed with the mdr1 probe, nuclear extracts from Hepa-1c1c7 and BP r c1 cells gave rise to the C1 complex, which was absent in Hepa-1c1c7 cells treated with 3-MC (Fig. 6A, lanes 9 -12), indicating that the protein factor present within C1 and which binds to the mdr1 promoter also binds to the p21 waf1/cip1 promoter. The addition of pAb421 to the binding reactions gave rise to the formation of a p53⅐pAb421⅐p21 C2 complex of low abundance in untreated Hepa-1c1c7 and BP r c1 nuclear extracts, indicating that these cells express basal levels of endogenous p53 (Fig. 6A, lanes 13  and 15). Moreover, the intensity of that complex was strongly increased in Hepa-1c1c7 cells but not in BP r c1 cells upon 3-MC treatment (Fig. 6A, compare lanes 14 and 16). The p53⅐pAb421 complex detected with the p21 probe was more abundant than that detected with the mdr1 probe (Fig. 6A, compare lanes 14  and 6), suggesting that the p53 binding site of the p21 waf1/cip1 promoter has a higher affinity for p53 than the one present in the mdr1 promoter.
Increased p53 Levels in Hepa-1c1c7 Cells upon 3-MC Treatment-It has been shown that p53 levels are increased in response to a variety of stresses, including DNA damage (39,40). To determine whether the exposure of hepatoma cells to 3-MC results in increased p53 levels, we analyzed by Western blotting total protein extracts prepared from Hepa-1c1c7 and BP r c1 cells treated or not with 3-MC, using antibody pAb421 (Fig. 7). This experiment showed that p53 expression is undetectable in total protein extracts from untreated Hepa-1c1c7 and BP r c1 cells (Fig. 7A, lanes 1, 3, and 5). However, its expression was detected in Hepa-1c1c7 cells after 12 h of exposure to 3-MC and further increased after 48 h of treatment (Fig.  7A, lanes 2 and 4). Increased p53 levels upon 3-MC treatment were not observed in BP r c1 cells (Fig. 7A, lane 5), demonstrating that an intact AhR⅐Arnt complex is required for p53 induction in response to 3-MC. Western blot analysis of the nuclear extracts used in the EMSA showed that low levels of p53 are present in the nuclei of the two untreated cell lines (Fig. 7B,  lanes 1 and 3) and that p53 expression is increased upon 3-MC treatment in Hepa-1c1c7 but not in BP r c1 cells (Fig. 7B, lanes  2 and 4, respectively). Taken together, our results indicate that the appearance of the p53⅐pAb421⅐mdr1 C2 complex detected in the EMSA (Fig. 6) correlates with an AhR⅐Arnt-dependent change in p53 levels in the cells.
The p53 Binding Site in the mdr1 Promoter Is Required for the Induction of mdr1 Transcription by 3-MC-To determine whether p53 is responsible for conferring 3-MC-inducible expression to mdr1, we abrogated p53 binding to the mdr1 promoter by introducing point mutations in the p53 binding site within the context of the Ϫ1165 mdr1 promoter CAT construct pmdr1. The C and G nucleotides of each half-site, known to be essential for binding of p53 to its DNA consensus sequence (37) were mutated to T and A, respectively, generating the two p53 mutant mdr1 promoter constructs pM1 and pM2 (Fig. 8A). These two constructs were transiently transfected into Hepa-1c1c7 cells along with the appropriate controls, and the trans-fectants were treated with 3-MC or with Me 2 SO for 48 h. The cellular extracts were prepared and assayed for CAT activity. This experiment showed that both the basal and inducible levels of CAT activity conferred by the wild-type mdr1 pro-FIG. 6. Binding of p53 to the mdr1 promoter. A, an EMSA was performed with nuclear extracts prepared from Hepa-1c1c7 (Hepa) and BP r c1 (BP r ) cells treated (ϩ) or not treated (Ϫ) with 3-MC (5 M) for 48 h. The nuclear extracts were assayed for binding to 32 P-labeled double-stranded oligonucleotides overlapping the p53 binding site in the mdr1 (lanes [1][2][3][4][5][6][7][8] or in the p21 waf1/cip1 (31) (lanes 9 -16) promoters. The binding reactions were carried out in the presence (lanes 5-8 and 13-16) or in the absence (lanes 1-4 and 9 -12) of the anti-p53 mAb pAb421. The complexes were separated on a native polyacrylamide gel. The gel was dried and exposed for 16 h with two intensifying screens. The C1 and C2 complexes are indicated on the left. The position of the free probe is also indicated (bracket). B, nuclear extracts from Hepa-1c1c7 cells treated (ϩ) or not treated (Ϫ) with 3-MC (5 M) for 48 h were assayed for binding to the mdr1-labeled probe in the absence (lanes 1 and 2) or in the presence of the ␣-p53 antibody pAb421 (lanes 3 and 4), of an ␣-Jun (lanes 5 and 6) or an ␣-Skn-1 (lanes 7 and 8) antibody.

FIG. 7. 3-MC Increases p53 levels in an AhR⅐Arnt-dependent manner.
A, total protein extracts were prepared from Hepa-1c1c7 (Hepa) and BP r c1 (BP r ) cells treated with 5 M 3-MC (ϩ) or with Me 2 SO (Ϫ). Cells were treated for 12 or 48 h (Hepa-1c1c7) or for 48 h (BP r c1). Protein extracts (75 g) were separated by SDS-polyacrylamide gel electrophoresis on a 10% acrylamide gel and transferred to a nitrocellulose membrane. The membrane was analyzed with the anti-p53 monoclonal antibody pAb421. The molecular mass standards (kDa) are shown on the left. B, nuclear extracts were prepared from Hepa-1c1c7 (Hepa) and BP r c1 (BP r ) cells treated with 5 M 3-MC (ϩ) or with Me 2 SO (Ϫ) for 48 h. Nuclear extracts (lanes 1, 3 and 4, 32 g; lane 2, 16 g) were analyzed as described for A. The position of p53 is indicated on the right of each membrane. The star at the right of B shows a nuclear protein of ϳ75 kDa detected by the pAb421 antibody. moter were completely abrogated (pM2) or strongly reduced (pM1) by mutations in the p53 binding site, resulting in levels of CAT activity similar to those conferred by the minimal mdr1 promoter in the p-93 transfectants (Fig. 8, B and C). Taken together, our results clearly demonstrate that the p53 binding site in the mdr1 promoter is essential for the basal and 3-MCinducible expression of mdr1 in hepatoma cells.
p53-mediated Induction of the mdr1 Promoter by Xenobiotic Compounds-Several metabolites of 3-MC possess high mutagenic activity (41,42) and thus could induce p53 through their DNA-damaging activity. To test this hypothesis, we have investigated the effect of three additional xenobiotic compounds with different genotoxic activities on p53 activation and mdr1 transcriptional induction: (i) B(a)P, a PAH and AhR ligand that, like 3-MC, is not in itself genotoxic but that is oxidized to reactive species by AhR⅐Arnt-regulated DME (12); (ii) TCDD, another AhR ligand that is nongenotoxic and resistant to metabolic breakdown (12); and (iii) DN, an anticancer drug and potent genotoxic agent that was recently shown to induce rat mdr1b expression via p53 (36).
Hepa-1c1c7 and BP r c1 cells were exposed to these three compounds as well as to 3-MC, and the level of p53 was determined by Western blotting (Fig. 9A). Treatment of Hepa-1c1c7 cells with B(a)P and DN resulted in increased p53 levels, similar to those reached upon 3-MC treatment, whereas TCDD had no effect on p53 expression (Fig. 9A, left panel). As for 3-MC, the increased levels of p53 in response to B(a)P required AhR⅐Arnt, since there was no induction of p53 by this compound in BP r c1 cells (Fig. 9A, right). Unlike 3-MC and B(a)P, however, DN was able to activate p53 and mdr1 in both cell lines. We then used a CAT assay to determine the effect of these compounds on the transcriptional activity of the mdr1 promoter (Fig. 9B). We found that in Hepa-1c1c7 cells, B(a)P and DN were as efficient as 3-MC in inducing mdr1 transcription (Fig. 9B, left). TCDD, however, had no effect on mdr1 transcription, although the cyp1a1 promoter was induced under the same conditions (data not shown). The absence of mdr1 induction by TCDD is in agreement with the results obtained by Teeter et al., which were unable to detect mdr1 induction in the liver of mice exposed to this compound (14). The induction of mdr1 by 3-MC and B(a)P was lost in BP r c1 cells, whereas DN was efficient at inducing mdr1 transcription in the two cell lines (Fig. 9B). These results, showing that the levels of p53 correlate with the induction of the mdr1 promoter, further confirm that the induction of mdr1 is mediated by p53. Indeed, there was no induction with any of the compounds tested when the pM2 mdr1 promoter construct carrying a mutation in the p53 binding site was used for the transfection (data not shown). Taken together, these data suggest that the activation of p53 and the subsequent induction of mdr1 are caused by a genotoxic stress resulting from the metabolism of 3-MC and B(a)P by AhR⅐Arnt-regulated DME. The ability of intrinsically genotoxic DN to induce p53 and mdr1 independently of the AhR⅐Arnt complex together with the inability of nongenotoxic TCDD to induce p53 and mdr1 further support this proposition.

DISCUSSION
In the present report, we have used a panel of wild-type, and AhR-and Arnt-defective hepatoma cell lines to investigate the transcriptional regulation of the murine mdr1 gene by 3-MC and other xenobiotic compounds. We show that mdr1 is induced at the transcriptional level by exposure of the cells to 3-MC and that this induction depends upon a functional AhR⅐Arnt complex (Figs. 3 and 4). These characteristics are shared by the two phase I genes cyp1a1 and cyp1a2 as well as by the four phase II genes nmo1 (NAD(P)H:menadione oxidoreductase), ahd4 (aldehyde dehydrogenase), ugt1a6 (UDPglucuronosyltransferase), and gsta1 Ya (glutathione transferase) (43). Together, these genes form the so-called dioxininducible mouse Ah battery, which plays an important role in environmental toxicity, cancer, and oxidative stress (43). These findings are compatible with the proposition that mdr1 could belong to the Ah battery in the mouse. However, different observations indicate that this is not the case. First, TCDD, a prototypical AhR ligand, does not induce mdr1 transcription (Fig. 9). Second, the kinetics of induction of cyp1a1 and mdr1 by 3-MC are different; while induction of cyp1a1 is readily detectable after 3 h of treatment, induction of mdr1 is only observed after 12 h of cell exposure to 3-MC (data not shown), suggesting that the two genes are induced by different mechanisms. Finally, our analysis of the mdr1 promoter deletions demonstrates that two potential XREs that were identified by sequence analysis in the mdr1 promoter are not required for the induction of mdr1 by 3-MC (Fig. 5), consistent with an indirect role for the AhR⅐Arnt complex in the induction process. It is likely that 3-MC, upon binding to AhR, induces DME regulated by AhR⅐Arnt, leading to its own metabolism, and that one or more resulting metabolites are responsible for the subsequent induction of mdr1 via an AhR⅐Arnt-independent pathway.
Our results indicate that this AhR⅐Arnt-independent pathway involves p53. Activation of p53 occurs in response to a number of cellular stresses, including DNA damage, and leads to the activation of several genes whose product trigger cell cycle arrest, apoptosis, or DNA repair (44). Several PAH metabolites possess high mutagenic activity that can induce p53 through their DNA-damaging activity (41,42). B(a)P treatment of murine 3T3 cells has been shown to result in DNA damage associated with elevated levels of nuclear p53 (45). Likewise, treatment of normal human fibroblasts with B(a)P-7,8-diol-9,10-epoxide, a highly reactive metabolite of B(a)P, results in elevated p53 levels (46). Moreover, B(a)P has been shown to activate the human p53 promoter through induction of the transcriptional activator NF-B (47). The induction of p53 upon B(a)P bears biological significance, since p53 has been shown to be required for the efficient repair of B(a)P-7,8-diol-9,10-epoxide adducts in human cells and to play a protective role against the teratogenic effects of B(a)P in mice (48 -50). Our data showing that p53 activation by 3-MC and B(a)P requires an intact AhR⅐Arnt complex demonstrates a pivotal role for AhR in the activation of p53 by PAHs. It also suggests that, in addition to its own direct targets, AhR indirectly regulates different p53 transcriptional targets in response to PAHs. The recent demonstration that c-Ha-ras activation by B(a)P requires an intact AhR further substantiates the widespread consequences of AhR activation upon cell exposure to PAHs (51).
Based on our present results, we can propose the following model for the AhR⅐Arnt-and p53-mediated transactivation of mdr1 in response to genotoxic stress (Fig. 10). Treatment of a cell with the AhR ligands TCDD, 3-MC, and B(a)P activates transcription of the genes coding for DME regulated by the AhR⅐Arnt complex. Upon metabolic activation, genotoxic metabolites of 3-MC and B(a)P are generated (3-MC* and B(a)P*) that can cause DNA damage, such as 1-sulfooxy-3-MC and B(a)P-7,8-diol-9,10-epoxide, that can form DNA adducts and thus act as mutagens (42,52). TCDD, the prototypical AhR ligand, is resistant to metabolic breakdown and is nongenotoxic. Experiments in AhR knockout mice have shown that TCDD-induced toxicity is mediated in most part by AhR, but the precise mechanism(s) by which this toxicity is exerted are still unclear (53). On the other hand, DN has intrinsic genotoxic activity and can induce DNA damage independently of the AhR⅐Arnt pathway. Upon DNA damage, p53 is activated and stabilized, resulting in the transcriptional induction of mdr1 (Fig. 10).
The combined results from the EMSA and Western blots yield interesting information about the mechanisms regulating mdr1 transcription. On the one hand, the EMSA shows that under normal conditions, an unknown protein within the C1 complex is bound to the p53 binding site in the mdr1 promoter but that this complex dissociates upon 3-MC treatment (Fig.  6A). This dissociation depends upon AhR⅐Arnt, since it occurs in Hepa-1c1c7 cells and not in BP r c1 cells, suggesting that the metabolic activation of 3-MC is required for the dissociation.
Similar results were obtained with the p21 waf1/cip1 probe, showing that the formation and dissociation of a C1 complex is not restricted to mdr1 but also extends to another p53 target. On the other hand, we observed in our Western blots that pAb421 detects, in addition to p53, a nuclear protein of ϳ75 kDa (Figs. 7 and 9). It is possible that this protein corresponds to p73, a recently discovered homologue of p53 (54). Indeed, it has been shown that pAb421 recognizes p73 in immunoprecipitation experiments (55). Interestingly, the intensity of the 75-kDa band was found to decrease upon treatment of the cells with 3-MC ( Fig. 7 and 9) in a manner that correlates with the decrease in the C1 complex upon 3-MC treatment. This observation, together with the ability of p73 to bind canonical p53 DNA binding sequences (54), suggests a link between p73 and the protein present within the C1 complex. However, the inability of pAb421 to supershift the C1 complex indicates that this is not the case. Also, p73 has been shown to be up-regulated upon DNA damage, not down-regulated (56,57). Studies aimed at identifying the C1 and p75 protein are currently under way.
The transcription factor NF-B mediates the cellular response to various stimuli, including oxidative stress, UV irradiation, DNA damage, and viral infections (58). NF-B binds to DNA sequence elements in the promoter of its target genes to activate their transcription (5Ј-GGGN 2-4 CC). It has been recently reported that the expression of the rat mdr1b gene is induced by the hepatocarcinogen 2-acetylaminofluorene through the generation of reactive oxygen species that activate NF-B signaling and binding to the mdr1b promoter (59). The metabolism of B(a)P also generates reactive oxygen species, and it has been shown that B(a)P activates transcription of the human p53 and cyclooxygenase-2 genes by inducing the transcriptional activity of NF-B (47,60). It is therefore likely that the NF-B pathway is also induced in hepatoma cells upon B(a)P exposure, in addition to the AhR and p53 pathways. Sequence analysis indicates the presence of a putative NF-B binding site in the mouse mdr1 promoter (5Ј-GGGAACC; positions Ϫ163 to Ϫ157). Interestingly, this site is adjacent to the p53 binding site and is located within the region conferring B(a)P induction of the mouse mdr1 promoter. It was recently shown that NF-B and p53 cooperatively bind to the p53 promoter to activate its transcription (61), suggesting that these two sites in the mdr1 promoter could also cooperate to regulate mdr1. However, it has been reported that p53 and NF-B also have antagonizing effects on transcription, most likely by competing for the transcriptional coactivator p300/CBP (62). Experiments are under way to assess a potential role for NF-B in the regulation of mdr1 in response to PAHs.
What is the functional significance of mdr1 up-regulation in response to genotoxic stress? Western blot analysis of Pgp in Hepa-1c1c7 treated with 3-MC showed that increased mdr1 transcription results in increased levels of Pgp in these cells (data not shown), indicating that Pgp can mediate some biological effects of p53 activation. A characteristic feature of Pgp is its ability to interact with a wide variety of structurally and mechanistically diverse compounds (8). It is therefore possible that mdr1 is activated by p53 as part of a global detoxification mechanism. In such a case, Pgp would function to export xenobiotic compounds from the cells, thus preventing their accumulation and eventual cellular damage. However, the demonstration that Pgp does not affect intracellular accumulation of B(a)P indicates that this is not the case, at least for this particular compound (63). Alternatively, it is possible that the biological effects carried out by Pgp upon genotoxic stress may be related to a yet unidentified function of the protein. Thus, the physiological consequences of p53-mediated Pgp induction in response to DNA damage remains to be determined.