The aryl hydrocarbon receptor constitutively represses c-myc ... - Nature

Oncogene (2005) 24, 7869–7881

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The aryl hydrocarbon receptor constitutively represses c-myc transcription in human mammary tumor cells Xinhai Yang1, Donghui Liu1, Tessa J Murray1, Geoffrey C Mitchell2, Eli V Hesterman2, Sibel I Karchner3, Rebeka R Merson3, Mark E Hahn3 and David H Sherr*,1 1 Department of Environmental Health, Boston University School of Public Health, Boston, MA, USA; 2Biology Department, Furman University, Greenville, SC, USA; 3Biology Department, Woods Hole Oceanographic Institute, Woods Hole, MA, USA

The aryl hydrocarbon receptor (AhR) is an environmental carcinogen-activated transcription factor associated with tumorigenesis. High levels of apparently active AhR characterize a variety of tumors, even in the absence of environmental ligands. Despite this association between transformation and AhR upregulation, little is known of the transcriptional consequences of constitutive AhR activation. Here, the effects of constitutively active and environmental ligand-induced AhR on c-myc, an oncogene whose promoter contains six AhR-binding sites (AhREs (aryl hydrocarbon response elements)), were investigated. A reporter containing the human c-myc promoter, with its six AhREs and two NF-jB-binding sites, was constructed. This vector, and variants with deletions in the NF-jB and/ or AhR-binding sites, was transfected into a human breast cancer cell line, Hs578T, which expresses high levels of apparently active, nuclear AhR. Results indicate that: (1) the AhR constitutively binds the c-myc promoter; (2) there is a low but significant baseline level of c-myc promoter activity, which is not regulated by NF-jB and is not affected by an environmental AhR ligand; (3) deletion of any one of the AhREs has no effect on constitutive reporter activity, while deletion of all six increases reporter activity approximately fivefold; (4) a similar increase in reporter activity occurs when constitutively active AhR is suppressed by transfection with an AhR repressor plasmid (AhRR); (5) AhRR transfection significantly increases background levels of endogenous c-myc mRNA and c-Myc protein. These results suggest that the AhR influences the expression of c-Myc, a protein critical to malignant transformation. Oncogene (2005) 24, 7869–7881. doi:10.1038/sj.onc.1208938; published online 1 August 2005 Keywords: aryl hydrocarbon receptor; c-Myc; mammary tumorigenesis

*Correspondence: DH Sherr, Department of Environmental Health, Boston University School of Public Health, 715 Albany St. (R-408), Boston, MA 02118, USA; E-mail: [email protected] Received 16 November 2004; revised 9 June 2005; accepted 10 June 2005; published online 1 August 2005

Introduction Members of the Per/aryl hydrocarbon nuclear transporter (ARNT)/Sim (PAS) family of transcription factors influence a variety of biologic activities including neurological development, hypoxia responses, angiogenesis, and circadian cycle regulation (Maltepe et al., 1997; Chan et al., 1999; Gu et al., 2000; Liu et al., 2003). One member of this family, the aryl hydrocarbon receptor (AhR), is particularly interesting in that it is activated by a variety of carcinogenic pollutants such as environmentally ubiquitous polycyclic aromatic hydrocarbons (PAH), dioxins (e.g. 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD)), and polychlorinated biphenyls (Poland et al., 1990; Dolwick et al., 1993; Chen and Perdew, 1994). In resting normal cells, the unliganded AhR exists primarily in a cytosolic complex consisting of the AhR, hsp90, p23, and an immunophilin-like molecule, ARA-9/ XAP2 (Carver et al., 1998; Meyer and Perdew, 1999). These and other factors regulate the cellular localization of the AhR and its transcriptional activity (Meyer and Perdew, 1999). Once bound by generally lipophilic pollutants, the AhR translocates to the nucleus where it binds to a second member of the PAS family, the ARNT, and to transcriptional coactivators or corepressors (Hankinson, 1995; Kumar et al., 1999; Nguyen et al., 1999; Beischlag et al., 2002; Rushing and Denison, 2002). The activated AhR complex then binds promoter regions of target genes at specific DNA-binding sites (aryl hydrocarbon response elements (AhREs)), thereby modulating gene transcription (Okey et al., 1994; Hankinson, 1995; Porter et al., 2001). A common outcome of AhR activation is the induction of genes such as those encoding the cytochrome P-4501A1, 1A2, and 1B1 monooxygenases (Poland et al., 1974; Whitlock, 1990; Carrier et al., 1992; Larsen et al., 1997). AhR-dependent upregulation of these enzymes results in increased metabolism of some AhR ligands such as PAH and the production of carcinogenic intermediates (Buters et al., 1999; Dertinger et al., 2001). In other cases, activation of the AhR results in transcriptional inhibition of genes such as those encoding the immunoglobulin heavy-chain (Sulentic et al., 2000) and estrogen-inducible p27, cathepsin D, and pS2 (Safe et al., 1998; Porter et al., 2001).

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While the AhR is most frequently studied in the context of environmental carcinogen exposure, a growing number of studies describe AhR upregulation in some rapidly growing normal cells and in tumors in the absence of exogenous ligands (Corton, 1996; Ma and Whitlock, 1996; Chang and Puga, 1998; Wang et al., 1999; Levine-Fridman et al., 2004). For example, AhR expression is upregulated in mitogen-activated fibroblasts (Vaziri et al., 1996), in mitogen-activated lymphocytes (Marcus et al., 1998), and in several rodent and human tumor types, including leukemias and mammary tumors (Trombino et al., 2000; Abdelrahim et al., 2003; Hayashibara et al., 2003). These results beg the question of what function the AhR is performing in these cells. This question is all the more significant in light of several studies associating AhR activation with cell growth (Ma and Whitlock, 1996; Weiss et al., 1996; Ge and Elferink, 1998; Puga et al., 2000, 2002; Abdelrahim et al., 2003; Ohtake et al., 2003; Thomsen et al., 2004) or apoptosis regulation (Yamaguchi et al., 1997; Matikainen et al., 2001, 2002; Ryu et al., 2003; Caruso et al., 2004). Indeed, the demonstrable influence of the AhR on at least tumor cell growth has motivated the targeting of this receptor/ transcription factor or its gene targets for cancer therapy (Safe, 2001; Koliopanos et al., 2002; Abdelrahim et al., 2003; Maecker et al., 2003; Zhang et al., 2003). Several laboratories have considered the ability of the AhR to modulate growth through interaction with other transcription factors including NF-kB (Tian et al., 1999; Kim et al., 2000a), Rb (Ge and Elferink, 1998; Puga et al., 2000), and estrogen receptors a and b (Ohtake et al., 2003; Wormke et al., 2003). Here, we evaluated the potential for the AhR to affect directly transcription of c-myc, an important oncogene that can regulate cell growth (Pelengaris and Khan, 2003) and apoptosis (Wu et al., 1996; Thompson, 1998; Ryu et al., 2003) and which is dysregulated in many tumor types including breast cancers (Escot et al., 1986; Leder et al., 1986; Borg et al., 1992; Pelengaris and Khan, 2003). AhRmediated regulation of c-myc seemed plausible given the presence of six AREs within a 3.2 kb region of the human c-myc promoter. To evaluate the role of the AhR in regulating baseline levels of c-myc in human tumors, a luciferase reporter construct driven by the human c-myc promoter was employed. The wild-type reporter gene, reporter variants mutated in the AhR and/or NF-kBbinding sites, and/or a plasmid encoding a potent AhR repressor (AhRR) were then transfected into a malignant human mammary tumor line, Hs578T, expressing high AhR levels, to determine the effect of a putatively active AhR on c-myc mRNA and c-Myc protein levels.

Results Constitutive nuclear AhR localization and binding to the c-myc promoter in Hs578T mammary tumor cells but not in a syngeneic normal mammary cell line A number of studies demonstrate AhR expression, usually at notably high levels, in rodent and human Oncogene

tumors (Ma and Whitlock, 1996; Weiss et al., 1996; Ge and Elferink, 1998; Puga et al., 2000, 2002; Trombino et al., 2000; Safe, 2001; Koliopanos et al., 2002; Abdelrahim et al., 2003; Hayashibara et al., 2003; Zhang et al., 2003; Thomsen et al., 2004). Furthermore, we have shown in a primary mammary tumor model that much of that AhR resides in the tumor cell nucleus, a result suggestive of constitutive AhR activation in the absence of exogenous ligands (Wang et al., 1995; Trombino et al., 2000). To develop a model system in which the effects of constitutively active human AhR on gene transcription in mammary tumors can be evaluated, AhR expression, localization, and/or function were evaluated in the malignant human mammary tumor cell line, Hs578T, and in a syngeneic normal myoepithelial cell line, Hs578Bst. Nontransformed Hs578Bst cells expressed low but significant levels of AhR protein, while malignant Hs578T cells expressed significantly higher levels of AhR (Figure 1a). Furthermore, approximately one-third of the AhR in Hs578T cells was located in the nucleus (Figure 1b), a result consistent with constitutive AhR activation. No nuclear AhR was detected in normal Hs578Bst cells, even when loading 120 mg nuclear protein extract/well and after long exposures (>5 s) of the film to the Western blot (Figure 1c). In considering possible gene targets for what appears to be active AhR in tumor cells, we noted the presence of six consensus AhREs within a 3.2 kb region of the human c-myc promoter (Figure 2). If the AhR affects c-myc gene transcription in the absence of exogenous ligands, then it would be predicted that the AhR would constitutively bind this promoter region. Chromosome immunoprecipitation (ChIP) experiments were performed to test this hypothesis. As seen in Figure 3a, a relatively strong c-myc-specific PCR signal was seen following immunoprecipitation with AhR-specific antibody and DNA amplification with c-myc-specific primers. Indeed, immunoprecipitation of the AhR complex resulted in a significant increase in the c-myc signal relative to the input control (Figure 3b, Po0.02). To determine if the constitutively active AhR binds to all AhRE-containing promoters uniformly, ChIP assays were performed with Hs578T cells using both CYP1A1and c-myc-specific primers. Interestingly, AhR from Hs578T cells did not bind to the endogenous CYP1A1 promoter region amplified by the primers to any appreciable extent unless the cells were pretreated with TCDD, a strong AhR activator (Figure 3c, left histograms). As in previous experiments (Figure 3b), the AhR constitutively bound to the c-myc promoter (Figure 3c, fifth histogram). However, no further increase in c-myc promoter binding was observed following TCDD treatment. These data suggest that the constitutively active AhR in this mammary tumor line preferentially binds to the endogenous c-myc promoter. AhRE-dependent regulation of the c-myc promoter The AhR can both induce (Matikainen et al., 2001, 2002; Nebert et al., 1991) and suppress gene transcription


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-Lamin A/C Hs578Bst Figure 1 Hs578T human mammary tumor cells express high levels of cytoplasmic and nuclear AhR. (a) Total AhR protein was extracted from nonmalignant Hs578Bst and from malignant Hs578T cells and 40 mg of extracts analysed by Western blotting. Blots were stripped and reprobed for a-tubulin to confirm equal loading of wells. Representative data from a total of three experiments are presented. (b) Cytoplasmic and nuclear cell extracts prepared from subconfluent monolayers of malignant Hs578T cells were analysed by Western immunoblotting with AhR-specific antibody following SDS–PAGE. Blots were stripped and reprobed for lamin A/C and a-tubulin to confirm purity of the nuclear and cytoplasmic cell fractions, respectively. Representative data from a total of three experiments are shown. (c) Cytoplasmic and nuclear cell extracts (120 mg) prepared from subconfluent monolayers of Hs578Bst cells were analysed by Western immunoblotting with AhR-specific antibody following SDS–PAGE. Blots were stripped and reprobed for lamin A/C and a-tubulin to confirm purity of the nuclear and cytoplasmic cell fractions, respectively. Extended exposure times (3–5 s) were used to detect extremely low AhR levels in Hs578Bst cells. Representative data from a total of three experiments are shown

(Sulentic et al., 1998; Wang et al., 1999, 2001). Therefore, a human c-myc promoter–reporter construct (pGL3-c-myc) was used to determine if constitutive AhR binding to the c-myc promoter effects an increase or decrease of c-myc levels. Since the 3.2 kb c-myc promoter region encompassing the six AhREs also contains two NF-kB-binding sites, and since NF-kB is well known for its ability to regulate c-myc (Duyao et al., 1990; Kessler et al., 1992), it also was important to evaluate the possible contribution of NF-kB to background levels of c-myc transactivation. To address these issues, pGL3-basic, a control plasmid containing a minimal promoter sequence, wild-type pGL3-c-myc, and a series of reporter constructs mutated in the NFkB- and/or the AhR-binding sites were transfected into Hs578T cells and reporter activity assayed 24 h later. Transfection of wild-type pGL3-c-myc into Hs578T cells resulted in approximately a 50% increase in the

background level of c-myc promoter activity as compared with cells transfected with the parental pGL3basic reporter plasmid (Figure 4a, first and second bars, Po0.001). Since mammary tumors, including Hs578T cells, frequently have elevated levels of transcriptionally active NF-kB (Kim et al., 2000a, b; Cao and Karin, 2003; Helbig et al., 2003), it was anticipated that deletion of both NF-kB-binding sites would result in a decrease in constitutive c-myc reporter activity. However, deletion of both NF-kB-binding sites (pGL3-NFkBKO) had no effect on reporter transactivation (Figure 4, third bar). Mutation of individual AhREs (pGL3-AhRE1Mut, pGL3-AhRE2Mut, pGL3-AhRE5Mut, pGL3-AhRE6Mut) or a pair of adjacent AhREs (pGL3-AhRE3,4Mut) following deletion of the two NF-kB-binding sites had no effect on constitutive c-myc reporter activity (Figure 4, bars 5–9). However, deletion of all six AhREs Oncogene


7872 -1155 NF-kB binding site 1 -1120 -919 AhRE1 AACGGTTTTTTTCACAAGGGTCTCTGCTGACTCCCC--//--TCCGTGTGGGAGGCGTG -893 -522 AhRE2 -495 -409 GGGGTGGGAC--//-- CACTAACATCCCACGCTCTGAACGCGCG--//--CAGCCTGGTACGC AhRE3 AhRE4 -372 -96 AhRE5 GCGTGGCGTGGCGGTGGGCGCGCAG--//--TCCCAAAGCAGAGGGCGTGGGGGAAAA -62 -10 +1 (P1) +16 +448 NF-kB binding site 2 GAAAAAAG--//--GACGGCTGAGGACCCCCGAGCTGTGCT--//--CTGCCCATTTGGGGACAC +476 +816 AhRE6 +841 TTCCCCGCCGC--//--ATGAATATATTCACGCTGACTCCCGG Figure 2 The human c-myc promoter contains six aryl hydrocarbon response elements (AhREs) and two NF-kB-binding sites. The locations of six consensus AhR binding motifs and two NF-kB-binding sites are indicated

(pGL3-AhR16Mut) resulted in a significant, 4–5-fold increase in reporter activity (fourth bar, Po0.001). These data suggest that constitutively active AhR represses baseline levels of c-myc transcription and that not all of the AhREs are required for AhR-mediated repression. Effect of a potent environmental AhR ligand, TCDD, on AhR-mediated repression of c-myc transactivation A number of AhR ligands are carcinogenic. Indeed, some AhR ligands are used in animal models of mammary gland tumorigenesis (Daniel and Joyce, 1984; Russo and Russo, 1987; Das et al., 1989; McDougal et al., 1997; Trombino et al., 2000). Although a principle mechanism for AhR ligand carcinogenicity is the oxidation of parent compounds into mutagenic metabolites (Christou et al., 1987; Nebert et al., 1991; Buters et al., 1999), a process mediated by AhR-regulated CYP1 enzymes, other mechanisms, including the dysregulation of oncogenes, have been considered (Puga et al., 1992). Therefore, it is conceivable that binding of tumor cell AhR by exogenous environmental pollutants would release transcriptionally repressive AhR from the c-myc promoter, thereby augmenting the contribution of c-myc to the transformation process. Alternatively, hyperactivation of the AhR with environmental ligands could stabilize the AhR-c-myc complex, further inhibiting c-myc transactivation. To test these possibilities, the effect of a potent AhR ligand, TCDD, on AhRmediated repression of c-myc reporter activity was evaluated. The effect of TCDD on a reporter construct known to be positively regulated by the AhR (i.e. pGudLuc) was evaluated first as a positive control. Transfection of pGudLuc into Hs578T cells resulted in a significant B10-fold increase in reporter activity relative to that observed in cells transfected with the parental pGL3-basic plasmid (Figure 5a, second bar, Po0.04). The addition of 1 nM TCDD further enhanced promoter activity (third bar; Po0.002). The significant level of constitutive reporter activity is not inconsistent with data obtained by ChIP analysis and demonstrating that constitutive AhR does not bind the endogenous Oncogene

human CYP1A1 promoter since the AhRE-containing pGudLuc reporter is derived from mice not humans, and since it has been modified and selected on the basis of maximal responsiveness to AhR ligands such as TCDD (Garrison et al., 1996; Ziccardi et al., 2000). In contrast, TCDD treatment of Hs578T cells transfected with pGL3-c-myc had no effect on the low but significant (Po0.002) levels of reporter activity (Figure 5b, third and fourth bars). As in previous experiments, Hs578T cell transfection with pGL3-cmyc16Mut significantly increased the level of constitutive reporter activity (fifth bar, Po0.001). As expected, in the absence of AhREs in the reporter construct, TCDD had no further effect on reporter activity (sixth bar). These results suggest that hyperactivation of the AhR with environmental chemicals in Hs578T cells has no effect on c-myc transcription. Inhibition of AhR activity derepressess c-myc promoter activity Experiments with pGL3-c-myc mutants are consistent with a role for the AhR in constitutively repressing c-myc transactivation. However, the formal possibility that deletion of NF-kB-binding sites and mutation of all six AhREs resulted in changes that could affect transcription factors other than NF-kB and AhR could not be ruled out. Therefore, a second approach to evaluating putative AhR repression of c-myc transcription was taken. An evolutionarily conserved AhRR has been described in several species (Mimura et al., 1999; Baba et al., 2001; Watanabe et al., 2001; Karchner et al., 2002; Korkalainen et al., 2004). The AhRR potently inhibits AhR-dependent CYP1A1 transcription by competing for the AhR binding partner ARNT and by blocking AhR–AhRE binding (Mimura et al., 1999; Karchner et al., 2002). AhRR–ARNT complexes are transcriptionally inactive (Mimura et al., 1999). Notably, AhRR derived from killifish (Fundulus heteroclitus) inhibits both human and mouse AhR-dependent transactivation in an AhR-specific manner (Karchner et al., 2002). In preliminary experiments, the F. heteroclitus AhRR (FhAhRR) was more effective at suppressing pGudLuc activity in Hs578T cells than a human AhRR


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Figure 4 Deletion of all six AhREs increases constitutive c-myc promoter activity. Hs578T cells were transfected with 0.05 mg phRL-TK and with 1.0 mg pGL3-basic, wild-type pGL3-c-Myc, a reporter construct in which the two NF-kB-binding sites were deleted (pGL3-NF-kBKO) or constructs in which the two NF-kB sites were deleted, and individual AhREs (pGL3-AhRE1Mut, pGL3-AhRE2Mut, pGL3-AhRE5Mut, pGL3-AhRE6Mut), a pair of AhREs (pGL3-AhRE3,4Mut) or all six AhREs (pGL3-AhRE1–6Mut) were mutated. After 24 h, cells were harvested and lysates assayed for Firefly and Renilla luciferase activity. Data pooled from seven experiments are presented as the average normalized Firefly luciferase activity7s.e. An asterisk (*) indicates a significant increase in normalized reporter activity relative to background activity observed following transfection with pGL3-basic, Po0.001. A hash sign (#) indicates a significant increase in reporter activity relative to that seen in cells transfected with wild-type pGL3-c-Myc, Po0.001

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Figure 3 The AhR constitutively associates with the c-myc promoter. ChIP was performed on untreated Hs578T cells as described in Materials and methods. (a) PCR of DNA from the input fraction, AhR immunoprecipitation fraction (IP), and a noantibody control. Representative data from a total of three ChIP experiments are presented. (b) Recovery of the c-myc promoter from equal quantities of DNA was measured by real-time PCR and normalized to the input fraction. Data are pooled from three experiments and expressed as mean7s.e. An asterisk (*) indicates a significant level of promoter binding relative to the input control, Po0.02. (c) Hs578T cells were treated for 20 min before ChIP analysis with AhR-specific antibody for immunoprecipitation and CYP1A1-specific and c-myc-specific primers. Data are pooled from three experiments and expressed as mean7s.e. A single (*) or double (**) asterisk indicates a significant level of promoter binding relative to the input control, Po0.03 or Po0.01, respectively

expression construct (not shown). Therefore, the FhAhRR construct was chosen to test the prediction that inhibition of constitutive AhR activity in Hs578T cells would derepress AhR-dependent c-myc promoter transactivation.

Significant levels of FhAhRR were detected in Hs578T cells 24 h after transfection with FhAhRR (Figure 6). As expected from previous experiments demonstrating AhRR specificity (Karchner et al., 2002), FhAhRR transfection had no effect on SV40 promoter-driven, pGL3-promoter reporter activity (Figure 7a). As in previous experiments, pGudLuc transfection resulted in a significant (B7-fold) increase in luciferase activity (Figure 7b, second bar) that was further augmented by the addition of TCDD (seventh bar). Cotransfection of cells with pGudLuc and 0.1, 0.5, or 1.0 mg FhAhRR ablated the background levels of reporter activity (bars 3–5, Po0.001) and significantly reduced the TCDDinduced reporter activity (bars 8–10, Po0.008). These data confirm that FhAhRR is a potent inhibitor of both the constitutive and inducible human AhR in these tumor cells. As in previous experiments, transfection of Hs578T cells with the wild-type pGL3-c-myc reporter resulted in a low level of reporter activity, which, in this series of experiments, was not significantly greater than the background activity in pGL-basic-transfected cells (Figure 7c, third bar). Importantly, cotransfection Oncogene


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of pGL3-c-myc with 1.0 mg FhAhRR significantly (Po0.007) increased reporter activity 3–4-fold (fourth bar). As expected, transfection with a construct in which all AhREs were mutated increased reporter activity (fifth bar, Po0.007) and cotransfection with the FhAhRR had no further effect on the pGL3-AhRE16Mut luciferase activity (sixth bar). These data are consistent with the conclusion that the AhR constitutively represses c-myc transactivation through its interaction with at least some of the AhREs.

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Figure 5 AhR activation with an exogenous ligand, TCDD, has no effect on c-myc reporter activity. (a) Subconfluent Hs578T cells were transfected with 0.1 mg of a control vector (pGL3-basic) or with 0.1 mg of a CYP1A1 promoter-driven Firefly luciferase gene (pGudLuc) and 0.5 mg of the Renilla luciferase vector phRL-TK. After 24 h, cultures were treated with vehicle (0.1% acetone) or with TCDD (109 M final concentration). Cells were harvested 24 h later and lysates assayed for Firefly and Renilla luciferase activity. Data pooled from four experiments are presented as the average normalized firefly luciferase activity7s.e. An asterisk (*) indicates a significant increase in normalized reporter activity relative to pGLbasic-transfected cells, Po0.04. A hash sign (#) indicates a significant increase relative to pGudLuc-transfected, vehicle-treated cells, Po0.002. (b) Cells were treated as above, except that 1.0 mg wild-type pGL3-c-myc or AhRE mutant pGL3-AhRE16Mut was substituted for the pGudLuc reporter. Data pooled from six experiments are presented as the average normalized Firefly luciferase activity7s.e. An asterisk (*) indicates a significant increase in normalized reporter activity relative to the activity in pGL3-basic-transfected cells, Po0.002. A hash sign (#) indicates a significant increase relative to pGL3-c-myc-transfected cells, Po0.001

While the data described above support regulation of tumor cell c-myc transcription by the AhR, they do not address the potential regulation of steady-state c-myc levels by the AhR in situ. To determine if constitutively active AhR affects the steady-state levels of c-myc, Hs578T cells were transfected with FhAhRR or control vector (pcDNA3.1) and c-myc mRNA levels quantified 24 h later by real-time PCR. Indeed, c-myc mRNA levels almost doubled following transfection with FhAhRR (Figure 8, Po0.05). Similarly, FhAhRR transfection significantly increased c-Myc protein levels approximately two-fold (Figure 9a and b, Po0.004). Although many factors contribute to maintenance of steady-state mRNA and protein levels, these data suggest that at least one contributor to the downregulation of c-myc mRNA and c-Myc protein is constitutively active AhR.

Discussion The potential for a constitutively active AhR to regulate c-Myc was evaluated in the present study. Much of this

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study was motivated by: (1) the clear association between aberrant c-Myc expression and dysregulated cell growth or apoptosis in several cancer cell types (Dean et al., 1988; Channavajhala and Seldin, 2002;

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Pelengaris and Khan, 2003), (2) our previous observations demonstrating potentially active, nuclear AhR in rodent primary mammary tumors (Trombino et al., 2000), and (3) the identification of six AhREs in the human c-myc promoter. As an extension of rodent studies, we first evaluated AhR expression and localization in human tumor cell lines. Consistent with rodent studies, normal human mammary cells, as represented by Hs578Bst cells, expressed low levels of AhR mRNA

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Figure 7 AhRR specifically inhibits constitutive pGudLuc and induces pGL3-c-myc promoter activity. (a) Hs578T cells were cotransfected with 1.0 mg of a Firefly luciferase reporter construct driven by the SV40 promoter (pGL3-promoter), 0.5 mg control vector (pcDNA3.1), or 0.5 mg pcDNA3-FhAhRR (FhAhRR). Cells were harvested 24 h later and assayed for reporter activity as above. Data pooled from five experiments are presented as the average normalized Firefly luciferase activity7s.e. (b) Hs578T cells were cotransfected in triplicate wells with control vectors pGL3-basic or pcDNA3.1, with 0.1 mg pGudLuc and 0.05 mg phRL-TK, and with titered concentrations of pcDNA3-FhAhRR (FhAhRR) as indicated. After 24 h, cultures were treated with 109 M TCDD, where indicated. Cells were harvested 24 h later and lysates assayed for Firefly and Renilla luciferase activity. Data pooled from three experiments are presented as the average normalized Firefly luciferase activity7s.e. An asterisk (*) indicates a significant decrease in normalized pGudLuc reporter activity in FhAhRRtransfeced cells relative to cells transfected with the control vector, pcDNA3.1, Po0.001. A hash sign (#) indicates a significant decrease in normalized pGudLuc reporter activity in TCDDtreated, FhAhRR-transfected cells relative to cells transfected with control vector, pcDNA3.1, and treated with TCDD, Po0.008. (c) Hs578T cells were transfected with1.0 mg pGL3-basic, wild-type pGL3-c-myc, or AhRE mutant (pGL3-AhRE16Mut) reporter plasmids, 0.05 mg phRL-TK, and, where indicated, 1.0 mg pcDNA3-FhAhRR (FhAhRR) or control plasmid pcDNA3.1. Cells were harvested 24 h later and lysates assayed for Firefly and Renilla luciferase activity. Data pooled from five experiments are presented as the average normalized Firefly luciferase activity7s.e. An asterisk indicates a significant increase in normalized reporter activity relative to cells transfected with pGL3-c-myc and control vector pcDNA3.1, Po0.007 Oncogene


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Figure 9 AhRR increases endogenous c-Myc protein. Hs578T cells were transfected with 0.5 mg control vector pcDNA3.1 or with 0.5 mg pcDNA3-FhAhRR (FhAhRR). After 24 h, cells were harvested, protein extracted, and c-Myc quantitated by Western blotting. (a) A representative Western blot (three experiments total). (b) c-Myc band densities were normalized to lamin band densities. Data are pooled from three experiments and are presented as the average normalized c-Myc levels7s.e. An asterisk (*) indicates a significant increase in c-Myc protein, Po0.004

(not shown) and protein (Figure 1). Similar low AhR mRNA levels were seen in two normal human mammary epithelial cell lines, 184L and 184S (not shown). The presence of the AhR in these cells, albeit at low levels, is consistent with the ability of AhR ligands to target these cells for transformation in model systems (Lee et al., 1986; Calaf and Russo, 1993; Jerry et al., 1994) and with the possibility that exposure to environmental AhR ligands plays a role in human mammary tumorigenesis (Ishibe et al., 1998; Negri et al., 2003). Just as was observed in AhR ligand-induced rodent tumors (Trombino et al., 2000), the malignant human mammary line, Hs578T, expresses high levels of AhR (Figure 1). Several other human tumor lines, including myeloma lines Hs Sultan and IM9 and mammary tumor lines CAMA-1, MCF-7, and MDA MB 231, also express high AhR mRNA levels comparable to those seen with Hs578T cells (not shown). Therefore, upregulation of the AhR appears to be a characteristic of the transformation process. Interestingly, recent studies with prostate carcinoma cells suggest that high AhR Oncogene

levels may be driven by Wnt signaling (Chesire et al., 2004), a pathway implicated in malignant transformation (Miyoshi et al., 2002; Li et al., 2003). Furthermore, nuclear localization of the AhR in Hs578T cells and in other human tumor cell lines (Singh et al., 1996; Ge and Elferink, 1998; Hayashibara et al., 2003) suggests that the AhR may be transcriptionally active, despite the absence of exogenous ligands. Indeed, the demonstration of constitutive AhR binding to the c-myc promoter (Figure 3) supports this conclusion. The absence of detectable nuclear AhR in nontransformed Hs578Bst cells further suggests that expression of nuclear AhR is a phenotype characteristic of transformed cells. It has been shown that steroid receptors, particularly those expressed in tumors, may become activated in the absence of cognate ligands (Weigel et al., 1995; Blaustein, 2004; Fowler et al., 2004). The data presented herein suggest that the same may be true for the AhR. The mechanism through which nuclear AhR expression and constitutive activity is enforced in Hs578T cells is not known. It has been suggested that the phosphorylation state of the AhR influences its cellular distribution (Singh et al., 1996). Additional in vitro studies indicate that accumulation of the AhR in subcellular compartments is affected by cofactors such as the immunophilinlike ARA9/XAP2 molecule, even in the absence of exogenous ligands (Meyer et al., 1998; Meyer and Perdew, 1999; LaPres, 2000, #1342). Similarly, ectopic expression of the AhR coactivator RIP140 increases AhRE-dependent reporter activity in the presence or absence of AhR ligand, suggesting that hyperexpression of some cofactors may increase either AhR nuclear localization or transcriptional activity (Kumar et al., 1999). An increase in nuclear AhR and the attendant induction of transcriptional activity may be a function of AhR nuclear accumulation in addition to increased nuclear translocation. For example, inhibition of AhR nuclear export with leptomycin B or by mutation of the nuclear export signal increases nuclear AhR levels and DNA binding (Davarinos and Pollenz, 1999). While any or all of these mechanisms may be invoked to induce AhR levels and activity in tumor cells, similar processes may occur in some activated normal cells. For example, AhR activation is effected in normal keratinocytes simply by converting adherent cell cultures to suspension cultures (Sadek and Allen-Hoffmann, 1994). Similarly, human B cells activated through CD40 or TLR-4 express constitutively active (i.e. nuclear) AhR (Allan and Sherr, 2005). Therefore, it seems reasonable to assume that ligand binding may be only one of several mechanisms of AhR activation. Significantly, the AhR in Hs578T cells constitutively binds the c-myc but not the CYP1A1 promoter region defined by our PCR primers (Figure 3c), suggesting a preferential gene tropism for constitutively active AhR. We have observed a precedent for this differential targeting by an AhR receptor, albeit following activation by exogenous ligands (Matikainen et al., 2001). 7,12-dimethylbenzo[a]anthracene (DMBA), a prototypic PAH, activated the AhR in human and murine oocytes and induced apoptosis mediated by an


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AhRE-dependent induction of the proapoptotic Bax gene (Matikainen et al., 2001). In contrast, TCDD, a higher affinity AhR ligand, had no effect on Bax transcription. The differential gene targeting by DMBAactivated AhR, as opposed to TCDD-activated AhR, was attributed to subtle differences in the sequences flanking the AhRE core sequence in TCDD-responsive CYP1A1 and TCDD-nonresponsive Bax promoters (Matikainen et al., 2001). Notably, the upstream and downstream flanking regions of the AhREs in the human CYP1A1 promoter region (Lai et al., 1996) are dissimilar to those flanking the six AhREs in the 3.2 kb region of the c-myc promoter. It is possible that these differences are responsible for the ability of a constitutively active AhR to bind to at least some AhREs in cmyc but not in CYP1A1. Alternatively, a constitutively active AhR may recruit a different set of cofactors than environmental ligandactivated AhR, thereby imparting a different hierarchy of gene activation/repression. Several protein chaperones, coactivators, and corepressors can modulate AhR function (Meyer et al., 1998; Kumar and Perdew, 1999; Kumar et al., 1999; Nguyen et al., 1999; Tohkin et al., 2000; Rushing and Denison, 2002; Hestermann and Brown, 2003; Fallone et al., 2004; Wang et al., 2004a; Beischlag and Perdew, 2005). Even other receptors, such as ER-a, can associate with and inhibit gene transactivation (Beischlag and Perdew, 2005). Conversely, other factors, for example, ERAP-140, can associate with and enhance AhR–DNA binding in TCDD-treated cells (Nguyen et al., 1999). Presumably, the constitutively active AhR in Hs578T cells is associated with a transcriptional corepressor(s), which inhibits c-myc transactivation. One such candidate is the silencing mediator for retinoic acid and thyroid hormone receptor (SMRT), which can associate with the AhR (Nguyen et al., 1999; Rushing and Denison, 2002) and suppress CYP1A1 transactivation (Fallone et al., 2004). However, SMRT does not appear to be involved in the current system since immunoprecipitation/Western immunoblotting experiments failed to detect AhR–SMRT complexes in untreated or TCDDtreated Hs578T cells (data not shown). In contrast, it is likely that ARNT is associated with constitutively active AhR in Hs578T cells since AhRR significantly lowers background pGudLuc reporter activity (Figure 6) and inhibits c-myc repression (Figures 7–9), and since the AhRR is believed to inhibit AhR activity primarily through ARNT sequestration (Mimura et al., 1999; Kikuchi et al., 2003). Analysis of other AhR-associated factors in this system is ongoing. In contrast with constitutively active AhR, hyperactivation of the AhR with TCDD significantly increased AhR binding to the CYP1A1 promoter (Figure 3c) and induced CYP1A1 promoter activity (Figure 5a), but had no effect on AhR binding to the c-myc promoter (Figure 3c) or on c-myc reporter activity (Figure 5b). (Previous reports indicated some difficulty in inducing CYP1A1 promoter-driven CAT reporter activity in human tumor cells ((Thomsen et al., 1994; Wang et al., 1996). However, we have noted that the

ease with which AhR-dependent reporter activity is induced is highly sensitive to culture conditions, with confluent or near-confluent monolayers downregulating their AhR levels and CYP1A1 inducibility.) The failure to affect c-myc promoter activity with TCDD indicates either that AhR binding to the promoter is maximal and transcription is maximally blocked even in the absence of exogenous ligand and/or that a TCDD/AhR complex does not target the same promoter region as that targeted by a constitutively active AhR complex. Indeed, the latter possibility would suggest that engagement of a constitutively active AhR by an exogenous ligand may block or divert the activity of a constitutively active AhR. An important outcome of AhR inhibition by AhRR transfection is a significant increase in steady-state levels of endogenous c-myc mRNA and c-Myc protein (Figures 8 and 9). That this increase in endogenous mRNA or protein was not as dramatic as the increase in c-myc reporter activity (Figure 7c) may reflect post-transcriptional and post-translational factors, which limit steady-state c-myc mRNA and c-Myc protein levels. Nevertheless, a two-fold increase in c-Myc is generally sufficient to effect biologic changes, at least in normal cells (Ryu et al., 2003). The biologic consequences of AhR downregulation in Hs578T cells are uncertain. Hs578T is a very rapidly growing line and it may be difficult to alter conditions, even doubling the level of c-Myc, such that cell growth would be accelerated. Nevertheless, we made several attempts to determine if downregulation of AhR would increase cell growth. In several experiments, transient transfection of FhAhRR did not affect Hs578T cell growth (data not shown). In addition, we constructed a GFP-tagged lentivirus vector into which the FhAhRR sequence was cloned. Despite a >90% transduction efficiency, and a documented decrease in AhR-driven pGudLuc reporter activity, FhAhRR-transduced cells grew at approximately the same rapid rate as cells transduced with the GFP-containing vector alone (data not shown). These results may reflect a profound loss of growth control in these rapidly dividing tumor cells. That is, alterations in genes that regulate cell growth downstream of c-myc may preclude changes in cell growth mediated by AhR-dependent c-myc downregulation. If this is the case, then it would be expected that AhR downregulation may affect growth of other, perhaps less advanced tumor cells. Indeed, recent studies with AhR siRNA indicate that, at least in one cell line, AhR downregulation increases the entry of cells into S phase (Abdelrahim et al., 2003). If AhR downregulation generally results in increased growth of nonmalignant cells through c-Myc, or for that matter through some other cell cycle regulator, then the considerable increase in AhR expression and nuclear localization seen in highly malignant cells such as Hs578T may represent a failed attempt by the AhR to limit growth of this particular cell line. Alternatively, or in addition, AhR-dependent changes in c-Myc levels could influence cell apoptosis. Increases in endogenous or ectopic c-Myc expression can either Oncogene


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enhance (Ruggero et al., 2004; Wang et al., 2004b) or inhibit apoptosis (Bellas and Sonenshein, 1999; Ryu et al., 2003), depending on the cell type studied. In primary mammary tumors and in Hs578T cells, c-Myc tends to induce apoptosis (Chen et al., 1998; Liao and Dickson, 2003). Consequently, the relatively high levels of constitutively active AhR may contribute to malignancy by suppressing c-Myc and increasing cell survival. This possibility is currently under investigation. Whether or not changes in cell growth or apoptosis can be demonstrated in Hs578T cells following AhR inhibition, the current studies suggest a novel level on which the AhR may affect tumorigenesis, that is, by regulating c-Myc levels through repression of c-myc transcription. Extension of this work to normal cells and to nonmalignant tumor cell lines in which changes in c-Myc levels may have more profound effects on cell function will help determine the significance of constitutive AhR activation and c-Myc regulation during the transformation process.

Materials and methods Cell culture The estrogen receptor-negative Hs578T tumor cell line (ATCC, Manassas, VA, USA) was derived from a human mammary carcinoma and is epithelial in origin (Hackett et al., 1977). The nontransformed Hs578Bst myoepithelial cell line was derived from normal tissue adjacent to the tumor from which Hs578T cells were derived (Hackett et al., 1977). Both lines were grown in a humidified, 5% CO2 atmosphere at 371C. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with glutamax-1 (L-alanyl-L-glutamine), supplemented with 10% fetal calf serum, penicillin (500 IU/ml), streptomycin (5 mg/ml), and L-glutamine (2 nM). All media components were purchased from Invitrogen (Carlsbad, CA, USA). Protein extraction and Western immunoblotting Total cell lysates were prepared from Hs578T and Hs578Bst cells by incubating washed cell pellets for 10 min in lysis buffer (50 mM KHPO4, pH 7.4, 5 mM DTT) and 10 ml/ml protease inhibitor cocktail (Sigma, St Louis, MO, USA) on ice. Following a 10 min centrifugation, protein concentrations of total cell lysates were quantified using Bio-Rad Protein Assay Reagent (Bio-Rad, Hercules, CA, USA). Nuclear and cytoplasmic fractions were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s instructions. Equal amounts of protein (typically 40 mg for Hs578T cells and up to 120 mg for Hs578Bst cells) were boiled for 5 min in 1 SDS–PAGE sample buffer (50 mM Tris buffer, pH 6.8, 10% glycerol, 2% SDS, 0.01% bromophenol blue and 1% b-mercapthoethanol) before SDS– PAGE electrophoresis through a 6.8% polyacrylamide gel and overnight transfer onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Following transfer, membranes were blocked with 5% skim milk powder in 1 TBS plus 0.05% Tween-20 (TBST). The primary antibody was either polyclonal rabbit anti-human AhR antibody (Santa Cruz, CA, USA) or mouse monoclonal anti-human c-Myc antibody (Santa Cruz, CA, USA) and the secondary antibody was HRP-linked rabbit Ig-specific goat antibody (Pierce, Rockford, IL, USA). Bands Oncogene

were detected using enhanced chemiluminescence substrate (Sigma, St Louis, MO, USA) and exposure to X-ray film (Fuji, Japan). Membranes were stripped with the Re-Blot Western Blot Recycling Kit (Chemicon, Temecula, CA, USA) and reblotted with b-actin-specific mAb (Sigma, St Louis, MO, USA) to confirm equal loading. Where indicated, blots were reprobed with a-tubulin- (Oncogene Research, Boston, MA, USA) or lamin A/C- (Novocastra Laboratories, UK) specific mAbs to confirm the purity of cytoplasmic and nuclear protein extracts, respectively. Chromosome immunoprecipitation ChIP and DNA quantification were performed as described previously (Hestermann and Brown, 2003). For gel electrophoresis, the region of the c-myc promoter 95–245 bp upstream of the transcription start site was amplified using the primers 50 -AGGCGCGCGTAGTTAATTCAT-30 and 50 -CGCCCT CTGCTTTGGGA-30 for 43 cycles (951C for 15 s, 581C for 60 s). The region of the CYP1A1 promoter 784–1156 bp upstream of the start site was amplified using the primers 50 -CACCCGCCACCCTTCGACAGTTCT-30 and 50 -CTCC CGGGGTGGCTAGTGCTTTGA-30 for 40 cycles (951C for 45 s, 581C for 45 s, and 701C for 60 s) as we described previously (Hestermann and Brown, 2003). In our hands, DNA is sheared within approximately 1 kb of the amplified fragment. Therefore, ChIP detects protein-bound DNA within approximately 1 kb of the region spanned by the primers. For quantification, equal amounts of DNA from each sample were amplified by real-time PCR using the same primers with SYBR Green master mix (Applied Biosystems, Bedford, MA, USA) in a DNA Engine Opticon 2 (MJ Research, Waltham, MA, USA). Reporter plasmids and site-directed mutagenesis The pGL3-c-myc reporter was constructed by cloning the HindIII (Blunted)–SacI human c-myc promoter fragment of pVCAT (kindly provided by Dr DL Levens; NCI) into the KpnI (Blunted)–SacI sites of the pGL-basic luciferase reporter plasmid (Promega, Madison, WI, USA). Deletion of two NFkB-binding sites in this plasmid was achieved by site-directed mutagenesis using two deoxyoligonucleotide primers: 50 -/ 5Phos/GAG TTA ACG GTT TTT TTC ACA ATG ACT CCC CCG GCT CGG-30 and 50 -/5Phos/GGC TAT TCT GCC CAT TTG CCC GCC GCT GCC AGG-30 . The resulting plasmid was designated ‘pGL3-NF-kBKO’. This plasmid was used as the template for further site-directed mutagenesis of AhREs as described (Matikainen et al., 2001). Plasmids in which each individual AhRE or pairs of AhREs (i.e. AhRE 3 and 4) were mutated (pGL3-AhRE1Mut, pGL3-AhRE2Mut, pGL3-AhRE3,4Mut, pGL3-AhRE5Mut, and pGL3-AhRE6Mut) were generated with the following primers: 50 -/5Phos/CCG TGT GGG AGG AAT GGG GGT GGG ACG-30 (pGL3AhRE1Mut), 50 -/5Phos/CCC TAT CTA CAC TAA CAT CCC ATT CTC TGA ACG CGC GCC-30 (pGL3-AhRE2Mut), 50 -/ 5Phos/GCA GCC TGG TAC GCG AAT GGA ATG GCG GTG GGC GCG C-30 (pGL3-AhRE3,4Mut), 50 -/5Phos/GGG TTC CCA AAG CAG AGG GAA TGG GCG AAA AGA AAA AAG ATC C-30 (pGL3-AhRE5Mut); 50 -/5Phos/CTG CCT TAT GAA TAT ATT CAT TCT GAC TCC CGG CCG GTC GG-30 (pGL3-AhRE6Mut). The positions of the mismatches are underlined. A plasmid in which all six AhREs were mutated (pGL3-AhRE16Mut) was produced by sequential mutation of each of the AhREs. All of the site-directed mutagenesis was conducted using the QuickChange multisitedirected mutagenesis kit (Stratagene Inc., La Jolla, CA, USA)


7879 according to the manufacturer’s instructions. DNA sequencing was performed on each plasmid to verify the deletions/ mutations and to confirm that no other sequence changes had occurred. AhR-dependent expression of the pGudLuc6.1-firefly luciferase reporter construct (pGudLuc) is driven by four AhREs derived from the CYP1A1 promoter (Han et al., 2004). This construct has been optimized for TCDD responsiveness and was kindly provided by Dr M Denison (UC Davis). Cloning and characterization of the FhAhRR expression vector has been described previously (Karchner et al., 2002). The product of this vector efficiently suppresses mammalian AhR activity induced by TCDD (Karchner et al., 2002). Transient transfections, TCDD treatment, and luciferase assays Hs578T cells (3 104/well) were plated in 12-well culture plates and cultured to 80% confluence. Lipofectamine 2000 transfection reagent (Invitrogen) was used according to the manufacturer’s instructions to transfect cells. The Renilla luciferase vector phRL-TK (0.05 mg) was cotransfected with Firefly luciferase reporter constructs (0.1 mg pGudLuc, 0.5–1.0 mg wild-type pGL3-c-Myc or mutant constructs). Where indicated, 0.5 mg of pcDNA-FhAhRR or control pcDNA3.1 (Invitrogen) was added to the transfection mixture. For each experiment, the amount of total DNA transfected was equilibrated with parental expression vectors. Cells were incubated overnight, washed twice with phosphate-buffered saline (PBS) (pH 7.2), and resuspended in 75 ml RPMI prior to luciferase analysis. Luciferase activity was determined with the Dual Glo Luciferase system (Promega, Madison, WI, USA), which allowed sequential reading of the Firefly and Renilla signals. Cells were lysed according to the manufacturer’s directions (Promega, Madison, WI, USA), transferred to 96-well white wall plates, and analysed using a Reporter Luminometer (Promega, Madison, WI, USA). The Renilla signal was read after quenching the Firefly output, thus allowing normalization between sample wells. The normalized Firefly luciferase signal is expressed relative to the Renilla signal. TCDD was obtained from Cambridge Isotopes Laboratories (Andover, MD, USA) at >99% purity and was maintained as a 1000 stock solution in anhydrous tissue culture grade dimethylsulfoxide (DMSO). TCDD (1.0 nM) in DMSO or DMSO alone (final volume 0.1%) was added to cultures 2 h after transfections and cells were incubated for an additional 24 h. Cells then were washed twice with PBS (pH 7.2) and resuspended in 75 ml RPMI for luciferase analysis. Quantitative c-myc-specific real-time PCR Hs578T cells were plated onto two 10 cm cell culture dishes (106 cell/well) and cultured to 80% confluence. Equal amounts of pcDNA-FhAhRR or parental vector pcDNA3.1 were added to separate plates and cells transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. After 24 h, adherent Hs578T cells were washed twice with PBS

(pH 7.2) and harvested. Total RNA was isolated using the Absolutely RNA RT–PCR Miniprep kit (Stratagene, La Jolla, CA, USA). RNA samples were treated with RNase-free DNase according to the manufacturer’s instructions. Total RNA was eluted from the columns with 60 ml RNase-free water and quantified by UV absorbance. First-strand cDNA was synthesized using 2 mg of each total RNA, random hexamers, and SuperscriptII reverse transcriptase according to the manufacturer’s instructions (Invitrogen). Real-time PCR amplification mixtures (25 ml) contained 1 ml template cDNA, 2 SYBR Green I Master Mix buffer (12.5 ml) (Applied Biosystems, Foster City, CA, USA), and 300 nM of a forward and reverse primer pair. The sequences for c-myc amplification in real-time PCR were described previously (Latil et al., 2000) and are as follows: sense, 50 -ACC ACC AGC AGC GAC TCT GA-30 ; antisense, 50 -TCC AGC AGA AGG TGA TCC AGA CT-30 . Human ribosomal RNA, amplified with previously published primer sequences (Nazarenko et al., 2002), was used for RNA normalization of the c-myc signal as described previously (Saez et al., 2003). The ribosomal RNA primers were as follows: sense, 50 -GAC TCA TTC GCC CTG TAA TTG GAA TGA GTC-30 ; antisense, 50 -CCA AGA TCC AAC TAC GAG CTT-30 . Reactions were run in an ABI PRISM 5700 Sequence Detector (Applied Biosystems) using the following standard cycling conditions: 10 min polymerase activation at 951C, 40 cycles for 15 s at 951C, and 601C for 60 s. Results for each experiment are calculated from three replicate PCR reactions. Threshold cycle (CT) values were collected at linearity. Relative mRNA expression was normalized against internal ribosomal controls. The parameter 2DCT , where DCT equals CT of the c-myc signal minus the CT of the endogenous ribosomal RNA control, was used to describe the relative levels of c-myc mRNA normalized to 18S rRNA. Data analyses Statistical analyses were performed with Statview (SAS Institute, Cary, NC, USA) or Excel. Data from a minimum of three experiments are presented as means7standard errors (s.e.). One-factor ANOVAs and a Fisher PLSD post hoc comparisons test or the Student’s t-test were used to determine significant differences.

Abbreviations AhR, aryl hydrocarbon receptor; AhRR, aryl hydrocarbon receptor repressor; ChIP, chromosome immunoprecipitation; DMBA, 7,12-dimethylbenzo[a]anthracene; DMSO, dimethylsulfoxide; PAH, polycyclic aromatic hydrocarbon; TCDD, 2,3,7,8 tetrachlrodibenzo-p-dioxin. Acknowledgements This work was supported by DAMD17-03-1-0406, NIEHS Grants PO1-ES11624, RO1-ES06086, and P42-ES07381.

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