Proc. Natl. Acad. Sci. USA Vol. 94, pp. 2581–2586, March 1997 Medical Sciences

The quinone reductase gene: A unique receptor-regulated gene that is activated by antiestrogens (electrophile response element͞tamoxifen͞antioxidant͞breast cancer)

MONICA M. MONTANO* AND BENITA S. KATZENELLENBOGEN*†‡

*Departments of Molecular and Integrative Physiology and †Cell and Structural Biology, University of Illinois and College of Medicine, Urbana, IL 61801-3704

Communicated by Jack Gorski, University of Wisconsin, Madison, WI, December 30, 1996 (received for review October 4, 1996)

ABSTRACT Antiestrogens are thought to exert most of 10 and 11). These widely distributed enzymes detoxify elec- their beneficial effects in by antagonizing the trophiles, thereby protecting cells against the toxic and neo- actions of estrogen. We report here that antiestrogens also plastic effects of carcinogens. stimulate the expression of quinone reductase (QR) [NAD- Using the technique of RNA differential display (12), we (P)H:quinone oxidoreductase, EC 1.6.99.2], which may pro- have identified in this report QR mRNA as a species that is vide protective effects against the toxicity and mutagenicity expressed at much higher levels in an MCF-7 breast cancer cell caused by quinones. QR is up-regulated by low concentrations subline that has been grown long term in the presence of the of antiestrogens (trans-hydroxytamoxifen, tamoxifen, and antiestrogen trans-hydroxytamoxifen (TOT) (13). We there- ICI182,780) in (ER)-containing breast can- fore undertook the examination of its regulation by antiestro- cer cells, and this increase is suppressed by estrogen via an gens as a possible basis for the proposed antioxidant action of ER-dependent mechanism. Since regulation of the QR gene, as tamoxifen. The molecular mechanisms for the induction of well as other genes involved in detoxification such as the phase 2 enzymes by antiestrogen were also explored in view of glutathione S-transferase Ya subunit (GST Ya) gene, is known their importance in devising strategies for chemoprotection to be mediated by an electrophile antioxidant response ele- ͞ against cancer. It has been reported that the induction of the ment (EpRE͞ARE), we examined the effects of antiestrogens GST Ya subunit (GST Ya) and QR genes is mediated through on a 41-bp electrophile responsive region derived from the GST Ya gene. Transfection of this EpRE-containing region an electrophile (or antioxidant) response element (EpRE or into ER-negative breast cancer cells in the presence or absence ARE) (14, 15), although the identity of the EpRE͞ARE of an expression vector for the human ER, as well as mu- enhancer binding protein(s) is not known. tagenesis studies, revealed that the EpRE-containing con- In our studies, we have observed that the QR gene shows struct was activated by antiestrogen to the same extent as by reversed pharmacology, being markedly up-regulated by an- tert-butylhydroquinone (TBHQ), a known activator of EpREs; tiestrogen and suppressed by estrogen in breast cancer cells. however, only the stimulation by antiestrogen, and not TBHQ, Our data indicate that there are two pathways for QR up- required ER and was repressed by estradiol, although activa- regulation, one that is antiestrogen modulated and estrogen tion by both inducers mapped to the same 10-bp EpRE receptor (ER) dependent, and a second that is stimulated by consensus sequence. Thus, there appear to be two pathways known electrophile inducers such as tert-butylhydroquinone for QR induction, one that is activated by electrophile induc- (TBHQ) and is ER independent. Transfection and mutagen- ers such as TBHQ and is ER independent, and a second that esis studies on gene constructs with the 41-bp EpRE- is antiestrogen regulated and ER dependent; both pathways containing region from the GST Ya gene indicate that anties- act through the EpRE. The anticancer action of antiestrogens trogen-mediated activation occurs at the transcriptional level may thus derive not only from the already well-known repres- via the EpRE and requires the ER. These observations have sion of estrogen-stimulated activities but also from the acti- broad implications regarding potential antiestrogen regulation vation of detoxifying enzymes, such as QR, that may contrib- of a variety of genes whose transcription is under the control ute to the beneficial antioxidant activity of antiestrogens. of EpRE͞AREs.

Tamoxifen is an anticancer drug that is widely used in the MATERIALS AND METHODS treatment of breast cancer (1–4). It is also being assessed as a preventive agent for this disease and for other potential Chemicals and Materials. Cell culture media were pur- benefits, such as protection against cardiovascular disease and chased from GIBCO. Calf serum was from HyClone and fetal osteoporosis (5, 6). It has been proposed that tamoxifen may calf serum (FCS) from Sigma. The antiestrogens ICI182,780 function as an anticancer drug, possibly by acting as an (ICI), tamoxifen, and TOT were kindly provided by Alan antioxidant (7–9). However, the basis for the antioxidant Wakeling and Zeneca Pharmaceuticals (Macclesfield, En- capabilities of tamoxifen has not been well characterized. gland). 12-Tetradecanoate 13-acetate (TPA), NADPH, men- Phase 2 detoxification enzymes such as NAD(P)H:(qui- adione, and cytochrome c were obtained from Sigma. TBHQ none-acceptor) oxidoreductase [quinone reductase (QR)], was obtained from Aldrich. glutathione S-transferases (GSTs), epoxide hydrolase, and UDP-glucuronosyltransferases are induced in cells by electro- philic compounds and phenolic antioxidants (reviewed in refs. Abbreviations: ER, estrogen receptor; E2, estradiol; QR, quinone reductase; TBHQ, tert-butylhydroquinone; EpRE͞ARE, electro- phile͞antioxidant response element; GST Ya, glutathione S- The publication costs of this article were defrayed in part by page charge transferase Ya subunit; TOT, trans-hydroxytamoxifen; GH, growth payment. This article must therefore be hereby marked ‘‘advertisement’’ in hormone; TPA, 12-tetradecanoate 13-acetate; TRE, TPA response accordance with 18 U.S.C. §1734 solely to indicate this fact. element; ␤-gal, ␤-galactosidase; ICI, ICI182,780. ‡To whom reprint requests should be addressed at: Department of Copyright ᭧ 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA Molecular and Integrative Physiology, University of Illinois, 524 0027-8424͞97͞942581-6$2.00͞0 Burrill Hall, 407 South Goodwin Avenue, Urbana, IL 61801. e-mail: PNAS is available online at http:͞͞www.pnas.org. [email protected].

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Plasmids. The growth hormone (GH) reporter gene con- inhibitor (GIBCO) for 30 min at 37ЊC. After phenol͞ structs p284GstYa-GH (containing the mouse GST Ya gene chloroform extraction and ethanol precipitation, three reverse minimal promoter region), p41–284GstYa-GH (containing transcription reactions were performed for each RNA sample the mouse GST Ya gene minimal promoter and the 5Ј using 0.2 ␮g of DNA-free total RNA in 1ϫ reverse transcrip- upstream 41-bp EpRE-containing region), and pTREX2– tion buffer (25 mM Tris⅐Cl, pH 8.3͞37.6 mM KCl͞1.5 mM 284GstYa-GH [containing the mouse GST Ya gene minimal MgCl2͞5mMDTT)and20␮M each of dATP, dCTP, dGTP, promoter region and two consensus TPA response element and dTTP, and 0.2 ␮MofHindIII restriction site-containing (TRE) sites] were kindly provided by Paul Talalay (The John one-base anchored oligo(dT) primers [either H-T11A (5Ј- Hopkins University School of Medicine) (16). AAGCTTTTTTTTTTTA-3Ј), H-T11C (5Ј-AAGCTT- Five reporter constructs, each containing mutations intro- TTTTTTTTTC-3Ј), or HT11G (5Ј-AAGCTTTTTTT- duced sequentially into the 41-bp fragment containing the TTTTG-3Ј)]. After the solution was heated at 65ЊC for 5 min EpRE, were constructed by site-directed mutagenesis (17), and cooled to 37ЊC, 100 units of Moloney murine leukemia with modification (18). The ScaI͞SacI fragment of p41– virus reverse transcriptase was added for 1 hr. PCRs were 284GstYa-GH was inserted into the SacI͞SmaI site of Blue- performed in reaction mixtures containing 0.1 vol of reverse script II SKϩ (Stratagene) to make p41–284GstYa-BSKϩ. transcription reaction mixture, 1ϫ PCR buffer (10 mM Tris⅐Cl, Mutagenic oligonucleotides were then annealed to single- pH 8.4͞50 mM KCl͞1.5 mM MgCl2͞0.001% gelatin), 2 ␮M stranded DNA generated using the f1 origin of replication in each of dATP, dCTP, dGTP, and dTTP, 0.2 ␮MofHindIII Bluescript II SKϩ. The mutagenic oligonucleotides used in restriction site-containing arbitrary 13-mer oligonucleotide five separate mutagenesis reactions were: 5Ј-CAGTGCCA- [either H-AP1 (5Ј-AAGCTTGATTGCC-3Ј), H-AP2 (5Ј- AGCTCTGCAGAAAAATGACATTGC-3Ј,5Ј-AGCTTA- AAGCTTCGACTGT-3Ј), H-AP3 (5Ј-AAGCTTTGGT- GCTTGGGTCCATGGTTGCTAATGGTG-3Ј,5Ј-TTGGA- CAG-3Ј), H-AP4 (5Ј-AAGCTTCTCAACG) or H-AP5 (5Ј- AATGACATTGCTCTTCGTGACAAAGCA-3Ј,5Ј-GACA- AAGCTTAGTAGGC-3Ј)], 0.2 ␮M of the corresponding Hin- TTGCTAATTTGGATCCAGCAACTTTGTC-3Ј, and 5Ј- dIII restriction site-containing one-base anchored oligo(dT) TAATGGTGACAAAGATCTTGTGTCGACTCTAG-3Ј. primer, 0.1 ␮Ci of 35S-dATP (1 Ci ϭ 37 GBq), and 1 unit of To make each of the five mutated p41–284GstYa-GH reporter AmpliTaq DNA polymerase (Perkin–Elmer͞Cetus). Light constructs (M1–M5) the NdeI͞SacI fragment of p41– mineral oil was overlaid and the PCR reactions were per- 284GstYa-GH was then replaced with the mutated NdeI͞SacI formed at 94ЊC for 30 sec and 40 cycles of 94ЊC for 30 sec, 40ЊC fragment of p41–284GstYa-BSKϩ. for 2 min, 72ЊC for 30 sec, followed by 72ЊC for 5 min. Stop The expression vector for the wild-type human ER and the buffer (95% formamide͞10 mM EDTA, pH 8.0͞0.09% xylene mutant ER that lacks activation function-2 activity (ERS554fs) cyanol͞0.09% bromophenol blue) was added to each sample has been described (19). The expression vector for the ER and heated at 80ЊC for 2 min before loading on 6% polyacryl- DNA binding mutant, ER (missing amino acids 185– DBDmut amide sequencing gels. After electrophoresis the gels were 251), was constructed by replacing the EagI EagI fragment ͞ exposed to Kodak XAR-5 film for 48 hr. Any band differen- from the wild-type pCMV-ER with the EagI EagI insert from ͞ tially expressed in parental vs. long-term TOT-maintained HE11, the latter plasmid kindly provided by Pierre Chambon cells was identified and the PCR was repeated to confirm the (20). The plasmid pCMV␤ (Clonetech, Palo Alto, CA), which findings. encodes the ␤-galactosidase (␤-gal) gene, was used as an Any cDNA species that was differentially expressed was internal control for transfection efficiency in all experiments. Cell Culture. MCF-7 human breast cancer cells were ac- recovered from the dried DNA sequencing gel (21) and quired from the Michigan Cancer Foundation. Cells were reamplified using the same primer set and PCR conditions as maintained in growth medium, which was minimum essential used in the mRNA display, except that the dNTP concentra- medium (MEM) plus phenol red supplemented with 5% tions were at 20 ␮M instead of 2 ␮M and no isotope was added. Ϫ12 Reamplified cDNA was run on a 1.2% agarose gel and purified heat-inactivated FCS, estradiol (E2) (10 M), and 10 mM Hepes. Prior to the experiments, cells were depleted of estro- using the QIAEX kit from Qiagen (Chatsworth, CA). Ream- gen by growth for 2 weeks in the same growth medium except plified cDNA was subsequently cloned into the PCRII vector using the TA cloning system from Invitrogen and sequenced without added E2. MCF-7 cells were then grown in MEM plus phenol red supplemented with 5% charcoal dextran-treated using the Sequenase kit (United States Biochemical). FCS (CDFCS) for 2 days, after which the cells were maintained Northern Blot Analysis. Gel-purified reamplified cDNA in improved MEM (IMEM) minus phenol red plus 5% CDFCS was random-primer labeled using the Ready-to-Go DNA for 6 days prior to use in experiments. All media included labeling kit from Pharmacia for Northern blot analysis to verify penicillin (100 units͞ml), streptomycin (100 ␮g͞ml), and gen- differential expression. Twenty micrograms of total RNA was tamycin (25 ␮g͞ml) (GIBCO). Long-term TOT-maintained separated by electrophoresis, transferred to nitrocellulose MCF-7 cells were derived from these parental MCF-7 cells by support, and hybridized with random-primer-labeled cDNA growth for over 6 months in the presence of 10Ϫ6 M TOT (13). (22). MDA-MB-231 human breast cancer cells were maintained Transfections. MDA-MB-231 cells were transfected as de- in Leibovitz’s L15 medium with 10 mM Hepes, 5% calf serum, scribed (23). Cells were seeded for transfection at 7.5 ϫ 105 per 100 units penicillin͞ml, 100 ␮g streptomycin͞ml, 25 ␮g gen- 60-mm dish in IMEM minus phenol red containing 5% CDCS. tamycin͞ml, 6 ng bovine insulin͞ml, 3.75 ng hydrocortisone͞ Cells were transfected by the CaPO4 coprecipitation method ml, and 16 ␮g glutathione͞ml. Cells were then grown in MEM 24 hr later with 3 ␮g of the GstYa-GH reporter constructs, 60 plus phenol red supplemented with 5% charcoal dextran- ng of ER expression vector, 100 ng of pCMV␤␤-gal internal treated calf serum (CDCS) for 2 days prior to use in experi- control plasmid, and 4.84 ␮g of pTZ19 carrier DNA. Cells ments. remained in contact with the precipitate for 5 hr and were then Cells used in all experiments were in the log phase of growth subjected to a 2-min glycerol shock (20% in IMEM minus and were at 30–50% of confluence. phenol red plus 5% CDCS). Cells were rinsed with Hanks’ Isolation of RNA. Total RNA was isolated using the RNA balanced salt solution and given fresh media with or without extraction kit from Pharmacia. hormones. Differential Display. Differential display was performed Media were collected 48 hr after hormone treatment for GH using the RNAimage kit from GenHunter (Nashville, TN). (reporter gene) assay. GH production was measured using the DNA-free RNA was obtained by treatment of total RNA with GH transient gene expression assay from Nichols Institute DNase I (Promega) in the presence of placental RNase (San Juan Capistrano, CA). Cells were harvested and pro- Downloaded by guest on September 26, 2021 Medical Sciences: Montano and Katzenellenbogen Proc. Natl. Acad. Sci. USA 94 (1997) 2583

cessed to measure ␤-gal activity, to normalize for transfection efficiency, as described (24). QR Assay. MCF-7 cells were plated at 2.5 ϫ 105 cells per 100-mm plate in IMEM minus phenol red containing 5% CDFCS. Hormone treatments were started the day after plating. For extended treatment regimens, cells were fed with fresh media containing a hormone(s) every 2 days. Before harvesting, the cell monolayer was washed twice with ice-cold phosphate-buffered saline without calcium and magnesium after removal of media. The cells were scraped from the plates and collected by centrifugation (800 ϫ g for 5 min). The cell pellet from each flask was homogenized briefly in 50 mM phosphate buffer (pH 7.5). The homogenate was centrifuged at 14,000 rpm for 15 min at 4ЊC and the supernatant was removed and frozen at Ϫ80ЊC until assayed for QR activity. To assay for QR enzyme activity by the spectrophotometric method, menadione was used as a substrate and cytochrome c as the terminal electron acceptor (25). After the mixing of 10 ␮M menadione, 70 ␮M cytochrome c, and cytosol in pH 7.5 buffer, the tube contents were transferred to a cuvette and the FIG. 1. MCF-7 breast cancer cells grown long term in the presence baseline A550 was determined in a spectrophotometer. Follow- of TOT express elevated levels of QR RNA. (A) Differential display ing addition of NADPH (final concentration, 0.5 mM) and gel showing cDNA species from parental and long-term TOT- rapid mixing of the contents, the linear change in absorbance maintained MCF-7 cells. Differential display was performed as de- at 550 nm was recorded at 30-sec periods. The enzyme activity scribed. RNA samples from parental and long-term TOT-maintained MCF-7 cells were compared by differential display using a one-base was calculated as nanomoles of cytrochrome c reduced per min anchored primer, H-T11G (5Ј-AAGCTTTTTTTTTTTG-3Ј), in con- and the specific QR activity is expressed as units (nanomoles junction with a 13-mer arbitrary primer, H-AP1 (5Ј-AAGCTTGAT- of cytochrome c reduced per min) per mg of protein. TGCC-3Ј). QR partial cDNA (indicated by arrow) was expressed at a much higher level in long-term TOT-maintained MCF-7 cells and was subsequently recovered, reamplified, and gel-purified as described. RESULTS (B) Northern blot analysis to verify differential expression of QR Induction of QR Activity in MCF-7 Cells by Antiestrogens. RNA. Total RNA was collected from MCF-7 parental cells and cells Ϫ6 Through the technique of differential RNA display, we ob- grown for over 6 months in 10 M TOT. Equal amounts (20 ␮g) of served QR to be a species that was present at a much higher total RNA were separated by electrophoresis as described. The blot was probed with the random-primer-labeled QR partial cDNA. As an level in an MCF-7 breast cancer cell subline that had been RNA loading control the same blot was reprobed with 36B4 cDNA. grown long term (over 6 months) in our laboratory in the presence of high (10Ϫ6 M) levels of the antiestrogen TOT, as Induction of EpRE Enhancer Activity in 231 Breast Cancer compared with the parental MCF-7 cells grown in the absence Cells. To examine more definitively the role of the ER in the of TOT (Fig. 1). The nucleotide sequence of the differentially antiestrogen-mediated induction, an ER-negative breast can- displayed cDNA species showed 96% homology with exon 6 of cer cell line, MDA-MB-231, was used. Since regulation of the the gene for human NAD(P)H:quinone oxidoreductase. As QR gene, as well as other genes involved in detoxification such shown in the Northern blot in Fig. 1 using this cDNA as a as the GST Ya gene, is known to be mediated through ARE probe, we found that QR RNA was present at 8 times greater or EpRE (14, 15), we used reporter constructs containing the abundance in the long-term TOT-maintained cells than in the EpRE enhancer region. Cells were transfected with a p41– parental MCF-7 cells. That the differentially expressed mes- 284GstYa-GH reporter construct (with the 41-bp EpRE- sage is the transcription product of the QR gene is supported containing 5Ј upstream region from the murine GST Ya gene by the 4-fold higher QR enzyme activity in the long-term and its homologous promoter fused to the human growth tamoxifen-maintained cell line (data not shown). We therefore hormone structural sequences) in the absence or presence of examined the ability of antiestrogen to evoke increases in QR an expression vector for the wild-type ER or for mutant forms activity over time when parental MCF-7 cells were treated with of the ER lacking DNA binding ability (ER ) or tran- antiestrogens. DBDmut As shown in Fig. 2, a 3- to 4-fold increase in QR activity was scriptional activity (ERS554fs). observed upon treatment with the antiestrogens TOT, tamox- A significant increase in transcriptional activity of the ifen, and ICI. The induction of QR activity by antiestrogens EpRE-containing reporter construct in the presence of TOT occurred in a dose-dependent manner (Fig. 2A), and maximal was observed in 231 cells only when cells were cotransfected stimulation was obtained with a relatively low concentration of with an expression vector for the wild-type ER (Fig. 4). Cells the antiestrogens (10Ϫ8 M for TOT and ICI, Ϸ10Ϫ7 M for transfected with the empty control pCMV vector (Fig. 4 Right), tamoxifen). The concentrations of antiestrogen required for lacking the ER cDNA, did not show an increase in GH stimulation of QR activity were substantially lower than those production in response to antiestrogen. Maximal induction of needed for stimulation by a previously identified potent in- GH production by antiestrogens was comparable to that ducer of QR activity in other systems, TBHQ. Maximal evoked by TBHQ. Of note, however, TBHQ stimulation of the induction of QR activity occurred after 3–4 days of treatment reporter gene construct did not require the ER and was equally with the antiestrogens (Fig. 2B), and the time course and robust in the presence or absence of ER. Consistent with the magnitude of induction, was similar to that achieved by TBHQ. lack of any significant increase in QR enzyme activity in the Of note, no increase in QR activity was observed in the presence of E2 in MCF-7 cells (Fig. 2), no increase in the presence of E2 (Fig. 2) or the tumor promoter phorbol ester transcriptional activity of EpRE-containing constructs was TPA (Fig. 2A). However, as shown in Fig. 3, the antiestrogen- observed with E2. Rather, TOT-mediated induction of EpRE- induced increase in QR activity was fully inhibited by E2. These reporter transcriptional activity was repressed in the presence observations suggest that the modulation of QR activity by of E2, consistent with an important role for the ER in the antiestrogens is mediated by the ER. In contrast, E2 did not antiestrogen-mediated activation. E2, by presumably binding to affect the induction of QR by TBHQ (Fig. 3). the ER in a manner competitive with antiestrogen, inhibited Downloaded by guest on September 26, 2021 2584 Medical Sciences: Montano and Katzenellenbogen Proc. Natl. Acad. Sci. USA 94 (1997)

FIG.3.E2 inhibits antiestrogen-mediated induction of NAD- (P)H:quinone oxidoreductase enzyme activity in MCF-7 cells. MCF-7 cells were treated with different concentrations of TOT and ICI or Ϫ5 with 10 M TBHQ with or without E2 at the concentrations indicated. Cells were harvested after 4 days of treatment. Cytosolic extracts were assayed for QR activity as described. A similar extent of inhibition of QR activity by TOT and ICI was observed with 10Ϫ6 M and 10Ϫ7 M Ϫ6 Ϫ8 E2, and therefore only 10 M and 10 ME2data are shown. Values are the means Ϯ SE from three separate experiments.

gene transactivation providing further evidence that functional ER is required (Fig. 4). The absence of stimulation by antiestrogen of a reporter construct lacking the 41-bp EpRE-containing fragment indi- cates that TOT stimulation of Gst Ya transcriptional activity requires this region (Fig. 5A). Because the EpRE͞AREisvery similar to a TRE, and TOT can have regulatory effects on

FIG. 2. Antiestrogens, but not estrogen, induce NAD(P)H:quinone oxidoreductase enzyme activity in MCF-7 breast cancer cells: Con- centration-dependence and time course. (A) Effect of different com- pounds on QR activity in MCF-7 cells. Cells were treated with different concentrations of E2, TOT, tamoxifen (TAM), ICI, TBHQ, or TPA. Cells were harvested after 4 days of treatment. (B) Time course of induction of QR activity in MCF-7 breast cancer cells. MCF-7 cells were treated with the indicated concentrations of E2, TOT, ICI, or TBHQ. Cells were then harvested at the indicated time points. Cytosolic extracts were assayed for QR activity as described. Values are the means Ϯ SE from three separate experiments.

the TOT-mediated increase in transcriptional activity. Anties- trogen-mediated transcriptional activation was not observed FIG. 4. Antiestrogen induction of EpRE-GstYa-GH reporter gene with an ER DNA binding mutant, which lacks most (amino activity in 231 breast cancer cells is mediated by the ER. 231 cells were transfected with the p41–284GstYa-GH reporter construct along with acids 185–251) of the ER DNA binding domain and therefore an expression vector for the wild-type human ER, an expression vector does not bind to DNA, suggesting the requirement for an for a DNA binding mutant of the ER (ERDBDmut, missing amino acids interaction of the ER either directly with DNA elements 185–251), an expression vector for a mutant ER that lacks activation within the 41-bp EpRE-containing region or with an ERE- function-2 activity (ERS554fs), or the empty expression vector missing containing gene, whose stimulation is required for production the ER cDNA (pCMV). The cells were also transfected with a ␤-gal of͞or regulation of a protein factor that directly binds to the internal control reporter to correct for transfection efficiency. Cells EpRE-containing enhancer region and is responsible for the were then treated for 48 hr with varying concentrations of TOT, E2, or TBHQ as indicated. Media were collected for measurement of GH antiestrogen stimulation. Likewise, a transcriptionally inactive levels as described. The cells were then harvested for measurement of ER (S554fs), which binds antiestrogen but lacks activation ␤-gal activity. Values are the means Ϯ SE from three separate function-2 activity (19), failed to stimulate EpRE reporter experiments. Downloaded by guest on September 26, 2021 Medical Sciences: Montano and Katzenellenbogen Proc. Natl. Acad. Sci. USA 94 (1997) 2585

response to TBHQ or TOT. These findings, shown in Fig. 5C, indicate that loss of stimulation by antiestrogen or by TBHQ map identically to the EpRE element.

DISCUSSION We have shown that QR, a phase 2 detoxification enzyme, is markedly induced by antiestrogens in breast cancer cells. Moreover, the antiestrogens stimulated reporter gene tran- scription from EpRE͞ARE-containing constructs, and mu- tagenesis and other studies showed that the stimulation by antiestrogen and by TBHQ mapped identically to the 10-bp EpRE element. The transcriptional activation by antiestrogens appears to be mediated by the ER and requires a DNA- binding, transcriptionally active ER. Thus, antiestrogens may function as anticancer agents not merely by inhibiting estro- gen-mediated activation of gene transcription, but also by being able to activate a separate subset of genes, which may have beneficial effects. Our experiments indicate that QR can be activated by ER-independent and ER-dependent pathways. Activation by antiestrogens depends on the presence of a functional ER and requires much lower doses of compound (10Ϫ10–10Ϫ8 M) than stimulation by known activators of QR such as TBHQ (10Ϫ6– 10Ϫ5 M). While the signals between these two pathways may be integrated at some level, the ER-dependent pathway—which may involve induction or regulation of some protein that regulates the EpRE—must be different in part from the electrophile-dependent pathway used by TBHQ, where the ER is not at all required for QR stimulation. Electrophilic metab- olites are known to be generated from tamoxifen and E2; however, it is not clear that they would be generated from TOT, ICI164,384, or tamoxifen more so than from E2, which reverses the activation, nor are they likely to be produced in sufficient quantities from the low concentrations of these antiestrogens used in our experiments. Therefore, we think it is unlikely that the generation of electrophilic metabolites can account for the ER-dependent activity of the antiestrogens. FIG. 5. Antiestrogen induction of GstYa-GH reporter activity is mediated by an EpRE. 231 cells were transfected with an expression The EpRE-binding proteins that mediate induction of phase vector for the ER along with (A) pGstYa-GH reporter construct 2 genes by phenolic antioxidants have not been identified, but (missing the 41-bp EpRE-containing fragment), (B) TREX2- they do not appear to be AP1 (Fos͞Jun) proteins (28–30). GstYa-GH reporter construct, or (C) mutated p41–284GstYa-GH Likewise, regulation through the EpRE by antiestrogens that reporter constructs. The nucleotide sequence of the 41-bp EpRE- we have observed in these studies does not appear to be due containing fragment. The consensus EpRE, ETS binding site, and to endogenous AP1 activity. Our analyses indicate that al- GCA box are also indicated. The mutated nucleotides are indicated by though the ER is required for antiestrogen-mediated induction the boxed regions in mutants 1–5. The cells were also transfected with of transcriptional activity of EpRE-containing genes, the ER the ␤-gal internal reporter to correct for transfection efficiency. They were then treated with TOT (10Ϫ7 M) or TBHQ (10Ϫ5 M) for 48 hr. is not required for TBHQ-mediated activation. Furthermore, Media were collected for measurement of GH levels and the cells were the 41-bp EpRE-containing fragment shares no homology then harvested for measurement of ␤-gal activity as described. Values with an estrogen response element. The time course of en- are the means Ϯ SE from three separate experiments. dogenous QR enzyme activation by the antiestrogens and by TBHQ (Fig. 2), as well as the time course for QR mRNA transcription through TRE sites in some cells (26), we also increases (by 24 hr) and for stimulation of EpRE-reporter gene examined whether TOT treatment could elicit an increase in activity by both agents (increases first observed at 36 hr, data transcriptional activity through a TRE site. While TPA was not shown), suggest that these may be secondary, rather than able to stimulate transcriptional activity from the TRE- primary, gene transcriptional effects. Thus, the antiestrogen– containing reporter construct, no increase in transcriptional ER complex may enhance the production of (or activity of) activity was elicited by TOT or TBHQ (Fig. 5B). Thus, stimulatory factors that bind to the EpRE or promote the TOT-mediated induction of QR activity is unlikely to be release of inhibitory factors. Further studies will be needed to attributable to endogenous AP1 cell activity. completely elucidate the mechanisms of antiestrogen regula- To document that stimulation by the antiestrogen-occupied tion. ER observed in Fig. 4 was mediated through the EpRE within The QR gene is unusual in that it is an antiestrogen- the Gst Ya 41-bp region, we did oligonucleotide mutagenesis stimulated gene and shows ‘‘reversed pharmacology,’’ being over this region (Fig. 5C). Introduction of specific base pair induced in ER-containing cells by antiestrogen treatment and changes at the EpREs (M2 and M4) eliminated the response suppressed when the ER is occupied with estrogen. Indeed, to both TBHQ and TOT. Mutation of the GCA box (M5), a very few genes have been found to be up-regulated by anties- sequence that is highly conserved among EpRE-containing trogens, transforming growth factor ␤3 (31) and an unchar- fragments of different genes but is of unknown function acterized secreted protein of Ϸ39 kDa (32, 33) being the only (reviewed in ref. 27), greatly decreased both TBHQ and two identified to date. TOT-induced transcriptional activity. Mutations at other sites Increased QR activity in the presence of antiestrogens may within the 41-bp fragment (M1 and M3) had little effect on the have several important consequences. Although regulation of Downloaded by guest on September 26, 2021 2586 Medical Sciences: Montano and Katzenellenbogen Proc. Natl. Acad. Sci. USA 94 (1997)

the effectiveness of cancer chemotherapeutic agents is com- 12. Liang, P. & Pardee, A. B. (1992) Science 257, 967–971. plex, antiestrogen treatment might enhance the sensitivity of 13. Herman, M. E. & Katzenellenbogen, B. S. (1996) J. Steroid cells to those agents that are activated by quinone reduction, Biochem. Mol. Biol. 59, 121–134. such as mitomycin C and aziridylbenzoquinones (34). Also, 14. Friling, R. S., Bensimon, A., Tichauer, Y. & Daniel, V. (1990) being a two-electron reductant, QR can also provide protec- Proc. Natl. Acad. Sci. USA 87, 6258–6262. 15. Rushmore, T. H., Morton, M. R. & Pickett, C. B. (1991) J. Biol. tion against the deleterious effects of reactive oxygen species Chem. 266, 11632–11639. (35). This may contribute to the beneficial effects of anties- 16. Prestera, T. & Talalay, P. (1995) Proc. Natl. Acad. Sci. USA 92, trogens in cancer therapy and in chemoprevention. In addition, 8965–8969. the potential for antiestrogens to modulate the activity of 17. Kunkel, T. A., Roberts, J. D. & Zakour, R. A. (1987) Methods EpRE-containing genes has broad implications regarding the Enzymol. 154, 367–382. regulation of many phase 2 detoxification enzymes and other 18. Mead, D., Szczesna-Skorupa, E. & Kemper, B. (1986) Protein genes whose transcription is under the control of EpRE͞ Eng. 1, 67–74. AREs. 19. Wrenn, C. K. & Katzenellenbogen, B. S. (1993) J. Biol. Chem. 268, 24089–24098. We thank Paul Talalay (The School of 20. Kumar, V., Green, S., Staub, A. & Chambon, P. (1986) EMBO Medicine) and Pierre Chambon (Institut National de la Sante´etdela J. 5, 2231–2236. Recherche Me´dicale,Illkirch, France) for providing plasmids and 21. Liang, P., Averboukh, L. & Pardee, A. B. (1993) Nucleic Acids John Katzenellenbogen (University of Illinois) for helpful discussions. Res. 21, 3269–3275. This work was supported by National Institutes of Health Grant 22. Cho, H., Ng, P. A. & Katzenellenbogen, B. S. (1991) Mol. En- CA18119 and U.S. Army Breast Cancer Program Grant DAMD17- docrinol. 5, 1323–1330. 94-J-4205 (B.S.K.) and by a postdoctoral fellowship from the Susan G. 23. Montano, M. M., Mu¨ller, V., Trobaugh, A. & Katzenellenbogen, Komen Breast Cancer Foundation (M.M.M.). B. S. (1995) Mol. Endocrinol. 9, 814–825. 24. Reese, J. C. & Katzenellenbogen, B. S. (1991) J. Biol. Chem. 266, 1. Katzenellenbogen, B. S. (1991) J. Natl. Cancer. Inst. 83, 1434– 10880–10887. 1435. 25. Jaiswal, A. K., McBride, O. W., Adesnik, M. & Nebert, D. W. 2. Read, L. D. & Katzenellenbogen, B. S. (1991) in Genes, Onco- (1988) J. Biol. Chem. 263, 13572–13578. genes, and Hormones: Advances in Cellular and Molecular Biology 26. Webb, P., Lopez, G. N., Uht, R. M. & Kushner, P. J. (1995) Mol. of Breast Cancer, eds. Dickson, R. B. & Lippman, M. E. (Kluwer, Endocrinol. 9, 443–456. Boston), pp. 277–299. 27. Jaiswal, A. K. (1994) Biochem. Pharmacol. 48, 439–444. 3. Wakeling, A. E. (1995) Biochem. Pharmacol. 49, 1545–1549. 28. Nguyen, T., Rushmore, T. H. & Pickett, C. B. (1994) J. Biol. 4. Osborne, C. K., Elledge, R. M. & Fuqua, S. A. W. (1996) Sci. Am. Chem. 269, 13656–13662. Sci. Med. 3, 32–41. 29. Wang, B. & Williamson, G. (1994) Biochim. Biophys. Acta 1219, 5. McDonald, C. C. & Stewart, H. J. (1991) Br. Med. J. 303, 435– 645–652. 437. 30. Yoshioka, K., Deng, T., Cavigelli, M. & Karin, M. (1995) Proc. 6. Love, R. R., Richard, M. D., Mazess, R. B., Barden, H. S., Ep- Natl. Acad. Sci. USA 92, 4972–4976. stein, S., Newcomb, P. A., Jordan, V. C., Carbone, P. P. & 31. Yang, N. N., Venugopalan, M., Hardikar, S. & Glasebrook, A. DeMets, D. L. (1992) N. Engl. J. Med. 326, 852–856. (1996) Science 273, 1222–1225. 7. Bhimani, R. S., Troll, W., Grunberger, D. & Frenkel, K. (1993) 32. Bronzert, D., Silverman, S. & Lippman, M. (1987) Cancer Res. 47, Cancer Res. 53, 4528–4533. 1234–1238. 8. Ahotupa, M., Hirsimaki, P., Parssinen, R. & Mantyla, E. (1994) 33. Sheen, Y. Y. & Katzenellenbogen, B. S. (1987) Endocrinology Carcinogenesis 15, 863–868. 120, 1140–1151. 9. Wiseman, H. (1994) Trends Pharmacol. Sci. 15, 83–88. 34. Ross, D., Siegel, D., Beall, H., Prakash, A. S., Mulcahy, R. T. & 10. Talalay, P. (1989) Adv. Enzyme Regul. 28, 237–250. Gibson, N. W. (1993) Cancer Metastasis Rev. 12, 83–101. 11. Prestera, T., Zhang, Y., Spencer, S. R., Wilczak, C. A. & Talalay, 35. Cadenas, E. (1995) in Oxidative Stress and Antioxidant Defenses in P. (1993) Adv. Enzyme Regul. 33, 281–296. Biology, ed. Ahmad, S. (Chapman and Hall, New York), pp. 1–61. Downloaded by guest on September 26, 2021