Proc. Natl. Acad. Sci. USA Vol. 94, pp. 2581–2586, March 1997 Medical Sciences The quinone reductase gene: A unique estrogen receptor-regulated gene that is activated by antiestrogens (electrophile response elementytamoxifenyantioxidantybreast 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 breast cancer 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 estrogen receptor (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- y against cancer. It has been reported that the induction of the ment (EpREyARE), 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 EpREyARE 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 EpREyAREs. 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; EpREyARE, electro- phileyantioxidant 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; b-gal, b-galactosidase; ICI, ICI182,780. ‡To whom reprint requests should be addressed at: Department of Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA Molecular and Integrative Physiology, University of Illinois, 524 0027-8424y97y942581-6$2.00y0 Burrill Hall, 407 South Goodwin Avenue, Urbana, IL 61801. e-mail: PNAS is available online at http:yywww.pnas.org. [email protected]. 2581 Downloaded by guest on September 26, 2021 2582 Medical Sciences: Montano and Katzenellenbogen Proc. Natl. Acad. Sci. USA 94 (1997) Plasmids. The growth hormone (GH) reporter gene con- inhibitor (GIBCO) for 30 min at 378C. After phenoly 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 59 using 0.2 mg of DNA-free total RNA in 13 reverse transcrip- upstream 41-bp EpRE-containing region), and pTREX2– tion buffer (25 mM TriszCl, pH 8.3y37.6 mM KCly1.5 mM 284GstYa-GH [containing the mouse GST Ya gene minimal MgCl2y5mMDTT)and20mM each of dATP, dCTP, dGTP, promoter region and two consensus TPA response element and dTTP, and 0.2 mMofHindIII restriction site-containing (TRE) sites] were kindly provided by Paul Talalay (The John one-base anchored oligo(dT) primers [either H-T11A (59- Hopkins University School of Medicine) (16). AAGCTTTTTTTTTTTA-39), H-T11C (59-AAGCTT- Five reporter constructs, each containing mutations intro- TTTTTTTTTC-39), or HT11G (59-AAGCTTTTTTT- duced sequentially into the 41-bp fragment containing the TTTTG-39)]. After the solution was heated at 658C for 5 min EpRE, were constructed by site-directed mutagenesis (17), and cooled to 378C, 100 units of Moloney murine leukemia with modification (18). The ScaIySacI fragment of p41– virus reverse transcriptase was added for 1 hr. PCRs were 284GstYa-GH was inserted into the SacIySmaI site of Blue- performed in reaction mixtures containing 0.1 vol of reverse script II SK1 (Stratagene) to make p41–284GstYa-BSK1. transcription reaction mixture, 13 PCR buffer (10 mM TriszCl, Mutagenic oligonucleotides were then annealed to single- pH 8.4y50 mM KCly1.5 mM MgCl2y0.001% gelatin), 2 mM stranded DNA generated using the f1 origin of replication in each of dATP, dCTP, dGTP, and dTTP, 0.2 mMofHindIII Bluescript II SK1. The mutagenic oligonucleotides used in restriction site-containing arbitrary 13-mer oligonucleotide five separate mutagenesis reactions were: 59-CAGTGCCA- [either H-AP1 (59-AAGCTTGATTGCC-39), H-AP2 (59- AGCTCTGCAGAAAAATGACATTGC-39,59-AGCTTA- AAGCTTCGACTGT-39), H-AP3 (59-AAGCTTTGGT- GCTTGGGTCCATGGTTGCTAATGGTG-39,59-TTGGA- CAG-39), H-AP4 (59-AAGCTTCTCAACG) or H-AP5 (59- AATGACATTGCTCTTCGTGACAAAGCA-39,59-GACA- AAGCTTAGTAGGC-39)], 0.2 mM of the corresponding Hin- TTGCTAATTTGGATCCAGCAACTTTGTC-39, and 59- dIII restriction site-containing one-base anchored oligo(dT) TAATGGTGACAAAGATCTTGTGTCGACTCTAG-39. primer, 0.1 mCi of 35S-dATP (1 Ci 5 37 GBq), and 1 unit of To make each of the five mutated p41–284GstYa-GH reporter AmpliTaq DNA polymerase (Perkin–ElmeryCetus). Light constructs (M1–M5) the NdeIySacI fragment of p41– mineral oil was overlaid and the PCR reactions were per- 284GstYa-GH was then replaced with the mutated NdeIySacI formed at 948C for 30 sec and 40 cycles of 948C for 30 sec, 408C fragment of p41–284GstYa-BSK1. for 2 min, 728C for 30 sec, followed by 728C for 5 min. Stop The expression vector for the wild-type human ER and the buffer (95% formamidey10 mM EDTA, pH 8.0y0.09% xylene mutant ER that lacks activation function-2 activity (ERS554fs) cyanoly0.09% bromophenol blue) was added to each sample has been described (19). The expression vector for the ER and heated at 808C 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 y exposed to Kodak XAR-5 film for 48 hr.