AU8924398A - Differential ligand activation of estrogen receptors eralpha and erbeta at ap1 sites - Google Patents

Description

WO 99/11760 PCT/US98/18030 1 0 DIFFERENTIAL LIGAND ACTIVATION OF ESTROGEN RECEPTORS ERoc AND ERP AT AP1 SITES CROSS-REFERENCE TO RELATED INVENTIONS 5 [ Not Applicable ] This invention was made with the Government support under Grant No. GM 50872, awarded by the National Institutes ofHealth. The Government of the United States of America may have certain rights in this invention. 10 BACKGROUND OF THE INVENTION Estrogens, antiestrogens, and other nuclear transcription factor ligands are used in a wide variety of therapeutic contexts. Thus, for example, estrogens are used in the treatment of osteoporosis and other aspects (e.g., vasomotor instability) of menopause, in the treatment of hypoestrogenism, and in the regulation of fertility. 15 Antiestrogens are used in the treatment of cancer. Tamoxifen, for example, is an antiestrogen that is used in breast cancer chemotherapy and is believed to function as an antitumor agent by inhibiting the action ofthe estrogen receptor (ER) in breast tissue (see, e.g., (Sutherland et al. (1987) Cancer Treat. Revs., 15: 183-194). Glucocorticoids are used in the treatment of pure red cell anemia, acute renal failure due to acute 20 glomerulonephritis or vasculitis, lymphocytic leukemias, lymphomas, and other conditions. Progestins or progestational agents such as medroxyprogesterone or megestrol acetate are used in the treatment of endometrial carcinoma and breast carcinoma, and are used in the regulation of fertility. It has long been known that nuclear transcription factor ligands may have 25 profound and contradictory effects upon patients depending on physiological context. For example, estrogen and estrogen agonists may have beneficial effects, such as preventing osteoporosis and reducing serum cholesterol (Love, et al. (1992) New Eng. J Med. 326: 852-856; Love, etal. (1990) J. Natl. CancerInst. 82: 1327-1332). Conversely, agonistic activity may also be harmful. Tamoxifen for example sometimes increases endometrial 30 tumor incidence (lino et al. (1991) Cancer Treat. & Res. 53: 228-237) or switches from WO 99/11760 PCT/US98/18030 2 0 inhibition to stimulation of estrogen dependent growth in breast tumor progression (Parker (1992), Cancer Surveys 14: Growth Regulation by Nuclear Hormone Receptors. Cold Spring Harbor Laboratory Press). The related benzothiophene analog raloxifene (Figure 1A) has been reported to retain the antiestrogen properties of tamoxifen in breast tissue and to show 5 minimal estrogen effects in the uterus; in addition, it has potentially beneficial estrogen like effects (in nonreproductive tissue such as bone and cardiovascular tissue (Jones et al. (1984) J. Med Chem., 27: 1057-1066; Black et al. (1994) J. Clin. Invest., 93: 63-69; Sato et al. (1996) FASEB J., 10: 905-912; Yang et al. (1996) Endocrinol., 137: 2075 2084; Yang et al., (1996) Science, 273: 1222-1225). 10 One explanation for these tissue-specific actions ofantiestrogens is that the ligand-bound ER has different transactivation properties when bound to different types ofDNA enhancer elements. The estrogen receptor (ER) has been shown to mediate gene transcription both from the classical estrogen response element (ERE) and from an AP 1 enhancer element that requires ligand and the AP1 transcription factors Fos and Jun for 15 transcriptional activation (Fig. IB). In transactivation experiments, tamoxifen inhibits the transcription of genes that are regulated by a classical ERE, but like the natural estrogen hormone 173-estradiol [E 2 (Fig. 1 A)], tamoxifen activates the transcription of genes that are under the control of an API element (Webb, et al (1995) Mol. Endo., 9: 443-456). At the end of 1995, a second ER (ER3) was cloned from a rat prostate 20 cDNA library (Kuiper et al. (1996) Proc. Natl. Acad. Sci., USA, 93: 5925-5930). The human (Mosselman et al. (1996) FEBS Lett., 392: 49-53) and mouse (Tremblay et al. (1997) Mol. Endocrinol., 11: 353-365) homologs have also been cloned. The first identified ER has been renamed ERot (Kuiper et al. (1996) supra.). The existence of two ERs was postulated to present a potential new mechanism tissue-specific estrogen 25 regulation. From the foregoing, it is clear that the activity and regulation of nuclear transcription factor ligands, especially estrogens, is complex and the use of various transcription factor ligands can lead to contradictory and often adverse consequences. Thus, when electing to use a nuclear transcription factor ligand in a therapeutic context, 30 it is desirable to elucidate as precisely as possible the various modes of action (biological WO 99/11760 PCT/US98/18030 3 0 activities) of the agent(s) under consideration. Similarly, it has long been known that various environmental compounds have estrogenic and possibly antiestrogenic activity. When evaluating the impact of such environmental estrogens and/or antiestrogens, it is desirable to evaluate their effect on all metabolic pathways in which they might prove active. 5 SUMMARY OF THE INVENTION The present invention provides methods to rapidly and effectively screen compounds for their ability to activate or inactivate gene transcription in a previously unknown regulatory pathway: an estrogen receptor beta (ER3)-mediated AP 1 pathway. This invention is premised, in part, on the surprising discovery that ER13 is capable of 10 intereacting with AP 1 to induce transcription of a gene under AP 1 control. Even more surprising was the discovery that ER3-mediated AP 1 interactions can produce results significantly different than ERa-mediated AP 1 interactions. For example, estradiol, which activates gene expression through an ERx-mediated AP 1 interaction, actually inhibits gene activation through an ER1-mediated AP I interaction. 15 In one embodiment, this invention provides methods of screening test compounds for differential ERo-mediated and ER3-mediated activation at an AP 1 site. The methods typically involve providing a first cell comprising an estrogen receptor 13 (ER3), an AP 1 protein, and a construct comprising a promoter comprising an AP 1 site which regulates expression of a first reporter gene. The first cell is contacted with the test 20 compound and the expression of the first reporter gene is compared with ERa-mediated expression of a gene at an AP 1 site in response to the same test compound. The cell can contain a heterologous estrogen receptor beta (ER3) and preferred ER3s comprise an amino acid seqeunce of SEQ ID NO: 3 or SEQ ID NO: 5. The cell can also contain a heterologous AP1 protein. Preferred reporter genes used in this assay include 25 chloramphenicol acetyl transferase (CAT), luciferase, 13 -galactosidase (13-gal), alkaline phosphatase, horse radish peroxidase (HRP), growth hormone (GH), and green fluorescent protein (GFP) with a luciferase gene or a green fluorescent protein (gene) being preferred. The test compound can be a compound known or suspected to have anti estrogenic activity. The method can be one in which the ERa-mediated expression of a 30 gene at an AP1 site is determined by providing a second cell comprising an estrogen WO 99/11760 PCT/US98/18030 4 0 receptor a (ERa), AP I proteins, and a construct comprising a promoter comprising an AP1 site which regulates expression of a second reporter gene. The second cell is contacted with the test compound; and expression of the second reporter gene is detected. One preferred standard estrogen response element is from the Xenopus vitellogenin A2 gene. The second reporter gene and the first reporter gene can be the same species of 5 reporter gene. The cell and the second cell are the same cell. In one embodiment, this invention provides methods screening a test compound for the ability to activate or inhibit estrogen receptor beta (ER3) mediated gene activation at an API site. The methods typically involve providing a first cell comprising an estrogen receptor 13 (ER3), API proteins, and a construct comprising a promoter 10 comprising an API site which regulates expression of a first reporter gene. The cell is contacted with a test compound and expression of the first reporter gene is detected. The cell can contain a native or heterologous estrogen receptor beta (ER3). In a preferred embodiment, the ERP3 the amino acid sequence of Sequence ID No: 3 or Sequence ID No: 5. The first cell can also contain a heterologous AP1 protein (e.g,. jun and/or fos). 15 Virtually any reporter gene may be used. Preferred reporter genes include, but are not limited to chloramphenicol acetyl transferase (CAT), luciferase, 3 -galactosidase (13-gal), alkaline phosphatase, horse radish peroxidase (HRP), or green fluorescent protein (GFP) with a luciferase or a green fluorescent protein (GFP) being most preferred. Virtually any compound can be screened according to the methods of this invention. However, 20 preferred test compounds are compounds known to have anti-estrogenic activity. In another embodiment, the above method can further involve providing a second cell comprising an estrogen receptor a (ERa), AP 1 proteins, and a construct comprising a promoter comprising an API site which regulates expression of a second reporter gene. The second cell is contacted with the test compound and the expression 25 of the second reporter gene is then detected. In addition, or alternatively, the above method can involve providing a third cell comprising an estrogen receptor a (ERa), and a construct comprising a promoter comprising a standard estrogen response element (ERE) which regulates expression of a third reporter gene. The third cell is contacted with the test compound; and expression of the third reporter gene is then detected. One 30 standard estrogen response element can be from the Xenopus vitellogenin A2 gene.

WO 99/11760 PCT/US98/18030 5 0 Additionally or alternatively, the above method can also involve providing a fourth cell comprising an estrogen receptor P3 (ERP3), and a construct comprising a promoter comprising a standard estrogen response element (ERE) which regulates expression of a fourth reporter gene. The fourth cell is contacted with the test compound and expression ofthe fourth reporter gene is detected. Again the standard estrogen response element can 5 be from the Xenopus vitellogenin A2 gene. In one embodiment, the first cell and said third cell are the same cell, while in another embodiment, the first cell and said fourth cell are the same cell. Any of the above-described assays can be run to detect or identify inhibitors that block compounds that activate ER3-mediated AP 1 gene transcription. This 10 typically involves performing the assays as described above, but, in addition, contacting the first cell with a second compound, in addition to the test compound, wherein said second compound is known to activate transcription through estrogen receptor 03 (ER3) mediated gene activation at an API1 site. Detecting then comprises detecting test compound mediated decrease in said estrogen receptor 03 (ER3) mediated gene activation 15 at an API1 site. In a particularly preferred embodiment, the detecting can involve comparing the expression of the first reporter gene in the presence of the test compound and the second compound with the expression of the reporter gene in the presence of the second compound without the test compound. In one embodiment, the second compound known to activate transcription 20 through estrogen receptor 13 (ER3) mediated gene activation at an AP 1 site is identified by a method involving providing a second cell comprising an estrogen receptor 13 (ER3), and API protein, and a construct comprising a promoter comprising an AP1 site that regulates expression of a second reporter gene. The second cell is contacted with the second compound and the expression of the second reporter gene is detected where an 25 increase in expression of the second reporter gene produced by the compound indicates that said second compound activates transcription through ER3 at an AP 1 site. The assays ofthis invention can also be used to detect or identify inhibitors that block compounds that inhibit ER3-mediated AP 1 gene transcription. These methods involve performing the assays as described above, while additionally contacting the first 30 cell with a second compound, in addition to the test compound, where the second WO 99/11760 PCT/US98/18030 6 0 compound is known to inhibit transcription through estrogen receptor 3 (ER3) mediated activity at an AP 1 site. Expression of the reporter gene is detected where the detection comprises detecting test compound mediated increase in estrogen receptor 03 (ER3) mediated gene activation at an AP 1 site. The detecting can involve comparing expression of the first reporter gene in the presence of both the second compound and the test 5 compound with expression of the first reporter gene in the presence of the second compound without the test compound. The second compound known to inhibit transcription through estrogen receptor 03 (ER3) mediated gene activation at an AP 1 site can be identified by providing a second cell comprising an estrogen receptor 03 (ERIB), and AP 1 protein, and a construct 10 comprising a promoter comprising an AP1 site that regulates expression of a second reporter gene. The second cell is contacted with the second compound; and expression of the second reporter gene is detected. A decrease in expression of said second reporter gene produced by the second compound indicates that the second compound inhibits transcription through ER3 at the AP I site. 15 This invention also provides for any ofthe cells described above or herein. In one embodiment the cell comprises an estrogen receptor 13 (ER3), an AP 1 protein (e.g., jun or fos), and a construct comprising a promoter comprising an API1 site which regulates expression of a first reporter gene. The cell can additionally include a receptor for a nuclear transcription factor ligand preferably for a nuclear transcription factor ligand 20 other than estrogen. The cell preferably contains a heterologous ERD3, more preferably an ER3 comprising an amino acid sequence of Sequence ID No: 3 or Sequence ID No: 5. The AP I protein can be a native API protein or a heterologous AP I protein. The reporter gene can be one selected from the group consisting of chloramphenicol acetyl transferase (CAT), luciferase, 03 -galactosidase (03-gal), alkaline phosphatase, horse radish 25 peroxidase (HRP), and green fluorescent protein (GFP), but in particulary preferred embodiment, the reporter gene encodes a luciferase or a green fluorescent protein (GFP). The cell can additionaly include a standard estrogen response element (ERE) which regulates expression of a second reporter gene. One preferred standard estrogen response element is from the Xenopus vitellogenin A2 gene. Preferred cells of this invention are 30 mammalian cells and particularly preferred cells are derived from breast tissue or from WO 99/11760 PCT/US98/18030 7 0 uterine tissue. The cells may be neoplastic cells. Any of the above-described assays can be run to detect or identify inhibitors that block compounds that activate ER3-mediated API gene transcription. In still another embodiment, this invention provides methods of screening a nuclear transcription factor ligand for the ability to modulate estrogen receptor 03 5 mediated activation or inactivation of transcription at an AP 1 site. The methods involve providing a first cell containing an estrogen receptor 03 (ER3), an AP 1 protein, a receptor for the nuclear transcription factor ligand, and a construct comprising a promoter comprising an API1 site which regulates expression of a first reporter gene. The cell is contacted with the transcription factor ligand and with a compound having ER3 mediated 10 activity at the API site. Expression of the first reporter gene is then detected. The method can further involve providing a second cell containing an estrogen receptor 03 (ER3), a receptor for the nuclear transcription factor ligand, and a construct comprising a promoter comprising an estrogen response element (ERE) that regulates expression of a second reporter gene. The second cell is contacted with the 15 transcription factor ligand and with the compound having AP-1 mediated estrogenic activity and expression of the second reporter gene is detected. The first and second cells can be the same or different. Alternatively, or in addition, the method can further involve providing a second cell containing a cognate receptor of the transcription factor ligand, and a 20 promoter comprising a response element for the cognate receptor that regulates expression of a second reporter gene. The second cell is contacted with the transcription factor ligand and with the compound having compound having ER3 mediated activity at said API site expression of the second reporter gene is detected. Again, the first and second cells can be the same or different cells. 25 In any of the above-described methods the nuclear transcription factor ligand can be selected from the group consisting of a glucocorticoid, a progestin, vitamin D, retinoic acid, a an androgen, a mineralcorticoid, and a prostaglandin. Similarly, the cognate receptor can be selected from the group consisting of an estrogen receptor, a glucocorticoid receptor, a progestin PR-A receptor, and progestin PR-B receptor, 30 androgen receptor, a mineralcorticoid receptor, and a prostaglandin receptor. In a WO 99/11760 PCT/US98/18030 8 0 particularly preferred embodiment, the ER3 comprises an amino acid sequence of Figure 5 or Figure 6A. The ER3 can be a heterologous ER3. Similarly, the receptor for the nuclear transcription factor ligand can be heterologous to the cell. The cell can express an API protein (e.g., jun or fos) from a heterologous DNA. In one particularly preferred embodiment, the nuclear transcription factor is a progestin; and said receptor for the 5 nuclear transcription factor ligand is a progestin receptor. In another preferred embodiment, the nuclear transcription factor is a glucocorticoid and said receptor for said nuclear transcription factor ligand is a GR receptor. This invention also provides methods of screening an agent for the ability to alter modulation of estrogen receptor 03 (ER3) activation or inactivation of 10 transcription at an AP 1 site by a nuclear transcription factor ligand. The methods involve providing a first cell containing an estrogen receptor 3 (ER3), an AP 1 protein, a receptor for the nuclear transcription factor ligand, and a promoter comprising an AP 1 site which regulates expression of a first reporter gene. The first cell is contacted with the transcription factor ligand, with a compound having ERP3 mediated activity at an AP 1 site, 15 and with the agent and expression of the first reporter gene is detected. This method can further involve providing a second cell containing an estrogen receptor 3 (ER3), a receptor for the nuclear transcription factor ligand, and a promoter comprising an estrogen response element (ERE) that regulates expression of a 20 second reporter gene. The second cell is contacted with the transcription factor ligand and with the compound having AP-1 mediated estrogenic activity and expression of the reporter gene is detected. The first and second cell can be the same cell or different cells. The nuclear transcription factor can be one selected from the group consisting of a glucocorticoid, a progestin, vitamin D, retinoic acid, an androgen, a mineralcorticoid, a 25 prostaglandin. Similarly, the nuclear transcription factor ligand is selected from the group consisting of an estrogen receptor, a glucocorticoid receptor, a progestin PR-A receptor, progestin PR-B receptor, an androgen receptor, a mineralcorticoid receptor, and a prostaglandin receptor. Again, in any of the assays described herein, the ER3 can be a heterologous ERP3 and in a preferred embodiment, the ERP3 comprises an amino acid 30 sequence of Sequence ID No: 3 or Sequence ID No: 5 or is encoded by a nucleic acid WO 99/11760 PCT/US98/18030 9 0 sequence of Sequence ID No: 3 or Sequence ID No: 6. The API protein(s) and/or the receptor for the nuclear transcription factor ligand can also be native to the cell or heterologous. In one particularly preferred embodiment, the nuclear transcription factor is a progestin; and the receptor for said nuclear transcription factor ligand is a progestin receptor, while in another preferred embodiment, the nuclear transcription factor is a 5 glucocorticoid and the receptor for said nuclear transcription factor ligand is a GR receptor. This invention also provides kits for screening a compound for the ability to activate or inhibit estrogen receptor 03 (ERP3) mediated gene activation at an AP 1 site. The kits can include a container containing a cell comprising an estrogen receptor 03 10 (ER3), an API protein (e.g., jun and/or fos), and a construct comprising a promoter comprising an AP 1 site which regulates expression of a first reporter gene. The cell ofthe kits can further a receptor for a nuclear transcription factor ligand, preferably a nuclear transcription factor ligand other than estrogen. The kits can also further include instructional materials containing protocols for the practice of any of the assay methods 15 described herein. DEFINITIONS The terms "activate transcription" or "inhibit transcription" as used herein refer to the upregulation of transcription of a gene or the downregulation of transcription of a gene. It will be appreciation that either complete, or partial, "turning on" or "turning 20 off' are is regarded herein as activation or inhibition, respectively. Activation and inhibition of transcription are typically measured with respect to a control or controls where the control or controls involve a similar treatment lacking the compound or agent in question and/or contain a standard agent (e.g., E 2 or tamoxifen). It will also be appreciated that there may exist a baseline level of transcription (e.g, of a particular 25 reporter gene) even where an assay cell of this invention is "unstimulated" (e.g. the receptor in question is unliganded), i.e., without exogenously supplied ligand). In this case, it may be possible to see inhibition without necessarily applying exogenous activator see, e.g., Example 1). As used herein an antiestrogen is a compound that substantially inhibits 30 estrogen activity as measured in an assay for estrogenic activity, for example, cellular WO 99/11760 PCT/US98/18030 10 0 assays as described in Webb et al. Mol. Endocrinol., 6:157-167 (1993). More generally, a "transcription factor antagonist" is a compound that substantially inhibits transcription factor activity as measured in a standard assay for that transcription factor activity. A "nuclear transcription factor" as used herein refers to members of the nuclear transcription factor superfamily. This is a family of receptors that are capable of 5 entering the nucleus of a cell and once there, effecting the up-regulation or down regulation of one or more genes. A "nuclear transcription factor ligand" is a compound that binds to a nuclear transcription factor. Preferred nuclear transcription factors are typically steroid receptors, however, the group is not so limited. Nuclear transcription factor ligands include, but are not limited to estrogen, progestins, androgens, 10 mineralcorticoids, glucocorticoids, retinoic acid, vitamin D, and prostaglandins. Transcription factor ligands also include analogues of naturally occurring factors and blocking agents (antagonists) of such factors. Transcription factors also include, as they are identified, the ligands that bind orphan receptors (those nuclear transcription factors which have been identified by sequence homology, but whose ligand is yet unidentified). 15 It will be appreciated that when used in the context of a modulator of estrogen activity, the nuclear transcription factor ligand is typically one other than estrogen (or other than the estrogen or estrogen agonist whose activity is being modulated). Nuclear transcription factors typically mediate their activity through binding of a cognate receptor in the cell nucleus. The term cognate receptor" refers to a receptor of the type that is 20 typically bound by the transcription ligand in question. Thus, the cognate receptor for an estrogen is an estrogen receptor, the cognate receptor for a glucocorticoid is a glucocorticoid receptor, the receptor for a progestin is a progestin receptor, and so forth. The cognate receptor includes the native (naturally occurring) form as well as modified receptors. 25 The phrase estrogen receptor beta (ER3)-mediated activation or inactivation of gene transcription at an AP1 site refers to the activation or inactivation of a gene (e.g., a reporter gene) under control of an AP1 site by the interaction of that AP1 site with a liganded ER3 receptor. Similarly ERot-mediated activation or inactivation refers to gene regulation mediated by the interaction of ERc. Inactivation or inactivation 30 at an ERE refers to activation or inactivation of a gene under control of an ERE.

WO 99/11760 PCT/US98/18030 11 0 The phrase "differential ERa-mediated and ERB-mediated activation at an AP 1 site" refers to differences between ERa- and ERO-mediated gene activation at an AP1 site in response to the same ligand. Differential activation can be reflected in significant differences in levels of gene activation or inactivation by the same ligand depending on whether it interacts with ERa or ERP. Differential activation can also 5 reflect differences in the "sign" of gene activation. Thus differential activation can refer to ER3-mediated activation oftranscription at an AP 1 site and ERa-mediated inactivation of gene transcription at an API1 site in response to the same ligand. Conversely, differential activation can refer to ERP-mediated inactivation of transcription at an AP 1 site and ERo-mediated activation of gene transcription at an AP 1 site in response to the 10 same ligand. APl-mediated estrogenic/agonist activity, as used herein, refers to activation of a gene under the control of an AP 1 site (also referred to as an AP 1 response element) mediated by the interaction of a nuclear transcription factor with the AP 1 site. When used in reference to ER mediated activation of a gene controlled by the AP 1 site, 15 the pathway is referred to as the indirect estrogen response (in contrast to the classical estrogen response which is mediated through an ERE). A general description of the AP I site is found in Angel & Kann, Biochem. Biophys. Acta., 1072: 129-157 (1991) and Angel, et al., Cell, 49: 729-739 (1987). A "compound having API1 mediated estrogenic activity" refers to a 20 compound that, when present in a cell containing a gene under control of an AP 1 site and AP 1 proteins, activates transcription of the gene under control of the API1 site. A "compound having the ability to inactivate or inhibit estrogen receptor beta (ER3) mediated gene activation at an AP 1 site refers to a compound that is capable of upregulating or downregulating transcription of a gene under the control of an AP 1 site 25 through its interaction (e.g., binding) of an ER3. The phrases "modulate estrogen activation" or "modulation of estrogen activation" refer to alteration of the estrogen induced expression of a particular gene. Where the phrase additionally recites "at an AP1 site or at an ERE" the phrase refers to alteration of the level of estrogen induced expression of one or more genes under control 30 of the API site or ERE site respectively. The phrase "detecting expression" when used WO 99/11760 PCT/US98/18030 12 0 with reference to a reporter gene refers to detection of presence or absence of expression of the reporter gene or to quantification of expression level of the reporter gene. The quantification can be either an absolute measurement or a relative measurement (e.g., in comparison to another expressed gene). The term "operably linked" refers to functional linkage between a nucleic 5 acid expression control sequence (such as a promoter, signal sequence, or transcription factor binding site) and a second nucleic acid sequence, wherein the expression control sequence affects transcription and/or translation of the nucleic acid corresponding to the second sequence. The term "recombinant" when used with reference to a cell indicates that 10 the cell replicates a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous nucleic acid. Recombinant cells can express genes that are not found within the native (non-recombinant) form of the cell. Recombinant cells can also express genes found in the native form of the cell wherein the genes are modified and re introduced into the cell by artificial means. Recombinant expression refers to the 15 expression of the heterologous nucleic acid by such a recombinant cell. A "heterologous nucleic acid", as used herein, is one that originates from a foreign source (or species) or, if from the same source, is modified from its original form. Thus, a heterologous nucleic acid operably linked to a promoter is from a source different from that from which the promoter was derived, or, if from the same source, is 20 modified from its original form. Modification of the heterologous sequence may occur, e.g., by treating the DNA with a restriction enzyme to generate a DNA fragment that is capable of being operably linked to the promoter. Techniques such as site-directed mutagenesis are also useful for modifying a heterologous sequence. Similarly, a "heterologous protein" refers to a protein that originates from a foreign source (e.g., 25 different cell or species) or, if from the same source, is modified from its original form, or is expressed from a heterologous nucleic acid. A "recombinant expression cassette" or simply an "expression cassette" is a nucleic acid construct, generated recombinantly or synthetically, with nucleic acid elements that are capable of effecting expression of a structural gene in hosts compatible 30 with such sequences. Expression cassettes include at least promoters and optionally, WO 99/11760 PCT/US98/18030 13 0 transcription termination signals. Typically, the recombinant expression cassette includes a nucleic acid to be transcribed (e.g., a nucleic acid encoding a desired polypeptide), and a promoter. Additional factors necessary or helpful in effecting expression may also be used as described herein. For example, an expression cassette can also include nucleotide sequences that encode a signal sequence that directs secretion of an expressed protein 5 from the host cell. Xenoestrogens are defined here to include any compound having estrogenic activity in the assays described herein, which is derived from a source outside the human body. Environmental compounds as used herein can be derived from a wide variety of sources including plants, soil, water, foods. They also include synthetic 10 compounds such as chlorinated organics, polycyclic aromatic hydrocarbons, herbicides, pesticides, pharmaceuticals and the like. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 A illustrates the structure of five estrogen receptor (ER) ligands: Estradiol (E 2 ), diethylastilbestrol (DES), ICI 184,384, raloxifene (Ral), and tamoxifen 15 (Tam). Figure lB illustrates two estrogen receptor (ER) response elements: a simple (classical) estrogen response element (ERE) and an ER dependent AP 1 element described also in USSN 08/410,807, in USSN 60/051,309, and by Webb etal(1995)Mol. Endo., 9: 443-456. 20 Figure 2 illustrates ER3 action at an estrogen response element (ERE). HeLa cells were transfected with an ERE-regulated luciferase reporter plasmid and an expression vector for rat ER3 as described herein. Transfected cells were treated with the ligands (E2, 0.1 [tM; DES, 1 pM, Ral, 1 tM, tamoxifen 5 [tM; and ICI, 1 tM) or an ethyl alcohol (EtOH) vehicle control. All assays were done with at least triplicate transfections. 25 Error bars show deviations between wells from a single representative transfection. Figure 3 illustrates ERa(x action at an AP 1 element. HeLa cells were transfected with an AP 1 reporter plasmid and an ERa expression plasmid and treated with the five ligands (see, e.g., Figure 2). Ligand concentrations were E2, 0.1 tM; DES, 1 gM,; Ral, 1 tM; Tam, 5 gM, and ICI, 1 tM. Error bars are as in Figure 2.

WO 99/11760 PCT/US98/18030 14 0 Figure 4 illustrates ERP activation and inhibition at AP 1. (A) ER3 action at an AP 1 response element. HeLa cells were transfected with an AP 1 reporter plasmid and a rat ERP expression plasmid as described herein. Transfected cells were treated with the following ligand concentrations: E 2 , 0.1 gM; DES, 1 gM; Ral, 1 gM, Tam, 5 gM; and ICI, 1 /M. (B) Dose response of raloxifene induction with ER3 at an AP1 element. 5 HeLa cells transfected as described for A were treated with the indicated range of raloxifene concentrations. (D) Comparative inhibition of raloxifene induction by E 2 and DES. HeLa cells were transfected as described for (A) and treated with ligands. The left panel shows transactivation induction by raloxifene (1 gM), the lack of induction by E 2 (0.1 /M) and induction to the amount observed with the control (no ligand added). The 10 right panel shows the dose dependence of inhibition of raloxifene (1 PM) induction by DES (solid line) and E 2 (Dashed line). (D) Raloxifene overriding E 2 inhibition. HeLa cells were transfected as described for (A) and treated with ligands. The left panel shows the transcription induction resulting from the vehicle control (EtOH), Ral (1 pM) plus E 2 (10 nM), and E 2 (10 nM) alone. The right panel shows the dose dependence of raloxifene 15 induction in the presence of E 2 (10 nM). Figure 5 illustrates ligand-dependent ER3 activity in three cell types; Ishikawa cells, MCF7 cells and MDA453 cells. (A) Ligand-dependent ER3 action at an AP 1 element in Ishikawa cells. Ishikawa cells were transfected with an AP 1-regulated luciferase reporter plasmid and an ERI3 expression plasmid. Transfected cells were treated 20 with one or two ligands as indicated (E 2 , 0.1 aM; DES, 1 jM; Ral, 1 pM, Tam, 5 [M; and ICI, 1 pM; or an EtOH vehicle (control)). (B) Ligand dependent ER3 action at an AP 1 element in MCF7 cells. MCF7 cells were treated and analyzed as described for (A). Ligand dependent ER3 action at an AP1 element in MDA453 cells. MDA453 cells were treated and analyzed as described for (A). 25 DETAILED DESCRIPTION Antiestrogens are therapeutic agents for the treatment and possible prevention of breast cancer. Tamoxifen (Figure 1A), for example, is an antiestrogen that is used in breast cancer chemotherapy and is believed to function as an antitumor agent by inhibiting the action of the estrogen receptor (ER) in breast tissue (Grainger et al. 30 (1996) Nature Med., 2: 381-385). Paradoxically, tamoxifen appears to function as an WO 99/11760 PCT/US98/18030 15 0 estrogen-like ligand in uterine tissue, and this tissue-specific iatrogenic effect may explain the increased risk of uterine cancer that is observed with prolonged tamoxifen therapy (Kedar et al. (1994) Lancet, 343: 1318-1321). The related benzothiophene analog raloxifene (Fig. lA) has been reported to retain the antiestrogen properties of tamoxifen in breast tissue and to show minimal 5 estrogen effects in the uterus; in addition, it has potentially beneficial estrogen-like effects (in nonreproductive tissue such as bone and cardiovascular tissue (Jones et al. (1984) J Med. Chem., 27: 1057-1066; Black et al. (1994) J. Clin. Invest., 93: 63-69; Sato et al. (1996) FASEB J., 10: 905-912; Yang et al. (1996) Endocrinol., 137: 2075-2084; Yang etal., (1996) Science, 273:1222-1225)). One explanation for these tissue-specific actions 10 of antiestrogens is that the ligand-bound ER may have different transactivation properties when bound to different types of DNA enhancer elements. The classical estrogen response element (ERE) is composed of two inverted hexanucleotide repeats, and ligand-bound ER binds to the ERE as a homodimer (Fig. IB). The ER also mediates gene transcription from an AP 1 enhancer element that 15 requires ligand and the AP 1 transcription factors Fos and Jun for transcriptional activation (Fig. I B) (Umayahara et al. (1994) . Biol. Chem., 269:16433-16442). In transactivation experiments, tamoxifen inhibits the transcription of genes that are regulated by a classical ERE, but like the natural estrogen hormone 17b-estradiol [E 2 (Fig. lA)], tamoxifen activates the transcription of genes that are under the control of an AP 1 element (Webb 20 et al. (1995)Mol. Endocrinol., 9: 443-456). At the end of 1995, a second ER (ER3) was cloned from a rat prostate cDNA library (Kuiper et al. (1996) Proc. Natl. Acad. Sci. USA, 93: 5925-5930). The human (Mosselman et al. (1996) FEBS Lett., 392: 49-53) and mouse (Tremblay et al. (1997) Mol. Endocrinol., 11: 353-365) homologs were also cloned. The first identified 25 ER has been renamed ERac (Kuiper et al. (1996) supra.). It was a discovery of this invention that ER3 presents another source of tissue-specific estrogen regulation, particularly as mediated through the API1 site. In particular, it was a discovery of this invention that ERac and ER3 respond differently to certain ligands at an AP I element. The results described herein suggest different regulatory functions for the two ER 30 subtypes. This invention thus provides materials and methods for screening for WO 99/11760 PCT/US98/18030 16 0 compounds that exhibit differential activity depending on whether their activity is mediated through ERa or ER3. In addition, this invention provides materials and methods for determining whether a compound is capable of activate or inhibit estrogen receptor 03 (ER3) mediated gene activation (transcription) at an AP 1 site. I. Screening Methods and Compositions. 5 It was a discovery of this ER3 can interact with a AP 1 site to activate or inactivate expression (e.g.transcription) of a gene under the control of the AP1 site. Moreover, it was a particularly surprising discovery that putative estrogens can actually demonstrate "antiestrogenic" activity in an ERP/API1 pathway (where antiestrogenic activity in this context is as compared to the activity of an estrogen in the classical 10 ERat/ERE pathway). Thus, where an estrogen would activate transcription in an ERct/ERE pathway the estrogen inactivates transcription in an ERac/AP1 pathway. Conversely, putative antiestrogens can demonstrate estrogenic activity in an ER3/AP 1 pathway. This invention thus provides methods for detecting antiestrogenic activity of putative estrogens, or for detecting estrogenic activity of putative antiestrogens. More 15 generally, as explained below, this invention provides methods of screening compounds for the ability to activate or inhibit estrogen receptor 03 (ER3) mediated gene activation at an AP1 site. This allows identification of previously unsuspected environmental estrogens or antiestrogens or for screening of compounds for those that have desirable estrogenic or antiestrogenic properties. Such compounds are expected to be useful for 20 the treatment or the prevention of various cancers (e.g.breast cancer, ovarian cancer, endometrial cancer) and other diseases (e.g. endometriosis) mediated by estrogen. A) Screening for ER3 mediated API activation or inhibition. This invention provides efficient ways to screen large numbers of test compounds for the ability to activate or inhibit estrogen receptor 03 (ER3) mediated gene 25 activation at an AP 1 site. In one embodiment, the methods utilize a cell containing an estrogen receptor beta (ER3), an AP 1 protein, and a construct comprising a promoter and reporter gene under the control of an AP 1 site such that ERP3 interaction with the AP 1 site, can increase or inhibit expression (e.g., transcription) of the reporter gene. The cell is contacted with one or more compounds whose ER3 activity at AP1 it is desired to 30 evaluate. In a preferred embodiment, the expression level of the reporter gene in the cell WO 99/11760 PCT/US98/18030 17 0 contacted with the compound is compared to the expression level of a cell contacted by a control (e.g., identical culture conditions lacking the test compound and/or with a reference compound e.g., estradiol or tamoxifen). A decrease in expression level of the reporter gene indicates that the test compound inhibits ER3-mediated expression (transcription) at an AP 1, site, while an increase in expression level of the reporter gene 5 indicates that the test compound activates ER3-mediated expression (transcription) at an AP I site. The criteria used to evaluate a change in expression level of the reporter gene in this assay, and the other assays described herein, are those standard in the art. Thus, for example, a statistically significant difference in expression level between the test 10 and control experiments are scored as a valid change. In a preferred embodiment, the expression level may change by a factor 1.5 or more, preferably by factor of 2 or more, more preferably by a factor of 4 or more, and most preferably by a factor of 5 or even 10 or more. Screening for differential ERa and ERP3 mediated activity. 15 It will be appreciated that using the methods of this invention, the ability of compounds to activate or inhibit ER3-mediated transcription at an AP I site can be compared to the ability of those compounds to activate or inhibit ER3-mediated activity at an ERE site or to the ability of those compounds to activate or inhibit ERa-mediated activity at an APlor ERE. In this manner, compounds having a highly specific mode of 20 activity across a wide tissue distribution, or alternatively compounds having a highly variable mode of activity can be identified. Four preferred estrogen receptor based assays are illustrated in Table 1. These correspond to ERa-mediated ERE activity, ERa-mediated API activity, ERI3 mediated ERE activity, and ERO3-mediated AP 1 activity. It was a discovery ofthe present 25 invention that various compounds exhibit differential activity in these various assays.

WO 99/11760 PCT/US98/18030 18 0 Table 1. Illustration of estrogen receptor based assays. ER ER ERE/reporter Cla 3 gene ssical pathway classical pathway 5 AP1/reporter Ind P3 gene irect pathway indirect pathway This is illustrated in Table 2, where it can be seen that estrogen activates transcription in both the classical response (at an ERE) and in the indirect response (at an 10 AP 1) when the interaction is mediated by ERa. In contrast, estrogen acts as an inhibitor of transcription at AP I when the interaction is mediated by ER3. In contrast, the estrogen antagonist tamoxifen appears to always act as an inhibitor at an ERE, but an activator of transcription at an AP 1 site. Moreover, the activity of ER3 does not appear to be tissue restricted. 15 WO 99/11760 PCT/US98/18030 19 0 Table 2. Illustration of the activity of estradiol (E 2 ) and an estrogen antagonist (tamoxifen) in each of the ER assays. ER ER ERE/reporter gene Ac Ac 5 Estradiol tivates tivates Inh Inh Tamoxifen ibits ibits AP1/reporter gene Ac Inh 10 Estradiol tivates ibits Ac Ac Tamoxifen tivates tivates The assay for ERO-mediated API activity is described above. The 15 remaining assays are performed in an analogous manner. Thus, the ERc-mediated activity assays simply involve substituting ERa for ERO, and the ERE activity assays simply involve substituting the ERE/reporter gene construct for the AP1/reporter gene construct. The ERa assays (both for ERE and API activity) are described in detail in USSN 08/410,807, in USSN 60/051,309, and by Webb et al (1995) Mol. Endo., 9: 443-456). 20 The assay for ER3-mediated ERE activity utilizes a cell containing an estrogen receptor beta (ERI3), and a construct comprising a promoter and reporter gene under the control of an ERE site such that ERO interaction with the ERE site, can increase or inhibit expression (e.g., transcription) of the reporter gene. The cell is contacted with one or more compounds whose ER3 activity at an ERE it is desired to evaluate. In a 25 preferred embodiment, the expression level of the reporter gene in the cell contacted with the compound is compared to the expression level of a cell contacted by a control (e.g., identical culture conditions lacking the test compound and/or with a reference compound e.g., estradiol or tamoxifen). A decrease in expression level of the reporter gene indicates that the test compound inhibits ERO-mediated expression (transcription) at an ERE, while WO 99/11760 PCT/US98/18030 20 0 an increase in expression level of the reporter gene indicates that the test compound activates ER3-mediated expression (transcription) at an ERE site. While, in a preferred embodiment, each assay is performed in a separate cell, it will be appreciated that API and ERE assays can be combined and performed in a single cell. In this case, the AP1/reporter gene construct preferably utilizes a different 5 reporter gene than the ERE/reporter gene construct so that AP 1 activation or inactivation can be distinguished from ERE activation or inactivation. Screening for inhibitor activity. The above-describe assays can also be used to identify (screen for) compounds that inhibit other compounds which have ERa-mediated or ERO3-mediated 10 activity an ERE or at an AP- 1 site. These assays are performed in the same manner as the assays described above. In this instance, however, the cell is contacted with two compounds, a test compound that is being screened for inhibitory activity and a second compound for which an inhibitor (or alternatively an agonist) is sought. Thus, for example, where it is desired to identify a test compound having 15 ER3-mediated estrogen inhibitory activity at an AP 1 site, the cell containing ER3, an AP 1 protein, and a reporter gene under control of an AP 1 site is contacted with estrogen and the test compound. If the compound inhibits the characteristic ER3-mediated estrogen activity at AP 1, the compound is an inhibitor. It should be noted that in this case, ERO3 mediated estrogen activity at AP 1 inhibits transcription, thus an estrogen inhibitor in this 20 context actually increases ERO3-mediated transcription at AP1. This is illustrated in Example 1, where it is shown that tamoxifen is one such inhibitor. Inhibitors, or agonists, of ER3-mediated or ERa-mediated estrogenic or antiestrogenic activity at ERE and at AP 1 can be screened in an analogous manner. D) Screening for environmental estrogens or antiestrogens. 25 As indicated above, this invention allows for screening of test compounds for estrogenic or antiestrogenic activity mediated through ER3 or ERa at an ERE or at an AP1 site. The assays are particularly useful for screening environmental compounds for estrogenic or antiestrogenic activity. Environmental compounds having estrogenic activity are referred to here as xenoestrogens. Xenoestrogens include any compound 30 derived from a source outside the human body, having estrogenic activity in the assays WO 99/11760 PCT/US98/18030 21 0 described herein. Environmental compounds as used herein can be derived from a wide variety of sources including plants, soil, water, foods. They also include synthetic compounds such as chlorinated organics, polycyclic aromatic hydrocarbons, herbicides, pesticides, pharmaceuticals and the like. It will be appreciated that environmental estrogens often are only weakly 5 active. Consequently, particularly when testing an environmental compound for estrogenic or antiestrogenic activity, it is often desirably to maximize sensitivity of the assay. This may be accomplished by using cells that produce the methods typically comprise cultured cells that produce high levels of the human estrogen receptor (ERc or ERP3). Such cells include, but are not limited to MCF-7 cells (ATCC No. HTB 22), MDA453 cells (ATCC 10 No. HTB 131), ZR-75-1 cells (ATCC No. CRL 1500) or ERC1 cells described in Kushner et al. (1990) Mol. Endocrinol., 4:1465-1473, and ERC2 and ERC3 cells as described by Webb et al. (1993) Mol. Endocrinol., 6:157-167. It is also known that environmental estrogens may show synergistic activity in combination. Thus, in one embodiment, two or more suspected environmental 15 estrogens are assayed according to the above methods in combination. It will be recognized, however, that such combined testing is not limited simply to environmental estrogens but rather, any combination of agents can be screened simultaneously. Screening for transcription factor modulation of ER3 activity at AP 1. It has been demonstrated that various nuclear transcription factors (e.g., 20 progesterone, glucocorticoids, etc.) interact with the ERot-mediated estrogenic activity at the API site (see, e.g., USSN 60/051,309). It is believed that ER3 is also capable of such interactions at AP 1. Thus, in another embodiment, this invention provides assays (methods of screening) nuclear transcription factor ligands, and putative or known transcription factor ligand agonists or antagonists for the ability to modulate ER3 25 mediated activation or inactivation of transcription at an AP 1 site. These assays are performed in the same manner as the assays described above, however the assay cell additionally contains a receptor for a second nuclear transcription ligand (preferably a ligand other than estrogen). Thus, the cell contains an estrogen receptor beta (ER3), an AP 1 protein, a receptor for a second nuclear 30 transcription factor ligand, and a construct comprising a promoter comprising an AP 1 site WO 99/11760 PCT/US98/18030 22 0 which regulates expression of a reporter gene. The cell is contacted with both a transcription factor ligand that is to be screened and with a compound having ER3 mediated activity at an AP 1 site. Alteration of the typical activity (level of API regulated reporter gene expression) ofthe compound having ERO3-mediated activity at an AP 1 site by the presence 5 of the compound being screened (the test transcription factor ligand) indicates that the screened compound is capable of modulating an ERO3-mediated AP I response of the compound having ERP-mediated activity at an API1 site. Preferred second nuclear transcription factor ligands include, but are not limited to glucocorticoids, progestins, vitamin D, retinoic acid, androgens, mineralcorticoids, and prostaglandins. 10 Similarly, inhibitors, or agonists, of the test compound can be screened by running the same assay in the presence of the inhibitor that is to be screened. II. Cell Types The assay methods of this invention provide methods for evaluating the ability of a test, or control, compound to activate or inhibit transcription through 15 interaction with a transcription factor receptor (e.g., estrogen receptor). Thus, in a preferred embodiment, the cells used in the assays of this invention preferably contain at least one transcription factor receptor. For example, where it is desired to screen for activity of a compound mediated by the estrogen receptor cc (ERa) cells are preferably provided that contain ERoa 20 and where it is desired to screen for activity of a compound mediated by estrogen receptor 03 (ER3) cells are preferably provided that contain ER3. Where it is desired to screen for the ability of a nuclear transcription factor ligand modulate estrogen receptor (a or 03) mediated activation or inactivation of transcription at an AP 1 site, the cell preferably include, in addition to the particular ERa 25 or ERJ3 at least a second nuclear transcription factor receptor (e.g., glucocorticoid receptor (GR)). Cells that naturally express one or more of the desired receptor types can be used in the assays of this invention. Alternatively, cells can be modified (e.g., through recombinant DNA techniques) to express ERa and/or ER3 and/or the transcription factor receptor of choice.

WO 99/11760 PCT/US98/18030 23 0 Suitable cells for practicing the methods of this invention include, but are not limited to cells derived from a uterine cervical adenocarcinoma (HeLa) , a hypothalamic cell line (GTI-1 (Mellon et al. (1990) Neuron, 5: 1-10), MCF-7 cells (ATCC No. HTB 22), MDA453 cells (ATCC No. HTB 131), ZR-75-1 cells (ATCC No. CRL 1500) or ERC1 cells described in Kushner et al., Mol. Endocrinol., 4:1465-1473 5 (1990). ERC2 and ERC3 cells as described by Webb, et al. Mol. Endocrinol., 6:157-167 (1993). It will be appreciated that the invention is not limited to practice in mammalian cells and may be practiced, for example in yeast and insect cells, transfected with the appropriate genes and recombinant constructs. A) Cells naturally expressing two or more receptor types. 10 Many cells that express a second transcription factor receptor in addition to the estrogen receptor (ER) are well known to those of skill in the art. Thus, for example, in the uterus there is evidence that ER and glucocorticoid receptors (GR) co exist in the endometrium (Prodi et al. (1979) Tumor. 65: 241-253). In the brain, maps of ER and GR immunoreactivity and mRNA localization suggest co-localization in certain 15 cerebral nuclei such as the paraventricular nucleus ofthe hypothalamus, the hypothalamic arcuate nucleus, and the central nucleus of the amygdala (Fuxe et al. (1985) Endocrinol., 118: 1803-1812; Simerly et al. (1990) J Comp. Neurol. 294: 76-95). In bone, ER and have been found in cultured osteoblast-like cells (Liesegang et al. (1994) J. Andrology, 14: 194-199). ER has also been demonstrated in osteoclasts (Oursler et al. (1994) Proc. 20 Natl. Acad. Sci., USA, 91: 5227-5231) and data suggest that the glucocorticoid dexamethasone (Dex) regulates metabolism in these cells (Wong (1979) J Biol. Chem., 254: 6337-6340) raising the possibility that osteoclasts contain functional GR as well. In addition, numerous tumor cell lines have been demonstrated to have both ER and GR (Ewing et al. (1989) Int. J. Cancer., 44: 744-752. 25 B) Cells recombinantly modified to express two or more receptor types. Cells normally lacking the ERa or ERI3 or other transcription factor cognate receptors can be recombinantly modified to express one or more of the desired receptors. Typically this involves transfecting the cell with an expression cassette comprising a nucleic acid encoding the receptor of interest and culturing the cell under 30 conditions where the receptor is expressed (e.g., in the presence of an appropriate inducer WO 99/11760 PCT/US98/18030 24 0 if the promoter regulating expression of the receptor is inducible). Typically, the cassette is selected to provide constitutive expression of the receptor. A cell that naturally expresses one receptor need only be modified to express the second receptor. However, if the cell expresses neither receptor, it may be transfected with expression cassettes expressing both receptors. Even where a cell 5 naturally expresses one or both receptors, it may be recombinantly modified to express those receptors at a higher level (e.g., by introducing expression cassettes encoding the receptor(s) whose expression level it is desired to increase). The cells need not contain "native" receptors, but may be modified to provide truncated or chimeric receptors to provide increased affinity and/or sensitivity of 10 the assay. Thus, for example, Berry, etal.(1990), EMBOJ, 9:2811-2818, describe the production of cells containing truncated or chimeric ER receptors. Methods of modifying cells to express particular receptors are well known to those of skill in the art. Thus, for example, cells modified to express high levels of estrogen receptor are described by Kushner et al. (1990), Mol. Endocrinol., 4:1465-1473. 15 See also Hirst et al. (1990) Mol. Endocrinol., 4: 162-170). Transfection of cells to express ERoa is described below, in the Examples, and in US SN 08/410,807. Transfection of cells to express ERi3 is described herein, and transfection of cells to express glucocorticoid receptors (GR), progestin receptors (PR), and other receptors is described in copending USSN 60/043,059. 20 C) Cells Containing API proteins. In assays that involve screening for transcription factor receptor mediated activation or inactivation oftranscription at AP 1, the cells preferably contain one or more AP 1 proteins (the Jun or Fos proteins or other members of that protein family, see Bohmaan, et al. (1987) Science, 238: 1386-1392) in addition to the transcription factor 25 receptor(s). The cells can naturally express the AP 1 protein(s) or they can be modified (e.g., by transfection with a suitable expression cassette) to express a heterologous AP 1 protein. Methods of expressing AP I proteins are well known to those of skill in the art (see, e.g., Turner et al. (1989) Science, 243:1689-1694 and Cohen et al. (1989) Genes WO 99/11760 PCT/US98/18030 25 0 & Dev., 3: 173-184, and Example 1). Cells that naturally express one or more API proteins may still be so modified to increase intracellular jun and/or fos levels. III) Expression of Nuclear Transcription Factor Receptors. As explained above the assays of this invention utilize cells containing one or more nuclear transcription factor receptors (e.g., ERa, ER3, GR, PR, etc.) an estrogen 5 receptor and a receptor for a nuclear transcription factor (typically a transcription factor other than estrogen). The factor can be one that is expressed endogenously by the cell or, alternatively, the cell can be modified (e.g., a recombinant cell) so that it expresses the receptor. A) Estrogen Receptor Alpha (ERa) 10 An estrogen receptor, as used herein, includes an estrogen receptor alpha (ERa) in its native (naturally occurring) form as well as modified estrogen receptors. Numerous modifications of estrogen receptors are known to those of skill in the art. These include, but are not limited to VP 16-ER, V-ER, a chimeric receptor comprising the strong VP 16 transcriptional activation domain linked to the amino terminus of the ER, V 15 ER in which the ER DNA binding domain (DBD) is deleted, H 11 an ER lacking the DNA binding domain, and the like (see e.g., Kumar et al., Cell, 51: 941-951 (1987) and Elliston et al. (1990) JBiol Chem 265:11517-21). Means ofrecombinantly expressing the estrogen receptor alpha (ERa) are well known to those of skill in the art (see, e.g., US SN 08/410,807 and Webb et al (1995) 20 Mol. Endocrinol., 9: 443-456). B) Estrogen Receptor Beta (ERP3). Estrogen receptor beta (ER3) is a second estrogen receptor (ER) cloned from a rat prostate cDNA library (Kuiper et al. (1996) Proc. Natl. Acad. Sci. USA, 93: 5925-5930). Subsequently the human (Mosselman et al. (1996) FEBSLett., 392: 49-53) 25 and mouse (Tremblay et al. (1997) Mol. Endocrinol., 11: 353-365) homologs were cloned. Accordingly, the original estrogen receptor (ER) has been renamed ERa (Kuiper et al. (1996) supra.). Using the known sequence information one of skill in the art can routinely construct vectors that express an ER3 when transfected into a suitable host cell. Detailed WO 99/11760 PCT/US98/18030 26 0 protocols for the preparation of an ER3 vector can be found in Kuiper et al. (1996) Proc. Natl. Acad. Sci. USA, 93: 5925-5930 and in WO 97/09348. It will be appreciated that exist a number of different estrogen beta receptors comprising various splice variants, mutations, and so forth. It will be appreciated that ER3 as used herein is intended to include all ER3 variants. However, in 5 a preferred embodiment, the ER3 variants used in this invention correspond to the so called "intermediate length" ER3 variants such as those described in WO 97/09348. Particularly preferred ER3 variants are shown in sequence listings 3, 4, and 5 herein which correspond to figures 1 and 13A and 13B of WO 97/09348, C) Nuclear transcription factor ligand and cognate receptor 10 As indicated above, the in addition to the estrogen receptor (ERaC and/or ER3), the cells can contain a cognate receptor for a nuclear transcription factor ligand whose interaction (preferably a cognate receptor other than an estrogen receptor). As used herein, the term "cognate receptor" refers to a receptor of the type that is typically bound by the transcription factor ligand in question. Thus, the cognate receptor for an 15 estrogen is an estrogen receptor, the cognate receptor for a glucocorticoid is a glucocorticoid receptor, the receptor for a progestin is a progestin receptor, and so forth. As with the estrogen receptor, the cognate receptor includes the native (naturally occurring ) form as well as modified receptors. Natural and modified cognate receptors for nuclear transcription factor 20 ligands, particularly for steroid nuclear transcription factors, are well known to those of skill in the art. These include, but are not limited to the glucocorticoid receptors, the progestin receptors (e.g., PR-A, PR-B (see, e.g., Law et al. (1987) Proc. Natl. Acad. Sci. USA 84: 2877-2881; Wei et al. (1988) Mol. Endo. 2: 62-72; and Kushner et al. (1990) Mol. Endocrinol, 4:1465-1473), vitamin D receptors, mineralcorticoid receptors, 25 androgen receptors, and thyroid hormone receptors (see, Mangelsdorf (1995) Cell, 83: 835-839). IV. ERE and API Reporter Constructs The cells of this invention preferably contain (e.g., are transfected with) nucleic acid constructs comprising one or more reporter genes under the control of a 30 response element (either the AP 1 site or estrogen response element (ERE)). Where two WO 99/11760 PCT/US98/18030 27 0 different response elements are monitored in a single cell, two different reporter genes are used. Thus, for example, one gene can reports transcription induced by the classical estrogen response system (ERE), while the other gene reports transcription induced by the indirect (AP 1) estrogen response. The two reporter genes and response elements are typically placed in separate cells, but the methods can also be used with both constructs 5 in the same cell. A) AP1/Reporter construct. In one embodiment the methods of this invention involve providing a cell containing an estrogen receptor (ERa. or ERi3), and a promoter comprising an AP I site that regulates expression of a reporter gene (also referred to herein as the reporter gene 10 for the indirect estrogen response pathway (see, e.g., USSN 08/410,807 and Webb et al (1995) Mol. Endocrinol., 9: 443-456). The reporter gene for the indirect estrogen response pathway contains an API1 site preferably upstream of the target promoter and capable of regulating (i.e., operably linked to) that promoter. AP 1 site are sites that are bound by AP 1 (the Jun and 15 Fos proteins) or other members of that protein family. In a preferred embodiment, the consensus API site (or API response element) is TGA(C/G)TCA (SEQ ID NO: 1). One of skill would recognize that the particular API1 site used is not a critical aspect ofthe invention. Any sequence capable ofbeing bound by AP 1 or members of that family and regulating a promoter is suitable. This would include promoters which 20 encompass a naturally occurring AP 1 site. Typical promoters include, but are not restricted to metalloprotease genes such as stromelysin, gelatinase, matrilysin, and the human collagenase gene. Alternatively promoters may be constructed which contain a non-naturally occurring AP1, or related, binding site. This facilitates the creation of reporter gene 25 systems that are not typically found under the control of AP 1. In addition, promoters may be constructed which contain multiple copies of the API1 site thereby increasing the sensitivity or possibly modulating the response the reporter gene system. B) ERE/Reporter Construct The methods of this invention can also involve providing a cell containing 30 a promoter comprising an estrogen response element that regulates expression of a WO 99/11760 PCT/US98/18030 28 0 reporter gene (also referred to herein as the reporter gene for the direct or classical estrogen response pathway (see, e.g., U.S.S.N. 08/410,807 and Webb, etal. (1995)Mol. Endo., 9: 443-456). This permits detection of the "direct" (classical) estrogen response and evaluation of the interaction or modulation of the classical response by the nuclear transcription factor ligand. 5 Typically, the estrogen response element (ERE) is upstream of the target promoter and capable of regulating that promoter. In a preferred embodiment the ERE may be the consensus estrogen response element AGGTCACAGTGACCT (SEQ ID NO: 2) from the Xenopus vitellogenin A2 gene. The particular ERE used in the cell is not a critical aspect of the invention and the present invention is not limited to the use of any 10 one particular ERE. Suitable EREs are well known to those of skill. For instance, other sources of naturally occurring EREs include the vitellogenin B2 gene, the chicken ovalbumin gene, and the PS2 gene. Alternatively, non-naturally occurring EREs may be inserted into particular promoters. The consensus ERE from theXenopus vitellogenin A2 gene is widely used for this purpose, but other EREs may be used as well. 15 C) Reporter Gene(s) The present invention is not limited to a particular reporter gene. Any gene that expresses an easily assayable product will provide a suitable indicator for the present assay. Suitable reporter genes are well known to those of skill in the art. Examples of reporter genes include, but are not limited to CAT (chloramphenicol acetyl 20 transferase) (Alton and Vapnek (1979) Nature 282: 864-869), luciferase, and other enzyme detection systems, such as beta-galactosidase; firefly luciferase (deWet et al. (1987) Mol. Cell. Biol. 7:725-737); bacterial luciferase (Engebrecht et al. (1984) Proc. Natl. Acad. Sci., USA, 1: 4154-4158; Baldwin etal. (1984) Biochemistry 23:3663-3667); alkaline phosphatase (Toh et al. (1989) Eur. J. Biochem.182: 231-238; Hall et al. (1983) 25 J. Mol. Appl. Gen. 2: 101), and green fluorescent protein. One of skill will recognize that various recombinant constructs comprising the AP-1 site can be used in combination with any promoter and reporter gene compatible with the cell being used. The promoter will preferably be one susceptible to regulation by the AP I site. 30 D) Construction of the Promoter/Reporter Expression Cassette.

WO 99/11760 PCT/US98/18030 29 0 The promoter/reporter expression cassettes and, other expression cassettes (constructs) described herein, can be constructed according to ordinary methods well known to those of skill in the art. Construction of these cassettes is variously exemplified in Example 1, in USSN 08/410,807, in Webb et al. (1995)Mol. Endo. 9: 443-456, and in other references cited herein. 5 The constructs can all be created using standard amplification and cloning methodologies well known to those of skill in the art. Examples of these techniques and instructions sufficient to direct persons of skill through many cloning exercises are found in Berger and Kimmel, Guide to Molecular Cloning Techniques: Methods inEnzymology, 152 Academic Press, Inc., San Diego, CA; Sambrook et al. (1989) Molecular Cloning 10 A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY,; Current Protocols in Molecular Biology, Ausubel et al., eds., Current Protocols, ajoint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel); Cashion etal., U.S. Patent No: 5,017,478; and Carr, European Patent No. 0,246,864. Examples oftechniques sufficient to direct persons 15 of skill through in vitro amplification methods are found in Berger supra., Sambrook supra., and Ausubel supra., as well as Mullis et aL, (1987) U.S. Patent No. 4,683,202; Innis et al. (1990) PCR Protocols A Guide to Methods andApplications, Academic Press Inc. San Diego, CA; Arnheim & Levinson (October 1, 1990) C&EN36-47; The Journal Of NIH Research (1991) 3: 81-94; Kwoh et al. (1989) Proc. Natl. Acad Sci. USA 86: 20 1173; Guatelli etal. (1990)Proc. Natl. Acad. Sci. USA 87, 1874; Lomell etal. (1989) J. Clin. Chem., 35: 1826; Landegren et al., (1988) Science, 241: 1077-1080; Van Brunt (1990) Biotechnology, 8: 291-294; Wu and Wallace, (1989) Gene, 4: 560; and Barringer et al. (1990) Gene, 89: 117. V. ER3-mediated Activation through tethered coativactors. 25 In still another embodiment, ER3 can mediate gene activation through virtually any response element using a tethered transcription factor coactivator strategy. The methods involve contacting a nucleic acid that includes the gene of interest operably linked to a response element with a tethered coactivator. The tethered coactivator is composed of a polypeptide that comprises an activation function derived from a 30 transcriptional coactivator, and a DNA binding moiety that is capable of specifically WO 99/11760 PCT/US98/18030 30 0 binding to the response element. The tethered coactivator is contacted with an activated transcription factor polypeptide (e.g., ER3) that includes an activation function derived from a transcription factor. The contacting of the tethered coactivator with the activated transcription factor polypeptide stimulates expression of the gene. The transcription factor can be, for example, a nuclear hormone receptor such as the estrogen receptor or 5 the estrogen receptor beta, or an AP 1 transcription factor, however, in a preferred embodiment, the transcription factor is ER3. Detailed protocols for the tethered transcription factor activation strategy are provided in copending USSN 60/043,059. VI. Detection of the reporter genes. Detection of the reporter genes of this invention is by standard methods 10 well known to those of skill in the art. Where the reporter gene is detected through its enzymatic activity this typically involves providing the enzyme with its appropriate substrate and detecting the reaction product (e.g., light produced by luciferase). The detection may involve simply detecting presence or absence of reporter gene produce, or alternatively, detection may involve quantification of the level of expression of reporter 15 gene products. The quantification can be absolute quantification, or alternatively, can be comparative e.g., with respect to the expression levels of one or more "housekeeping" genes. Methods of quantifying the expression levels of particular reporter genes are well known to those of skill in the art. It will be appreciated that such detection can be performed "manually" or may be automated e.g., as in a high-throughput screening 20 system. High throughput assays for the presence, absence, or quantification ofgene expression (e.g., via the detection ofthe transcribed nucleic acid (mRNA) or the detection of gene expression (protein product)) are well known to those of skill in the art. Thus, for example, U.S. Patent 5,559,410 discloses high throughput screening methods for proteins, 25 U.S. Patent 5,585,639 discloses high throughput screening methods for nucleic acid binding (i.e., in arrays), while U.S. Patents 5,576,220 and 5,541,061 disclose high throughput methods of screening for ligand/antibody binding. In addition, high throughput screening systems are commercially available (see, e.g., Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; 30 Beckman Instruments, Inc. Fullerton, CA; Precision Systems, Inc., Natick, MA, etc.).

WO 99/11760 PCT/UJS98/18030 31 0 These systems typically automate entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. Compounds to be Screened. 5 It will be appreciated that virtually any compound can be screened by the methods of this invention. Such compounds include, but are not limited to known or suspected estrogens or antiestrogens including environmental estrogens or environmental antiestrogens as described above. It will be appreciated that compounds are expected to be show the most 10 estrogenic or antiestrogenic activity if they are capable of penetrating to the nucleus of a cell and binding to a transcription factor receptor (e.g., ERca or ER3). Such compounds are often lipophilic or capable of entering cells passively through pores or gates, through active transport, or through endocytosis. Particularly preferred compounds include, but are not limited to, steroid compounds or steroid analogs. 15 VIII. Assay Kits In another embodiment, this invention provides kits for the practice of the methods of this invention. The kits preferably include one or more containers containing the cells described herein for the practice of the assays of this invention. Thus, for example, the cells may include, but are not limited to, cells containing an estrogen 20 receptor 03 (ER3), AP 1 protein(s), and a construct comprising a promoter comprising an AP 1 site which regulates expression of a first reporter gene, or such cells additionally containing a receptor for a nuclear transcription factor ligand other than estrogen. The AP1/recporter gene and the ERE/reporter gene constructs can be in separate cells or together in the same cell. The cells may additionally express high levels of AP 1 proteins 25 such as fos and/orjun. Alternatively, or in addition, the kits can contain the AP1/reporter gene and/or the ERE/reporter gene constructs described herein and/or the ERot, ERO3, or other nuclear transcription factor receptor vectors. The kits may optionally contain any of the buffers, reagents, culture media, culture plates, reporter gene detection reagents, and so forth that are useful for the practice of the methods of this invention.

WO 99/11760 PCT/US98/18030 32 0 In addition, the kits may include instructional materials containing directions (i.e., protocols) for the practice of the assay methods of this invention. While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not 5 limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials. EXAMPLES The following examples are offered to illustrate, but not to limit the present 10 invention. Example 1 Comparison of the Transactivation Properties of ERc and ER3 This example describes the investigation of the transactivation properties of ERoc and ER3 with a panel of five ER ligands with the use of a reporter gene under the 15 control of either a classical ERE or an AP 1 element. The results presented herein show that ERcc and ER3 respond differently to certain ligands at an AP I element suggesting different regulatory functions for the two ER subtypes. Screening Methods 20 The transactivation properties of ERa and ERP3 were compared with a panel of five estrogen receptor (ER) ligands using a reporter gene under the control of either a classical estrogen response element (ERE) or an AP 1 element. The ERE and AP 1 driven luciferase reporter plasmids (EREII-LucGl45 and Acoll78, respectively) and the ERca expression plasmid (pSG5-HEO) were used as described in Webb et al. (1995)Mol. 25 Endocrinol., 9:443-456, and in USSN 08/410,807 now issued as U.S. Patent, _ The rat ERP3 expression vector has been previously described (Kuiper et al. (1996) Proc. Natl. Acad. Sci. USA, 93: 5925-5930). The full-length human ER3 cDNA which was isolated from an ovarian cDNA library and found to be identical to the 30 previously reported partial cDNA clone (Mosselman et al. (1996) FEBSLett., 392: 49- WO 99/11760 PCT/US98/18030 33 0 53) was cloned into the pCMV5 eukaryotic expression vector and the resulting ERP3 expression vector was used for these experiments (see, Kuiper et al. (1996) Proc. Natl. Acad. Sci. USA, 93: 5925-5930). The ligands used to compare ERoc and ER3 transactivation properties included the estrogens 3-estradiol (E 2 ) and diethylstilbestrol (DES) and the antiestrogens Imperial Chemical Industries (ICI) 164384, tamoxifen, and 5 raloxifene. Raloxifene was synthesized according to published procedure (Jones et al. (1984) J. Med. Chem., 27: 1057). Structure and purity were verified by 'H nuclear magnetic resonance (NMR), 8 C NMR, ultraviolet thin layer chromatography, and high resolution mass spectrometry. ICI 164384 was obtained from a private source and the other compounds were obtained from commercial sources. 10 The experiments were conducted by transfecting HeLa cells with either an ERoc or ERI3 expression plasmid along with a reporter plasmid that contained a luciferase gene under the transcriptional control of an estrogen response element (ERE). Cells were grown in Nunc Delta Surface tissue culture plates to a density of not more than 5 x 10' per cm 2 . Cells were grown in 0.1 gm sterile filtered DME-F-12 15 Coon's Modified Medium (Sigma Cell Culture) with 15 mM Hepes, 0.438 g/L L glutamine, 1.338 g/L NaHCO 3 , 10% Seru-Max 4 (an iron supplemented, formula fed newborn calf serum, Sigma Cell culture; from a lot tested for low estrogenic activity), 0.05 mg/mL Gentamycin, 100 mg/ml Streptomycin SO 4 , and 100 units/ml penicillin "G". Ishikawa cells were grown in a medium containing 100 nM tamoxifen and 20 MCF-7 cells were grown in medium containing 10 nM estradiol. For the transfection assays, cells were suspended 0./5 ml ofelectroporation buffer in 0.4 cm gap electroporation cuvettes (BioRad) at 106 to 2 x 106 cells per cuvette. The electroporation buffer was prepared as a solution of 500 ml phosphate buffered saline (PBS), 5 ml of 10% glucose, and 50 lL of Biobrene. Five ag of reporter plasmid and 6 25 ag ofER expression plasmid were added and the cuvette was agitated to facilitate mixing of the solution and homogeneous cell distribution in the cuvette. Cells were then immediately transfected by electroporation with a BioRad GenePulser electroporation apparatus at a potential of 0.25 kV and a capacitance of 960 PF. To the electroporation cuvettes was added 1 ML growth medium (described above).

WO 99/11760 PCT/US98/18030 34 0 The transfected cells for one experiment were pooled and carefully resuspended in growth medium at a density of 8 x 104- 1.6 x 10s cells/mL. After a homogenous cell distribution was obtained by thorough mixing cells were plated on Nunc 6-well dishes at 2 mL per well. After 2 h of incubation hormones were added and the medium was mixed by gentle swirling. Cells were then incubated in the presence of 5 hormone for 40-48 hours. Growth medium was removed from the wells, and the cells were washed with Mg 2 ' and Ca 2 ' free PBS, and then they were lysed chemically with 0.2 mL of 100 mM potassium phosphate buffer (pH 7.5) containing 0.2% Triton X- 100 and 1 mM DTT). The plates were then frozen to -80*C, thawed and scraped with a rubber policeman to 10 loosen and break up cell fragments. The lysate was centrifuged in a microfuge for 2 min, 0.1 mL of the supernatant was combined with 0.3 mL luciferase assay solution, and the chemiluminescence was measured immediately for a period of 10 s. The luciferase assay solution consisted of 25 nM glycylglycine, 15 mM MgSO4, 4 mM EGTA, 15 mM potassium phosphate at pH 7.8, with the addition ofDTT 15 to a final concentration of 1 mM, ATP to a final concentration of 2 mM and luciferin (Analytical Luminescence Laboratories) to a final concentration of200 gM shortly before commencing the assay. Luminescence measurements were performed on a Monolight 1500 (Analytical Luminescence Laboratories). The relative light units reported here were adjusted to a scale of 100 for uniformity. 20 The data were collected using the HIEO ER variant. HEO shows reduced transactivation response from the unliganded receptor compared with the wild-type ER resulting in clearer ligand-induced transactivation data. Each experiment with ERa was also checked with the wild-type ER (HEGO), and the general ligand induction trends were found to the same as those obtained with HEO. The only difference was that the ligand 25 induced transactivation responses were lower with HEGO than with the control (no ligand added). Transactivation experiments were performed with both rat and human ER3 and identical trends in ligand behavior and similar induction levels were seen with both ERP3s in HeLa cells. The data shown in Figure 2B and Figure 4 were obtained with the 30 rat ER3 expression plasmid.

WO 99/11760 PCT/US98/18030 35 0 Experiments and Results The transactivation properties of ERa and ER3 at a classical ERE in response to the estrogens E 2 and diethylstilbestrol (DES) and the antiestrogens Imperial Chemical Industries (ICI) 164384, tamoxifen, and raloxifene were first investigated. Both 5 ERa (18) and ER3 (Fig. 2) showed the same transactivation profiles with the panel of ligands. E 2 and DES stimulated luciferase production 10-fold over ICI 164384, raloxifene, tamoxifen, and the control (no ligand added). The antiestrogens blocked E 2 stimulation in ligand competition experiments. Next, the ligand-induced transactivation behavior of ERa and ER3 at an 10 AP 1 site was examined. With ER, all five ligands stimulated luciferase transcription, including the antiestrogens ICI 164384, tamoxifen, and raloxifene (Fig. 3). This stimulation was dependent on transfected ER, as cells transfected with only the reporter plasmid showed no induction of reporter transcription. Of the five ligands, raloxifene induced transcription the least, showing twofold induction compared with the sixfold 15 inductions typically seen with E 2 and tamoxifen. The raloxifene-induced transactivation was dose dependent with a concentration value required for one-half maximal activation

(EC

50 ) of about 1 nM. In addition, raloxifene reduced the activation caused by E 2 in a dose-dependent manner to the amount observed with raloxifene alone, demonstrating that raloxifene induction is weaker than induction by E 2 and that raloxifene-induced 20 transactivation results from binding to ERa. If E 2 is classified as a full activator of ERa at an API1 element (ERa-AP1), then raloxifene functions as a partial activator and tamoxifen functions as a full activator. In contrast to the results seen with ERa-AP 1, a difference in the ligand activation profile of ERO at an AP1 element (ERO3-AP1) was observed. In cells 25 transfected with ERP, treatment with the estrogens E 2 and DES did not increase luciferase transcription over the control (no ligand added), whereas treatment with the antiestrogens ICI 164384, raloxifene, and tamoxifen increased luciferase transcription (Fig. 4A). This transcription activation required transfected ER3, as wells that were transfected with only the reporter plasmid did not show transcriptional activation by the antiestrogens. The 30 transcriptional activation caused by raloxifene was dose dependent with an ECs 5 0 value of WO 99/11760 PCT/US98/18030 36 0 about 50 nM (Fig. 4B). In ligand competition experiments, both E 2 and DES were able to block the raloxifene induction, and both estrogen ligands were able to reduce raloxifene induction to the basal level of transcription in a dose-dependent manner with concentration values required for one-half maximal inhibition of 1 to 10 nM (Fig. 4C). In a different ligand competition experiment, the inhibitory effect on 5 transcription resulting from E 2 treatment could be overcome by higher concentrations of raloxifene in a dose-dependent manner (Fig. 4D). Thus, it appears that the pharmacology of ER ligands is reversed at an AP 1 element with ER3; with ERO-AP1, the antiestrogens act as transcription activators, and the estrogens act as transcription inhibitors. It was next investigated whether the action ofERO3-AP 1 could be observed 10 in cell lines derived from estrogen target tissues such as the uterus and breast. Transactivation assays for ER3-AP 1 were performed in Ishikawa cells (a human uterine cell line) (Fig. 5A) and in MCF7 (Fig. 5B) and MDA453 (Fig. Sc) human breast cancer cells. (The human ERP was used for transactivation in these cells.) In each of these cell lines, the ligands acted the same as they did in the HeLa cells; the three antiestrogens 15 activated and the estrogens inhibited ER3-dependent transcription from an AP 1 site (Fig. 5). No induction was seen with cells that were not transfected with the ER expression plasmid, indicating that the antiestrogen induction required ERP3. Antiestrogen induction in the breast cell lines was higher than that observed in HeLa cells. Transfected MCF7 cells treated with raloxifene gave a 20- to 80-fold transactivation response over the 20 control (no ligand added). In addition, raloxifene and ICI 164384 induced transcription more than tamoxifen in the breast cell lines(Fig. 5, B and C). MCF7 cells did not appear to contain high concentrations of endogenous ERO mRNA (Kuiper et al. (1997) Endocrinol., 138: 553); however, the results suggest that the additional transactivation machinery required for ERO3-AP1 function is present in 25 these cells. With two of these target tissue cell lines, E 2 treatment reduced the amount of transcription to less than that seen with the control (no ligand added). In MDA453 (Fig. 5C) and Ishikawa cells (Fig. 5A), E 2 treatment resulted in a consistent 40 to 75% reduction of reporter transcription levels compared with the control. This effect was also observed in ligand competition experiments (Fig. 5, A and C); E 2 and DES blocked 30 raloxifene induction and reduced the amount of transcription to less than that seen for the WO 99/11760 PCT/US98/18030 37 0 control. Thus, when ERP3 is bound by the estrogen hormone E 2 or the synthetic estrogen DES, it functions as a negative regulator of genes controlled by an ER-dependent API1 element. The ER is the only known member of the steroidal subfamily of nuclear receptors that has different subtypes (Mangeldorf et al. (1996) Cell, 83: 835-839). 5 Nuclear receptors that respond to nonsteroidal hormones that have different known subtypes include the thyroid receptor (TRa and TR3), the retinoic acid receptor (RARa, RAR3, and RARy), and the retinoid X receptor (RXRa, RXR3, and RXRy) (Mangelsdorf et al. (1996) Cell, 83: 841-850). The results presented herein demonstrate that two nuclear receptor subtypes can respond in opposite regulatory modes to the natural 10 hormone from the same DNA response element. Moreover, the ligand-induced responses with ERP3 at an AP 1 site provide an example of negative transcriptional regulation by the natural hormone and strong positive regulation by synthetic antiestrogens. (The genes for transforming growth factor and quinone reductase are ER-regulated genes controlled by promoters containing nonclassical EREs that are activated by antiestrogens. However, 15 the action of ERP at either of these promoters has not been reported. The action of ERa on the quinone reductase gene shows a similar ligand profile to that of ERP at an AP1 site; antiestrogens are transcription activators, and E 2 is a transcription inhibitor. If signaling from ER-dependent AP1 elements occurs in estrogen target tissues, the finding herein that ERa and ER3 respond differently to ligands at API sites 20 reveals a potential control mechanism for transcriptional regulation ofestrogen-responsive genes and adds a layer of complexity in analyzing the pharmacology of antiestrogen therapeutics. The role of E 2 complexed to ER3 would be to turn off the transcription of these genes, whereas the antiestrogens raloxifene, tamoxifen, and ICI 164384 could override this blockade and activate gene transcription. It will be T helpful to search for 25 genes in estrogen target tissues that are transcriptionally regulated by ER3 at an AP 1 site and to characterize the phenotype of cells in which these genes are activated. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereofwill 30 be suggested to persons skilled in the art and are to be included within the spirit and WO 99/11760 PCT/US98/18030 38 0 purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA (ii) TITLE OF INVENTION: DIFFERENTIAL LIGAND ACTIVATION OF ESTROGEN RECEPTORS ERalpha AND ERbeta AT AP1 SITES (iii) NUMBER OF SEQUENCES: 6 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Fulbright & Jaworski L.L.P. (B) STREET: 865 S. Figueroa Street, 29 th Floor (C) CITY: Los Angeles (D) STATE: California (E) COUNTRY: USA (F) ZIP: 90017-2576 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: BERLINER, Robert (B) REGISTRATION NUMBER: 20,121 (C) REFERENCE/DOCKET NUMBER: 5555-497 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 213-892-9200 (B) TELEFAX: 213-680-4518 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: (A) NAME/KEY: (B) LOCATION: 1..7 (D) OTHER INFORMATION: /note= "AP1 response element" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: TGASTCA 7 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base pairs (B) TYPE: nucleic acid

Claims (71)

1. A method of screening a test compound for differential ERa-mediated and ERO-mediated activation at an AP 1 site, said method comprising the steps of: a) providing a first cell comprising an estrogen receptor 03 (ER3), an API protein, and a construct comprising a promoter comprising an AP 1 site which regulates 5 expression of a first reporter gene; b) contacting said first cell with said test compound; and c) comparing the expression of said first reporter gene with the ERt mediated expression of a gene at an AP 1 site.

2. The method of claim 1, wherein said first cell contains a heterologous 10 estrogen receptor beta (ER3).

3. The method of claim 1, wherein said ER3 comprises an amino acid seqeunce of SEQ ID NO: 3 or SEQ ID NO: 4.

4. The method of claim 1, wherein said cell contains a heterologous API1 protein. 15

5. The method of claim 1, wherein said reporter gene is selected from the group consisting ofchloramphenicol acetyl transferase (CAT), luciferase, 03 -galactosidase (3-gal), alkaline phosphatase, horse radish peroxidase (HRP), growth hormone (GH), and green fluorescent protein (GFP).

6. The method of claim 5, wherein said reporter gene encodes a luciferase 20 or a green fluorescent protein (GFP).

7. The method of claim 1, wherein said test compound is a test compound known to have anti-estrogenic activity.

8. The method of claim 1, wherein said ERt-mediated expression ofa gene at an API site is determined by: 25 d) providing a second cell comprising an estrogen receptor a (ERa), AP 1 proteins, and a construct comprising a promoter comprising an AP 1 site which regulates expression of a second reporter gene; e) contacting said second cell with said test compound; and f) detecting expression of said second reporter gene WO 99/11760 PCT/US98/18030 47 0

9. The method of claim 8, wherein said standard estrogen response element is from the Xenopus vitellogenin A2 gene.

10. The method of claim 8, wherein said second reporter gene and said first reporter gene are the same reporter genes.

11. The method of claim 8, wherein said first cell and said second cell are 5 the same cell.

12. A method of screening a test compound for the ability to activate or inhibit estrogen receptor 3 (ER3) mediated gene activation at an AP 1 site, said method comprising the steps of: a) providing a first cell comprising an estrogen receptor 3 (ER3), API1 10 proteins, and a construct comprising a promoter comprising an AP 1 site which regulates expression of a first reporter gene; b) contacting said first cell with said test compound; and c) detecting expression of said first reporter gene.

13. The method of claim 12, wherein said first cell contains a heterologous 15 estrogen receptor 3 (ER3).

14. The method of claim 12, wherein said ER3 comprises the amino acid sequence of Seq ID No: 3 or Seq ID NO: 5.

15. The method of claim 14, wherein said first cell contains a heterologous API protein. 20

16. The method of claim 12, wherein said reporter gene is selected from the group consisting of chloramphenicol acetyl transferase (CAT), luciferase, 03 galactosidase (03-gal), alkaline phosphatase, horse radish peroxidase (HRP), growth hormone (GH), and green fluorescent protein (GFP).

17. The method of claim 16, wherein said reporter gene encodes a 25 luciferase or a green fluorescent protein (GFP).

18. The method of claim 12, wherein said test compound is a test compound known to have anti-estrogenic activity.

19. The method of claim 12, further comprising the steps of: WO 99/11760 PCT/US98/18030 48 0 d) providing a second cell comprising an estrogen receptor ac (ERa), AP1 proteins, and a construct comprising a promoter comprising an AP I site which regulates expression of a second reporter gene; e) contacting said second cell with said test compound; and f) detecting expression of said second reporter gene. 5

20. The method of claim 12, further comprising the steps of: d) providing a third cell comprising an estrogen receptor a (ERa), and a construct comprising a promoter comprising a standard estrogen response element (ERE) which regulates expression of a third reporter gene; e) contacting said third cell with said test compound; and 10 f) detecting expression of said third reporter gene.

21. The method of claim 20, wherein said standard estrogen response element is from the Xenopus vitellogenin A2 gene.

22. The method of claim 12, further comprising the steps of: d) providing a fourth cell comprising an estrogen receptor 03 (ER3), and 15 a construct comprising a promoter comprising a standard estrogen response element (ERE) which regulates expression of a fourth reporter gene; e) contacting said fourth cell with said test compound; and f) detecting expression of said fourth reporter gene.

23. The method of claim 22, wherein said standard estrogen response 20 element is from the Xenopus vitellogenin A2 gene.

24. The method of claim 20, wherein said first cell and said third cell are the same cell.

25. The method of claim 22, wherein said first cell and said fourth cell are the same cell. 25

26. The method of claim 12, further comprising contacting said first cell with a second compound, in addition to said test compound, wherein said second compound is known to activate transcription through estrogen receptor 03 (ERP3) mediated gene activation at an AP 1 site; wherein said detecting comprises detecting test compound mediated 30 decrease in said estrogen receptor 03 (ER3) mediated gene activation at an AP 1 site. WO 99/11760 PCT/US98/18030 49 0

27. The method of claim 26, wherein said detecting comprises comparing the expression of said first reporter gene in the presence of the test compound and the second compound with the expression of said first reporter gene in the presence of the second compound without the test compound.

28. The method of claim 26, wherein said second compound known to 5 activate transcription through estrogen receptor 03 (ER3) mediated gene activation at an AP 1 site is identified by a method comprising the steps of: a) providing a second cell comprising an estrogen receptor 03 (ER3), and AP 1 protein, and a construct comprising a promoter comprising an AP 1 site that regulates expression of a second reporter gene; 10 b) contacting said second cell with second compound; and c) detecting the expression of said second reporter gene, wherein an increase in expression of said second reporter gene produced by said compound indicates that said second compound activates transcription through ER3 at said AP 1 site.

29. The method of claim 12, further comprising contacting said first cell 15 with a second compound, in addition to said test compound, wherein said second compound is known to inhibit transcription through estrogen receptor 03 (ER3) mediated activity at an AP 1 site; and wherein said detecting comprises detecting test compound mediated increase in estrogen receptor 03 (ER3) mediated gene activation at an AP 1 site.

30. The method of claim 29, wherein said detecting comprises comparing 20 the expression of said first reporter gene in the presence of said second compound and said test compound with the expression of said first reporter gene in the presence of said second compound without said test compound.

31. The method of claim 29, wherein said second compound known to inhibit transcription through estrogen receptor 03 (ER3) mediated gene activation at an 25 API site is identified by a method comprising the steps of: a) providing a second cell comprising an estrogen receptor 03 (ER3), and AP 1 protein, and a construct comprising a promoter comprising an AP 1 site that regulates expression of a second reporter gene; b) contacting said second cell with second compound; and WO 99/11760 PCT/US98/18030 50 0 c) detecting the expression of said second reporter gene, wherein a decrease in expression of said second reporter gene produced by said compound indicates that said second compound inhibits transcription through ER3 at said AP I site.

32. A cell comprising an estrogen receptor 03 (ER3), AP1 proteins, and a construct comprising a promoter comprising an AP I site which regulates expression of 5 a first reporter gene.

33. The cell of claim 32, wherein said cell further contains a receptor for a nuclear transcription factor ligand other than estrogen.

34. The cell of claim 32, wherein said cell contains a heterologous estrogen receptor 13 (ER3). 10

35. The cell of claim 32, wherein said cell contains a heterologous AP 1 protein.

36. The cell of claim 32, wherein said heterologous API protein is c-jun.

37. The cell of claim 32, wherein said first reporter gene is selected from the group consisting of chloramphenicol acetyl transferase (CAT), luciferase, 03 15 galactosidase (3-gal), alkaline phosphatase, horse radish peroxidase (HRP), growth hormone (GH), and green fluorescent protein (GFP).

38. The cell of claim 37, wherein said reporter gene encodes a luciferase or a green fluorescent protein (GFP).

39. The cell of claim 38, wherein said cell further comprises a construct 20 comprising a promoter comprising a standard estrogen response element (ERE) which regulates expression of a second reporter gene.

40. The cell of claim 39, wherein said standard estrogen response element is from the Xenopus vitellogenin A2 gene.

41. The cell of claim 32, wherein said cell is a mammalian cell. 25

42. The cell of claim 41, wherein said cell is derived from breast tissue or from uterine tissue.

43. A method of screening a nuclear transcription factor ligand for the ability to modulate estrogen receptor 03 mediated activation or inactivation oftranscription at an AP 1 site, said method comprising the steps of: WO 99/11760 PCT/US98/18030 51 0 a) providing a first cell containing an estrogen receptor 03 (ER3), an AP I protein, a receptor for said nuclear transcription factor ligand, and a construct comprising a promoter comprising an AP 1 site which regulates expression of a first reporter gene; b) contacting said first cell with said transcription factor ligand and with a compound having ERO mediated activity at said AP I site; and 5 c) detecting expression of said first reporter gene.

44. The method of claim 43, further comprising the steps of: d) providing a second cell containing an estrogen receptor 03 (ER3), a receptor for said nuclear transcription factor ligand, and a construct comprising a promoter comprising an estrogen response element (ERE) that regulates expression of a 10 second reporter gene; e) contacting said second cell with said transcription factor ligand and with said compound having AP-1 mediated estrogenic activity; and f) detecting expression of said second reporter gene.

45. The method of claim 44, wherein said first cell and said second cell are 15 the same cell.

46. The method of claim 43, further comprising the steps of: d) providing a second cell containing a cognate receptor of said transcription factor ligand, and a promoter comprising a response element for said cognate receptor that regulates expression of a second reporter gene; 20 e) contacting said second cell with said transcription factor ligand and with said compound having compound having ER3 mediated activity at said AP 1 site; and f) detecting expression of said second reporter gene.

47. The method of claim 46, wherein said first cell and said second cell are the same cell. 25

48. The method of claim 43, wherein said nuclear transcription factor ligand is selected from the group consisting of a glucocorticoid, a progestin, vitamin D, retinoic acid, a an androgen, a mineralcorticoid, and a prostaglandin.

49. The method of claim 46, wherein said cognate receptor is selected from the group consisting of an estrogen receptor, a glucocorticoid receptor, a progestin WO 99/11760 PCT/US98/18030 52 0 PR-A receptor, and progestin PR-B receptor, androgen receptor, a mineralcorticoid receptor, and a prostaglandin receptor.

50. The method of claim 43, wherein said ERP comprises an amino acid sequence of Seq ID No: 3 or SEQ ID No: 5.

51. The method of claim 43, wherein said estrogen receptor ER3 is 5 heterologous to said cell.

52. The method of claim 43, wherein said receptor for said nuclear transcription factor ligand is heterologous to said cell.

53. The method of claim 43, wherein said cell expresses an AP 1 protein from a heterologous DNA. 10

54. The method of claim 53, wherein said API protein is c-jun.

55. The method of claim 43, wherein said nuclear transcription factor is a progestin; and said receptor for said nuclear transcription factor ligand is a progestin receptor.

56. The method of claim 43, wherein said nuclear transcription factor is 15 a glucocorticoid and said receptor for said nuclear transcription factor ligand is a GR receptor. WO 99/11760 PCT/US98/18030 53 0

57. A method of screening an agent for the ability to alter modulation of estrogen receptor 03 (ER3) activation or inactivation of transcription at an AP 1 site by a nuclear transcription factor ligand, said method comprising the steps of: a) providing a first cell containing an estrogen receptor 03 (ER3), an AP 1 protein, a receptor for said nuclear transcription factor ligand, and a promoter comprising 5 an AP 1 site which regulates expression of a first reporter gene; b) contacting said first cell with said transcription factor ligand, with a compound having ER3 mediated activity at an AP 1 site, and with said agent; and c) detecting expression of said first reporter gene.

58. The method of claim 57, further comprising the steps of: 10 d) providing a second cell containing an estrogen receptor 3 (ER3), a receptor for said nuclear transcription factor ligand, and a promoter comprising an estrogen response element (ERE) that regulates expression of a second reporter gene; e) contacting said second cell with said transcription factor ligand and with said compound having AP-1 mediated estrogenic activity; and 15 f) detecting expression of said second reporter gene.

59. The method of claim 58, wherein said first cell and said second cell are the same cell.

60. The method of claim 57, wherein said nuclear transcription factor is selected from the group consisting of a glucocorticoid, a progestin, vitamin D, retinoic 20 acid, an androgen, a mineralcorticoid, a prostaglandin.

61. The method of claim 57, wherein said a receptor for said nuclear transcription factor ligand is selected from the group consisting of an estrogen receptor, a glucocorticoid receptor, a progestin PR-A receptor, progestin PR-B receptor, an androgen receptor, a mineralcorticoid receptor, and a prostaglandin receptor. 25

62. The method of claim 57, wherein said cell contains a heterologous estrogen receptor 03 (ERP3).

63. The method of claim 57, wherein said cell expresses a heterologous receptor for said nuclear transcription factor ligand.

64. The method of claim 57, wherein said cell contains a heterologous 30 AP1 protein. WO 99/11760 PCT/US98/18030 54 0

65. The method of claim 64, wherein said AP I protein is c-jun.

66. The method of claim 57, wherein said nuclear transcription factor is a progestin; and said receptor for said nuclear transcription factor ligand is a progestin receptor.

67. The method of claim 57, wherein said nuclear transcription factor is 5 a glucocorticoid and said receptor for said nuclear transcription factor ligand is a GR receptor.

68. A kit for screening a compound for the ability to activate or inhibit estrogen receptor 03 (ER3) mediated gene activation at an AP 1 site, said kit comprising a container containing a cell comprising an estrogen receptor 03 (ER3), an API1 protein, 10 and a construct comprising a promoter comprising an AP 1 site which regulates expression of a first reporter gene.

69. The kit ofclaim 68, further comprising instruction materials containing protocols for the practice of the assay methods of claims 1, 9, 10, 12, 16, or 18.

70. The kit of claim 68, wherein said cell further comprises a receptor for 15 a nuclear transcription factor ligand other than estrogen.

71. The kit ofclaim 68, further comprising instruction materials containing protocols for the practice of the assay methods of claims 29, 30, 32, 42, or 43.