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WO1999011760A1 - ACTIVATION DIFFERENTIELLE DES LIGANDS DES RECEPTEURS ERα ET ERβ DE L'OESTROGENE DANS LES SITES AP1 - Google Patents

ACTIVATION DIFFERENTIELLE DES LIGANDS DES RECEPTEURS ERα ET ERβ DE L'OESTROGENE DANS LES SITES AP1 Download PDF

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WO1999011760A1
WO1999011760A1 PCT/US1998/018030 US9818030W WO9911760A1 WO 1999011760 A1 WO1999011760 A1 WO 1999011760A1 US 9818030 W US9818030 W US 9818030W WO 9911760 A1 WO9911760 A1 WO 9911760A1
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WIPO (PCT)
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cell
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arg
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PCT/US1998/018030
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English (en)
Inventor
Peter J. Kushner
Jan-Ake Gustafsson
George G. J. M. Kuiper
Stefan Nilsson
Kolja Paech
Thomas S. Scanlan
Paul Webb
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The Regents Of The University Of California
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Priority to AU89243/98A priority Critical patent/AU757348B2/en
Priority to EP98941104A priority patent/EP1009807A4/fr
Priority to JP2000508772A priority patent/JP2001514843A/ja
Priority to CA002301143A priority patent/CA2301143A1/fr
Priority to IL13481198A priority patent/IL134811A0/xx
Publication of WO1999011760A1 publication Critical patent/WO1999011760A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/743—Steroid hormones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/723—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems

Definitions

  • Estrogens, antiestrogens, and other nuclear transcription factor ligands are used in a wide variety of therapeutic contexts.
  • 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.
  • 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 of the estrogen receptor (ER) in breast tissue (see, e.g., (Sutherland et al. (1987) Cancer Treat. Revs., 15: 183-194).
  • ER estrogen receptor
  • Glucocorticoids are used in the treatment of pure red cell anemia, acute renal failure due to acute 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.
  • nuclear transcription factor ligands may have profound and contradictory effects upon patients depending on physiological context.
  • 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. Cancer Inst. 82: 1327-1332).
  • agonistic activity may also be harmful. Tamoxifen for example sometimes increases endometrial tumor incidence (lino et al. (1991) Cancer Treat. &Res. 53: 228-237) or switches from 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).
  • ER estrogen receptor
  • Fig. IB the classical estrogen response element
  • tamoxifen inhibits the transcription of genes that are regulated by a classical ERE, but like the natural estrogen hormone 17 ⁇ -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).
  • ER ⁇ a second ER was cloned from a rat prostate cDNA library (Kuiper et al. (1996) Proc. Natl. Acad. Set, 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 ER ⁇ (Kuiper et al. ( 1996) supra. ) .
  • the existence of two ERs was postulated to present a potential new mechanism tissue-specific estrogen regulation.
  • 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 (ER ⁇ )-mediated API pathway.
  • ER ⁇ estrogen receptor beta
  • This invention is premised, in part, on the surprising discovery that ER ⁇ is capable of intereacting with API to induce transcription of a gene under API control. Even more surprising was the discovery that ER ⁇ -mediated API interactions can produce results significantly different than ER ⁇ -mediated API interactions. For example, estradiol, which activates gene expression through an ER ⁇ -mediated API interaction, actually inhibits gene activation through an ER ⁇ -mediated API interaction.
  • this invention provides methods of screening test compounds for differential ER ⁇ -mediated and ER ⁇ -mediated activation at an API site.
  • the methods typically involve providing a first cell comprising an estrogen receptor ⁇ (ER ⁇ ), an API protein, and a construct comprising a promoter comprising an API site which regulates expression of a first reporter gene.
  • the first cell is contacted with the test compound and the expression of the first reporter gene is compared with ER ⁇ -mediated expression of a gene at an API site in response to the same test compound.
  • the cell can contain a heterologous estrogen receptor beta (ER ⁇ ) and preferred ER ⁇ s comprise an amino acid seqeunce of SEQ ID NO: 3 or SEQ ID NO: 5.
  • the cell can also contain a heterologous API protein.
  • Preferred reporter genes used in this assay include chloramphenicol acetyl transferase (CAT), luciferase, ⁇ -galactosidase ( ⁇ -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 antiestrogenic activity.
  • the method can be one in which the ER ⁇ -mediated expression of a gene at an API site is determined by providing a second cell comprising an estrogen receptor ⁇ (ER ⁇ ), API 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 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 reporter gene.
  • the cell and the second cell are the same cell.
  • this invention provides methods screening a test compound for the ability to activate or inhibit estrogen receptor beta (ER ⁇ ) mediated gene activation at an API site.
  • the methods typically involve providing a first cell comprising an estrogen receptor ⁇ (ER ⁇ ), API proteins, and a construct comprising a promoter 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 (ER ⁇ ).
  • the ER ⁇ the amino acid sequence of Sequence ID No: 3 or Sequence ID No: 5.
  • the first cell can also contain a heterologous API protein (e.g,. jun and/or fos). Virtually any reporter gene may be used.
  • Preferred reporter genes include, but are not limited to chloramphenicol acetyl transferase (CAT), luciferase, ⁇ -galactosidase ( ⁇ -gal), alkaline phosphatase, horse radish peroxidase (HRP), or green fluorescent protein (GFP) with a luciferase or a green fluorescent protein (GFP) being most preferred.
  • CAT chloramphenicol acetyl transferase
  • ⁇ -gal ⁇ -galactosidase
  • HRP horse radish peroxidase
  • GFP green fluorescent protein
  • Virtually any compound can be screened according to the methods of this invention.
  • preferred test compounds are compounds known to have anti-estrogenic activity.
  • the above method can further involve providing a second cell comprising an estrogen receptor ⁇ (ER ⁇ ), API 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 of the second reporter gene is then detected.
  • the above method can involve providing a third cell comprising an estrogen receptor ⁇ (ER ⁇ ), 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 standard estrogen response element can be from the Xenopus vitellogenin A2 gene.
  • the above method can also involve providing a fourth cell comprising an estrogen receptor ⁇ (ER ⁇ ), 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 of the fourth reporter gene is detected.
  • the standard estrogen response element can be from the Xenopus vitellogenin A2 gene.
  • 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 ER ⁇ -mediated AP 1 gene transcription. This 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 ⁇ (ER ⁇ ) mediated gene activation at an API site. Detecting then comprises detecting test compound mediated decrease in said estrogen receptor ⁇ (ER ⁇ ) mediated gene activation at an API 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.
  • the second compound known to activate transcription through estrogen receptor ⁇ (ER ⁇ ) mediated gene activation at an API site is identified by a method involving providing a second cell comprising an estrogen receptor ⁇ (ER ⁇ ), and API protein, and a construct comprising a promoter comprising an API 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 increase in expression of the second reporter gene produced by the compound indicates that said second compound activates transcription through ER ⁇ at an API site.
  • the assays of this invention can also be used to detect or identify inhibitors that block compounds that inhibit ER ⁇ -mediated AP 1 gene transcription. These methods involve performing the assays as described above, while additionally contacting the first cell with a second compound, in addition to the test compound, where the second compound is known to inhibit transcription through estrogen receptor ⁇ (ER ⁇ ) mediated activity at an API site. Expression of the reporter gene is detected where the detection comprises detecting test compound mediated increase in estrogen receptor ⁇ (ER ⁇ ) mediated gene activation at an API site. The detecting can involve comparing expression of the first reporter gene in the presence of both the second compound and the test 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 ⁇ (ER ⁇ ) mediated gene activation at an API site can be identified by providing a second cell comprising an estrogen receptor ⁇ (ER ⁇ ), and API protein, and a construct comprising a promoter comprising an API 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 ER ⁇ at the API site.
  • This invention also provides for any of the cells described above or herein.
  • the cell comprises an estrogen receptor ⁇ (ER ⁇ ), an AP 1 protein (e.g. , jun or fos), and a construct comprising a promoter comprising an API 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 other than estrogen.
  • the cell preferably contains a heterologous ER ⁇ , more preferably an ER ⁇ comprising an amino acid sequence of Sequence ID No: 3 or Sequence ID No: 5.
  • the API protein can be a native API protein or a heterologous API protein.
  • the reporter gene can be one selected from the group consisting of chloramphenicol acetyl transferase (CAT), luciferase, ⁇ -galactosidase ( ⁇ -gal), alkaline phosphatase, horse radish 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 mammalian cells and particularly preferred cells are derived from breast tissue or from 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 ER ⁇ -mediated API gene transcription.
  • this invention provides methods of screening a nuclear transcription factor ligand for the ability to modulate estrogen receptor ⁇ mediated activation or inactivation of transcription at an AP 1 site.
  • the methods involve providing a first cell containing an estrogen receptor ⁇ (ER ⁇ ), an API protein, a receptor for the nuclear transcription factor ligand, and a construct comprising a promoter comprising an API site which regulates expression of a first reporter gene.
  • the cell is contacted with the transcription factor ligand and with a compound having ER ⁇ mediated 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 ⁇ (ER ⁇ ), 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.
  • ER ⁇ estrogen receptor ⁇
  • EEE estrogen response element
  • the second cell is contacted with the 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.
  • the method can further involve providing a second cell containing a cognate receptor of the transcription factor ligand, and a 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 ER ⁇ mediated activity at said API site expression of the second reporter gene is detected.
  • the first and second cells can be the same or different cells.
  • 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.
  • 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, androgen receptor, a mineralcorticoid receptor, and a prostaglandin receptor.
  • the ER ⁇ comprises an amino acid sequence of Figure 5 or Figure 6 A.
  • the ER ⁇ can be a heterologous ER ⁇ .
  • 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.
  • the nuclear transcription factor is a progestin; and said receptor for the nuclear transcription factor ligand is a progestin receptor.
  • 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 ⁇ (ER ⁇ ) activation or inactivation of transcription at an AP 1 site by a nuclear transcription factor ligand.
  • the methods involve providing a first cell containing an estrogen receptor ⁇ (ER ⁇ ), an API protein, a receptor for the nuclear transcription factor ligand, and a promoter comprising an API site which regulates expression of a first reporter gene.
  • the first cell is contacted with the transcription factor ligand, with a compound having ER ⁇ mediated activity at an AP 1 site, 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 ⁇ (ER ⁇ ), a receptor for the nuclear transcription factor ligand, and a promoter comprising an estrogen response element (ERE) that regulates expression of a 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 prostaglandin.
  • 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.
  • the ER ⁇ can be a heterologous ER ⁇ and in a preferred embodiment, the ER ⁇ comprises an amino acid sequence of Sequence ID No: 3 or Sequence ID No: 5 or is encoded by a nucleic acid 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.
  • 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 glucocorticoid and the receptor for said nuclear transcription factor ligand is a GR receptor.
  • kits for screening a compound for the ability to activate or inhibit estrogen receptor ⁇ (ER ⁇ ) mediated gene activation at an API site can include a container containing a cell comprising an estrogen receptor ⁇ (ER ⁇ ), an API protein (e.g., jun and/or fos), and a construct comprising a promoter comprising an API site which regulates expression of a first reporter gene.
  • the cell of the 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 described herein.
  • 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 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 reporter gene) even where an assay cell of this invention is "unstimulated” (e.g. the receptor in question is unliganded), t ' .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).
  • an antiestrogen is a compound that substantially inhibits estrogen activity as measured in an assay for estrogenic activity, for example, cellular 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 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, 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). 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.
  • cognate receptor refers to a receptor of the type that is typically bound by the transcription ligand in question.
  • 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
  • the cognate receptor includes the native (naturally occurring) form as well as modified receptors.
  • estrogen receptor beta (ER ⁇ )-mediated activation or inactivation of gene transcription at an API site refers to the activation or inactivation of a gene (e.g., a reporter gene) under control of an API site by the interaction of that API site with a liganded ER ⁇ receptor.
  • ER ⁇ -mediated activation or inactivation refers to gene regulation mediated by the interaction of ER ⁇ .
  • Inactivation or inactivation at an ERE refers to activation or inactivation of a gene under control of an ERE.
  • API site refers to differences between ER ⁇ - and ER ⁇ -mediated gene activation at an API 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 ER ⁇ or ER ⁇ . Differential activation can also reflect differences in the "sign" of gene activation. Thus differential activation can refer to ER ⁇ -mediated activation of transcription at an AP 1 site and ER ⁇ -mediated inactivation of gene transcription at an API site in response to the same ligand. Conversely, differential activation can refer to ER ⁇ -mediated inactivation of transcription at an API site and ER ⁇ -mediated activation of gene transcription at an API site in response to the same ligand.
  • API -mediated estrogenic/agonist activity refers to activation of a gene under the control of an API site (also referred to as an API response element) mediated by the interaction of a nuclear transcription factor with the API site.
  • an API site also referred to as an API response element
  • 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 API site is found in Angel & Kann, Biochem. Biophys. Ada., 1072: 129-157 (1991) and Angel, et al, Cell, 49: 729-739 (1987).
  • a "compound having API mediated estrogenic activity” refers to a compound that, when present in a cell containing a gene under control of an AP 1 site and API proteins, activates transcription of the gene under control of the API site.
  • a "compound having the ability to inactivate or inhibit estrogen receptor beta (ER ⁇ ) mediated gene activation at an API site refers to a compound that is capable of upregulating or downregulating transcription of a gene under the control of an AP 1 site through its interaction (e.g., binding) of an ER ⁇ .
  • 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 API site or at an ERE” the phrase refers to alteration of the level of estrogen induced expression of one or more genes under control of the API site or ERE site respectively.
  • detecting expression when used 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).
  • operably linked refers to functional linkage between a nucleic 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.
  • a nucleic acid expression control sequence such as a promoter, signal sequence, or transcription factor binding site
  • Recombinant when used with reference to a cell indicates that 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 expression of the heterologous nucleic acid by such a recombinant cell.
  • heterologous nucleic acid is one that originates from a foreign source (or species) or, if from the same source, is modified from its original form.
  • 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 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.
  • heterologous protein refers to a protein that originates from a foreign source (e.g., 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 with such sequences.
  • Expression cassettes include at least promoters and optionally, transcription termination signals.
  • 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.
  • an expression cassette can also include nucleotide sequences that encode a signal sequence that directs secretion of an expressed protein 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 compounds such as chlorinated organics, polycyclic aromatic hydrocarbons, herbicides, pesticides, pharmaceuticals and the like.
  • Figure 1 A illustrates the structure of five estrogen receptor (ER) ligands: Estradiol (E 2 ), diethylastilbestrol (DES), ICI 184,384, raloxifene (Ral), and tamoxifen (Tam).
  • ER estrogen receptor
  • Figure IB illustrates two estrogen receptor (ER) response elements: a simple (classical) estrogen response element (ERE) and an ER dependent API element described also in USSN 08/410,807, in USSN 60/051,309, and by Webb etal (1995) Mol. Endo., 9: 443-456.
  • Figure 2 illustrates ER ⁇ action at an estrogen response element (ERE).
  • HeLa cells were transfected with an ERE-regulated luciferase reporter plasmid and an expression vector for rat ER ⁇ as described herein.
  • Transfected cells were treated with the ligands (E2, 0.1 ⁇ M; DES, 1 ⁇ M, Ral, 1 ⁇ M, tamoxifen 5 ⁇ M; and ICI, 1 ⁇ M) or an ethyl alcohol (EtOH) vehicle control. All assays were done with at least triplicate transfections. Error bars show deviations between wells from a single representative transfection.
  • Figure 3 illustrates ER ⁇ action at an API element.
  • HeLa cells were transfected with an AP 1 reporter plasmid and an ER ⁇ expression plasmid and treated with the five ligands (see, e.g., Figure 2).
  • Ligand concentrations were E2, 0.1 ⁇ M; DES, 1 ⁇ M,; Ral, 1 ⁇ M; Tam, 5 ⁇ M, and ICI, l ⁇ M. Error bars are as in Figure 2.
  • Figure 4 illustrates ER ⁇ activation and inhibition at AP 1.
  • A ER ⁇ action at an API response element. HeLa cells were transfected with an API reporter plasmid and a rat ER ⁇ expression plasmid as described herein.
  • Transfected cells were treated with the following ligand concentrations: E 2 , 0.1 ⁇ M; DES, 1 ⁇ M; Ral, 1 ⁇ M, Tam, 5 ⁇ M; and ICI, 1 ⁇ M.
  • B Dose response of raloxifene induction with ER ⁇ at an API element. 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 ⁇ M), the lack of induction by E 2 (0.1 ⁇ M) and induction to the amount observed with the control (no ligand added).
  • the right panel shows the dose dependence of inhibition of raloxifene (1 ⁇ M) induction by DES (solid line) and E 2 (Dashed line).
  • 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 ⁇ M) plus E 2 (10 nM), and E 2 (10 nM) alone.
  • the right panel shows the dose dependence of raloxifene induction in the presence of E 2 ( 10 nM) .
  • Figure 5 illustrates ligand-dependent ER ⁇ activity in three cell types; Ishikawa cells, MCF7 cells and MDA453 cells.
  • A Ligand-dependent ER ⁇ action at an API element in Ishikawa cells. Ishikawa cells were transfected with an API -regulated luciferase reporter plasmid and an ER ⁇ expression plasmid. Transfected cells were treated with one or two ligands as indicated (E 2 , 0.1 ⁇ M; DES, 1 ⁇ M; Ral, 1 ⁇ M, Tam, 5 ⁇ M; and ICI, 1 ⁇ M; or an EtOH vehicle (control)).
  • B Ligand dependent ER ⁇ action at an API element in MCF7 cells. MCF7 cells were treated and analyzed as described for (A). Ligand dependent ER ⁇ action at an API element inMDA453 cells. MDA453 cells were treated and analyzed as described for (A). DETAILED DESCRIPTION
  • Tamoxifens are therapeutic agents for the treatment and possible prevention of breast cancer.
  • Tamoxifen (Figure 1 A), 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. (1996) Nature Med., 2: 381-385).
  • ER estrogen receptor
  • Fig. 1 A The related benzothiophene analog raloxifene (Fig. 1 A) has been reported to retain the antiestrogen properties of tamoxifen in breast tissue and to show 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. C . Invest., 93: 63-69; Sato et al. (1996) FASEB J., 10: 905-912; Yang etal. (1996) Endocrinol, 137: 2075-2084; Yang etal, (1996) Science, 273: 1222-1225)).
  • tissue-specific actions 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 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 API enhancer element that requires ligand and the AP 1 transcription factors Fos and Jun for transcriptionai activation (Fig. lB)(Umayaharaet ⁇ /. (1994)J.5 o/. Chem., 269: 16433-16442).
  • tamoxifen inhibits the transcription of genes that are regulated by a classical ERE, but like the natural estrogen hormone 17b-estradiol [E 2 (Fig. 1A)], tamoxifen activates the transcription of genes that are under the control of an API element (Webb et al. (1995) Mol. Endocrinol, 9: 443-456).
  • ER ⁇ a second ER was cloned from a rat prostate cDNA library (Kuiper et al. (1996) Proc. Natl. Acad. Sci.USA, 93: 5925-5930).
  • the first identified ER has been renamed ER ⁇ (Kuiper et al. (1996) supra.).
  • ER ⁇ presents another source of tissue-specific estrogen regulation, particularly as mediated through the API site.
  • ER ⁇ and ER ⁇ respond differently to certain ligands at an API element.
  • the results described herein suggest different regulatory functions for the two ER subtypes.
  • This invention thus provides materials and methods for screening for compounds that exhibit differential activity depending on whether their activity is mediated through ER ⁇ or ER ⁇ .
  • this invention provides materials and methods for determining whether a compound is capable of activate or inhibit estrogen receptor ⁇ (ER ⁇ ) mediated gene activation (transcription) at an API site.
  • ER ⁇ can interact with a API site to activate or inactivate expression (e.g.transcription) of a gene under the control of the API site.
  • putative estrogens can actually demonstrate "antiestrogenic" activity in an ER ⁇ / API pathway (where antiestrogenic activity in this context is as compared to the activity of an estrogen in the classical ER ⁇ /ERE pathway).
  • an estrogen would activate transcription in an ER ⁇ /ERE pathway the estrogen inactivates transcription in an ER ⁇ / API pathway.
  • putative antiestrogens can demonstrate estrogenic activity in an ER ⁇ / API pathway.
  • This invention thus provides methods for detecting antiestrogenic activity of putative estrogens, or for detecting estrogenic activity of putative antiestrogens. More generally, as explained below, this invention provides methods of screening compounds for the ability to activate or inhibit estrogen receptor ⁇ (ER ⁇ ) mediated gene activation at an API 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 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.
  • This invention provides efficient ways to screen large numbers of test compounds for the ability to activate or inhibit estrogen receptor ⁇ (ER ⁇ ) mediated gene activation at an API site.
  • the methods utilize a cell containing an estrogen receptor beta (ER ⁇ ), an AP 1 protein, and a construct comprising a promoter and reporter gene under the control of an API site such that ER ⁇ interaction with the API site, can increase or inhibit expression (e.g., transcription) of the reporter gene.
  • the cell is contacted with one or more compounds whose ER ⁇ activity at API it is desired to evaluate.
  • 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 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 ER ⁇ -mediated expression (transcription) at an API, site, while an increase in expression level of the reporter gene indicates that the test compound activates ER ⁇ -mediated expression (transcription) at an API 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 and control experiments are scored as a valid change.
  • 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.
  • estrogen activates transcription in both the classical response (at an ERE) and in the indirect response (at an API) when the interaction is mediated by ER ⁇ .
  • estrogen acts as an inhibitor of transcription at AP 1 when the interaction is mediated by ER ⁇ .
  • the estrogen antagonist tamoxifen appears to always act as an inhibitor at an ERE, but an activator of transcription at an API site.
  • the activity of ER ⁇ does not appear to be tissue restricted.
  • the assay for ER ⁇ -mediated API activity is described above.
  • the remaining assays are performed in an analogous manner.
  • the ER ⁇ -mediated activity assays simply involve substituting ER ⁇ for ER ⁇
  • the ERE activity assays simply involve substituting the ERE/reporter gene construct for the AP 1/reporter gene construct.
  • the ER ⁇ 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).
  • the assay for ER ⁇ -mediated ERE activity utilizes a cell containing an estrogen receptor beta (ER ⁇ ), and a construct comprising a promoter and reporter gene under the control of an ERE site such that ER ⁇ 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 ER ⁇ activity at an ERE it is desired to evaluate.
  • 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 ER ⁇ -mediated expression (transcription) at an ERE, while an increase in expression level of the reporter gene indicates that the test compound activates ER ⁇ -mediated expression (transcription) at an ERE site.
  • each assay is performed in a separate cell
  • API and ERE assays can be combined and performed in a single cell.
  • the API/reporter gene construct preferably utilizes a different reporter gene than the ERE/reporter gene construct so that AP 1 activation or inactivation can be distinguished from ERE activation or inactivation.
  • the above-describe assays can also be used to identify (screen for) compounds that inhibit other compounds which have ER ⁇ -mediated or ER ⁇ -mediated 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.
  • the cell containing ER ⁇ , an AP 1 protein, and a reporter gene under control of an API site is contacted with estrogen and the test compound. If the compound inhibits the characteristic ER ⁇ -mediated estrogen activity at API, the compound is an inhibitor. It should be noted that in this case, ER ⁇ - mediated estrogen activity at API inhibits transcription, thus an estrogen inhibitor in this context actually increases ER ⁇ -mediated transcription at API. This is illustrated in Example 1, where it is shown that tamoxifen is one such inhibitor.
  • Inhibitors, or agonists, of ER ⁇ -mediated or ER ⁇ -mediated estrogenic or antiestrogenic activity at ERE and at API can be screened in an analogous manner.
  • this invention allows for screening of test compounds for estrogenic or antiestrogenic activity mediated through ER ⁇ or ER ⁇ at an ERE or at an API 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 derived from a source outside the human body, having estrogenic activity in the assays 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.
  • environmental estrogens may show synergistic activity in combination.
  • two or more suspected environmental 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.
  • 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 ER ⁇ - mediated activation or inactivation of transcription at an API site.
  • the assay cell additionally contains a receptor for a second nuclear transcription ligand (preferably a ligand other than estrogen).
  • a receptor for a second nuclear transcription ligand preferably a ligand other than estrogen.
  • the cell contains an estrogen receptor beta (ER ⁇ ), an API protein, a receptor for a second nuclear transcription factor ligand, and a construct comprising a promoter comprising an AP 1 site 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 ER ⁇ mediated activity at an API site.
  • Preferred second nuclear transcription factor ligands include, but are not limited to glucocorticoids, progestins, vitamin D, retinoic acid, androgens, mineralcorticoids, and prostaglandins.
  • 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.
  • the assay methods of this invention provide methods for evaluating the ability of a test, or control, compound to activate or inhibit transcription through interaction with a transcription factor receptor (e.g., estrogen receptor).
  • a transcription factor receptor e.g., estrogen receptor
  • the cells used in the assays of this invention preferably contain at least one transcription factor receptor.
  • ER ⁇ estrogen receptor ⁇
  • ER ⁇ estrogen receptor ⁇
  • the cell preferably include, in addition to the particular ER ⁇ or ER ⁇ at least a second nuclear transcription factor receptor (e.g., glucocorticoid receptor (GR)).
  • a nuclear transcription factor receptor e.g., glucocorticoid receptor (GR)
  • GR glucocorticoid receptor
  • 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 (GT1-1 (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 (1990). ERC2 and ERC3 cells as described by Webb, etal. 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.
  • Cells normally lacking the ER ⁇ or ER ⁇ 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 conditions where the receptor is expressed (e.g. , in the presence of an appropriate inducer 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 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 the assay.
  • “native” receptors for example, Berry, et ⁇ /.(1990), EMBOJ., 9: 2811-2818, describe the production of cells containing truncated or chimeric ER receptors.
  • the cells preferably contain one or more API 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 receptor(s).
  • API proteins the Jun or Fos proteins or other members of that protein family, see Bohmaan, et al. (1987) Science, 238: 1386-1392
  • the cells can naturally express the API protein(s) or they can be modified (e.g., by transfection with a suitable expression cassette) to express a heterologous API protein.
  • Methods of expressing API 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 & 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.
  • Ill Expression of Nuclear Transcription Factor Receptors.
  • the assays of this invention utilize cells containing one or more nuclear transcription factor receptors (e.g.
  • an estrogen 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.
  • Estrogen Receptor Alpha An estrogen receptor, as used herein, includes an estrogen receptor alpha
  • VP16-ER V-ER
  • a chimeric receptor comprising the strong VP 16 transcriptionai activation domain linked to the amino terminus of the ER
  • V- ER in which the ER DNA binding domain (DBD) is deleted, HI 1 an ER lacking the DNA binding domain, and the like
  • DBD ER DNA binding domain
  • ER ⁇ estrogen receptor alpha
  • Estrogen receptor beta 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. ( ⁇ 996)FEBSLett, 392: 49-53) and mouse (Tremblay et al. (1997) Mol. Endocrinol, 11 : 353-365) homologs were cloned. Accordingly, the original estrogen receptor (ER) has been renamed ER ⁇ (Kuiper et al. (1996) supra.).
  • ER ⁇ as used herein is intended to include all ER ⁇ variants. However, in a preferred embodiment, the ER ⁇ variants used in this invention correspond to the so called “intermediate length" ER ⁇ variants such as those described in WO 97/09348.
  • ER ⁇ variants are shown in sequence listings 3, 4, and 5 herein which correspond to figures 1 and 13A and 13B of WO 97/09348,
  • the cells can contain a cognate receptor for a nuclear transcription factor ligand whose interaction (preferably a cognate receptor other than an estrogen receptor).
  • a cognate receptor refers to a receptor of the type that is typically bound by the transcription factor ligand in question.
  • 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.
  • Natural and modified cognate receptors for nuclear transcription factor 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., aw etal. (1987) Proc. Natl. Acad. Sci.
  • 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 response element (either the API site or estrogen response element (ERE)).
  • a response element either the API site or estrogen response element (ERE)
  • EEE estrogen response element
  • two different reporter genes are used.
  • 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 in the same cell.
  • the methods of this invention involve providing a cell containing an estrogen receptor (ER ⁇ or ER ⁇ ), and a promoter comprising an API site that regulates expression of a reporter gene (also referred to herein as the reporter gene for the indirect estrogen response pathway (see, e.g., USSN 08/410,807 and Webb et al
  • the reporter gene for the indirect estrogen response pathway contains an
  • API site preferably upstream of the target promoter and capable of regulating (t.e., operably linked to) that promoter.
  • API site are sites that are bound by API (the Jun and Fos proteins) or other members of that protein family.
  • the consensus API site (or API response element) is TGA(C/G)TCA (SEQ ID NO: 1).
  • any sequence capable of being bound by AP 1 or members of that family and regulating a promoter is suitable. This would include promoters which encompass a naturally occurring API site. Typical promoters include, but are not restricted to metalloprotease genes such as stromelysin, gelatinase, matrilysin, and the human collagenase gene.
  • promoters may be constructed which contain a non-naturally occurring API, or related, binding site. This facilitates the creation of reporter gene systems that are not typically found under the control of AP 1.
  • promoters may be constructed which contain multiple copies of the API site thereby increasing the sensitivity or possibly modulating the response the reporter gene system.
  • the methods of this invention can also involve providing a cell containing a promoter comprising an estrogen response element that regulates expression of a 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).
  • a reporter gene also referred to herein as the reporter gene for the direct or classical estrogen response pathway
  • 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.
  • the estrogen response element is upstream of the target promoter and capable of regulating that promoter.
  • 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 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 t eXenopus vitellogenin A2 gene is widely used for this purpose, but other EREs may be used as well.
  • 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 transferase) (Alton and Vapnek (1979) N ⁇ twre 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.
  • 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. 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 B erger and Kimmel, Guide to Molecular Cloning Techniques: Methods in Enzymology, 152 Academic Press, Inc., San Diego, CA; Sambrook et al.
  • ER ⁇ 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 transcriptionai coactivator, and a DNA binding moiety that is capable of specifically binding to the response element.
  • the tethered coactivator is contacted with an activated transcription factor polypeptide (e.g., ER ⁇ ) that includes an activation function derived from a transcription factor.
  • an activated transcription factor polypeptide e.g., ER ⁇
  • 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 the estrogen receptor beta, or an API transcription factor, however, in a preferred embodiment, the transcription factor is ER ⁇ .
  • 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 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 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.
  • compounds are expected to be show the most 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., ER ⁇ or ER ⁇ ). 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. VIII. Assay Kits
  • kits for the practice of the methods of this invention preferably include one or more containers containing the cells described herein for the practice of the assays of this invention.
  • the cells may include, but are not limited to, cells containing an estrogen receptor ⁇ (ER ⁇ ), AP 1 protein(s), and a construct comprising a promoter comprising an API 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 APl/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 API proteins such as fos and/or jun.
  • kits can contain the AP 1/reporter gene and/or the ERE/reporter gene constructs described herein and/or the ER ⁇ , ER ⁇ , 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.
  • 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 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.
  • This example describes the investigation of the transactivation properties of ER ⁇ and ER ⁇ with a panel of five ER ligands with the use of a reporter gene under the control of either a classical ERE or an API element.
  • the results presented herein show that ER ⁇ and ER ⁇ respond differently to certain ligands at an API element suggesting different regulatory functions for the two ER subtypes.
  • the rat ER ⁇ expression vector has been previously described (Kuiper et al. (1996) Proc. Natl. Acad. Sci.USA, 93: 5925-5930).
  • the full-length human ER ⁇ cDNA which was isolated from an ovarian cDNA library and found to be identical to the previously reported partial cDNA clone (Mosselman et al. (1996) FEBSLett., 392: 49- 53) was cloned into the pCMV5 eukaryotic expression vector and the resulting ER ⁇ 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 ER ⁇ and ER ⁇ transactivation properties included the estrogens ⁇ -estradiol (E 2 ) and diethylstilbestrol (DES) and the antiestrogens Imperial Chemical Industries (ICI) 164384, tamoxifen, and 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), 18 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. The experiments were conducted by transfecting HeLa cells with either an estrogens ⁇ -estradiol (E 2 ) and diethylstilbestrol (DES) and the antiestrogens Imperial Chemical Industries (ICI) 164384, tamoxifen, and raloxifene.
  • ER ⁇ or ER ⁇ expression plasmid along with a reporter plasmid that contained a luciferase gene under the transcriptionai control of an estrogen response element (ERE).
  • EEE estrogen response element
  • Cells were grown inNunc Delta Surface tissue culture plates to a density of not more than 5 x 10 4 per cm 2 .
  • Cells were grown in 0.1 ⁇ m sterile filtered DME-F-12 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),
  • Ishikawa cells were grown in a medium containing 100 nM tamoxifen and MCF-7 cells were grown in medium containing 10 nM estradiol.
  • cells were suspended 0./5 ml of electroporation buffer in 0.4 cm gap electroporation cuvettes (BioRad) at 10 6 to 2 x 10 6 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 ⁇ L of Biobrene. Five ⁇ g of reporter plasmid and 6 ⁇ g of ER expression plasmid were added and the cuvette was agitated to facilitate mixing of the solution and homogeneous cell distribution in the cuvette.
  • 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 of DTT to a final concentration of 1 mM, ATP to a final concentration of 2 mM and luciferin (Analytical Luminescence Laboratories) to a final concentration of 200 ⁇ M 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. The data were collected using the HEO ER variant.
  • HEO shows reduced transactivation response from the unliganded receptor compared with the wild-type ER resulting in clearer ligand-induced transactivation data.
  • ER ⁇ 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- induced transactivation responses were lower with HEGO than with the control (no ligand added).
  • ER ⁇ and ER ⁇ at a classical ERE were first investigated. Both ER ⁇ (18) and ER ⁇ (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.
  • ER ⁇ and ER ⁇ at an API site were examined.
  • 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.
  • raloxifene induced transcription the least, showing twofold induction compared with the sixfold inductions typically seen with E 2 and tamoxifen.
  • raloxifene-induced transactivation was dose dependent with a concentration value required for one-half maximal activation (EC 50 ) of about 1 nM.
  • 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 transactivation results from binding to ER ⁇ . If E 2 is classified as a full activator of ER ⁇ at an API element (ER ⁇ -APl), then raloxifene functions as a partial activator and tamoxifen functions as a full activator.
  • raloxifene The transcriptionai activation caused by raloxifene was dose dependent with an EC 50 value of about 50 nM (Fig. 4B).
  • E 2 and DES were able to block the raloxifene induction
  • 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).
  • ER ⁇ - AP 1 was investigated whether the action of ER ⁇ - AP 1 could be observed in cell lines derived from estrogen target tissues such as the uterus and breast.
  • Transactivation assays for ER ⁇ -APl were performed in Ishikawa cells (a human uterine cell line) (Fig. 5 A) and in MCF7 (Fig. 5B) and MDA453 (Fig. 5c) human breast cancer cells. (The human ER ⁇ was used for transactivation in these cells.)
  • the ligands acted the same as they did in the HeLa cells; the three antiestrogens activated and the estrogens inhibited ER ⁇ -dependent transcription from an AP 1 site (Fig. 5).
  • MCF7 cells did not appear to contain high concentrations of endogenous ER ⁇ mRNA (Kuiper et al. (1997) Endocrinol, 138: 553); however, the results suggest that the additional transactivation machinery required for ER ⁇ -APl function is present in these cells.
  • E 2 treatment reduced the amount of transcription to less than that seen with the control (no ligand added).
  • Fig. 5C Fig. 5C
  • Ishikawa cells Fig. 5A
  • E 2 and DES blocked raloxifene induction and reduced the amount of transcription to less than that seen for the control.
  • ER ⁇ 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 API 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).
  • Nuclear receptors that respond to nonsteroidal hormones that have different known subtypes include the thyroid receptor (TR ⁇ and TR ⁇ ), the retinoic acid receptor (RAR ⁇ , RAR ⁇ , and RAR ⁇ ), and the retinoid X receptor (RXR ⁇ , RXR ⁇ , and RXR ⁇ ) (Mangelsdorf et al. (1996) Cell, 83: 841-850).
  • TR ⁇ and TR ⁇ the thyroid receptor
  • RAR ⁇ , RAR ⁇ , and RAR ⁇ the retinoic acid receptor
  • RXR ⁇ , RXR ⁇ , and RXR ⁇ retinoid X receptor
  • the ligand-induced responses with ER ⁇ at an API site provide an example of negative transcriptionai 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.
  • the action of ER ⁇ at either of these promoters has not been reported.
  • the action of ER ⁇ on the quinone reductase gene shows a similar ligand profile to that of ER ⁇ at an API site; antiestrogens are transcription activators, and E 2 is a transcription inhibitor.
  • MOLECULE TYPE DNA (genomic)

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Abstract

L'invention porte sur des procédés de criblage de composés d'essai pour déterminer leur capacité d'activer ou d'inhiber au niveau d'un site AP1 l'activation du gène induite par le récepteur β(ERβ) de l'oestrogène. Lesdits procédés consistent en particulier à obtenir une cellule comprenant le récepteur β(ERβ) de l'oestrogène, des protéines AP1, et un produit d'assemblage comportant un promoteur comprenant un site AP1 régulant l'expression d'un premier gène reporter. La cellule est mise en contact avec le composé d'essai et on peut détecter des modifications du niveau d'expression du gène reporter indiquant su ledit composé active la transcription, inactive la transcription, ou n'a pas d'effet sur le site AP1.
PCT/US1998/018030 1997-09-04 1998-08-31 ACTIVATION DIFFERENTIELLE DES LIGANDS DES RECEPTEURS ERα ET ERβ DE L'OESTROGENE DANS LES SITES AP1 WO1999011760A1 (fr)

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AU89243/98A AU757348B2 (en) 1997-09-04 1998-08-31 Differential ligand activation of estrogen receptors ERalpha and ERbeta at AP1 sites
EP98941104A EP1009807A4 (fr) 1997-09-04 1998-08-31 ACTIVATION DIFFERENTIELLE DES LIGANDS DES RECEPTEURS ER$g(a) ET ER$g(b) DE L'OESTROGENE DANS LES SITES AP1
JP2000508772A JP2001514843A (ja) 1997-09-04 1998-08-31 ＡＰ１部位でのエストロゲンレセプターＥＲαおよびＥＲβの弁別的なリガンド活性化
CA002301143A CA2301143A1 (fr) 1997-09-04 1998-08-31 Activation differentielle des ligands des recepteurs er.alpha. et er.beta. de l'oestrogene dans les sites ap1
IL13481198A IL134811A0 (en) 1997-09-04 1998-08-31 A CELL CONTAINING AN ESTROGEN RECEPTOR β AND A METHOD OF SCREENING UTILIZING THE SAME

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US92370897A 1997-09-04 1997-09-04
US08/923,708 1997-09-04

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WO2000037681A1 (fr) * 1998-12-18 2000-06-29 American Home Products Corporation DOSAGE BIOLOGIQUE PERMETTANT D'IDENTIFIER DES MODULATEURS SELECTIFS DES RECEPTEURS DES OESTROGENES β/$g(a)
WO2000061230A3 (fr) * 1999-04-09 2001-07-12 Karobio Ab Recepteur des oestrogenes et les os
WO2001055726A1 (fr) * 2000-01-25 2001-08-02 Glaxo Group Limited Analyse de voie de signalisation d'un recepteur de glucocorticoides
WO2002048360A1 (fr) * 2000-12-14 2002-06-20 Sumitomo Chemical Company, Limited Genes du recepteur des oestrogenes et leur utilisation
WO2002052010A1 (fr) * 2000-12-25 2002-07-04 Sumitomo Chemical Company, Limited Genes de recepteur d'oestrogenes et utilisation de ceux-ci
EP1012177A4 (fr) * 1997-09-08 2004-10-06 Merck & Co Inc Recepteur d'oestrogene
EP1457571A4 (fr) * 2001-12-13 2004-12-29 Otsuka Pharma Co Ltd Procede d'essai biologique d'un gene rapporteur
EP1400810A3 (fr) * 1999-04-09 2005-03-02 Karo Bio Ab Récepteurs des oestrogènes et les maladies de l'os
US6994967B1 (en) * 2000-07-05 2006-02-07 California Institute Of Technology Transcription factor regulators and methods for screening for same
US7157568B1 (en) 1997-08-05 2007-01-02 American Home Products Corporation Human estrogen receptor-β
US7166438B2 (en) 2001-11-07 2007-01-23 Schering Ag In Vitro screening for ligands of the estrogen receptor
US20120270799A1 (en) * 2011-04-21 2012-10-25 Taipei Medical University METHOD OF IDENTIFYING A CANDIDATE COMPOUND WHICH MAY INHIBIT a9-nAchR OVEREXPRESSION OR ESTROGEN RECEPTOR-DEPENDENT TRANSCRIPTION IN NICOTINE-DERIVED-COMPOUND-INDUCED BREAST CANCER CELLS

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ATE133267T1 (de) * 1990-09-21 1996-02-15 Salk Inst For Biological Studi Durch protoonkogenischen proteinkomplex ap-1 kontrollierte verfahren
US5723291A (en) * 1993-09-01 1998-03-03 The Regents Of The University Of California Methods for screening compounds for estrogenic activity

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MOSSELMAN S., POLMAN J., DIJKEMA R.: "ERBETA: IDENTIFICATION AND CHARACTERIZATION OF A NOVEL HUMAN ESTROGEN RECEPTOR.", FEBS LETTERS., ELSEVIER, AMSTERDAM., NL, vol. 392., 19 August 1996 (1996-08-19), NL, pages 49 - 53., XP002915394, ISSN: 0014-5793, DOI: 10.1016/0014-5793(96)00782-X *
See also references of EP1009807A4 *
WEBB P., ET AL.: "TAMOXIFEN ACTIVATION OF THE ESTROGEN RECEPTOR/AP-1 PATHWAY: POTENTIAL ORIGIN FOR THE CELL-SPECIFIC ESTROGEN-LIKE EFFECTS OF ANTIESTROGENS.", MOLECULAR ENDOCRINOLOGY, THE ENDOCRINE SOCIETY, US, vol. 09., no. 04., 1 April 1995 (1995-04-01), US, pages 443 - 456., XP002915393, ISSN: 0888-8809, DOI: 10.1210/me.9.4.443 *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7157568B1 (en) 1997-08-05 2007-01-02 American Home Products Corporation Human estrogen receptor-β
EP1012177A4 (fr) * 1997-09-08 2004-10-06 Merck & Co Inc Recepteur d'oestrogene
WO2000037681A1 (fr) * 1998-12-18 2000-06-29 American Home Products Corporation DOSAGE BIOLOGIQUE PERMETTANT D'IDENTIFIER DES MODULATEURS SELECTIFS DES RECEPTEURS DES OESTROGENES β/$g(a)
EP1400810A3 (fr) * 1999-04-09 2005-03-02 Karo Bio Ab Récepteurs des oestrogènes et les maladies de l'os
WO2000061230A3 (fr) * 1999-04-09 2001-07-12 Karobio Ab Recepteur des oestrogenes et les os
WO2001055726A1 (fr) * 2000-01-25 2001-08-02 Glaxo Group Limited Analyse de voie de signalisation d'un recepteur de glucocorticoides
US6994967B1 (en) * 2000-07-05 2006-02-07 California Institute Of Technology Transcription factor regulators and methods for screening for same
US7432078B2 (en) 2000-12-14 2008-10-07 Sumitomo Chemical Company Limited Estrogen receptor genes and utilization thereof
WO2002048360A1 (fr) * 2000-12-14 2002-06-20 Sumitomo Chemical Company, Limited Genes du recepteur des oestrogenes et leur utilisation
WO2002052010A1 (fr) * 2000-12-25 2002-07-04 Sumitomo Chemical Company, Limited Genes de recepteur d'oestrogenes et utilisation de ceux-ci
US7419800B2 (en) 2000-12-25 2008-09-02 Sumitomo Chemical Company Limited Estrogen receptor genes
US7166438B2 (en) 2001-11-07 2007-01-23 Schering Ag In Vitro screening for ligands of the estrogen receptor
EP1457571A4 (fr) * 2001-12-13 2004-12-29 Otsuka Pharma Co Ltd Procede d'essai biologique d'un gene rapporteur
US8609342B2 (en) 2001-12-13 2013-12-17 Otsuka Pharmaceutical Co., Ltd. Reporter gene assay method
US20120270799A1 (en) * 2011-04-21 2012-10-25 Taipei Medical University METHOD OF IDENTIFYING A CANDIDATE COMPOUND WHICH MAY INHIBIT a9-nAchR OVEREXPRESSION OR ESTROGEN RECEPTOR-DEPENDENT TRANSCRIPTION IN NICOTINE-DERIVED-COMPOUND-INDUCED BREAST CANCER CELLS
US8980571B2 (en) * 2011-04-21 2015-03-17 Taipei Medical University Method of identifying a candidate compound which may inhibit α9-nAchR overexpression or estrogen receptor-dependent transcription in nicotine-derived-compound-induced breast cancer cells

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