WO1999005171A1 - NOVEL ESTROGEN RECEPTOR β AND ISOFORMS - Google Patents
NOVEL ESTROGEN RECEPTOR β AND ISOFORMS Download PDFInfo
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- WO1999005171A1 WO1999005171A1 PCT/US1998/015540 US9815540W WO9905171A1 WO 1999005171 A1 WO1999005171 A1 WO 1999005171A1 US 9815540 W US9815540 W US 9815540W WO 9905171 A1 WO9905171 A1 WO 9905171A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
- C07K14/721—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/08—Drugs for disorders of the urinary system of the prostate
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70567—Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to a novel complete mammalian estrogen receptor ⁇ , referred to as ER ⁇ c , its polypeptide sequence, the nucleic acid sequence encoding ER ⁇ c and methods of making or expressing ER ⁇ c .
- the present invention also relates to methods of screening for drugs which modulate the interaction of estrogens and ER ⁇ c as well as methods of diagnosing and/or treating diseases involving ER ⁇ c or its isoforms.
- This application is related to U.S. Provisional applications 60/053,869 and 60/054,210, which are herein incorporated by reference.
- Estrogens are a class of naturally occurring steroid hormones which are produced in the ovaries and other tissues of the body including the testis. Estrogens are known to directly influence the growth, differentiation and function of specific target tissues and organs in humans and animals. These specific tissues and organs also include the mammary gland, uterus, prostate, pituitary, brain and liver. Estrogens also play an important role in bone maintenance and in the cardiovascular system, where estrogens have certain cardio-protective effects. In bone, both osteoclasts and osteoblasts have been reported to respond to estrogens with estrogen withdrawal leading to increased turnover and bone loss. A variety of naturally occurring and chemically synthesized estrogens have been identified and characterized, perhaps the best known of which is the endogenous estrogen, estradiol-17 beta (also known as E 2 ).
- Estrogens act by binding to the ligand binding domain (LB D) of an intracellular protein identified as an "estrogen receptor" (ER).
- ER ligand binding domain
- the presence of this intracellular ER provides and accounts for both cell proliferation and protein synthesis by estrogen-dependent cells.
- the estrogen receptor In the absence of the estrogen hormone, the estrogen receptor is biologically inactive both in vivo and in vitro; and, if the cells or tissues are homogenized and fractionated into cytosol and nuclear fractions, the estrogen receptor is found in the nucleus and may also be detected the cytosol.
- the known estrogen receptors are members of the well studied family of gene regulatory proteins referred to as the steroid hormone receptor family.
- Nuclear receptors such as steroid hormone receptors, have a modular structure with six distinct regions.
- the N-terminal domain is the A/B region which includes a non-ligand dependent activation function (See Fig. la).
- the C region is the DNA binding domain (DBD).
- the D region contains nuclear localization signals.
- the E domain contains the ligand binding domain (LBD) and the ligand-dependent transaction function.
- LBD ligand binding domain
- the central DBD is typically about 100 amino acids.
- estrogen receptors are activated by the binding of estrogen to the C-terminal LBD.
- the receptor proteins enable cells to respond to various lipid-soluble hormones by activating or repressing specific genes, through the interaction between the steroid hormone and its receptor.
- Steroid hormone receptors are distinguishable from other nuclear receptors in a number of respects, including the nature of their ligands, their association (in the unliganded state) with a repertoire of heat-shock proteins and the fact that they may bind to hormone response elements as homodimers.
- TR thyroid hormone receptors
- TRs have a dual regulatory role: in the presence of hormone they function as transcriptional activators, whereas in the absence of hormone, TRs are response element (TRE) specific transcriptional repressors.
- the first estrogen receptor discovered was ER ⁇ , which was known for the past ten years merely as ER.
- the human ER (hER) is composed of 595 amino acids in its unbound state and is approximately 67,000 Daltons. In the absence of estrogen-binding, the ER ⁇ protein can be located in vitro within the cytosol.
- PI represents the major ER ⁇ transcriptional start site.
- the PI start site is predominantly utilized in human mammary epithelial cells (HMEC) and is the major start site in ER ⁇ -positive human breast carcinomas. Multiple start sites have been identified for the P0 promoter.
- Studies of the murine ER ⁇ gene identified 10 start sites spanning approximately 60 bases, and there is a start site at - 1,994 (from the PI start site) in human cells, which would agree closely with the major murine P0 start site.
- Transcription from the P0 promoter is characteristic of human endometrial tissue and can account for 12 to 33% of ER ⁇ transcription in breast carcinoma cells.
- the ⁇ R ⁇ protein can be found in various molecular forms with sedimentation coefficients of 8S, 5S or 4S as determined by sucrose density gradient analysis.
- the 8S form of ⁇ R ⁇ protein is believed to be the inactivated, untransformed form of ⁇ R ⁇ protein associated with the unbound, inactive state of estrogen receptor in the absence of estrogen.
- the 4S ⁇ R ⁇ protein is a monomeric protein molecule that can be generated from the 8S form in vitro.
- the 4S form binds to both nuclei and DNA-cellulose in vitro; it is generally termed the "activated but untransformed" estrogen receptor protein.
- the 5S form of ⁇ R ⁇ is a dimeric protein molecule, which is created by the conversion of the 4S ER ⁇ protein via a bimolecular reaction. It is generally believed that the 5S form of ER ⁇ protein is both "activated and transformed," and therefore is the biologically active entity which binds to the DNA within the nuclei. Moreover, it is also this 5S form which is found associated with the nuclei subsequent to the administration of estradiol in vivo. Already it has been demonstrated that both ER ⁇ and ER ⁇ can form heterodimers (Kuiper and Gustafsson, FEBS 410: 87 (1997)).
- mRNA new messenger RNA
- mRNA new messenger RNA
- ribosomes then translate the mRNA into new proteins; the hormone/receptor protein complex can also down-regulate mRNA transcription.
- ER ⁇ the localization of ER ⁇ along with the manner in which it modulates transcription will be at least grossly similar to ER ⁇ ; however, affinities for certain DNA sequences, as well as receptor ligands likely will differ between ER ⁇ and ER ⁇ , as there is a 97% and 60% identity respectively between the DBD and LBD sequences between the two estrogen receptors. Tremblay et al, (1997).
- Tamoxifen a substituted triphenylethylene antiestrogen
- Gallo et al. Semin. Oncol. 24: SI (1997).
- the expression of the receptor is usually associated with a better prognosis and is less metastatic.
- Bonetti et al. Breast Cancer Res. Treat. 38(3): 289 (1996).
- the tumors are either ER-negative or contain splice variants that are commonly biologically inactive.
- SARMs selective estrogen receptor modulators
- Estrogen receptors are also present in human and rat prostate, as evidenced by ligand binding studies. In contrast to androgen receptors, the major part of the estrogen receptors are localized in the stroma of the rat prostate, although the epithelial cells of the secreting alveoli contain ER.
- Estrogens are, in addition to androgens, implicated in the growth of the prostate, and consequently estrogens have been implicated in the pathogenesis of benign prostatic hyperplasia.
- Habenich et al J. Steroid Biochem. Mol Biol. 44: 557 (1993); Kuiper et al, PNAS 93: 5930 (1996).
- Diethylstilbesterol (DES) a stilbene estrogen with an increased affinity for ER, is used to treat prostatic hyperplasia and carcinoma. Goethuys et al, Am. J. Clin. Oncol. 20(1): 40 (1997); Aprikian et al, Cancer 71(12): 3952 (1993). Therefore, identifying the tissues and diseases that express ER ⁇ likely will prove helpful in the treatment of diseases involving ER ⁇ .
- Estrogen has also been demonstrated to prevent osteoporosis.
- Postmenopausal osteoporosis the most common bone disease in the developed world, is associated with estrogen deficiency. This deficiency increases generation and activity of osteoclasts, large multi-nuclear cells involved with bone resorption.
- Estrogen has been demonstrated to down-regulate osteoclast formation and function.
- Tamoxifen has been demonstrated to possess estrogenic effects on bone resorption likely through tamoxifen- induced osteoclast apoptosis.
- ER ⁇ Following the cloning of estrogen receptor ⁇ (ER ⁇ ) 10 years ago, there was general acceptance that only one ER gene existed and consequently only one subtype of ER, ER ⁇ . This contrasted sharply with other members of the nuclear receptor superfamily, where multiple forms have been reported, e.g., thyroid hormone receptor (TR) ⁇ and ⁇ and retinoic acid receptor (RAR) ⁇ , ⁇ , and ⁇ .
- TR thyroid hormone receptor
- RAR retinoic acid receptor
- ER ⁇ was partially isolated from cDNA libraries from human testis, mouse ovaries and rat prostate, which are not generally considered to be major estrogen target tissues.
- the estrogen receptor subtype initially discovered was termed ER ⁇ , but for purposes of this invention will be termed the incomplete ER ⁇ (ER ⁇ j ) to differentiate it from the complete ER ⁇ (ER ⁇ c or ER ⁇ -3) of the present invention, or the three claimed alternatively spliced isoforms (ER ⁇ -1, ER ⁇ -2 and ER ⁇ -4) of this invention.
- ER ⁇ -3 refers to the sequence as isolated from mouse ovaries or its analogous sequence in other mammalian species.
- ER ⁇ c refers to the sequence that encodes the complete ER ⁇ , which includes the novel 192 bp at the 5' terminus of exon 1 and the newly described exon 5B;
- ER ⁇ c includes ER ⁇ -3, the complete sequence that encodes the nine exons of murine ER ⁇ .
- ER ⁇ i5 as characterized using the clones obtained from mouse ovary tissue, encodes a protein that has a molecular weight of approximately 62 kDa and has a 60 kilobase (Kb) gene size.
- ER ⁇ j Clone 29
- EAE estrogen response element
- ER ⁇ j was isolated in an effort to clone and characterize novel nuclear receptors or unknown isoforms of existing receptors.
- Degenerate primers were designed based on conserved regions within the DBD and LBD of nuclear receptors. Using these primers in conjunction with Polymerase Chain Reaction (PCR), rat prostate mRNA was amplified.
- PCR Polymerase Chain Reaction
- One targeted tissue was the prostate, an organ of interest given the high incidence of prostate cancer and benign prostatic hyperplasia. Nearly all prostate tumors eventually become androgen-independent, at which point they are beyond clinical control. Kuiper et al, (1996).
- TR2 and TR4 human testicular receptors 2 and 4
- ER estrogen receptor
- ERR1 and ERR2 are examples of orphan nuclear genes whose receptors are expressed in the prostate. Kuiper et al, (1996).
- ER ⁇ Tissue expression of ER ⁇ , revealed additional differences from ER ⁇ expression. Some tissues contain exclusively ER ⁇ (i.e., uterus, pituitary, epididymis, and kidney). Other tissues display equal or greater levels of ER ⁇ , RNA and may be expressed preferentially in the different cell types of an organ (i.e., ovary and prostate). Kuiper et al, (1996). In brain, ER ⁇ j appears to be a conspicuous fraction of the ER subtype RNA. Although Northern blots did not detect ER ⁇ , expression in peripheral blood lymphocytes, the initial PCR fragment of ER ⁇ , cloned by Mosselman was acquired from these cells.
- the ER ⁇ , subtype may play a significant role in estrogen action in brain, ovary, prostate, hypothalamus and possibly other tissues.
- the order of competition for physiological estrogens and stilbene estrogens, which form a diphenolic resonance structure, for ER ⁇ versus the ER ⁇ , isoform was also observed to vary. Kuiper et al, (1997).
- ER ⁇ OHT displayed no agonistic activity on ER ⁇ ,. Tremblay et al, (1997). Therefore, once the underlying ER subtype responsible for a particular disease state is determined (e.g., ER ⁇ positive breast cancer), one may have a more accurate means of prognosticating the estrogen receptor related disease outcome; one may accurately follow therapies; one may develop gene specific and isoform specific therapies targeting diseases influenced by ER ⁇ and/or ER ⁇ ; and one may provide for opportunities for varying the aggressiveness of the therapy.
- the present invention is based, in part, on the isolation and identification of the complete murine (m) estrogen receptor ⁇ gene (mER ⁇ -3) and two alternatively spliced isoforms, e.g., mER ⁇ -1 and mER ⁇ -2 and a third isoform isolated from rat (r) ovaries, rER ⁇ -4. More broadly, the invention relates to the corresponding ER ⁇ c gene (including the human gene) and to certain mammalian receptors (denoted herein as ER ⁇ -1, ER ⁇ -2, ER ⁇ -3 and ER ⁇ -4). The ER ⁇ , sequence has been published by other laboratories, which had prematurely claimed that ER ⁇ , represented the complete ER ⁇ gene (ER ⁇ c ).
- the present invention further provides nucleic acid molecules that encode the mER ⁇ -1, mER ⁇ -2, mER ⁇ -3 and mER ⁇ -4 proteins.
- Such nucleic acid molecules can be in an isolated form or can be operably linked to expression control elements or vector sequences.
- the present invention also provides methods of identifying other alternatively spliced forms of the mER ⁇ -3, the analogous mER ⁇ -3 and corresponding ER ⁇ c as expressed in different animal species or additional ER subtypes.
- the nucleic acid sequence of mER ⁇ -3 can be used as a probe or to generate PCR primers to identify nucleic acid molecules that encode other members of the ER ⁇ c family of proteins.
- the nucleic acid molecules encoding mER ⁇ -1, mER ⁇ -2, mER ⁇ -3 or rER ⁇ -4 can be used to identify and isolate the ER ⁇ -3 gene or corresponding ER ⁇ c in other mammalian species, and has been used to isolate the ER ⁇ -3 analog in human DNA.
- the present invention further provides antibodies that recognize and bind to the
- ER ⁇ c protein or the mER ⁇ -3 protein or its isoforms can be either polyclonal or monoclonal. Particularly preferred are antibodies that are specific for the complete receptor protein, ER ⁇ c , as opposed to antibodies against the previously known receptors, e.g., ER ⁇ and ER ⁇ ,. More specifically, the invention claims an anti-peptide antibody that distinguishes between ER ⁇ , and ER ⁇ c . Antibodies that bind to the ER ⁇ c protein can be utilized in a variety of diagnostic and prognostic formats and therapeutic methods. Alternatively, antibodies that can distinguish between the complete form, ER ⁇ c , and its isoforms may also be useful for purposes of diagnosis and treatment of ER ⁇ subtype based disease.
- the present invention further provides methods for reducing, blocking or augmenting the association of an estrogen and other agonists and antagonists with the ER ⁇ c protein.
- an ER ⁇ -3 protein with a cytoplasmic signaling partner such as estradiol
- estradiol can be blocked or reduced by contacting the ER ⁇ -3 protein with a compound that blocks the binding of estradiol or other estrogen-like agonists or antagonists (e.g., estrogens, stilbene estrogens or triphenylethylene antiestrogens).
- estradiol or other estrogen-like agonists or antagonists e.g., estrogens, stilbene estrogens or triphenylethylene antiestrogens.
- the association of the ligand with ER ⁇ can also be influenced, in theory, by the dimer partner. Therefore, identifying agents that modulate ER ⁇ dimerization may pose another means of manipul
- Blocking the interaction between the ligand and ER ⁇ -3 or one of its isoforms can be used to modulate biological and pathological processes that require such a ligand bound complex to mediate transcription. Such methods and agents can be used to modulate cellular proliferation, differentiation, DNA synthesis or cell cycle distribution.
- the present invention further provides methods for isolating ER ⁇ c or ER ⁇ c protein isoforms (e.g., ER ⁇ -1, ER ⁇ -2, ER ⁇ -3 and ER ⁇ -4) that regulate transcription.
- ER ⁇ -3 ligand binding partners e.g., estrogen, are isolated using the ER ⁇ -3 protein or ligand binding portions thereof.
- the DNA sequences that the ER ⁇ -3 protein binds can be determined, for example, utilizing electrophoretic mobility shift assays (EMSA), yeast two hybrid assays, or by affinity selection and degenerate ERE consensus sequences using the DNA binding domains (DBDs) of ER ⁇ c or its isoforms.
- ESA electrophoretic mobility shift assays
- yeast two hybrid assays or by affinity selection and degenerate ERE consensus sequences using the DNA binding domains (DBDs) of ER ⁇ c or its isoforms.
- the invention also describes methods to screen compounds that can distinguish between ER ⁇ and ER ⁇ c and its isoforms (e.g., ER ⁇ -1, ER ⁇ -2, ER ⁇ -3 and ER ⁇ -4).
- These methods will include methods of determining whether the compound binds and either functionally acts as an agonist or an antagonist with regard to each estrogen receptor.
- One method to determine whether compounds act in an agonistic or antagonistic fashion would use ER ⁇ c in a yeast two hybrid system. Such methods have been previously employed to test the interaction of certain drugs with ER ⁇ and recognized by those of ordinary skill in the art. See Ichinose et al, Gene 188: 95 (1997); Collins et al, Steroids 62: 365 (1997); Jackson et al, Mol. Endocrinol. 11 : 693 (1997). The biological and pathological processes that require estrogen/ER ⁇ c complex can be modulated further by using gene therapy methods.
- an ER ⁇ -3 gene can be introduced into a mammal deficient for ER ⁇ -3 protein to correct the genetic deficiency; peptide modulators of ER ⁇ -3 activity can be produced within a target cell using genetic transfection methods to introduce into the target cells nucleic acid molecules encoding the modulators; and the ER ⁇ -3 gene can be introduced or deleted in a non-human mammal to produce animal models expressing ER ⁇ -3 gene abnormalities or delete the gene entirely (e.g., knock-out mice).
- ER ⁇ -3 transgenic animals is particularly useful for identifying agents in vivo that modulate ER ⁇ -3 activity and perhaps even other genes that encode proteins that influence ER ⁇ -3 actions.
- nucleic acids for antisense and triple helix therapies and interventions are also expressly contemplated.
- Figure 1 Nucleotide sequence, deduced amino acid sequence and putative domain structure of the complete murine ER ⁇ c gene (mER ⁇ -3)
- Fig. 1 (a) Illustrates the location of each of the nine exons comprising the clone of the complete murine ER ⁇ c , mER ⁇ -3, and the splicing domains that yield the different alternatively spliced isoforms of mER ⁇ -3.
- the numbers directly above the lines signifying the exons represented by terminal nucleotides of the exon.
- the sizes of the nine exons in base pairs (bp) and the encoded amino acid (a.a.) sequence for each of the exons and splice variants derived from mouse ovaries is indicated.
- the 1 ,704 nucleotides of mER ⁇ -3 encodes a 567 amino acid protein.
- the letters (A through F) refer to regions of homology shared by all members of the steroid receptor super family. Green et al. , Cold Spring Harbor Symposia on Quantitative Biology 51 (2): 751 -8 (1986).
- Region C corresponds to the DNA binding domain (DBD).
- Region E is the ligand binding domain (LBD).
- LBD ligand binding domain
- the newly described exon 5B lies within the LBD. Exon 5B starts with GTCCTCA and stops with CCCAAG.
- the isolated isoform mER ⁇ -2 is 1,533 bp and lacks both exon 3 and exon 5B.
- Isoform rER ⁇ -4 isolated from rat tissue, is 1,570 bp. Although rER ⁇ -4 possesses the new exon 5B, it lacks exon 6. The loss of exon 6 results in a frame shift that causes translation to terminate at a stop codon located in exon 7.
- Fig. 1 (b). The full length sequence of murine mER ⁇ c (mER ⁇ -3 clone). The additional sequence included in all mER ⁇ clones (mER ⁇ -1, mER ⁇ -2 ), as well as the alternatively spliced rat isoform, (rER ⁇ -4) is noted in underlined bold type. The sequence included in the ninth exon, exon 5B, is presented in lower case letters beginning at base 1,149.
- FIG. 2 Amino acid sequences of the alternatively spliced isoforms of the mER ⁇ -3 Fig. 2 (a). Deduced amino acid sequences for alternative splice variant mER ⁇ -1. The polypeptide sequence shared by all 3 of the alternatively spliced isoforms is indicated by the underlined sequence in bold characters. The mER ⁇ -1 protein contains 549 amino acid residues.
- Fig. 2 (b). Deduced amino acid sequences for alternative splice variant mER ⁇ -2.
- the alternatively spliced mER ⁇ -2 is 510 amino acid residues in length.
- Fig. 2 (c). Deduced amino acid sequences for a rat alternative splice variant rER ⁇ -4. This splice variant was obtained from rat ovaries. The deletion of exon 6 produces a frame shift causing a truncation that terminates 13 amino acids beyond the translated exon 5B; the resulting rER ⁇ -4 protein likely is 414 residues long.
- the italicized, underlined, bold characters represent the polypeptide encoded by the novel 192 nucleotides located at the 5' terminus of exon 1.
- the characters indicated in bold and underlined represent the polypeptide encoded by exon 5B.
- the "*" refers to a translated stop codon.
- FIG. 3 (b) Western blot of human ovary, mouse ovary, rat ovary, ROS 17/2.8 cells, and murine primary osteoblasts protein extracts probed with antibody 1068 pre- immune sera.
- the protein extracts of each lane of both Figures 3(a) and 3(b) are: lane 1, human ovary; lane 2, mouse ovary; lane 3, rat ovary; lane 4, ROS 17/2.8 cells; lane 5, ROS 17/2.8 cells treated with 100 nM estradiol for 16 hours; lane 6, murine primary osteoblasts.
- RNA from rat ovarian and ROS 17/2.8 cells amplified for 35 cycles using an oligo that can detect rER ⁇ .
- Each lane in Fig. 4 (a) contains PCR products derived from the following types of RNA: lane 1, control, no RNA; lane 2, rat ovarian RNA (0.1 ⁇ g); lane 3, ROS 17/2.8 cells (0.1 ⁇ g); lane 4, rat ovarian RNA control (0.1 ⁇ g), no reverse transcriptase (RT); and lane 5, ROS 17/2.8 total RNA (0.1 ⁇ g), no RT.
- RNA 4 contains the following types and amounts of RNA: lane 1, control, no RNA; lane 2, rat ovarian RNA (2 ng); lane 3, ROS 17/2.8 total RNA (0.1 ⁇ g), lane 4, total (cultured) bone marrow RNA (0.1 ⁇ g); lane 5, total cultured bone marrow RNA (0.1 ⁇ g) where the cells had been treated with estradiol for 16 hours; lane 6, total RNA from primary osteoblasts in co-culture (0.1 ⁇ g); lanes 7-11, control reactions without reverse transcriptase (RT) for lanes 2-6, respectively.
- FIG. 5 Gel Shift Assay Fig. 5(a). Gel shift analysis of mER ⁇ -3. The receptor-DNA complex was disrupted using the anti-peptide antibody 1067, which recognizes polypeptides encoded by exon 5B.
- Fig. 5(b) Gel shift analysis of the human alpha form of the estrogen receptor (ER ⁇ ). Disruption of the ER ⁇ -DNA complex was assayed using the two anti-peptide antibodies specific to exon 5B.
- Both Fig. 5 (a) and (b) contain the following: lanes 1 and 2, extract alone; antibody 1067, lanes 3 and 4; antibody 1067 pre-immune serum, lanes 5 and 6; antibody 1068, lanes 7 and 8; antibody 1068 pre-immune serum, lanes 9 and 10; lanes 11 and 12 are control lanes that contain 16 ⁇ g of untransfected COS-7 nuclear extract.
- FIG. 6 Comparison of mER ⁇ -3 protein with the murine ER ⁇
- the upper sequence is the protein sequence of mER ⁇ -3, whereas the lower sequence is that of the mouse (m) mER ⁇ .
- " between the matched sequences indicates residue identity.
- the ":” between the matched sequences represents similar amino acids.
- the ".” observed in the sequences is a "gap” added by the sequence alignment program.
- the lines bisecting the paired sequences delineate the six domains (A-F) found in ER ⁇ c and ER ⁇ . There is 99% similarity and 97% identity between the C domains, which contain the DBD, of the two murine estrogen receptor subtypes. There is 79% similarity and 59% identity between the E domains, which contains the LBD.
- the upper paired sequence (which starts at nucleotide 151) is the nucleotide sequence of mER ⁇ -3, whereas the lower sequence is the nucleotide sequence o ⁇ mER ⁇ , published by Tremblay et al, (1997).
- Reporter constructs expressing ER ⁇ -1 (BI), ER ⁇ -3 (B3), ER ⁇ (alpha), or both ER ⁇ -1 and ER ⁇ -3 (B1+B3) were exposed to clomiphene, diethylstilbesterol (DES), 4 OH-tamoxifen (4-OHT), or 17 ⁇ estradiol (E2). Expression was standardized to ER ⁇ response to 100 nM drug.
- FIG. 9 Transactivation Profiles - cV2ERE
- the four panels display the ability of the different estrogen receptors to transactivate cV2ERE.
- ER ⁇ ER Alpha
- murine ER ⁇ -1 mER- BI
- murine ER ⁇ -3 mER-B3
- coexpression of both murine ER ⁇ -1 and ER ⁇ -3 isoforms mER B1+B3 in COS-7 cells to E2, clomid, DES and 4-OHT were compared.
- Ovaries upper panels
- uteri middle panels
- E-15 rat embryos were serially sectioned and probed using anti-sense (left panels) and sense (right panels) probes from ER ⁇ and ER ⁇ . Cervical spine is shown in the lower panels.
- Estrogen receptors are members of the nuclear hormone receptor family. Biologically, these proteins are intracellular receptors which mediate the effects of steroid hormones. Upon hormone binding, estrogen receptors control the transcriptional expression of certain hormone-responsive genes. This involves the binding of the receptors, often in homo- or heterodimeric form, to specific sequences, hormone response elements, located in the target gene promoter.
- compositions and methods of this invention provide for the screening of candidate compounds to be used to treat ER ⁇ c related diseases.
- the compositions are based on the isolation of an ER ⁇ c sequence, ER ⁇ -3, and the three alternatively spliced isoforms, ER ⁇ -1, ER ⁇ -2 and ER ⁇ -4. Additionally, these compositions can be used to screen for ER ⁇ c based disease to facilitate disease prognosis and to monitor disease- related aberrant expression of ER ⁇ c or its isoforms.
- the specific embodiments disclosed in this invention relate to the isolation of the nucleic acid sequence that encodes the ER ⁇ c gene, ER ⁇ -3.
- the murine (m) form of ER ⁇ -3 is composed of 1,704 base pairs (bp) from the ATG start codon to TGA (Fig. la and b) and encodes a 567 amino acid protein; this sequence contains nine exons, including the newly described exon 5B, which is located in the region encoding the LBD.
- Also isolated were three alternatively spliced isoforms: mER ⁇ -1, mER ⁇ -2 and rER ⁇ -4.
- mER ⁇ -1 is 1,650 bp and encodes a 549 residue long polypeptide; ER ⁇ -1 lacks exon 5B (see Figs, la and 2a).
- mER ⁇ -2 is composed of 1,533 base pairs (bp); it lacks both exon 5B and exon 3, which contains 117 bp (see Figs. 1 a and 2b).
- the sequence encoding rER ⁇ -4, an alternatively spliced isoform isolated from rat ovaries, is composed of 1,570 bp; it contains exon 5B, and the 54 bp it comprises, but exon 6, which contains 134 bp, has been deleted (see Figs, la and 2c).
- the methods of using the nucleic acid sequences of ER ⁇ -3 or its isoforms include determination of what tissues express ER ⁇ c and its isoforms (e.g., ER ⁇ -1, ER ⁇ -2 and ER ⁇ -4), function characterization for the proteins and nucleic acid sequences of ER ⁇ -3 and its isoforms, development of methods to recombinantly express ER ⁇ c nucleic acid molecules and their associated protein products, development of an ER ⁇ -3 reporter system, identification of ER ⁇ -3 ligands such as estrogen that influence ER ⁇ -3 or its isoforms and identification of compounds that modulate the influence exerted by ER ⁇ -3 or an isoform thereof on transcriptional regulation of other genes and determining the corresponding physiological effects of such influence.
- ER ⁇ c and its isoforms e.g., ER ⁇ -1, ER ⁇ -2 and ER ⁇ -4
- function characterization for the proteins and nucleic acid sequences of ER ⁇ -3 and its isoforms e.g.,
- RT-PCR reverse transcriptase
- 5' RACE rapid amplification of cDNA ends
- Genomic primers wee used for RT-PCR on mouse ovary RNAs to clone murein (m) mER ⁇ -1, mER ⁇ -2 and mER ⁇ -3.
- the sequences for mER ⁇ -1, mER ⁇ -3 and the rat (r) isoform rER ⁇ -4 were obtained by 5' RACE using the Marathon system and a different set of primers.
- the primers and vectors chosen to isolate and clone these sequences would have been commonly known to an individual skilled in the art.
- the nucleic acid sequence information for ER ⁇ -3 predicts a 567 amino acid protein with a molecular weight of approximately 63 kDa, instead of 54 kD predicted for ER ⁇ j.
- the heretofore unknown mER ⁇ -3 gene or portions thereof can be used as probes. These probes should be of at least 18 nucleotides and preferably should be redundant for one or more sequences encoding the ER ⁇ -3 protein; the probes are to be designed from the ER ⁇ c amino acid sequence and should account for the degenerate genetic code.
- An appropriate cDNA library such as that for ovary, testes or prostate cells, may then be screened with the probes for cDNAs which hybridize under standard conditions to one or more of the probe compositions.
- the cDNAs may then be isolated and sequenced to determine whether they code for the ER ⁇ c protein. In this manner, the cDNA encoding the human ER ⁇ c protein or other mammalian ER ⁇ c genes and their respective species specific isoforms may be isolated.
- nucleic acid sequences can be isolated by probing a DNA library such as that for prostate, ovary or testes, which is comprised of either genomic DNA or cDNA.
- Libraries may be from either commercial sources or prepared from mammalian tissue by techniques known to those skilled in the art.
- the preferred cDNA libraries are human cDNA libraries which are available from commercial sources such as Stratagene.
- the DNA libraries can be probed by plaque hybridization using oligonucleotide probes of at least 20 nucleic acid residues in length, which are complementary to unique sequences of murine or other ER ⁇ -3 genes.
- the preferred probes are the sequences for Primer 1 and Primer 2.
- the nucleic acid probes may be labeled to facilitate isolation of the hybridized clones. Labeling can be by any of the techniques known to those skilled in the art, but typically the probes are labeled with [ 32 P] using terminal deoxynucleotidyl- transferase as disclosed in Sambrook et al, (1989).
- PCR polymerase chain reaction
- in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of ER ⁇ -3 DNA or ER ⁇ -3 mRNA in tissue samples, for nucleic acid sequencing, or for other purposes.
- PCR PROTOCOLS A GUIDE TO METHODS AND APPLICATIONS (Innis, M, Gelfand, D., Sninsky, J. and White, T., eds.), Academic Press, San Diego (1990), incorporated herein by reference.
- nucleic acid molecules that encode ER ⁇ - 1, and the related ER ⁇ -3 isoform proteins herein described, preferably in isolated form.
- nucleic acid is defined as RNA or DNA that encodes a ER ⁇ -3 polypeptide, or is complementary to nucleic acid sequence encoding such peptides, or hybridizes to such nucleic acid and remains stably bound to it under appropriate stringency conditions, or encodes a polypeptide sharing at least 75% sequence identity, preferably at least 80%, and more preferably at least 85%, with the peptide sequences.
- genomic DNA e.g., genomic DNA, cDNA, mRNA and antisense molecules, as well as nucleic acids based on alternative backbone or including alternative bases whether derived from natural sources or synthesized.
- a hybridizing or complementary nucleic acid is defined further as being novel and nonobvious over any prior art nucleic acid including that encodes, hybridizes under stringent conditions or other appropriate stringency conditions, or is complementary to a nucleic acid encoding an ER ⁇ -3 protein according to the present invention.
- “Stringent conditions” are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl, 0.0015 M sodium titrate, 0.1 % SDS at 50°C; or (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin (BSA), 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C.
- BSA bovine serum albumin
- polyvinylpyrrolidone 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C.
- Another example is use of 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1%) SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2 x SSC and 0.1% SDS.
- a skilled artisan can readily determine and vary the stringency conditions appropriately to obtain a clear and detectable hybridization signal.
- the complete estrogen receptor ⁇ such as mER ⁇ -3, contains nine exons.
- the three isoforms that have been isolated include mER ⁇ -1, mER ⁇ -2, mER ⁇ -3 and the alternatively spliced isoform from rat ovaries, rER ⁇ -4.
- mER ⁇ -1 is 1,650 bp; it contains the previously identified eight exons, lacks the new exon 5B, but contains the previously undescribed 192 bp located at the 5' end of exon 1 (see Figs, la and 2a).
- nucleotide 1,244 in exon 6 of the mER ⁇ -3 sequence is an adenine whereas in the sequence by Tremblay et al, (1997) it is a guanine (nucleotide 1,009).
- mER ⁇ -2 contains 1,533 base pairs (bp); ER ⁇ -2 lacks both exon 3 and exon 5B (see Figs, la and 2b).
- rER ⁇ -4 includes 1,570 bp and has exon 5B, but exon 6 is deleted (see Figs, la and 2c).
- the full length mER ⁇ -3 contains a previously unidentified 192 nucleotides at its 5' terminus as well as the sequences of exon 5B and exon 6. All three isoforms, as well as mER ⁇ -3, contain the novel 192 bp located at the 5' terminus of exon 1.
- One embodiment of this invention includes using ER ⁇ -3 nucleic acid sequences containing the heretofore unknown 192 bp or 54 bp (exon 5B) domains or portions thereof and placing these sequences in appropriate vectors for purposes of replication. Such vectors can then be introduced into the appropriate cell expression systems to express the proteins for use either in an assay system or to help to characterize the function of particular portions of the ER ⁇ c gene or its corresponding protein. Characterization of the ER ⁇ c protein can be performed by creating mutants, using antibodies that recognize specific domains on ER ⁇ c and using polypeptide sequences to specific regions of the protein to determine their function through competition assays. This invention proposes using such techniques to characterize the specific functions of the sequences or isoforms containing the novel 192 bp and/or exon 5B (54 bp) sequences.
- Another method of characterizing ER ⁇ c and its isoform proteins includes the use of antibodies to map out specific functional domains on the ER ⁇ c protein, including the LBD, the dimerization site, and the DNA binding domain (DBD) of the ER ⁇ c protein. Antibodies could also be utilized to determine whether the ER ⁇ c or its isoforms is in a functional or non-functional conformation. C. Creating antibodies to ER ⁇ . protein sequences
- Antibodies are useful in several areas, including determining tissue expression of ER ⁇ c , such as ER ⁇ -3 or its isoforms (e.g., ER ⁇ -1, ER ⁇ -2 or ER ⁇ -4), and determining the functional domains of ER ⁇ -3 or its isoforms.
- tissue expression of ER ⁇ c such as ER ⁇ -3 or its isoforms (e.g., ER ⁇ -1, ER ⁇ -2 or ER ⁇ -4), and determining the functional domains of ER ⁇ -3 or its isoforms.
- tissue expression of ER ⁇ c such as ER ⁇ -3 or its isoforms (e.g., ER ⁇ -1, ER ⁇ -2 or ER ⁇ -4), and determining the functional domains of ER ⁇ -3 or its isoforms.
- Another embodiment of this invention includes using polypeptides to create antibodies. Polypeptide sequences can be assessed using computer software to determine the antigenicity of certain polypeptide sequences for the purpose of creating antibodies to these ER ⁇ c specific poly
- One antibody that has been created is an anti-peptide antibody that can distinguish between the mER ⁇ -3 and ER ⁇ j .
- Other antibodies can be created to distinguish between the ER ⁇ -3 isoforms, in addition to being able to distinguish between the active and inactive states of ER ⁇ resulting from allosteric-induced ligand interactions with the receptor.
- the anti-peptide antibodies that distinguish between ER ⁇ -3 and ER ⁇ j were prepared using conventional methods and were raised to the polypeptide sequence encoded by exon 5B with a cysteine group at the amino terminus: N - CSSEDPHWHVAQTKSAVPR - OH (Antibodies 1067 and 1068). This antibody contains all of the exon 5B polypeptide.
- the Jameson- Wo If antigenicity program determined that this polypeptide possesses a high degree of antigenicity.
- This program or the Hopp and Wood algorithm can also be employed to determine sequences of antigenicity in the novel amino terminus of ER ⁇ -3 and its isoforms to develop additional antibodies.
- Antibodies 1069 and 1070 Two other antibodies were created that recognize both ER ⁇ -3 and ER ⁇ j. These antibodies (Antibodies 1069 and 1070) were created against the following sequence: N - CSSTEDSKNKESSQ - OH. This polypeptide sequence is located in the carboxy terminus of the published rat ER ⁇ j . Kuiper et al, (1996 and 1997). Antibodies 1067 and 1068 or 1069 and 1070 were obtained from the eggs of different chickens.
- ER ⁇ -3 polypeptide sequences An alternative method to create antibodies to ER ⁇ -3 polypeptide sequences involves isolating ER ⁇ -3 proteins and digesting them with various proteases. The cleavage fragments can then be purified by size and used to raise antibodies against specific portions of ER ⁇ -3. Finally, ER ⁇ -3 polypeptide sequences can be created recombinantly through fusion protein techniques. ER ⁇ -3 polypeptide sequences can be expressed by fusing the desired ER ⁇ -3 nucleotide sequence to, for example, the gene expressing glutathione S-transferase (GST).
- GST glutathione S-transferase
- the expressed ER ⁇ -3 polypeptide sequences created as a fusion ER ⁇ -3/GST fusion product can then be used to create antibodies to the specific portion of ER ⁇ -3 encoded in the ER ⁇ -3 containing fusion gene construct.
- Antibodies raised to such recombinant proteins can be either monoclonal or polyclonal and such preparation techniques are generally known. Polyclonal antibodies 1067, 1068, 1069 and 1070 were raised in chickens. Other animals could also be utilized. Pre-immune sera was purified from 2-3 eggs collected prior to hen immunization.
- Immunizations were prepared with 2 mg of antigen conjugated to 2 mg Imject Keyhole limpet hemocyanin (KLH) via maleimide to the extra cysteine residue located at the amino terminus of each peptide as recommended in the manufacturer's (Pierce) instructions.
- the coupled carrier-antigen complex (0.5 ml) was emulsified with Complete Freund's adjuvant (0.5 ml) and 1.0 ml was used for the initial injection.
- the chickens were subsequently boosted every 2 weeks with coupled immunogen as described by Aves Laboratory, except that Incomplete Freund's Adjuvant was used.
- Six eggs were collected and the IgY was purified from the yolks.
- Other immunoglobulin isotypes and isotype subclasses can also be used (e.g., IgG,, IgG 2 , IgM).
- Monoclonal antibodies may be obtained by various techniques familiar to those skilled in the art. Briefly, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see, Kohler and Milstein, Eur. J. Immunol. 6: 511 (1976)). Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses or other methods well known to those of ordinary skill in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired antigen specificity and affinity. The yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host.
- peptide specific antibodies such as antibodies 1067 and 1068
- suitable mammalian hosts e.g., chickens or rabbits
- suitable mammalian hosts e.g., chickens or rabbits
- suitable mammalian hosts e.g., chickens or rabbits
- suitable mammalian hosts e.g., chickens or rabbits
- suitable mammalian hosts e.g., chickens or rabbits
- suitable mammalian hosts e.g., chickens or rabbits
- Methods for preparing immunogenic conjugates with carriers such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH) or other carrier proteins are well known in the art.
- BSA bovine serum albumin
- KLH keyhole limpet hemocyanin
- direct conjugation using, for example, carbodiimide reagents may be effective.
- linking reagents such as those supplied by Pierce Chemical Co., Rockford, IL, may be desirable to provide accessibility to the hapten.
- the hapten peptides can be extended at either the amino or carboxy terminus with a cysteine (Cys) residue or interspersed with cysteine residues, for example, to facilitate linking to a carrier.
- Administration of the immunogens is conducted generally by injection over a suitable time period and with use of a suitable adjuvant, as is generally understood in the art. During the immunization schedule, titers of antibodies are taken to determine adequacy of antibody formation.
- Immortalized cell lines which secrete the desired monoclonal antibodies may be prepared using the standard method of Kohler and Milstein or with modifications which effect immortalization of lymphocytes or spleen cells, as is generally known. Kohler and Milstein, (1976). The immortalized cell lines secreting the desired antibodies are screened by immunoassay in which the antigen is the peptide hapten or is the ER ⁇ c protein itself.
- the cells can be cultured either in vitro or by production from ascites fluid.
- the desired monoclonal antibodies are then recovered from the culture supernatant or from the ascites supernatant. Fragments of the monoclonal or the polyclonal antisera which contain the immunologically significant portion can be used as antagonists, as well as the intact antibodies.
- Use of immunologically reactive fragments, such as the Fv, Fab, Fab', or F(ab') 2 fragments, is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin.
- the antibodies or fragments may also be produced, using current technology, by recombinant means.
- Regions that bind specifically to the desired regions of the receptor can also be produced in the context of chimeras with multiple species origin.
- the ER ⁇ c specific antibody can be humanized antibodies or human antibodies, as described in U. S. Patent No. 5,585,089 by Queen et al. See also Riechmann et al, Nature 332: 323 (1988).
- ER ⁇ c protein may be involved in certain cancers, it would be useful to create bispecific antibodies capable of recognizing both the ER ⁇ c protein and, for example, cytotoxic T cells to facilitate the killing of tumor cells which may be useful in treating cancer. Berg et al. , PNAS 88: 4723 (1991).
- the antibodies thus produced are useful not only as modulators of ER ⁇ c -estrogen interaction, but are also useful in immunoassays to detect ER ⁇ c protein or its isoforms and for the purification of ER ⁇ c protein or its protein isoforms.
- immunoassays to detect the ER ⁇ c protein or its alternatively spliced isoforms.
- Immunoassays can be used to qualitatively and quantitatively analyze the ER ⁇ c protein.
- a general overview of the applicable technology can be found in Harlow and Lane, (1988).
- ER ⁇ c protein or a fragment or isoform thereof is expressed in transfected cells, preferably bacterial cells, and purified as generally described above and in the examples.
- the product is then injected into a mammal capable of producing antibodies.
- Either monoclonal or polyclonal antibodies specific for the gene product can be used in various immunoassays; such assays include enzyme linked immunoabsorbant assays (ELISAs), competitive immunoassays, radioimmunoassays, Western blots (Fig. 3), indirect immunofluorescent assays, gel shift assays (Fig. 5) and the like.
- ELISAs enzyme linked immunoabsorbant assays
- competitive immunoassays include radioimmunoassays, Western blots (Fig. 3), indirect immuno
- One embodiment of this invention utilizes ER ⁇ c polypeptide sequences to assay their ability to interfere with ER ⁇ c protein mediated transcription regulation.
- interference can be created by preventing ER ⁇ c activating agents, such as estradiol, from binding to the ER ⁇ c protein.
- a polypeptide could be designed to inhibit dimerization and subsequent signaling from occurring.
- Such polypeptides could be created using peptide synthesizers or by creating fusion protein expressing gene constructs or other expression systems for either prokaryotic or eukaryotic cell systems.
- the expression of natural or synthetic nucleic acids encoding mammalian ER ⁇ c will typically be achieved by operably linking the gene or cDNA to a promoter (which is either constitutive or inducible) and incorporating it into an expression vector.
- the vectors preferably are suitable for replication and integration in either prokaryotes or eukaryotes.
- Typical cloning vectors contain transcription and translation terminators, initiation sequences and promoters useful for regulation of the expression of the ER ⁇ c gene.
- the vectors may also comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the plasmid in both eukaryotes and prokaryotes, i.e., shuttle vectors and selectable markers for both prokaryotic and eukaryotic systems.
- cloned genes in bacteria are also well known.
- selectable markers in DNA vectors transformed in E. coli is also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline or chloramphenicol.
- Suitable eukaryote hosts may include plant cells, insect cells, mammalian cells, yeast and filamentous fungi.
- the baculovirus/insect cell system is used for gene expression.
- the protein encoded by the ER ⁇ c gene which can be produced by recombinant DNA technology, may be purified by standard techniques well known to those of skill in the art. Recombinantly produced ER ⁇ c can be directly expressed or expressed as a fusion protein. The protein is then purified by a combination of cell lysis (e.g., sonication) and affinity chromatography. For fusion products, subsequent digestion of the fusion protein with an appropriate proteolytic enzyme releases the desired ER ⁇ c , its isoforms or a fragment thereof.
- cell lysis e.g., sonication
- affinity chromatography affinity chromatography
- the purified ER ⁇ c when described as “isolated” and or “substantially pure”, describes a protein that has been separated from components which naturally accompany it. Typically, a monomeric protein is substantially pure when at least about 85% or more of a sample exhibits a single polypeptide backbone. Minor variants or chemical modifications may typically share the same polypeptide sequence. Depending on the purification procedure, purities of 85%, and preferably over 95% pure are possible. Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band on a polyacrylamide gel upon staining. For certain purposes, high resolution will be needed and high performance liquid chromatography (HPLC) or a similar means for purification utilized.
- HPLC high performance liquid chromatography
- the ER ⁇ c protein or its isoforms of this invention may be purified to substantial purity by standard techniques well known in the art, including selective precipitation with such substances as ammonium sulfate, column chromatography, immunopurification methods, and others. See, for instance, R. Scopes, PROTEIN PURIFICATION: PRINCIPLES AND PRACTICE, Springer- Verlag: New York (1982).
- ER ⁇ c polypeptides or isoform polypeptides could then be used in various assays, such as gel shift assays or yeast two hybrid systems wherein these polypeptide sequences can be tested to observe their binding ability to the hormone response elements (HRE) on DNA sequences, dimerization binding ability, and agonist/antagonist binding ability.
- HRE hormone response elements
- probes can be synthesized either using polymerase chain reaction (PCR) techniques or using in vitro transcription, of which both techniques are known to skilled artisans. These probes, which are typically radiolabeled, can be utilized to determine which tissues express a particular ER ⁇ c transcript either via Northem blot analysis or dot blots of RNA samples or by Southern blots wherein the mRNA has been reverse transcribed into DNA, which is then further amplified using polymerase chain reaction (PCR) as demonstrated in Fig. 4.
- PCR polymerase chain reaction
- Southern analysis of DNA is also useful in determining whether the ER ⁇ c gene is present or disrupted. For example, it is known that ER ⁇ is disrupted in certain breast tumors; such information may in turn be beneficial in determining the course of chemotherapy to be utilized on a patient. Using nucleic acid sequences unique to the ER ⁇ c , it can be readily determined what tissues express the gene.
- the present invention also provides methods for detecting the presence, absence and or abnormal expression o ⁇ ER ⁇ c gene products in a physiological specimen, as well as in other tissue samples.
- One method for evaluating the presence or absence of ER ⁇ c in a sample involves a Southern transfer and is well known to those of skill in the art (Fig. 4). Briefly, the digested genomic DNA is run on agarose slab gels in buffer and transferred to membranes. Hybridization is carried out using the probes discussed above. Visualization of the hybridized portions allows the qualitative determination of the presence or absence of the ER ⁇ c gene or its isoforms. Southern blotting will also distinguish, depending on the stringency conditions used for hybridization, whether the ER ⁇ c gene is normal or contains gene deletions or rearrangements.
- RNA messenger RNA
- This procedure is also well known in the art. See Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold
- the mRNA is isolated from a given cell sample using an acid guanidinium-phenol-chloroform extraction method. The mRNA is then electrophoresed to separate the mRNA species, and the mRNA is transferred from the gel to a nitrocellulose membrane. Labeled probes are used to identify the presence or absence of the ER ⁇ c transcript.
- An alternative means for determining the level of expression of the ER ⁇ c gene is in situ hybridization. In situ hybridization assays are well known and are generally described in Angerer et al, Methods Enzymol.. 152: 649 (1987). This hybridization technique has already been used to study ER ⁇ j expression in rat hypothalamus.
- This invention relates to recombinant sequences that express the entire ER ⁇ c gene, its isoforms or portions thereof that include the newly described 5' terminus or the newly described 54 bp of exon 5B, as described in Figures 1 and 2.
- the invention includes all methods of expressing such recombinant constructs both in prokaryotic and eukaryotic replication systems, which would have been known to one skilled in the art.
- rDNA deoxyribonucleic acid
- rRNA ribonucleic acid
- the invention also relates to a method of introducing the recombinant full length form of ER ⁇ 0 such as ER ⁇ -3 or one of the other isolated isoforms, ER ⁇ -1, ER ⁇ -2 or ER ⁇ -4 into non-ER ⁇ -3 expressing cells and assaying the effect said rDNA and its associated protein product have on transcriptional regulation.
- ER ⁇ -3 e.g., ER ⁇ -1, ER ⁇ -2 or ER ⁇ -4
- the present invention further provides host cells transformed or transfected with a nucleic acid molecule encoding an ER ⁇ -3 protein.
- the host cell can be either prokaryotic or eukaryotic.
- Eukaryotic cells useful for expression of a ER ⁇ -3 protein are not limited, so long as the cell line is compatible with cell culture methods and with the propagation of the expression vector and expression of the ER ⁇ -3 gene product.
- Preferred eukaryotic host cells include, but are not limited to, yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic cell line. Particularly preferred eukaryotic host cells include insect cells. Any prokaryotic host can be used to express an ER ⁇ -3 encoding recombinant DNA (rDNA) molecule.
- the preferred prokaryotic host is E. coli.
- Transformation of appropriate cell hosts with a rDNA molecule of the present invention is accomplished by well known methods that typically depend on the type of vector used and host system employed. With regard to transformation of prokaryotic host cells, electroporation and salt treatment methods are typically employed, see, for example, Cohen et al, PNAS 69: 2110 (1972); and Maniatis et al, (1982); Sambrook et al, (1989); or CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, F. Ausubel et al, ed. Greene Publishing and Wiley-Interscience, New York (1987).
- Utilization of the full-length, fragments, or isoforms of ER ⁇ -3 to dete ⁇ riine their ability to regulate the formation of colonies in soft agar is useful in assessing whether a particular isoform of the ER ⁇ -3 gene is responsible for cellular proliferation and or differentiation.
- the ability of a particular ER ⁇ -3 isoform to spur proliferation and/or differentiation may in turn correspond to the gene's involvement in certain ER ⁇ associated diseases.
- the isoforms may be used in transfected cell lines to assay [ 3 H]- thymidine incorporation to test the effect of a particular ER ⁇ c isoform on DNA synthesis.
- Fluorescent activated cell sorting FACS
- FACS Fluorescent activated cell sorting
- transfected cells could be examined for morphological changes due to the expression of different ER ⁇ c isoforms.
- This invention also describes the methods used to express the ER ⁇ c protein, such as by using recombinant DNA (rDNA) of the ER ⁇ -3 gene, such as using its novel isoforms (ER ⁇ -1, ER ⁇ -2 and ER ⁇ -4) or portions thereof.
- rDNA recombinant DNA
- Methods for generating rDNA molecules are well known in the art, for example, see Sambrook et al. , (1989).
- the preferred rDNA molecules would contain an ER ⁇ -3 encoding DNA or a DNA encoding one of its isoforms operably linked to expression control sequences and/or vector sequences.
- vector and or expression control sequences to which one of the ER ⁇ c nucleic acid molecules of the present invention is operably linked depends directly, as is well known in the art, on the functional properties desired, e.g. , protein expression, and the host cell to be transformed.
- Any vector contemplated by the present invention should be at least capable of directing replication, insertion into the eukaryote's chromosome or replicating extrachromasomally in a prokaryote, and preferably also expression of the ER ⁇ -3 protein encoded in the rDNA molecule.
- Expression control elements that are used for regulating the expression of an operably linked protein encoding sequence are known in the art and include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, and other regulatory elements.
- the inducible promoter is readily controlled, such as being responsive to a nutrient in the host cell's medium.
- the vector containing a ER ⁇ -3 encoding nucleic acid molecule will include a prokaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
- a prokaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
- a prokaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
- vectors that include a prokaryotic replicon may also include genes which confer such detect
- Vectors that include a prokaryotic replicon can further include a prokaryotic or viral promoter capable of directing the expression (transcription and translation) of the ER ⁇ - 3 gene sequences in a bacterial host cell, such as E. coli.
- a promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.
- Typical of such vector plasmids are pUC8, pUC9, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, CA), pPL and pKK223 available from Pharmacia, Piscataway, N. J.
- Expression vectors compatible with eukaryotic cells can also be used to form rDNA molecules that contain ER ⁇ -3 sequences.
- Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired DNA segment. Typical of such vectors are pSVL and pKSV-10 (Pharmacia), pBPV-l/pML2d (International Biotechnologies, Inc.), pTDTl (ATCC, #31255), the vector pCDM8 described herein, and like eukaryotic expression vectors.
- Eukaryotic cell expression vectors used to construct the rDNA molecules of the present invention may further include a selectable marker that is effective in an eukaryotic cell, preferably a drug resistance selection marker.
- a preferred drug resistance marker is the gene for which expression results in neomycin resistance, . e. , the neomycin phosphotransferase (neo) gene as described by Southem et al. , J. Mol. Anal. Genet. 1 : 327 (1982).
- Another embodiment of the present invention is the use of ER ⁇ c nucleic acid sequences to measure changes in cells' mRNA concentrations.
- Methods of quantitatively and/or qualitatively assessing mRNA levels includes Northem blotting, in situ hybridization, nucleic acid hybridization and RT-PCR. Raval, J. Pharmacol. Toxicol. Methods 32(3): 125 (1994).
- the mRNA may be reverse transcribed into DNA and the DNA expanded using PCR.
- RT-PCR Reverse transcription PCR
- This in situ labeling technique which would employ labeled nucleic acid sequences capable of hybridizing to ER ⁇ c mRNA or its alternatively spliced isoforms and subsequent detection by a imaging device, would be useful in localizing tissues that have increased or decreased expression of ER ⁇ c or its isoforms' mRNA.
- This technique also would be commonly known to individuals skilled in the art. For example, see Guldenaar et al, Br n Res.700 (1-2): 107 (1995).
- the presence and amount of transcription and expression of ER ⁇ -3 or its isoforms may be determined, as a measure of the expression of ER ⁇ -3 protein, as well as other proteins for which transcription is regulated by the ER ⁇ -3 protein.
- This information is related to the aggressive nature of a particular cancer, the change in the nature of the cancer in relation to treatments, such as irradiation, chemotherapy, or surgery, the metastatic nature of the cancer, as well as the aggressiveness of metastases, and the like.
- treatments such as irradiation, chemotherapy, or surgery
- the metastatic nature of the cancer as well as the aggressiveness of metastases, and the like.
- Maas et al Cancer Lett. 97(1): 107 (1995), which discussed changes of specific mRNA levels in breast cancer cells using RT-PCR after treatment with different anti-cancer agents.
- This relationship may be useful for deterrnining the level of therapeutic treatment, monitoring the response of the tumor (or other ER ⁇ c related diseases) to the therapeutic treatment, and in
- Another embodiment of the present invention provides methods for identifying agents that inhibit or block the association of an estrogen or estrogen-like agonists/antagonists with ER ⁇ c protein.
- estrogen can be mixed with the ER ⁇ c protein or a cellular extract containing the ER ⁇ c , in the presence and absence of the compound to be tested. After mixing under conditions that allow association of the estrogen or estrogen-like agonist/antagonist with ER ⁇ c , the two mixtures are analyzed and compared to determine if the compound augmented, reduced or completely blocked the association of the estrogen or estrogen-like agonist/antagonist with the ER ⁇ . protein or its isoforms. Agents that block or reduce the association of an estrogen or estrogen-like agonist/antagonist with the ER ⁇ c protein will be identified as decreasing the concentration of estrogen-ER ⁇ c binding present in the sample containing the tested compound.
- the receptor protein likely must undergo allosteric change in its conformation before the estrogen-ER ⁇ c complex has the ability to bind to DNA. Once inside the nucleus, the activated receptor initiates transcription of genetic information from the DNA to mRNA, which is in turn a template for the linking of amino acids into proteins.
- the antiestrogen effects produced by drugs such as tamoxifen appear to be one of preventing the estrogen receptor from interacting with DNA in the nucleus to stimulate RNA and protein synthesis. This action initiates a block in the synthesis of macromolecules such as proteins, causing cell damage and the ultimate death of the cell.
- Antiestrogens are believed to be lipophilic molecules having a portion of the molecule which resembles naturally occurring estrogens. This portion of the antiestrogen selectively binds to the estrogen receptors.
- the antiestrogens however, have a side chain arm (e.g., dimethylaminophenyl ethoxy) which distorts the three-dimensional configuration of the estrogen receptor preventing translocation of the receptor to the nucleus.
- a side chain arm e.g., dimethylaminophenyl ethoxy
- Another method of deteirnining whether candidate reagents inhibit estrogen action on the complete estrogen receptor ⁇ subtype would be by deteirnining whether ER ⁇ c has undergone an allosteric transformation as a result of interacting with a candidate reagent such that ER ⁇ c or its isoforms can no longer combine with the native substrate, estrogen.
- Changes in the conformation of ER ⁇ c or homodimers of ER ⁇ c can be detected using antibodies, either monoclonal or polyclonal, to conformational epitopes that exist on ER ⁇ c or homodimers of the receptor.
- Antibodies were used to determine the functional state of ER ⁇ and a similar method could be used in deteirnining whether compounds augment transformation into the activated allosteric conformation or inhibit the conformation all together. See Wotiz et al , U.S. Patent No. 5,312,752 (1994).
- Antibodies can not only be used to determine whether the ER ⁇ c is functionally in an active or inactive state. Antibodies could also be screened to determine whether their binding to either the ligand or to the receptor itself enhanced the binding of the ligand to the receptor. Methods of deterrnining said enhancement are known to the art. See Aguilar et al. Mol. Cell. Biochem. 136(1): 35 (1994). Another method of deterrnining whether a particular reagent augments or inhibits dimerization of ER ⁇ c or augments or inhibits ER ⁇ c from assuming the activated state would be to utilize a yeast two hybrid system.
- Yeast two hybrid systems have been successfully used to determine whether ER ⁇ dimerization is ligand-dependent (Wang et al, J. Biol. Chem. 270(40): 23,322 (1992)); to isolate agents such as proteins or antibodies that enhance transcriptional activity of hormone receptors (Onate et al, Science 270(5240): 1354 (1995)); to isolate compounds that are antagonistic to ER ⁇ c action in a manner comparable to what has been done with ER ⁇ (Ichinose et al. , (1997) and Collins et al. , (1997)); and to determine whether ER ⁇ c can form heterodimers in a manner analogous to what has been observed for retinoic acid receptors. See for further discussion Forman et al, Cell 81(4): 541 (1995) and Walfish et al, PNAS 94(8): 3697 (1997).
- Another method to screen agents is to use a reporter gene such as ⁇ -galactosidase ( ⁇ - gal) or luciferase.
- ⁇ - gal ⁇ -galactosidase
- luciferase ⁇ -galactosidase
- cV2ERE estrogen responsive element
- Compounds that are assayed by the above methods can be randomly selected or rationally selected or designed.
- an agent is said to be randomly selected when the agent is chosen arbitrarily, without considering the specific sequences involved in the association of the estrogen or estrogen-like agonist/antagonist to the ER ⁇ c protein.
- An example of such randomly selected agents is the use a chemical library, a peptide combinatorial library or a growth broth of an organism.
- an agent is said to be rationally selected or designed when the agent is chosen on a non-random basis which takes into account the sequence of the target site and/or its conformation in connection with the agent's action.
- Agents can be rationally selected or rationally designed by utilizing the peptide sequences that recognize and bind to either the estrogen or estrogen-like agonist/antagonist or to the steroid hormone binding site on the ER ⁇ c protein.
- the agents of this embodiment can be, by way of example, peptides or other small molecules, antibodies (e.g. , monoclonal or polyclonal), fragments of antibodies (e.g., Fv), or drugs with antiestrogenic or estrogenic activity (e.g., narigenin, kaempferide, phloretin, biochanin A, flavone, ICI 182,780, raloxifene, tamoxifen, [6-hydroxy-3-[4-[2-(l- piperid ⁇ yl)ethoxy]phenoxy]-2-]4-hydroxybenzo[b]thiophene, raloxifene HCl, and ethynyl estradiol).
- drugs with antiestrogenic or estrogenic activity e.g., narigenin, kaempferide, phloretin, biochanin A, flavone, ICI 182,780, raloxifene, tamoxif
- One class of compounds of the present invention includes polypeptide agents whose amino acid sequences are chosen based on the amino acid sequence of the ER ⁇ c LBD.
- the peptide agents of the invention can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as is known in the art.
- rDNAs encoding these polypeptides may be synthesized using commercially available oligonucleotide synthesis instrumentation and produced recombinantly using standard recombinant production systems. These rDNA molecules can then be utilized to recombinantly express polypeptides that bind to the ER ⁇ -3 protein or its isoforms. The production using solid phase peptide synthesis is necessitated if non-recombinantly produced polypeptide sequences are to be used.
- agents that affect ER ⁇ c signaling can be provided alone, or in combination with additional agents that modulate a particular pathological process.
- an agent of the present invention that reduces or otherwise modulates ER ⁇ c transcriptional regulation, by blocking estrogen or other agonist/antagonists from binding and tiansforming the ER ⁇ c protein or its isoforms into an active state can be administered in combination with other similar agents.
- two agents are said to be adudiissered in combination when the two agents are administered simultaneously or are administered independently in a fashion such that the agents will act at the same time.
- the agents of the present invention can be administered via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route.
- the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
- the present invention further provides compositions containing one or more agents which block transcriptional regulation by the ER ⁇ -1 protein. While individual needs may vary, determination of optimal ranges of effective amounts of each component is vvithin the skill of the art.
- compositions of the present invention may contain pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically for delivery to the site of action.
- suitable formulations for parenteral adnrinistration include aqueous solutions of the active compounds in water- soluble form, for example, water-soluble salts.
- suspensions of the active compounds as appropriate oily injection suspensions may be a ⁇ inistered.
- Suitable lipophilic solvents or vehicles include fatty oils (e.g., sesame oil) or synthetic fatty acid esters (e.g., ethyl oleate or triglycerides).
- Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran.
- Antiestrogens are typically characterized as having limited solubility, therefore the use of agents such as dimethylformamide increases the solubility of such agonists/antagonists thus increasing their effect on, in this instance, ER ⁇ c or its isoforms.
- the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the agent for delivery into the cell.
- agents e.g., acetone and polyethylene glycol 4000
- acetone and polyethylene glycol 4000 may be required to enhance the drug's solubility.
- the pharmaceutical formulation for systemic a ⁇ rninistration according to the invention may be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulations may be used simultaneously to achieve systemic a ⁇ inistration of the active ingredient.
- Suitable formulations for oral adrninistration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.
- the compounds of this invention may be used alone or in combination with other therapeutic or diagnostic agents.
- the compounds of this invention may be co-administered along with other compounds typically prescribed for these conditions according to generally accepted medical practice.
- ER ⁇ c gene for example ER ⁇ -3 gene and the ER ⁇ -3 protein can also serve as a target for gene therapy in a variety of contexts.
- ER ⁇ -3 deficient animals can be generated using standard knock-out procedures to inactivate a ER ⁇ - 3 gene.
- a non-human mammal e.g., a mouse or a rat
- ER ⁇ -3 gene is inactivated or deleted. This can be accomplished using a variety procedures known in the art, such as targeted recombination.
- the ER ⁇ -3 deficient animal can be used to (1) identify biological and pathological processes mediated by the ER ⁇ -3 gene; (2) identify proteins and other genes that interact with ER ⁇ -3; (3) identify agents that can be exogenously supplied to overcome ER ⁇ -3 deficiency; and (4) serve as an appropriate screen for identifying mutations within ER ⁇ -3 gene that increase or decrease activity.
- human ER ⁇ c deficiencies or mutations can be corrected by supplying to a patient a genetic construct encoding the necessary ER ⁇ c protein.
- a variety of techniques are presently available, and others are being developed, for introducing nucleic acid molecules into human subjects to correct genetic deficiencies and mutations. Such methods can be readily adapted to employ the ER ⁇ c encoding nucleic acid molecules of the present invention.
- genetic therapy can be used as a means for modulating an ER ⁇ c mediated biological or pathological process.
- a genetic expression unit that encodes a modulator of ER ⁇ c mediated transcriptional regulation, such as a nucleic acid molecule that is antisense to the ER ⁇ c mRNA.
- tissue specific co-activators or co-repressors could be identified and introduced into a recipient to augment modulation of ER ⁇ c or its isoforms.
- Such a modulator can either be constitutively produced or inducible within a cell or specific target cell. This allows a continual or inducible supply of a therapeutic agent within the patient.
- the invention includes specifically prepared immunogens, polyclonal antisera and monoclonal antibodies which bind specifically to the DBD of ER ⁇ c or its isoforms, and immunoassays employing these site-specific antibodies with cellular samples on a functional and correlative test basis, as described above.
- Example 1 Cloning of the complete murine mER ⁇ - 3 cDNA
- the mER ⁇ -3 clone was twice isolated using two separate procedures: (1 ) reverse transcriptase PCR (RT-PCR) of mRNA, and (2) amplification from a mouse embryonic stem (ES) cell genomic DNA library.
- RT-PCR reverse transcriptase PCR
- ES mouse embryonic stem
- oligonucleotides were: 5'- ATG ACA TTC TAC AGT CCT GCT GTG ATG-3* (Primer 1) and 5'-GAA GTG AGC ATC CCT CTT TGC GTT TGG-3' (Primer 2). Using these oligonucleotides five clones were obtained.
- Two primers were chosen in these genomic DNAs, one around the first ATG, which is 192 bp upstream from the published ATG (Kuiper et al, (1996); Mosselman et al, (1996); and Tremblay et al, (1997)), 5'-TCT CTG AGA GCA TCA TGT CC-3' (Primer 3), and one around the TGA, 5'-CAG CCT GGC CGT CAC TGT GA-3' (Primer 4).
- the RT-PCR was performed on 10 and 100 ng samples of mouse ovary RNA using the TitanTM RT-PCR System of Boehringer Mannheim according to manufacturer's instructions.
- the amplified products obtained using Primers 3 and 4 underwent a second amplification using: 5'-TGC TCT AGA CCA CCA TGT CCA TCT GTG CCT CT-3' (Primer 5) and 5'-CCG GAA TTC TCA CTG TGA CTG GAG GTT CTG 3' (Primer 6).
- the products obtained using Primers 6 and 7 were then inserted into Bluescript® vector. The same conditions were used to clone mER ⁇ -1, mER ⁇ -2 and mER ⁇ - 3.
- the mER ⁇ -3 clone was also isolated from mRNA using the Marathon RT-PCR system from Clontech.
- poly A+ RNA was prepared from total RNA derived from mouse ovaries according to the methods described in Sambrook et al, (1989). Approximately 0.5 ⁇ g of the poly A+ RNA was reverse transcribed using 200 U Superscript II exogenase- (exo-) using the Marathon cDNA synthesis primer, 5 -TTC TAG AAT TCA GCG GCC GC(T 30 )-3', according to manufacturer instructions (GIBCO). The second strand synthesis and all subsequent steps, except PCR, were performed according to the conditions described by Marathon.
- the cDNA (0.5 ⁇ l of a 10 ⁇ l reaction) was then amplified using the Marathon adaptor primer, 5'-CCA TCC TAA TAC GAC TCA CTA TAG GC-3', with one of two gene specific reverse primers in the presence of Advantage Taq polymerase: 5'-GCA GTA GCT CCT TCA CCC G-3' (Primer 7) or 5'-GCA CTT CAT GCT GAG CAG-3' (Primer 8).
- thermocycling program was used to amplify the two products: (1) 5 cycles, 30 sec at 94°C, 4 min at 72°C; (2) 5 cycles, 30 sec at 94°C; (3) 25 cycles , 4 min at 70°C; and (4) 20 sec at 94°C, 4 min at 68°C.
- Single, predominant amplicons corresponding to the 5' end of the cDNA were then digested with restriction enzymes, cloned and sequenced. The clone was then inserted into a Bluescript® vector as described above.
- nucleic acid and the amino acid sequences were deduced for the complete estrogen receptor ⁇ sequence (see Figs, la and b).
- nucleotide 1,244 an adenine, in exon 6 of the mER ⁇ -3 sequence differs from the guanine (nucleotide 1,009) found in the sequence by Tremblay et al, (1997).
- the mER ⁇ -3 gene is 1,704 nucleotides long and encodes a 567 amino acid protein.
- Example 2 Isolation of three alternatively spliced isoforms
- mER ⁇ -3 two other alternatively spliced murine forms of mER ⁇ -3 were identified (mER ⁇ -1 and mER ⁇ -2,) as well as a fourth alternatively spliced isoform isolated from rat ovaries, rER ⁇ -4.
- the first alternatively spliced form of m.ER ⁇ -3, m.ER ⁇ -1 contains the novel 192 bp at the 5' terminus of exon 1, but lacks the 54 bp of exon 5B; it is 1,650 nucleotides in length and putatively encodes a 549 amino acid long polypeptide (Fig. 2a).
- Preliminary data indicates that the mER ⁇ -1 isoform may be more active than the full length mER ⁇ -3.
- the mER ⁇ -1 isoform was isolated using both methods described for the isolation of mER ⁇ -3.
- Isoform mER ⁇ -2 is composed of 1,533 bp, which would encode 510 amino acids (Fig. 2b); mER ⁇ -2 lacks exon 3, which contains 117 bp.
- the mER ⁇ -2 isoform was isolated only from the mouse ES cell genomic library.
- Isoform rER ⁇ -4 was obtained from rat (r) ovaries whereas mER ⁇ -1 and mER ⁇ -2 as well as the full length m.ER ⁇ -3 were obtained from mouse (m) ovaries; it is 1,570 nucleotides in length and contains exon 5B, but exon 6 is deleted. Exon 6 is comprised (as shown in Fig. la) of 134 bp.
- the putative protein product of rER ⁇ -4 would be 414 amino acids (Fig. 2c). All the nucleic acid sequences discussed relate to the coding regions and sequences for the corresponding mRNAs would be longer in both their 5' and 3' regions. It is likely that the published incomplete estrogen receptor ⁇ genes (ER ⁇ ) isolated from human, rat and mouse libraries are splice variants of this complete form, which in mice is mER ⁇ -3, and contains the 54 bp of exon 5B and the 192 bp located at the 5' terminus of exon 1. Mosselman et al, (1996); Kuiper et ⁇ /.,(1996); and Tremblay et al, (1997).
- All four sequences contain the 192 bp located at the 5' teiminus of exon 1 and not described in the previously published sequences. Id. A sequence similar to the novel 192 bp region located in the 5' terminus of exon 1 may also exist in human ER ⁇ c and its isoforms.
- the alternatively spliced isoforms (e.g., mER ⁇ -1, mER ⁇ -2 and rER ⁇ -4) of the full length murine ER ⁇ c gene, m.ER ⁇ -3, were twice isolated using the same two different procedures used to acquire mER ⁇ -3.
- the primers used in both Examples 1 and 2 were selected based on the assumption that variants, if any, would occur within the boundaries of these selected primers. Historically, similar primers have produced analogous results with ER ⁇ .
- the isoforms were isolated the DNA sequences could be sequenced and the amino acid sequence encoded by each could be determined.
- the proteins for the three alternatively spliced isoforms are shown in Figures 2a, 2b and 2c.
- Anti-peptide antibodies raised against a sequence specific to the mouse ER ⁇ c specifically recognized a protein of 64 kDa in ovary and in bone, as well as in other tissues.
- Two anti-peptide antibodies were raised in chickens to N- CSSEDPHWHVAQTKSAVPR-OH (Antibodies 1067 and 1068); this polypeptide is encoded by exon 5B and recognizes mER ⁇ -3 as well as the isoforms that express the exon 5B coding region.
- Antibody 1067 and 1068 were obtained from the eggs of two different chickens, as were antibodies 1069 and 1070.
- the blots were probed using a 1 : 1,000 dilution of the chicken antisera to mER ⁇ -3 (Antibody 1068) in conjunction with a 1:1 ,000 dilution of a secondary antibody conjugated to horseradish peroxidase (Promega).
- the proteins were visualized using the ECL chemiluminescent substrate, and exposed to film (BMR film, Kodak) for one minute.
- Figure 3 shows the results of the Western blot obtained using Antibody 1068, which detects the polypeptide encoded by exon 5B.
- Total protein 60 ⁇ g was resolved by electrophoresis. The proteins were transferred to nitrocellulose membrane and probed with a 1 : 1,000 dilution of Antibody 1068 (Fig. 3 a).
- Figure 3(b) is the blot probed with antibody 1068 pre-immune sera.
- the protein extracts of each lane of both Figures 3(a) and 3(b) are: lane 1, human ovary; lane 2, mouse ovary; lane 3, rat ovary; lane 4, ROS 17/2.8 cells; lane 5, ROS 17/2.8 cells treated with 100 nM estradiol for 16 hours; lane 6, murine primary osteoblasts.
- the antibody specifically recognizes a 64 kDa protein, which closely approximates the predicted size of mER ⁇ -3.
- the question mark refers to a protein migrating at approximately 58 kDa that may be immune specific but is otherwise unidentified.
- ROS 17/2.8 cells are a line characterized by Gideon Rodan; it is a rat osteoblast-like osteosarcoma cell line (ROS).
- ROS 17/2.8 cells are a rat osteoblast-like osteosarcoma cell line (ROS).
- the rat cDNA was then amplified by PCR in 100 ⁇ l reactions using 2 U Taq polymerase and 1 ⁇ M 5*-GTC AAG TGT GGA TCC AGG-3' (Primer 9; beginning at base 924 of Accession U57439 and corresponding to base 700 of mER ⁇ -3) and 5'-GCT CAC TAG CAC ATT GGG-3' (Primer 10; begirining at base 1,130 of rER ⁇ j by Kuiper et al, Accession U57439, and corresponding to base 906 of mER ⁇ -3) per each individual reaction.
- Products were amplified using 25-40 cycles of the following amplification program: 90°C x 1 min; 55°C x 45 sec; 72°C x 2 min. The product was allowed to be extended at 72°C x 5 min at the end of the program. Following amplification, the PCR products were resolved in a 4% NuSieve agarose (FMP) TBE gel; the DNA was transferred to nylon membranes (Boehringer Mannheim) and cross-linked by UV irradiation for Southem analysis.
- FMP NuSieve agarose
- Figure 4 is an autoradiograph of Southem blot of rat ER ⁇ (rER ⁇ ) products amplified by RT-PCR. Total RNA from a variety of tissues was reverse transcribed, amplified by PCR, transferred to nylon membranes and probed using a 32 P labeled mER ⁇ -3 oligonucleotide. Figure 4 (a) was amplified for 35 cycles.
- Each lane in Figure 4 (a) contains the following types and amounts of RNA: lane 1 , control, no RNA; lane 2, rat ovarian RNA (0.1 ⁇ g); lane 3, ROS 17/2.8 cells (0.1 ⁇ g); lane 4, rat ovarian RNA control (0.1 ⁇ g), no reverse transcriptase (RT); and lane 5, ROS 17/2.8 total RNA (0.1 ⁇ g), no RT.
- Figure 4 (b) is a Southem blot of total RNA.
- the ER ⁇ products were amplified for 25 cycles by RT-PCR.
- Each lane in Figure 4 (b) contains the following types and amounts of RNA: lane 1, control, no RNA; lane 2, rat ovarian RNA (2 ng); lane 3, ROS 17/2.8 total RNA (0.1 ⁇ g), lane 4, total (cultured) bone marrow RNA (0.1 ⁇ g); lane 5, total cultured bone marrow RNA (0.1 ⁇ g) where the cells had been treated with estradiol for 16 hours; lane 6, total RNA from primary osteoblasts in co-culture (0.1 ⁇ g); lanes 7-11, control reactions for lanes 2-6, respectively.
- Discrimination analysis for the relative expression of ER ⁇ c isoforms may be done utilizing random primers and reverse transcriptase (RT) to synthesize the cDNA from various rat or mouse or other mammalian tissues.
- the cDNAs so obtained are then amplified by PCR using the completely homologous rat and mouse primers 5'-GTC AAG TGT GGA TCC AGG-3' (Primer 9), which corresponds to base 700 of mER ⁇ -3 or base 924 of Rattus norvegicus estrogen receptor ⁇ mRNA, accession U57439 (Kuiper et al, 1996), and 5'-GCA CTT CAT GCT GAG CAG-3' (Primer 8) corresponding to base 1,554 of mER ⁇ -3 and 1,724 of accession U57439.
- the PCR products are purified and digested with Fsp I, a restriction endonuclease with a consensus site within exon 5B (TGCGCA at base 1,176 of mER ⁇ and also present in rER ⁇ -4). Digestion of the mouse or rat amplicons bearing the exon 5B sequence thus yields smaller products.
- the digested PCR products are resolved by agarose gel electrophoresis, transferred to nylon membranes, and probed with complementary oligonucleotide probes specific to either rat or murine sequences, or both.
- the specific sizes of the hybridized DNA present determines what isoform is present in a particular tissue or cell sample. Additionally, the intensity of the band allows quantitation of the relative abundance of the isoform(s) in a particular sample.
- Example 5 Gel Shift Assays Gel shift analysis of mER ⁇ -3 is demonstrated in Figure 5(a). The results obtained by the mER ⁇ -3 gel shift (Fig. 5a) were compared to that obtained for the human estrogen receptor alpha (ER ⁇ ) form, as displayed in Fig. 5(b). The receptor-DNA complexes formed were then disrupted using anti-peptide antibodies directed toward the novel exon 5B (Antibodies 1067 and 1068). Nuclear extracts (16 ⁇ g) derived from COS-7 cells transfected with expression plasmids containing mER ⁇ -3 (Fig. 5a) or human alpha estrogen receptor (pHEGO) (Fig.
- the lanes for both Fig. 5 (a) and (b) contain the following: lanes 1 and 2, extract alone; antibody 1067, lanes 3 and 4; antibody 1067 pre-immune serum, lanes 5 and 6; antibody 1068, lanes 7 and 8; antibody 1068 pre-immune serum, lanes 9 and 10; lanes 11 and 12 are control lanes that contain 16 ⁇ g of untransfected COS-7 nuclear extract.
- Example 6 Relative Affinities of Various Estrogens for ER Subtypes
- the experimental data displayed in Table I demonstrates the different affinities that various estrogens have for the ER ⁇ subtypes (e.g., ER ⁇ , ER ⁇ -1 and ER ⁇ -3).
- E2 binding affinity was determined by incubating transfected COS-7 cell cytosol with different concentrations of [ 3 H]-E2 (0-200nM) and with or without unlabeled E2 500X for 4 h at 4 °C in 40 mM Tris HCl pH 7.4, 150 mM KCl, PMSF 0.1 mM, DTT 2 mM.
- COS-7 cells were transfected as described in Example 7. Bound receptor was separated by the hydroxy apatite method (Obourn et al. Biochemistry 32(24): 6229-6236 (1993)) or the ligand was removed by the dextran coated charcoal method (Garcia et al, Mol. Endocrinol. 6(12): 2071-2078 (1992)), and bound hormone measured by liquid scintillation counting. Dissociation constants (kd) were obtained by Scatchard plots. Similarly, the relative affinities of different estrogenic ligands were determined using 1 nM receptor and 5 nM [ 3 H]-E2 with or without various concentrations of the described competitor steroids.
- Table I shows the different affinities of estrogens to human ER ⁇ , mouse ER ⁇ -1 and mouse ER ⁇ -3 (which contains exon 5B). As indicated, the affinity of the different estrogens varies as to the receptor. The larger the number, the greater the affinity the estrogen has for the estrogen receptor target. Diethylstilbestrol (DES) has a greater affinity for the ER ⁇ isoforms than for ER ⁇ . TABLE I. Relative Affinities of Various Estrogens for ER Subtypes
- the method used in this experiment can be utilized for screening reagents with different affinities for each of the ER ⁇ isoforms and comparing them to the ER ⁇ for determination of the affinity a particular drug may have for the other estrogen receptor proteins and their isoforms.
- Example 7 Transactivation Profiles of ER ⁇ - 1 and ER ⁇ -3 Isoforms This experiment assessed the effect of ER ⁇ - 1 and ER ⁇ -3 isoforms when expressed both individually and when expressed together as compared to the effect of ER ⁇ .
- the ability of estrogens to stimulate transcription via an estrogen response element (ERE) functionally linked to tk-C AT was measured by transient transfection of the expression vectors for mER ⁇ -1 and mER ⁇ -3 in COS-7 cells. For transfection, COS-7 cells were seeded into six-well plates in phenol red free, low glucose DMEM.
- the cells were transfected using lipofectamine according to the manufacturer's instructions (GIBCO-BRL).
- the expression constructs were transfected with a total of 2 ⁇ g DNA containing 500 ng of reporter, 100-500 ng expression plasmid, and the remainder (1-1.4 ⁇ g) as pBluescript as a carrier DNA.
- the cells were washed with DMEM and replaced with fresh medium containing drug ( 17- ⁇ estradiol, 4-hydroxy tamoxifen, clomiphene or DES at 1-300 nM concentrations) or vehicle (ethanol).
- mER ⁇ is capable of stimulating transcription from a reporter containing a canonical responsive element.
- the mER ⁇ -1 can stimulate transcription to approximately 50-70%) of that observed in similar cells transfected with the ER ⁇ construct, pHEGO, at estradiol concentrations of 100 nM.
- the mER ⁇ -3 isoform is capable of stimulating transcription to only approximately 40% of that observed in pHEGO at the 100 nM drug concentration.
- mER ⁇ -1 has a transactivation profile similar to ER ⁇ when exposed to E2, clomiphene (clomid), diethylstilbestrol (DES) and 4-OHT.
- the mER ⁇ -3 isoform has a decreased ability to transactivate cV2ERE as compared to either ER ⁇ or mER ⁇ -1.
- the transactivation activity is reduced when the isoforms are co- expressed (Fig. 8, panel indicated as mER B 1+B3).
- the assay utilized in this example can be similarly used to deteimine what agents can modulate homodimers of ER ⁇ isoforms, as well as heterodimers of the ER ⁇ isoforms or heterodimers composed of ER ⁇ and ER ⁇ isoforms.
- Figure 9 demonstrates that ER ⁇ -1 (displayed as BI in Fig. 9) and ER ⁇ -3 (B3) both possess similar activity when exposed to clomiphene, DES, 4-OHT, and E2. However when ER ⁇ -1 and ER ⁇ -3 are co-expressed in a reporter system, their activity is down regulated as compared to individual expression of the ER ⁇ isoforms or to ER ⁇ . This assay system can be utilized to screen other estrogens or compounds that modulate the activity of the various ER ⁇ isoforms.
- Example 8 In situ Hybridization of various tissues In situ hybridization analysis was performed using anti-sense cRNA probes to both the ER ⁇ and ER ⁇ to localize the message for each of the ER subtypes.
- the tissue was treated with 0.1 M TEA, pH 8.0, plus 0.25% acetic anhydride for 10 min at room temperature, rinsed three times in 2X SSC, dehydrated through a series of alcohols and air dried.
- cRNA riboprobes corresponding to the ER ⁇ or ER ⁇ -3 isoforms were prepared and used to probe tissue sections. The hybridization solution was removed, the sections washed and air dried.
- an 801 base pair insert corresponding to the ligand binding domain of the mER ⁇ -1 plasmid was linearized using the restriction enzyme ApaLI and transcribed using RNA polymerase in vitro in the presence of [ 35 S]-UTP and [ 35 S]-CTP according to methods of Goldstein et al, Neuroscience 71(1): 243 (1996).
- the riboprobes were purified by ethanol precipitation, and the dried tissue sections hybridized with probe in hybridization buffer overnight at 55°C. The hybridization solution was removed, the sections were incubated briefly with RNase, then washed, dehydrated, and air dried. The dried sections were exposed to film for normalization of subsequent exposure times and dipped in NTB3 emulsion to determine the cellular and anatomical localization of each mRNA.
- ER ⁇ is expressed in ossification center that appear to correspond with mesenchymal condensation zones in developing rat bone (12 days), especially in the spine (Fig. 10).
- the ER ⁇ message is observed in developing Graffian follicles (GA), but not in resorbing follicles (FA) undergoing atresia (Fig. 10, top panel, antisense).
- GA Graffian follicles
- FA resorbing follicles
- Fig. 10, top panel, antisense the ER ⁇ message receptor was only abundant within the uterine tube (not shown).
- ER ⁇ mRNA was observed to be widely expressed throughout the uterus (Fig. 10, middle panel, antisense).
- M mesenchymal ossification
- Controls corresponding to serial sections hybridized using sense riboprobe controls are also shown in the panels on the right.
- Example 9 Methods of Screening for Drugs A. Phosphorylation of ER ⁇ . Most of the members of the steroid receptor superfamily, including ER ⁇ , undergo post-translational modifications (e.g., phosphorylation) as a function of their basal state or in response to ligand binding. With ER ⁇ , there are a variety of sites on the molecule that are phosphorylated in response to ligand binding. Post-translational modification of mER ⁇ or human ER ⁇ can be accomplished using the same methods as previously utilized for ER ⁇ .
- post-translational modifications e.g., phosphorylation
- Methods of analyzing phosphorylation include transient or stable expression of the various cDNA constructs in COS-7 cells, or by immunoprecipitation of [ 32 P]-labeled ER ⁇ from cells metabolically labeled with [ 32 P]-orthophosphate. Tryptic maps from ligand stimulated or unstimulated cells can be obtained using ER ⁇ proteins isolated by immunoprecipitation of the mER ⁇ or human ER ⁇ molecule using our antibodies (e.g. , directed towards products of exon 5B such as the antibody used to obtain Fig. 3) or commercially available antibodies.
- a triple-myc tag or GST tag can also be linked to the carboxyl or amino termini by cloning the appropriate coding sequence into the expression plasmid.
- the expressed (phosphorylated) protein can then be immunoprecipitated using a very reliable, and commercially available anti-myc antibody (if using the triple-myc tag) or anti-GST antibodies.
- exon 5B amino acid residues can be substituted with other residues to prevent phosphorylation.
- exon 5B which is unique to mER ⁇ -3, is located within a region of the molecule that otherwise is extremely hydrophobic.
- the exon 5B region is unusually hydrophilic and contains a consensus casein kinase II (CKH) phosphorylation site (VLDRSSEDP) that arises as direct consequence of the location of the exon 5-exon 5B-splice junction.
- CKH consensus casein kinase II
- VLDRSSEDP consensus casein kinase II phosphorylation site
- Many of the steroid receptors, including the ER ⁇ subtype, are phosphorylated on CKII sites.
- the serines present in the portion of ER ⁇ encoded by exon 5B can be substituted with alanine residues (or other uncharged amino acids) or with residues which mimic constitutively phosphorylated molecules (e.g. , aspartic acid residues).
- ER ⁇ can be utilized in screening and isolating drugs which modulate the activity of the various ER ⁇ isoforms.
- these mutant forms of ER ⁇ or polypeptide fragments containing this region can themselves be tested for agonist or antagonist activity in the ER ⁇ signal pathways.
- B. Domain Switching The amino terminus of the ER ⁇ contains an autonomous transcriptional activity (AF-1) that is only fully active when "integrated" with the ligand-dependent transcriptional domain (AF-2) present within the ligand binding domain of the ER ⁇ molecule. While these domains have yet to be described for the ER ⁇ molecule, the high degree of sequence homology at the protein level between ER ⁇ and ER ⁇ molecules logically suggests that ER ⁇ is similarly organized.
- LBD ligand binding domain
- epitope tags such as GST and myc.
- Such constructs can then be utilized to identify, in whole cell lysates or other expression models such as expression libraries, proteins that functionally alter the transcriptional responsiveness of the ER complex.
- specific integrator molecules may be found using the LBD of mER ⁇ -3 fused to such convenient epitope tags as probes for protein-protein interactions.
- Such proteins can then alter the transcriptional responsiveness of the functional ER complex, (defined as the homo-dimers of ER ⁇ 3 with ER ⁇ 3 or hetero-dimers of ER ⁇ 3 with ER ⁇ l, or ER ⁇ 3 with ER ⁇ ) portion of the amino terminus fused with such epitope tags as probes for proteins that interact with the ER-complex.
- These complexes in turn can be used in dmg screening assays to identify drags which modulate ER ⁇ isoform activity.
- the complexes themselves may be used to regulate pathways mediated by estrogen receptors.
- PCR PROTOCOLS A GUIDE TO METHODS AND APPLICA ⁇ ONS, Innis, M, Gelfand, D.,
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Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP98937169A EP1001984A1 (en) | 1997-07-28 | 1998-07-28 | NOVEL ESTROGEN RECEPTOR $g(b) AND ISOFORMS |
CA002298053A CA2298053A1 (en) | 1997-07-28 | 1998-07-28 | Novel estrogen receptor .beta. and isoforms |
JP2000504164A JP2001510691A (en) | 1997-07-28 | 1998-07-28 | Novel estrogen receptor beta and its isoform proteins |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US5386997P | 1997-07-28 | 1997-07-28 | |
US60/053,869 | 1997-07-28 | ||
US5421097P | 1997-07-30 | 1997-07-30 | |
US60/054,210 | 1997-07-30 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1999005171A1 true WO1999005171A1 (en) | 1999-02-04 |
Family
ID=26732327
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1998/015540 WO1999005171A1 (en) | 1997-07-28 | 1998-07-28 | NOVEL ESTROGEN RECEPTOR β AND ISOFORMS |
PCT/US1998/015539 WO1999005170A1 (en) | 1997-07-28 | 1998-07-28 | NOVEL ESTROGEN β RECEPTOR AND ISOFORMS |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1998/015539 WO1999005170A1 (en) | 1997-07-28 | 1998-07-28 | NOVEL ESTROGEN β RECEPTOR AND ISOFORMS |
Country Status (4)
Country | Link |
---|---|
EP (2) | EP1001983A1 (en) |
JP (2) | JP2001510690A (en) |
CA (2) | CA2298053A1 (en) |
WO (2) | WO1999005171A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6222015B1 (en) | 1997-09-08 | 2001-04-24 | Merck & Co., Inc. | Estrogen receptor |
US7947463B2 (en) * | 2000-05-10 | 2011-05-24 | Signe Biopharma, Inc. | Compositions and methods for the diagnosis, treatment and prevention of steroid hormone responsive cancers |
-
1998
- 1998-07-28 EP EP98937168A patent/EP1001983A1/en not_active Withdrawn
- 1998-07-28 EP EP98937169A patent/EP1001984A1/en not_active Withdrawn
- 1998-07-28 JP JP2000504163A patent/JP2001510690A/en active Pending
- 1998-07-28 WO PCT/US1998/015540 patent/WO1999005171A1/en not_active Application Discontinuation
- 1998-07-28 JP JP2000504164A patent/JP2001510691A/en active Pending
- 1998-07-28 CA CA002298053A patent/CA2298053A1/en not_active Abandoned
- 1998-07-28 CA CA002297907A patent/CA2297907A1/en not_active Abandoned
- 1998-07-28 WO PCT/US1998/015539 patent/WO1999005170A1/en not_active Application Discontinuation
Non-Patent Citations (3)
Title |
---|
KUIPER G. G. J. M., ET AL.: "CLONING OF A NOVEL ESTROGEN RECEPTOR EXPRESSED IN RAT PROSTATE AND OVARY.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 93., 11 June 1996 (1996-06-11), US, pages 5925 - 5930., XP002914538, ISSN: 0027-8424, DOI: 10.1073/pnas.93.12.5925 * |
MOSSELMAN S., POLMAN J., DIJKEMA R.: "ERBETA: IDENTIFICATION AND CHARACTERIZATION OF A NOVEL HUMAN ESTROGEN RECEPTOR.", FEBS LETTERS., ELSEVIER, AMSTERDAM., NL, vol. 392., no. 01., 19 August 1996 (1996-08-19), NL, pages 49 - 53., XP002914536, ISSN: 0014-5793, DOI: 10.1016/0014-5793(96)00782-X * |
TREMBLAY G. B., ET AL.: "CLONING, CHROMOSOMAL LOCALIZATION, AND FUNCTIONAL ANALYSIS OF THE MURINE ESTROGEN RECEPTOR BETA.", MOLECULAR ENDOCRINOLOGY, THE ENDOCRINE SOCIETY, US, vol. 11., no. 03., 1 March 1997 (1997-03-01), US, pages 353 - 365., XP002914537, ISSN: 0888-8809, DOI: 10.1210/me.11.3.353 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6222015B1 (en) | 1997-09-08 | 2001-04-24 | Merck & Co., Inc. | Estrogen receptor |
US6458551B1 (en) | 1997-09-08 | 2002-10-01 | Merck & Co., Inc. | Estrogen receptor |
US6562592B1 (en) | 1997-09-08 | 2003-05-13 | Merck & Co., Inc. | Polynucleotide encoding estrogen receptor |
US7947463B2 (en) * | 2000-05-10 | 2011-05-24 | Signe Biopharma, Inc. | Compositions and methods for the diagnosis, treatment and prevention of steroid hormone responsive cancers |
US8563249B2 (en) | 2000-05-10 | 2013-10-22 | Signe Biopharma Inc. | Receptor gene screening for detecting or diagnosing cancer |
Also Published As
Publication number | Publication date |
---|---|
CA2297907A1 (en) | 1999-02-04 |
WO1999005170A8 (en) | 1999-04-15 |
EP1001983A1 (en) | 2000-05-24 |
JP2001510690A (en) | 2001-08-07 |
CA2298053A1 (en) | 1999-02-04 |
EP1001984A1 (en) | 2000-05-24 |
JP2001510691A (en) | 2001-08-07 |
WO1999005170A1 (en) | 1999-02-04 |
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