WO1997029184A1 - Estrogen response element binding proteins and nucleotides encoding therefor - Google Patents
Estrogen response element binding proteins and nucleotides encoding therefor Download PDFInfo
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- WO1997029184A1 WO1997029184A1 PCT/US1997/002208 US9702208W WO9729184A1 WO 1997029184 A1 WO1997029184 A1 WO 1997029184A1 US 9702208 W US9702208 W US 9702208W WO 9729184 A1 WO9729184 A1 WO 9729184A1
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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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 invention is in the field of estrogen signaling. Portions of the invention described herein were made in the course of research supported in part by NIH grants AR37399 and DK07682. The Government may have certain rights in this invention. 0
- Estrogens and other related steroids that bind to estrogen receptors have profound 5 physiological effects in humans and other animals. These effects include decreasing the rate of bone resorp ion, altering the circulating levels of clotting factors, stimulating breast development, and many others. Detailed descriptions of the physiological effects of estrogens can be ft found in many standard text books, including Goodman and Gilman's. The Pharmacological Basis of Therapeutics. Ei ⁇ ht Edition and Goodman, et al . Pergaman Press Elmsford, NY (1990) .
- Estrogens are known to bind to an intracellular estrogen _ receptor (ER) .
- Intracellular estrogen receptors are known to belong to a superfamily of intracellular receptors that comprise DNA binding domains. Estrogen receptors bind to the consensus estrogen response element 5' -AGGTCACAGTGACCT, see Tsai, et al. , Annu. Rev. Biochem 63:451-486 (1994).
- the invention described herein relates to the discovery of a novel protein that inhibits the binding of estrogen receptor to the estrogen response element (ERE) . By inhibiting the binding of the estrogen receptor to estrogen response element, it is possible to modulate the expression _ of genes regulated by estrogen via estrogen receptors. For example, estrogen response element binding protein may be used to modulate fertility.
- estrogen and estrogen receptors are known to play a significant role in the etiology of many diseases such as osteoporosis and cancer, e.g. estrogen responsive tumors. It is thus of interest to provide proteins that interact with estrogen response elements and polynucleotides encoding such proteins.
- the invention relates to the discovery and purification of novel estrogen response element binding proteins (ERE-BP) and the isolation of polynucleotide sequences encoding the proteins.
- Estrogen response element binding proteins are of interest because they may mediate the high levels steroid hormones observed in new world primates.
- Estrogen response element binding proteins are distinct from the estrogen receptor and other intracellular receptors, e.g. estrogen receptor.
- Estrogen response element binding proteins can interfere with the biological activity of the estrogen receptor and other related intracellular receptor proteins.
- Estrogen response element binding proteins of the present invention can bind to a DNA sequence known as the estrogen response element that is conserved (or partially conserved) among the regulatory regions of estrogen regulated genes.
- One aspect of the invention is to provide substantially purified estrogen response element binding proteins.
- the purified proteins may be obtained from either recombinant cells or naturally occurring cells.
- the purified estrogen response element binding proteins of the invention may be mammalian in origin. Estrogen response element binding proteins derived from primates, including human and Calli thrix jacchus (common marmoset) , are examples of the various estrogen response element binding proteins specifically provided for in the present application.
- the invention also provides allelic variants and biologically active derivatives of naturally occurring estrogen response element binding proteins.
- Another aspect of the invention is to provide polynucleotides encoding the estrogen response element binding proteins of the invention and to provide polynucleotides complementary to polynucleotide coding strand.
- the polynucleotides of the invention may be used to provide for the recombinant expression of estrogen response element binding proteins.
- the polynucleotides of the invention may also be used for genetic therapy purposes so as to treat diseases related to estrogen receptors and ligands that bind to estrogen receptors.
- the present invention also provides polynucleotides for use as hybridization probes and amplification primers for the detection of naturally occurring polynucleotides encoding estrogen response element binding proteins.
- Another aspect of the invention is to provide antibodies capable of binding to the estrogen response element binding proteins of the invention.
- the antibodies may be polyclonal or monoclonal.
- the invention also provides methods of using the subject antibodies to detect and measure expression of estrogen response element binding protein either in vi tro or in vivo.
- Another aspect of the invention is to provide assays for the detection or screening of therapeutic compounds that interfere with the interaction between estrogen response element binding protein and estrogen response elements.
- the assays of the invention comprise the step of measuring the effect of a compound of interest on binding between estrogen response element binding protein and an estrogen response element. Binding may be measured in a variety of ways, including the use of labeled estrogen response element binding protein or labeled DNA sequences comprising an estrogen response binding element. 4.0.
- Estrogen response element binding proteins are of interest because, inter alia, they modulate the activity of estrogen receptors. Estrogen response element binding proteins are distinct from the estrogen receptor. Estrogen response element binding proteins can interfere with the activity of the estrogen receptor by binding to the same DNA sequence, i.e., the estrogen receptor binding element. Thus by regulating the intracellular levels of the subject estrogen response element binding proteins, physiological effects of interest may be obtained. Such effects may be used to modulate fertility, or to treat a variety of diseases involving the signaling at intracellular receptors including osteoporosis, glucocorticoid mediated disorders, hypercalcemia, granuloma forming diseases, and estrogen responsive tumors or proliferative disorders.
- the estrogen response element binding proteins of the invention have the biological activity of specifically binding to estrogen response element DNA sequences, including the estrogen response element consensus sequence and estrogen response element from one or more genes.
- the estrogen response element binding proteins of the invention may be isolated from a variety of mammalian animal species. Preferred mammalian species for isolation are primates, humans and new world primates being particularly preferred. Although humans and old world primates do not produce large enough quantities of estrogen response element binding protein to manifest the elevated steroid hormone phenomenon seen in new world primates, humans and old world primates (as well as other mammals) are believed to produce estrogen response element binding proteins.
- the invention also contemplates allelic variants of estrogen response element binding protein.
- Estrogen response element binding proteins may be prepared from a variety of mammalian tissues; however, leukocytes and cell lines established from blood leukocytes are preferred non-recombinant sources of estrogen response element binding proteins.
- estrogen response element binding proteins are obtained from recombinant host cells genetically engineered to express significant quantities of estrogen response element binding proteins.
- Estrogen response element binding proteins may be isolated from non-recombinant cells in a variety of ways well known to a person of ordinary skill in the art. One example of such an isolation method is provided below in the examples section. Methods for purifying recombinant proteins from genetically engineered host cells vary with the host cell type and are well known to persons of ordinary skill in the art.
- estrogen response element binding protein suggests that the protein isolated from B95-8 cell, (see examples section) has a relative molecular weight of about 44-45 kDa (kilodaltons) , as determined by SDS-PAGE.
- estrogen response element binding protein refers not only to proteins having the amino acid residue sequence of naturally occurring estrogen response element binding proteins but also refers to functional derivatives and variants of naturally occurring estrogen response element binding protein.
- a “functional derivative" of a native polypeptide is a compound having a qualitative biological activity in common with the native polypeptide.
- a functional derivative of a native estrogen response element binding protein is a compound that has a qualitative biological activity in common with a native estrogen response element binding protein, e.g., binding to the ERE and other cognate ligands.
- the functional derivative of ERE-BP is distinct and largely unrelated to ER as evidenced by the fact that the functional derivative will specifically bind the ERE, and, like ERE-BP, will preferably not specifically binding 17 / 3- estradiol as measured by a electromobility shift assays
- “Functional derivatives” include, but are not limited to, fragments of native polypeptides from any animal species (including humans) , and derivatives of native (human and non- human) polypeptides and their fragments, provided that they have a biological activity in common with a respective native polypeptide. "Fragments” comprise regions within the sequence of a mature native polypeptide.
- the term “derivative” is used to define amino acid sequence and glycosylated variants, and covalent modifications of a native polypeptide, whereas the term “variant” refers to amino acid sequence and glycosylated variants within this definition.
- the functional derivatives are polypeptides which have at least about 65% amino acid sequence identity, more preferably about 75% amino acid sequence identify, even more preferably at least 85% amino acid sequence identity, most preferably at least about 95% amino acid sequence identity with the sequence of a corresponding native polypeptide.
- the functional derivatives of a native estrogen response element binding protein retain or mimic the region or regions within the native polypeptide sequence that directly participate in ligand binding.
- An additional feature of such functional derivatives is that they are not substantially bound or hindered by anti-ER antibodies as demonstrated by EMSA.
- Functional derivatives of ERE-BP also include chemically modified or derivatized molecules derived from ERE-BP.
- the phrase "functional derivative” further and specifically includes peptides and small organic molecules having a qualitative biological activity in common with a native estrogen response element binding protein.
- Identity or “homology” with respect to a native polypeptide and its functional derivative is defined herein as the percentage of amino acid residues in the candidate sequence that are identical to the corresponding residues of a native polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Neither N- or C-terminal extensions nor insertions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known in the art.
- amino acid sequence variants of native estrogen response element binding proteins and estrogen response element binding protein fragments are prepared by methods known in the art by introducing appropriate nucleotide changes into a native or variant estrogen response element binding protein encoding DNA, or by in vitro synthesis of the desired polypeptide. There are two principal variables in the construction of amino acid sequence variants: the location of the mutation site and the nature of the mutation.
- the amino acid sequence variants of estrogen response element protein are preferably constructed by mutating the ERE-BP encoding DNA to generate corresponding ERE-BP amino acid sequence variants that do not occur in nature.
- Such mutants may be engineered, for example, as frame- shift mutations that result in an altered reading frame and early termination of translation to produce a truncated ERE- BP molecule.
- in-frame deletions may be made in the ERE-BP gene that effectively result in the removal of discrete portions of ERE-BP.
- amino acid sequence deletions generally range from about l to 30 residues, more preferably about 1 to 10 residues, and are typically, but not necessarily, contiguous. Deletions are generally introduced in regions that are not directly involved in ligand binding.
- amino acid alterations can be made at sites that differ in estrogen response element binding proteins from various species, or in highly conserved regions, depending on the goal to be achieved. Sites at such locations will typically be modified in series, e.g. by (1) substituting first with conservative choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue or residues, or (3) inserting residues of the same or different class adjacent to the located site, or combinations of options 1-3.
- alanine scanning Cunningham and Wells, Science 244. 1081-1085 (1989) .
- a residue or group of target resides is identified and substituted by alanine or polyalanine.
- Those domains demonstrating functional sensitivity to the alanine substitutions are then refined by introducing further or other substituents at or for the sites of alanine substitution.
- the gene encoding an estrogen response element binding protein variant can, for example, be obtained by chemical synthesis.
- DNA encoding an estrogen response element binding protein amino acid sequence variant is prepared by site-directed mutagenesis of DNA that encodes are earlier prepared variant or a nonvariant version of the estrogen response element binding protein.
- Site-directed (site-specific) mutagenesis allows the production of estrogen response element binding protein variants through the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
- a primer of about 20 to 50 nucleotides in length is preferred, with at least about 5 to 10 residues on both sides of the junction of the sequence being altered.
- site-specific mutagenesis In general, the techniques of site-specific mutagenesis are well known in the art, as exemplified by publications such as, Edelman et al . , ⁇ NA 2:183 (1983) .
- the site-specific mutagenesis technique typically employs a phage vector that exists in both a single-stranded and double-stranded form.
- Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. This and other phage vectors are commercially available and their use is well known to those skilled in the art.
- a versatile and efficient procedure for the construction of oligodeoxyribonucleotide directed site- specific mutations in DNA fragments using M13-derived vectors was published by Zoller, M.J.
- plasmid vectors that contain a single-stranded phage origin of replication may be employed to obtain single- stranded DNA.
- nucleotide substitutions are introduced by synthesizing the appropriate DNA fragment in vitro, and amplifying it by PCR procedures known in the art.
- site-specific mutagenesis may be performed by first obtaining a single-stranded vector that includes within its sequence a DNA sequence that encodes the relevant protein.
- An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically, for example, by the method of Crea et al . , Proc. Natl. Acad. Sci. USA 75, 5765 (1978) .
- This primer is then annealed with the single-stranded protein sequence-containing vector, and subjected to DNA-polymerizing enzymes such as, E. coli polymerase I Klenow fragment, to complete the synthesis of the mutation-bearing strand.
- DNA-polymerizing enzymes such as, E. coli polymerase I Klenow fragment
- This heteroduplex vector is then used to transform appropriate host cells such as HBlOl cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement. Thereafter, the mutated region may be removed and placed in an appropriate expression vector for protein production.
- the PCR technique may also be used in creating amino acid sequence variants of an estrogen response element binding protein.
- one of the primers is designed to overlap the position of the mutation and to contain the mutation; the sequence of the other primer must be identical to a stretch of sequence of the opposite strand of the plasmid, but this sequence can be located anywhere along the plasmid DNA. It is preferred, however, that the sequence of the second primer is located within 200 nucleotides from that of the first, such that in the end the entire amplified region of DNA bounded by the primes can be easily sequenced.
- PCR amplification using a primer pair like the one just described results in a population of DNA fragments that differ at the position of the mutation specified by the primer, and possibly at other positions, as template copying is somewhat error-prone.
- Naturally-occurring amino acids may be divided into groups based on common side chain properties:
- hydrophobic norleucine, met, ala, val, leu, ile
- Conservative substitutions involve exchanging a member within one group for another member within the same group, whereas non-conservative substitutions will entail exchanging a member of one of these classes for another (see generally Orcutt, B.C. and Dayhoff, M.O., Scoring Matrices, PIN Report MAT-0285, February 1985) .
- Variants obtained by non- conservative substitutions are expected to result in significant changes in the biological properties/function of the obtained variant, and may result in estrogen response element binding protein variants which block estrogen response element binding protein biological activities, i.e., binding to estrogen response elements.
- Amino acid positions that are conserved among various species are generally substituted in a relatively conservative manner if the goal is to retain biological function.
- Amino acid insertions include amino- and/or carboxyl- terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
- Intrasequence insertions i.e. insertions within the estrogen response element binding protein amino acid sequence
- terminal insertions include the estrogen response element binding proteins with an N-terminal methionyl residue, an artifact of direct expression in bacterial recombinant cell culture, and fusion of a heterologous N-terminal signal sequence to the N-terminus of the estrogen response element binding protein to facilitate the secretion of the mature estrogen response element binding protein from recombinant host cells.
- signal sequences will generally be obtained from, and thus homologous to, the intended host cell species. Suitable sequences include STII or Ipp for E. coli, alpha factor for yeast, and viral signals such as herpes gD for mammalian cells.
- insertional variants of the native estrogen response element binding protein molecules include the fusion of the N- or C-terminus of an estrogen response element binding protein to immunogenic polypeptides, e.g. bacterial polypeptides such as beta-lactamase or an enzyme encoded by the E. coli trp locus, or yeast protein, and C-terminal fusions with proteins having a long half-life such as i munoglobulin regions (preferably immunoglobulin constant regions) , albumin, or ferritin, as described in PCT published application WO 89/02922.
- immunogenic polypeptides e.g. bacterial polypeptides such as beta-lactamase or an enzyme encoded by the E. coli trp locus, or yeast protein
- C-terminal fusions with proteins having a long half-life such as i munoglobulin regions (preferably immunoglobulin constant regions) , albumin, or ferritin, as described in PCT published application WO 89/02922.
- the genetic therapy methods of the invention comprise the step of introducing a vector for the expression of estrogen response element binding protein (or inhibitory anti-sense RNA) into a patient cell.
- the patient cell may be either in the patient, i.e., in vivo genetic therapy, or external to the patient and subsequently reintroduced into the patient, i.e., in vi tro genetic therapy.
- Diseases that may be treated by the subject genetic therapy methods include osteoporosis, vitamin D toxicity, glucocorticoid hormone overproduction, sex steroid hormone over expression and under expression, hypercalcemia (attributable to vitamin D over expression) , granuloma forming diseases and the like.
- the subject invention provides methods for the treatment of a variety of diseases characterized by undesirably high levels of estrogen or other steroids that can bind to estrogen response element binding proteins of the invention.
- treatment e.g., symptoms related to disease, sensitivity to environmental or factors, normal aging, and the like
- treatment e.g., chemotherapy, or “medicinal use” used herein shall refer to any and all uses of the claimed compositions which remedy a disease state or symptoms, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever.
- an appropriate dosage of ERE-BP, or a functional derivative thereof may be determined by any of several well established methodologies. For instance, animal studies are commonly used to determine the maximal tolerable dose, or MTD, of bioactive agent per kilogram weight. In general, at least one of the animal species tested is mammalian. Those skilled in the art regularly extrapolate doses for efficacy and avoiding toxicity to other species, including human. Before human studies of efficacy are undertaken, Phase I clinical studies in normal subjects help establish safe doses.
- the ERE-BP may prepared and maintained under sterile conditions and thus avoid microbial contamination.
- Compositions comprising ERE- BPs may also be sterile filtered prior to use.
- antimicrobial agents may also be added.
- Antimicrobial agents which may be used, generally in amounts of up to about 3% w/v, preferably from about 0.5 to 2.5%, of the total formulation, include, but are not limited to, methylparaben, ethylparaben, propylparaben, butylparaben, phenol, dehydroacetic acid, phenylethyl alcohol, sodium benzoate, sorbic acid, thymol, thimerosal, sodium dehydroacetate, benzyl alcohol, cresol, p-chloro-m-cresol, chlorobutanol, phenylmercuric acetate, phenylmercuric borate, phenylmercurie nitrate and benzylalkonium chloride.
- anti-microbial additives will either enhance the biochemical properties of ERE-BP, or will be inert with respect ERE-BP activity.
- another agent may be substituted which effects ERE-BP function to a lesser extent.
- compositions comprising ERE-BPs as active components may be introduced in vivo by any of a number of established methods.
- the agent may be administered by inhalation; by subcutaneous (sub-q) ; intravenous (I.V.), intraperitoneal (I.P.) , or intramuscular (I.M.) injection; rectally, as a topically applied agent (transdermal patch, ointments, creams, salves, eye drops, and the like), or directly injected into tissue such as tumors or other organs, or in or around the viscera.
- subcutaneous subcutaneous
- I.V. intravenous
- I.P. intraperitoneal
- I.M. intramuscular
- ERE-BPs are conjugated to ligands that facilitate the delivery of ERE-BPs to specifically targeted cells or tissues.
- targeting ligands include, but are not limited to, giycoproteins, polysaccharides, lectins, cell receptors or surface markers (or fragments thereof) antibodies (or fragments thereof) , apatmeric oligonucleotides, and the like.
- Another aspect of the invention is to provide assays useful for determining if a compound of interest can bind to estrogen response element binding proteins so as to interfere with the binding of an estrogen response element to estrogen response element receptor proteins.
- the assay comprises the steps of measuring the binding of a compound of interest to an estrogen response element binding protein.
- Either the intracellular binding protein or the compound of interest to be assayed may be labeled with a detectable label, e.g., a radioactive or fluorescent label, so as to provide for the detection of complex formation between the compound of interest and the estrogen response element binding protein.
- the assays involve measuring the interference, i.e., competitive binding, of a compound of interest with the binding interaction between an estrogen response element binding protein and an estrogen response element.
- the effect of increasing quantities of a compound of interest on the formation of complexes between radioactivity labeled ERE and an estrogen response element binding protein may be measured by quantifying the formation of labeled ligand- estrogen response element binding protein complex formation. Additional methods of measuring ligand binding to estrogen response element binding proteins can be found in the example section below.
- Polyclonal antibodies to estrogen response element binding proteins generally are raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of an estrogen response element binding protein and an adjuvant. Evolutionarily conserved proteins often share a high degree of interspecies homology. This high level of homology may render a given protein substantially nonimmunogenic when used during attempts to generate polyclonal antisera against a given protein. In such situations, it may be useful to conjugate the protein (e.g., estrogen response element binding protein) or a fragment containing the target amino acid sequence to a protein that is immunogenic in the species to be immunized, e.g.
- the protein e.g., estrogen response element binding protein
- a fragment containing the target amino acid sequence e.g.
- a bifunctional or derivatizing agent for example maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues) , N-hydroxysuccinimide (through lysine resides) , glutaraldehyde, succinic anhydride, S0C1 2 , or RjNsCsNR, where R and R ⁇ are different alkyl groups.
- denatured protein may be used as an immunizing agen .
- Animals are immunized against the immunogenic conjugates or derivatives by combining 1 mg or 1 ⁇ g of conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites.
- 1 mg or 1 ⁇ g of conjugate for rabbits or mice, respectively
- 3 volumes of Freund's complete adjuvant injecting the solution intradermally at multiple sites.
- the animals are boosted with 1/5 to 1/10 the original amount of conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites.
- 7 to 14 days later the animals are bled and the serum is assayed for anti-estrogen response element binding proteins antibody titer. Animals are boosted until the titer plateaus.
- the animal is boosted with a conjugate of the same estrogen response element binding protein that has been conjugated to a different protein and/or through a different cross-linking reagent.
- Conjugates can also be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are used to enhance the immune response.
- Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts.
- the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.
- the anti-estrogen response element binding protein monoclonal antibodies of the invention may be made using the hybridoma method first described by Kohler & Milstein, Nature 256 :495 (1975) , or may be made by recombinant DNA methods [Cabilly, et al, U.S. Pat. No. 4,816,567] .
- lymphocytes In the hybridoma method, a mouse or other appropriate host animal, such a hamster is immunized as hereinabove described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (academic Press, 1986)] .
- suitable fusing agent such as polyethylene glycol
- the anti-estrogen response element binding protein specific antibodies of the invention have a number of uses.
- the antibodies may be used to purify estrogen response element binding proteins from either recombinant or non- recombinant cells.
- the subject antibodies may be used to detect and/or quantify the presence of estrogen response element binding proteins in tissue samples, e.g., from blood, skin, and the like. Quantitative measurements of estrogen response element binding proteins may be used diagnostically for those diseases and physiological or genetic conditions that have been correlated with particular levels of estrogen response element binding protein expression levels.
- the B-lymphoblastoid cell line B95-8 and MLA-144 were obtained from the American Type Culture Collection (ATCC,
- Postnuclear extracts of each cell line were prepared as previously described in Gacad et al . J. Clin. Invest. 87:996- 1001 (1991) . Briefly, confluent B95-8 cells were harvested by trypsinization and MLA-144 cells by centrifugation, respectively. Harvested cells were washed twice in ice-cold phosphate-buffered saline (PBS) and twice with ETD buffer (1 mM EDTA, 10 mM Tris-HCl, 5 mM dithiothreitol [DTT] . P H 7.4) containing 1 mM phenylmethylsulfonyflouride (PMSF) .
- PBS ice-cold phosphate-buffered saline
- ETD buffer 1 mM EDTA, 10 mM Tris-HCl, 5 mM dithiothreitol [DTT] .
- PMSF phenylmethylsulfon
- the cells pellets were then resuspended in ETD buffer and homogenized on ice in five 10-second bursts. Nuclei, with associated nuclear steroid receptor proteins, were pelleted at 4000xg for 30 minutes at 4°C. The supernatant of this spin was filtered through a Microcon-30 filter (molecular weight cut off 30 kDa; Amicon, Beverly, MA) for the purposes of desalting and concentrating prior to being aliquoted and stored at-70°C.
- a Microcon-30 filter molecular weight cut off 30 kDa; Amicon, Beverly, MA
- Nuclear extracts were prepared as described by Zervitz and Akusjarvi Gene Anal . Tech 6:101-109 (1989) .
- Cultured B95-8 and MLA-144 cells were harvested as previously described. Cells were washed twice in ice-cold PBS, washed once in buffer A (250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM spermine) , resuspended in buffer A and allowed to swell at room temperature for 5 minutes. Lysis of the cell membrane was achieved by addition of lysolecithin to a concentration of 400 mg/ml and gentle repetitive inversion of the cells for 90 seconds.
- Cells membrane lysis was terminated by addition of two volumes of ice-cold buffer B (buffer A containing 3% BSA) . All subsequent steps were performed at 4°C. Nuclei were initially pelleted at lOOOxg for 30 seconds and then repelleted at 25,000xg for 60 seconds. The resultant nuclear pellet was resuspended in buffer C (20 mM HEPES [pH 7.9] , 0.6 M KCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 25% [v/v] glycerol) and the nuclear membrane disrupted by repetitive passage through a 23-gauge needle.
- buffer C (20 mM HEPES [pH 7.9] , 0.6 M KCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 25% [v/v] glycerol
- the homogenate was gently stirred on ice for 30 minutes and then centrifuged for 30 minutes at 25,000xg.
- the supernatant, designated nuclear extract was dialyzed against buffer D (20 mM HEPES [pH 7.9] , 0.1 M KCl, 0.2 M EDTA, 0.5 mM DTT, 0.5 mM DTT, 0.5 mM PMSF in 20% glycerol) for 30 hours with three exchanges.
- the nuclear extract was cleared by centrifugation at 25,000xg for 20 minutes, aliquoted, and stored at 70°C.
- the protein concentration of nuclear and postnuclear extracts were determined by the method of Bradford Anal. Biochem 72:248-254 (1976) . 5.3.
- Electromobilitv Shift As ⁇ av ESA
- Synthetic oligonucleotides representing various forms of consensus steroid response elements were either prepared at the Molecular Biology Core Facility of the Cedars-Sinai Research Institute or purchased from Promega (Madison, Wl) . Sequences for the various oligonucleotides were as follows: consensus estrogen response element (ERE) 5'- CTAGAAAGTCAGGTCACAGTGACCTGATCAAT-3' : ERE half-site 5'- CTAGAAAGTCAG2 ⁇ SACAGGATCAAT-3' ; AGGTCA half-site direct repeat (DR3> 5' -CTAGTGCTCGGGTAGAGGTCACAGAGGTCACTCGACTCGT-3' ; osteopontin vitamin D response element (VDRE) 5'- CTAGTGGGGCTCGGGTAGGGGTTCA-CGAGGTTCACTCGACTCGT-3' : and the irrelevant CTF/NFl probe 5' -CCTTTGGCATGCTGCCAATATG-3' .
- EEE consensus estrogen response element
- Single-strand oligonucleotides were annealed with their complementary sequence s and radiolabeled with "P-ATP (DuPont-New England Nuclear, Boston, MA) by T4 Kinase (GIBCO- BRL, Grand Island, NY) to a specific activity of 10' cpm/ ⁇ g DNA.
- Postnuclear or nuclear extracts (10 ⁇ g protein) preincubated (or not) for 1 hour at 23°C with 100 nM 17 / 3- estradiol, 25 OHD3 or 1,25- (OH) 2 D3, were incubated with 2 ⁇ g poly-dl-dC (Boehringer-Mannheim, Indianapolis, IN) and 20 mM HEPES (pH 7.9), 100 mM KCl, 5 mM MgCl 2 in 10% glycerol on ice for 15 minutes, "P-labeled probe was added and the incubation continued at room temperature for 15 minutes. Samples of this reaction were subjected to 6% polyacrylamide gel electrophore ⁇ es in 0.5 x TBE buffer at 100V.
- Cellular extracts were prepared identically from both the representative NWP B95-8 and OWP MLA cell line. Prior to extraction, cells were ruptured and intact nuclei separated from cell cytosol in a "low-salt" buffer in order to separate the ER, associated with the nuclear pellet, Tzu Ker an, et al . Mol . Endrocrin S:21-30 (1994), from the post-nuclear supernatant that is enriched in intracellular vitamin D binding protein under these conditions Gacad et al . Bone Min. Res. 8:27-35 (1993) . The high-salt" nuclear extract was adjusted to 0.1 M KCl by dialysis, while the "low-salt” nuclear extract was adjusted to same salt concentration by dilution.
- the post-nuclear and nuclear extracts were matched for protein concentration and used in EMSA in the presence or absence of added human ER.
- the specific DNA binding potential of nuclear and post-nuclear extracts of steroid- resistant new world primate cells and steroid-responsive old world primate cells was studied using a panel of synthetically prepared oligonucleotides including the consensus ERE (estrogen response element) (AGGTCAcagTGACCT) .
- AGGTCAcagTGACCT consensus ERE (estrogen response element)
- post-nuclear extract of steroid-resistant B95-8 cells contained a protein(s) (labeled ERE-BP (estrogen response element-binding protein) which retarded the mobility of the labeled estrogen response element.
- IVD-BP vitamin D binding protein
- ERE-BP Competitively Disrupts the ER-ERE Interaction _ ⁇ ____
- the estrogen response element binding protein present in NWP cell extracts is functionally capable of rendering the cell estrogen resistant in vivo. Consequently, ERE-BP should: [1] be able to bind to the ERE with comparable (or greater) affinity than the ER and/or be present in much higher concentrations than the ER in the cell; and [2] be able to interrupt the normal interaction of the ER with the ERE.
- the consensus ERE is characterized by an inverted repeat of the six-base pair half site motif AGGTCA (18) .
- AGGTCA six-base pair half site motif
- AGGTCA half site motif were used to compete away labeled ERE- ERE-BP binding.
- the list of unlabeled competitive oligonucleotides containing the AGGTCA half site included the consensus ERE, TRE, TRE palindrome (TREpal) , RXRE, and COUP- TF1E. Of these only a 100-fold excess of ERE completely eliminated labeled ERE-ERE-BP binding.
- a synthetic oligonucleotide DR 3 2 harboring a direct repeat of the AGGTCA half site separated by the trinucleotide cag found in the consensus ERE, was nearly as efficient as the ERE in competing with the labeled ERE probe, and this competitive potential was not related to the sequence flanking the half sites.
- This oligonucleotide was as effective as the consensus ERE in competing with the labeled probe, indicating the ERE-BP may bind to a single core element as a monomer.
- the DNA affinity column was prepared essentially as described by Kadondaga and Tijan (16) .
- Two gel-purified oligodeoxynucleotides (30-mers containing 26 nucleotides of complementary sequence and having four base-pair, cohesive ends; 5' -GATCCTA-GAAAGT-CAGGTCACAGGATCAAT-3 ' and 5' - GATCATTGATCCTGTGACCTGACTTTC-TAG-3' ) were synthesized. 220 ⁇ g of each oligonucleotide was annealed, 5'-phosphorylated with [gamma- 3 P] ATP to ascertain coupling efficiency, and ligated.
- the resultant DNA oligomers were then coupled to cyanogen bromide (CNBr) -activated Sepharose 4B (Pharmacia, Piscataway, NJ) .
- CNBr cyanogen bromide
- Sepharose 4B Pharmacia, Piscataway, NJ
- the DNA-coupled resin was placed in a 2 ml poly-prep column (BIO-Rad, Hercules, CA) for chromatography.
- the column was equilibrated in elution buffer (25 mM Hepes [pH 7.6], 12.5 mM MgCl 2 , 1 mM DTT, 20% glycerol, and 0.1% nonidet P-40) containing 0.1 M KCl.
- the B95-8 cell extract (13-20 mg) prepared as above) was solubilized in the elution buffer containing nonspecific competitor poly dldC (4 mg/ml) and added to the column by gravity flow (12 ml/hour) . Protein was then eluted from the column in a stepwise gradient fashion by the successive addition of ten 10 mL aliquots of elution buffer containing 0.1-1.0 M KCl. A fraction (2 ml) of each column eluate were concentrated and desalted through a Microcon-30 filter and ERE binding capacity assessed by EMSA. An aliquot of each fraction was used to determine total protein concentration. The constituent proteins in each fraction were resolved on a 10% SDS-PAGE and identified by silver staining.
- ERE-BP estrogen response element binding protein
- the above experiments have shed insight into the functional aspects of the identified estrogen response element binding protein(s) (ERE-BP) in vi tro.
- the ERE-BP does not appear to be related to the ER.
- Both the ER-ERE and ERE-BP-ERE complexes may be observed as distinct bands in the same gel with the apparent molecular mass of the ERE-BP-ERE complex being less than that of the ER-ERE complex; data suggest that ERE-BP has a molecular mass in the range of 44- 45 kDa.
- the ER and ERE-BP compete for binding to the ERE.
- ERE-BP and ER are distinct entities. Not withstanding the fact that there is a distinct species difference between human and non-human primates, particularly NWP, ERE-BP is not super shifted by anti-ER antibody. Nor is the ERE-BP complex altered by pre- exposure to 170-estradiol, the ligand of choice for the ER. Finally, whereas the preferred mode of interaction of the ER with the ERE is as a ligand-transformed homodimer, Tsai, et al . , Annu. Rev. Biochem 63:451-486 (1994). ERE-BP may interact with the ERE as a monomer. This is supported by the fact that the labeled ERE-BP-ERE complex can be effectively competed away by addition of the hexanucleotide half-site AGGTCA alone or by a direct repeat of this half-site.
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Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
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JP9528750A JP2000504576A (en) | 1996-02-12 | 1997-02-12 | Estrogen response element binding proteins and nucleotides encoding the same |
EP97907610A EP0880583A4 (en) | 1996-02-12 | 1997-02-12 | Estrogen response element binding proteins and nucleotides encoding therefor |
AU19573/97A AU1957397A (en) | 1996-02-12 | 1997-02-12 | Estrogen response element binding proteins and nucleotides encoding therefor |
Applications Claiming Priority (2)
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US1148296P | 1996-02-12 | 1996-02-12 | |
US60/011,482 | 1996-02-12 |
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WO1997029184A1 true WO1997029184A1 (en) | 1997-08-14 |
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PCT/US1997/002208 WO1997029184A1 (en) | 1996-02-12 | 1997-02-12 | Estrogen response element binding proteins and nucleotides encoding therefor |
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EP (1) | EP0880583A4 (en) |
JP (1) | JP2000504576A (en) |
AU (1) | AU1957397A (en) |
CA (1) | CA2245806A1 (en) |
WO (1) | WO1997029184A1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0939084A1 (en) * | 1998-02-11 | 1999-09-01 | Ramanath B. Rao | An estrogen binding proteinaceous substance, its possible role in estrogen action, and potential use. |
WO2001074846A2 (en) * | 2000-04-03 | 2001-10-11 | Yeda Research And Development Co. Ltd. | Peptides mimicking the biological activity of steroid hormones and their uses |
US6739280B2 (en) | 2001-11-26 | 2004-05-25 | Rogenald J. Keller | Boat with swing seating |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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US5563035A (en) * | 1994-06-16 | 1996-10-08 | The Board Of Trustees Of The Leland Stanford Junior University | Estrogen receptor regulation and its uses |
Family Cites Families (1)
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US5312752A (en) * | 1989-07-31 | 1994-05-17 | Trustees Of Boston University | Specific antibodies against the DNA-binding domain of and immunoassays to determine the presence and functional status of estrogen receptor proteins |
-
1997
- 1997-02-12 WO PCT/US1997/002208 patent/WO1997029184A1/en not_active Application Discontinuation
- 1997-02-12 EP EP97907610A patent/EP0880583A4/en not_active Withdrawn
- 1997-02-12 JP JP9528750A patent/JP2000504576A/en active Pending
- 1997-02-12 CA CA002245806A patent/CA2245806A1/en not_active Abandoned
- 1997-02-12 AU AU19573/97A patent/AU1957397A/en not_active Abandoned
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US5563035A (en) * | 1994-06-16 | 1996-10-08 | The Board Of Trustees Of The Leland Stanford Junior University | Estrogen receptor regulation and its uses |
Non-Patent Citations (7)
Title |
---|
ARNOLD S F, VOROJEIKINA D P, NOTIDES A C: "PHOSPHORYLATION OF TYROSINE 537 ON THE HUMAN ESTROGEN RECEPTOR IS REQUIRED FOR BINDING TO AN ESTROGEN RESPONSE ELEMENT", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 270, no. 50, 15 December 1995 (1995-12-15), US, pages 30205 - 30212, XP002941971, ISSN: 0021-9258, DOI: 10.1074/jbc.270.4.1850 * |
CHEN H, ET AL.: "VITAMIN D AND GONODAL STEROID-RESISTANT NEW WORLD PRIMATE CELLS EXPRESS AN INTRACELLULAR PROTEIN WHICH COMPETES WITH THE ESTROGEN RECEPTOR FOR BINDING TO THE ESTROGEN RESPONSE ELEMENT", JOURNAL OF CLINICAL INVESTIGATION, AMERICAN SOCIETY FOR CLINICAL INVESTIGATION, US, vol. 99, no. 04, 1 January 1997 (1997-01-01), US, pages 669 - 675, XP000983368, ISSN: 0021-9738, DOI: 10.1172/JCI119210 * |
GRAY W G, GORSKI J: "IDENTIFICATION AND CHARACTERIZATION OF AN ESTROGEN-RESPONSIVE ELEMENT BINDING PROTEIN REPRESSED BY ESTRADIOL", BIOCHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 35, no. 36, 1 January 1996 (1996-01-01), US, pages 11685 - 11692, XP002943807, ISSN: 0006-2960, DOI: 10.1021/bi960068k * |
HUGHES M J, ET AL.: "PURIFICATION AND CHARACTERIZATION OF A PROTEIN FROM HELA CELLS THATBINDS WITH HIGH AFFINITY TO THE ESTROGEN RESPONSE ELEMENT, GGTCAGCGTGACC", BIOCHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 28, no. 23, 1 January 1989 (1989-01-01), US, pages 9137 - 9142, XP002943808, ISSN: 0006-2960, DOI: 10.1021/bi00449a027 * |
INANO K, ET AL.: "HEAT SHOCK PROTEIN 90 STRONGLY STIMULATES THE BINDING OF PURIFIED ESTROGEN RECEPTOR TO ITS RESPONSIVE ELEMENT", JOURNAL OF BIOCHEMISTRY, OXFORD UNIVERSITY PRESS, GB, vol. 116, no. 04, 1 January 1994 (1994-01-01), GB, pages 759 - 766, XP002943809, ISSN: 0021-924X * |
NARDULLI AM, GREENE G L, SHAPIRO D J: "HUMAN ESTROGEN RECEPTOR BOUND TO AN ESTROGEN RESPONSE ELEMENT BENDSDNA", MOLECULAR ENDOCRINOLOGY, THE ENDOCRINE SOCIETY, US, 1 January 1993 (1993-01-01), US, pages 331 - 340, XP002941984, ISSN: 0888-8809, DOI: 10.1210/me.7.3.331 * |
See also references of EP0880583A4 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0939084A1 (en) * | 1998-02-11 | 1999-09-01 | Ramanath B. Rao | An estrogen binding proteinaceous substance, its possible role in estrogen action, and potential use. |
WO2001074846A2 (en) * | 2000-04-03 | 2001-10-11 | Yeda Research And Development Co. Ltd. | Peptides mimicking the biological activity of steroid hormones and their uses |
WO2001074846A3 (en) * | 2000-04-03 | 2003-01-09 | Yeda Res & Dev | Peptides mimicking the biological activity of steroid hormones and their uses |
US6739280B2 (en) | 2001-11-26 | 2004-05-25 | Rogenald J. Keller | Boat with swing seating |
Also Published As
Publication number | Publication date |
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EP0880583A1 (en) | 1998-12-02 |
EP0880583A4 (en) | 2001-03-21 |
JP2000504576A (en) | 2000-04-18 |
CA2245806A1 (en) | 1997-08-14 |
AU1957397A (en) | 1997-08-28 |
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