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WO1999015544A1 - SEQUENCES DE NUCLEOTIDES ET D'ACIDES AMINES DU GENE brx ET PRODUIT GENIQUE DE CE DERNIER ET UTILISATIONS - Google Patents

SEQUENCES DE NUCLEOTIDES ET D'ACIDES AMINES DU GENE brx ET PRODUIT GENIQUE DE CE DERNIER ET UTILISATIONS Download PDF

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WO1999015544A1
WO1999015544A1 PCT/US1998/019782 US9819782W WO9915544A1 WO 1999015544 A1 WO1999015544 A1 WO 1999015544A1 US 9819782 W US9819782 W US 9819782W WO 9915544 A1 WO9915544 A1 WO 9915544A1
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brx
nucleic acid
protein
seq
cells
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Domenica M. Rubino
James Segers
Paul H. Driggers
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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
    • C07K14/46—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • This invention pertains to the fields of oncology and reproductive biology. It generally relates to oncogenes, particularly their use to diagnose disease and in therapeutic regimens.
  • this invention pertains to the discovery of a gene, brx, and its gene product, Brx, implicated in the etiology of breast and ovarian cancers.
  • the invention relates to methods of using brx and Brx to diagnose and treat certain cancers and diseases and disorders of reproductive and immune tissues.
  • Ovarian cancer has the highest mortality rate of gynecological cancer.
  • Some cancerous conditions may be arrested, if diagnosed and treated in a timely fashion, and the life of the patient thereby prolonged.
  • the cancer is discovered after it has advanced to the point that the fatal progress of the disease cannot be reversed, and the patient's life cannot be saved.
  • it is important to develop methods for the early diagnosis and treatment of cancer.
  • proliferative disorders in reproductive tissues such as endometriosis affect many women and abnormal response of such tissues to hormone regulation may result in implantation abnormalities and/or luteal insufficiency.
  • Some existing methods of cancer diagnosis and treatment are targeted to tumor-specific proteins. Proteins that are specifically associated with certain tumors have been described as early as 1965. Garrett and Kurtz, Medical Clinics of North America 70: 1295-1306 (1986) defined the ideal biological tumor marker as one that is a) specific without false-positive results, b) sensitive without false-negative results and c) capable of demonstrating an absolute correlation with the extent of disease.
  • tumor-associated proteins or tumor markers would be a powerful tool in detecting malignancies if they were present only on tumor cells. However, although most of the currently identified tumor markers are present in high yields in tumor cells, they are also found in non-tumor cells.
  • NHR nuclear hormone receptor
  • T3R nuclear hormone receptor
  • VDR nuclear hormone receptor
  • RAR PPAR
  • RXR RXR
  • ER nuclear hormone receptor
  • Rho proteins have not been shown to be directly involved in ER activation.
  • Rho proteins act as binary molecular switches through incompletely defined interactions with specific target effector proteins to direct transcription, cell proliferation, cell adhesion, actin cytoskeletal organization, protein kinase signalling (Watanabe et al. , Science 271 :645-648 (1996)) and oncogenesis (Bos, Cancer Res. 49: 4682-4689 (1989)).
  • Substantial evidence suggests that signals generated by p21 GTPases (especially p21 ras ) are transmitted to the nucleus. Hill et al. , Cell SI: 1159-1170 (1995); Minden et al. , Cell 81: 1147-1157 (1995); Settleman et ⁇ /.
  • Rho in nuclear hormone receptor signalling was suggested by the description of p 190, a rho/rac GAP, which exists in both nuclear and cytoplasmic compartments, transduces signals from p21 ras to the nucleus (Settleman et al., Cell 69: 539-549 (1992)), and may suppress glucocorticoid receptor transcription (LeClerc et al., J. Biol. Chem. 266: 17333-17340 (1991)).
  • Estrogen and progesterone receptor status of breast tumors has been shown to be of prognostic importance, but there are few tests for ovarian cancer, thus necessitating the identification of new prognostic markers for this condition.
  • the present invention addresses this and other current problems.
  • It is a primary object of this invention is to provide novel proteins, and their corresponding DNA, mRNA and amino acid sequences, which encode growth regulatory proteins or oncoproteins useful in the regulation of cell proliferation in cell cultures and in vivo, particularly sequences active in reproductive epithelium, immune cells, ovary, breast cells, and spleen.
  • One object of this invention to provide generally safe and specific therapeutic and prophylactic methods and products useful for controlling growth disorders, including cancer in reproductive and immune tissues.
  • the present invention is based on the discovery of novel receptor-binding proteins in breast cell lines.
  • a breast cancer cDNA expression library (ZR75-1; Clontech) was screened using a modified method of interaction cloning; a recombinant, epitope-tagged, bacterially expressed nuclear receptor (RXR); and commercially available antibodies directed against the epitope (FLAG-IBI). The screening yielded several clones, one of which (clone 2.10) contained a 1.8 kB fragment.
  • the brx sequence has been mapped by FISH to chromosome 15q25-26. Some regions of chromosome 15q have been associated with breast cancer. Northern analysis showed that brx message was expressed in ovary, breast, breast cancer cell lines, testis, spleen, and immune cells, but not in liver and pancreas. Labelled Brx bound specifically to GST-RXR ⁇ , ER ⁇ , and other NHRs including TR and PPAR. Brx also bound to ER/3 (Rubino and Driggers). Brx bound to nuclear hormone receptors, including the estrogen receptor, through a novel carboxyl region.
  • Brx augmented ligand-dependent gene activation by the estrogen receptors a (Rubino, et al) and ⁇ ( Rubino and Driggers, unpublished) and was localized to nuclear and cytoplasmic cellular compartments.
  • Brx binds to transcription factors known to be involved in mitogenesis: c-Jun, c-Fos, Atf-2 and activates the serum response element, and cAMP response element (CRE) DNA regulatory elements regulating genes involved in cell proliferation.
  • Brx was found to bind to the Rho GTPase family members, Rho A and Cdc42Hs in vitro. Furthermore, we expect that Brx will bind to isoforms of TR, PPAR, ER and RXR as well as other nuclear hormone receptors: androgen receptor
  • AR progesterone receptor
  • PR progesterone receptor
  • GR glucocorticoid receptor
  • RAR retinoic acid receptor
  • the present invention comprises a novel protein, Brx and conservatively modified variants.
  • a preferred embodiment is a human protein consisting essentially of SEQ ID NO: l.
  • Human Brx contains a diacylglycerol binding domain and homology to the Rho guanine-nucleotide-exchange (Rho-GEF) protein, Dbl.
  • Rho-GEF Rho guanine-nucleotide-exchange
  • the full length cDNA contains a region of sequence identity to Ibc (nt. 1857-3105), a partial cDNA associated with acute blast crisis of chronic myelogenous leukemia (Toksoz, Williams 1994).
  • this invention provides for an isolated peptide consisting essentially of at least 10, preferably at least 50, more preferably at least 100, and most preferably at least 200 unique contiguous amino acid residues of SEQ ID NO: 1.
  • the polypeptides of this invention can include conservative substitutions of any of the above-described polypeptides.
  • this invention provides for antibodies specific to
  • Particularly preferred antibodies specifically bind a polypeptide comprising at least 10, 20, 40, 50, 100 or 200 contiguous amino acids, or any length between 20 contiguous amino acids up to the full length polypeptide encoded by a nucleic acid selected from the group consisting of nucleic acids that encode SEQ ID NO: 1, wherein said polypeptide, when presented as an antigen, elicits the production of an antibody which specifically binds to a polypeptide encoded by a nucleic acid selected from the group consisting of nucleic acids that encode SEQ ID NO: 1; and said polypeptide does not bind to antisera raised against a polypeptide of SEQ ID NO: 1 which has been fully immunosorbed with a polypeptide having SEQ ID NO: 1.
  • the antibody can be humanized or human.
  • the present invention comprises an isolated nucleic acid that encodes Brx, designated brx.
  • this invention provides an isolated human nucleic acid encoding a Brx protein or a unique subsequence thereof, wherein said nucleic acid specifically hybridizes, under stringent conditions, to a second nucleic acid consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2 or a unique subsequence in SEQ ID NO:2.
  • the isolated nucleic acid is at least about 30, 100, 200, 400, 800, 1,000, 2,000, 3,000, 4,000, 5,000, or 5,300 nucleotides in length.
  • the isolated human brx nucleic acid or a portion thereof is amplified from a cDNA or genomic DNA library using a pair of primers selected from the group consisting of SEQ ID NO: 3, 4, 5, 6, and 7.
  • the brx nucleic acid is identified by specific hybridization with any of the nucleic acids amplified from a genomic library using any of these primer pairs.
  • this invention provides for an isolated human nucleic acid sequence, wherein said nucleic acid preferably encodes a unique polypeptide subsequence of at least 10 contiguous amino acid residues of the polypeptide of SEQ ID NO: 1, or conservative substitutions of said polypeptide subsequence. Also included are isolated nucleic acid that encode sequences of preferably at least about 30, 50, 100, 200, 400, 600, 800, 1,000 or 1428 amino acids in length.
  • this invention provides an isolated nucleic acid encoding a unique Brx polypeptide comprising at least 10 contiguous amino acids from SEQ ID NO: l, wherein said polypeptide, when presented as an antigen, elicits the production of an antibody which specifically binds to a polypeptide sequence encoded by said nucleic acid.
  • this invention provides for vectors incorporating any of the above-described nucleic acids.
  • the vectors preferably include the above- described nucleic acid operably linked (under the control of ) a promoter; either constitutive or inducible.
  • the vector can also include an initiation and a termination codon.
  • the following vectors are envisioned for use, but not limited to: eukaryotic and prokaryotic expression and cloning vectors; gene therapy vectors which can be used in prokaryotic and/or eukaryotic cells.
  • This invention also provides for cells ⁇ e.g., recombinant cells such as hybridomas or triomas) expressing any of the above-described antibodies.
  • this invention provides for pharmacological compositions comprising a pharmaceutically acceptable carrier and a molecule selected from the group consisting of an vector encoding a Brx polypeptide or subsequence thereof, a Brx polypeptide or subsequence thereof, and an anti-Brx antibody as described herein.
  • the DNA sequences of the present invention can be used in a variety of ways in accordance with the present invention. For example, they can be used as DNA probes to screen other cDNA and genomic DNA libraries so as to select by hybridization other DNA sequences that code for proteins related to SEQ ID NO: l .
  • the DNA sequences of the present invention coding for all or part of a human Brx can be used as DNA probes to screen other cDNA and genomic DNA libraries to select by hybridization DNA sequences that code for the Brx from other species.
  • the present invention further concerns a method for detecting a nucleic acid sequence coding for all or part of a Brx protein having SEQ ID NO: 1 or a related nucleic acid sequence comprising contacting the nucleic acid sequence with a detectable marker which binds specifically to at least a portion of the nucleic acid sequence, and detecting the marker so bound.
  • the presence of bound marker indicates the presence of the nucleic acid sequence.
  • the nucleic acid sequence is a DNA sequence having all or part of the nucleotide sequence substantially as shown in SEQ ID NO:2.
  • kits for the detection and/or quantification of a brx gene or gene product can include a container containing one or more of any of the above identified nucleic acids, amplification primers, and antibodies with or without labels, free, or bound to a solid support as described herein.
  • kits can also include instructions for the use of one or more of these reagents in any of the assays described herein.
  • This invention also provides for methods of detecting a predisposition to breast or ovarian cancer and other proliferative disorders of reproductive and immune tissues.
  • the methods include the steps of i) providing a biological sample of the organism; and ii) detecting the presence, absence and/or level of expression of a brx gene or gene product in the sample.
  • detecting can involve detecting the presence or absence, or quantifying a brx gene or subsequence thereof including any of the above-described nucleic acids.
  • the detecting can also involve detecting the presence or absence or quantifying a Brx polypeptide or subsequence thereof including any of the above-described polypeptides.
  • the detecting can involve detecting the presence or absence of normal or abnormal brx nucleic acids or Brx polypeptides.
  • This invention also provides therapeutic methods. These include a methods of diagnosing, preventing or treating cancers and other proliferative diseases of reproductive epithelium, immune cells, ovary, breast, and spleen, particularly in a mammal such as a human patient.
  • the methods can involve transfecting cells of the mammal with a vector encoding a Brx such that the cells express a functional Brx polypeptide as described herein.
  • the transfection can be in vivo or ex vivo. Ex vivo transfection is preferably followed by re-infusion of the cells back into the organism as described herein.
  • the methods are preferably performed on mammals such as mice, rats, rabbits, sheep, goats, pigs, more preferably on primates including human patients.
  • FIG. 1A is a schematic diagram of the Brx-encoding nucleic acid brx.
  • FIG. IB is a comparison of diacylglycerol binding site in brx with protein kinase C (pkc) and related oncogenes: Lfc, vav, a-raf, and unc 13. conserveed residues are shaded.
  • FIG. 2A-C are northern hybridization analyses of expression of brx mRNA in different tissues, including normal and malignant.
  • Fig. 3 A illustrates the binding of Brx to nuclear hormone receptors. Lysates of [55S]methionine-labeled in vitro translated ER (HEGO), RXR (murine RXRP), PPAR (murine PPAR(x), luciferase (Luc), or TR (thyroid hormone receptor (x) were incubated with bacterially expressed GST-Brx, or GST alone, coupled to glutathione-Sepharose beads. Bound proteins were resolved using SDS PAGE. Input lanes show one tenth of added lysates. No binding was seen to the control, [ 35 S]-labeled luciferase.
  • Fig. 3B illustrates the binding of Brx to truncated estrogen receptors. Lysates of [ 5 S] -labeled wild type or truncated estrogen receptor proteins were tested as in Fig. 3a. ER- ⁇ ABC (deleted amino acids 1-268), ER- ⁇ DEF (deleted amino acids 268-595),
  • ER- ⁇ F deoxyribonucleic acid
  • Luc luciferase control
  • Fig. 4 A shows the augmentation of estrogen receptor-mediated reporter activity by Brx.
  • Expression plasmids encoding ER (HEGO) and Brx (RSV-Brx), or control (RSN-0) were added to Ishikawa endometrial cells with an ERE-t£ luciferase reporter plasmid (top panel) or a tk luciferase control reporter (bottom panel) and harvested after 24 hours.
  • Estradiol (10 nM, black bars) or vehicle control (stippled bars) was added as shown. Experiments were performed in triplicate and repeated three separate times. Luciferase activity represents fold induction (mean ⁇ _ S.D.) over control (RSV-0 with tk luciferase).
  • Fig. 4B illustrates that Brx dependent activation of ER is inhibited by N17 Cdc42Hs.
  • Ishikawa cells were transfected with pRSV-ER, pRSV-FLAG-Brx and an ERE-tk- luciferase reporter; in addition to decreasing concentrations of expression plasmids for dominant negative mutants pCEV-N17Racl (N17Racl), pCEV-N19RhoA (N19RhoA), and pCEV-N17Cdc42Hs (N17Cdc42) as denoted by triangles.
  • Dominant negative mutant expression vectors were added to 500, 250, or 0 ng.
  • pCEV Concentrations of pCEV were kept constant at 500 ng by the addition of pCEVO.
  • Cells were treated with lOnM estradiol (+) or vehicle control (-) as indicated.
  • Fig. 4C is a graph of % maximal induction of luciferase activity over a control which shows that 4-hydroxytamoxifen abolishes Brx-dependent activation of the estrogen receptor.
  • Ishikawa cells were transfected with pRSV-FLAG Brx and ERE-tk- luciferase reporter. Expression plasmids for either ER (+), or pPCKR2 control plasmid lacking ER (-) were added as shown. Cells were treated with lOnM estradiol
  • Cells were harvested 20 hours after addition of ligand. Three independent experiments were performed in triplicate. Values represent % of maximal induction of luciferase activity (mean) over control (ER with pRSV-FLAG-Brx and estradiol.) Error bars represent standard deviations.
  • Figs. 5A, B, C, D are photographs depicting intracellular localization of FLAG- Brx in Ishikawa cells.
  • Ishikawa cells were transfected with an expression plasmid, FLAG-Brx and analyzed 24 hours following transfection using an anti-FLAG primary antibody and C3-conjugated secondary antibody. Staining was observed in the nucleus (A) ( ⁇ 5% of cells), cytoplasm (B), and throughout transfected cells (C), but not in cells transfected with RSV-0 control plasmid (D).
  • a cell line is said to be “malignant” if, when the cell line is injected into a host animal, the host animal develops tumors or cancers that are anaplastic, invasive, and/or metastatic.
  • a "human” tumor is comprised of cells that have human chromosomes. Such tumors include those in a human patient, and tumors resulting from the introduction of a human malignant cell line into a non-human host animal if cells from such tumors have human chromosomes.
  • the terms "treating cancer", “therapy”, and the like mean generally a treatment that causes any improvement in a mammal having a cancer wherein the improvement can be ascribed to treatment with a Brx peptide. The improvement can be either subjective or objective.
  • the patient may note improved vigor or vitality or decreased pain as subjective symptoms of improvement or response to therapy.
  • the clinician may notice a decrease in tumor size or tumor burden based on physical exam, laboratory parameters, tumor markers, or radiographic findings.
  • the term "effective amount" means a dosage sufficient to produce a desired result.
  • the desired result can be subjective or objective improvement in the recipient of the dosage, a decrease in tumor size, a decrease in the rate of growth of cancer cells, or a decrease in metastasis.
  • “Inhibiting the growth of cancer cells” is evaluated by any accepted method of measuring whether growth of the cancer cells has been slowed or diminished. This includes direct observation and indirect evaluation such as subjective symptoms or objective signs as discussed below.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al. , Nucleic Acid Res. 79:5081 (1991); Ohtsuka et al. , J. Biol. Chem. 260:2605-2608 (1985); and Cassol et al, 1992; Rossolini et al , Mol. Cell. Probes 8: 91-98 (1994)).
  • nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
  • a “conservative substitution”, when describing a protein refers to a change in the amino acid composition of the protein that does not substantially alter the protein's activity.
  • “conservatively modified variations” of a particular amino acid sequence refers to amino acid substitutions of those amino acids that are not critical for protein activity or substitution of amino acids with other amino acids having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitutions of even critical amino acids do not substantially alter activity.
  • Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another:
  • a nucleic acid sequence encoding refers to a nucleic acid which contains sequence information for a structural RNA such as rRNA, a tRNA, or the primary amino acid sequence of a specific protein or peptide, or a binding site for a trans-acting regulatory agent. This phrase specifically encompasses degenerate codons
  • Nucleic acid probes may be DNA or RNA fragments.
  • DNA fragments can be prepared, for example, by digesting plasmid DNA, or by use of PCR, or synthesized by either the phosphoramidite method described by Beaucage and Carruthers, Tetrahedron Lett. 22: 1859-1862 (1981), or by the triester method according to Matteucci, et al. , J. Am. Chem. Soc, 103:3185 (1981), both incorporated herein by reference.
  • a double stranded fragment may then be obtained, if desired, by annealing the chemically synthesized single strands together under appropriate conditions or by synthesizing the complementary strand using DNA polymerase with an appropriate primer sequence.
  • a specific sequence for a nucleic acid probe is given, it is understood that the complementary strand is also identified and included. The complementary strand will work equally well in situations where the target is a double- stranded nucleic acid.
  • the phrase "selectively hybridizing to” refers to a nucleic acid probe that hybridizes, duplexes or binds only to a particular target DNA or RNA sequence when the target sequences are present in a preparation of total cellular DNA or RNA.
  • “Complementary” or “target” nucleic acid sequences refer to those nucleic acid sequences which selectively hybridize to a nucleic acid probe. Proper annealing conditions depend, for example, upon a probe's length, base composition, and the number of mismatches and their position on the probe, and must often be determined empirically. For discussions of nucleic acid probe design and annealing conditions, see, for example, Sambrook et al. , MOLECULAR CLONING: A LABORATORY MANUAL (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989) or CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, F. Ausubel et al. , ed.
  • sequence refers to a sequence of nucleic acids or amino acids that comprise a part of a longer sequence of nucleic acids or amino acids (e.g., polypeptide) respectively.
  • polypeptides comprises a sequence with at least 70% sequence identity to a reference sequence, or preferably 80% , or more preferably 85 % sequence identity to the reference sequence, or most preferably 90% identity over a comparison window of about 10-20 amino acid residues.
  • An indication that two polypeptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide.
  • a polypeptide is substantially identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution.
  • nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.
  • Stringent hybridization conditions refers to conditions under which a nucleic acid probe will hybridize substantially only to its target sequence in a mixture of sequences that includes its target sequence. Stringent conditions are sequence dependent. Generally, stringent conditions are selected to be about 5° lower than the thermal melting point (T m ) of the specific sequence, at a defined pH and ionic strength. The T m is the temperature (at a defined pH and ionic strength) at which 50% of the target sequence hybridizes to a complementary probe. Typically, stringent conditions will be those in which the salt concentration is at most 0.2 M at pH 7 and the temperature is at least 55°C for sequences longer that 50 nucleotides and at least 42°C for sequences of about 10 to about 20 nucleotides.
  • Lower stringency refers to conditions in which the salt concentration is greater than 0.2M at pH 7 and/or the temperature is less than or equal to 54°C.
  • Other factors including, among others, base composition, size of complementary strands, presence of organic solvents and extent of base mismatching) may significantly affect the stringency of hybridization, and the combination of parameters is more important than the absolute measure of any one.
  • reference sequence is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing, such as the nucleic acid sequence of SEQ ID NO: 2, or may comprise a complete cDNA or gene sequence.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (USA) 85:2444, or by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, WI).
  • substantially identical or “substantial sequence identity” as applied to nucleic acid sequences and as used herein and denote a characteristic of a polynucleotide sequence, wherein the poly nucleotide comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, and more preferably at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 25-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.
  • the reference sequence may be a subset of a larger sequence.
  • the terms "substantial identity” or “substantial sequence identity” mean that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 70 percent sequence identity, preferably at least 80 percent sequence identity, more preferably at least 90 percent sequence identity, and most preferably at least 95 percent amino acid identity or more.
  • Perfectage amino acid identity or “percentage amino acid sequence identity” refers to a comparison of the amino acids of two polypeptides which, when optimally aligned, have approximately the designated percentage of the same amino acids. For example, "95% amino acid identity” refers to a comparison of the amino acids of two polypeptides which when optimally aligned have 95% amino acid identity.
  • residue positions which are not identical differ by conservative amino acid substitutions. Because the substituted amino acids have similar properties, the substitutions do not change the functional properties of the polypeptides.
  • the phrase "specifically binds to an antibody” or “specifically immunoreactive with” when referring to a protein or peptide, refers to a binding reaction which is determinative of the presence of the protein in the presence of a heterogeneous population of proteins and other biologies.
  • the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample.
  • Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. B. Detailed Description 1.
  • Brx proteins Interaction cloning was used to screen a breast cancer ZR75-1 cDNA expression library with recombinant RXR/3 protein which yielded a partial cDNA (1.8Kb) used to screen two human cDNA libraries (breast and testis) to generate a full length cDNA clone encoding the protein of the present invention, Brx.
  • the amino acid sequence of human Brx is SEQ ID NO: l.
  • a murine brx has been detected, and it is probable that other animal species (e.g., primates, rats, rabbits, pigs, cows, etc.) will have a related protein.
  • the present invention comprises the protein of SEQ ID NO: l and conservatively modified variants thereof.
  • the invention comprises proteins that specifically bind antibodies raised against the protein of SEQ ID NO: l, and that possess all or most of the biological properties of Brx: e.g., the ability to specifically bind to GST-RXR/3, ER or other NHRs, binding through a novel carboxyl region, the ability to enhance activation by the estrogen receptor, localized homology to Rho-GEF oncoproteins, and a molecular weight of about 168 kd.
  • Nucleic acids encoding Brx proteins Nucleic acids that encode Brx may be obtained by generating genomic or cDNA libraries, subcloning the library into expression vectors, labelling probes, DNA hybridization, and the like, as described in Sambrook, et al.
  • DNA encoding a Brx or a peptide fragment thereof is identified in either cDNA or genomic libraries by its ability to hybridize with nucleic acid probes, for example on Southern blots, and these DNA regions are isolated by standard methods familiar to those of skill in the art. See Sambrook, et al.
  • the nucleic acid sequences of the invention are typically identical to or show substantial sequence identity (determined as described below) to the nucleic acid sequence of SEQ ID. No. 2.
  • Nucleic acids encoding Brx or a peptide fragment thereof will typically hybridize under stringent hybridization conditions to sequences that are complementary to unique sequences in SEQ ID NO: 2.
  • PCR polymerase chain reaction
  • oligonucleotide primers complementary to the two 3' borders of the DNA region to be amplified are synthesized.
  • the polymerase chain reaction is then carried out using the two primers. See PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS. (Innis, M, Gelfand, D., Sninsky, J. and White, T., eds.), Academic Press, San Diego (1990).
  • Primers can be selected to amplify the entire regions encoding a full-length Brx or to amplify smaller DNA segments as desired.
  • oligonucleotides having the following sequences can be used in a PCR protocol herein to amplify regions of DNA' s encoding Brx or peptide fragments thereof.
  • oligonucleotide probes can be prepared from sequence obtained. These probes can then be used to isolate DNA's encoding Brx or a peptide fragment thereof.
  • Oligonucleotides for use as probes are optionally chemically synthesized using the solid phase phosphoramidite triester method of Beaucage and Carruthers, Tetrahedron Lett.
  • Such mutations may be either degenerate, i.e. , the mutation does not change the amino acid sequence encoded by the mutated codon, or non-degenerate, i.e. , the mutation changes the amino acid sequence encoded by the mutated codon.
  • modified DNA sequences may be prepared, for example, by mutating SEQ ID NO: 2 so that the mutation results in the deletion, substitution, insertion, inversion or addition of one or more amino acids in the encoded polypeptide using various methods known in the art. For example, the methods of site-directed mutagenesis described in Taylor et al, Nucl. Acids Res. 13, 8749-8764 (1985) and Kunkel, Proc. Natl. Acad. Sci.
  • kits for site-directed mutagenesis may be purchased from commercial vendors.
  • a kit for performing site-directed mutagenesis may be purchased from Amersham Corp. (Arlington Heights, 111.).
  • Both degenerate and non-degenerate mutations may be advantageous in producing or using the polypeptides of the present invention. For example, these mutations may permit higher levels of production, easier purification, or provide additional restriction endonuclease recognition sites. All such modified DNAs (and the encoded polypeptide molecules) are included within the scope of the present invention.
  • nucleic acid is expressed in a variety of recombinantly engineered cells to ascertain that the isolated nucleic acid indeed encodes the desired Brx or a peptide fragment thereof.
  • the expression of natural or synthetic nucleic acids is typically achieved by operably linking a nucleic acid of interest to a promoter (which is either constitutive or inducible), incorporating the construct into an expression vector, and introducing the vector into a suitable host cell.
  • Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular nucleic acid.
  • the vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both, (e.g. , shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems.
  • Vectors are suitable for replication and integration in prokaryotes, eukaryotes, or preferably both. See, Giliman and Smith (1979), Gene, 8:81-97; Roberts et al. (1987), Nature, 328:731-734; Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology 152,
  • nucleic acids used in the present method can be isolated from natural sources, obtained from such sources as ATCC or GenBank libraries, or prepared by synthetic methods. Synthetic nucleic acids can be prepared by a variety of solution or solid phase methods.
  • nucleic acids there are several well-known methods of introducing nucleic acids into bacterial and animal cells, any of which may be used in the present invention. These include: calcium phosphate precipitation, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA, DEAE dextran, receptor-mediated endocytosis, electroporation, micro-injection of the DNA directly into the cells, infection with viral vectors, etc.
  • nucleic acids can be to any cell grown in culture, whether of bacterial, plant or animal origin, vertebrate or invertebrate, and of any tissue or type.
  • concentration of nucleic acid varies widely depending on the particular application, but is generally between about 1 ⁇ mol and about 10 mmol.
  • Treatment of the cells with the nucleic acid is generally carried out at physiological temperatures (about 37 °C) for periods of time of from about 1 to 48 hours, preferably of from about 2 to 4 hours.
  • a nucleic acid is added to 60-80% confluent plated cells having a cell density of from about 10 3 to about 10 5 cells/mL, more preferably about 2 x 10 4 cells/mL.
  • the concentration of the suspension added to the cells is preferably of from about 0.01 to 0.2 ⁇ g/mL, more preferably about 0.1 ⁇ g/mL.
  • compositions and methods of the present invention are used to transfer genes into a wide variety of cell types, in vivo and in vitro. Although any prokaryotic or eukaryotic cells may be used, preferred cells include Ishikawa human endometrial cells. c. Detection and quantification of presence and expression of brx or an oligonucleotide fragment thereof, or Brx or a peptide fragment thereof
  • Brx-encoding nucleic acids and Brx proteins and peptide fragments are detected and quantified herein by any of a number of means well known to those of skill in the art. These include analytic biochemical methods such as spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, and various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, and the like.
  • the detection of nucleic acids proceeds by well known methods such as Southern analysis, northern analysis, gel electrophoresis,
  • the present invention provides methods for detecting DNA or RNA encoding Brx or a peptide fragment thereof.
  • a variety of methods for specific DNA and RNA measurement using nucleic acid hybridization techniques are known to those of skill in the art. See Sambrook, et al. ; NUCLEIC ACID HYBRIDIZATION, A PRACTICAL
  • one method for evaluating the presence or absence of DNA encoding Brx or a peptide fragment thereof in a sample involves a Southern transfer.
  • nucleic acid probes are designed based on the nucleic acid sequences encoding Brx or a peptide fragment thereof (See SEQ ID NOs: l and 2)
  • the probes can be full length or less than the full length of the nucleic acid sequence encoding the Brx. Shorter probes are empirically tested for specificity.
  • nucleic acid probes are 20 bases or longer in length. (See Sambrook, et al. for methods of selecting nucleic acid probe sequences for use in nucleic acid hybridization.)
  • Visualization of the hybridized portions allows the qualitative determination of the presence or absence of DNA encoding Brx or a peptide fragment thereof.
  • a Northern transfer may be used for the detection of mRNA encoding Brx or a peptide fragment thereof.
  • 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. As with the Southern blots, labeled probes are used to identify the presence or absence of brx.
  • Sandwich assays are commercially useful hybridization assays for detecting or isolating nucleic acid sequences. Such assays utilize a "capture" nucleic acid covalently immobilized to a solid support and a labelled “signal" nucleic acid in solution. The clinical sample will provide the target nucleic acid. The "capture” nucleic acid and “signal” nucleic acid probe hybridize with the target nucleic acid to form a "sandwich” hybridization complex. To be effective, the signal nucleic acid cannot hybridize with the capture nucleic acid. Typically, labelled signal nucleic acids are used to detect hybridization.
  • Complementary nucleic acids or signal nucleic acids may be labelled by any one of several methods typically used to detect the presence of hybridized polynucleotides. The most common method of detection is the use of autoradiography with 3 H, 125 I, 35 S, 14 C, or 32 P-labelled probes or the like. Other labels include ligands which bind to labelled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labelled ligand.
  • Detection of a hybridization complex may require the binding of a signal generating complex to a duplex of target and probe polynucleotides or nucleic acids. Typically, such binding occurs through ligand and anti-ligand interactions as between a ligand-conjugated probe and an anti-ligand conjugated with a signal.
  • the label may also allow indirect detection of the hybridization complex.
  • the label is a hapten or antigen
  • the sample can be detected by using antibodies.
  • a signal is generated by attaching fluorescent or enzyme molecules to the antibodies or, in some cases, by attachment to a radioactive label.
  • the sensitivity of the hybridization assays may be enhanced through use of a nucleic acid amplification system which multiplies the target nucleic acid being detected, in vitro amplification techniques suitable for amplifying sequences for use as molecular probes or for generating nucleic acid fragments for subsequent subcloning are known.
  • in vitro amplification techniques suitable for amplifying sequences for use as molecular probes or for generating nucleic acid fragments for subsequent subcloning are known.
  • examples of techniques sufficient to direct persons of skill through such in vitro amplification methods including the polymerase chain reaction (PCR) the ligase chain reaction (LCR), Q Beta-replicase amplification and other RNA polymerase mediated techniques (e.g. , NASBA) are found in Berger, Sambrook, and Ausubel, as well as Mullis et al. (1987), U.S. Patent No.
  • nucleic acid sequence based amplification NASBATM, Cangene, Mississauga, Ontario
  • Q Beta Replicase systems can be used to directly identify mutants where the PCR or LCR primers are designed to be extended or ligated only when a select sequence is present.
  • the select sequences can be generally amplified using, for example, nonspecific PCR primers and the amplified target region later probed for a specific sequence indicative of a mutation.
  • Oligonucleotides for use as probes are typically synthesized chemically according to the solid phase phosphoramidite triester method described by Beaucage and Caruthers,
  • Brx or a peptide fragment thereof may be detected or quantified by a variety of methods. Preferred methods involve the use of specific antibodies.
  • polypeptide of SEQ ID NO: 1 or a fragment thereof is isolated as described herein.
  • recombinant protein is produced in a transformed cell line.
  • An inbred strain of mice or rabbits is immunized with the protein of SEQ ID NO: 1 or a peptide thereof, using a standard adjuvant, such as Freund's adjuvant, and a standard immunization protocol.
  • a synthetic peptide derived from the sequences disclosed herein and conjugated to a carrier protein can be used an immunogen.
  • Polyclonal sera are collected and titered against the immunogen protein in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support.
  • Polyclonal antisera with a titer of 10 4 or greater are selected and tested for their cross reactivity against non-Brx or even Brx from other cell types or species or a peptide fragment thereof, using a competitive binding immunoassay.
  • Specific monoclonal and polyclonal antibodies and antisera will usually bind with a K D of at least about 0.1 mM, more usually at least about 1 ⁇ M, preferably at least about .1 ⁇ M or better, and most preferably, .01 ⁇ M or better. .
  • a number of immunogens may be used to produce antibodies specifically reactive with Brx or a peptide fragment thereof.
  • Recombinant protein is the preferred immunogen for the production of monoclonal or polyclonal antibodies.
  • Naturally occurring protein may also be used either in pure or impure form.
  • Synthetic peptides made using the Brx or a peptide fragment thereof sequences described herein may also used as an immunogen for the production of antibodies to the protein.
  • Recombinant protein can be expressed in eukaryotic or prokaryotic cells as described above, and purified as generally described above. The product is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies may be generated, for subsequent use in immunoassay s to measure the protein.
  • an immunogen preferably a purified protein such as Brx or a peptide fragment thereof is mixed with an adjuvant and injected into an animal of choice (e.g., a mouse, rat, rabbit, pig, goat, cow, horse, chicken, etc .) at intervals of 1-4 weeks.
  • the immunogen may be conjugated to a carrier protein can be used an immunogen.
  • the animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the Brx or a peptide fragment thereof.
  • blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be done if desired. (See Harlow and Lane, supra).
  • Polyclonal sera are collected and titered against the immunogen protein in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support.
  • Polyclonal antisera with a titer of 10 4 or greater are selected and tested for their cross reactivity against non-Brx, using a competitive binding immunoassay such as the one described in Harlow and Lane, supra, at pages 570-573.
  • polypeptide immunogens Such polyclonal antisera have been prepared by immunization of rabbits with the following polypeptide immunogens:
  • Peptide 1 LRDGRPSWPSARRRCSRGSRTWKRSGRSSS antisera #2741-
  • 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. 6:511-519 (1976), incorporated herein by reference) . Alternative methods of immortalization include transformation with Epstein
  • Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and 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.
  • a particular protein can be measured by a variety of immunoassay methods.
  • immunoassay methods for a review of immunological and immunoassay procedures in general, see BASIC AND CLINICAL IMMUNOLOGY, 7th Edition (D. Stites and A. Terr ,eds.) 1991.
  • the immunoassays of the present invention can be performed in any of several configurations, which are reviewed extensively in ENZYME IMMUNOASSAY, E.T.
  • Immunoassays to Brx or a peptide fragment thereof of the present invention may use a polyclonal antiserum which was raised to the protein of SEQ ID NO: l or a fragment thereof. This antiserum is selected to have low crossreactivity against other (non-Brx or Brx) proteins and any such crossreactivity is removed by immunoabsorption prior to use in the immunoassay.
  • Immunoassays in the competitive binding format can be used for the crossreactivity determinations.
  • the protein of SEQ ID NO: l can be immobilized to a solid support. Proteins (Brx-like, or non-Brx, or unknown) are added to the assay which compete with the binding of the antisera to the immobilized antigen. The ability of the above proteins to compete with the binding anti-BRX antisera or antibodies to the immobilized protein is compared to the protein of SEQ ID NO: l, or a peptide thereof. The percent crossreactivity for the above proteins is calculated, using standard calculations. Those antisera with less than 10% crossreactivity with each of the proteins listed above are selected and pooled. Any cross-reacting antibodies are optionally removed from the pooled antisera by immunoabsorption with the above-listed proteins.
  • the immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay as described herein to compare the binding of a second protein to that of the immunogen protein or peptide, the Brx of SEQ ID NO: 1.
  • the two proteins are each assayed at a wide range of concentrations and the amount of each protein required to inhibit 50 % of the binding of the antisera to the immobilized protein is determined. If the amount of the second protein required is less than 10 times the amount of the protein of SEQ ID NO: 1 that is required, then the second protein is said to specifically bind to an antibody generated to an immunogen consisting of the protein of SEQ ID NO: l.
  • the presence of a desired polypeptide (including peptide, transcript, or enzymatic digestion product) in a sample may be detected and quantified using Western blot analysis.
  • the technique generally comprises separating sample products by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the sample with labeling antibodies that specifically bind to the analyte protein.
  • the labeling antibodies specifically bind to analyte on the solid support.
  • These antibodies are directly labeled, or alternatively are subsequently detected using labeling agents such as antibodies (e.g. , labeled sheep anti- mouse antibodies where the antibody to an analyte is a murine antibody) that specifically bind to the labeling antibody.
  • the polypeptides of this invention may be purified to substantial purity by standard techniques, 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), inco ⁇ orated herein by reference.
  • the Brx proteins and polypeptides produced by recombinant DNA technology may be purified by a combination of cell lysis (e.g. , sonication) and affinity chromatography or immunoprecipitation with a specific antibody to Brx or a peptide fragment thereof.
  • cell lysis e.g. , sonication
  • affinity chromatography e.g. chromatography
  • immunoprecipitation e.g., antigene fragment of a specific antibody to Brx or a peptide fragment thereof.
  • subsequent digestion of the fusion protein with an appropriate proteolytic enzyme releases the desired polypeptide.
  • the proteins may
  • compositions for administration will commonly comprise a solution of the Brx polypeptide, antibody or antibody chimera/ fusion dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier.
  • a pharmaceutically acceptable carrier preferably an aqueous carrier.
  • aqueous carriers can be used, e.g. , buffered saline and the like. These solutions are sterile and generally free of undesirable matter.
  • These compositions may be sterilized by conventional, well known sterilization techniques.
  • the Brx proteins, peptides thereof, or ligands or antagonists of Brx or brx are provided in powder form, or in a pharmaceutically acceptable solution such as an aqueous solution, often a saline solution.
  • the Brx may be combined with conventional excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium, carbonate, and the like.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • the concentration of chimeric molecule in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs.
  • the resulting compositions may be in the form of a solution, suspension, tablets, pill, capsule, powder, gel, cream, lotion, ointment, aerosol or the like.
  • the pharmaceutical composition or medium that comprises Brx or a Brx agonist or antagonist is administered orally, parenterally, enterically, gastrically, topically, subcutaneously, locally or systemically. It is recognized that the Brx polypeptides and related compounds described above, when administered orally, must be protected from digestion. This is typically accomplished either by complexing the protein with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the protein in an appropriately resistant carrier such as a liposome. Means of protecting proteins from digestion are well known in the art.
  • a typical pharmaceutical composition for intravenous administration would be about 0.1 to 10 mg per patient per day. Dosages from 0.1 up to about 100 mg per patient per day may be used, particularly when the drug is administered to a secluded site and not into the blood stream, such as into a body cavity or into a lumen of an organ. Substantially higher dosages are possible in topical administration. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington 's Pharmaceutical Science, 15th ed. , Mack Publishing Company, Easton, Pennsylvania (1980).
  • compositions containing the present Brx polypeptides, antibodies or antibody chimer/ fusions, or a cocktail thereof (i.e. , with other proteins), can be administered for therapeutic treatments.
  • compositions are administered to a patient suffering from a disease (e.g., Brx or basal cell carcinoma) in an amount sufficient to cure or at least partially arrest the disease and its complications.
  • a disease e.g., Brx or basal cell carcinoma
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose. " Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health.
  • compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of the proteins of this invention to effectively treat the patient. 6. Diagnostic applications
  • brx may be a tumor suppressor gene, and that alterations in the expression pattern of the brx gene may be associated with malignancy.
  • Brx expression in cancer tissue sections is of prognostic significance. Characterization of Brx will likely lead to the development of screening tests for malignancies such as breast and ovarian malignancies. Accordingly, the methods of detecting the presence, absence, level of expression, and tissue-distribution of brx or Brx that are mentioned above and below are used to detect disease states mediated by the expression of brx. In particular, these methods permit the identification of ER resistant tumors, and the analysis of ER function in tumor progression and treatment. 7. Drug Screening Assays
  • the methods of detecting the presence, absence, level of expression, and tissue-distribution of brx or Brx that are mentioned above and below are used to detect molecules and administration regimes that interact with and/or alter the pattern of expression of brx, and give rise to defined phenotypes.
  • the drug will be administered to an organism, tissue, or cell that expresses or is engineered to express brx.
  • Expression levels, or expression products will be assayed as described herein and the results compared with results from to organisms, tissues, or cells similarly treated, but without the drug being tested.
  • the Brx, Brx fragments, and Brx binding compounds detected by the methods of the invention are used to treat cancer patients.
  • the claimed methods are effective against a range of different cancer types, including not limited to tumors of immune and reproductive tissues, e.g., germinal epithelium of both ovary and testis, mammary epithelium, and epithelial and stromal cells of the endometrium.
  • cells derived from the tumor are grown in tissue culture.
  • the growth inhibiting effect may be assessed using a number of commonly used assays, such as cell counts, or radioactive thymidine incorporation, or a methylcellulose assay (Lunardi-Iskandar et al. , Gin. Exp. Immunol. , 60:285-293
  • the above compounds can be injected intratumorly: the tumor, the placement of the needle and release of the contents of the syringe may be visualized either by direct observation (for easily accessible mmors such as surface tumors or tumors easily exposed by surgical techniques), by endoscopic visualization, or by electromagnetic imaging techniques such as ultrasound, magnetic resonance imaging (MRI), CT scans.
  • the compounds can also be administered via injection into the bloodstream using a cannula or catheter; the vein or artery is selected to maximize delivery of cells to the tumor or affected tissue.
  • cystic or vesicular tumors or tissues the compounds may be delivered intracystically or intravesicularly.
  • the compounds will be administered under the guidance of a physician.
  • concentration of compounds to be administered at a given time and to a given patient will vary from 0.1 ⁇ g-100 mg and preferably 0.1-10 mg per day per patient.
  • the dosage to be administered is the amount necessary to reduce cancer cell growth and/or to destroy cancer cells and/or preferably to eradicate the cancer.
  • the exact number is a function of the size and compactness (or diffuseness) of the particular transformed cell mass to be treated, and the distance or accessibility of the tissue to be treated from the point of administration of the cells. More than one administration may be necessary.
  • the supervising physician will monitor the progress of the treatment, and will determine whether a given administration is successful and sufficient, or whether subsequent administrations are needed.
  • Tumor regression and other parameters of successful treatment are assessed by methods known to persons of skill in the art. This includes any imaging techniques that are capable of visualizing cancerous tissues (e.g. , MRI), biopsies, methods for assessing metabolites produced by the cancer tissue or affected tissue in question, the subjective well-being of the patient, etc.
  • cDNAs packageable brx nucleic acids
  • These packageable nucleic acids can be inserted into any of a number of well known vectors for the transfection and transformation of target cells and organisms as described below.
  • the nucleic acids are transfected into cells, ex vivo or in vivo, through the interaction of the vector and the target cell.
  • the brx cDNA under the control of a promoter, then expresses the Brx protein, thereby mitigating the effects of absent brx genes or partial inactivation of the brx gene or abnormal expression of the brx gene.
  • Such gene therapy procedures have been used to correct acquired and inherited genetic defects, cancer, and viral infection in a number of contexts.
  • the ability to express artificial genes in humans facilitates the prevention and/or cure of many important human diseases, including many diseases which are not amenable to treatment by other therapies.
  • Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof. See, e.g., Buchscher et al. (1992) J. Virol. 66(5) 2731-2739; Johann et al. (1992) J. Virol. 66 (5): 1635-1640 (1992); Sommerfelt et al , (1990) Virol. 176:58-59; Wilson et al.
  • MiLV murine leukemia virus
  • GaLV gibbon ape leukemia virus
  • SIV Simian Immuno deficiency virus
  • HAV human immuno deficiency virus
  • AAV-based vectors are also used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and in in vivo and ex vivo gene therapy procedures. See, West et al (1987) Virology 160:38-47; Carter et al (1989) U.S. Patent No. 4,797,368; Carter et al. WO 93/24641 (1993); Kotin (1994) Human Gene Therapy 5:793-801; Muzyczka (1994) J. Clin. Invst. 94: 1351 and Samulski (supra) for an overview of AAV vectors. Construction of recombinant AAV vectors are described in a number of publications, including Lebkowski, U.S. Pat. No.
  • RXR retinoid X receptor
  • Overlapping cDNA clones were consistent with a 5.3 kilobase cDNA (SEQ ID NO:2) encoding a 1428 amino acid, 168 kilodalton MW protein (SEQ ID NO: l).
  • GenBank A search of nucleotide sequences in GenBank revealed the gene to be distinct from proteins reported to bind nuclear hormone receptors. Chen & Evans, Nature 371 ', 454-457 (1995); Horlein et al. Nature 377, 397-404 (1995); Onate et al , Science 270, 1354-1357 (1995); Jacq et al , Cell 79, 107-117 (1994); Cavailles et al , EMBO J. 14, 3741-3751 (1995).
  • FIG. 1 A is a schematic diagram of the b ⁇ r-encoding nucleic acid.
  • the cDNA was divided into regions 1-5, based on homology with existing proteins.
  • the solid lines at the top of the diagram represent the overlapping cDNA clones used to sequence the entire coding region.
  • the dashed line indicates sequence also confirmed by 5' RACE ("Rapid Amplification of DNA Ends"), following the procedure in the manufacturer's instructions (Clontech, Palo Alto, CA). Novel regions are shown in black.
  • the bar at the bottom represents different regions of the coding sequence.
  • Region 1 contains the N-terminal methionine preceded by four in-frame stop codons.
  • Region 2 is homologous to the carboxyterminus of Ht 31 partial cDNA (nucleotides 1782-3036; Carr et al, J. Biol. Chem. 266, 14188-14192 (1991)), a type II cAMP-dependent protein kinase A-anchoring protein (Carr. et al, J. Biol. Chem. 266,
  • Brx protein decreased as breast mmors became increasingly malignant, and the cells became less differentiated. This decrease in Brx expression in malignancies, coupled with the observation that a region of Brx is linked to oncogenesis
  • Fig. 4A shows that overexpression of brx markedly augmented activity of an estrogen response element (ERE) containing reporter construct in the presence, but not absence, of ligand. Furthermore, activation was specific for ERE-containing reporter constructs and was not seen with a reporter construct lacking an ERE.
  • ERE estrogen response element
  • Ishikawa cells were cultured in 12 well plates in DMEM/F-12 with 5% charcoal-stripped fetal bovine serum. 50 ng of HEGO, 0.5 ⁇ g of Brx expression plasmid, and 1.0 ⁇ g of ERE-t& luciferase were added to Ishikawa cells with Lipofectamine as per manufacturer's directions (Gibco-BRL). Cells were harvested after 24 hours and luciferase activity was determined as described. Segars et al, Molec. Cell. Biol. 13: 2258-2268 (1993).
  • Brx expression plasmid was constructed by introducing into RSV-PBK (Stratagene) an EcoRl fragment of brx containing the carboxyl region of Brx Expression plasmids encoding ER (HEGO) and Brx (RSV-Brx), or control (RSV-0), were added to Ishikawa endometrial cells with an ERE-t& luciferase reporter plasmid (Fig. 4 A) or a tk luciferase control reporter (Fig. 4 A) and harvested after 24 hours. Estradiol (10 nM, black bars) or vehicle control (stippled bars) was added as shown. Experiments were performed in triplicate and repeated three separate times.
  • Luciferase activity represents fold induction (mean _+ S.D.) over control (RSV-0 with tk luciferase). This activation by Brx was inhibited by 4-hydroxytomoxifen. See Fig. 4C.
  • Brx is a novel member of the Dbl (Rho-GEF) family that is capable of augmenting ligand-dependent estrogen receptor function in reproductive tissues.
  • the expression of brx in breast tissues, the augmentation of ligand dependent ER activation, and homology to the Dbl oncogene (Rho-GEF) emphasize the importance of further study of Brx in the pathogenesis of cancers, including breast cancer.
  • the results suggest that Brx represents a novel effector protein that may couple Rho and ER signalling pathways.
  • Brx is a novel member of the Dbl (Rho-GEF) family that is capable of augmenting ligand-dependent estrogen receptor function in reproductive tissues.
  • Brx in breast tissues, the augmentation of ligand dependent ER activation, and homology to the Dbl oncogene (Rho-GEF) emphasize the role of Brx in the pathogenesis of reproductive and immune cancers, including breast cancer. Brx may also have an important role in the etiology of other proliferative disorders of reproductive and immune tissues.

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Abstract

L'invention concerne un gène, brx, et son produit génique, Brx, impliqués dans l'étiologie des cancers mammaires et ovariens. L'invention comprend en outre des méthodes d'utilisation de brx et de Brx pour diagnostiquer et traiter les maladies prolifératives des tissus reproducteurs et immunitaires, notamment les cancers.
PCT/US1998/019782 1997-09-23 1998-09-23 SEQUENCES DE NUCLEOTIDES ET D'ACIDES AMINES DU GENE brx ET PRODUIT GENIQUE DE CE DERNIER ET UTILISATIONS Ceased WO1999015544A1 (fr)

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AU95747/98A AU9574798A (en) 1997-09-23 1998-09-23 Nucleotide and amino acid sequences of the (brx) gene and gene product, and sesthereof

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EP1186319A1 (fr) * 2000-09-08 2002-03-13 Alfred Prof. Dr. Nordheim Utilisation d' un modulateur de SRF dans le traitement des maladies liées au SRF

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Title
KITAMURA K., ET AL.: "EXPRESSION PATTERNS OF BRXL (RIEG GENE), SONIC HEDGEHOG, NKX2.2, DLXL AND ARX DURING ZONA LIMITANS INTRATHALAMICA AND EMBRYONIC VENTRAL LATERAL GENICULATE NUCLEAR FORMATION.", MECHANISMS OF DEVELOPMENT., ELSEVIER SCIENCE IRELAND LTD., IE, vol. 67., 1 September 1997 (1997-09-01), IE, pages 83 - 96., XP002915649, ISSN: 0925-4773, DOI: 10.1016/S0925-4773(97)00110-X *
RUBINO D., ET AL.: "CHARACTERIZATION OF BRX, A NOVEL DBL FAMILY MEMBER THAT MODULATES ESTROGEN RECEPTOR ACTION.", ONCOGENE, NATURE PUBLISHING GROUP, GB, vol. 16., 1 May 1998 (1998-05-01), GB, pages 2513 - 2526., XP002915648, ISSN: 0950-9232, DOI: 10.1038/sj.onc.1201783 *
SIMMLER M.-C., ET AL.: "LOCALIZATION AND EXPRESSION ANALYSIS OF A NOVEL CONSERVED BRAIN EXPRESSED TRANSCRIPT, BRX/BRX, LYING WITHIN THE XIC/XIC CANDIDATE REGION.", MAMMALIAN GENOME, SPRINGER NEW YORK LLC, US, vol. 08., 1 June 1997 (1997-06-01), US, pages 760 - 766., XP002916037, ISSN: 0938-8990, DOI: 10.1007/s003359900561 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1186319A1 (fr) * 2000-09-08 2002-03-13 Alfred Prof. Dr. Nordheim Utilisation d' un modulateur de SRF dans le traitement des maladies liées au SRF
WO2002020092A1 (fr) * 2000-09-08 2002-03-14 Alfred Nordheim Utilisation du modulateur du facteur de réponse sérique (srf) pour traiter des pathologies en relation avec le srf

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