MXPA99000406A - Brca1 compositions and methods for the diagnosis and treatment of breast cancer - Google Patents

Abstract

Disclosed are methods and compositions relating to the diagnosis and treatment of breast and related cancers. Compositions and methods for the detection of the BRCA1 gene product in vivo and in vitro are disclosed, as well as methods for diagnosing aberrant localization of BRCA1 protein in cells using anti-BRCA1 antibodies. Also disclosed are methods for identifying BRCA1-associated proteins which function in the proper translocation of the BRCA1 gene product to the cell nucleus.

Description

COMPOSITIONS OF BRCAl AND METHODS FOR THE DIAGNOSIS AND TREATMENT OF BREAST CANCER Background of the Invention The present application is a continuous application of the United States Provisional Application serial number 60 / 015,863 filed July 8, 1996, the content of which is fully and specifically incorporated into this specification by reference. The United States government has certain rights in the present invention under grants DA57317, CA58318, P50-CA58183 and 059-CA58183 from the National Cancer Institute.

Technical Field of the Invention The present invention relates, generally, to the field of molecular biology, and more specifically, certain embodiments relate to methods and compositions that include the BRCA1 compositions and methods for the diagnosis and treatment of cancer. mom. Methods and compositions useful for various pharmacological and immunological applications are described.

Description of the State of the Art Breast cancer Breast cancer is the most common of the deadly malignancies that afflict women in developed countries. The etiology of breast cancer includes a complex interaction of genetic, hormonal and dietary factors that overlap the physiological state of the patient. An extensive genetic analysis of tumors of the breast has detected several alterations in the expression of genes associated with the disease. At the molecular level, in addition to the frequently observed gene amplification (Escot et al., 1986, Lidereau et al., 1988, Slamon et al., 1987, van de Vijver et al., 1987, Varley et al., 1988) , it is thought that the development of tumors of the breast is a consequence of the unmasking by mutation of one or more recessive genes. Two genetic events serve to inactivate a recessive locus, and the resulting reduction to homozygosity of altered alleles has been proposed as an essential step in tumorigenesis. A loss of heterozygosity (LOH) has been observed in six different regions of the human genome including lq, 3p, l lp, 13q, 17p and 17q chromosomes, in a high percentage of primary-type breast cancers (Ali et al., 1987, Chen et al., 1989, Devilee et al., 1990, Devilee et al., 1989, Lundberg et al., 1987, Mackay et al., 1988a, Mackay et al., 1988b, Futreal et al., 1992 ), and these allelic losses in tumor tissues suggest the location of possible tumor suppressor genes. Recently, the great expectation generated by the cloning of the BRCAl family gene for breast and ovarian cancer on chromosome 17q (Miki et al., 1994) has been moderated somewhat by the fact that mutations of the gene in cancer have not been found of sporadic breast (Futreal; cois., 1994, Friedman ^ cois., 1994, Shattuck-Eiders et al., 1995) -although BRCAl has been associated with just over 45% of site-specific hereditary breast cancers, and with 80% of families with breast and ovarian cancer (Easton et al., 1993), although no sporadic breast cancer has been found, and only about 10% of sporadic ovarian cancers harbor BRCAl mutations (Futreal et al., 1994, Hosking _y cois., 1995, Merajver > > cois., 1995).

In this way, the role of BRCA1 in the pathogenesis of sporadic breast cancers, which constitute approximately 95%, has been questioned (Vogelstein cois., 1994). of all breast cancers (Claus et al., 1991). This contrasts with most other tumor suppressors, such as RB and p53, in which mutations are observed in both familial and sporadic cancers (Bookstein and Lee, 1991, Levine et al., 1991). An explanation of the results of these investigations, contrary to established concepts regarding tumor suppressor genes, suggests that BRCAl is only inactivated in familial breast cancers (Boyd, 1995, Castilla jμcois, 1994), and only in a subset thereof, since it is known that other breast cancer genes, including BRCA2 on chromosome 13ql2-13 (Wooster et al., 1994; 1995), are found in different genetic loci. The pathogenesis of cancer is a multi-step process, and eventually different pathways can result in the same or similar consequences. There are precedents, such as Wilms tumor and hereditary nonpolyposis colorectal cancer, of different pathways for the same type of cancer (Vogelstein et al., 1994). For example, it has been shown that inactivation of both alleles of the WT1 tumor suppressor gene is important in a considerable proportion of hereditary Wilms tumors, especially those that occur as part of the Denys-Drash syndrome, but not in tumors of Sporadic Wilms (Pelletier et al., 1991, Coppes et al., 1993). Similar observations of inactivation of the mismatched msh2 mating repair gene of DNA in familial but non-sporadic colon cancers can be explained by the "mutant phenotype" generated by the msh2 mutation (Cleaver, 1994; Vogelstein et al., 1994). However, even without the inactivation of msh2, the same steps of tumorigenesis may occur, albeit less frequently or rapidly.

Role played by BRCA 1 The cloning of the BRCAl breast and ovarian cancer gene (Miki et al., 1994) was an important advance in breast cancer research. However, although BRCAl has been associated with more than 45% of site-specific hereditary breast cancers, and with 80% of families with breast and ovarian cancer (Easton et al., 1993), no breast cancer sporadic, and only about 10% of sporadic ovarian cancers harbor BRCAl mutations (Miki et al., 1994, Futreal et al., 1994, Friedman et al., 1994, Shattuck-Eiders et al., 1995; cois., 1995; Merajver ^ cois., 1995). In this way, the general function of BRCA1 in the pathogenesis of sporadic breast cancers, which constitute approximately 95% of these neoplasms (Claus. And co., 1991), has not been tested to date (Boyd, 1995; / cois., 1994). The complementary DNA of BRCA1 encodes a protein of 1863 amino acids whose predicted structure includes two zinc finger domains near the terminal domain NH2 and an acidic terminal domain COOH, and it is therefore speculated that the BRCA1 protein is a transcription factor (Miki > cois., 1994; Vogelstein &Kinzler, 1994). A year and a half after the BRCA1 was isolated (Miki et al., 1994), the 17q21 human chromosome gene, responsible for almost 50% of hereditary breast cancers, remains an enigma. Although BRCAl mutations have been clearly linked to hereditary breast cancer and ovarian cancer, it has been found that no sporadic breast cancer, and only 10% of sporadic ovarian cancers harbor BRCA1 mutations (Futreal et al., 1994; Hosking et al., 1995; Merajver j .; cois .. 1995). Rather, it has been suggested that BRCA1 is functionally inactivated by displacement from its normal nuclear location to the cytoplasm in spontaneous cancers (Chen et al., 1995). It is assumed that the responsible defect is found in a protein necessary for the translocation of BRCA1 to the nucleus, since once marked, the wild-type exogenous BRCA1 is translocated in a similar manner in the breast cancer cell lineages (Chen et al. , nineteen ninety six). It has been speculated that BRCA1 is a transcription factor, based on the presence of an annular finger motif near the N-terminus and the C-terminal segment rich in acid residues (Miki et al., 1994). With the reported nuclear location of BRCAl, however, no direct evidence that BRCAl is a transcription factor has yet been presented.Data on in situ hybridization suggest that BRCAl may play a critical role in the growth and cellular differentiation, since the BRCAl mRNA seems to be expressed in a general manner in all developing mouse embryos, with an especially high activity that seems correlated with tissues that undergo rapid proliferation and differentiation (Lane et al., 1995; cois., 1995) Another congruent fact is that the homozygous deletion of BRCA1 in mice is fatal in early embryogenesis (Gowen et al., 1996, Chia-Yang Liu et al.). that the results of these investigations suggest potential functions for BRCAl, a detailed characterization of the BRCAl function at the molecular level has not been achieved due to the lack of well-characterized antibodies and of sufficient purified protein to perform in vitro function assays. In this way, there are discrepancies in the literature regarding the size and cellular location of the BRCAl. Although two groups have found that BRCA1 is a 220 kDa nuclear protein (Chen et al., 1995; Scully et al., 1996), other investigators who used similar antibodies suggest that BRCA1 is a nuclear protein of 190 kDa (Gudas et al., 1995; Tensen et al., 1996).

Family Inheritance of Breast Cancer It is widely believed that BRCA1, located on chromosome 17q21, is responsible for approximately 50% of familial breast and ovarian cancers.

Based on the presence of a zinc finger type motif and an acid activation domain, it has been speculated that BRCA1 is a transcription factor (Miki et al., 1994). However, to date it has not been documented that it has any function of activating or repressing genes. BRCA1 may play a role in cell growth and differentiation, since its mRNA is widely expressed in developing embryos, and is especially high in tissues where cells proliferate and rapidly differentiate (Lañe et al., 1995; Marquis et al. cois., 1995). Despite the report of a case of a woman with two mutated alleles (Boyd ^ cois., 1995), the homozygous deletion of the BRCA1 gene in mice is deadly in early embryogenesis (Gowan et al., 1996: Liu et al. nineteen ninety six). The level of expression and phosphorylation by the cdk2 kinase is regulated during the cell cycle (Chen et al., 1996). In general, the data are compatible with the role of BRCAl in the regulation of cell proliferation and differentiation.

BRCAl tumor suppressor function It is paradoxical, although it is a tumor suppressor gene, BRCAl mutations are clearly related to hereditary breast and ovarian cancers, but they are almost never found in sporadic tumors (Miki et al., 1994; Futreal et al. 1994, Hosking et al., 1995). One suggestion is that, although genetically intact, BRCA may be inactivated functionally by misplacing it from the nuclear to the cytoplasmic compartments in sporadic breast cancer cells (Chen et al., 1995, 1996). However, the problem lies in nuclear transport, retention or cytoplasmic confinement, since the exogenous wild type BCRA1 protein labeled with epitope is also in the cytoplasm, in at least two lineages of breast cancer cells ( Chen et al., 1996).

Since BRCA1 has a molecular mass of approximately 220 kDa, it probably translocates actively from the cytoplasm to the nucleus through direct interactions with the receptor of nuclear localization signals or through indirect interactions with other NLS-containing proteins (Hicks and Rikhel, 1995; Dingwall and Laskey, 1991). The direct importation of caryophilic proteins through the nuclear pore complex requires energy (Newmeyer and Forbes, 1988; Richardson et al., 1988) and a nuclear localization sequence (NLS) located on the transport substrate (Dingwall et al. ., 1982; Kalderon et al., 1984) to which a cytosolic receptor complex, the importin-a and the importin-β, is attached (Górlich et al., 1994, 1995). A GTP-binding protein, the RAN, mediates the energy-dependent translocation of the substrate-receptor complex through the nuclear pore complex (Moore and Blobel, 1993). After the translocation, the importin-ß dissociates from the complex in the vicinity of the internal aspect of the nuclear cover, while the importin-a accompanies the substrate to the sites where it operates (Gorlich J. Cois, 1995). Contrary to the nuclear location reported by us (Chen et al., 1995; 1996) and others (Scully et al., 1996), there is a published report that indicates that BRCA1 is a secreted protein (Jensen et al., 1996). As the subcellular location of proteins is a fundamental aspect of their function, it is important to reinforce the data regarding the location of BRCA1 in normal and cancerous cells. More importantly, the subcellular compartment where BRCA1 is located is also a critical issue regarding the role it plays in breast tumorigenesis.

Deficiencies of Previous Inventions Accordingly, previous inventions lack novel methods and compositions to facilitate the diagnosis and treatment of breast cancer. Methods and compositions to determine the subcellular location of BRCA1 proteins in normal cells and in those suspected of being cancerous are not available. Methods and compositions that include specific proteins associated with BRCA1, to which the correct translocation of the BRCA1 gene product to the nucleus of the cell, are also necessary.

Summary of the Invention. The present invention allows to overcome one or more of these and other disadvantages inherent in previous inventions, by providing novel compositions and methods for the diagnosis and treatment of breast cancer. In one of its aspects, the invention offers a method for locating a BRCA1 protein or peptide in the cell. In general, the method consists of contacting the cell with a labeled antibody that specifically binds to BRCA1 or a protein or peptide associated with BRCA1, under conditions effective to allow the formation of immune complexes; and to determine the location of immune complexes in the cell. When these complexes are located in the cell cytoplasm, this is an indication of the existence of metastasis or primary cancer in the cell. This information is useful for the detection and early examination of cancers, and in particular, of breast and ovarian cancers, which the inventors have shown to be correlated with the subcellular protein cytoplasmic location of BRCAl. Preferably, the cells should be human, and in particular, cells of the breast or ovary. Yet another object of the invention is a method for identifying breast or ovarian cancer cells in a sample. In general, the method consists in obtaining an ovarian or breast tumor cell that is suspected to be cancerous, and in determining the subcellular location of a BRCA1 protein or peptide in the tumor cell. As stated above, the inventors demonstrated that the subcellular location of the BRCA1 protein or peptide in the cellular cytoplasm is indicative of the presence of cancer. A method for predicting the cancer susceptibility of an ovarian or breast cell is also described. In general, the method consists of identifying BRCA1 in the cell, or a BRCA I protein or peptide located in the cytoplasm, in which case the presence of the protein or peptide in the cytoplasm is an indication of the cell's susceptibility to cancer .

Compositions of Nucleic Acid. The invention provides nucleic acid sequences encoding a protein associated with BRCAl (BAP). As used herein, the BAP gene means a nucleic acid sequence encoding a protein associated with BRCA1. A preferential nucleic acid sequence encoding a BAP gene is the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1. It is expected that the nucleic acid sequence of the gene coding for BAP will vary, but that the sequence variation of the nucleic acid does not prevent hybridization between sequences encoding BAP in each sample under stringent hybridization conditions. As used herein, a variant BAP strain means any polypeptide encoded, in whole or in part, by a nucleic acid sequence that hybridizes under stringent hybridization conditions to give a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1. In the present invention, it is also understood that BAP means a polypeptide that reacts immunologically with the antibodies generated against the BAP protein of SEQ ID NO: 1. Likewise, BRCAl is understood to mean a polypeptide capable of generating antibodies immunologically reactive with BRCA1 and BRCA1-like gene products, and BAP is understood to mean a polypeptide capable of generating immunologically reactive antibodies to a BAP encoded by a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: l . As used in this specification, an active fragment of BAP includes BAPs that are modified by conventional techniques, such as for example by addition, deletion or substitution, and said active fragment has fundamentally the same structure and function as BAP as described herein, determining its antigenicity by conventional methods. With respect to BAP. the present invention relates to DNA segments, which can practically be isolated from any bacterial source, which are free of total genomic DNA and which encode proteins with activity similar to BAP. DNA segments encoding BAP-like species may also encode proteins, polypeptides, subunits, functional domains, and the like. As used herein, the term "DNA segment" refers to a DNA molecule that has been isolated and is free of total genomic DNA of a specific species. Accordingly, a DNA segment encoding BAP refers to a DNA segment that contains BAP coding sequences and yet is isolated from DNA or purified from the total genomic DNA of the species from which the DNA segment is obtained. Included within the term "DNA segment" are DNA segments and smaller fragments of those segments, in addition to recombinant vectors, including, for example, plasmids, cosmids, phagemids, bacteriophages, viruses and the like. Similarly, a DNA segment that includes an isolated or purified BAP gene, refers to a DNA segment that includes BAP coding sequences and, in certain aspects, regulatory sequences, substantially isolated from other protein coding sequences or genes that they occur naturally. In this regard, the term "gene" is used for simplicity to refer to a functional protein, a polypeptide or a peptide coding unit. As will be understood by those skilled in the art, this functional term includes both genomic and extragenomic sequences and sequences encoded in plasmids and small segments constructed of gene that express, or can be adapted to express, proteins, polypeptides or peptides. These segments can be isolated naturally, or synthetically modified by the hand of man. "Substantially isolated from other coding sequences" means that the gene in question, in this case a gene encoding BAP, makes up the majority of the coding region of the DNA segment, and that the DNA segment does not contain large portions of DNA natural encoder, such as large chromosomal fragments or other functional genes or polypeptide coding regions. Of course this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man. It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional amino acids with N or C termini, or 5 'or 3' sequences, and yet they will remain essentially as set forth in any of the sequences described. in this report, as long as the sequence conforms to the previously established criteria, including the preservation of biological protein activity, as regards protein expression. The addition of terminal sequences applies in particular to nucleic acid sequences which may include, for example, various non-coding sequences that surround 5 'or 3' portions of the coding region, or may include various regulatory or structural genes above or below Of course, the present invention also encompasses DNA segments complementary, or essentially complementary, to the set sequence SEQ ID NO: 1. The "complementary" nucleic acid sequences are those capable of base pairing according to the standard rules of complementarity of Watson and Crick. As used herein, the term "complementary sequences" means substantially complementary nucleic acid sequences, as could be evaluated by the same nucleotide comparison as set forth above, or which are defined as being capable of hybridizing to a nucleic acid segment encoding the amino acid sequence of SEQ ID NO: 1, under relatively stringent conditions, such as those described in this report. The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional enzymatic restriction sites, multiple cloning sites, other coding segments , and the like, so that their total length can be very variable. Therefore, it is envisaged to employ a nucleic acid fragment of almost any length, and preferably the total length will be limited by the ease of preparation and use in the recombinant DNA protocol that is used. For example, nucleic acid fragments can be prepared which include a short contiguous elongation identical or complementary to a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1, such as for example about 14 nucleotides with an approximate length of 10,000 or more. approximately 5,000 base pairs in length, preferably, in certain cases, with length segments of 3,000. It is also considered that DNA segments with total lengths of about 2,000, about 1,000, about 500, about 200, about 100, and about 50 base pairs in length (including all intermediate lengths) will be useful. It will be immediately understood that in this type of context the term "intermediate lengths" means any length between the figures mentioned, such as 14, 1 5, 16, 17, 18, 19, 20, etc.; 21, 22, 23, etc .; 30, 31, 32, etc .; 50, 51, 52, 53, etc .; 100, 101, 102, 103, etc .; 150, 151, 152, 1 53, etc .; including all integers in the ranges of 200 to 500; from 500 to 1,000; from 1,000 to 2,000; from 2,000 to 3,000; from 3,000 to 5,000; from 5,000 to 10,000, up to and including sequences of approximately 12,001, 12,002, 13,001, 13,002 and other similar. It will also be understood that this invention is not limited to the specific nucleic acid or amino acid sequences described herein. Therefore, recombinant vectors and isolated segments of DNA may variously include the BAP coding regions themselves, coding regions that exhibit select alterations or modifications in the basic coding region, or that encode larger polypeptides than however, they include BAP coding regions, or that encode functionally equivalent proteins or peptides having variant amino acid sequences. If desired, fused proteins and peptides can also be prepared, for example, in which the BAP coding regions are aligned within the same expression unit with other proteins or peptides having the desired functions, such as for example purification or immunodetection (for example, that can be purified by affinity chromatography and by enzymatic labeling of coding regions, respectively). Recombinant vectors constitute another additional aspect of the present invention. It is considered that those vectors in which the coding portion of the DNA segment, whether it encodes a full-length protein or a minor peptide, will be placed under the control of a promoter will be especially useful. The promoter can be in the form of a promoter naturally associated with a BAP gene, which can be obtained by isolating the 5 'non-coding sequences located above the coding segment, for example, using recombinant cloning, PCRGM technology or both, in relation with the compositions described herein. In other embodiments it is envisioned that certain advantages will be obtained by placing the DNA coding segment under the control of a recombinant or heterologous promoter. As used herein, the recombinant or heterologous promoter refers to a promoter that is not normally associated with the BAP gene in its natural environment. Such promoters may include BAP promoters normally associated with other genes, promoters isolated from any bacterial, viral, eukaryotic, or mammalian cell, or promoters of both types. Naturally, it is important to employ a promoter that efficiently directs the expression of the DNA segment in the cell type, organism or even animal, chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those skilled in molecular biology; for example, see Sambrook et al., 1989. The promoters employed may be constitutive or inducible, and are employed under the appropriate conditions to direct the high level expression of the introduced DNA segment, so that it is advantageous for production. large-scale protein or recombinant peptides. The prokaryotic expression of nucleic acid segments of the present invention can be carried out by methods known to those skilled in the art, and probably comprises expression vectors and promoter sequences such as those provided by tac, rp, lac. lacUVS or T7. To achieve the expression of BAP recombinant proteins, similar to BAP, BRCA1, or BRCA1-like in eukaryotic cells, various expression systems known to those skilled in the art are available. An exemplary eukaryotic promoter system whose use is envisioned in high level expression, is the Pichia expression vector system (Pharmacia LKB Biotechnology). With respect to the expression embodiments for preparing recombinant BAP, BRCAl, other related peptides, or all of them, it is expected to employ more frequently longer DNA segments, preferentially using DNA segments that encode all BAP or BRCAl domains or functional, epitopes, ligand binding domains, subunits , etc. However, it will be appreciated that the use of shorter DNA segments to direct the expression of BAP or BRCAl peptides or epitope internal regions, such as those used to generate anti-BAP or anti-BRCAl antibodies, also falls within the scope of the invention. present invention. It is believed that DNA segments encoding peptide antigens of about 15 to about 100 amino acids in length, or preferentially about 15 to about 50 amino acids in length, will be particularly useful. The BAP gene and the DNA segments can also be used in connection with somatic expression in an animal or to create a transgenic animal. Again, in such embodiments, provision is particularly made for the use of a recombinant vector which directs the expression of the entire length or of the active BAP protein. In particular, it is envisioned that the expression of a BAP transgene in animals will be useful in the production of anti-BAP antibodies for use in passive immunization methods and treatment of specific breast cancers.

Recombinant Expression of BAP and BRCAl As used herein, the term "engineered" or "recombinant" cell is used to refer to a cell into which a recombinant gene has been introduced, such as the gene encoding BAP or to BRCAl. Therefore, engineered cells can not be differentiated from wild-type cells that do not contain a gene introduced recombinantly. Consequently, the cells obtained by engineering are cells that have a gene or several introduced by the hand of man. Genes introduced in recombinant form are in the form of a single structural gene, a whole genomic clone comprising a structural gene and the accompanying DNA, or an operon or other functional segment of nucleic acid that may also include genes placed either above of the promoter, below, or in both sites, regulatory elements, or a structural gene itself, or even genes that are not naturally associated with the structural gene of specific interest. When the introduction of a recombinant version of one or more of the aforementioned genes is required, it is important to introduce the gene so that it is under the control of a promoter that effectively directs the expression of the gene in the cell type chosen to effect the engineering process. In general, it is convenient to use a promoter that allows the constitutive (constant) expression of the gene of interest. Commonly used constitutive eukaryotic promoters include viral promoters, such as the cytomegalovirus (CMV) promoter, the long terminal repeat sequence of Rous sarcoma (LTR), or the early SV40 genetic promoter. The use of these constitutive promoters will ensure a constant and high level of expression of the introduced genes. The inventors have observed that the level of expression from the introduced genes of interest varies in different genes, and in genes isolated from different strains or bacteria. Thus, the level of expression of a specific recombinant gene can be chosen by evaluating the different genes derived from each transfection experiment; Once the lineage has been chosen, the constitutive promoter ensures that the desired level of expression is maintained permanently. It is also possible to use specific promoters for the type of cell that undergoes the engineering process, such as the insulin promoter in the insulinoma cell lines, or the prolactin or growth hormone promoters in the cell lines of the anterior pituitary gland. The recombinant genetic fusion GST-BRCA1 ABg / Il described herein was deposited in the American Collection of Crop Types in E coli DH5 I MF 'according to the terms of the Budapest Treaty, and was assigned the following access number: AT CC 98100.

Immunodetection kits In other additional embodiments, the present invention includes methods of immunodetection and the associated kits. It is envisaged that the proteins or peptides of the invention are used to detect antibodies having reactivity with them, or alternatively, the antibodies prepared according to the present invention can be used to detect BAP or BRCAl peptides. The kits can also be used for purification of antigens or antibodies, as appropriate. In general, preferred immunodetection methods include first obtaining a sample suspected of containing an antibody reactive with BAP or BRCAl, such as a biological sample from a patient, and contacting said sample with a first BAP peptide or BRCAl under effective conditions to allow the formation of an immune complex (primary immune complex). Next, the presence of the primary immune complexes that are formed is detected.

To contact the chosen sample with the peptide of BAP or BRCAl under effective conditions to allow the formation of immune complexes (primary)In general, it is sufficient to simply add the protein or peptide composition to the sample. The sample is then incubated long enough to allow the aggregated antigens to form immune complexes, that is, to bind with the antibodies present in the sample. After this period, the composition of the sample, which can be a tissue cut, or an ELISA plate, a dot blot test. or western blot, it is generally washed to eliminate non-specific bound antigen species, allowing only species specifically linked within immune complexes to be detected. The detection of the formation of an immune complex is well known in this field and is achieved by applying various methods known to those skilled in the art. The detection of primary immune complexes is generally based on detecting a marker compound, which may be a radioactive, fluorescent, biological or enzymatic marker, with enzymatic detectors such as alkaline phosphatase, urease, horseradish peroxidase and glucose oxidase. The antigen that is used in particular can be linked to a detectable marker, and in that case simply said marker will be detected, which will allow to determine the amount of bound antigen present in the composition.

Another alternative is to detect the primary immune complexes by a second binding ligand that binds to a detectable label, which has binding affinity towards the first protein or peptide. The second binding ligand is usually an antibody, which is called the "secondary" antibody. The primary immune complexes are contacted with the labeled secondary binding ligand or antibody under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes. Next, the secondary immune complexes are generally washed to remove the labeled secondary antibodies non-specifically bound and to detect the remaining linked labels. For diagnostic purposes, it is proposed to use practically any sample suspected of containing the antibodies of interest. These samples may include clinical samples obtained from the patient, such as blood or serum samples, cerebrospinal fluid, synovitis. or bronchoalveolar, ear swabs, sputum samples, middle ear fluid, or even urine samples. In addition, it is envisaged that these embodiments are applicable to non-clinical samples, such as, for example, to titrate antibody samples, to select hybridomas, and other similar processes. Another alternative is to use clinical samples from veterinary sources that may include domestic animals, such as cattle, sheep and goats. It is also possible to use samples from feline, canine and equine sources, according to the methods described herein. In related embodiments, the present invention provides for the preparation of kits useful for detecting the presence of antibodies specific for BAP or BRCAl in a sample. Generally, kits according to the present invention will include a suitable protein or peptide, together with an immunodetection reagent, and a means for containing the protein or peptide and the reagent. The immunodetection reagent will generally consist of a label associated with a BAP or BRAC1 peptide. or associated with a secondary binding ligand. Some examples of ligands may be a secondary antibody directed against the first BAP or BRCAl peptide or antibody, or an avidin or biotin (or streptavidin) ligand having an associated label. Detectable labels linked with antibodies that have binding affinity to a human antibody are also considered, for example, in protocols where the first reagent is a BAP or BRCA1 peptide that is used to bind a reactive antibody from a human sample. . Of course, as mentioned above, various types of markers are known in the field, and all of them can be used in conjunction with the present invention. The kits may contain conjugates of antigen or antibody-marker, either in fully conjugated form, in the form of intermediates, or as separate entities that are to be conjugated by the user of the kit. The containers generally include at least one vial, test tube, flask, bottle, syringe, or some other type of container in which the antigen can be placed and assigned appropriately. When a second linker ligand is included, the kit will generally also contain a second vial or other container in which this ligand or antibody is placed. The kits of the present invention will also generally include a method for containing vials in closed confinement for commercial sale, such as, for example, blown or injection molded plastic containers into which the desired vials are placed. The BRCA1 6B4 hybridoma described herein as a producer of mAbs against BRCA1 (BRCA1BG1) was also deposited in the American Collection of Crop Types according to the indications of the Budapest Treaty and assigned the following accession number: ATCC HB-12146. The isotype of this antibody is IgGl, k. Other antibodies specific for BRCAl that are believed to be useful in the practice of the present invention include BRCA1N and BRCA1, and are described in detail in Section 5.

Formulation of the Vaccine and Compositions It is expected that to achieve an "immunologically effective formulation" it may be convenient to administer BRCA1 or BRCA1-associated protein to the human or animal subject in a pharmaceutically acceptable composition, including an immunologically effective amount of BRCA1 or protein. associated with BRCAl mixed with other excipients, vehicles, or diluents, which improve or in some way alter the stimulation of the responses of B cells, T cells, or cells of both types, or immunologically inert salts, acids and organic bases, carbohydrates and similar products, that promote the stability of this type of samples. Immunostimulatory excipients, also called coadjuvants, can include aluminum salts (often called Alumbres), simple or complex fatty acids, and sterol compounds, physiologically acceptable oils, polymeric carbohydrates, toxins from chemically or genetically modified proteins and various combinations of particles or of emulsions thereof. Along with these mixtures, the formula will include BRCAl, peptides derived from BRCAl, or one or more BAPs, or each variant in case more than one is present, in concentrations of about 0.0001 to 0.1 milligrams, or preferably from 0.001 to 0.1 milligrams, or better yet, less than 0.1 milligrams per dose. It is also envisaged to obtain, by engineering, attenuated organisms to express recombinant BRCAl gene products or a pro tein associated with BRCAl and these in themselves may be vehicles for administering the invention. Especially preferred are attenuated bacterial species, such as Mycobacterium, and in particular M. bovis, M. smegmatis, or BCG. Another alternative is to employ smallpox, polio, adenovirus or other viruses, and bacteria from species such as Salmonella or Shigella, together with the methods and compositions described herein. It has been shown that naked DNA technology, often called genetic immunization, is adequate as a protection against infectious organisms. This type of DNA segments can be used in various ways, including naked DNA and plasmid DNA, and can be administered to the subject in various ways including parenteral, mucous inoculations and the use of "genetic guns" of the microprojectile type. The use of nucleic acid compositions with the BRCA1 or BAP gene of the present invention, for immunization techniques of this type, is proposed as being useful for the formulation of antibodies directed against proteins of this type. Those skilled in the art will recognize that an optimal dosage schedule of a vaccination regimen may include up to five or six, but preferably three to five, or even better, one to three administrations of the immunizing entity supplied. at intervals of only two to four weeks, or longer, as of five to ten years and occasionally at even greater intervals.

Transformed Host Cells and Recombinant Vectors Specific aspects of the invention relate to the use of plasmid vectors for the cloning and expression of recombinant peptides and epitopes of specific peptides comprising either BRCA1 or BRCA1 epitopes associated with native proteins or with mutations at specific sites . The generation of recombinant vectors, the transformation of host cells, and the expression of recombinant proteins is well known to those skilled in the art. Prokaryotic hosts are preferred for the expression of the peptide compositions of the present invention. An example of a preferred prokaryotic host is E. coli, and in particular E. coli strains ATCC69791, BL21 (DE3), JM101, XL1-Blue®, RR1, LE392, B, P'776 (ATCC No. 31537) and W3110 (F, ", prototrophic, ATCC273325) Another alternative for the recombinant expression of the genetic constructs described herein is to employ species of Enterobacter i? Ceas such as Salmonella lyphimurium and Serratia marcescens, or even other gram-negative hosts including several species of Pseudomonas. In general, plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are used in connection with these hosts. The vector generally carries a replication site and also marker sequences that allow the selection of phenotypes in the transformed cells. For example, E. coli can be transformed in typical form using vectors such as pBR322, or any of its derivatives (Bolivar et al., 1977). PBR322 contains genes for resistance to ampicillin and tetracycline, and is thus an easy method to identify transformed cells. PBR322, its derivatives and other microbial or bacteriophage plasmids, may also contain, or be modified to contain, promoters that can be used by the microbial organism for the expression of endogenous proteins. In addition, phage vectors containing replicon and control sequences compatible with the host microorganism can be used as transformation vectors in connection with these hosts. For example, the bacteriophage GEMI M-1 1 can be used to make a recombinant vector that can be used to transform susceptible host cells such as E. coli LE392. The promoters that are most frequently used to construct recombinant DNA include the lactose-and-lactamase (penicillinase) promoter systems (Chang et al., 1978).; Itakura cois., 1977; Goeddel et al., 1979) or the tryptophan (trp) promoter system (Goeddel et al., 1980). The use of recombinant and native promoters is well known to those skilled in the art, and the details with respect to nucleotide sequences and specific methodologies are in the public domain, which will allow skillful workers to construct specific recombinant vectors and delivery systems. expression for the purpose of producing the compositions of the present invention.

In addition to the preferred embodiment expression in prokaryotes, eukaryotic microbes may also be employed as yeast cultures for the methods described herein. Saccharomyces cerevisiae, or common baker's yeast, is one of the most commonly used eukaryotic microorganisms, although other species can also be used in this type of eukaryotic expression systems. For example, for expression in Saccharomyces, plasmid YRp7 is often used (Stinchcomb et al., 1979, Kingsman cois., 1979, Tschemper et al. 1980). This plasmid already contains the trpL gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, such as ATCC no. 44076 or PEP4-1 (Jones, 1977). The presence of the trpL lesion as a characteristic of the genome of the yeast host cell then constitutes an efficient means to detect transformation by growth in the absence of tryptophan. Suitable promoter sequences in yeast vectors include promoters for 3-phosphoglycerate kinase (Hitzeman et al., 1980) and other glycolytic enzymes (Hess; / cois., 1968; Holland et al., 1978), such as enolase , glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase. pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase and glucokinase. By constructing plasmids suitable for expression, the termination sequences associated with these genes are also linked within the 3 'expression vector of the sequence that is desired to be expressed, to achieve polyadenylation of the mRNA and termination. Other promoters that have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, glyceraldehyde-3-phosphate dehydrogenase mentioned above and the enzymes that allow the use of maltose and galactose. Any plasmid vector containing a promoter compatible with yeast, an origin of replication, and termination sequences is suitable. In addition to microorganisms, cell cultures derived from multicellular organisms can also be used as hosts for the routine practice of the described methods. In principle, any cell culture of this type works, whether it comes from a vertebrate or invertebrate culture. However, there is increased interest in vertebrate cells, and the spread of vertebrate cells in cultures (tissue cultures) has become the routine procedure in recent years. Some examples of useful host cell lineages of this type are VERO and HeLa cells, Chinese hamster ovary cell lineages (CHO) and cell lines Wl 38, BHK, COS-7,293 and MDCK. Expression vectors for cells of this type generally include (if necessary) an origin of replication, a promoter located on the front of the gene to be expressed, together with the necessary binding sites on the ribosomes, the sites of RNA splicing, polyadenylation site and transcription termination sequences. The control functions of expression vectors for use in mammalian cells are often provided by viral material. For example, the promoters that are often employed are derived from polyoma, Adenovirus 2, and very often Simian virus 40 (SV40). The early and late promoters of the SV40 virus are very useful, because both can easily be obtained from the virus in the form of a fragment that also contains the SV40 viral origin of replication (Fiers et al., 1978). It is also possible to use smaller or larger SV40 fragments, as long as a sequence of approximately 250 base pairs is included that extends from the Hindlll site to the Bgll site located at the origin of viral replication. In addition, it is also possible and often desirable to use promoter or control sequences normally associated with the desired genetic sequence, as long as these control sequences are compatible with the host cell systems.

The origin of replication is obtained by either constructing the vector to include an exogenous origin, such as that which can be derived from SV40 or another viral source (such as Polyoma, Adenovirus, VSV, BVP) or can be provided by the mechanism of chromosomal replication of the host cell. When the vector is integrated into the chromosome of the host cell, the latter is often sufficient. It will be further understood that some of the polypeptides may be present in amounts below the limits of detection by the Coomassie brilliant blue staining procedure, which is usually employed in the analysis of SDS / PAGE gels, or that their presence may be masked by an inactive M polypeptide, similar. Although not necessary for the routine practice of the present invention, provision is made for the possibility of using other detection techniques in an advantageous manner to visualize the polypeptides of specific interest. Immunological techniques such as Westerm blot using enzymatically labeled antibodies, with radiolabels, or with fluorescent markers described herein, are considered to be of specific utility in this regard. Another alternative is to detect the peptides of the present invention using antibodies of the present invention combined with secondary antibodies with affinity for primary antibodies of this type. This secondary antibody can be labeled enzymatically, with radiolabel, or alternatively with fluorescent label or colloidal gold. The methods for labeling and detection that are employed in two-step secondary antibody techniques of this type are well known to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS The drawings are part of the present specification, and are included to more fully demonstrate certain aspects of the present invention. The invention will be better understood by reference to one or more of these drawings in combination with the detailed description of the specific embodiments presented herein. FIG. ÍA. Schematic drawing showing 3 spliced cDNAs that are used to construct full-length BRCAl cDNA. The three BRCAl cDNA regions that are used to label the GST fusion proteins are also delineated. FIG. IB. Identification of BRCAl as a 220-kDa protein in human cells. Whole cell extracts labeled with 35S methionine (1 X 10 7 cells / field) were subjected to immunoprecipitation with excess pre-immune serum (Field 1) or with anti-BRCAl polyclonal antibodies (Fields 2-7). Fields 2, 4 and 6 are single-step immunoprecipitates. Fields 3, 5 and 7 are double immunoprecipitation. Gray arrow: potential coprecipitated protein. FIG. 2A. Comparison of in vitro mobility of translational BRCAl with HBL100 cells. Fields I, 2: BRCAl of HBL100 cells (1 X 107 / field) precipitated with anti-BRCAl. In field 2, the extract was treated with CIP before immunoprecipitation. In fields 3, 4 and 5: BRCAl transferred in vitro (l / 20th of the total product) immunoprecipitated with each of the three antisera. FIG. 2B. Comparison of the mobility of recombinant BRCA1 derived from baculovirus with that of BRCAl of HBL100 cells. Fields 1, 2: HBL100 cells (0.5 X 106 / field). Field 3: SF9 cells not infected. Fields 4-7: infected SF9 cells. Fields 1 and 4: immunoprecipitated preimmune serum. Fields 2, 3 and 5: immunoprecipitated with anti-BRCAl. Field 6: immunoprecipitated with anti-BVRCAlBgl. Field 7: immunoprecipitated with anti-BRCAlN. Immunoprecipitates were detected by western blot and by probing with monoclonal anti-BRCAl MAb6B4. FIG. 3A. The expression of BRCA1 and phosphorylation is dependent on the cell cycle. Aliquots of synchronized T24 cell extracts were taken, separated by SDS-PAGE, subjected to western blotting and probed as follows: Upper Group: 1 x 10 7 cells / field probed to detect BRCAl using anti-BRCAl antiserum. Intermediate group: 1 x 106 cells / field probed to detect lORB using MAb 11D7. Lower group: 5 x 103 cells / field probed to detect p 84 with anti-N5-3. Field 1 (U) unsynchronized cells; Field 2 (Gl) 1 hour after the release; Field 3 (G l 1) 1 1 hours after release; Field 4 (G18) 1 8 hours after release; Field 5 (G24) 24 hours after the release; Field 6 (G33) 33 hours after the release; Field 7 (M) cells treated with nocodazole (0.4 g / ml for 8 hours). FIG. 3B. The expression of BRCA1 and phosphorylation depends on the cell cycle. Phosphorylation of BRCAl. T24 cells (2 x 102 / field) synchronized as described above, were pulsed with 300 Ci, 2P ortho-phosphate for 4 hours in phosphate-free medium and then harvested in a buffer for lysis and immunoprecipitated. with anti-BRCAl. Field 8: Immunoprecipitation with pre-immune serum. Fields 9-14: immunoprecipitation with anti-BRCAl. The upper and lower groups of this figure are different exposures of the same gel. FIG. 3C. The expression and phosphorylation of BRCAl depend on the cell cycle. FACS analysis of the cells synchronized at the points in time tested using the percentage of distribution of the cells in the various stages of the cell cycle. FIG. 3D, FIG. 3E, FIG. 3F, FIG. 3G, FIG. 3H, FIG. 31, FIG. 3J, FIG. 3K, F1G. 3L. FIG. 3M, FIG. 3N, FIG. 3O. The expression and phosphorylation of BRCAl is cycle dependent. Fluorescence staining for BRCAl during the cell cycle. FIG. 3D, F1G. 3F. FIG. 3H, FIG. 3J, FIG. 3L, FIG. 3N: DAP1 stain for DNA.

FIG. 3E, FIG. 3G, FIG. 31, FIG. 3K, FIG. 3M, FIG. 30: Indirect immunofluorescence staining for BRCAl using anti-BRCAl antibody as primary and FITC conjugated sheep-anti body anti-mouse antibody as secondary. FIG. 3D, FIG. 3E: 11 hours after release (Gl 1); FIG. 3F, FIG. 3G: 24 hours after release (G24); FIG. 3H, FIG. 31: 33 hours after the release (G33); FIG. 3J, FIG. 3K: metaphase; FIG. 3L, FIG. 3M: telophase; FIG. 3N, FIG. 30: cells reentering Gl. FIG. 4. Phosphorylation of BRCAl with various cyclin-dependent kinases. Extracts of HBL100 cells were precipitated with various anticycline / cyclin dependent kinase antibodies, as shown. The precipitates were incubated with kinase buffer in the presence of [? - > 2 PJATP and then washed and dissociated. The resulting supernatants were reprecipitated with antiBRCA1 separated by SDS-PAGE and the gels were dried and autoradiographed. FIG. 5A. Identification of the BRCAl. The diploid cells of human breast epithelium (HBL100, approximately 1 x IO7 cells per field) were incubated with ^ S-methionine (fields 1 to 6), [> 2P] phosphoric acid (fields 7 and 8). The proteins of the lysates were subsequently immunoprecipitated with a surplus of pre-immune mouse serum (fields 1. 4 and 8) or with mouse polyclonal anti-BRCAl (field 2), separated by electrophoresis in SDS-polyacrylamide gel and autoradiographed. The arrows indicate the proteins that co-immunoprecipitated with the anti-BRCAl serum. The immunoprecip proteins were dissociated from the anti-BRCAl and immunoprecipitated again with excess of the same antibody to visualize only the BRCAl (field 3). The same protein was immunoprecipitated with two different antibodies, antiBRCAl (field 5) and C20 (field 6). A protein species labeled with [32 P] phosphate, also immunoprecipitated with anti-BRCAl (field 7), but not with pre-immune serum (field 8). FIG. 5B. Detection of full-length BRCAl in normal mammary epithelial cells and breast cancer cell lineages. The established cell lineages were obtained from the American Collection of Tissue Culture Types. Malignant cells from plural effusion immediately after being obtained from the patients were washed with 50:50 Ham's F-12-Dulbecco modified Eagle's medium (DMEM) and frozen in liquid nitrogen without circulation, in the same medium, with 50% of calf fetal serum (FCS) and 10% dimethyl sulfoxide. Prior to fixation for immunostaining, the cells were washed and then plated for 12 hours in Ham's F-12-DMEM plus 10% FCS. Viable cells were placed on glass coverslips and fixed as established for cell lineages. The human breast cell lineages (5 x 106 cells per field) were labeled with [2 P] phosphoric acid. Field 1 was lysed of immunoprecipitated HBL100 with preimmune mouse serum. The cell lysates from fields 2 to 11 were immunoprecipitated with anti BRCA1: field 2, T47D; field 3, MCF7; field 4, MB468; field 5, MB 175-7; field 6, MB-361M; field 7, MB-231; field 8, MB-435S; field 9, MB41 5; field 10, HS578T; and field 1 1, HBLI OO. 5 μm thickness sections chosen randomly from breast cancer biopsies, paraffin-embedded and formalin-fixed from the inventors' tumor cell bank, to effect immunostaining by a modification of the avidin-biotin-peroxidase complex method of radish (ABC) (Hsu et al., 1981). Anti-BRCA1 was used at a 1: 100 dilution. The two cases of invasive breast cancer that did not show cytollasmic or nuclear immunostaining for BRCA1 did not give positive immunostaining with the MiB1 nuclear proliferation antigen either. FIG. 5C. Full-length BRCA1 is expressed in lineages of tumor cells derived from other tissues besides the breast. The lineages of human cells (approximately 2 x 1 06 per field) were metabolically labeled with '"' S-methionine.

A lysate was immunoprecipitated with pre-immune serum (field 1) and all others with anti-BRCAl (fields 2 to 12). Cell lines: fields 1 and 2, T24 (transitional cell carcinoma (TCC) of the bladder); field 3, 5637 (BCC of the bladder); field 4, DU 145 (prostate carcinoma); field 5, CAOV3 (ovarian carcinoma); field 6, RD (rhabdomyosarcoma); field 7, HCT1 16 (colon cancer); field 8, SW620 (colon cancer); field 9, C41 1 (cervical carcinoma); field 10, MS751 (cervical carcinoma); field 11, SAOS-2 (osteosarcoma); and field 12, U20S (osteosarcoma). FIG. 6A. Location of BRCAl in normal cells and breast cancer cells. Fractionation of cells 1-1 BL 100. Cells (1.5 x 107) were labeled with S-methionine; 5 x 106 cells were left unfractionated (total or T, field 1) and the rest were separated into the fractions: nuclear (N, field 2), cytoplasmic (C, field 3) and membrane (M, field 4) ( Chen et al., 1995; Abrams et al., 1982). To control the fractionation procedure, pl 10RD was used as a marker of the nuclear distribution and GST for the cytoplasmic distribution. Small aliquots were incubated with GST beads, separated by SDS-PAGE and stained with Coomassie Brilliant Blue to visualize the expected 26-kDa band of glutathione-S-transferase (GST). FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E, FIG. 6F, FIG. 6G, FIG. 6H, FIG. 61. Location of BRCAl in normal and cancerous cells of the breast. Detection of BRCAl in the nucleus of intact HBL100 cells by indirect immunofluorescence staining. (FIG 6B, F1G 6D, FIG 6F, FIG 6H) DAP1 stain to label the nuclei; (FIG 6C, F1G 6E, FIG 6G, FIG 61) immunofluorescence staining of the same cells. Indirect immunofluorescence procedures have been previously described (Durfee et al., 1994). In summary, the cells developed on slides were fixed with 4% formaldehyde and 0.1% Triton X-100® in phosphate-buffered saline (PBS) and permeabilized with 0.05% Saponin in water. The fixed cells were subsequently blocked with 10% normal goat serum plus 0.5%) NP-40R in PBS, incubated with primary anti-BRCAl polyclonal antiserum (dilution 1: 1000), washed and incubated with goat antibody, for mouse immunoglobulin G labeled with fluorescein. At the end of the incubation, with the secondary antibody, a drop of 4,6-diamidino-2-phenolindol propidium (DAPI) was added to the cells for 10 minutes to stain the DNA. Then, the cells were observed and photographed with a fluorescence microscope (FIG 6B and FIG 6C). Preimmune serum as primary antibody; (FIG 6D and FIG 6E) antiBRCAl as primary antibody; (FTG.6F and FIG.6G) antiBRCA1 preabsorbed with GST antigen; (FIG 6H and FIG 61) anti-BRCA1 preabsorbed with GST-BRCA1 fusion protein. FIG. 6J, FIG. 6K, FIG. 6L, FIG. 6M, FIG. 6N, FIG. 6O, FIG. 6P, FIG. 6Q. Location of BRCAl in normal and cancerous cells of the breast. Detection of BRCAl in nuclei of cell lineages derived from other tissues besides the breast (FIG 6.T, FIG 6L, FTG 6N, FIG 6P) DAP1 staining; (FIG 6K, F1G 6M, F1G 60, FIG 6Q) BRCAl stain. (FIG.6I and F1G.6K) DU145 cells (prostate cancer); (FIG 6L and FIG 6M) RAT2 fibroblasts; (FIG 6N and FIG 60) T24 cells (BCC of the bladder); (FIG 6P and FIG 6Q) CV1 cells (monkey kidney epithelium). FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E, FIG. 7F, FIG. 7G, FIG. 7H, F1G. 71, FIG. 7J, FIG. 7K, FIG. 7L, FIG. 7M, FIG. 7N, FIG. 7O, FIG. 7P. Location of BRCAl in normal and cancerous cells of the breast. Cytoplasmic location of BRCAl in breast cancer cells. FIG. 7A to FIG. 7H breast cancer lineage T47D; (FIG 7K and FIG 7L) MCF7 breast cancer lineage; (FIG 7M and FIG 7N) cells from the primary malignant effusion No. 22550; (FIG 70 and FIG 7P) cells from primary spill No. 23 159. (FIG 7a, F1G 7C, FIG 7E, FIG 7G, FIG 71, FIG 7K, FIG 7M, FIG. 70) DAP1 staining; (F1G.7B) Preimmune serum as primary antibody; (F1G. 7D) anti-BRCAl polyclonal primary antiserum; (FIG.7F) antiBRCA1 preabsorbed with GST; (FIG.7H) antiBRCA1 absorbed with GST-BRCA1 fusion protein; (FIG 71 to FIG 7P) primary antibody anti BRCAl absorbed with glutathione-S-transferase. The expansion is the same in the FJG. 7B to Fig. 7D. FIG. 8A. Cuts of primary breast cancer stained to detect BRCAl by the immunoperoxidase method. The 5 m thick sections of randomly selected, formalin-fixed and paraffin-embedded breast cancer biopsies, which were obtained from the tumor bank of the inventors, were immunostained by a modification of the avidin-biotin complex method -radish peroxidase (ABC) (Hsu et al., 1981). The anti-BRCA1 was used at a 1: 100 dilution. The two cases of invasive breast cancer that did not demonstrate cytoplasmic or nuclear immunological staining for BRCA1 did show positive immunological staining for the MiB1 nuclear proliferation antigen. BRCA1 was located in both the cytoplasm and the nucleus. FIG. 8B. Cuts of primary breast cancer stained to detect BRCAl by the immunoperoxidase method. The 5-m-thick sections of randomly selected, formalin-fixed, paraffin-embedded breast cancer biopsies obtained from the tumor bank of the inventors were immunostained by a modification of the avidin-biotin complex method. -radish peroxidase (ABC) (Hsu et al., 1981). Anti-BRCA1 was used at a dilution of 1: 100. The two cases of invasive breast cancer that did not show cytoplasmic or nuclear immunostaining for BRCA1 did show positive immunostaining for the nuclear proliferation antigen MiBl. The BRCAl was located only in the cytoplasm. FIG. 8C. Cuts of primary breast cancer stained to detect BRCAl by the immunoperoxidase method. The 5 μm-thick slices of randomly selected breast cancer biopsies, formalin jigs and paraffin-embedded samples obtained from the tumor bank of the inventors were immunostained by a modification of the avidin-biotin complex method. -radish peroxidase (ABC) (Hsu et al., 1981). Anti-BRCA1 was used at a dilution of 1: 100. The two cases of invasive breast cancer that did not show cytoplasmic or nuclear immunostaining for BRCA1 did show positive immunostaining for the nuclear proliferation antigen MiBl. No staining of BRCAl is observed. The dark, small, round signals in all sections are nuclei of stromal cells and lymphocytes. Original amplification, 400X. FIG. 9. Mutant constructs with NLS deletion are shown. In the scheme, the positions and sequences of three possible NLS motifs in the BRCA1 are indicated, together with the respective changes introduced in each one by PCR-based mutagenesis. The constructs shown were cloned, within the framework with the FLAG epitope, into the vector pCEP4, which directs the expression of high level cDNAs inserted under the control of the late CMV major promoter. FIG. 10. Identification of a / raws-activation domain in the BRCAl using the yeast system "of a hybrid". The various fragments of BRCAl shown are fused within the framework with the DNA GAL4 binding domain expressed in the yeast vector pAS. These constructs were subsequently transferred to yeast strain Y153, which harbors a GAL4 reporter gene that responds to b-galactosidase. The b-galactosidase activity was determined either qualitatively by smears of the transformants on the plates and by performing colony elevation assay or quantitatively by CPRG assay. These tests have been previously described by the inventors (Durfee et al., 1993). FIG. 11. Schematic showing the BRCAl regions that were used as bait in the detection of yeast hybrid closes. The putative Zn finger, the NLS motifs and the // * c v-activation domain of the BRCAl are illustrated in a schema for BRCAl cDNA. The positions of the two regions used as bait in the detection of the two yeast hybrids are shown below it. These regions were cloned into the frame with a Gal4 DNA binding domain of the yeast pAS expression vector. The numbers above the bars represent the positions of the amino acids within the sequence.

Detailed Description of the Preferred Embodiment of the Invention.

Therapeutic and Diagnostic Cases for BAP or BRCAl The therapeutic kits that include in a suitable container a BAP or BRCAl composition of the present invention in pharmaceutically acceptable formulation, constitute another aspect of the invention. The composition of BAP or BRCAl can be native BAP or BRCAl, BAP or BRCAl truncated, BAP or BRCAl with mutation at a specific site, or epitopes of peptides encoded with BAP or BRCAl, or also antibodies that bind with native BAP or BRCAl, BAP or BRCAl truncated, BAP or BRCAl with mutation in a specific site, or epitopes of peptides encoded with BAP or BRCAl. In other embodiments, the BAP or BRCA1 composition can be nucleic acid segments encoding BAP or BRCA1, BAP or BRCA1 truncated, BAP or BRCA1 with mutation at a specific site, or peptide epitopes encoded with BAP or BRCA1. These nucleic acid segments can be DNA or RNA, and can be segments of native, recombinant or mutagenized nucleic acid. The kits may include a single container containing the BAP or BRCAl composition. If desired, the container may contain a sterile pharmaceutically acceptable excipient having the BRCA1 or BAP composition associated with it and, optionally, a detectable label or an imaging agent. The formulation may be in the form of a gelatinous composition, such as, for example, a collagenous composition of BRCAl or BAP, or it may even be in a more liquid form which nevertheless results in a gel-like composition when administered to the body. In these cases, the container can be a syringe, a pipette or other similar device by which the composition of BRCA1 or BAP can be applied to a specific site. However, the single container may contain a dry or lyophilized mixture of the BRCAl or BAP composition, which requires or does not require wetting before use. Another alternative is that the kits of the invention include a different container for each component. In this case, one container will contain the composition of BAP or BRCAl, either as a sterile DNA solution or in lyophilized form, and the other container will contain the matrix that may or may not need pre-wetting with sterile solution, or having gelatinous, liquid form or injectable. The kits may also include a second or third container for containing a sterile and pharmaceutically acceptable diluent or solvent buffer solution. This solution may be necessary to formulate the BAP or BRCA component I in a form more suitable for application to the body, such as, for example, topical preparation or alternatively, oral, parenteral or intravenous preparation. However, it is necessary to observe that all the components of the kit can be provided in dry form (lyophilized) that will "wet" upon contact with body fluids. Thus, the presence of some type of pharmaceutically acceptable buffer or solvent is not always a requirement in the kits of the invention. Said cases may also contain a second or third container in which a pharmaceutically acceptable composition agent detectable by imaging is found. The container will generally be a container of the type of a vial, test tube, flask, vial, syringe or any other container in which the components of the case may be placed. The matrix and genetic components may also be included in the form of aliquots in smaller containers, if desired. The kits of the present invention may also include a method for containing the individual containers in closed confinement for commercial sale, such as, for example, blown or injection molded plastic containers in which the desired vials or syringes are placed.

Regardless of the number of containers, the kits and the invention may also include or be packaged with an instrument to help introduce the composition of the genetic matrix into the body of the animal. This instrument can be a syringe, pipette, forceps, or any suitable apparatus approved in medicine.

Affinity Chromatography Affinity chromatography is generally based on recognizing a protein by a substance that can be a ligand or an antibody. The material of the column can be synthesized by covalent bonding between a binding molecule, such as, for example, an activated dye, with an insoluble matrix. Then, the column material is allowed to adsorb the desired substance from the solution. Next, the conditions are changed to others that do not allow the link to occur, and the substrate is eluted. The requirements for successful affinity chromatography are: 1) that the matrix specifically adsorbs the molecules of interest; 2) that the other pollutants do not adsorb; 3) that the ligand mates without altering its binding activity; 4) that the ligand binds with sufficient strength to the matrix; and 5) that it is possible to elute the molecules of interest without destroying them.

One of the preferred embodiments of the present invention is the affinity chromatography method for purifying the antibodies from the solution, in which the matrix contains BAP or BRCAl, or another alternative is to employ epitopes of peptides derived from BAP or BRCAl, covalently linked to a suitable matrix, such as, for example, Sepharose CL6B or CL4B. This matrix binds the antibodies of the present invention directly and allows them to be separated by elution with a suitable gradient, such as salt, GuHCl, pH or urea. Another preferred embodiment of the present invention is an affinity chromatography method for the purification of BAP, BRCAl or epitopes of related peptides from the solution. The matrix is linked to the amino acid compositions of the present invention directly, and allows them to be separated by elution with a suitable buffer solution, as described above.

Methods for Nucleic Acid Delivery and DNA Transfection In certain embodiments it is envisioned that the nucleic acid segments described herein are employed to effect transfection of suitable host cells. The technology for introducing DNA into cells is well known to those skilled in the art. Four general methods for providing a nucleic segment to cells have been described: (1) chemical methods (Graham and Van der Eb, 1973); (2) physical methods such as microinjection (Capecchi, 1980), eletroporation (Wong and Neumann, 1982; Fromm et al., 1985) and the gun gene (Yang et al. 1990); (3) viral vectors (Clapp, 1993; Eglitis and Anderson, 1988); and (4) mechanism mediated by receiver (Curiel et al., 1991; Wagner et al., 1992).

Liposomes and Nanocapsules In certain embodiments, the inventors envisage the use of liposomes, nanocapsules, or both, for the introduction of specific peptides or nucleic acid segments to the host cells. These formulations may be preferred for the introduction of pharmaceutically acceptable formulations of the nucleic acids, peptides, antibodies or all of them described herein. The formation and use of liposomes are generally known to those skilled in the art (see for example, Couvreur et al., 1977. which describes the use of liposomes and nanocapsules in antibiotic-directed therapy for bacterial infections and diseases) . Recently, liposomes with better serum stability and circulating half-life were developed (Gabizon and Papahadjopoulos, 1988, Alien and Choun, 1987). Nanocapsules generally trap compounds in a stable and reproducible manner (Henry-Michelland et al., 1987). To avoid side effects due to intracellular polymer overload, ultrafine particles of this type (approximately 0.1 μm in size) should be designed using polymers that can degrade in vivo. In the present invention, the use of biodegradable polyalkyl cyanoacrylate nanoparticles that meet these requirements is contemplated, and these particles are easily manufactured as described previously (Couvreur et al., 1977).; 1988). Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar bilayer vesicles of concentric multilamellar bilayers (also called multilamellar vesicles (MLVs).) In general, MLVs have diameters of approximately 25 mm to μm. The sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters ranging from 200 to 500 A, which contain aqueous solution in the internal part.In addition to the teachings of Couvreur et al. (1988), the The following information can be used to generate liposomal formulations: Phospholipids form various structures other than liposomes when dispersed in water, depending on the molar ratio between lipid and water.In low ratios, the liposome is the preferred structure. Physical characteristics of liposomes depend on pH, ionic strength and the presence of cati Divalent ones Liposomes have low permeability to ionic and polar substances, but at high temperatures they undergo a phase transition that significantly alters their permeability. The phase transition consists of a change from the ordered structure with close packing known as the gel state, to a loose and less ordered packing structure, called the liquid state. This occurs at a characteristic phase transition temperature and results in an increase in permeability to ions, sugars and drugs. Liposomes interact with cells by four distinct mechanisms: endocytosis by phagocytic cells of the reticuloendothelial system, such as macrophages and neutrophils; adsorption to the cell surface, either by weak and non-specific hydrophobic or electrostatic forces, or by specific interactions with cell surface components; fusion with the cellular plasma membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of the liposomal content into the cytoplasm; and by transferring the liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome content. It is often difficult to determine what mechanism is produced, and sometimes more than one may occur simultaneously.

Methods to prepare BAP. BRCAl, AND ABS ANTI-BAP or ANTI-BRCA1 In another aspect, the present invention relates to an antibody that exhibits immunoreactivity to a polypeptide of the invention. As stated above, one of the applications of BRCA1 and of epitope peptides derived from BRCA1 or BAP or epitope peptides derived from BAP according to the present invention is the generation of antibodies. The reference to antibodies throughout the specification includes polyclonal and monoclonal antibodies (mAbs), and parts thereof, either alone or conjugated with other entities. Parts of antibodies include Fab and F (ab) 2 fragments and single chain antibodies. The antibodies can be manufactured in vivo in suitable laboratory animals, or in vitro using recombinant DNA techniques. In a preferred embodiment, the antibody is a polyclonal antibody.

Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogen that includes a polypeptide of the present invention, and collecting antisera from the immunized animal. Various animal species can be used for the production of antisera. In general, the animals that are used to produce antisera are rabbits, mice, rats, hamsters or guinea pigs. Due to the relatively abundant blood volume of rabbits, they are chosen more frequently for the production of polyclonal antibodies. Antibodies, both polyclonal and monoclonal, specific for BRCA1 and epitopes derived from BRCA1, or alternatively BAC and epitopes derived from BAP, are prepared by conventional immunization techniques, as those skilled in the art know. The composition containing antigenic epitopes of BRCA1 s and specific BAPs described herein can be used to immunize one or more experimental animals, such as a rabbit or a mouse, which will then proceed to produce specific antibodies against the BAP peptides or BRCAl. Polyclonal antisera are obtained after allowing sufficient time for the generation of antibodies, simply by bleeding the animal and preparing serum samples of the whole blood. ^ fc The amount of immunogen composition used to produce antibodies Monoclonal antibodies vary according to the nature of the immunogen, and also according to the animal that is used for the immunization. The immunogen is administered by various routes (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies is monitored by sampling the blood of the immunized animal at various times after immunization. A second injection of reinforcement. The process of administration of reinforcements and determination of the title is repeated until an adequate degree is obtained. When the desired level of immunogenicity is achieved, the immunized animal is bled to isolate the serum and store it, and the animal can be used to generate mAbs (see below), or both. One of the important factors described in the present invention is a polyclonal serum relatively homogeneous with respect to the specificity of the antibodies it contains. In general, polyclonal antisera are derived from a variety of different "ions", for example, B cells of different lineage. In contrast, mAbs are defined as coming from antibody producing cells and have as a common ancestor a B cell, and therefore are "mono" clonal. 20 By employing peptides as antigens to produce polyclonal sera, considerably less variation is expected in the clonal nature of the sera than in the use of a whole antigen. Unfortunately, when incomplete fragments of an epitope are present, the peptide often assumes multiple (and probably non-native) configurations. As a result, even short peptides produce polyclonal antisera with relatively plural specificities and, unfortunately, an antiserum that does not react or reacts little with the native molecule. Polyclonal antisera according to the present invention are produced against peptides that are predicted to include intact and whole epitopes. Therefore, it is believed that these epitopes are more stable in the immunological sense and, consequently, express a more constant immunological target for the immune system. According to this model, the number of potential B cell samples that will respond to this peptide is considerably lower and, therefore, the homogeneity of the resulting sera will be higher. In various embodiments, the present invention allows to obtain polyclonal antisera, in which the clonality, that is, the percentage of clone that reacts with the same molecular determinant, is at least 80% >; Even higher clonalities are predicted, up to 90 or 95%, or even higher. To obtain mAbs, an experimental animal, often a mouse, is initially immunized with a composition containing BRCAl. Then, after sufficient time to allow the generation of antibodies, a population of cells of the spleen or lymphatic cells of the animal is obtained. Spleen or lymphatic cells fuse with cell lineages, such as human or mouse myeloma strains, to produce hybridomas that secrete antibodies. These hybridomas are isolated to obtain individual measurements that are then subjected to detection of antibody production against the desired peptide. Following immunization, spleen cells are removed and fused according to a standard fusion protocol with plasmacytoma cells to produce hybridomas that secrete mAbs against BAP or BRCAl. Hybridomas that produce mAbs against the selected antigens are identified by standard techniques, such as ELISA and Western blot methods. A hybridoma culture is performed in liquid media and the culture supernatants are purified to obtain specific mAbs for BAP or BRCAl. It is proposed that the mAbs of the present invention will also find useful application in immunochemical methods such as ELISA and Western blot methods, and also in other methods that use antibodies specific for BAPs or BRCAl as methods of immunoprecipitation, immunocytological methods, etc. In particular, BAP or BRCAl antibodies can be used in immunoabsorbent protocols to purify native or recombinant BAPs, like BRCAl, or peptide species derived from BAP or BRCA 1, or natural or synthetic variants thereof. The antibodies described herein can be used in antibody cloning protocols to obtain cDNAs or genes encoding BAPs or BRCAls from other species or organisms, or to identify proteins that have a high homology with BAP or BRCAl. They can also be used in inhibition studies to analyze the effects of BAP or BRCAl on cells, tissues, or on the entire animal. Anti-BRCA I and anti-BAP antibodies will also be useful in immunolocalization studies to analyze the distribution of BRCA1 or BAP proteins in different physiological conditions. An especially useful application of antibodies of this type is the purification of native or recombinant BAPs or BRCAl, for example, using an antibody affinity column. The way to implement all these immunological techniques, taking into account the present description, is known to the persons skilled in the art.

Recombinant Expression of BAP or BRCAl Recombinant expressions expressing the BAP or BRCAl nucleic acid segments can be used to prepare purified recombinant BRCAl (rBRCAl), peptide antigens derived from purified rBRCAl, or alternatively, purified recombinant BAP (rBAP), peptide antigens derived from purified rBAP, and also mutant recombinant protein species or variants in large amounts. It is proposed that the antigens chosen and the variants thereof are very useful for the diagnosis and treatment of breast cancer. For example, it is proposed to use the rBAPs, the rBRCAls, the peptide variants thereof, the antibodies against these rBAPs or rBRCAl s, or all of them, in immunological assays to detect the location of BAP or BRCAl in vivo, or as vaccines or immunotherapy for the treatment of breast cancers. Additionally, by applying techniques such as DNA mutagenesis, the present invention allows the easy preparation of so-called "second generation" molecules which have modified or simplified protein structures. Second-generation proteins generally share one or more properties in common with the full-length antigen, such as, for example, an antigenic / immunogenic specific epitope internal sequence. Epitope sequences can be obtained in relatively short molecules prepared by knowledge of the peptide, or coding sequential DNA information. Variant molecules of this type are not only derived from selected immunogenic / antigenic regions of the protein structure, but additionally or alternatively, may include one or more functionally equivalent amino acids chosen based on similarities or even differences with respect to the natural sequence.

Antibody Compositions and Formulations Thereof The methods for preparing and characterizing antibodies are well known to those skilled in the art (see, for example, Harlow and Lane, (1988), incorporated herein by reference). The methods for generating mABs usually start in the same way as those for preparing polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immunizing the animal with an immunogenic composition according to the present invention and collecting the antiserum from the immunized animal. Very different species can be used for the production of antisera. In general, the animals that are used to produce anti-antiserum are rabbits, mice, rats, hamsters, guinea pigs or goats. Since rabbits have a relatively abundant blood volume, these animals are preferred for the production of polyclonal antibodies.

As is well known in the art, a given composition may have variable immunogenicity. Therefore, it is often necessary to boost the host's immune system, which can be achieved by coupling a peptide or polypeptide immunogen to a carrier. Some examples of preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins can also be used as carriers, such as ovalbumin, mouse serum albumin, or rabbit serum albumin. Methods for conjugating a polypeptide with a transport protein are well known in the art and include glutaraldehyde, ester / 77-maleimidobenzoyl-? Idroxysuccinimide, carbodiimide and bis-diazotized benzidine. The mAbs are readily prepared by well-known techniques such as those included in US Pat. No. 4,196,265, incorporated herein by reference. In general, this technique consists of immunizing a suitable animal with a selected immunogenic composition, such as, for example, a protein, polypeptide, or peptide purified in whole or in part. The immunizing composition is administered efficiently to stimulate the antibody producing cells. Preferably rodents are used as mice and rats; however, it is also possible to use rabbits, sheep or frog cells. The use of rats provides certain advantages (Goding, 1986), although the use of mice is preferred, and BALB / c mice are the most used because they generally produce a higher percentage of stable fusions. After fusion, somatic cells with potential for antibody production, specifically B lymphocytes (B cells), are selected for use in the mAb generation protocol. These cells are obtained by biopsy of the spleen, tonsils or lymph nodes, or a peripheral blood sample. The use of spleen cells and peripheral blood is preferred, since the former are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because the peripheral blood is easily obtained. A group of animals is often immunized and the spleen of the animal with the highest antibody concentration is removed to obtain lymphocytes from the spleen by homogenizing the spleen with a syringe. Characteristically, the spleen of an immunized mouse contains approximately 5 X 10 7 to 2 X 10 8 lymphocytes. The B lymphocytes producing antibodies from the immunized animal are then fused with cells derived from an immortal myeloma cell, which is generally of the same species as the immunized animal. Myeloma cell lineages suitable for use in fusion procedures for hybridoma production, preferably should not be antibody producers, because they have a high fusion efficiency and have enzymatic deficiencies that prevent them from developing in certain selective media that only allow the development of fused cells (hybridomas). It is possible to employ various myeloma cells, as known to those skilled in the art (Goding, 1986; Campbell, 1984). For example, if the immunized animal is a mouse, P3-X63 / Ag8, X63-Ag8.653, NSI-l .Ag 4 1, Sp210-Agl4, FO, NSO / U, MPC-11-, MPC1 can be used. 1 -X45-GTG 1 .7 and S194-5XX0 Bul; for rats, R210.RCY3, Y3-Ag 1.2.3, I4983F and 4B210 can be used; and U-266, GM-1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful with respect to human cell fusions. A preferred murine myeloma cell is derived from the NS-1 myeloma cell lineage (also called P3-NS-l -Ag4-l), which is easily obtained from the NIGMS Human Genetic Mutation Cell Repository by requesting the cell line with number of GM3573 deposit. Another lineage of mouse myeloma cells that can be used is mouse murine myeloma resistant to 8-azaguanine and not producing SO2 / 0. Methods for generating hybrids of lymph node or spleen-producing cells of antibodies and myeloma cells generally involve mixing somatic cells with myeloma cells in a ratio of 2: 1, although this ratio can vary from about 20: 1 to about 1: 1, respectively, in the presence of one or more substances (chemical or electrical) that favor the fusion of cell membranes. Fusion methods have been described using Sendai virus (Kohler and Milstein, 1974; 1976), and others using polyethylene glycol (PEG), for example with 37% (V / V) PEG, by Gefter et al. (1977). It is also appropriate to employ methods of electrically induced fusion (Goding, 1986). Fusion procedures usually produce viable hybrids at low frequencies, from about 1 X 10"6 to about 1 X 10" 8. However, this is not a problem, since the viable fused hybrids differ from the original non-fused cells (particularly unfused myeloma cells which would normally continue to divide indefinitely) by culture in a selective medium. Said selective medium is usually one that contains some substance that blocks the de novo synthesis of nucleotides in the tissue of the culture medium. Some examples of preferred substances are aminopterin, methotrexate and azaserin. Aminopterin and methotrexate block the de novo synthesis of pinins and pyrimidines, while azaserin blocks only the synthesis of purines. When aminopterin or methotrexate is used, the medium is supplemented with hypoxanthine and thymidine with a nucleotide medium (HAT medium). When azaserin is used, the medium is supplemented with hypoxanthine. The preferred means of selection is HAT. Only cells capable of functioning in nucleotide rescue routes manage to survive in the HAT medium. The myeloma cells have a defect in the key enzymes of the salvage route, that is, the hypoxanthine phosphoribosyl transferase (HPRT), so they do not survive. B cells can function in this way but they have a limited life span in the culture and generally die within approximately two weeks. Therefore, the only cells that survive in the selective media are hybrids formed from B cells and myeloma. This type of culture provides a population of hybridomas from which specific hybridomas are selected. In general, the selection of hybridomas is carried out by culturing the cells by dilution of single clone in microtiter plates, followed by testing the individual clonal supernatants (after a lapse of two to three weeks) to determine if they have the desired reactivity. The assay should be sensitive, simple and rapid, such as, for example, radioimmunoassay, immunoenzymatic assay, cytotoxicity assay, plaque assay, dot immunoblot assay and the like. The selected hybridomas are serially diluted and cloned with lineages of individual-type antibody-producing cells; Such statements can subsequently be propagated indefinitely to produce mAbs. Cell lineages are exploited for mAb production in two fundamental ways. A sample of the hybridoma (often peritoneal cavity) is injected from a histocompatible animal of the type used to obtain the somatic and myeloma cells for the original fusion. The injected animal develops tumors that secrete the specific mAb produced by the hybrid of the fused cell. Animal body fluids, such as serum or ascites fluid, can be used to obtain high concentration mAbs. Individual cell lineages can also be cultured in vitro, in which case mAbs are naturally secreted into the culture medium, where they are easily obtained in high concentrations. The mAbs produced by any of the mentioned methods are further purified, if required, by filtration, centrifugation and by various chromatographic methods such as HPLC or affinity chromatography.

Immunological Assays As mentioned, it is proposed that the native peptides and derivatives in synthetic form and the epitopes of the peptides of the invention be useful as immunogens, as for example for the development of vaccines, and as antigens in immunological assays for the detection of reactive antibodies. Taking into account the immunological assays first, in its simplest and most direct sense, the preferred immunological assays of the invention include the various types of the enzyme-linked immunosorbent assay (ELISA), known to those skilled in the art. However, it will be readily appreciated that the utility of proteins and peptides derived from BRCA1 is not limited to this type of assays and that other useful embodiments include RIAs and other assays and methods not linked to enzymatic methods. In preferred ELISA assays, proteins or peptides that incorporate BAP, rBAP. BRCAl, rBRCAl, or protein antigenic sequences derived from BAP or BRCAl are immobilized on a given surface, in particular a surface with protein affinity, such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, the well is generally bound or coated with a non-specific protein known to be antigenically neutral to test antisera, such as bovine serum albumin (BSA). ) or casein. This allows the non-specific adsorption sites to be blocked on the immobilizing surface and therefore reduces the background caused by the antiserum being non-specifically bound on the surface. After the binding of the antigenic material to the well, the coating with a non-reactive material to reduce the background, and the washing to remove the unbound material, the immobilizing surface is brought into contact with the antiserum or the clinical or biological extract that is going to test in order to achieve the formation of the immune complex (antigen / antibody). These conditions, preferably, include diluting the antiserum with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS) / Tween®. These added substances also tend to help reduce the non-specific fund. The layered serum is allowed to incubate, for example, 2 to 4 hours at a temperature of preferably 25 ° to about 27 ° C. After incubation, the surface that was in contact with the antiserum is washed to remove the material that did not form the immunocomplex. A common washing procedure is to use PBS / Tween® or a borate buffer solution as a wash solution. After the formation of specific immunocomplexes between the test sample and the bound antigen, and subsequent washing, it is determined what amount of immune complex was formed by subjecting the complex to a second antibody having specificity towards the former. Of course, since the test sample is generally of human origin, the second antibody used will preferably be one that has specificity for human antibodies. To have a detection method, the second antibody preferably will have an associated detectable label, such as an enzyme label which will generate a signal, such as color formation, by incubating it with a suitable chromogenic substrate. Thus, for example, it can be contacted with the surface bound to the antiserum and incubated with anti-human IgG conjugated with peroxidase or urease for a period of time and under conditions that favor the formation of the immunocomplex (e.g. hours at room temperature in a solution containing PBS, such as PBS-Tween®). After incubation with the enzyme-labeled second antibody and subsequent washing to remove the unbound material, the amount of the label is quantified by incubation with a chromogenic substrate such as urea and bromocresol purple or acid 2., 2'-azino-di (3-ethyl-benzthiazolin) -6-sulphonic acid (ABTS) and H202, in the case that peroxidase has been used as an enzymatic marker. Quantification is carried out by measuring the degree of color generation, for example, using a visible spectrum spectrophotometer. ELISA tests can be used in conjunction with the invention. In an ELISA of this type, the proteins or peptides incorporating the antigenic sequences of the present invention are immobilized on a selected surface, preferably a surface having protein affinity, such as the wells of a polystyrene microtiter plate. After washing to remove the incompletely adsorbed material, it is convenient to bind or coat the wells of the assay plate with a non-specific protein known to be antigenically neutral with respect to test antisera, such as bovine serum albumin (BSA). ), casein, or a milk powder solution. This allows to block non-specific adsorption sites on the immobilization surface and therefore reduces the background caused by the non-specific binding of antisera to the surface.

Immunoprecipitation The anti-BRCA1 and anti-BAP antibodies of the present invention are particularly useful for isolating BRCA1 and BAP antigens by immunoprecipitation. Immunoprecipitation consists of separating the component of the target antigen from a complex sample, and is used to discriminate or isolate very small amounts of protein. In an alternate embodiment, the antibodies of the present invention are useful for the close juxtaposition of two antigens. This is particularly useful for increasing the localized concentration of antigens, i.e., enzyme-substrate pairs.

Western Blot Analysis The composition of the present invention will be very useful in immunoblot or Western blot analyzes. Anti-BRCAl and anti-BAP antibodies can be used as high affinity primary reagents to identify immobilized proteins on a solid support matrix, such as nitrocellulose, nylon, or combinations thereof. Together with the immunoprecipitation followed by gel electrophoresis, these can be used as single pass reagents to detect antigens against which the secondary reagents used in the detection of the antigen cause an adverse background. This is particularly useful when the antigens studied are immunoglobulins (which prevents the use of immunoglobulins that bind to the components of the bacterial cell wall), when the antigens studied cross-react with the detection substance or migrate to a molecular weight relative as a cross reaction signal. Immunological detection methods together with Western blot techniques (including secondary antibodies labeled enzymatically, radioactively or fluorescently against the toxin part) are considered particularly useful in this respect.

Vaccines The present invention provides for the preparation of vaccines for use in active and passive immunization embodiments. The proposed immunogenic compositions that can be used as a vaccine are easily prepared directly from the novel immunogenic proteins, the peptide epitopes described herein, or both. Preferably the antigenic material is dialysed extensively to remove undesirable low molecular weight molecules, lyophilized, or both, to have a more immediate formulation in a desirable vehicle. The preparation of vaccines containing peptide sequences as active ingredients is generally known to those skilled in the art, as set forth in U.S. Patents 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all incorporated herein by reference. In general these vaccines are prepared as injectables, either in solution or as liquid suspensions, in solid form to be dissolved or suspended in liquid before injection. The preparation can also be emulsified. The active immunogenic ingredient is often mixed with pharmaceutically acceptable excipients and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or similar products or combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH-buffering agents, or adjuvants that increase the efficacy of vaccines.

A composition that includes BAP, BRCA1 and proteins derived from BRCA1, native or modified epitope peptides, or all of them, could be the basis of vaccines for humans. The preparation of compositions of this type essentially free of endotoxins is achieved following the published methodology; for example, U.S. Patent 4,271,147 (incorporated herein by reference), describes methods for the preparation of Neisseria meningitidis membrane proteins for use in vaccines. BAP-based vaccines, BRCA1, BRCA1-derived epitope and BAP-derived epitope, can be administered conventionally parenterally, by injection, and, for example, subcutaneously or intramuscularly. Additional formulations suitable for other types of administration include suppositories and, in certain cases, oral formulations. In the case of suppositories, traditional binders and carriers can include, for example, polyalkylene glycols or triglycerides: these suppositories can be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, and preferably from 1 to 2%. Oral formulations include excipients of normal use, such as, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, all of pharmaceutical grade, and similar compounds. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulas or powders containing from 10 to 95% of the active ingredient, and preferably from 25 to 70%. The proteins can be formulated in the vaccine in neutral form or as salts. Pharmaceutically acceptable salts include salts obtained by the addition of acids (formed with the free amine groups of the peptide) and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids of the acetic, oxalic, tartaric type, mandélico and others similar. The salts formed with the free carboxylic groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ctylamine ethanol, histidine, procaine and the like. The vaccines can be administered in a manner compatible with the formulation of the dose and in such amount as to be therapeutically effective and immunogenic. The amount that is administered will depend on the subject receiving the treatment, including for example, the ability of the individual's immune system to synthesize antibodies and the degree of protection desired. The precise amounts of active ingredient that need to be administered can easily be determined by trained doctors. However, suitable dose ranges are in the order of several hundred micrograms of active ingredient per vaccine. Suitable regimens for initial administration and booster injections are also variable, but generally consist of the initial administration followed by subsequent inoculations or other administrations. The type of application is very variable. Any of the conventional methods for administration of vaccines is applicable. These are considered to include oral application on a physiologically acceptable solid base, or parenteral use by injection or similar method of a physiologically acceptable dispersion. The dose of the vaccine will depend on the route of administration and will vary according to the size of the host. The various methods to achieve the adjuvant effect of the vaccine include the use of substances such as aluminum hydroxide or aluminum phosphate (alum), commonly used from 0.05 to 0.1% in phosphate buffered saline, a mixture with synthetic polymers of sugars ( Carbopol ®) used as a 0.25% solution), protein aggregation in the vaccine by heat treatment at temperatures from about 70 to about 101 ° C for periods from 30 seconds to 2 minutes, respectively. Aggregation by reactivation with F (ab) antibodies to albumin treated with pepsin, mixing with bacterial cells such as C. parvum or endotoxins or lipopolysaccharide components of gram-negative bacteria, emulsion in physiologically acceptable oily vehicles as inanimate monooleate (Aracel-A ™) or the emulsion with 20 percent perfluorocarbon solution (Fluosol-DA IM), used as a block substitute, it can also be used. In many cases it will be desirable to employ multiple administrations of the vaccine, generally not exceeding six immunizations and, more frequently, not exceeding four immunizations, and preferably one or more, and usually at least three. immunizations. Immunizations will normally be carried out at intervals of two to twelve weeks and more usually at intervals of three to five weeks. The periodic reinforcements at intervals of 1 to 5 years, usually every 3 years, are advisable to maintain the protective levels of the antibodies. The course of immunization can be followed by antibody tests against the antigens of the supernatant. These assays can be carried out by labeling with conventional markers, such as radionuclides, enzymes, fluorescent substances and the like. These techniques are well known and are described in various patents, such as, for example, U.S. Patent Nos. 3,791, 932; 4,174,384 and 3,949,064, which constitute an illustration of this type of tests. Of course, taking into account the new technology of DNA vaccines, it will be understood that practically all vaccination regimens of this type will be suitable for use with DNA vectors and constructions, as described by Ulmer et al. (1993), Tang et al. .. (1992), Cox et al .. (1993), Fynan et al .. (1993), Wang et al., (1993a, 1993b) and Whitton et al .. (1993), each of which incorporated in this memory as a reference. In addition to the parenteral routes for DNA inoculation, including intramuscular and intravenous injections, mucosal vaccination is also envisaged, which is verified by administering drops of the DNA composition to the nasal cavities or the trachea. It is envisaged in particular the use of a genetic pistol to provide a quantity of DNA to the epidermis, which allows an effective immunization (Fynan et al., 1993).

Pharmaceutical Compositions The pharmaceutical compositions described herein can be administered orally, for example, using an inert diluent or an edible assimilable carrier, or included in a soft or hard type gelatin capsule, or can be compressed to form tablets or directly incorporated to the diet food. For oral therapeutic administration, the active components can be incorporated with excipients and used in the form of non-digestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and similar vehicles. This type of compositions and preparations must contain at least 0.1% > of the active compound. Of course, the percentage of the compositions and preparations may be varied and, conveniently, may be from 2 to about 60% of the weight of the unit. The amount of active components in the therapeutically useful compositions of this type will be such as to allow the appropriate dose to be obtained. Tablets, troches, pills, capsules and similar vehicles may also contain the following: a binder such as tragacanth gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and some sweetening substance such as sucrose, lactose or saccharin or a flavoring agent, such as peppermint, pyroxy oil or cherry flavor may be added. When the unit dose form is a capsule, it may contain, in addition to the aforementioned materials, a liquid carrier. Various other materials may be present as coatings or to modify the physical form of the unit dose in some other way. For example, tablets, pills or capsules may be coated with lacquer, sugar, or both. The elixir syrup may contain the active compounds of sucrose, as a sweetening agent, methyl and propyl syrups as preservatives, a dye and a flavoring such as cherry or orange flavor. Of course, any material that is used to prepare the unit dose must be pharmaceutically pure and substantially non-toxic in the amounts used. In addition, the active compounds can be incorporated into sustained release preparations and formulations. The active components can also be administered parenterally or intraperitoneally. Solutions of the active components can be prepared as free base or pharmacologically acceptable salts in water suitably mixed with some surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations will contain a condom to prevent the development of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and liquid, so that it can be easily introduced by means of a syringe. It must be stable under the conditions of manufacture and storage and be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (such as, for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity will be maintained, for example, by using a coating such as lecithin, maintaining the necessary particle size in the case of dispersions, and using surfactants. The action of organisms will be avoided by using various antibacterial and antifungal substances, such as, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases it will be preferable to include isotonic substances, such as sugars or sodium chloride. Prolonged absorption of the injectable compositions will be achieved by the use in the compositions of substances delaying absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active components in the necessary amount of the suitable solvent with the various ingredients listed above, as required, followed by sterilization by filtration. In general, the dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and the other necessary ingredients, among those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation will be the vacuum drying and freeze drying techniques which result in the powder of the active ingredient plus any additional desired ingredient of the solution previously filtered in sterile form thereof. As used herein, the words "pharmaceutically acceptable carrier" include any and all solvents, dispersion media, coatings, antibacterial and antifungal substances, substances for delaying absorption and isotonic and the like. The use of means and substances of this type together with substances with pharmaceutical activity is well known in this field. The use of any conventional medium or substance is envisaged, except in cases where it is incompatible with the active ingredient. Supplementary active ingredients can also be incorporated into the compositions. For oral prophylaxis, the polypeptide can be incorporated with excipients and used in the form of mouthwash and non-ingestible toothpaste. The mouthwash can be prepared by incorporating the active ingredient in the necessary amount and in a suitable solvent, such as a solution of sodium borate (Dobell's solution). Another alternative is to incorporate the active ingredient into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate. The active ingredient can also be dispersed in toothpastes, including: gels, pastes, powders and grouts. The active ingredient may be added in a therapeutically effective amount to a toothpaste which may include water, binders, abrasives, flavoring substances, foaming and wetting substances. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic reaction or harmful reaction of a similar type when administered to humans. The preparation of an aqueous composition containing a protein as an active ingredient is well known in the art. In general, these compositions are prepared as injectables, either as liquid solutions or suspensions; they can also be prepared in solid forms suitable for dissolving or suspending in a liquid before injecting. The preparation can also be emulsified. The composition can be formulated in neutral or salt form. Pharmaceutically acceptable salts include salts obtained by addition of acid (formed with the free amine groups of the protein) and which are formed with inorganic acids, such as, for example, hydrochloric or phosphoric acid, or organic acids, such as, for example, acetic, oxalic , tartaric, mandélico and others similar. The salts formed with the free carboxylic groups can also be derived from inorganic bases, such as, for example, sodium, potassium, ammonium, calcium or iron hydroxides, and from organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like. After the formulation, the solutions will be administered in a manner compatible with the formulated dose and in such quantity as to be therapeutically effective. The formulations are easily administered in the various dosage forms as injectable solutions, drug release capsules and the like. For parenteral administration in aqueous solution, for example, the solution should be suitably buffered and if necessary the liquid diluent should first be isotonic with sufficient saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, the sterile aqueous media that can be used are those known to those skilled in the art, taking into account the present description. For example, a dose can be dissolved in 1 ml of isotonic NaCl solution and added to 100 ml of hypodermoclysis fluid or injected into the proposed site of channeling (see for example, "Remington's Pharmaceutical Sciences", 15th edition, pages 1035-1038). 1570-1580). Necessarily there will be some variation in the dose depending on the affection of the subject under treatment. The person in charge of the administration may in any case determine the appropriate dose for each subject. In addition, for administration to humans, the preparations must comply with the standards of sterility, pyrogenicity, general safety and purity imposed by the FDA Office of Biological Standards.

Internal Epitope Sequences The present invention is also directed to protein or peptide compositions, free of total cells and other peptides, which constitute a purified protein or peptide that incorporates an epitope that immunologically cross-reacts with one or more of the antibodies of the epitope. present invention. As used herein, the term "incorporating an epitope (or epitopes) that immunologically produces cross-reactions with one or more anti-BRCA1 antibodies" refers to a peptide or protein antigen that includes a primary, secondary, or tertiary, similar to an epitope located within a BAP polypeptide or BRCAl. The level of similarity will generally be of such a degree that the monoclonal or polyclonal antibodies directed against the BAP or BRCA1 polypeptide will also bind, react with or otherwise recognize, the cross-reacting protein or peptide antigen. Various methods of immunological assay can be employed together with antibodies of this type, such as for example Western blot, ELISA, RIA and the like, all of which are well known to those skilled in the art. The identification of epitopes of BAP or BRCA1, such as those derived from BAP or BRCA1 or genetic products similar to BRCA1, their functional equivalents, or all of them, suitable for use in vaccines, is a relatively simple process. For example, the Hopp methods described in US Pat. No. 4,554,101, incorporated herein by reference, which teach to identify and prepare epitopes from amino acid sequences based on hydrophilicity can be used. The methods described in various articles and the software programs based on them are also useful for identifying epitopic internal sequences (see, for example, Jameson and Wolf, 1988, Wolf et al., 1988, U.S. Patent No. 4,554,101). The amino acid sequence of these "epitope internal sequences" may then be easily incorporated into the peptides, either by the application of peptide synthesis or by recombinant technology. The peptides which are preferably used according to the present invention, generally have from 5 to 25 amino acids in length and preferably from 8 to 20 amino acids in length. It is proposed that shorter antigenic peptide sequences are advantageous under certain circumstances, such as for preparing vaccines or immunological detection assays. Some advantages include ease of preparation and purification, relatively low cost, better reproducibility of production and advantageous biodistribution. It is proposed that the specific advantages of the present invention can be achieved through the preparation of synthetic peptides that include the extended, modified epitope and immunogenic internal sequences, or both, giving rise to a "universal" epitope peptide targeting BAP sequences , related to BAP, BRCAl or related to BRCAl. It is proposed that these regions represent those that are more likely to promote the stimulation of T cells or B cells in an animal and therefore result in the specific production of antibodies in animals of this type. The term "epitope internal sequence" as used herein, corresponds to a relatively short length of amino acids that is "complementary" and will therefore bind to the antigenic binding sites on epitopic antibodies specific for BAP or BRCAl. Additionally or alternately, an epitope internal sequence is that which produces antibodies that cross-react with antibodies directed against the compositions of the peptides of the present invention. It will be understood that within the context of the present disclosure the term "complementary" refers to amino acids or peptides that exhibit a mutual attractive force. In this way, certain epitope internal sequences of the present invention can be defined operatively in terms of their ability to compete against, or perhaps to displace the binding of the desired protein antigen with the corresponding antisera directed to the protein. In general, the size of the polypeptide antigen is not considered to be of crucial importance, as long as it is large enough to carry the sequence or internal sequences identified. The smallest internal sequence of utility that is expected in the present description is generally of the order of about 5 amino acids long and preferably with sequences of the order of 8 to 25 amino acids long. Thus, this size will generally correspond to that of the smaller peptide antigens prepared according to the invention. However, the size of the antigen may be greater when desired, as long as it contains the basic internal epitope sequence. The process of identifying epitope internal sequences is known to those skilled in the art, such as, for example, the method described in US Patent 4,544,101, incorporated herein by reference, which teaches to identify and prepare epitopes of amino acid sequences based on their hydrophilicity. In addition, various computer programs are available to predict the antigenic portions of proteins (see, for example, Jameson and Wolf, 1988).; Wolf et al., 1988). Computerized programs for analyzing the peptide sequence (eg, DNA Star® software, DNAStar, Inc. Madison, Wl) are also useful in the design of synthetic BRCA1 peptides and peptide analogs according to the present disclosure. The peptides that are prepared according to the present invention are ideal targets for use as vaccines or immunoreactive for the detection of BAP, BRCA1 and genes encoded by BAP or BRCA1, or alternatively to detect BAP, BRCA1 or BRCA1-like gene products. In this regard, specific advantages will be achieved by the preparation of synthetic peptides that include epitope / immunogenic internal sequences. These epitope internal sequences can be identified as hydrophilic, mobile regions, or both, of polypeptides that include a T-cell motif. It is known in this field, that these regions represent the areas most likely to promote B-cell stimulation. or T cells, and therefore lead to the production of specific antibodies. It is also relatively simple to confirm whether a protein or peptide cross-reacts immunologically or is a biologically functional equivalent of one or more epitopes of the described peptides. For this, specific assays are used, such as, for example, the single proposed epitope sequence, or more general detection methods, such as, for example, a set of synthetic peptides or randomly generated protein fragments. Detection assays can be used to identify either the equivalent antigens or the cross-reactive antibodies. In any case the principle is the same, that is, it is based on the competition between the antibodies and the antigens for the binding sites. Suitable competition assays that can be employed include protocols that are based on immunohistochemical assays, ELISA, RIA, Western blot, dot blot, and the like. In any of the competitive assays one of the components of the linkage, generally the known element, which may be a peptide derived from the BRCA1 or a known antibody, is labeled with a detectable label, and the test components, which are generally not labeled, they are tested to see their ability to reduce the amount of label bound to the corresponding antigen or antibody reagent.

As an example of embodiment, to carry out a competition study between BAP or BRCA1 and any test antigen, the BAP or BRCA1 is first labeled with a detectable marker, such as biotin, or a radioactive or fluorogenic enzyme label, for allow their subsequent identification. Then the labeled antigen is incubated with the other, the test is carried out and the antigen is examined in different proportions (for example, 1: 1, 1: 10 and 1: 100) and, after mixing it, the mixture is added to the an antibody of the present invention. Preferably, the known antibody is immobilized, for example, by fixing it to an ELISA plate. The ability of the mixture to bind to the antibody is determined by detecting the presence of the specifically linked label. This value is compared against a control value, in which no potentially competing (test) antigen is included during the incubation. The assay can be any of the various immunological assays based on hybridization and the reactive antigens are detected by identifying the label, for example, using streptavidin in the case of biotinylated antigens or by using a chromogenic substrate in the case of an enzymatic label, or simply by detecting a radioactive or fluorescent marker For example, an antigen that binds to the same antibody as BRCA1 will compete effectively for the link and therefore will significantly reduce the BRCA1 linkage, which will be evidenced as a decrease in the amount of marker detected. The reactivity of the labeled antigen, such as, for example, a BAP composition or BRCAl in the absence of test antigen, will be the high control value. The low control value is obtained by incubating the labeled antigen with an excess of unlabeled BAP or BRCAl antigen, in which case competition would occur that would reduce the degree of binding. The considerable reduction of the reactivity of the labeled antigen in the presence of a test antigen indicates that said test antigen produces "cross-reactivity", that is, that it has binding affinity towards the same antibody. In the terms of the present application "a significant reduction" can be defined as a reproducible (ie, consistently observed) reduction in the link. In addition to the peptidyl compounds described herein, we also contemplate the possibility of formulating sterically similar compounds to simulate the key portions of the peptide structure. These compounds, which may be called peptidomimetics, can be used in the same way as the peptides of the invention and therefore are their functional equivalents. A functional structural equivalent can be generated by the modeling and chemical design techniques known to those skilled in the art. It should be understood that all sterically similar constructions of this type are within the scope of the present invention. The synthesis of epitope sequences or peptides that include an antigenic epitope within their sequence is easily achieved by conventional synthesis techniques such as the solid phase method (ie, by using a commercially available peptide synthesizer, such as the Peptide Synthesizer Model 430A from Applied Biosystems). Aliquots of the peptide antigens synthesized in predetermined amounts are thus taken and stored in a conventional manner such as, for example, in aqueous solution or preferably in the lyophilized state or in powder form until they are used. In general, due to their relative stability, the peptides can be easily stored in aqueous solution for rather long periods if desired, for example up to six months or more, using practically any type of aqueous solution, without suffering appreciable degradation or loss of antigenic activity. Nevertheless, if a prolonged storage in aqueous state is foreseen, it is generally convenient to include substances such as buffer solutions of the Tris type or a phosphate buffer to maintain a pH from about 7.0 to 7.5. In addition, it is advisable to include substances that inhibit microbial development, such as sodium azide or Merthiolate. For prolonged storage in an aqueous state, it is advisable to store the solutions at 4 ° C, or preferably to freeze them. Of course, when the peptides are stored in a lyophilized or powdered state they can be stored practically for indefinite periods, for example in aliquots measured rehydratable with a predetermined amount of water (preferably distilled) or buffer solution before they are used.

Mutagenesis in Specific Sites Mutagenesis at specific sites is a useful technique for preparing individual peptides, or proteins or peptides functionally equivalent from the biological point of view, by specific mutagenesis of the underlying DNA. The technique, well known to those skilled in the art, also allows the possibility of preparing and testing variants of the sequence, for example, incorporating one or more of the above considerations, introducing one or more changes in the DNA nucleotide sequence. . Mutagenesis of specific sites allows the production of mutants by using specific sequences of oligonucleotides that encode the DNA sequence of the desired mutation, and in addition a sufficient number of adjacent nucleotides to obtain a primer sequence of sufficient size and complex sequence to form a stable duplex on both sides of the deletion junction that is traversed. In general, a primer is preferred from about 14 to 25 nucleotides in length, with about 5 to 10 residues on both sides of the junction of the sequence being altered.

In general, the site-specific mutagenesis technique is well known in the art, and is mentioned in various publications. As will be appreciated, this technique typically employs a phage vector that exists in both a single chain and a double chain form. Typical vectors useful for site-directed mutagenesis include vectors such as phage 1 3. These phages are readily available commercially and their use is generally known to those skilled in the art. Double-stranded plasmids are also routinely used in site-directed mutagenesis, which eliminates the transfer step of the gene of interest from the plasmid to the phage. In general, site-directed mutagenesis according to this specification is carried out by first obtaining a single chain vector or by dividing two strands of a double chain vector including within its sequence a DNA sequence encoding the peptide wanted. An oligonucleotide primer is prepared that carries the desired mutation sequence, generally synthetically. This primer then binds with the single chain vector and is subjected to DNA polymerizing enzymes, such as the Klenow fragment of E. coli polymerase I, to complete the synthesis of the chain carrying the mutation. In this way, a heteroduplex is formed in which one strand encodes the original sequence without mutation and the second strand carries the desired mutation. Then, this heteroduplex vector is used to transform suitable cells, such as, for example, E. Coli cells, and selections are included that include recombinant vectors that carry the ordering with the mutated sequence. The preparation of sequence variants of selected segments of peptide-encoding DNA using site-directed mutagenesis is provided as a method to produce potentially useful species and is not limiting in nature, since there are other ways in which they can obtain sequence variants of the peptides and the DNA sequences encoding them. For example, recombinant vectors encoding the desired peptide sequence can be treated with mutagenic substances such as hydroxylamine, to obtain sequence variants. The specific details regarding these methods and protocols can be found in the writings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991; Kuby, 1994; and Maniatis et al., 1982, each of which is incorporated herein by reference for that purpose. The PCR ™ -based chain splice extension (SOE) (Ho et al., 1989) for site-directed mutagenesis, is particularly preferred for effecting site-directed mutagenesis of the nucleic acid compositions of the present invention. PCR ™ techniques are well known to those skilled in the art, as described hereinabove. The SOE procedure consists of a two-step PCR ™ protocol, in which a complementary pair of internal primers (B and C) is used to introduce the appropriate changes in the nucleotides in the wild-type sequence. In two separate reactions, it is used on the sides of the primer A PCR (restriction site incorporated in the oligo) and the primer D (restriction site incorporated in the oligo), together with the primers B and C, respectively, to generate products AB and CD PCR ™. The PCRI M products are encoded by agarose gel electrophoresis, and the two spliced PCRrM fragments, AB and CD, are combined with the primers of sides A and B and used in a second PCRI M reaction. The amplified PCR ™ product it is purified on agarose gel, digested with the appropriate enzymes, ligated to an expression vector and transformed into E.coli cells JMl 01, XLl-Blue ™ (Stratagene, La Jolla, CA), JM105, or TG1 (Carter et al., 1985). Isolates are isolated and the mutations are confirmed by sequence of the isolated plasmids. Starting with the native genetic sequence of BRCAl, suitable sites and subclones can be manufactured from which site-specific mutagenesis is carried out.

Functional Equivalents from the Biological Viewpoint Modifications and changes in the structure of the peptides of the present invention and in the segments of the DNA encoding them can be made and still obtain a functional molecule that encodes a protein or peptide with the desired characteristics. Next, the way to modify the amino acids of a protein to create a second generation molecule equivalent or even improved is discussed. The amino acid changes are achieved by modifying the codons of the DNA sequence, according to Table 1. For example, certain amino acids are substituted by other amino acids within the structure of the protein without appreciable loss of the capacity of interactive linkage with structures, such as for example, with the antigen binding regions of the antibodies or with the binding sites on substrate molecules. As the ability to interact and the nature of the protein is what defines the biological functional activity of said protein, certain amino acid sequence substitutions can be made in the protein sequence and, of course, in the coding sequence of the underlying DNA, and However, obtain a protein with similar properties. Therefore, the inventors foresee the possibility of making various changes in the peptide sequences of the compositions described, or in the corresponding sequences of the DNA encoding said peptides, without appreciable loss of their usefulness or biological activity.

TABLE 1 Amino Acids Codons Alanine Wing A GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic Acid Asp D GAC GAU Glu Glutamic Acid Glu E GAA GAG Phenylalanine Phe F UUC uu Glycine Gly G GGC GGG GGU Histin His H CAC CAU Isoleucin lie I AUA AUC AUU Lysina Lys K AAA AAG Leucina Leu L UUA UUG CUA CUC CUG CU Methionine Met M AUG Asparagine Asn AAC AAU Proline Pro CCA CCC CCG CCU Glutamine Gln Q CAÁ CAG Arginine Arg R AGA AGG CGA CGC CGG CG Serina Ser S AGC AGU UCA UCC UCG UC Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan W UGG Tyrosine Tyr And UAC UAU When making these types of changes, it is convenient to consider the hydropathic index of the amino acids. The importance of the hydropathic index of the amino acid that gives it a biological function of interaction with the protein is understood by the persons skilled in the art (Kyte and Doolitle, 1982, incorporated herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules, such as, for example, enzymes, substrates, receptors, DNA, antibodies, antigens and other similar substances. Each amino acid is assigned a hydropathic index according to its hydrophobicity and charge characteristics (Kyte and Doolitle, 1982), these are: isoleucine (+4.5); valina (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); Alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); pro lina (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

Those skilled in the art know that it is possible to substitute certain amino acids for others with similar index or hydropathic number to obtain a protein with similar biological activity, that is, to obtain a functionally equivalent protein from the biological point of view. When making these types of changes, it is preferred to replace amino acids whose hydropathic indices differ by ± 2; in particular, those that differ by ± 1 are preferred, and those that differ by ± 0.5 are preferred. Those skilled in the art also understand that a substitution of similar amino acids can be made, depending on their hydrophilicity. US Patent 4,554,101 incorporated herein by reference indicates that the higher local average hydrophilicity of a protein, governed by the hydrophilicity of adjacent amino acids, correlates with some biological property of the protein. As detailed in U.S. Patent 4,554,101, the following hydrophilicity values are assigned to the amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+ 3.0 + 1); glutamate (+ 3.0 + 1); serine (+3.0); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 + 1); Alanine (-0.5); histidine (-0.5); cysteine (-1 .0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted by another with a value of near hydrophilicity to obtain a biologically equivalent, and in particular, immunologically equivalent protein. When making changes of this type, it is preferred to substitute amino acids whose hydrophilicity values differ by ± 2, but those that differ by ± 1, and preferably, those that differ by ± 0.5, are preferred.

As described above, amino acid substitutions are generally based on the relative similarity of the amino acid side chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size and the like. Examples of substitutions that take into account the above characteristics are well known to those skilled in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

Nuclear Transportation Nuclear transportation is a multi-step process. After synthesis in the cytoplasm, proteins that contain an active sequence of nuclear location (NLS) are transported to the nucleus, penetrating through the complexes of the pores found in the nuclear envelope (Silver, 1991). This process is carried out through various critical steps in which various proteins participate. First, the NLS of the proteins to be imported is recognized. Second, the proteins are taken to the nuclear pore complex. Third, the pore complex mediates the selective entry of these proteins. In addition, there are several examples of proteins that are retained in the cytoplasm by other proteins that release them for transport to the nucleus in response to specific signals. The glucocorticoid receptor interactions NF-kB / iBB and Hsp90 are paradigms of this type of regulation (Sánchez et al, 1985; Ghosh and Baltimore, 1990). The phosphorylation status of the protein also affects its location. For example, the yeast transcription factor PH04 can not be translocated to the nucleus when it is phosphorylated by the PHO850-PHO85-cyclin-CDK complex (O'Neill et al., 1996). The incorrect location of BRCA1 in advanced breast tumor cells may be due to a failure in one of these steps. However, it is unlikely that there is a major nuclear transport system defective in these tumor cells, since they are viable. Most likely, subtle regulators, such as the proteins needed to modify BRCA1 in order to expose their NLS, or some protein that specifically recognizes the BRCA1 NLS, do not work correctly.

Abbreviations cdk = cyclin-dependent kinase CIP = calf intestinal alkaline phosphatase GST = glutathione-S-transferase IP = immunoprecipitation FACS = fluorescence-activated cell sorting BSA = bovine serum albumin pc - post coitum EGF = epidermal development factor SDS = sodium dodecyl sulfate PAGE = electro fores is polyacrylamide gel EXAMPLES The following examples are included to demonstrate the preferred embodiments of the invention. Those skilled in the art will appreciate that the techniques described in the following examples represent techniques that the inventors have found to work well in the practice of the invention, and therefore can be considered to be the preferred modes of practice. However, the persons skilled in the art should appreciate, taking into account the present description, that it is possible to make various changes in the specific embodiments that are described and obtain similar or equal results, without departing from the spirit and scope of the present invention.

Example 1 - Characterization of brcal Nuclear Phosphoprotein This example describes how to isolate and characterize specific polyclonal antisera to different regions of the BRCAl protein. These are used to confirm that BRCA1 is a 220 kDa phosphoprotein of human cells, and that it is expressed and phosphorylated in a cell cycle dependent manner and is located in the nucleus of normal cells.

Materials and Methods Generation of Specific Antibodies to Human BRCAl Mouse polyclonal antisera were generated as previously described, using purified GST-BRCA1 fusion proteins, expressed as immunogens in bacteria (Durfee et al., 1994). Two of the antisera (anti-BRCAlBgl and anti-BRCAl, formed against amino acids 341-748 and 762-1315, respectively) are described in this specification. A third antiserum (anti-BRCAIN) was generated using a GST fusion protein encoding the first 302 amino acids of BRCA1. The anti-BRCAl monoclonal antibody (MAb 6B4) was developed against GST-BRCAlBgl by standardized methods (Harlow and Lane, 1988).

Generation of a full-length BRCAl cDNA A full-length BRCAl cDNA was obtained by using an exon 11 fragment generated by PCR to probe a human cDNA library (Zhu et al., 1995). Three spliced kidneys were identified, which together codified the entire cDNA. Convenient restriction sites were then employed within these to assemble a full-length clone (see FIG. IA) in pBSK (Stralagene, La Jolla, CA).

TRANSCRIPTION AND TRANSLATION ÍN V1TRO BRCAl translated in vitro was generated from cDNA using the transcription and translation kit of reticulocyte-lysate TNT (Promega, Madison, Wl) according to the manufacturer's instructions. Immunoprecipitations were performed using one-twentieth of the total reaction volume.

Immunoprecipitation and IP / Western and Western Analysis Immunoprecipitation was performed with the three polyclonal antisera against BRCA1 according to standardized protocols (Chen et al., 1989), using the various antisucros at 1: 1000 dilution. After immunoprecipitation, the proteins were separated on 6.5% polyacrylamide gels. Proteins labeled with "" S-methionine (300 mCi for 90 minutes in methionine-free medium) were detected by autoradiography. To perform the IP / Western studies, the immunoprecipitates were transferred to ImmobilonI membranes (Millipore, Bedford, A) and probed with MAb6B4 at a 1: 1000 dilution, according to standardized procedures (Durfee et al., 1994). The double immunoprecipitations were performed as described previously. To detect pl 10RB and p84 by Western analysis, MAb 11D7 and anti-N5-3, respectively, were used as primary antibodies, as described (Durfee cois, 1994). The treatment of the extracts with calf intestinal alkaline phosphatase was carried out before immunoprecipitation, as described (Zhy et al., 1995).

Indirect immunofluorescence Indirect immunofluorescence was performed as described (Chen et al., 1995; Durfee et al., 1994).

Production of BRCAl in Insect Cells The full-length BRCAl cDNA with a Notl site produced by engineering in an immediate 5 'position with respect to the initial methionine codon, was subcloned into pAcHLT-B (Pharmingen, San Diego, CA) using Noli and S al sites to generate a BRCAl labeled with poly-histidine, expressed from the baculovirus polyhedrin promoter. This construct was cotransfected to SF9 cells (obtained from ATTC, Rockville, MD) together with BaculoGold® viral DNA (Pharmingen), according to the manufacturer's protocols. Five days later, the culture medium was collected and a plaque assay was carried out according to the manufacturer's protocols. After one week, the recombinant plaques were identified and chosen. The production of BRCA1 was then detected in various viruses purified from the plates using nickel affinity chromatography to purify the protein expressed in the infected SF9 cells. Purified BRCAl was detected by Coomassie Brilliant Blue staining, or Western blot with MAb 6B4.

FACS analysis The cells (5 X 10") were triptinized, fixed in 10% cold ethanol, washed with PBS and resuspended in 1 ml of PBS containing 200 mg / ml of RNase A and 20 mg / ml of iodide of propidium for 30 minutes at 37 ° C. The analysis was carried out by flow cytometry using a FACSCalibur ® flow cytometer (Becton-Dickinson, San José, CA).

KINASE Assay The cells were lysed in Lysis 250 buffer solution, as described (Chen et al., 1989), and the extracts were immunoprecipitated with antibodies against various cyclins and cyclin dependent kinases (CDC2, CDK2, CDC4, cyclin. D, cyclin E, and cyclin A: all purchased from Santa Cruz Biotech, Santa Cruz, CA). The precipitates were washed and then resuspended in 50 ml of CDC2 kinase buffer (75 mM HEPES, pH 7.5, 100 mM MgCl2, 25 mM EGTA, 3 mM DTT, 1 mg / ml BSA) and incubated in the presence mCi [g-, 2P] ATP for 30 minutes at 30 ° C. The reactions were washed once with cold kinase buffer, and the complexes dissociated and re-immunoprecipitated with anti-BRCA1, as previously described (Chen et al., 1995). The resulting precipitates were separated by SDA-PAGE, dried on gel and a radiographic plate thereof was taken.

RESULTS Antibody Compositions Against Certain BRCAl Regions Mouse polyclonal antisera against human BRCA were generated using GST fusion proteins that encode three distinct regions of the BRCAl protein as immunogens (as schematically illustrated in FIG. 1A). As shown in FIG. IB (fields 2, 4 and 6), each of the three independent antisera immunoprecipitated a protein, which ran as a doublet band, with a molecular mass of 220 kDa, from whole cell lysates of HBL 100 cells (breast epithelium human) labeled with? -methionine. This protein was not observed using pre-immune serum (FIG. I B, field 1). As other proteins coinmunoprecipitate with the BRCA1, the specificity of each anti serum was further confirmed by a double immunoprecipitation protocol (Chen et al., 1995), in which the precipitates obtained on the first occasion were denatured to break up any protein complexes and they were re-immunoprecipitated with the same antibody. This stricter protocol resulted in the detection of the 220 kDa doublet by the three antisera (FIG. IB, fields 3, 5 and 7), strongly suggesting that only the doublet is specifically recognized by the three independent antisera and which most likely corresponds to the BRCAl. Many of the other bands detected in single pass immunoprecipitation are also observed with preimmune serum and therefore are likely to be non-specific. However, some of the bands besides the 220 kDa doublet are not detected with the pre-immune serum. These may be proteins that form complexes with BRCAl. The most important of these co-immunoprecipitate with the three antisera, as for example, the bands at 1 10 kDa (FIG.I B). To further confirm that the 220 kDa protein was BRCAl, a full-length cDNA was generated for the BRCA1. This was used to produce the in vitro translation of the genetic product that was then immunoprecipitated with the three antisera. As seen in the F1G. 2A, the three antisera recognize the 220 kDa protein that comes from the in vitro translation mixture that comigrates with the lower band of the doublet detected in the whole cell lysates (FIG 2A, fields 1, 3, 4, and 5 ). It was shown that the upper band incorporated radioactive sulphate, suggesting that it may be a phosphorylated form of BRCAl (Chen et al., 1995). Consistent with the above, the treatment of cellular lysates with calf intestinal alkaline phosphatase (CIP) before immunoprecipitation generated a single band, with the same mobility as the band of the original doublet that migrated more rapidly (FIG 2A, field 2).

BRCA1 is a 222 kDa Protein in Baculovirus Systems As the in vitro translated product is formed in quite small amounts, probably due to the size of the transcript, a baculovirus-based expression system was prepared for the BRCAl in order to generate Larger amounts of full length BRCAl that could be easily purified by nickel affinity chromatography. Various plaque-purified recombinant viruses were analyzed and found to produce a 220 kDa protein that could be isolated by nickel affinity chromatography from lysates of infected SF9 cells. This protein was not detected in lysates of non-infected SF9 cells. When the extracts of the infected SF9 cells were immunoprecipitated using each of the three antisera, and the immunoprecipitated protein was detected by probing a Western blot with the anti-BRCAl monoclonal antibody MAb 6B4, a 220 kDa protein was detected. migrated with endogenous BRCA1 from HBL100 cells (F1G.2B). This protein could not be detected in uninfected cells or by immunoprecipitation with pre-immune serum. The fact that the monoclonal antibody 6B4 against BRCA1 recognizes a 220 kDa protein in all immunoprecipitates is strong support for the conclusion that the three antisera recognize the same 220 kDa protein, which is BRCA1.

Expression of BRCAl follows the Cell Cycle It has been shown that the expression of high-level BRCA1 mRNA in mice correlates with tissues that undergo rapid proliferation combined with differentiation (Lañe et al., 1995, Marquis et al., 1995). Therefore, it was reasoned that the expression of BRCA1 probably varied according to the stage of the cell cycle. To investigate this possibility, the expression of BRCA1 in synchronized bladder carcinoma T24 cells was analyzed. These cells conveniently stop at G0 by contact inhibition and exhibit very high synchrony when re-sown at low density in a fresh medium. In FIG. 3A an IP / Western assay for BRCA l is illustrated using extracts made either at various times after the release of high density T24 cells, or from T24 cells stopped in M phase using nocodazole (0.4 mg / ml for 8 hours) . The distribution profile of the cell cycle at each point in time, determined by FACS analysis, is presented in FIG. 3B. The phosphorylation state of Rb in the same extracts was used as an additional indicator of cell cycle progression (FIG 3B, intermediate panel) and p84 staining (FIG 3A, lower panel) allowed to quantify the charge. Although BRCA1 is easily detected in non-synchronized cells (field 1), it is expressed at extremely low levels in the early G 1 phase, so that it is not detectable by Western analysis until 18 hours after release (field 4). This corresponds to the late Gl phase, since the cells still have a 2N DNA content according to the FACS analysis, but the Rb is already phosphorylated (FIG 3B, field 4). As the cells enter the S phase, BRCA expression l reaches a maximum rapidly (FIG 3 A, fields 5, 6). Although the level of expression decreases slightly in the M phase, it generally remains high (field 7).

Immunostaining of BRCA l During the Cell Cycle Since the alterations in the level of expression of BRCA1 were parallel to the advancement of the cell cycle, it was decided to determine if it was possible to detect differences in the immunostaining pattern. To do this, the T24 cells were again stopped in GO by high density and then stimulated to enter synchronously into the cell cycle by replacing them in a low density coverslip. At various times, the cells were fixed and stained as described previously (Chen et al., 1995; Durfee et al., 1994), to detect the expression of BRCA1 by indirect immunofluorescence and to detect DNA using DAPI. The results are presented in FIGS. 3D at 30. Eleven hours after the release, BRCAl is barely detected as homogenous nuclear staining (FIG 3D and F1G.3E). As the cells progress to the S phase (24 and 33 hours after the release) the staining is intensified (consistent with the data from the IP / Western assay) and acquires a dotted appearance (FIG 3F, FIG 3G, FIG 3H, FIG 31).

During mitosis, the stained BRCA1 appears to surround the chromosomes as they align on the metaphase plate (F1G 3.1 and 3K) and then separate (FIG 3L and FIG. 3M). As the cells re-enter Gl, a weak and homologous nuclear staining is observed (FIG 3N and F1G 30), confirming that the BRCAl is expressed throughout the cell cycle, although it was detected by Western analysis in the early Gl (F1G.3A).

Phosphorylation of BRCAl Depends on the Cell Cycle As shown in FIG. 3A, BRCA1 is phosphorylated in vivo and results in the detection of hypo- and hyperphosphorylated species in the form of the 220 kDa doublet by 15 immunoprecipitation. We investigated how this phosphorylation of cell cycle progression depends on pulse-marked T24 cells synchronized with 2P orthophosphate at various times after the release of high in GO or after high with nocodazole.

Then, the cell extracts were immunoprecipitated with anti-BRCAl. This analysis revealed that BRCA1 was phosphorylated in a cell cycle dependent manner, and parallel to its expression: which becomes evident in the middle and late part of G 1, reaches a maximum in S phase and then remains high throughout Phase M (FIG 3A and FfG 3D to FIG.

). Phosphorylation of BRCAl by Pro tein Specific KINASES Dependent of 25 Cyclins As the phosphorylation of BRCAl depends on the cell cycle, we proceeded to determine if any of the known protein kinases dependent on the cell cycle could phosphorylate BRCAl. For this, the cell lysates were immunoprecipitated with antibodies directed against several CDKs and cyclins. Then the precipitates were incubated in kinase buffer in the presence of? -, 2P] ATP. Finally, the precipitates were washed, dissociated and re-immunoprecipitated with anti-BRCAl antibodies. Then, the resulting precipitates were separated by SDS-PAGE, the gels were dried and autoradiography was obtained. The results of a typical study, which is presented in FIG. 4, show that BRCA1 is phosphorylated by cyclins D and A, both complexed with cdk2. This is consistent with the fact that the phosphorylation of BRCA1 starts in the middle part of Gl and that its phosphorylation continues during the S phase.

Comments The antisera specific for BRCA1 were characterized and used to analyze the expression of BRCA1 in normal cells. The results confirmed and extended the previous observation that BRCA1 is a 220 kDa nuclear phosphoprotein in normal cells. It was shown that polyclonal antisera, confronted against three distinct regions of the BRCA1 protein, all specifically recognized a 220 kDa protein in whole cell lysates. Immunostaining reconfirmed previous observations that BRCA1 is a nuclear protein in normal cells (Chen et al., 1995). Confirming that the 220-kDa protein is indeed full-length BRCA1, it was shown that both the BRCA1 translated and the recombinant BRCA1 expressed in vitro using the baculovirus system, co-migrated with the BRCA1 of the HBLIOO cells. In human cells BRCAl migrates as a doublet, and the upper band of said doublet is a phosphorylated form of BRCAl. The 220 kDa size is fully congruent with the predicted molecular weight for the full-length BRCAl and agrees with the above data (Chen et al., 1995; Scully et al., 1996). Other researchers report the detection of a 190 kDa protein using antibodies produced against the same immunogen, such as Scully and cois. (Scully et al., 1996; Gudas et al., 1995; .Tensen et al., 1996). It is reported that BRCAl undergoes alternative splicing (Miki et al., 1994) and it is possible that the 190 kDa species is a variant of alternating splicing of BRCA1 expressed in certain cell types. However, a protein of the same size was detected in cells transfected with a retrovirus expressing full-length BRCAl cD A (Holt et al., 1996). The same group of authors also described a recombinant BRCA1 derived from baculovirus with a molecular weight of 180 kDa (Jenseny cois., 1996). In the present invention, BRCA1 derived from baculovirus was expressed as a 220 kDa protein that co-migrated with the BRCA1 of FIBL100 cells. The fact is that this 220-kDa protein could be detected by three different criteria: nickel affinity chromatography, immunoprecipitation with three independent antisera specific for BRCA1, and Western blot with a monoclonal antibody specific for BRCA1, which is very unlikely that this protein has not been synthesized correctly. There are three possible explanations for these differences. First, to produce proteins using bacilovirus, it is required to introduce the cDNA of interest to the viral genome through homologous recombination. It is possible that an inaccurate recombination generates an incomplete protein; plaque-purified multiple viruses were examined and all were found to produce a 220 kDa protein. Second, it was observed that BRCA 1 is susceptible to proteolytic degradation and that lower molecular weight degradation products are occasionally observed in addition to the full length 220 kDa protein. This second possibility may explain the detection of a 220 kDa protein in the extracts of human cells. By last, perhaps the peptide antisera used in other studies are not specific for BRCA1, but for some other protein, such as the EGF receptor. Several types of evidence suggest that BRCAl plays a critical role in the regulation of cell growth and determination, at least in mice. BRCA nulicigoto mice l die in the development stage corresponding to the ovule cylinder (5-6 days pc), suggesting that BRCAl plays a certain role in determining the fate of early cells (Chia-Yang Liu et al., 1996 ). It is unclear if BRCAl plays a similar role in humans. A woman with normal development who carries nonsense mutations of germline lineage in both alleles of BRCAl was described (Boyd et al., 1995). Perhaps in humans there is a functional redundancy between the BRCA1 and another protein, or the different mutations exert variable effects. In this regard, it should be noted that it is still necessary to report homozygous-null individuals among the widely studied population of Ashkenazi Jews, who have a high incidence of breast and ovarian cancer due to a mutation of the founder BRCA (Friedman et al., 1995). In situ hybridization studies reveal a ubiquitous location of BRCA I in early mouse embryos and the time when this expression occurs, as well as that observed in breast epithelium during puberty, is consistent with the expression of BRCAl is higher in tissues with rapid development and differentiation (Goweny cois., 1996; Scully et al., 1996). Consistent with these observations, it was observed that BRCA1 is expressed in a cell cycle dependent manner. The initial expression is detected in the intermediate part of G l, at the restriction point, or just before it. The expression reaches a maximum in the S phase and remains high throughout the M phase, descending to low levels again in Gl. In parallel with the increase in expression observed as cells pass through G l and enter the S phase, BRCAl is apparently phosphorylated by cyclin D and A, both complexed with cdk2. The fact that BRCA1 is phosphorylated in parallel with its synthesis suggests that the phosphorylated species may be the active form of the protein and that its activity is regulated by cyclin-dependent kinases. Consistent with the above, there are two cdk phosphorylation consensus sites within the BRCAl.

Example 2 - Preparation of Antibodies to BRCAl To characterize BRCA l. the inventors generated polyclonal antibodies to BRCAl (anti-BRCAl) by forming a glutathione-S-transferase (GST) -BRCA1 fusion protein containing amino acids encoded by a 3 'portion of exon 1 1 of BRCAl.

Fusion Proteins To form fusion constructs of glutathione-S-transferase (GST) -BRCAl, PCR ™ was used to amplify the exon fragments of BRCA1 from the genomic DNA of W? 38 (human diploid lung). A fragment of approximately 1.9 kb was amplified with two primers of 27 nucleotides synthesized according to the published sequence of BRCA1: BRCA9 [5 '-TTGCAAACTGAAAGATCTGTAGAGAGT-3'] (SEQ ID NO: 2), above with respect to the BglU site and BRCA7 [ 5 '-TTCCAAGCCCGTTCCTCTTTCTTCCAT-3' J, (SEQ ID NO: 3) below with respect to a BamHl site. The amplified genomic DNA was subsequently digested with BglU and BamHl to form a 1.8 kb fragment from codon 762 to 13 1 5. This fragment was purified and subcloned into the GST expression vector pGEX-2T to form pGST-BRCA l. To form a second GST-BRCAl-Bgl plasmid, another 27 nucleotide primer was used, BRCA8 [5'-GATTTGAACACCACTGAGAAGCGTGCA-3 '] (SEQ ID NO: 4), starting at codon 245 and a BRCA9 primer to amplify a 3.2 kb fragment that encompassed almost all of exon 1 1. Then, this fragment was digested with BglII to form a 1.2 kb fragment from codon 341 to 748, which was subcloned to give a modified pGEX-2T. Each of the two fusion proteins was expressed in Escherichia coli and purified with glutathione-Sepharose beads to be used as an antigen in the mice. Then, the serum of the immunized mice was preabsorbed in GST affinity columns. The serum produced against the first GST-BRCAl protein was used in all the studies illustrated in the figures. Preimmune serum was obtained from the same mice and used at the same dilution. The anti-BRCAl serum immunoprecipitated specifically to a protein with a molecular mass of 220 kDa in human diploid breast epithelial cells HBL100, metabolically labeled with S-methionine (F1G.5 A). The protein migrated approximately according to the predicted size from the 1 863 amino acid sequence (Miki et al., 1994). As the anti-BRCAl serum coprecipitated at least five other proteins in addition to BRCA l. double immunoprecipitation including denaturation was carried out and only the 220 kDa protein was detected (FIG 5A, field 3). Immunoprecipitation was carried out by labeling the HBL100 cells with ° S methionine, lysing with Lysis-250 buffer, and immunoprecipitating them with anti-BRCA1, as previously described for the retinoblastoma protein (Chen et al., 1989). Immunoprecipitated proteins were boiled in denaturing buffer [20 mM Tris-HCl (pH 7.4), 50 mM NaCl, 1% SDS and 5 mM dithiothreitol] for 5 minutes, diluted 10-fold with 50-lysis buffer containing different detergents [20 M Tris-HCl (ph 7.5), 50 mM NaCl, 1% NP-40, and 1% deoxycholate], and were re-immunoprecipitated with anti-BRCAl in the same buffer solution. Subsequently, this double-immunoprecipitated protein was washed with Lysis-250 buffer before being separated by SDA-polyacrylamide gel electrophoresis (SDS-PAGE). Two additional polyclonal antibodies were used in similar studies. C20, directed against a COOH end epitope and BRCA1 -Bgl, produced against a fusion protein with sequences encoded by the portion closest to 5 'of exon 1 1, identified the same protein as the first antibody (FIG. 5A, field 6). The rabbit polyclonal antibody C20, produced against a synthetic peptide corresponding to amino acids 1843 to 1 862 of BRCAl. was purchased from Santa Cruz Biotechnology, Inc. When producing glutathione-S-transferase (GST) -BRCAl fusion constructs, PCR ™ was used to amplify two BRCAI exon fragments of genomic DNA from Wl 38 cells (human diploid lung) . A fragment of approximately 1.9 kb was amplified with two primers of 27 nucleotides synthesized according to the published BRCAl sequence: BRCA9 [5 '-TTGCAAACTGAAAGATCTGTAGAGAGT-3'] (SEQ ID NO: 2), above with respect to the BglII site, and BRCA7 [5 '-TTCCAAGCCCGTTCCTCTTTCTTCCAT-3]' (SEQ ID NO: 3), below with respect to a BamHl site. The amplified genomic DNA was subsequently digested with BglU and BamHI to form a 1.8 kb fragment from codon 762 to 1315. This fragment was purified and subcloned into the GST expression vector pGEX-2T to form pGST-BRCA1. To form a second plasmid. GST-BRCAl -Bgl, another 27 nucleotide primer BRCA8 was used | 5 '-GATTTGAACACCACTGAGAAGCGTGCA-3'] (SEQ ID NO: 4), starting at codon 245 and a BRCA9 primer to amplify a 3.2 kb fragment that included almost all of exon 1 1. This fragment was then digested with BglII to form a 1.2 kb fragment from codon 341 to 748, which was subcloned into modified pGEX-2T. Each of the two fusion proteins was expressed in Escherichia coli and purified with glutathione-Seiarose beads to be used as an antigen in mice. Then, the serum of the immunized mice was preabsorbed in GST affinity columns. The serum produced against the first GST-BTAC 1 protein was used in all the studies illustrated in the figures. Preimmune serum was obtained from the same mice and used at the same dilution. The same results were obtained by carrying out each step of the double immunoprecipitation with a different polyclonal antibody. These immunological results demonstrated that the 220 kDa protein is the genetic product of BRCA l. The immunoprecipitate of the lysate of ITBL100 cells labeled with [32 P] phosphoric acid contained a single species that migrated more slowly (field 7) and therefore this showed that BRCA1 is a phosphoprotein. BRCA1 is present not only in normal breast epithelial cells such as the HBL100 lineage, but in all the breast cancer lineages that were tested (FIG 5B). Apparently it is expressed almost intact in these cells, because the proteins identified by '2P labeling and immunoprecipitation with anti-BRCAl, all migrated in the gel to approximately 220 kDa. Therefore, BRCAl does not undergo mutation by truncation in most lineages of breast cancer cells. Apparently BRCAl is more abundant in tumor lineages derived from tissues other than the breast than in breast cancer lineages; it is more easily detected in bladder, cervical, colon and other types of cancer by labeling with 3S-methionine (F1G, 5C).

Subcellular Location of BRCAl To determine the subcellular location of BRCAl, the inventors fractionated HBL100 cells into their nuclear, cytoplasmic and membrane components (Chen et al., 1994, Abrams et al., 1989). BRCA1 was detected mainly in the nuclei of normal cells (ITG, 6A). In addition, indirect immunostaining of intact cells including HBL100, other diverse normal cell lineages, and tumor cells derived from tissues other than the breast or ovary, also indicated that the BRCA1 was located in the nuclei (FIG 6B to FIG 6Q; Table 2). In contrast, BRCA1 was detected mainly in the cytoplasm of almost all lineages of breast cancer cells that were tested (F1G, 6.1 to FIG, 6Q, Table 2). In 14 of the 17 cell lineages established from breast cancer, BRCAl staining was mainly cytoplasmic. In two other breast cancer lineages, both nuclear and cytoplasmic staining was observed in the same cells. In a lineage, MDA-MB361, which was originally derived from a brain metastasis (Cailleau et al., 1978), observed two distinct populations of cells: a less abundant fraction of more heterogeneous and larger cells, in which the BRCAl was located in the nucleus, and a more abundant fraction of more homogeneous and smaller cells in which BRCA1 was located in the cytoplasm. Similar results were obtained by cell fractionation in several of the same cell lineages. These results suggest that BRCA1 is aberrantly located in the cytoplasm of most lineages of ovarian and breast cancer cells. Next, primary cells from malignant pleural effusion and biopsy sections from patients with breast cancer were examined. In all primary malignant effusion cells obtained from 17 different patients, BRCA1 was also located mainly in the cytoplasm (FIG 7N and FIG 7P, Table 2). Other tumor cells developed in suspension (such as the CEM, FIL60 and Molt4 leukemia lineages) or metastatic to the pleura (K562 and U937) stained mainly in the nuclei (Table 2). Breast tumor cells in culture and those from malignant pleural effusion were all derived from advanced metastatic cancers. To determine if BRCA1 was also aberrantly located in the primary tumors, the inventors used the same anti-BRCAl polyclonal serum to stain the cells in tissue sections. A complete or partial location of BRCA I in the cytoplasm was demonstrated in most of the breast cancer cells (FIG 8A, F1G 8B and FIG 8C). In 50 biopsies, BRCAl staining was mainly cytoplasmic in 6 (12%), cytoplasmic and nuclear in variable degree in 34 (68%), and mainly nuclear in 10 (20% >), and absent in 2 ( 4%) (Table 2). These results indicate the abnormal subcellular location of BRCAl in primary and also distant metastatic breast tumors. The completely incorrect location of BRCAl seems more common in end-stage breast cancer, however it was observed to varying degrees in the vast majority of tumors in a randomized survey. 4% of tumors lacking BRCAl may represent familial cases; this percentage correlates well with the small and similar incidence of BRCA1 mutations in breast cancers of all types (Claus et al., 1991). Note that in the stromal cells and lymphocytes from the tumor of FIG. 8C, the staining of BRCAl is nuclear, while in the breast tumor cells of the same sections staining was not observed when using the same procedure.

TABLE 2 SINGLE-BRCA LOCATION l IN CELL LINE, PRIMARY TUMOR CELLS FROM PLEURAL MALIGNANT SPLASH AND TISSUE BLOOD COURTS ejido or tumor Cases (77) BRCA l Loe; jzión BRCA l of origin Nucleo plasma Ai nbos Absent Established lines Normal fibroblast 2 2 0 0 0 Renal epithelium 1 1 0 0 0 Bladder carcinoma 3 0 0 0 Cervical carcinoma 2 2 0 0 0 Leukemia or lymphoma 4 4 0 0 0 Osteosarcoma 2 2 0 0 0 Prostate carcinoma 1 1 0 0 0 Rhabdomyosarcoma 2 2 0 0 0 Breast epithelium 1 1 0 0 0 Breast adenocarcinoma 1 8 1 1 5 2 0 Ovarian carcinoma 3 1 2 0 0 Malignant effusions Breast adenocarcinoma 1 7 0 1 7 0 0 Ovarian carcinoma 8 0 8 0 0 Leukemia or lymphoma 2 2 0 0 0 Fixed sections of tissue Infiltrating lymphocytes 50 50 0 0 0 Breast carcinoma 50 8 5 34 2 The amino acid sequence of BRCAl does not have the typical bipartite nuclear location signals (NLSs) (Miki and cois, 1994), but it does contain at least two other putative NLSs (Boulikas, 1994). These signals, NKLKRKRRP (SEQ ID NO: 5), amino acids 419 to 427; and NRLRRKS (SEQ ID NO: 6), amino acids 609 to 615, are similar to the sequences observed in estrogens, progesterone and other streptococcal hormone receptor molecules (Boulikas, 1994; Arriza; cois., 1987; Danielsen et al. ., 1986, Green et al., 1986, Kastner et al., 1990, Lauder et al., 1991). To be activated and redistributed from a primarily cytoplasmic location to the nucleus, steroid hormone receptors require binding to their ligands, undergo configuration changes and probably dimerize (Forman and Samuels, 1990, Jensen, 1991). The BRCA1 may reach its normal location in the nucleus in a similar way, by dissociation of the proteins that anchor it to the cytoplasm, as a passenger along with other nuclear proteins, or after a modification to expose its own potential NLS. Similar transport mechanisms have been demonstrated for other transcription factors, including SV40 and c-Fos large T antigen (Schneider et al., 1988; Roux et al., 1990; Molí et al., 1991). Mutations of the molecules involved in the BRCA1 transport pathway from the site where it is synthesized to the sites of action in the nucleus, may be alternate ways to inactivate the same crucial protein in various sporadic breast cancers.

Example 3 - BRCAl Sequences Interactions with the Immunity Transport Signal Receptor Immunity-a The BRCA I gene product is a nuclear phosphoroprotein that is located aberrantly in the cytoplasm of most breast cancer cells. In an attempt to elucidate the potential nuclear transport mechanism of the BRCAl protein, three regions of highly charged basic residues ^ "KRKRRP308 (SEQ ID NO: 7), 606PKKNRLRRKS651 (SEQ ID NO: 8), and 65 I KKYN656 were identified. (SEQ TD N0: 9) as ^ fc potential nuclear location signals (NLSs). These three regions were imitated after 503KLP3508, 607KLS615, and 1, IKLA '', (', respectively.) The wild type and mutated proteins were labeled with the FLAG epitope expressed in human DU145 cells, and were detected with the monoclonal antibody M2. DU145, the KLP mutant can not be located in the nucleus RL, whereas the KLS mutant is found mainly in the cytoplasm and occasionally in the nucleus.The KLN protein is always located in the nucleus.Constantly, the hSRPla (importin-a ), a component of the NLS receptor complex, was identified in a detection of two yeast hybrids using BRCA1 as bait.The specificity of the interaction between BRCA1 and importane was demonstrated more extensively by showing that the regions), KRPRRP) S (SEQ ID 15 NO: 7) and 606PKKNRLRRKSÍ, I (SEQ TD NO: 8) are critical for this interaction, but not the region fo iKKKKYN '° 6 (SEQ TD NO: 9). To determine if the incorrect cytoplasmic location of endogenous BRCA1 in breast cancer cells is due to a deficiency of the cells, the wild-type BRCA1 protein marked with the FLAG epitope was ectopically expressed in six lineages of breast cancer cells. . The analysis showed that in all of them this protein was located in the cytoplasm of said cells. In contrast, the expression of the construction in four lineages of non-breast cancer cells resulted in a nuclear location. These data support the possibility that the incorrect location of the BRCA1 protein in breast cancer cells may be due to a defect in the cellular mechanism involved in nuclear import mediated by the NLS receptor.

Experimental Procedures Cell Culture and DNA Transfections Strains of human cells DU145 (prostate cancer), T24 (bladder cancer), T47D, ZR75 were cultured. MB231, MB468. MDA330, MCF7 (breast cancer), HBL100 (normal breast epithelial cells immortalized with SV40), and CV 1 (monkey kidney cell lineage) at 37 ° C in a humidified atmosphere containing 10% C02, on medium Modified Eagle type Dulbecce (DMEM, Life Technologies, Inc.) supplemented with 10% heat inactivated calf fetal serum (Hyclone Laboratories, Inc.) on plastic surfaces. Each 10 cm box of cells developed to the confluence of 60%) was transfected with 10 mg of plasmid DNA using the calcium phosphate method (Kingston, 1994). The calcium phosphate precipitate was left in the culture medium for 6-8 hours. Then, the medium was drained and the cells were refed with fresh medium.

NLS Mutagenesis To introduce mutations in the three putative nuclear location sequences of the BRCAl, a PCR ™ strategy was employed. In summary, the following external primers and internal primers with Hindlll restriction sites (underlined below) were used to form deletions within the framework of each LS sequence and the addition of a leucine residue. The external primers used for all NLS mutations were 5 '-GATTTGAACACCACTGAGAAGCGTGCA-3' [745 to 771 of BRCAl cDNA] (SEQ ID NO: 4) and 5'-CTTTAAGGACCCAGGTGGGCAGAGAA-3 '[2791 to 2765] (SEQ ID NOM) OR). For the KLP mutation the following internal primers were used. ÍA: 5'- CCTTTTAAGCTTTAATTTATTTGTGAAGGGGACGCTC-3 '[1521 to 1495] (SEQ ID NO: l l) and lb 5' -CCTTAAAGCTTCCTACATCAGGCCTTCASTCCTGA-3 (SEQ ID NO: 12). For the KLS mutation the following internal primers were used: 2A 5 '-CTCCCAAGCTTAGGTGCTTTTGAATTGTGGATATTT-3' [1830- 1 806] (SEQ TD NO: 13) and 2B 5 '-CCTCCCAAGCTTTCTTCTACCAGGCATATTCATGCGC-3' [1854 to 1879] (SEQ ID NO : 14). The KLN mutation was generated with the following internal primers: 3a 5 -CCTCCCAAGCTTTATCTCTTCACTGCTAGAACAACT-3 '[1978 to 2101] (SEQ ID NOT 5; and 3B 5 '-CCTCCCAAGCTTAACCAAATGCCAFTCAGGCACAGC-3' [1978 to 2101] (SEQ ID NO: 16). Plasmid BSK-BRCAla containing full-length BRCAl cDNA (Chen et al., 1996) was used as a standard for PCR ™ amplifications using each pair of internal and external primers. The resulting DNA fragments were gel purified and cut with Aflll and Hindlll to obtain the N-terminal cDNA fragments and with Kpnl and Hindlll to obtain the C-terminal fragments of the C-terminus. The fragments of the N and C termini were then used to replace the Afl fragment? / Kp l in the pBSK-BRCAla. The union of the indl site in each of the NLS sites generated deletions within the framework and additions of CTT codons for leucine residues. The AflWKpnl fragments of pBSK-BRCAl-KLP, KPS and T LN were then used to replace a similar fragment in the pCEP-FlagBRCAl expression vector (Chen et al., I 996) to generate PCEP-FlagBRCAKLI, pCEP-FlagBRCAl KLS and pCEP-FlagBRCAlkI N.

Transient Expression and Immunostaining Cells were transfected with pCEP-FlagBRCAl KLP, pCEP-FlagBRCAlKLS or pCEP-FlagBRCAlKLN for the expression of marked epitope NLS proteins (Flag, Kodak, IBI) and pCEP-FlagBRCA1 for wild-type BRCA1 tagged with Flag (flag). After the plate change and development on slides for 30 hours, the cells were fixed and indirectly immunostained with the M2 Flag mAb marker (Kodak, IBI), by the procedures described above (Mancini et al., 1994). . The microscopic images were obtained with a Hammamatsu Color Chilled 3CCD camera attached to a Zeiss Axiophot ™ fluorescence microscope. The digital files were digitally processed for presentation with Adobe Photoshop ®.

Immunoprecipitation and Western Blot assays BRCA I proteins were immunoprecipitated as described (Chen et al., 1995), using anti-mouse BRCA-Bgl antibodies (Chen et al., 1995). After the SDS / PAGE procedure. the epitope-tagged BRCAl protein was detected by Western blot assays using anti-Flag M2 mAB (Kodak, IBI) and the endogenous BRCAl was detected with the BRCA1-Bgl antibodies (Cheny cois., 1995).

Identification of the BRCAl Activation Domain The identification of an activation domain in the BRCAl was carried out by means of an assay of a yeast hybrid in the Y153 strain of Sacharomyces cerevisiae, which contains a lacZ reporter under the control of a promoter with GAL4 binding sites in the activation sequence that is above GALI (UASG) (Durfee et al., 1993). The BRCAl deletion constructs of FIG. 2A and FIG. 2B were obtained by Iraslational fusion of the GAL4 DNA binding domain (Keegan et al., 1986; Ma and PCashnc.187) in pAS (Durfee et al., 1993) with cDNA fragments obtained from pBSK-BRCAla (Chen et al., 1996a) using convenient restriction sites. The β-galactosidase activity was determined by the color of the colony and quantified using chlorophenyl red β-D-galactopyranoside in the assays as described previously (Durfec et al., 1993).

Detection of Two Yeast Hybrids A cDNA library prepared from human B lymphocytes was detected as previously described (Durfee cois .. 1993). The pAS-BRCA3.5 protein (see FIG 2A and FIG 2B) was used as "bait", and consisted of the amino acids 1 -1 142 of BRCAl fused to the GAL4 DNA binding domain (Keegan et al. ., 1986; Ma and PCashne, 1987) in the pAS plasmid (Durfee et al., 1993). Interactions between the BRCAI NLS and yeast-importin-a strain Y153 was co-transfected with pAS-BRCA3.5, pAS-KLP, pAS-KLS, or pAS-T LN and pACT-importina220_259 (see FIG. 3 A) and the assay was performed to detect β-galactosidase activity as described previously (Durfee et al., 1993). For the expression of importin-a, the cDNA coding for amino acids 220-529 was fused with the GAL4 activation domain (T eegan et al., 1986; Ma and PCashne, 1987) in pACT (Durfee et al., 1993). PAS-KLP, pAS-KLS, mad pAS-KLN was made by melting BRCAl,.,, 42 from pBSK-BRCAl-KLP, KLS and KLN with the DNA binding domain of GAL4 in pAS (Durfee et al, 1993) . The β-galactosidase activity was tested as described above.

RESULTS Nuclear Location Sequence of the BRCAl To begin the study of BRCAl nuclear transport, the NLS motifs were identified. Through the analysis of the amino acid sequence, three possible nuclear location sequences of the BRCAl were determined. The three regions of basic waste with high load are 503-KRKRRP-508 (SEQ IDNO: 7), 606-PKKNRLRRKS-615 (SEQ ID NO: 8) and 651-KKKKYN-656 (SEQ ID NO: 9) (FIG. 9). To determine if these sequences have any function at the nuclear location, a PRC ™ mutagenesis was performed that generated deletions within the framework and addition of a leucine residue at each site. Each of the mutated proteins was expressed in DU145 cells as fusions containing a Flag epitope at the N-terminus (Kodak, IBI) in plasmids pCEP-FlagBRCAlKLP, pCEP-FlagBRCAl KLS and pCEP-FlagBRCAlKLN. To demonstrate the expression of full length labeled protein in these studies, Western 1P assays were performed using anti-BRCAl and anti-Flag M2 antibodies for precipitation and detection. A BRCA1 protein with a Flag marker was expressed with the same mobility as the full-length endogenous BRCA1. The subcellular location of each of the mutated proteins and the wild-type proteins was determined by immunostaining with mAB anti-Flag mAB (Kodak, IBI). Consistent with previous studies, the wild-type Flag-BRCAl protein was located in the nucleus. The 651-KLN-656 mutation was also found in the nucleus, indicating that residues 651-KKKKYN-656 (SEQ ID NO: 9) are not important for the nuclear transport of Flag-BRCAlkLN. In contrast, mutations 503-KLP-508 and 607-KLS-615 both resulted in the cytoplasmic location of Flag-BRCAl KL_, and Flag-BRCAl KLS, indicating that both regions of basic waste are critical for nuclear import. In the course of these studies, it was observed that when the Flag-BRCAl KLS is expressed excessively, it sometimes reaches the nucleus. Occasional nuclear staining when this mutated protein has excessive expression may result from a slightly higher affinity towards the nuclear cytosolic receptor than that observed for the KLP mutation. We insist that the BRCAl I U, marked with Flag was never observed in the nucleus.

Identification of proteins that interact with BRCAl. Evidently, for the nuclear transport of BRCAl, interactions with other cellular proteins are required. The inventors chose to employ the yeast two-hybrid method to identify and clone the genes encoding proteins that interact with BRCA1. Since it has been proposed that BRCA1 is a transcription factor (Miki et al., 1994), it consequently creates transactivation activity. The presence of activity of this type would produce confusing results in a two-hybrid assay. To functionally identify the potential transactivation domains in the BRCA1, various domains of the BRCA1 protein were fused within the framework with the DNA binding domain of GAL4 (FIG 2A and FIG 2B) in the pAS plasmid (Durfee et al. ., 1993). If these fusion proteins contain an activation domain, they must activate the ß-galactosidase reporter that responds to UAS & in GAL4 (Durfee et al., 1993) after transfection to strain Y1 53. By this analysis the inventors defined a strong activation domain located between amino acids 1 142 and 1646 (FIG 2A and FIG 2B). This activation domain underwent deletion in BRCA3.5 (FIG 2A and FIG 2B), which only contains amino acids 1 -1 142 of BRCAl. The BRCA3.5 was then fused to the GAL4 DNA binding domain of the pAS vector as bait to detect proteins that interact with BRCA1 as described previously (Durfee et al., 1993). Four different isolates were isolated and their sequences were determined. When compared against GenBank ™, the inventors found that one of them is novel, another has homology with a protein containing an uncharacterised zinc finger domain and two more have sequence homology with previously cloned cDNAs (Table 3). Interestingly, the sequence of hBRAP21 is identical to that of the nuclear location signal receptor hSRPla (Weis et al., 1995), also known as importina-a (Górlich et al., 1994) or carioferin-a (Raduy cois). , nineteen ninety five).

TABLE 3 SUMMARY OF CLONES ENCODING PROTEINS THAT INTERACTION WITH BRCAl Insert Clone Size to Similarity activity with known β-galactosidase sequences hBRAP2 ++++ With the sequence conserved in S cerevi iae and Canorhabdilis elegans hBRAPI 2 +++ Part of a protein containing an uncharacterized Zinc finger domain hBRAP14 ++ New sequence hBRAP21 0.9 ++ Identical to the location signal receptor (hSRPl a / importina-a) The size of the insert is indicated in pairs of kilobases.

Interaction of the Importinaa with the BRCAI NLS To investigate the possible interaction of the BRCAI NLS with hSRPl to / importina-a and obtain additional evidence regarding the functional NLS of the BRCAl, a two-hybrid yeast assay was employed. This method exploits the ability of HSRPla / importin-a to interact with BRCAl M l42 in the yeast two-hybrid system and activate a ß-galactosidase gene responsive to UASC, from GAL4. In this assay, pAS-BRCA3.5 was used with amino acid sequence 1-1 142 of wild-type BRCAl, or the same region containing the NLS, KLP and KLS 5 and KLN mutation sequences cloned in the pAS expression vector (pAS-KLP, pASKLS and pASKLN). The importin-a region of amino acids 220 to 529, which is known to interact with BRCA1,.,, 42, was translationally fused with the activation domain of GAL4 in pACT (Durfee et al., 1993). In the two-hybrid assays, expression of pAS-BRCA3.5 encoding wild-type M M2 BRCA1 produced blue colonies and experienced '0 a 100-fold increase in β-galactosidase activity with respect to the non-transfected negative control Y153 cells. Consistent with the ability of Flag-BRCAl ^ LN to translocate to the nucleus, the pAS-KLN assay also resulted in blue colonies and a 150-fold increase in β-galactosidase activity. Consistent with the inability of Flag-BRCAl kL to be imported into the core, this The mutation of BRCA1,.,, L2 gave rise to white colonies and no increase in β-galactosidase was observed with respect to the background. Interestingly, the expression of pAS-KLS has shown a ten-fold increase in β-galactosidase activity with respect to the background. As mentioned previously, this increase in activity is congruent with immunostaining data for this mutation, indicating an occasional nuclear location when there is excessive expression of Flag-BRCAl KLS.

BRCA1 Cytoplasmic Location in Breast Cancer Cells We previously performed the transfection of an expression plasmid containing FLAG-labeled BRCAl to two lineages of 5-lactate cells, T47D and MB468, and to a lineage of non-breast epithelial cells immortalized, HBLIOO. FLAG-tagged BRCA1 protein was found in the cytoplasm of T47D and MB468 cells and in the nucleus of HBL100 cells by immunostaining with anti-FLAG M2 monoclonal antibody. To confirm this observation and to verify the expression of the full-length FLAG-tagged BRCAl protein, the inventors repeated this study using four lineages of non-breast cancer cells and six lineages of breast cancer cells, as indicated in Table 4 The nuclear location of the BRCA1 flag is observed in normal monkey kidney cells CV1 and in cells DU145, T24 and FIBL100 (Table 4). In contrast, the cytoplasmic location of this protein is observed in breast tumor cells ZR75 and MB231 and in MB468, MDA330 and MCF7 (Table 4). These data suggest an alteration of the transport or retention system of the proticain BRCA1 in breast cancer cells.

TABLE 4 SUMMARY OF FLAG-BRCA1 STAIN RESULTS Location Cell line Anti-BRCAl "'M2 CV1 Nb N DU145 NN H24 NN HBL100 NN T47D OC MB468 CC MDA330 N, C c MB231 C c ZR75 C c MCF7 N, CC '' Chen et al. (1995), included for comparison with M2 staining, bN, nucleus, CC, cytoplasm.

COMMENTS The identification of two regions of basic amino acids with charge between 503 and 507 and between 606 and 61 5, which are both crucial for the efficient nuclear transport of the BRCA1 protein, supports this concept more widely. The distance between these two motifs is much greater than the ten amino acids that separate bipartite sites from nucleoplasmin (Robbins et al., 1991). The structure and function of the NLSs of BRCAl is similar to that of other nuclear proteins in which two NLSs are at a greater distance than that observed in the polyoma T antigen (Richardson et al., 1986), in the NS1 protein. of influenza A virus (Greenspan et al., 1988), and on the adenovirus DNA binding protein (Morin et al., 1989). Although the inventors can not rule out the possibility that other sequences are also required for the translocation of BRCA1 from the cytoplasm to the nucleus, the NLS found at 503-507 is fundamental for said process. This observation was supported by the results indicating that the mutation of the NLS in 503-508 in BRCA completely abolished its interactions with the importin-a. The NLS at 606-615 of the BRCAl is less critical, since the mutation of this NLS did not totally decrease the nuclear import of BRCAl. The results obtained by the inventors indicate that the BRCAI is a nuclear protein with functional NLS, they differ from the report that indicates that this protein binds to the membrane and is secreted (Jensen et al., 1996). This discrepancy is intriguing, but it can be explained by the cross-reactivity of peptide antisera to the epidermal growth factor receptor (Wilson et al., 1996). Using mouse polyclonal antibodies specific for the BRCAl protein, the inventors observed constantly that BRCA1 is a 220 kDa nuclear protein that is located aberrantly in the cytoplasm in advanced breast cancer cells (Cheny et al., 1995; Chen et al., 1996b). However, Scully et al. (Scully et al., 1996) report that the 220 kDa BRCAl protein remains in the nucleus of some lineages of breast cancer cells. Although the exact reason for such discrepancy is unclear, the possibility of less specific antibodies, possible immunostaining artifacts, or both may not be excluded. By ectopic expression of the marked BRCA1 epitope protein and using the anti-FLAG M2 specific monoclonal antibody, the inventors avoided the difficulties in obtaining highly specific antibodies against BRCAl. With this completely different method, BRCAl labeled with wild-type FLAG expressed in breast cancer cells remains in the cytoplasm. This result also suggests that the incorrect location in breast cancer cells is not due to mutations of BRCAl itself. Rather, the aberrant location seems to be the result of alterations in the cells, perhaps at the level of BRCAl nuclear transport. In this regard, the demonstration in this case that the BRCA1 interacts with the importin-a subunit of the nuclear transport receptor complex could be an important indication. However, if there is a problem in the importin-a subunit or the importin-substrate complex, why does it manifest in breast epithelial cells? Does this indicate an unsuspected specificity of the importin-a towards the BRCAl? And, does the defect in BRCAl function reside in the cytoplasm or in the nucleus? Once the translocation through the nuclear pore complex is verified, it is reported that the importin-a accompanies the transport substrate to the areas of nuclear function (Gdrlich et al., 1995b). If there is any problem with the process of dissociation of the BRCA1 from the importin-a in the nucleus, the BRCA1 protein may be exported immediately from the nucleus, and as a result appear in the cytoplasmic location. Co-location studies similar to those performed in Gorlich et al., (Gorlich et al., 1995b) using breast cancer cells and mammary cells, could take into account this possibility. Another alternative possibility is that in breast cancer cells there is a problem in the regulation of nuclear transport of BRCAl. The known mechanisms for the regulation of nuclear trans-location (reviewed in Refs. 12 and 13) are the following: (a) phosphorylation / dephosphorylation, for example, c-rel and v-jun and proteins regulated by the cell cycle , such as the cyclin B-Cdk complex and pendulin; (b) cytoplasmic retention by masking the NLSs as seen in the dorsal, NF.KB, the glucocorticoid receptor, and the per iodicity protein, or (c) more general regulation at the level of the nuclear pore complex. Disturbances in the genetic products in any of these regulatory systems could potentially result in the cytoplasmic location of BRCAl in breast epithelial cells. The possibility is being investigated that some of the other proteins that interact with the BRCA1 identified in the detection of two hybrids, play this type of role in breast cancer cells. It is interesting that there are other reports of incorrect location in the cytoplasm of a nuclear tumor suppressor protein in breast cells and other types of cancer cells. In 27 cases of breast cancer examined, 37% presented cytoplasmic staining for p53, which was found to be wild-type by sequence determination (Molí et al., 1992). In another study, wild-type p53 located in the cytoplasm of lineages of human cervical carcinoma cells with human papillomavirus 18 or 16 integrated was found (Liang et al., 1993). Both studies suggest that the tumor suppressor function of normal p53, in certain cases, is inactivated by its incorrect location in the cytoplasm (Molí et al., 1992; Liang et al., 1993). These data are similar to the inventors' observations for BRCAl and seem to suggest a global alteration of subcellular compartmentalization in breast cancer cells. In the event that this is true, then BRCA1 and p53, probably with other regulatory proteins, are retained in the cytoplasm of these cells, and the composite effect of this may contribute to tumorigenesis.

Example 4-Identification of the Specific Proteins Needed for the Correct Location of BRCAl. The fragments of BRCAl that exclude any activity of / r < s'-intrinsic activation of potential type, are fused within the framework with the DNA binding domain of Gal4. These were used to detect a cDNA library, fused with the Gal4 activation domain, using a yeast strain containing a gene responsive to ß-galactosidase Gal4. The measurements were analyzed based on the following criteria. First, it was determined if they were in the chromosomal regions known to experience frequent LOH in breast cancer. Second, it was checked whether large-scale deletions of the genes were detectable by Southern DNA analysis that comes from tumor lineages in which the BRCA1 is located at the wrong site. And third, it was observed whether the most subtle mutations in these genes were detectable in the tumor lineages by PCR-based single chain configurational polymorphism assay (RT-PCR ™ / SSCP). The genes in which mutations are detected are strong candidates for genes responsible for the translocation of BRCA1 to the nucleus.

First, the protein patterns that co-immunoprecipitate with wild-type BRCA1 or with mutant proteins lacking the NLS motif were compared. Second, the proteins that co-immunoprecipitate with wild-type BRCA1 were compared in extracts from normal cells, but not in extracts of breast tumor lineages. The genes encoding these candidates as mediators of BRCAl nuclear transport were subsequently cloned by standard procedures. To confirm the specificity of the interaction and further define the region of the BRCA1 in which the identified proteins are linked, in vitro binding and co-immunoprecipitation assays were carried out in vivo using BRCA1 wild-type, NLS-deficient mutants and deletion mutants that span other BRCAl regions.

Example 5-Expression of Rescue Transport of BAPs from Endogenous BRCAl Expressed Ectopically in the Nucleus of Breast Tumor Cells. To determine whether the genes identified in aim 2 complement the BRCA1 location defect in breast cancer cells, it was necessary to clone full-length cDNAs. In addition, antibodies against the protein products were produced to allow their detection. If the mutations of the identified genes were responsible for the correct location of the BRCA1 in the tumor lineages, then these antibodies were also used for the detection of tumor samples. The identified cDNAs were expressed in lineages of breast cancer cells under the control of the tetracycline-inducible promoter, so that the expression was regulated by the presence of tetracycline in the culture medium. This avoids potentially toxic effects due to the expression of said proteins. The effect of their expression on the location of exogenous, endogenous wild type BRCA1, or both, was monitored by immunofluorescence and cell fractionation assay.

Example 6-Defective Transport is a Common Cause of Incorrect Location of BRCAl in Advanced Breast Tumor Cells. Wild-type BRCAl can be expressed in normal cells and in breast tumor lineages. If the protein fails to pass into the nucleus in breast tumor cells, this suggests that advanced breast tumor cells present a defective transport process for BRCAi, rather than mutations in BRCAl itself. This example was designed to confirm that a defective nuclear transport system is the common cause of the misplacement of the BRCA1 protein in advanced breast cancer cells. It was carried out analyzing the location of the BRCAl in 15 additional cell lines in which the BRCA1 is incorrectly located. Initially, the transiently transfected cells that express these constructionsiVE , were analyzed to determine the location of the protein by staining by indirect immunofluorescence of the anti-FLAG antibody. Subsequently, the location was confirmed by cell fractionation coupled to the Western blot analysis to detect the exogenous protein by SDS-PAGE in stably transfected lineages, as described previously (Chen et al., 1995). As described above, the protein associated with the nuclear matrix p85 (NS) was used as a control of the nuclear transport function. Immortalized breast epithelial cells (HBL100) and CVS1 cells were used as controls for the normal location of BRCAl. BRCA1 tagged proteins were constructed with deletions in each of the three NLS motifs and expressed in normal cells to identify the motif necessary for the transport of BRCAl to the nucleus. If the three putatNLS motifs are not responsible for such transport, it is possible to generate a series of mutants with systematic deletion and test them in a similar way to define the NLS functional motif. Once defined, the NLS mutants can be used as tools to detect the BRCAl-associated proteins identified in aim 2 to determine if any of them interact with the NLS. In order to determine which of the BRCAI NLSs is important for nuclear import, the inventors constructed three BRCAl deletion mutants, specifically eliminating one of the putatNLS motifs (amino acids: 500-508, 606-614 and 650). -655). These were cloned within the frame with the FLAG epitope, within the vector pCEP4 (FIG 9). The mutant proteins can be expressed transiently in normal cells, to determine their location by indirect immunofluorescence, using the anti-FLAG M2 antibody (Kodak). It is possible that any of the NLS motifs promote translocation of BRCAl and therefore mutants with double and triple deletion were also constructed to test this possibility. Based on previous experiences on the determination of NLSs of mitosin, the Rb-associated protein (Zhu et al., 1995), it was evident that the rules for determining functional NLS mot were not absolute and it was perhaps necessary to extend the spectrum of potential candidate NLS sequences. If this were true, a series of systematic mutants of BRCAl deletion could be generated and tested to determine their translocation capacity to the nucleus. These studies were useful to identify the crucial residues for the transport of BRCA1 to the nucleus.

Example 7-Specific BAPs are Required for the Right Location of the BRCAl. The yeast two-hybrid system has been used successfully to isolate a total of 25 Rb-associated proteins (Durfee cois., 1993). An advantage of this method is that it detects fairly weak interactions and is more sensitive than co-immunoprecipitation assays that depend on the efficiency of the metabolic labeling, the affinity of the antibody to the immunoprecipitation, and the half-life of the complex being assayed. . Two protein detections associated with BRCAl were initiated. As mentioned, it has previously been suggested that BRCA1 is a transcription factor. This is based on the presence of a putative zinc finger domain for DNA binding in the N-terminal region of the protein, three putative NLS motifs and a potential domain of acidic trans-activation towards the C-terminus. The nature of the assay with two yeast hybrids requires that the bait protein does not contain intrinsic activation-activation activity. Therefore, the inventors first detected a series of BRCAl deletion mutants to identify the regions containing said activity. As shown in FIG. 10, the region of approximately 600 amino acids towards the C-terminus contains strong activity of activation. Therefore, the inventors used BRCAl fragments that do not encode this region as bait to identify the associated proteins. They constructed two BRCA1 expression plasmids: pAS-BRCA2.5 (amino acids 303-1 142) and pAS-BRCA3.5 (amino acids 1-1 142) (FIG.1 1). These form hybrid molecules between the sequences for the DNA binding domain of the yeast Gal4 transcription factor (amino acids 1-147; [Keegan et al., 1986]) and portions of the BRCA1 proteins. These constructs were used to detect a cDNA library of human B lymphocytes, which was cloned into a second expression plasmid containing sequences for the Gal4 activation domain II (amino acids 768-881; [Ma and Ptasline, 1987]) . An additional detection is currently being carried out using a mouse embryonic cDNA library. As demonstrated for the first time with the Gal4-Gal80 interactions (Ma and Ptashne, 1988) and subsequently generalized (Fields and Song, 1989), if the two proteins expressed in the yeast can interact, the resulting complex will activate the transcription of promoters. containing Gal4 binding sites derived from the activating sequence found above with respect to the Gall gene. Previously, the inventors had successfully identified several important proteins that interact with the binding domain of the T-protein antigen of retinoblastoma by this method (Durfee et al., 1993). This method was rapidly disseminated to the whole community as a powerful tool to identify new protein-protein interactions. In the first detection, a total of four mice with high ß-galactosidase activity were selected. These 4 items were characterized more extensively (Table 5). One of the genes (hBRAP21) is identical, in the 400 bp region of known sequence, to the human hSRPla gene, recently identified as a human homolog of a Xenopus protein, the importin (Gorlich et al., 1994; Weis et al. , nineteen ninety five). The importin / hSRP 1 a compound works in the recognition of NLS and cooperates with other nuclear import proteins such as Ran / TC4, to promote the binding of NLS-containing peptides to the nuclear pore complex, where they undergo translocation through the envelope nuclear (Wcis et al., 1995). It is interesting that there appear to be at least 5 forms of importin, all of them closely related and differing only by a few amino acids (Gorlich et al., 1994). Its sequence is identical to the main form, at least in the region of 400 bp, whose sequence has been determined. However, there may be subtle differences in the region whose sequence has not yet been determined. The presence of diverse importin molecules with close relationship indicates the possibility that each one participates in the nuclear transport of specific subsets of proteins (Gorlicli et al., 1994).

TABLE 5 ISOLATED CLONES USING THE TWO YEAST HYBRIDS SYSTEM Clone Activity CPRG Size (Kb) Repeated Activity Similarity to cDNA Assay Sequences Known hBRAP2 3000-6000 0.8 ++++ Identical to a sequence conserved in S cerevisiae and C. elegans Part of a domain of hBRAPJ2 790-850 1 .2 zinc finger uncharacterized hBRAP14 220-240 ++ New sequence hBRAB21 150- 160 0.9 ++ Similar to the Nuclear Location Signal Receptor (hSRP l a / Importina) The analysis of BRCAl-associated proteins by co-immunoprecipitation constitutes a useful complement for the detection in two yeast hybrids described above. This method has two significant advantages over the system of two yeast hybrids. First, it is not restricted by the presence of a potential Irans-activation domain in BRCA l. Therefore, it allows to take into account the protein interactions with the full length BRCAl. This is important, because although protein defects interacting with the BRCA1 NLs are the main suspects of misplacing BRCA1 in advanced breast cancers, it is equally likely that proteins that interact with other BRCA1 regions are important. in that process, probably because they cause configuration changes in the BRCAl to reveal an NLS reason that would otherwise be hidden. Therefore, proteins that interact with other BRCA1 regions and were not present in the detection of two yeast hybrids are likely to be critical for the nuclear transport of BRCA1. For example, a protein that retains BRCA1 in the cytoplasm by phosphorylation, by action similar to that of PHO80-PH085-CDK on PHO4 (O'Neill et al., 1996), or by directly masking the NLS, similarly to the IkB action on NF-kB (Ghosh and Baltimore, 1990), does not necessarily have to link to the NLS of BRCAl. The second advantage of this method is that it allows a more directed search for proteins that participate in the transport of BRCAl than the detection in two yeast hybrids. At least five proteins with molecular weights of: 40, 41, 49, 95 and 140 kDa co-immunoprecipitate specifically with BRCA1 in OBL HBLI cells. Broader analyzes revealed the following: first, the proteins that co-immunoprecipitate with wild type BRCA1 were compared in normal cells and breast tumor cells. This allowed us to identify proteins that interact and potentially participate in the importation of BRCA1 and that may be defective in cancer cells. Second, differences in the co-immunoprecipitated proteins between the NLS mutants and the wild-type BRCA1 in normal cells were determined. This was done to identify proteins that interact specifically with the NLS of BRCAl. Together, these results allowed the identification of critical proteins for the nuclear import of BRCAl. Therefore, to ensure efficient co-immunoprecipitation, the inventors excessively expressed the FLAG-tagged BRCA protein in the cells by transfection. To avoid any potentially toxic effect of this expression, the tetracycline inducible system was employed. The FLAG marker is a convenient method for labeling exogenous proteins, since it is located at the N-terminus of the protein and therefore less likely to induce a steric hindrance between BRCA1 and the proteins with which it interacts. In addition, the high affinity monoclonal antibody against FLAG (M2, Kodak) is highly specific, which reduces the problems of contamination of the co-precipitate with related proteins that cross-react. The genes encoding the identified proteins were cloned according to procedures previously established in the laboratory for cloning of Rb-associated proteins (Qian et al., 1993). Briefly, sufficient protein was purified by co-immunoprecipitation to perform the determination of the peptide sequence. Comparison of the peptide sequences obtained with the GenBank database reveals whether the genes are novel and provides additional insights with respect to their functions. The novel genes were cloned by detection of a cDNA library with degenerate oligonucleotides, based on the obtained peptide sequences. The identity of the cloned genes and the co-immunoprecipitated proteins was confirmed in various ways. First, the SDS-PAGE mobility of the co-immunoprecipitated proteins and the in vitro transcribed / translated product of the cloned cDNAs was compared. Second, antibodies against the cloned genes were developed and tested for their recognition capacity of BRCAl-associated proteins in immunoprecipitation / Western studies. Third, in vitro and in vivo interaction of genes cloned with BRCAl was carried out.

Example 8-Generation of Transgenic Mice carrying the BRCAJ gene Flanked by LOXp. This strain was constructed using conventional genetic target techniques (Lee et al., 1992, Liu et al., 1996). A plasmid blank was constructed using a HindU-BamVU DNA fragment of 8.0 kb containing BRCA1 (exons 9, 10 and part of 1 1). First, a single loxP site derived from pGEM-30 (Gu et al., 1993) was inserted into a unique Sacl site within intron 9 using EcoRl binders. Next, a second set of loxP sites was inserted together with a neonomycin cassette derived from P12-neo [obtained from H. Gu, NH-I / NIAID] at a unique Xhol site within intron 10. The resulting plasmid it was called Brcal-loxP. A cassette pMCl -tk was placed at the 5 'end of the construct to generate the final target vector: Brcal-loxP ko. The construct was linearized by BamHl and transfected individually to ES cells [an early passage of El 4-1 (Flandyside et al., 1998)], as previously described (Lee et al., 1992; Liu et al. , nineteen ninety six). Colonies doubly resistant to G418 and FIAU were isolated, and their DNA was analyzed by Southern blot to identify the isolates containing site-specific integration of the Breal-floxP gene resulting from homologous recombination. Perhaps the presence of the Neo cassette in intron 10 altered Brcal expression by interfering with RNA processing. To eliminate this possibility, white ES cell transfection was performed with pIC-Cre (Sternberg et al., 1981) to initiate the transient expression of Cí e recombinase. Under these conditions, it was expected that Cre-mediated excision occurred only once in certain cells (Gu et al., 1994). As there are three possible recombination events, a 100-percent detection after transfection was used to generate the desired recombinant, with Neo cassette deletion, but not exon 10.

The ES cells with the desired location of loxP sites were injected independently to the C57BL / 6 blastocysts, which were implanted in the uteri of pseudo-pregnant female nurse mice F, [CBA X C57BL / 6] to generate chimeras. The transmission of the germinal lineage was tested by carrying out a new cross of male chimeric mice, with C57BL / 6 females. Because loxP sites were inserted into intron sequences, they were not expected to affect the function of the endogenous Brcal gene (Ou et al., 1994). Germline lineage chimeras were used to establish heterozygous and homozygous Brcal-loxP transgenic lineages.

Example 9 - Analysis of the Delegation Elect of BRCAl on the Development and Functioning of the Mammary Gland. The ability to conditionally and temporally initiate excision of the Brcal gene in mouse breast epithelial cells provided a unique opportunity to investigate the role of this gene. In virgin females, the transgenes driven by the WAP promoter can be cyclically activated during estrus in 30% of the alveolar secretion structures (Robinson et al., 1995). At the onset of pregnancy, the number of alveoli recruited for differentiation and activation of WAP transgenes begins to increase approximately on day 15 and reaches a maximum during lactation (Robinson et al., 1995). As discussed above, by regulating the expression of RT rt with the WAP promoter, it should be possible to initiate homozygous excision of the Brcal gene by treating the females with doxycycline during estrus, or at any time after the middle part of pregnancy. After treatment with doxycycline, the inventors carried out a detailed and complete histological examination of the mammary glands of the control animals and of the animals that received the treatment. In addition, the inventors planned to include an examination of differentiation markers as expression of endogenous milk protein gene. Something concerning with regard to this method was the asynchrony in the development of alveolar secretory units, monitored by WAP expression (Robinson et al., 1995). For example, if the Brcal gene undergoes deletion in the majority of the epithelial cells of only 30% of the secretory units during estrus [assuming a high expression efficiency and Cre excision], how would this affect the development of the total of the gland? Evidently it was very difficult to predict the result and it was necessary to prove it experimentally. There was also concern regarding the fate of Brea l'1 'cells in the subsequent right and proessor, although there is evidence that they persist as cells that do not express themselves, but they do differ [(Robinson et al., 1995) and the references included there]. As the recruitment of secretory subunits increases during the course of pregnancy, this is a minor concern, although the fate of the Brcal cells during the involution after weaning, in case the glands advanced to that stage, was completely unknown. and the effect of the Brcal non-functional protein on this process was also unknown.

Example 10 - Generation of Double Transgenic Mice carrying the BRCA1 -LOXp and the Recombinasa Inducible by Tetracycline. Transgenic lineages homozygous with high expression of Cre recombinase were paired, as described above, with homozygous Brcal-loxP mice. Initially, this generated obligate heterozygotes for both WPA-rtTA and for Cre Brcal-loxP. These F heterozygotes were paired to generate a F2 population in which 1 out of every 16 mice was expected to be pirozygous for both alleles. The mating of these animals generated homozygotes bound WVA-rt-TA; Cte / Brcal -loxP in generation F-., Which were then used for directed excision studies. Doxycycline was administered to female mice in the drinking water (or, alternatively, used subcutaneously slow-release tablets, Innovative Research of America) to induce the expression of Cre recombinase. Initially, doxycycline was administered at four points; estrus, late pregnancy (days 15-18) and day 3 of lactation. Previous studies showed that the WAP promoter activates the reporter gene expression at these points in time. Therefore, the inventors waited for the expression of rtTA to be induced at this time (Robinson et al., 1995). By treatment with doxycycline, the rtTa should activate the expression of Cre recombinase, which in turn would catalyze the excision directed towards exon 10 of Brcal. The negative controls were females of the same litter not treated with doxycycline. To confirm that exon 10 of Brcal underwent deletion after the induction of Cre, histological sections were made from the mammary glands of the mice at each time point of the treatment. They were then examined for Brcal by standardized immunohistochemical methods employing BRCA1 antibodies that specifically recognize the C-terminal region of exon 11 (Chen et al., 1996b). Another alternative is to employ microdissection followed by PCR ™ analysis as described previously (Liu et al., 1996) to directly detect the genomic deletion of exon 10. After confirming that the excision event could be induced, the groups were followed up. mice to observe the development of the mammary glands and the genesis of tumors, as described below. After the treatment of W? ArtTA; C? ABcal-loxP females with doxycycline, the breasts were prepared for the examination as follows. In virgins of 4 months without treatment or treated with doxycycline during estrus, tissues (8 breasts per animal) were collected for analysis according to the plan shown. In pregnant animals, treatment with doxycycline was administered beginning on day 14 and tissues were collected according to a pre-established schedule. Using standardized histological procedures (Liu et al., 1996), serial sections of four breasts per animal were analyzed to determine the abnormal development of the glands (such as the ductal tree or the alveolar structures), and to determine the increment or total decrease in the number of secretory units compared to controls. To confirm the deletion of the Brcal gene, a microdissection of PCR ™ was carried out in individual alveolar secretion units (Liu et al., 1996). For this analysis, two breasts per animal were used. If the Brcal deletion produced disturbance of the mammary gland development, then the inventors would probably find abnormalities in the mammary epithelial cells when carrying out the histochemical studies. The differentiation markers commonly employed are milk protein genes, specifically for the purposes of the inventors (b-casein and WDNMl [early pregnancy], and acid-whey protein and a-lactalbumin (subsequently, near the end of gestation) (Robinson and cois, 1995) Two breasts per animal were used as a tissue source for immunohistochemical studies (antibodies can be obtained from L. Henninghausen, NIH-NIDDK), or in situ hybridization.

Example 1 1 - Tumor Development Analysis Although the inventors predicted that females treated with doxycycline would develop breast tumors, they could not predict how long it would take for the tumor to form. Once the experimental animals were treated with doxycycline, both they and the untreated controls underwent routine examination for breast tumors. In case of tumor development, the tissues were examined histologically. PCR ™ microdissection (Liu et al., 1996) and immunohistochemistry with BRCA1 antibodies (Chen et al., 1996b) were used to verify if the tumors were derived from cells in which Brcal had undergone deletion. The degree of penetration of the tumor formation was determined by determining the percentage of treated animals that developed tumors, and comparing it against the controls without treatment. To constitute a useful model, the penetration should be high and ideally, tumor formation should occur at the predictable time and after the alteration of Brcal. It was also important to compare the number of tumor foci with the frequency of induced homozygous deletion. Once the occurrence of tumors has been characterized, the dynamics of tumor development is determined by certifying the mice to perform the histological examination of the breasts at various points in time, from the initial event of deletion, to the moment in which the declared tumors They were detectable. In this way, it would be possible to document the formation of tumors from their earliest stages to the last stages of the malignancy, including metastasis. This method was recently used successfully to document the dynamics of melanotrophic tumor formation in Rb + - mice (Nikitin and Lee, 1996).

Example 12 - Interactions of BRCA1 with hBRAP12 In detections performed on two yeast hybrids using BRCA1 (without its activation domain) as bait, the inventors identified four cDNAs encoding putative interaction proteins with BRCA1 (Table 6).

TABLE 6 SUMMARY OF CLONES THAT CODIFY PROTEINS THAT INTERACT WITH BRCA l Clone Size and Activity of Similarity with Insertion (Kb) ß- galactosidase Known Sequences hBRAP2 0.8 ++++ Sequence conserved in S. cerevisiae and C. elegans hBRAP12 1. 2 +++ Part of a protein domain with zinc finger hBRAP 14 1. 1 ++ None known hBRAO21 0.9 ++ Nuclear Location Signal Receptor (hSRPl to / importina-a) The primary amino acid sequence of the hBRAP12 protein is shown in SEQ ID NO: 1. It is remarkable that it contains eight zinc fingers of the C2IT2 class, which are highly homologous to each other, but are not homologous with any other protein, including the zinc finger domains of GenBank. To determine the subcellular location of hBRAP12, translational fusion of full-length cDNA with green fluorescent protein was performed. The subcellular location of the fusion protein is nuclear in CV1 and HBLIOO cells. The fact that T? BRAP12 is a nuclear protein is congruent with its interaction with BRCAl. The in vitro interactions were confirmed by pull-down assays with GST as previously applied for protein interactions E2F-retinoblastoma (Shan et al., 1992). In vivo interactions between BRCA1 and hBRAP12 were tested using reciprocal immunoprecipitation combined with Western blot (Shan et al., 1992). The interaction of hBRAP12 with BRCAl was evaluated at two levels. First, their ability to associate in vitro was tested by testing the ability of GST-hBRAP12 bound with glutathione-agarose beads, to bind to labeled 5S-methionine, BRCA1 translated in vitro, or BRCA1 made in baculovirus (Chen et al., 1996b ). The negative control was GST alone. The inverse study was also performed using different fragments of GST-BRCAl fusion proteins linked with glutathione beads (Chen et al., 1996b) and hBRAP12 transferred in vitro, as previously described (Shan et al., 1992). Second, the in vivo interaction of hBRAP12 and BRCAl was tested by co-immunoprecipitation assays of whole cell extracts (Shan et al., 1992) using antibodies generated against hBRAP12 and those available for BRCAl (Chen et al., 1995). Highly specific monoclonal antibodies to BRCAl produced against two different epitopes in exon 1 1 of BRCAl can also be used in in vivo and in vitro binding assays. Mouse antiserum against hBRAP12 was generated using GST-hBRAP12 fusion proteins expressed in E. coli as described previously (Chen et al., 1995). Before using the hBRAP12 antiserum, it was preabsorbed with GST-glutathione beads (Chen et al., 1995). Co-immunoprecipitation assays were carried out using cells that co-express sufficient amounts of endogenous proteins, or using cells co-transfected with expression vectors for BRCA1 and labeled hBRAPl2 (FLAG) or unlabeled. To accomplish this goal, a series of hBRAP12 deletion mutants was generated, including the N and C termini and zinc finger domains and their binding to full-length BRCA1 was tested, as described above. The initial hypothesis of the inventors was that, in addition to the linkage with DNA, the zinc finger domain is also important for the interaction of BRCAl. In any case, these studies should be effective in determining the region of hBRAP12 that is required for BRCAl binding. Using a strategy similar to that described in the previous paragraph, the deletion mutant assay of BRCA I was performed to determine its ability to interact with GST-hBRAP12 in an in vitro binding assay, as described above. Apparently, in this interaction, the annular finger domain of BRCAl was important, and GST fusions have been prepared for the N wild-type region of BRCAl or the same region with a single point mutation (substituyendp T for G) that give as a result a change from Cys61 to Gly, found in a case of familial breast cancer (Johannsson et al., 1996). This mutation alters the ANNULAR finger domain of BRCAl and if it participates in protein-protein interactions, it should be negative for interactions with liBRAP 12. If hBRAP12 is a DNA-binding transcription factor, it should recognize a sequence DNA specific. To identify this sequence, the method of random sequence selection and PCR ™ was used (Perkins et al., 1991; Blackwell and Weintraub, 1990) to define the consensus of the DNA binding site for BRCAl. It is important that the identification of the cognate binding site for hBRAP12 allows performing the functional test of activating function of hBRAP12, as described. The identification of the cognate binding site is also important to identify the target genes for BRAP12 below. Complementary DNAs encoding hBRAP12, hBRAP12-Zs (the zinc finger domain only) DZn-hBRAP12 (minus the zinc finger), were translationally fused with GST using the pGEX-3X vector. The cDNAs for these constructions were generated by standard PCR ™ and cloning methodologies. After expression in E. coli, the bacterial lysates were incubated with glutathione and agarose beads and extensively washed to remove non-specific binding proteins. After quantification of the protein binding of GST-hBRAP12, GST-hBRAP12-Zx and DZn-hBRAP12, the respective beads were used to detect the random sequence in the DNA library. For the selection and amplification of the hBRAP12 binding sites, 10 mg of randomly chosen oligonucleotides were incubated with beads linked with 10 mg of GST-DZn-hBRAP12 protein in a binding buffer solution containing ZnSO4 and 100 mg of tRNA as non-specific competitor. The objective of this first incubation was to eliminate the DNA that is bound in a non-specific manner. Then either GST-hBRAP or GST-l? BRAP12Zs were incubated with the linked random DNA library. After extensive washing with a low salt buffer, the DNA was eluted with a high salt buffer (1.0 M NaCl) and precipitated with ethanol in the presence of carrier tRNA. The recovered DNA was subsequently subjected to PCR ™ amplification and the binding process was repeated four times. After the fifth selection and PCR ™ process, the DNA was detached with BamH1 and HindIII, purified on 15% acrylamide gels and ligated with the pBSK vector (Stratagene). Colonies were chosen at random and the plasmid DNA sequence of minipreparations was determined following standardized procedures. By means of this sequence analysis, the consensus consensus sequences were identified. The progress of the selection was monitored using gel displacement analysis (Shan et al., 1992). For this, the PCR ™ reaction was carried out with an end-labeled primer with 2p-gATP using T4 polynucleotide kinase. The specific link per binding consensus site was determined by gel displacement analysis using specific and non-specific competing oligonucleotides (Shan et al., 1992), DNAasal fingerprints (Cao et al., 1988), or both, with proteins of wild type T? BRAP12 or with DZn-hBRAP12. Another alternative to affinity purification is to employ electrophoretic mobility shift to isolate DNA fragments (Kinzler and Vogesltein, 1989; Caubin et al., 1994) which is amplified by PCR ™ and then selected and amplified four more times. The resulting fragments are digested and ligated with pBSK, as described above.

TABLE 7 SUMMARY OF THE RESULTS OF BRCAl LOCATION BY DIFFERENT METHODS Location of Anti-BRCA Cell Lines l flag-BRCA l GFP-BRCA 1 mAB 17F8 Non-breast cancer cell lines CV1 N N N N DU145 N N N / D N T24 N N N N Saos-2 N N / A N N 5637 N N / A N / A N HBL100 N N N N Breast cancer cell lines T47D C c C c MB468 C c C c MDA330 N, a c N / A N / A MB231 c c C C ZR75 c c N / D c MCF7 N.C * c C c MB435S c N / D C c MB415 c N / D c c SKBR-3 c N / D N / A c MB175-7 c N / D C c Hs578T c N / D N / D c MB361 c N / D C c BT483 C N / C C C BT20 C N / D N / D C N: nucleus C: cytoplasm N / A: not effected *: a small portion of cells shows nuclear location.

Example 13 - Amino Acid Sequence of the BRCAl-Associated Protein Derived from hBRAP12. A full-length cDNA for hBRAP12 was isolated from a human fibroblast library using the partial cDNA of a two-hybrid yeast detection as a hybridization probe. The eight fingers of zinc start at residue 208 and end at 431. The residues of Cys and His in each finger are indicated in bold. hBRAP primary sequence (SEQ ID NO: l) MIQAQESITLEDVAVDFTWEEWQLLGAAQKDLYRDVMLENYSNLVAVGYQASK PDALFKLEQGEQPWTIEDGIHSGACSDIWKVDHVLERLQSESLVNRRKPCHEH DAFENIVHCSKSQFLLGQNHDIFDLRGKSLKSNLTLVNQSKGYEIKNSVEFTG NGDSFLHANHERLHTAIKFPASQKLISTKSQFISPKHQKTRKLEKHFIVCSECG KAFIKKSWLTDHVMHTGEKPHRCSLCEKAFSRKFMLTEHQRTHNTGEKPYECP ECGKAFLKKSRLNIHQKTHTGEKPYICSECGKGFIQKGNLIVHQRIHTGEKPY ICNECGKGFIQKTCLIAHQRFHTGKTPFVCSECGKSCSQKSGLIKHQRIHTGE KPFECSECGKAFSTKQKLIVHQRTHTGERPYGCNECGKAFAYMSCLVKHKRIH TREKQEAAKVENPPAERHSSLHTSDVMQEKNSANGATTQVPSVAPQTSLNISG LLANRNVVLVGQPVVRCAASGDNSGFAQDRNLVNAVNVVVPSVINYVLFYVTE NP REFERENCES The following bibliographical citations and those mentioned above are incorporated in the pertinent part as reference for the memory for the reasons cited in the text that are found above: US Patent 3,791,932. U.S. Patent 3,949,064. U.S. Patent 4,174,384. U.S. Patent 4,196,265. U.S. Patent 4,271,147. U.S. Patent 4,554,101. U.S. Patent 4,578,770. U.S. Patent 4,596,792. U.S. Patent 4,599,230. U.S. Patent 4,599,231. U.S. Patent 4,601, 903. U.S. Patent 4,608,251. U.S. Patent 4,683,195. U.S. Patent 4,683,202. U.S. Patent 4,952,496. U.S. Patent 5, 168,050. Abel, Xy, Yin, Lyons, Meisler, Weber "Mouse Brcal: localization, sequence analysis and identification of evolutionary conserved domains" (Mouse brcal: location, sequence analysis and identification of domains conserved by evolution), Hum. Genet., 4: 2265-2273, 1995. Adelman et al., DNA, 2/3: 183-193, 1983.

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All of the compositions and methods described and claimed herein may be made and executed without undue experimentation having regard to the present disclosure. Although the compositions and methods of this invention have been described in terms of preferred embodiments, it will be obvious to those skilled in the art that variations of the composition, methods and steps or sequence of steps of the described methods may be applied. in this memory, without departing from the concept, spirit and scope of the invention. More specifically, it will be obvious that certain substances that are chemically and physiologically related can be substituted for the substances described herein to achieve the same or similar results. All obvious substitutes and similar modifications for the persons skilled in the art are considered to be included within the spirit, scope and concept of the invention, as defined in the claims set forth below. Accordingly, the exclusive rights that are desired to be patented are described in the following claims. 7. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT (A) NAME: Board of Regents, The University of Texas System (B) STREET: 201 W. 7th (C) ) CITY: Austin (D) STATE: Texas (E) COUNTRY: United States (F) ZIP CODE: 78701 (G) TELEPHONE: (512) 418-3000 (H) TELEFAX: (512) 474-7577 (ii) TITLE OF THE INVENTION: COMPOSITIONS OF BRCAl AND METHODS FOR THE DIAGNOSIS AND TREATMENT OF BREAST CANCER (iii) NUMBER OF SEQUENCES: 16 (iv) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIA: Floppy disk (B) COMPUTER: IBM compatible with PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patent in process no. 1 .0, version no. 1.30 (EPO). (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 532 amino acids (B) TYPE: amino acid (C) CHAIN: (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: Met He Gln Ala Gln Glu Ser He Thr Leu Glu Asp Val Ala Val Asp 1 5 10 15 Phe Thr Trp Glu Glu Trp Gln Leu Leu Gly Ala Wing Gln Lys Asp Leu 20 25 30 Tyr Arg Asp Val Met Leu Glu Asn Tyr Ser Asn Leu Val Wing Val Gly 35 40 45 Tyr Gln Wing Ser Lys Pro Asp Wing Leu Phe Lys Leu Glu Gln Gly Glu 50 55 60 Gln Pro Trp Thr He Glu Asp Gly He His Ser Gly Wing Cys Ser Asp 65 70 75 80 lie Trp Lys Val Asp His Val Leu Glu Arg Leu Gln Ser Glu Ser Leu 85 90 95 Val Asn Arg Arg Lys Pro Cys His Glu His Asp Ala Phe Glu Asn He 100 105 110 Val His Cys Ser Lys Ser Gln Phe Leu Leu Gly Gln Asn His Asp He 115 120 125 Phe Asp Leu Arg Gly Lys Ser Leu Lys Ser Asn Leu Thr Leu Val Asn 130 135 140 Gln Ser Lys Gly Tyr Glu He Lys Asn Ser Val Glu Phe Thr Gly Asn 145 150 155 160 Gly Asp Ser Phe Leu His Wing Asn His Glu Arg Leu His Thr Wing He 165 1 70 175 Lys Phe Pro Wing Ser Gln Lys Leu He Ser Thr Lys Ser Gln Pl e He 180 185 190 Ser Pro Lys His Gln Lys Thr Arg Lys Leu Glu Lys His His Val Cys 195 200 205 Ser Glu Cys Gly Lys Wing Phe He Lys Lys Ser Trp Leu Thr Asp His 210 215 220 Gln Val Met His Thr Gly Glu Lys Pro His Arg Cys Ser Leu Cys Glu 225 230 235 240 Lys Wing Phe Ser Arg Lys Phe Met Leu Thr Glu His Gln Arg Thr His 245 250 255 Thr Gly Glu Lys Pro Tyr Glu Cys Pro Glu Cys Gly Lys Wing Phe Leu 260 265 270 Lys Lys Ser Arg Leu Asn He His Gln Lys Thr His Tlir Gly Glu Lys 275 280 285 Pro Tyr He Cys Ser Glu Cys Gly Lys Gly Phe He Gln Lys Gly Asn 290 295 300 Leu He Val His Gln Arg He His Thr Gly Glu LysPro Tyr He Cys 305 310 315 320 Asn Glu Cys Gly Lys Gly Phe He Gln Lys Thr Cys Leu He Ala His 325 Gln Arg Phe His Thr Gly Lys Thr Pro Phe Val Cys Ser Glu Cys Gly 340 345 350 Lys Ser Cys Ser Gln Lys Ser Gly Leu He Lys His Gln Arg He His 355 360 365 Thr Gly Glu Lys Pro Phe Glu Cys Ser Glu Cys Gly Lys Wing Phe Ser 370 375 380 Thr Lys Gln Lys Leu He Val His Gln Arg Thr His Thr Gly Glu Arg 385 390 395 400 Pro Tyr Gly Cys Asn Glu Cys Gly Lys Wing Phe Wing Tyr Met Ser Cys 405 410 415 Leu Val Lys His Lys Arg I le His Thr Arg Glu Lys Gln Glu Ala Wing 420 425 430 Lys Val Glu Asn Pro Pro Wing Glu Arg His Ser Ser Leu His Thr Ser 435 440 445 Asp Val Met Gln Glu Lys Asn Ser Wing Asn Gly Wing Thr Thr Gln Val 450 455 460 Pro Ser Val Wing Pro Gln Thr Ser Leu Asn He Ser Gly Leu Leu Ala 465 470 475 480 Asn Arg Asn Val Val Leu Val Gly Gln Pro Val Val Arg Cys Ala Ala 485 490 495 Ser Gly Asp Asn Ser Gly Phc Wing Gln Asp Arg Asn Leu Val Asn Wing 500 505 510 Val Asn Val Val Val Pro Ser Val He Asn Tyr Val Leu Phe Tyr Val 515 520 525 Thr Glu Asn Pro 530 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) CHAIN: single (D) TOPOLOGY: linear (xi) ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: TTGCAAACTG AAAGATCTGT AGAGAGT (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 base pairs (B) TYPE: acid nucleic (C) CHAIN: single (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 3: TTCCAAGCCC GTTCCTCTTT CTTCCAT (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRING: single (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 4: GATTTGAACA CCACTGAGAA GCGTGCA (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acids (C) CHAIN: (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO :5: Asn Lys Leu Lys Arg Lys Arg Arg Pro 1 5 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) CHAIN: ( D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: Asn Arg Leu Arg Arg Lys Ser 1 5 (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) ) LENGTH: 6 amino acids (B) TYPE: amino acid (C) CHAIN: (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 7: Lys Arg Lys Arg Arg Pro 1 5 (2) INFORMATION FOR THE SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) CHAIN: (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: Pro Lys Lys Asn Arg Leu Arg Arg Lys Ser I 5 10 (2) INFORMATION FOR SEQ 1D N0: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) ) CHAIN: (D) TOPOLOGY: linear (xi) DESCRI PTION OF THE SEQUENCE: SEQ ID NO: 9: Lys Lys Lys Lys Tyr Asn 1 5 (2) INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) CHAIN: single (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 10: CTTTAAGGAC CCAGGTGGGC AGAGAA (2) INFORMATION FOR SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE : (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRING: single (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 1 1: CCTTTTAAGC TTTAATTTAT TTGTGAAGGG GACGCTC (2) ) INFORMATION FOR SEQ ID NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) CHAIN: single (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12 CCTTAAAGCT TCCTACATCA GGCCTTCATC CTGA (2) INFORMATION FOR SEQ ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 base pairs (B) TYPE: acid n loop (C) STRING: single (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 13: CTCCCAAGCT TAGGTGCTTT TGAATTGTGG ATATTT (2) INFORMATION FOR SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE : (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) STRING: single (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 14: CCTCCCAAGC TTTCTTCTAC CAGGCATATT CATGCGC (2) INFORMATION FOR SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) CHAIN: single (D) TOPOLOGY: linear (ix) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15: CCTCCCAAGC TTTATCTCTT CACTGCTAGA ACAACT (2) INFORMATION FOR SEQ ID NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) ) CHAIN: single (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 16: CCTCCCAAGC TTAACCAAAT GCCAGTCAGG CAC AGC

Claims (47)

1. CLAIMS 1. A polynucleotide comprising a gene encoding a BRCA1-associated protein or peptide that includes a contiguous amino acid sequence of SEQ ID NO: 1.
2. 2. The polynucleotide of claim 1, positioned under the control of a promoter .
3. 3. The polynucleotide of claim 1, further comprising a recombinant vector.
4. 4. The polynucleotide of claim 1, further defined as a DNA segment.
5. 5. The polynucleotide of claim 1, further defined as a segment of RNA.
6. 6. A recombinant host cell comprising the DNA segment encoding a protein or peptide associated with BRCA1.
7. 7. The recombinant host cell of claim 6, further defined as a bacterial host cell.
8. 8. The recombinant host cell of claim 7, wherein the bacterial host cell is E. coli.
9. 9. The recombinant host cell of claim 6, wherein the DNA segment is introduced into the cell by means of a recombinant vector. The recombinant host cell of claim 6, wherein the host cell expresses the DNA segment to produce the protein or peptide associated with the encoded BRCAJ. The recombinant host cell of claim 6, wherein the protein or peptide associated with BRCA1 expressed comprises a contiguous amino acid sequence of SEQ JD NO: 2. 12. A method in which a DNA segment encoding a BRCA1-associated protein or peptide is used comprising the following steps: (a) preparing a recombinant vector in which a segment of DNA encoding a protein is positioned or peptide associated with BRCAl under the control of a promoter; (b) introducing said recombinant vector into a recombinant host cell; (c) culturing the recombinant host cell under conditions effective to allow expression of an encoded BRCA1-associated protein or peptide; and (d) collecting said protein or peptide associated with the BRCA I expressed. 13. A method for detecting a polynucleotide that encodes a BRCA1-associated protein or peptide, comprising the following steps: (a) obtaining a sample of nucleic acids suspected of encoding the BRCA-associated protein or peptide l; (b) contacting said nucleic acid samples with a polynucleotide comprising a contiguous sequence of nucleic acids of SEQ ID NO: I under conditions effective to allow hybridization of substantially complementary nucleic acids; and (c) detecting the hybridized complementary nucleic acids that are formed. The method of claim 13, wherein the sample of contacted nucleic acids is located within a cell. The method of claim 13, wherein the nucleic acid sample is separated from a cell prior to contact. 16. The method of claim 13, wherein the nucleic acid sample is DNA. 17. The method of claim 13, wherein the nucleic acid sample is RNA. The method of claim 13, wherein said polynucleotide further comprises a detectable label, and the hybridized complementary nucleic acids are detected by detection of said marker. 19. The method of claim 1 8, wherein the nucleic acid segment comprises a radio-enzymatic or fluorescent marker. 20. A nucleic acid detection kit comprising, in a suitable container, an isolated polynucleotide encoding a protein or peptide associated with BRCA I and a detection reagent. 21. The nucleic acid detection kit of claim 23, further comprising an unrelated polynucleotide that is used as a control. 22. The nucleic acid detection kit of claim 20, further comprising a restriction enzyme. 23. The nucleic acid detection kit of claim 20, comprising one or more nucleic acid sequences encoding one or more contiguous amino acid sequences of SEQ ID NO: 1. 24. The nucleic acid detection kit of claim 20, wherein the detection reagent is a detectable label that is connected to said polynucleotide. 25. A protein or peptide composition, free of total bacterial cells, comprising a protein or peptide associated with purified BRCAl, including a contiguous amino acid sequence of SEQ ID NO: 1, or a sequence of peptides with nuclear location that has the sequence of SEQ ID NO: 9 or SEQ ID NO: 10. 26. The composition of claim 25, which comprises a peptide having the amino acid sequence SEQ ID NO: l. 27. The composition of claim 25, prepared by the method of claim 12. 28. An antibody produced by the hybridoma ATCC HB-12164, or an antibody that binds to the same epitope as said antibody. 29. The antibody of claim 28, wherein said antibody is selected from the group consisting of aBRCAl, aBRCAIN and aBRCA16B4. 30. The antibody of claim 29, obtained from the hybridoma ATCC HB-12146.

   31. The antibody of claim 28, wherein the antibody is ligated to a detectable marker. 32. The antibody of claim 31, wherein the antibody is ligated with a radioactive label, a fluorogenic marker, a nuclear magnetic spin resonance marker, biotin, or an enzyme that generates a colored product upon contact with a substrate. chromogenic The antibody of claim 32, wherein the antibody is ligated with an enzyme such as alkaline phosphatase, hydroperoxidase or glucose oxidase. 34. The antibody of claim 28, wherein the antibody is a monoclonal antibody. 35. The antibody of claim 28, wherein the antibody is a polyclonal antiserum.

   36. A method for detecting a BRCA1 protein or peptide in a biological sample, comprising the following steps: (a) obtaining a biological sample suspected of containing a BRCA1 protein or peptide; (b) contacting said sample with a first antibody that binds to the BRCA1 protein or peptide under conditions effective to allow the formation of immune complexes; and (c) detecting the immune complexes formed in this manner. 37. An immunodetection kit including, in a suitable container, a BRCA1 protein or peptide, or a first antibody that binds to a BRCA1 protein or peptide, and an immunodetection reagent. 38. The immunodetection kit of claim 37, wherein the immunodetection reagent is a detectable label that is connected to said protein, peptide or said first antibody. 39. The immunodetection kit of claim 37, wherein the immunodetection reagent is a detectable label that is connected to a second antibody having binding affinity for said protein, peptide or said first antibody. 40. The immunodetection kit of claim 37, wherein the immunodetection reagent is a detectable label that is connected to a second antibody that has binding affinity to a human antibody. 41. A method for generating an immune response, comprising administering to an animal a pharmaceutical composition comprising an immunologically effective amount of BRCA1 or a protein or peptide composition associated with BRCA1.

   42. A method for locating the BRCA1 protein or peptide in a cell, which comprises contacting said cell with a labeled antibody that specifically binds to said BRCA1 protein or peptide under conditions effective to allow the formation of immune complexes; and determining the location of said immune complexes in said cell. 43. The method of claim 42, wherein said complexes are located in the cytoplasm of said cell. 44. The method of claim 43, wherein the location of said complexes in said cytoplasm indicates metastasis or primary cancer of said cell. 45. The method of claim 42, wherein said cell is a human cell.

   46. The method of claim 45, wherein said human cell is an ovarian or breast cell. 47. A method for identifying the breast or ovarian cancer cell, which consists of: (a) obtaining a breast or ovarian tumor cell suspected of being cancerous; and (b) determining the subcellular location of a BRCA1 protein or peptide in said tumor cell, wherein the subcellular location of said BRCA1 protein or peptide in the cytoplasm of said cell indicates the presence of said cancer cell. 8. A method for predicting the susceptibility to cancer of an ovarian or breast cell, which includes identifying in cytoplasmic location in said cell, BRCA1 or BRCA1-associated protein or peptide, wherein the presence of said protein or peptide in the cytoplasm is indicative of the susceptibility of said cell to cancer. A modified BRCA1 protein or peptide composition that lacks one or more amino acid sequences selected from the group consisting of the sequences SEQ JD NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.