Polypeptide (versions) acting in complex with androgen receptor, nucleic acid (versions), vector (versions) and hostcell (versions)

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and genetic engineering. Polypeptide with sequence SEQ ID NO:3 binds with androgen receptor and increases capability of androgen receptor to transactivate androgen-sensitive gene. Also claimed are nucleic acid encoding such polypeptide, and vector containing such nucleic acid. Group of inventions can be used for creation of transgenic animals, which express gene of protein acting in complex with androgen receptor, serving as models for elaboration of medications for cancer treatment.

EFFECT: increase in androgen receptor capability to transactivate androgen-sensitive gene.

24 cl

 

This application is a partial continuation of application U.S. serial number 09/781693, filed February 12, 2001, and provisional application U.S. serial number 60/262312, filed January 17, 2001, and claims priority on the basis of these applications, the contents of which are incorporated in this description by reference.

BACKGROUND of INVENTION

Was identified a number of genes expressed in tumor cells more intensely than in healthy. I believe that the identification of such genes will provide targets for drugs in the development of anti-cancer drugs and cancer diagnostics. In the tumor cells of the liver, the number of steroid receptors (e.g., androgen receptor) is higher than in adjacent healthy liver cells.

Steroid hormones usually have physiological effects by binding with their specific nuclear receptors to form complexes, which, in turn, act as transcription factors. These complexes are associated with a specific nucleotide sequence elements that are sensitive to steroids) in the promoters sensitive to steroids genes, promoting the transcription of these genes.

SUMMARY of INVENTION

This invention is based on the discovery of the mouse gene, codereuse the protein, acting in complex with the androgen receptor (ARCAP)that is 85% identical to the human ARCAP. It was found that human ARCAP is expressed in hepatoma cells at a higher level (compared to adjacent normal cells), binds to the androgen receptor and enhances the ability of the receptor to transactivate androgen-sensitive gene. As the homologue of the human gene ARCAP, a murine gene ARCAP can be used to obtain transgenic mouse, which serves as an animal model for drug development for the treatment of cancer (e.g., liver cancer).

Full-size cDNA of mouse ARCAP (denoted SEQ ID NO:1), with the underlined start - and stop-codons below:

The nucleotide sequence encoding murine protein ARCAP (i.e. from the start ATG codon to codon immediately preceding the stop codon in SEQ ID NO:1), denoted SEQ ID NO:2. Mouse protein ARCAP (denoted SEQ ID NO:3)encoded by the above-mentioned cDNA, the following:

Accordingly, the hallmark of this invention is a substantially pure polypeptide or protein comprising amino acid sequence, m is Nisha least 70% (e.g., at least 75, 80, 85, 90, 95, 98, or 100%) identical to SEQ ID NO:3. If the polypeptide includes a sequence 100% identical to SEQ ID NO:3, the polypeptide can contain up to 30 conservative amino acid substitutions. The invention also includes an essentially pure polypeptide encoded by a nucleic acid which hybridizes in stringent conditions with a probe having the sequence of SEQ ID NO:2. The polypeptide binds to the androgen receptor and increases the ability of the androgen receptor to transactivate gene, sensitive to androgens. These polypeptides can be used to generate antibodies against ARCAP (either monoclonal or polyclonal). These antibodies, in turn, can be used to detect the presence and distribution of ARCAP in tissues and cell compartments. For example, such antibodies can be used to diagnose cancer of the liver tissue by detecting the presence of expression or overexpression ARCAP in the tissue. In addition, they can be used to treat cancer (e.g., liver cancer), as described below.

Further, the hallmark of this invention is an isolated nucleic acid encoding a polypeptide of the present invention, a vector comprising a nucleic acid of the present invention, and a cell in an animal model or in Kul is ur), containing nucleic acid of the present invention. An example of nucleic acid within the present invention includes an isolated nucleic acid chain which hybridizes in harsh environments with single-stranded probe having the sequence of SEQ ID NO:2 or a sequence complementary to SEQ ID NO:2. The length of such a nucleic acid can be at least 15 (e.g., at least, 30, 50, 100, 200, 500 or 1000) nucleotides. These nucleic acids, vectors and cells can be used to produce animal models, cancer diagnosis (e.g., liver cancer) or to obtain polypeptides of the present invention. For example, nucleic acids of the present invention can be used to diagnose liver cancer by determining the presence in the tissue or cell expression or overexpression of mRNA ARCAP. Nucleic acids can be used as primers in detection methods based on PCR or as labeled probes in nucleotide blots (e.g., Northern-blots).

The hallmark of this invention is a transgenic animal (e.g., a rodent, a mouse)whose genome comprises a transgene containing the nucleic acid of the present invention, and shows an increased sensitivity to androgen compared with wild-type animals. Transgenic animal which may be obtained by introducing a nucleic acid of the present invention in the cell so that what nucleic acids obtained the transcript, and the transcript is translated into protein. These transgenic animals can be used as models for drug development for the treatment of cancer (e.g., liver cancer).

In addition, the hallmark of this invention is a method of producing the polypeptide of the present invention by culturing the above-described cells, the implementation of the expression of the polypeptide in the cell and separating the polypeptide from the culture.

Further, the hallmark of this invention is the method of determining the content in the animal sample (e.g. blood sample) of cancer cells. This method comprises obtaining a sample from an animal (such rodent like a mouse) and determining the level of gene expression ARCAP in the specified pattern. If the level of expression of the gene ARCAP in the sample is higher than in the normal sample, this indicates that the animal sample contains cancer cells (e.g. tumor cells of the liver). This method can be used for diagnosing cancer or monitoring cancer treatment in animal model.

In addition, the scope of the present invention includes a method of treating cancer (e.g., liver cancer) in animals (for example, such a rodent like a mouse). This method includes determining whether the animal cancer cell, expressyou the her gene ARCAP, and treatment of the animal a composition that blocks the binding of ARCAP with the androgen receptor or reduces the ability of the androgen receptor to transactivate androgen-sensitive gene. The composition may contain the antibody of the present invention, or antisense nucleic acid of the present invention. This method can be used for screening drugs (including used for gene therapy) and testing their effectiveness in animal models.

The term "essentially pure"as used in this description and in relation to a specific polypeptide means that the polypeptide is essentially free of other biological macromolecules. Essentially pure polypeptide has at least 75% (e.g., at least 80, 85, 95 or 99%) purity on a dry weight basis. Purity may be determined using any appropriate standard method, for example by column chromatography, polyacrylamide gel electrophoresis or analysis by HPLC.

"Conservative amino acid substitution" is a substitution, wherein the amino acid residue is substituted by another residue having a chemically similar side chain. In this field of technology is defined family of amino acid residues having similar side chains. These families include AMI is ocelote with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, Proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

Hybridization in the "stringent conditions" means hybridization at 65°C, 0.5 X SSC, followed by washing at 45°C, 0.1 X SSC.

"Percent identity" of two amino acid sequences or of two nucleic acids is determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990), modified in Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). This algorithm is included in the program NBLAST and XBLAST Altschul et al. (J. Mol. Biol. 215:403-410, 1990). Nucleotide BLAST search is performed using the NBLAST program, score=100, word length=12. Protein BLAST search is performed using the XBLAST program, score=50, word length=3. If two sequences are gaps, using Gapped BLAST, as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When using the programs BLAST and Gapped BLAST apply parameter values default from the respective programs is (for example, XBLAST and NBLAST). Cm. www.ncbi.nlm.nih.gov.

"Isolated nucleic acid" is a nucleic acid the structure of which is not identical to the structure of any naturally occurring nucleic acid or structure of any fragment of a naturally occurring genomic nucleic acid, which unites more than three separate genes. Therefore, the term covers, for example, (a) a DNA which has the sequence of part of a naturally occurring molecule in the genomic DNA, but not adjacent to both coding sequences that flank that part of the molecule in the genome of the organism in which it occurs in nature; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in such a way that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (C) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or restricttions fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e. a gene encoding a hybrid protein. In particular, this definition excludes nucleic acid present in the mixtures of different (i) DNA molecules, (ii) transfected cells, or (iii) the cell to the ons for example, those which are found in the library DNA, such as cDNA library or genomic DNA library.

Other features or advantages of the present invention will become apparent from the following detailed description and from the claims.

DETAILED DESCRIPTION

This invention relates to a transgenic animal other than human, which Express the gene ARCAP, and to methods of use of animals in drug development for treatment or prevention of liver cancer.

In this description, "transgenic animal that differs from person"includes parental transgenic animal other than human, and their offspring, as well as cells and tissues of these animals. Transgenic animal other than human, may be farm animals, such as pigs, goats, sheep, cows, horses and rabbits, rodents, such as rats, Guinea pigs and mice and non-human primates, such as baboons, monkeys, and chimpanzees. Especially the use of transgenic pigs and mice.

Non-human transgenic animal of the present invention contains a nucleic acid encoding a protein ARCAP, integrated into the genome of the animal. In this description, the term "protein ARCAP" refers to a protein ARCAP wild-type or functionally equivalent variant, such as a fragment of alertmessage protein.

It is believed that it may be useful to identify the sequence of introns, if any, in the genome and include them in a nucleic acid encoding a protein ARCAP.

In the transgenic animals of the present invention, non-human nucleic acid operatively linked to a regulatory element, which can activate gene expression ARCAP, for example, in the liver. In this description, the term "operatively linked" refers to this location regulatory element and a nucleic acid, which promotes transcription of the nucleic acid. The regulatory element can be a specific for liver promoter, such as RISK and the albumin promoter.

The hallmark of this invention are expression vectors that are suitable for producing transgenic animals of the present invention, different from man. Expression vectors may include a promoter that is able to mediate expression in the liver, operatively associated with a nucleic acid that encodes a protein ARCAP, as described above.

For introducing expression vectors in non-human animals receiving lines - the founders of the transgenic animals of the present invention, non-human, you can use a variety of methods known in this field. Such methods include, n is limited to, pronuclear microinjection (U.S. patent No. 4873191), retrovirus-mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci. USA, 82:6148, 1985), the positioning of the gene in embryonic stem cells (Thompson et al., Cell 56:313, 1989), electroporation of embryos (Lo, Mol. Cell. Biol., 3:1803, 1983) and the transformation of somatic cells in vitro, followed by transplantation of the nucleus (Wilmut et al., Nature, 385(6619):810-813, 1997; Wakayama et al., Nature, 394:369-374, 1998). In one example, the expression vector introduced by the method of microinjection into the oocyte or embryo is non-human animal, or in embryonic stem cells non-human animal.

After obtaining a transgenic animal other than human (e.g., transgenic mice, obtained in accordance with the Current Protocol (Wiley, USA) and Manuplate the Mouse Embryo (Hogan Beddington Costantini and Lacy, CSHL Press), gene expression ARCAP can be estimated using standard methods. In order to determine whether there was integration of the transgene, an initial screening using the methods southern blot or PCR. Description southern analysis can show, for example, in Sambrook et al., 1989, "Molecular Cloning, A Laboratory Manual", second edition. Cold Spring Harbor Press, Plainview; NY, in sections 9.37-9.52. The expression of a nucleic acid that encodes a protein ARCAP, in the tissues of transgenic animals other than humans can be assessed using techniques that include, but are not ogranichivayas what they analysis of tissue samples obtained from the animal, by means of Northern blot analysis by hybridization in sutu and PCR with reverse transcriptase (RT-PCR).

Transgenic animals of the present invention, non-human, can be used as models of liver cancer. In particular, these animals can be used to identify compounds or compositions that are effective for the treatment or prevention of liver cancer. The compounds or compositions can be identified by introducing a test compound or composition of the transgenic animal of the present invention, other than the person, or by bringing the test compound or composition in contact with the body tissue (e.g., liver) or cells (e.g., liver cells)obtained from a transgenic animal other than human. The effects provided by the test compound or composition on liver cancer transgenic animal other than human, organ, tissue or cells, undergo evaluation. For example, a transgenic animal other than human, you can determine the size of the cancer based on clinical and pathological identification. Test compounds or compositions that reduce the symptoms of cancer, can be effective for the treatment or prevention of cancer.

The pharmaceutical compositions can be recip who are on the basis of the test compounds by mixing these compounds with pharmaceutically acceptable non-toxic excipients or carriers, and they can be introduced transgenic animals of the present invention, non-human, in any way. For example, you can use parenteral routes, such as subcutaneous, intramuscular, intravascular, subcutaneous, intranasal introduction, introduction by inhalation, intrathecal or intraperitoneal, and enteral methods, such as sublingual, oral or rectal administration

It is believed that a person skilled in the art based on the descriptions given here may use the present invention to the full extent without additional development. All citybuses here publications included in this description by reference in their entirety.

OTHER EMBODIMENTS

All the features disclosed in this specification may be combined in any combination. Every sign, disclosed herein, can be replaced by an alternative that serves for the same, equivalent or similar purpose. Thus, unless otherwise stated, each open sign is only an example of the total number of equivalent or similar features.

Proceeding from the above description, the person skilled in the art can easily set the essential characteristics of this invention and, without departing from its spirit and scope, can make various changes and modifications to this invention, adapting it to different applications and conditions. Thus, other embodiments are also included in the scope the following claims.

1. The polypeptide containing the amino acid sequence that is at least 70% identical to SEQ ID NO:3, where the polypeptide binds to the androgen receptor and increases the ability of the androgen receptor to transactivate androgen-sensitive gene.

2. The polypeptide according to claim 1, where the amino acid sequence at least 80% identical to SEQ ID NO:3.

3. The polypeptide according to claim 2, where the amino acid sequence at least 90% identical to SEQ ID NO:3.

4. The polypeptide according to claim 3, where the amino acid sequence at least 95% identical to SEQ ID NO:3.

5. The polypeptide according to claim 4, where the amino acid sequence represents SEQ ID NO:3.

6. The polypeptide containing the amino acid sequence of SEQ ID NO:3 with up to 30 conservative amino acid substitutions, where the specified polypeptide binds to the receptor for the m androgens and increases the ability of the androgen receptor to transactivate androgen-sensitive gene.

7. The polypeptide encoded by the nucleic acid, which hybridizes in stringent conditions with a probe having the sequence of SEQ ID NO:2, where the polypeptide binds to the androgen receptor and increases the ability of the androgen receptor to transactivate androgen-sensitive gene.

8. The selected nucleic acid encoding a polypeptide according to claim 1.

9. Nucleic acid of claim 8, where the amino acid sequence represents SEQ ID NO:3.

10. The selected nucleic acid encoding a polypeptide according to claim 6.

11. The selected nucleic acid containing a chain, which hybridizes in harsh environments with single-stranded probe having the sequence of SEQ ID NO:2, or a sequence complementary to SEQ ID NO:2.

12. Nucleic acid according to claim 11, where the nucleic acid encodes a polypeptide that binds to the androgen receptor and increases the ability of the androgen receptor to transactivate androgen-sensitive gene.

13. Nucleic acid according to item 12, where the amino acid sequence of the polypeptide comprises SEQ ID NO:3.

14. Nucleic acid according to claim 11, where the chain length is at least 15 nucleotides.

15. The expression vector containing the nucleic acid of claim 8.

16. The vector according to item 15, where the encoded amino acid sequence represents SEQ ID NO:3.

18. The expression vector comprising the nucleic acid according to item 11.

19. Vector on p, where this nucleic acid encodes a polypeptide that binds to the androgen receptor and increases the ability of the androgen receptor to transactivate androgen-sensitive gene.

20. A host cell containing the nucleic acid of claim 8.

21. The cell according to claim 20, where the encoded amino acid sequence represents SEQ ID NO:3.

22. A host cell containing the nucleic acid of claim 10.

23. A host cell containing the nucleic acid according to item 11.

24. The cell according to item 23, where this nucleic acid encodes a polypeptide that binds to the androgen receptor and increases the ability of the androgen receptor to transactivate androgen-sensitive gene.



 

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