WO2003008561A2 - Genes associes a l'hyperplasie benigne de la prostate - Google Patents

Genes associes a l'hyperplasie benigne de la prostate Download PDF

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WO2003008561A2
WO2003008561A2 PCT/US2002/023210 US0223210W WO03008561A2 WO 2003008561 A2 WO2003008561 A2 WO 2003008561A2 US 0223210 W US0223210 W US 0223210W WO 03008561 A2 WO03008561 A2 WO 03008561A2
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protein
seq
nucleic acid
expression
agent
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PCT/US2002/023210
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WO2003008561A3 (fr
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Prakash Kulkarni
William E. Munger
Robert H. Getzenberg
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Gene Logic, Inc.
Japan Tobacco, Inc.
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Priority to AU2002317553A priority Critical patent/AU2002317553A1/en
Publication of WO2003008561A2 publication Critical patent/WO2003008561A2/fr
Publication of WO2003008561A3 publication Critical patent/WO2003008561A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the invention relates generally to the changes in gene expression in prostate tissue removed from male patients with benign prostatic hyperplasia (BPH).
  • BPH benign prostatic hyperplasia
  • the invention specifically relates to a human gene family which is differentially expressed in BPH tissue compared to normal prostate tissue.
  • Benign Prostatic Hyperplasia BPH is the most common benign tumor in men over the age of 60. It is estimated that one in four men living to the age of 80 will require treatment for this disease. BPH is usually noted clinically after the age of 50, the incidence increasing with age, but as many as two thirds of men between the ages of 40 and 49 demonstrate histological evidence of the disease.
  • the anatomic location of the prostate at the bladder neck enveloping the urethra plays an important role in the pathology of BPH, including bladder outlet obstruction.
  • Two prostate components are thought to play a role in bladder outlet obstruction. The first is the relative increase in prostate tissue mass. The second component is the prostatic smooth muscle tone.
  • the causative factors of BPH in man have been intensively studied (see Ziada et al (1999) Urology 53, 1-6). In general, the two most important factors appear to be aging and the presence of functional testes. Although these factors appear to be key to the development of BPH, both appear to be nonspecific.
  • the invention includes an isolated nucleic acid molecule that encodes a protein that is expressed in benign prostatic hype ⁇ lasia (BPH) and that exhibits at least about 70% nucleotide sequence identity over the entire contiguous sequence of SEQ ID NO: 1, 3 or 5.
  • the nucleic acids can be operably linked to one or more expression control elements.
  • the invention also includes a vector comprising an isolated nucleic acid molecules of the invention and a host cell transformed to contain the nucleic acid molecules and/or vectors.
  • the host cell can be either a prokaryotic or eukaryotic host cell.
  • the invention further includes a method for producing a polypeptide comprising culturing a host cell transformed with the nucleic acid molecules of the invention under conditions in which the protein encoded by said nucleic acid molecule is expressed.
  • the invention inlcudes an isolated polypeptide or protein encoded by the aforementioned nucleic acids.
  • the invention further includes an isolated polypeptide or protein selected from the group consisting of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4 or 6; an isolated polypeptide comprising a fragment of at least 10 amino acids of SEQ ID NO: 2, 4 or 6, an isolated polypeptide comprising conservative amino acid substitutions of SEQ ID NO: 2, 4 or 6; an isolated polypeptide comprising naturally occurring amino acid sequence variants of SEQ ID NO: 2, 4 or 6; and an isolated polypeptide exhibiting at least about 70% amino acid sequence identity with SEQ ID NO: 2, 4 or 6.
  • the invention includes an isolated antibody or antigen-binding antibody fragment that binds to a polypeptide comprising SEQ ID NO: 2, 4 or 6 or fragments thereof.
  • the antibody can be either a monoclonal or polyclonal antibody.
  • the invention includes a method of identifying an agent which modulates the expression of a nucleic acid encoding a protein comprising SEQ ID NO: 2, 4 or 6 comprising exposing cells which express the nucleic acid to the agent; and determining whether the agent modulates expression of said nucleic acid, thereby identifying an agent which modulates the expression of a nucleic acid encoding the protein.
  • the invention further includes a method of identifying an agent which modulates the level of, or at least one activity of, a protein comprising SEQ ID NO: 2, 4 or 6 comprising exposing cells which express the protein to the agent; and determining whether the agent modulates the level of, or at least one activity of, said protein, thereby identifying an agent which modulates the level of or at least one activity of the protein.
  • the invention also includes a method of identifying binding partners for a protein comprising SEQ ID NO: 2, 4 or 6 comprising exposing said protein to a potential binding partner; and determining if the potential binding partner binds to said protein, thereby identifying binding partners for the protein.
  • the invention further includes a method of modulating the expression of a nucleic acid encoding a protein comprising SEQ ID NO: 2, 4 or 6 comprising administering an effective amount of an agent which modulates the expression of the nucleic acid.
  • the invention includes a method of modulating at least one activity of a protein comprising SEQ ID NO: 2, 4 or 6 comprising administering an effective amount of an agent which modulates at least one activity of the protein.
  • the modulation is effective for the treatment of benign prostatic hyperplasia and the expression and/or activity is up-regulated.
  • the invention includes a method of diagnosing a disease state in a subject, comprising determining the level of expression of a nucleic acid molecule or protein of the invention.
  • the disease state is benign prostatic hyperplasia with or without symptoms.
  • the invention includes a non-human transgenic animal modified to contain a nucleic acid molecules of the invention.
  • the nucleic acid molecule in the transgenic animal can contain a mutation that prevents expression of the encoded protein.
  • the invention includes a composition for inhibiting benign prostatic hyperplasia comprising a diluent and a polypeptide selected from the group consisting of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4 or 6 or fragment thereof.
  • Figure 1 shows the expression of Clone N94303 (SEQ ID NO: 1 or 3) as analyzed using an Affymetrix GeneChip in normal human control prostate and BPH samples, including BPH samples from men without symptoms (without), BPH samples from men who were diagnosed with prostate cancer (Cancer) and BPH samples from men with symptoms (With). In all cases, the subregion of the prostate analyzed in the normal and BPH patient samples was the transitional zone. Averages are shown with error bars representing the standard deviation. N94303 is down-regulated in samples from symptomatic BPH patients and patients with BPH and cancer versus normal control patients.
  • Figure 2 shows the results of semi-quantitative PCR (40 cycles) for the expression of N94303 (SEQ ID NO: 1 or 3) in prostate tissue samples from normal men and from patients with BPH, including BPH samples from men without symptoms, BPH samples from men who were diagnosed with prostate cancer and BPH samples from men with symptoms.
  • N94303 SEQ ID NO: 1 or 3
  • Figure 1 the subregion of the prostate analyzed in the normal and BPH patient samples was the transitional zone.
  • Each sample was assayed at least 3 times and normalized to an internal reference (Cyclophilin A), and the mean and standard deviation from the mean (vertical bars) are shown for each sample. The group average is shown at the right of each group.
  • the data show that expression of SEQ ID NO: 1 or 3 is down-regulated in patients who have BPH with symptoms or BPH with cancer.
  • Figure 3 shows the expression of Clone AA599331 (SEQ ID NO: 5) as analyzed using an Affymetrix GeneChip in normal human control prostate and BPH samples, including BPH samples from men without symptoms (without), BPH samples from men who were diagnosed with prostate cancer (Cancer) and BPH samples from men with symptoms (With). In all cases, the subregion of the prostate analyzed in the normal and BPH patient samples was the transitional zone. Averages are shown with error bars representing the standard deviation. AA599331 is down-regulated in samples from symptomatic BPH patients and patients with BPH and cancer versus normal control patients.
  • Figures 4 and 5 show the results of semi-quantitative PCR (40 cycles) for the expression of AA599331 (SEQ ID NO: 5) in prostate tissue samples from normal men and from patients with BPH, including BPH samples from men without symptoms, BPH samples from men who were diagnosed with prostate cancer and BPH samples from men with symptoms.
  • the subregion of the prostate analyzed in the normal and BPH patient samples was the transitional zone.
  • Each sample was assayed at least 3 times and normalized to an internal reference (Cyclophilin A), and the mean and SDM are shown for each sample. The group average is shown at the right of each group.
  • the data show that expression of SEQ ID NO: 5 is down-regulated in patients who have BPH with I symptoms or BPH with cancer.
  • Figure 6 shows the results of a semi-quantitative PCR assay in which the expression level of AA599331 was measured across a panel of normal human tissues. For each tissue type, the mean +/- SDM is shown as a vertical bar for samples obtained from 3 or more normal individuals. Expression levels in prostate tissue are higher than in all the other tissues tested.
  • Figure 7 illustrates the nucleotide sequence differences among the splice variants in this gene family.
  • N94303, clone HF (SEQ ID NO: 3), contains the longest sequence in the 5' untranslated region.
  • N94303, clone FG7 (SEQ ID NO: 1) contains the longest sequence in the 3' untranslated region, a middle portion of which is spliced out in SEQ ID NO: 3. All three sequences share a region of 312 nucleotides within their coding regions.
  • An additional common sequence of 48 nucleotides is upstream of the coding region in SEQ ID NO: 5, but within the coding region in SEQ ID NO: 1 and 3.
  • the present invention is based in part on the identification of a new gene family that is differentially expressed in human BPH tissue compared to normal human prostate tissue.
  • This gene family corresponds to the human cDNA of SEQ ID NO: 1, 3 and 5.
  • Genes that encode the human protein of SEQ ID NO: 2, 4 and 6 may also be found in other animal species, particularly mammalian species.
  • genes and proteins of the invention may be used as diagnostic agents or markers to detect BPH or the progression of BPH in a sample. They can also serve as a target for agents that modulate gene expression or activity. For example, agents may be identified that modulate biological processes associated with prostate growth, including the hyperplastic process of BPH.
  • the present invention is further based on the development of methods for isolating binding partners that bind to the proteins.
  • Probes based on the proteins are used as capture probes to isolate potential binding partners, such as other proteins.
  • Dominant negative proteins, DNA encoding these proteins, antibodies to these proteins, peptide fragments of these proteins or mimics of these proteins may be introduced into cells to affect function.
  • the proteins provide novel targets for the screening of synthetic small molecules and combinatorial or naturally occurring compound libraries to discover novel therapeutics to regulate prostate function.
  • the Proteins Associated with BPH The Proteins Associated with BPH
  • the present invention provides isolated proteins, allelic variants of the proteins, and conservative amino acid substitutions of the proteins.
  • the "protein” or “polypeptide” refers, in part, to a protein that has the human amino acid sequence depicted in SEQ ID NO: 2, 4 or 6.
  • the terms also refer to naturally occurring allelic variants and proteins that have a slightly different amino acid sequence than that specifically recited above. Allelic variants, though possessing a slightly different amino acid sequence than those recited above, will still have the same or similar biological functions associated with these proteins.
  • the family of proteins related to the human amino acid sequence of SEQ ID NO: 2, 4 or 6 refers to proteins that have been isolated from organisms in addition to humans. The methods used to identify and isolate other members of the family of proteins related to these proteins are described below.
  • the proteins of the present invention are preferably in isolated form.
  • a protein is said to be isolated when physical, mechanical or chemical methods are employed to remove the protein from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated protein.
  • the proteins of the present invention further include insertion, deletion or conservative amino acid substitution variants of SEQ ID NO: 2, 4 or 6.
  • a conservative variant refers to alterations in the amino acid sequence that do not adversely affect the biological functions of the protein.
  • a substitution, insertion or deletion is said to adversely affect the protein when the altered sequence prevents or disrupts a biological function associated with the protein.
  • the overall charge, structure or hydrophobic/hydrophilic properties of the protein can be altered without adversely affecting a biological activity.
  • the amino acid sequence can be altered, for example to render the peptide more hydrophobic or hydrophilic, without adversely affecting the biological activities of the protein.
  • allelic variants, the conservative substitution variants, and the members of the protein family will have an amino acid sequence having at least about 50, 60, 70 or 75% amino acid sequence identity with the sequence set forth in SEQ ID NO: 2, 4 or 6, more preferably at least about 80%, even more preferably at least about 90-95%, and most preferably at least about 95-98% sequence identity.
  • Identity or homology with respect to such sequences is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the known peptides, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity (see section B for the relevant parameters). Fusion proteins, or N-terminal, C-terminal or internal extensions, deletions, or insertions into the peptide sequence shall not be construed as affecting homology.
  • the proteins of the present invention include molecules having the amino acid sequence disclosed in SEQ ID NO: 2, 4 or 6; fragments thereof having a consecutive sequence of at least about 3, 4, 5, 6, 10, 15, 20, 25, 30, 35 or more amino acid residues of these proteins; amino acid sequence variants wherein one or more amino acid residues has been inserted N- or C-terminal to, or within, the disclosed coding sequence; and amino acid sequence variants of the disclosed sequence, or their fragments as defined above, that have been substituted by at least one residue.
  • Such fragments also referred to as peptides or polypeptides, may contain antigenic regions, functional regions of the protein identified as regions of the amino acid sequence which correspond to known protein domains, as well as regions of pronounced hydrophilicity. The regions are all easily identifiable by using commonly available protein sequence analysis software such as Mac Vector (Oxford Molecular).
  • Contemplated variants further include those containing predetermined mutations
  • proteins of other animal species including but not limited to rabbit, mouse, rat, porcine, bovine, ovine, equine and non-human primate species, and the alleles or other naturally occurring variants of the family of proteins; and derivatives wherein the protein has been covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid (for example a detectable moiety such as an enzyme or radioisotope).
  • compositions comprising a protein or polypeptide of the invention and a diluent.
  • Suitable diluents can be aqueous or non-aqueous solvents or a combination thereof, and can comprise additional components, for example water-soluble salts or glycerol, that contribute to the stability, solubility, activity, and/or storage of the protein or polypeptide.
  • members of the family of proteins can be used: (1) to identify agents which modulate the level of or at least one activity of the protein, (2) to identify binding partners for the protein, (3) as an antigen to raise polyclonal or monoclonal antibodies, (4) as a therapeutic agent or target and (5) as a diagnostic agent or marker of BPH and other hyperplastic diseases.
  • nucleic acid is defined as RNA or DNA that encodes a protein or peptide as defined above, is complementary to a nucleic acid sequence encoding such peptides, hybridizes to the nucleic acid of SEQ ID NO: 1, 3 or 5 and remains stably bound to it under appropriate stringency conditions, encodes a polypeptide sharing at least about 50, 60, 70 or 75%, preferably at least about 80%, more preferably at least about 85%, and most preferably at least about 90-95% or 95-98% or more identity with the peptide sequence of SEQ ID NO: 2 or 4 or exhibits at least 50, 60, 70 or 75%, preferably at least about 80%, more preferably at least about 85%, and even more preferably at least about 90-95% or 95-98% or more nucleotide sequence
  • genomic DNA e.g., genomic DNA, cDNA, mRNA and antisense molecules, as well as nucleic acids based on alternative backbones or including alternative bases, whether derived from natural sources or synthesized.
  • hybridizing or complementary nucleic acids are defined further as being novel and unobvious over any prior art nucleic acid including that which encodes, hybridizes under appropriate stringency conditions, or is complementary to nucleic acid encoding a protein according to the present invention.
  • BLAST Basic Local Alignment Search Tool
  • blastp, blastn, blasts, tblastn and tblastx Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402; Karlin et al. (1990) Proc. Natl. Acad. Sci. USA 87, 2264-2268, both fully incorporated by reference
  • the approach used by the BLAST program is to first consider similar segments, with and without gaps, between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a preselected threshold of significance.
  • a preselected threshold of significance For a discussion of basic issues in similarity searching of sequence databases (see, e.g., Altschul et al. (1994) Nature Genetics 6, 119- 129 which is fully incorporated by reference).
  • the search parameters for histogram, descriptions, alignments, expect i.e., the statistical significance threshold for reporting matches against database sequences
  • cutoff i.e., the statistical significance threshold for reporting matches against database sequences
  • matrix and filter low complexity
  • the default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al. (1992) Proc. Natl. Acad. Sci. USA 89, 10915-10919, fully incorporated by reference), recommended for query sequences over 85 nucleotides or amino acids in length.
  • the scoring matrix is set by the ratios of M (i.e., the reward score for a pair of matching residues) to N (i.e., the penalty score for mismatching residues), wherein the default values for M and N are 5 and -4, respectively.
  • “Stringent conditions” are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50 C, or (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42 C.
  • a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42 C.
  • Another example is hybridization in 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfate at 42 C, with washes at 42 C in 0.2x SSC and 0.1% SDS.
  • a skilled artisan can readily determine and vary the stringency conditions appropriately to obtain a clear and detectable hybridization signal.
  • Preferred molecules are those that hybridize under the above conditions to the complement of SEQ ID NO: 1, 3 or 5 and which encode a functional protein.
  • hybridizing molecules are those that hybridize under the above conditions to the complement strand of the open reading frame of SEQ ID NO: 1, 3 or 5.
  • a nucleic acid molecule is said to be "isolated” when the nucleic acid molecule is substantially separated from contaminant nucleic acid molecules encoding other polypeptides.
  • the present invention further provides fragments of the encoding nucleic acid molecule.
  • a fragment of an encoding nucleic acid molecule refers to a small portion of the entire protein coding sequence. The size of the fragment will be determined by the intended use. For example, if the fragment is chosen so as to encode an active portion of the protein, the fragment will need to be large enough to encode the functional region(s) of the protein. For instance, fragments which encode peptides corresponding to predicted antigenic regions may be prepared. If the fragment is to be used as a nucleic acid probe or PCR primer, then the fragment length is chosen so as to obtain a relatively small number of false positives during probing/priming (see the discussion in Section H).
  • Fragments of the encoding nucleic acid molecules of the present invention i.e., synthetic oligonucleotides
  • PCR polymerase chain reaction
  • Fragments of the encoding nucleic acid molecules of the present invention can easily be synthesized by chemical techniques, for example, the phosphoramidite method of Matteucci et al. (1981) J. Am. Chem. Soc. 103, 3185-3191, or using automated synthesis methods, hi addition, larger DNA segments can readily be prepared by well known methods, such as synthesis of a group of oligonucleotides that define various modular segments of the gene, followed by ligation of oligonucleotides to build the complete modified gene.
  • the encoding nucleic acid molecules of the present invention may further be modified so as to contain a detectable label for diagnostic and probe purposes.
  • a detectable label for diagnostic and probe purposes.
  • labels include, but are not limited to, biotin, radiolabeled nucleotides and the like. A skilled artisan can readily employ any such label to obtain labeled variants of the nucleic acid molecules of the invention.
  • nucleic acid molecule having SEQ ID NO: 1, 3 or 5 allows a skilled artisan to isolate nucleic acid molecules that encode other members of the protein family in addition to the sequences herein described. Further, the presently disclosed nucleic acid molecules allow a skilled artisan to isolate nucleic acid molecules that encode other members of the family of proteins in addition to the proteins having SEQ ID NO: 2, 4 or 6.
  • polyclonal antiserum from mammals such as rabbits immunized with the purified protein (as described below) or monoclonal antibodies can be used to probe a mammalian cDNA or genomic expression library, such as lambda gtll library, to obtain the appropriate coding sequence for other members of the protein family.
  • the cloned cDNA sequence can be expressed as a fusion protein, expressed directly using its own control sequences, or expressed by constructions using control sequences appropriate to the particular host used for expression of the enzyme.
  • coding sequence herein described can be synthesized and used as a probe to retrieve DNA encoding a member of the protein family from any mammalian organism. Oligomers containing approximately 18-20 nucleotides (encoding about a 6-7 amino acid stretch) are prepared and used to screen genomic DNA or cDNA libraries to obtain hybridization under stringent conditions or conditions of sufficient stringency to eliminate an undue level of false positives.
  • pairs of oligonucleotide primers can be prepared for use in a polymerase chain reaction (PCR) to selectively clone an encoding nucleic acid molecule.
  • PCR polymerase chain reaction
  • a PCR denature/anneal/extend cycle for using such PCR primers is well known in the art and can readily be adapted for use in isolating other encoding nucleic acid molecules.
  • Nucleic acid molecules encoding other members of the protein family may also be identified in existing genomic or other sequence information using any available computational method, including but not limited to: PSI-BLAST (Altschul et al. (1997)
  • the present invention further provides recombinant DNA molecules (rDNAs) that contain a coding sequence.
  • a rDNA molecule is a DNA molecule that has been subjected to molecular manipulation in situ. Methods for generating rDNA molecules are well l ⁇ iown in the art, for example, see Sambrook et al. (2001) Molecular Cloning - A
  • a coding DNA sequence is operably linked to expression control sequences and/or vector sequences.
  • vector and/or expression control sequences to which one of the protein family encoding sequences of the present invention is operably linked depends directly, as is well known in the art, on the functional properties desired, e.g., protein expression, and the host cell to be transformed.
  • a vector contemplated by the present invention is at least capable of directing the replication or insertion into the host chromosome, and preferably also expression, of the structural gene included in the rDNA molecule.
  • Expression control elements that are used for regulating the expression of an operably linked protein encoding sequence are l ⁇ iown in the art and include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, and other regulatory elements.
  • the inducible promoter is readily controlled, such as being responsive to a nutrient in the host cell's medium.
  • the vector containing a coding nucleic acid molecule will include a prokaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
  • a prokaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
  • a prokaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith
  • Vectors that include a prokaryotic replicon can further include a prokaryotic or bacteriophage promoter capable of directing the expression (transcription and translation) of the coding gene sequences in a bacterial host cell, such as E. coli.
  • a promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.
  • Typical of such vector plasmids are pUC8, pUC9, pBR322 and pBR329 available from BioRad Laboratories (Richmond, CA), pPL and pKK223 available from Pharmacia (Piscataway, NJ).
  • Expression vectors compatible with eukaryotic cells can also be used to form rDNA molecules that contain a coding sequence.
  • Eukaryotic cell expression vectors including viral vectors, are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired DNA segment. Typical of such vectors are pSVL and pKSV-10 (Pharmacia), pBPV-l/pML2d (International Biotechnologies, Inc.), pTDTl (ATCC #31255), the vector pCDM ⁇ described herein, and the like eukaryotic expression vectors. Vectors may be modified to include prostate cell specific promoters if needed.
  • Eukaryotic cell expression vectors used to construct the rDNA molecules of the present invention may further include a selectable marker that is effective in an eukaryotic cell, preferably a drug resistance selection marker.
  • a preferred drug resistance marker is the gene whose expression results in neomycin resistance, i.e., the neomycin phosphotransferase (neo) gene (Southern et al. (1982) J. Mol. Anal. Genet. 1, 327-341).
  • the selectable marker can be present on a separate plasmid, and the two vectors are introduced by co-transfection of the host cell, and selected by culturing in the appropriate drug for the selectable marker.
  • the present invention further provides host cells transformed with a nucleic acid molecule that encodes a protein of the present invention.
  • the host cell can be either prokaryotic or eukaryotic.
  • Eukaryotic cells useful for expression of a protein of the invention are not limited, so long as the cell line is compatible with cell culture methods and compatible with the propagation of the expression vector and expression of the gene product.
  • Preferred ⁇ eukaryotic host cells include, but are not limited to, yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human cell line.
  • Preferred eukaryotic host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells (NTH/3T3) available from the ATCC as CRL 1658, baby hamster kidney cells (BHK), and the like eukaryotic tissue culture cell lines. Any prokaryotic host can be used to express a rDNA molecule encoding a protein of the invention.
  • the preferred prokaryotic host is E. coli.
  • Transformation of appropriate cell hosts with a rDNA molecule of the present invention is accomplished by well known methods that typically depend on the type of vector used and host system employed.
  • electroporation and salt treatment methods are typically employed (see, e.g., Cohen et al. (1972) Proc. Natl. Acad. Sci. USA 69, 2110; Sambrook et al. (2001) supra).
  • electroporation, cationic lipid or salt treatment methods are typically employed (see, e.g., Graham et al. (1973) Virol. 52, 456-459; Wigler et al. (1979) Proc. Natl. Acad. Sci. USA 76, 1373-1376).
  • Successfully transformed cells i.e., cells that contain a rDNA molecule of the present invention
  • cells resulting from the introduction of an rDNA of the present invention can be cloned to produce single colonies. Cells from those colonies can be harvested, lysed and their DNA content examined for the presence of the rDNA using a method such as that described by Southern (1975) J. Mol. Biol. 98, 503-504; Berent et al. (1985) Biotech 3, 208-209, or the proteins produced from the cell assayed via an immunological method.
  • the present invention further provides methods for producing a protein of the invention using nucleic acid molecules herein described.
  • the production of a recombinant form of a protein typically involves the following steps:
  • a nucleic acid molecule that encodes a protein of the invention, such as a nucleic acid molecule comprising, consisting essentially of, or consisting of the coding sequence of SEQ ID NO: 1, 3 or 5; or nucleotides 64-1083 or 64-1080 of SEQ ID NO: 1; or nucleotides 776-1795 or 776-1792 of SEQ ID NO: 3; or nucleotides 1218-1613 or 1218-1610 of SEQ ID NO: 5. If the encoding sequence is uninterrupted by introns, as are these open- reading-frames, it is directly suitable for expression in any host.
  • the nucleic acid molecule is then preferably placed in operable linkage with suitable control sequences, as described above, to form an expression unit containing the protein open reading frame.
  • the expression unit is used to transform a suitable host and the transformed host is cultured under conditions that allow the production of the recombinant protein.
  • the recombinant protein is isolated from the medium or from the cells; recovery and purification of the protein may not be necessary in some instances where some impurities may be tolerated.
  • the desired coding sequences may be obtained from genomic fragments and used directly in appropriate hosts.
  • the construction of expression vectors that are operable in a variety of hosts is accomplished using appropriate replicons and control sequences, as set forth above.
  • the control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the gene and were discussed in detail earlier.
  • Suitable restriction sites can, if not normally available, be added to the ends of the coding sequence so as to provide an excisable gene to insert into these vectors.
  • a skilled artisan can readily adapt any host/expression system known in the art for use with the nucleic acid molecules of the invention to produce recombinant protein.
  • Another embodiment of the present invention provides methods for isolating and identifying binding partners of proteins of the invention, hi general, a protein of the invention is mixed with a potential binding partner or an extract or fraction of a cell under conditions that allow the association of potential binding partners with the protein of the invention. After mixing, peptides, polypeptides, proteins or other molecules that have become associated with a protein of the invention are separated from the mixture. The binding partner that bound to the protein of the invention can then be removed and further analyzed. To identify and isolate a binding partner, the entire protein, for instance a protein comprising the entire amino acid sequence of SEQ ID NO: 2, 4 or 6 can be used. Alternatively, a fragment of the protein can be used.
  • a cellular extract refers to a preparation or fraction which is made from a lysed or disrupted cell.
  • the preferred source of cellular extracts will be cells derived from human prostate tissue or cells, for instance, biopsy tissue or tissue culture cells from BPH.
  • cellular extracts may be prepared from normal tissue or available cell lines, particularly prostate derived cell lines.
  • a variety of methods can be used to obtain an extract of a cell.
  • Cells can be disrupted using either physical or chemical disruption methods. Examples of physical disruption methods include, but are not limited to, sonication and mechanical shearing. Examples of chemical lysis methods include, but are not limited to, detergent lysis and enzyme lysis.
  • a skilled artisan can readily adapt methods for preparing cellular extracts in order to obtain extracts for use in the present methods.
  • the extract is mixed with the protein of the invention under conditions in which association of the protein with the binding partner can occur.
  • a variety of conditions can be used, the most preferred being conditions that closely resemble conditions found in the cytoplasm of a human cell.
  • Features such as osmolarity, pH, temperature, and the concentration of cellular extract used, can be varied to optimize the association of the protein with the binding partner.
  • the bound complex is separated from the mixture.
  • a variety of techniques can be utilized to separate the mixture. For example, antibodies specific to a protein of the invention can be used to immunoprecipitate the binding partner complex. Alternatively, standard chemical separation techniques such as chromatography and density/sediment centrifugation can be used.
  • the binding partner can be dissociated from the complex using conventional methods. For example, dissociation can be accomplished by altering the salt concentration or pH of the mixture.
  • the protein of the invention can be immobilized on a solid support.
  • the protein can be attached to a nitrocellulose matrix or acrylic beads. Attachment of the protein to a solid support aids in separating peptide/binding partner pairs from other constituents found in the extract.
  • the identified binding partners can be either a single protein or a complex made up of two or more proteins. Alternatively, binding partners may be identified using a Far- Western assay according to the procedures of Takayama et al. (1997) Methods Mol. Biol. 69, 171-184 or Sauder et al. (1996) J. Gen. Virol. 77, 991-996 or identified through the use of epitope tagged proteins or GST fusion proteins.
  • the nucleic acid molecules of the invention can be used in a yeast two- hybrid system or other in vivo protein-protein detection system.
  • the yeast two-hybrid system has been used to identify other protein partner pairs and can readily be adapted to employ the nucleic acid molecules herein described.
  • Another embodiment of the present invention provides methods for identifying agents that modulate the expression of a nucleic acid encoding a protein of the invention such as a protein having the amino acid sequence of SEQ ID NO: 2, 4 or 6.
  • Such assays may utilize any available means of monitoring for changes in the expression level of the nucleic acids of the invention.
  • an agent is said to modulate the expression of a nucleic acid of the invention if it is capable of up- or down-regulating expression of the nucleic acid in a cell.
  • cell lines that contain reporter gene fusions between nucleotides from within the open reading frame defined by nucleotides 64-1083 of SEQ ID NO: 1, or nucleotides 776-1795 of SEQ ID NO: 3, or nucleotides 1218-1613 of SEQ ID NO: 5, and/or the 5 'and/or 3 ' regulatory elements and any assayable fusion partner may be prepared.
  • Numerous assayable fusion partners are known and readily available including the firefly luciferase gene and the gene encoding chloramphenicol acetyltransferase (Alam et al. (1990) Anal. Biochem. 188, 245-254).
  • Cell lines containing the reporter gene fusions are then exposed to the agent to be tested under appropriate conditions and time. Differential expression of the reporter gene between samples exposed to the agent and control samples identifies agents which modulate the expression of a nucleic acid of the invention. Additional assay formats may be used to monitor the ability of the agent to modulate the expression of a nucleic acid encoding a protein of the invention, such as the protein having SEQ ID NO: 2, 4 or 6. For instance, mRNA expression may be monitored directly by hybridization to the nucleic acids of the invention. Cell lines are exposed to the agent to be tested under appropriate conditions and time and total RNA or mRNA is isolated by standard procedures such those disclosed in Sambrook et al. (2001) Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory Press.
  • the preferred cells will be those derived from human prostate tissue, for instance, prostate biopsy tissue or cultured prostate cells from normal or BPH patients, for example BRF-55T cells (immortalized human prostate cells obtained from an individual with BPH, see Iype et al. (1998) Int. J. Oncol. 12, 257-263). Alternatively, other available cells or cell lines may be used.
  • Probes to detect differences in RNA expression levels between cells exposed to the agent and control cells may be prepared from the nucleic acids of the invention. It is preferable, but not necessary, to design probes which hybridize only with target nucleic acids under conditions of high stringency. Only highly complementary nucleic acid hybrids form under conditions of high stringency. Accordingly, the stringency of the assay conditions determines the amount of complementarity which should exist between two nucleic acid strands in order to form a hybrid. Stringency should be chosen to maximize the difference in stability between the probe:target hybrid and probe:non-target hybrids. Probes may be designed from the nucleic acids of the invention through methods l ⁇ iown in the art.
  • the G+C content of the probe and the probe length can affect probe binding to its target sequence.
  • Methods to optimize probe specificity are commonly available in Sambrook et al. (2001) supra or Ausubel et al. (1999) Short Protocols in Molecular Biology, John Wiley & Sons.
  • Hybridization conditions are modified using l ⁇ iown methods, such as those described by Sambrook et al. and Ausubel et al. as required for each probe.
  • Hybridization of total cellular RNA or RNA enriched for polyA RNA can be accomplished in any available format.
  • total cellular RNA or RNA enriched for polyA RNA can be affixed to a solid support and the solid support exposed to at least one probe comprising at least one, or part of one of the sequences of the invention under conditions in which the probe will specifically hybridize.
  • nucleic acid fragments comprising at least one, or part of one of the sequences of the invention can be affixed to a solid support, such as a silicon chip, porous glass wafer or membrane.
  • the solid support can then be exposed to total cellular RNA or polyA RNA from a sample under conditions in which the affixed sequences will specifically hybridize.
  • Such solid supports and hybridization methods are widely available, for example, those disclosed by Beattie (1995) WO 95/11755.
  • RNA sample from an untreated cell population and from a cell population exposed to the agent
  • agents which up- or down-regulate the expression of a nucleic acid encoding the protein having the sequence of SEQ ID NO: 2, 4 or 6 are identified.
  • Hybridization for qualitative and quantitative analysis of mRNA may also be carried out by using a RNase Protection Assay (z ' .e., RP A, see Ma et ⁇ /. (1996) Methods 10, 273- 238).
  • an expression vehicle comprising cDNA encoding the gene product and a phage specific DNA dependent RNA polymerase promoter (e.g., T7, T3 or SP6 RNA polymerase) is linearized at the 3' end of the cDNA molecule, downstream from the phage promoter, wherein such a linearized molecule is subsequently used as a template for synthesis of a labeled antisense transcript of the cDNA by in vitro transcription.
  • a phage specific DNA dependent RNA polymerase promoter e.g., T7, T3 or SP6 RNA polymerase
  • the labeled transcript is then hybridized to a mixture of isolated RNA (i.e., total or fractionated mRNA) by incubation at 45°C overnight in a buffer comprising 80% formamide, 40 mM Pipes, pH 6.4, 0.4 M NaCl and 1 mM EDTA.
  • the resulting hybrids are then digested in a buffer comprising 40 ⁇ g/ml ribonuclease A and 2 ⁇ g/ml ribonuclease. After deactivation and extraction of extraneous proteins, the samples are loaded onto urea/polyacrylamide gels for analysis.
  • cells or cell lines are first identified which express the gene products of the invention physiologically.
  • Cell and/or cell lines so identified would be expected to comprise the necessary cellular machinery such that the fidelity of modulation of the transcriptional apparatus is maintained with regard to exogenous contact of agent with appropriate surface transduction mechanisms and/or the cytosolic cascades.
  • such cells or cell lines would be transduced or transfected with an expression vehicle (e.g., a plasmid or viral vector) construct comprising an operable non-translated 5 'promoter-containing end of the structural gene encoding the instant gene products fused to one or more antigenic fragments, which are peculiar to the instant gene products, wherein said fragments are under the transcriptional control of said promoter and are expressed as polypeptides whose molecular weight can be distinguished from the naturally occurring polypeptides or may further comprise an immunologically distinct tag or other detectable marker.
  • an expression vehicle e.g., a plasmid or viral vector
  • Cells or cell lines transduced or transfected as outlined above are then contacted with agents under appropriate conditions.
  • the agent in a pharmaceutically acceptable excipient is contacted with cells in an aqueous physiological buffer such as phosphate buffered saline (PBS) at physiological pH, Eagles balanced salt solution (BSS) at physiological pH, PBS or BSS comprising serum or conditioned media comprising PBS or BSS and/or serum incubated at 37°C.
  • PBS phosphate buffered saline
  • BSS Eagles balanced salt solution
  • Said conditions may be modulated as deemed necessary by one of skill in the art.
  • the cells will be disrupted and the polypeptides of the lysate are fractionated such that a polypeptide fraction is pooled and contacted with an antibody to be further processed by immunological assay (e.g., ELISA, immunoprecipitation or Western blot).
  • immunological assay e.g., ELISA, immunoprecipitation or Western blot.
  • the pool of proteins isolated from the "agent-contacted” sample will be compared with a control sample where only the excipient is contacted with the cells and an increase or decrease in the immunologically generated signal from the "agent-contacted” sample compared to the confrol will be used to distinguish the effectiveness of the agent.
  • Another embodiment of the present invention provides methods for identifying agents that modulate the level or at least one activity of a protein of the invention such as the protein having the amino acid sequence of SEQ ID NO: 2, 4 or 6. Such methods or assays may utilize any means of monitoring or detecting the desired activity.
  • the relative amounts of a protein of the invention between a cell population that has been exposed to the agent to be tested compared to an un-exposed control cell population may be assayed.
  • probes such as specific antibodies are used to monitor the differential expression of the protein in the different cell populations.
  • Cell lines or populations are exposed to the agent to be tested under appropriate conditions and time.
  • Cellular lysates may be prepared from the exposed cell line or population and a control, unexposed cell line or population. The cellular lysates are then analyzed with the probe.
  • Antibody probes are prepared by immunizing suitable mammalian hosts in appropriate immunization protocols using the peptides, polypeptides or proteins of the invention if they are of sufficient length, or, if desired, or if required to enhance immunogenicity, conjugated to suitable carriers. Methods for preparing immunogenic conjugates with carriers such as BSA, KLH, or other carrier proteins are well known in the art. In some circumstances, direct conjugation using, for example, carbodiimide reagents may be effective; in other instances linking reagents such as those supplied by Pierce Chemical Co. (Rockford, IL), may be desirable to provide accessibility to the hapten.
  • the hapten peptides can be extended at either the amino or carboxy terminus with a cysteine residue or interspersed with cysteine residues, for example, to facilitate linking to a carrier.
  • Administration of the immunogens is conducted generally by injection over a suitable time period and with use of suitable adjuvants, as is generally understood in the art.
  • titers of antibodies are taken to determine adequacy of antibody formation.
  • Immortalized cell lines which secrete the desired monoclonal antibodies may be prepared using the standard method of Kohler and Milstein (1975) Nature 256, 495-497 or modifications which effect immortalization of lymphocytes or spleen cells, as is generally known.
  • the immortalized cell lines secreting the desired antibodies are screened by immunoassay in which the antigen is the peptide hapten, polypeptide or protein.
  • the cells can be cultured either in vitro or by production in ascites fluid.
  • the desired monoclonal antibodies are then recovered from the culture supernatant or from the ascites supernatant. Fragments of the monoclonal antibodies or the polyclonal antisera which contain the immunologically significant (antigen-binding) portion can be used as antagonists, as well as the intact antibodies.
  • Use of immunologically reactive (antigen- binding) antibody fragments, such as the Fab, Fab' or F(ab') 2 fragments is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin.
  • the antibodies or antigen-binding fragments may also be produced, using current technology, by recombinant means.
  • Antibody regions that bind specifically to the desired regions of the protein can also be produced in the context of chimeras with multiple species origin, such as humanized antibodies.
  • Agents that are assayed in the above method can be randomly selected or rationally selected or designed.
  • an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences involved in the association of a protein of the invention alone or with its associated substrates, binding partners, etc.
  • An example of randomly selected agents is the use a chemical library or a peptide combinatorial library, or a growth broth of an organism.
  • an agent is said to be rationally selected or designed when the agent is chosen on a nonrandom basis which takes into account the sequence of the target site and/or its conformation in connection with the agent's action.
  • Agents can be rationally selected or rationally designed by utilizing the peptide sequences that make up these sites.
  • a rationally selected peptide agent can be a peptide whose amino acid sequence is identical to or a derivative of any functional consensus site.
  • the agents of the present invention can be, as examples, peptides, small molecules, vitamin derivatives, as well as carbohydrates. Dominant negative proteins, DNAs encoding these proteins, antibodies to these proteins, peptide fragments of these proteins or mimics of these proteins may be introduced into cells to affect function. "Mimic” used herein refers to the modification of a region or several regions of a peptide molecule to provide a structure chemically different from the parent peptide but topographically and functionally similar to the parent peptide (see Grant (1995) Molecular Biology and Biotechnology, VCH Publishers, 659-664). A skilled artisan can readily recognize that there is no limit as to the structural nature of the agents of the present invention.
  • the peptide agents of the invention can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as is known in the art.
  • the DNA encoding these peptides may be synthesized using commercially available oligonucleotide synthesis instrumentation and produced recombinantly using standard recombinant production systems. The production using solid phase peptide synthesis is necessitated if non-gene-encoded amino acids are to be included.
  • Another class of agents of the present invention are antibodies immunoreactive with critical positions of proteins of the invention.
  • Antibody agents are obtained by immunization of suitable mammalian subjects with peptides, containing as antigenic regions, those portions of the protein intended to be targeted by the antibodies.
  • the proteins and nucleic acids of the invention are differentially expressed in BPH tissue.
  • Agents that up- or down- regulate or modulate the expression of the protein or at least one activity of the protein, such as agonists or antagonists, of may be used to modulate biological and pathologic processes associated with the protein's function and activity.
  • a subject can be any mammal, so long as the mammal is in need of modulation of a pathological or biological process mediated by a protein of the invention.
  • the term "mammal" is defined as an individual belonging to the class Mammalia.
  • the invention is particularly useful in the treatment of human subjects.
  • Pathological processes refer to a category of biological processes which produce a deleterious effect.
  • expression of a protein of the invention may be associated with prostate cell growth or hyperplasia.
  • an agent is said to modulate a pathological process when the agent reduces the degree or severity of the process.
  • BPH may be prevented or disease progression modulated by the administration of agents which up- or down-regulate or modulate in some way the expression or at least one activity of a protein of the invention.
  • agents of the present invention can be provided alone, or in combination with other agents that modulate a particular pathological process.
  • an agent of the present invention can be administered in combination with other known drugs.
  • two agents are said to be administered in combination when the two agents are administered simultaneously or are administered independently in a fashion such that the agents will act at the same time.
  • the agents of the present invention can be administered via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route.
  • the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • the present invention further provides compositions containing one or more agents which modulate expression or at least one activity of a protein of the invention. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. Typical dosages comprise 0.1 to 100 ⁇ g/kg body weight. The preferred dosages comprise 0.1 to 10 ⁇ g/kg body weight. The most preferred dosages comprise 0.1 to 1 ⁇ g/kg body weight.
  • compositions of the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically for delivery to the site of action.
  • suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts.
  • suspensions of the active compounds as appropriate oily injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran.
  • the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the agent for delivery into the cell.
  • the pharmaceutical formulation for systemic administration according to the invention may be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulations may be used simultaneously to achieve systemic administration of the active ingredient.
  • Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.
  • the compounds of this invention may be used alone or in combination, or in combination with other therapeutic or diagnostic agents.
  • the compounds of this invention may be coadministered along with other compounds typically prescribed for these conditions according to generally accepted medical practice.
  • the compounds of this invention can be utilized in vivo, ordinarily in mammals, such as humans, sheep, horses, cattle, pigs, dogs, cats, rats and mice, or in vitro.
  • the genes and proteins of the invention are differentially expressed in BPH tissue compared to normal prostate tissue, the genes and proteins of the invention may be used to diagnose or monitor BPH, prostate function, or to track disease progression.
  • One means of diagnosing BPH using the nucleic acid molecules or proteins of the invention involves obtaining prostate tissue from living subjects. Obtaining tissue samples from living sources is problematic for tissues such as prostate. However, due to the nature of the treatment paradigms for BPH, biopsy may be necessary. When possible, urine, blood or peripheral lymphocyte samples may be used as the tissue sample in the assay.
  • genes which are up-regulated in the affected tissue for example the prostate
  • lymphocytes which may be isolated from whole blood.
  • nucleic acid probes comprising all or at least part of the sequence of SEQ ID NO : 1 , 3 or 5 may be used to determine the expression of a nucleic acid molecule in forensic/pathology specimens.
  • nucleic acid assays may be carried out by any means of conducting a transcriptional profiling analysis, a addition to nucleic acid analysis, forensic methods of the invention may target the proteins of the invention, particularly a protein comprising SEQ ID NO: 2, 4 or 6, to determine up- or down-regulation of the genes (Shiverick et al (1975) Biochim. Biophys. Acta 393, 124-133).
  • Methods of the invention may involve treatment of tissues with collagenases or other proteases to make the tissue amenable to cell lysis (Semenov et al. (1987) Biull. Eksp. Biol. Med. 104, 113-116). Further, it is possible to obtain biopsy samples from different regions of the prostate for analysis.
  • Assays to detect nucleic acid or protein molecules of the invention may be in any available format.
  • Typical assays for nucleic acid molecules include hybridization or PCR based formats.
  • Typical assays for the detection of proteins, polypeptides or peptides of the invention include the use of antibody probes in any available format such as in situ binding assays, etc. (see Harlow & Lane (1988) Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory Press).
  • assays are carried-out with appropriate controls.
  • the above methods may also be used in other diagnostic protocols, including protocols and methods to detect disease states in other tissues or organs, for example in tissues in which expression of a nucleic acid molecule of the invention is detected.
  • Transgenic animals containing mutant, knock-out or modified genes corresponding to the cDNA sequence of SEQ ID NO: 1, 3 or 5, or the open reading frame encoding the polypeptide sequence of SEQ ID NO: 2, 4 or 6, or fragments thereof having a consecutive sequence of at least about 3, 4, 5, 6, 10, 15, 20, 25, 30, 35 or more amino acid residues, are also included in the invention.
  • Transgenic animals are genetically modified animals into which recombinant, exogenous or cloned genetic material has been experimentally transferred. Such genetic material is often referred to as a "transgene" in the art.
  • the nucleic acid sequence of the transgene in this case a form of SEQ ID NO: 1, 3 or 5, may be integrated either at a locus of a genome where that particular nucleic acid sequence is not otherwise normally found or at the normal locus for the transgene.
  • the transgene may consist of nucleic acid sequences derived from the genome of the same species or of a different species than the species of the target animal.
  • transgenic animals in which all or a portion of a gene comprising SEQ ID NO: 1, 3 or 5 is deleted may be constructed.
  • the gene corresponding to SEQ JJD NO: 1, 3 or 5 contains one or more introns
  • the entire gene- all exons, introns and the regulatory sequences- may be deleted.
  • less than the entire gene may be deleted.
  • a single exon and/or infron may be deleted, so as to create an animal expressing a modified version of a protein of the invention.
  • germ cell line transgenic animal refers to a transgenic animal in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability of the transgenic animal to transfer the genetic information to offspring. If such offspring in fact possess some or all of that alteration or genetic information, then they too are transgenic animals.
  • the alteration or genetic information may be foreign to the species of animal to which the recipient belongs, foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene.
  • Transgenic animals can be produced by a variety of different methods including transfection, electroporation, microinjection, gene targeting in embryonic stem cells and recombinant viral and refroviral infection (see, e.g., U.S. Patent 4,736,866; U.S. Patent 5,602,307; Mullins et al. (1993) Hypertension 22, 630-633; Brenin et al. (1997) Surg. Oncol. 6, 99-110; Tuan (1997) Recombinant Gene Expression Protocols (Methods in Molecular Biology, Vol. 62), Humana Press.
  • mice A number of recombinant or transgenic mice have been produced, including those which express an activated oncogene sequence (U.S. Patent 4,736,866); express simian SV40 T-antigen (U.S. Patent 5,728,915); lack the expression of interferon regulatory factor 1 (E F- 1) (U.S. Patent 5,731,490); exhibit dopaminergic dysfunction (U.S. Patent 5,723,719); express at least one human gene which participates in blood pressure control (U.S. Patent 5,731,489); display greater similarity to the conditions existing in naturally occurring Alzheimer's disease (U.S. Patent 5,720,936); have a reduced capacity to mediate cellular adhesion (U.S.
  • Patent 5,602,307 possess a bovine growth hormone gene (Clutter et al. (1996) Genetics 143, 1753-1760); or, are capable of generating a fully human antibody response (McCarthy (1997) Lancet 349, 405-406).
  • mice and rats remain the animals of choice for most transgenic experimentation, in some instances it is preferable or even necessary to use alternative animal species.
  • Transgenic procedures have been successfully utilized in a variety of non-murine animals, including sheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits, cows and guinea pigs (see, e.g., Kim et al. (1997) Mol. Reprod. Dev. 46, 515-526; Houdebine (1995) Reprod. Nufr. Dev. 35, 609-617; Petters (1994) Reprod. Fertil. Dev. 6, 643-645; Schnieke et al. (1997) Science 278, 2130-2133; and Amoah (1997) J. Animal Science 75, 578-585).
  • the method of introduction of nucleic acid fragments into recombination competent mammalian cells can be by any method which favors co-transformation of multiple nucleic acid molecules.
  • Detailed procedures for producing transgenic animals are readily available to one skilled in the art, including the disclosures in U.S. Patent 5,489,743 and U.S. Patent 5,602,307.
  • Human tissue was obtained from the transitional zone of the prostate in biopsy samples from normal individuals and from patients with BPH or prostate cancer. BPH was defined histologically in all samples. Normal tissue and asymptomatic BPH samples came from individuals who died of trauma, and did not report symptoms. Patients having BPH with symptoms were defined as those with a need for frequent urination; in these patients a radical prostatectomy had been performed. Prostate cancer patients provided age-matched tissue samples for symptomatic BPH patients, but were without symptoms and without cancer in the transitional zone under histological examination. Microarray sample preparation was conducted with minor modifications, following the protocols set forth in the Affymetrix GeneChip ® Expression Analysis Manual. Frozen tissue was ground to a powder using a Spex Certiprep 6800 Freezer Mill.
  • RNA was extracted from ground tissue or cultured cells with Trizol ® (GibcoBRL) utilizing the manufacturer's protocol. The total RNA yield for each tissue sample was 200-500 ⁇ g per 300 mg tissue weight. mRNA was isolated using the Oligotex mRNA Midi kit ® (Qiagen) followed by ethanol precipitation. Double stranded cDNA was generated from mRNA using the Superscript Choice ® system (GibcoBRL). First strand cDNA synthesis was primed with a T7-(dT24) oligonucleotide. The cDNA was phenol-chloroform extracted and ethanol precipitated to a final concentration of 1 ⁇ g/ ⁇ l. From 2 ⁇ g of cDNA, cRNA was synthesized using Ambion's T7 MegaScript in vitro Transcription Kit ® .
  • cRNA was fragmented (5' fragmentation buffer consisting of 200 mM Tris-acetate (pH 8.1), 500 mM KOAc, 150 mM MgOAc) for thirty-five minutes at 94°C. Following the Affymetrix protocol, 55 ⁇ g of fragmented cRNA was hybridized on a Human chip set and the HuGeneFL array for twenty-four hours at 60 rpm in a 45°C hybridization oven.
  • the chips were washed and stained with Streptavidrn Phycoerythrin (SAPE) (Molecular Probes) in Affymetrix fluidics stations. To amplify staining, SAPE solution was added twice with an anti-streptavidin biotinylated antibody (Vector Laboratories) staining step in between.
  • SAPE Streptavidrn Phycoerythrin
  • Hybridization to the probe arrays was detected by fluorometric scanning (Hewlett Packard Gene Array Scanner). Data was analyzed using Affymetrix GeneChip version 3.0 and Expression Data Mining Tool (EDMT) software (version 1.0).
  • EDMT Expression Data Mining Tool
  • Example 2 Cloning of Full Length Human cDNA Corresponding to Differentially Expressed mRNA
  • the full length cDNA having SEQ ID NO: 5 was obtained by the oligo-pulling method. Briefly, a gene-specific oligo was designed based on the sequence of AA599331. The oligo was labeled with biotin and used to hybridize to 2 ⁇ g of single stranded plasmid DNA (cDNA recombinants) from a placental cDNA library following the procedures of Sambrook et al. (2001) supra. The hybridized cDNA were separated by streptavidin-conjugated beads and eluted by heating.
  • the eluted cDNA was converted to double stranded plasmid DNA and used to transform E. coli cells (DH10B), and the longest cDNA was screened. After positive selection was confirmed by PCR using gene-specific primers, the cDNA clone was subjected to DNA sequencing.
  • the full length cDNA having SEQ ED NO: 1 or 3 was obtained by an iterative PCR method known as Rapid-ScreenTM Arrayed cDNA Library Panels (OriGene Technologies, Inc.).
  • a gene specific oligo was created based on the sequence N94303, and a Rapid- ScreenTM microarray cDNA library panel ("master plate”), formatted in a 96-well tray containing plasmid DNA from approximately 500,000 clones was screened by PCR. Each well of the master plate contained approximately 5,000 clones. The presence of PCR products in each well was determined using agarose gel electrophoresis. For each well from the master plate, there was a corresponding second cDNA library (“subplate”) with the 5,000 clones parsed into 96 wells, each well containing about 50 clones. For each positive well identified in master plate library screening, the corresponding subplate was screened.
  • SEQ ED NO: 1 corresponds to a clone that was isolated from a human heart cDNA library (OriGene).
  • SEQ ED NO: 3 corresponds to a clone that was isolated from a human liver cDNA library (OriGene).
  • clones were confirmed by sequence analysis and by Northern blot analysis.
  • a commercially available Northern blot (ClonTech) was used to determine the size of the transcript(s) that corresponded to a specific clone. Clone inserts matched in size with the Northern blot transcripts.
  • a labeled probe designed from the N94303 sequence a 4.1 kb transcript was identified that corresponded to the 3.4 and 4.0 kb inserts of the N94303 clones.
  • a labeled probe designed from the AA599331 sequence a 1.6 kb transcript was identified which corresponded to the 1.8 kb insert of AA599331.
  • SEQ ED NO: 1 The nucleotide sequence of the full-length human cDNAs corresponding to the differentially regulated mRNA detected above is set forth in SEQ ED NO: 1, 3 and 5.
  • the cDNA comprises 3943 base pairs (3915 base pairs and a polyA tail).
  • This sequence is similar but not identical to cDNA "FLJ13607 fis" (GenBank accession AK023669) from human placenta.
  • the AK023669 sequence lacks the first 15 bases of the sequence of the instant invention, but shows homology over 2466 of 3943 nucleotides of SEQ ED NO: 1.
  • AK000192 was submitted to GenBank without any functional data, tissue expression results or coding region indicated and was not recognized as being associated with BPH.
  • the cDNA comprises 3377 base pairs (3345 base pairs and a polyA tail), while SEQ ED NO: 5 comprises 1665 base pairs (1648 base pairs and a polyA tail).
  • the amino acid sequence corresponding to a predicted protein encoded by SEQ ED NO: 1 is set forth in SEQ ED NO: 2. This sequence is similar in part to an "uncharacterized bone marrow protein" (GenBank Accession AF217515).
  • the "uncharacterized bone marrow protein” sequence shows homology to the sequence set forth in SEQ ED NO: 2 over 179 of 339 amino acids.
  • SEQ ED NO: 4 An open reading frame within the cDNA nucleotide sequence of SEQ ED NO: 3, at nucleotides 776-1792 (776-1795 including the stop codon), also encodes a protein of 339 amino acids.
  • the amino acid sequence corresponding to a predicted protein encoded by SEQ ED NO: 3 is set forth in SEQ ED NO: 4.
  • the protein sequence of SEQ ED NO: 4 is identical to that of SEQ ED NO: 2, although the nucleic acid sequences encoding these proteins differ outside the coding region.
  • SEQ ED NO: 1 and 3 are similar, but not identical to, a sequence disclosed in PCT Publication WO 00/65067, GenBank Accession AX041977. The sequence differences are upstream and downstream of the open reading frame.
  • SEQ ED NO: 5 An open reading frame within the cDNA nucleotide sequence of SEQ ED NO: 5, at nucleotides 1218-1610 (1218-1613 including the stop codon), encodes a protein of 131 amino acids.
  • the amino acid sequence corresponding to a predicted protein encoded by SEQ ED NO: 5 is set forth in SEQ ED NO: 6.
  • the protein sequence of SEQ ED NO: 6 shows partial homology to SEQ ED NO: 2 and 4, the first 104 of 131 amino acids, although the nucleic acid sequences encoding these proteins differ outside the coding region.
  • SEQ ED NO: 5 is similar, but not identical to, a sequence disclosed in PCT Publication WO 01/42467, GenBank Accession AX188302. The sequence differences are upstream and downstream of the open reading frame.
  • SEQ ED NO: 1, 3 and 5 are also similar, but not identical to, human "chromosome 16 clone PR11-340B7" (GenBank accession AC009079). The AC009079 sequence does not disclose an open reading frame or exon/intron boundary positions. Additionally, it lacks the first 12 bases of SEQ ED NO: 5.
  • AC009079 is 199,851 nucleotides in length.
  • SEQ ED NO: 5 Because the coding region of SEQ ED NO: 5 begins at nucleotide 1218, the region of similarity to this GenBank sequence is almost entirely outside the coding region. Furthermore, AC009079 was submitted to GenBank without any functional data, tissue expression results or coding region indicated and was not recognized as being associated with BPH. SEQ ED NO: 6 shows partial similarity to GenBank Accession AF217515, the "uncharacterized bone marrow protein" discussed above.
  • Figures 2, 4 and 5 show the results of semi-quantitative PCR analysis of expression levels of mRNA corresponding to SEQ ED NO: 1 or 3 ( Figure 2) or SEQ ED NO: 5 ( Figures 4 and 5) in prostate tissue samples from normal and diseased human patients. These data also show a significant decrease in N94303 and AA599331 expression in prostate sample from patients with BPH with symptoms versus normal prostate samples.
  • Real time PCR detection was accomplished by the use of the ABI PRISM 7700 Sequence Detection System. The 7700 measures the fluorescence intensity of the sample each cycle and is able to detect the presence of specific amplicons within the PCR reaction.
  • Cyclophilin A detection was performed using Perkin Elmer kit (part 402869) according to the manufacturer's directions. Primers were designed from SEQ ED NO: 1, 3 or 5 using Primer Express, a program developed by PE to efficiently find primers and probes for specific sequences. These primers were used in conjunction with FAM (carboxyfluorescein, Molecular Probes) to measure the expression level of mRNA corresponding to SEQ ED NO: 1, 3 or 5, which was normalized to the Cyclophilin A level in each sample.
  • FAM carboxyfluorescein, Molecular Probes
  • the mean +/- SDM is shown as a vertical bar for samples obtained from experiments done in triplicate. Expression is strongest in the prostate, with moderate levels detected in the heart, testes, bone marrow, liver, placenta, skeletal muscle and stomach. Low levels of expression were also observed in a number of other tissues, although all tissues tested had at least a slight amount detectable.
  • N94303 or AA599331 DNA or of protein products derived from N94303 or AA599331 is likely to be of diagnostic significance in inflammatory responses in tissues in which N94303 or AA599331 is found, and 94303 or AA599331 nucleotides or protein products derived fromN94303 or AA599331 are potential targets for therapeutic intervention.
  • the expression level of mRNA corresponding to SEQ ED NO: 1, 3 or 5 is determined in prostate tissue biopsy samples, in urine or in lymphocytes from blood samples, as described in Example 1 and Figures 1 and 3, i.e., by screening mRNA samples on a GeneChip, or as described in Example 2 and Figures 2, 4 and 5, i.e., by semi-quantitative PCR analysis using the fluorescent detection system.
  • samples from non- prostate hyperplastic tissues in malignant or non-malignant states may also be analyzed.
  • Tissue samples from patients with BPH with symptoms and from normal subjects may be used as positive and negative controls.
  • Using an Affymetrix GeneChip set a level of expression lower than that of the normal control is indicative of BPH or a likelihood of developing BPH.
  • a level of expression below the expression level in normal prostate tissue is indicative of BPH or a likelihood of developing BPH .
  • the down-regulation of N94303 or AA599331 in prostate could specifically identify cases in which inflammation is occurring, which would have additional diagnostic and prognostic implications and utility. More generally for inflammatory conditions, the tissue distribution results indicate that the down-regulation of N94303 or AA599331 is likely to be of diagnostic significance in inflammatory responses in tissues in which these genes are found, and they are a potential target for therapeutic intervention.

Abstract

L'invention concerne, d'une manière générale, les changements d'expression des gênes dans l'hyperplasie bénigne de la prostate. Plus particulièrement, cette invention se rapporte à une famille des gênes humains qui correspond à des espèces d'ARNm qui sont exprimées différentiellement dans l'HBP (hypertrophie bénigne de la prostate) par rapport aux tissus prostatiques normaux.
PCT/US2002/023210 2001-07-19 2002-07-19 Genes associes a l'hyperplasie benigne de la prostate WO2003008561A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006007134A2 (fr) * 2004-06-18 2006-01-19 University Of Pittsburgh Jm-27 constituant un marqueur de l'hypertrophie benigne de la prostate

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [Online] 26 June 2001 OTA ET AL., XP002963841 Database accession no. (AAH18147) *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006007134A2 (fr) * 2004-06-18 2006-01-19 University Of Pittsburgh Jm-27 constituant un marqueur de l'hypertrophie benigne de la prostate
WO2006007134A3 (fr) * 2004-06-18 2006-06-01 Univ Pittsburgh Jm-27 constituant un marqueur de l'hypertrophie benigne de la prostate

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