WO2000065067A2 - Polynucleotides, polypeptides specifiques a la prostate, et leurs procedes d'utilisation - Google Patents

Polynucleotides, polypeptides specifiques a la prostate, et leurs procedes d'utilisation Download PDF

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WO2000065067A2
WO2000065067A2 PCT/US2000/010920 US0010920W WO0065067A2 WO 2000065067 A2 WO2000065067 A2 WO 2000065067A2 US 0010920 W US0010920 W US 0010920W WO 0065067 A2 WO0065067 A2 WO 0065067A2
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prostate
seq
tmprss2
polypeptide
arsdrl
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PCT/US2000/010920
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WO2000065067A3 (fr
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Peter S. Nelson
Leroy Hood
Biaoyang Lin
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University Of Washington
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • This invention relates generally to prostate cancer and, more specifically, to androgen-regulated, prostate-specific, nucleic acid molecules, proteins and antibodies that can be used to diagnose and treat prostate cancer Background of the Invention
  • PSA prostate specific antigen
  • BPH benign prostatic hyperplasia
  • Androgens are a class of C19 steroids that are essential for the development, growth, and maintenance of the prostate -Androgens exert their effects on the prostate target cells via the intracellular androgen receptor (AR)
  • AR intracellular androgen receptor
  • the AR facilitates androgen- induced regulation of genes involved in cellular proliferation and differentiation.
  • the present invention relates to isolated, recombinant polypeptides that are expressed in the prostate gland, and to isolated polynucleotide sequences which are predominantly expressed in the prostate gland, such as the sequences designated SEQ ID NO 1, which encodes ARSDR1, a short chain dehydrogenase/reductase having the ammo acid sequence SEQ ID NO 2, polynucleotide SEQ ID NO 3, which encodes TMPRSS2, a se ⁇ ne protease having the amino acid sequence SEQ ID NO 4. polynucleotide SEQ -ID NO 5, which encodes PART-1, a polypeptide of unknown function having the amino acid sequence SEQ ID NO 1, which encodes ARSDR1, a short chain dehydrogenase/reductase having the ammo acid sequence SEQ ID NO 2, polynucleotide SEQ ID NO 3, which encodes TMPRSS2, a se ⁇ ne protease having the amino acid sequence SEQ ID NO 4. polynucleotide S
  • the present invention provides an isolated polynucleotide capable of hybridizing under stringent conditions to at least 15 contiguous nucleotides of a polynucleotide sequence selected from the group consisting of SEQ ID NO 1, SEQ ID NO 2,
  • the present invention provides a substantially pure polynucleotide probe comprising at least 15 contiguous nucleotides of a polynucleotide sequence selected from the group consisting of SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO
  • the present invention provides a substantially pure polypeptide comprising substantially an ammo acid sequence selected from the group consisting of the sequences shown as SEQ ID NO 2, SEQ ID NO 6, and functional fragments thereof
  • the invention further provides a method of diagnosing or predicting the susceptibility of a prostate neoplastic condition in an individual suspected of having a neoplastic condition of the prostate The method is performed by
  • the method can be performed by obtaining a prostate cell sample from the individual, determining an expression level of one of the inventive polypeptides in the prostate cell sample, and comparing the prostate expression level to a normal expression level of the corresponding inventive polypeptide from normal prostate cells or from an androgen- dependent cell line
  • a 2-fold or more increase in expression of the inventive polypeptide from the prostate cell sample from the individual compared to the normal expression level indicates the presence of a prostate neoplastic condition.
  • This invention is directed to prostate localized polypeptides and encoding polynucleotide molecules Promoter and regulatory regions of the prostate expressed transcripts are also included More specifically, four different androgen-responsive polynucleotides and polypeptides are provided' a polynucleotide having the nucleotide sequence shown in SEQ ID NO 1, that encodes ARSDR1, a short-chain dehydrogenase/reductase 1 having the polypeptide sequence of SEQ ID NO.2; a polynucleotide having the nucleotide sequence shown in SEQ ID NO:3 that encodes TMPRSS2, a prostate-specific serine protease having the amino acid sequence presented in SEQ ID NO:4, and two polynucleotides having the nucleic acid sequences represented in SEQ ID NOS: 5 and 7, respectively, that encode polypeptides of unknown function- Polynucleotide SEQ ID NOS: 5 encodes a polypeptide
  • polypeptides encoded by the androgen-responsive polynucleotides of the present invention are useful as both diagnostic markers for neoplastic conditions of the prostate and as targets for therapy- Polynucleotides corresponding to the expressed transcripts or promoters and regulatory regions are similarly applicable in both diagnostic and therapeutic procedures.
  • the invention is directed to polynucleotide transcripts of an androgen regulated polynucleotide encoded by one of the nucleotide sequences shown in SEQ ID NOS:l, 3, 5, and 7.
  • the invention also pertains to 5' promoter and regulatory regions shown in SEQ ID NO:8 (nucleotides 1 to 3, 113), SEQ ID NO:9, SEQ ID NOT 1 and a 3' untranslated region (UTR) of TMPRSS2 (SEQ ID NO: 10).
  • inventive polynucleotides, fragments of the polynucleotides and short ohgonucleotides corresponding to unique sequences are useful in a variety of diagnostic procedures which employ probe hybridization methods
  • One advantage of employing nucleic acid hybridization in diagnostic procedures is that very little sample can be used because the analyte nucleic acid can be amplified to many copies by, for example, polymerase chain reaction (PCR) or other well known methods in the art for polynucleotide amplification and synthesis.
  • PCR polymerase chain reaction
  • the invention is directed to substantially pure polypeptides and functional fragments thereof that are encoded by the polynucleotides of the invention
  • inventive polypeptides can be used to prepare antibodies ARSDR1, TMPRSS2, and PART-1 specific antibodies can be used, following a variety of methods that are well known in the art, to diagnose prostate cancer
  • the invention is directed to methods for diagnosing prostate neoplastic conditions
  • the short-chain dehydrogenase/reductase of the invention is primarily expressed in prostate cells and becomes elevated in response to androgens
  • the polynucleotide sequences of the present invention are applicable alone or in combination with other molecules, as a specific marker for prostate cells and prostate neoplastic conditions
  • the term "nucleotide” means a monome ⁇ c unit of DNA or RNA containing a sugar moiety (pentose), a phosphate and a nitrogenous heterocyclic base
  • the base is linked to the sugar moiety via the glycosidic carbon (V carbon of pentose) and that combination of base and sugar is called a nucleoside
  • the base characterizes the nucleotide with the four bases of DNA being adenine ("A"), guanine ("G”), cytosine ("C”) and thymine (“T”) Inosine (“I”) is a synthetic base that
  • nucleotide sequences described herein comprise a linear array of nucleotides connected by phosphodiester bonds between the 3' and 5' carbons of adjacent pentoses
  • Oligonucleotide refers to short length single or double stranded sequences of deoxyribonucleotides linked via phosphodiester bonds
  • the ohgonucleotides are chemically synthesized by known methods and purified, for example, on polyacrylamide gels.
  • hybridize under stringent conditions means that a polynucleotide molecule that has hybridized to a target polynucleotide molecule immobilized on a DNA or RNA blot (such as a Southern blot or Northern blot) remains hybridized to the immobilized target molecule on the blot during washing of the blot under stringent conditions
  • exemplary hybridization conditions are- hybridization at 65°C in 5 0 X SSC, 1% sodium dodecyl sulfate, for 16 hours (lower stringency hybridizations preferably utilize 6 0 X SSC, 1% sodium dodecyl sulfate, at 20°C to 30°C for 16 hours)
  • Exemplary very high stringency conditions for washing DNA or RNA blots are two washes of fifteen minutes each at 20°C to 30°C in 2 0 X SSC, followed by two washes of twenty minutes each at 65°C in 0.5
  • polynucleotide refers to a deoxy ⁇ bonucleic acid (DNA) or ⁇ bonucleic acid (RNA) molecule that can optionally include one or more non-native nucleotides, having, for example one or more modifications to the base, sugar, or phosphate portion, or can include a modified phosphodiester linkage
  • polynucleotide includes both single-stranded and double-stranded polynucleotide molecules, which can represent the sense strand anti-sense strand, or both, and includes linear, circular and branched conformations
  • Exemplary polynucleotides include genomic DNA, cDNA, mRNA and ohgonucleotides, corresponding to either the coding or non-coding portion of the molecule
  • a polynucleotide of the invention can additionally contain, if desired, a detectable moiety such as a radiolabel, fluorochrome, ferromagnetic substance, luminescent tag or a detect
  • the term "isolated" in regard to a polynucleotide of the invention is intended to mean a polynucleotide whose structure is not identical to that of any naturally occurring polynucleotide or to that of any fragment of a naturally occurring genomic polynucleotide spanning more than three separate genes
  • the term therefore includes, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs, (b) a polynucleotide incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring extrachromosomally replicating DNA or genomic DNA, (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or
  • ARSDRl refers to a polypeptide termed androgen regulated short-chain dehydrogenase/reductase 1, which has substantially the same amino acid sequence as shown in SEQ ID NO 2 ARSDRl is a member of the short-chain dehydrogenase/reductase superfamily and is predominantly expressed in normal and neoplastic prostate epithelium
  • the ARSDRl polypeptide is encoded by an approximately 2.5 kb message having the nucleic acid sequence represented in SEQ ID NOT
  • the ARSDRl promoter and regulatory region is approximately 3 1 kb in size and has the sequence shown as nucleotides 1 to 3,1 13 of SEQ ID NO 8 (genomic nucleotide sequence of ARSDRl)
  • the ARSDRl promoter contains an androgen response element (ARE) at nucleotides 2,246 to 2,2559 of SEQ ID NO 8 (Roche et al , Mol.
  • ARE androgen response element
  • TMPRSS2 is intended to refer to a polypeptide having substantially the same amino acid sequence as presented in SEQ ID NO 4
  • the TMPRSS2 polypeptide sequence was also previously described by Paoloni-Giacobino et al., Genomics 44 309-329 (1997) Briefly, TMPRSS2 is an androgen-regulated serine protease expressed in normal and neoplastic prostate epithelium
  • the TMPRSS2 polypeptide is encoded by an approximately 3 8 kb message having the nucleic acid sequence shown in SEQ ID NO 3
  • the TMPRSS2 promoter and regulatory region is approximately 0.9 kb in size and has the nucleotide sequence presented in SEQ ID NO:9
  • the TMPRSS2 promoter region contains an androgen response element (ARE) at nucleotides 576 to 590 of SEQ ID NO 9
  • PART-1 refers to a polypeptide termed prostate androgen-regulated transcript, which has substantially the same amino acid sequence as shown in SEQ ID NO 6 PART-1 is encoded by an androgen-regulated cDNA whose nucleotide sequence is represented in SEQ ID NO 5 The PART-1 polypeptide is encoded by an approximately 2 1 kb message The promoter and regulatory region of the polynucleotide encoding PART-1 is contained in an about 2 kb base pair region having the sequence shown in SEQ ID NO 1 1
  • the PART-1 promoter region contains a putative binding site for the homeo-domain containing protein Pbx-la (Van Dijk et al , Proc. Natl. Acad. Sci 90:6061-6065 (1993)) at nucleotides 536 to 544 of SEQ ID NO. l 1
  • fragment as used in reference to a substantially pure polynucleotide of the present invention is intended to refer to a portion of the polynucleotide molecule having the ability to selectively hybridize with the parent polynucleotide molecule
  • selectively hybridize refers to an ability to bind the parent polynucleotide molecule without substantial cross-reactivity with a molecule that is not the parent polynucleotide molecule Therefore, the term includes specific hybridization where there is little or no detectable cross-reactivity with other polynucleotide molecules.
  • a fragment of a polynucleotide of the invention can be used, for example, as a PCR primer to selectively amplify a nucleic acid molecule of the invention, as a selective primer for 5' or 3' RACE to determine additional 5' or 3' sequence of a polynucleotide molecule of the invention, as a selective probe to identify or isolate a polynucleotide of the invention on a RNA or DNA blot, or genomic or cDNA library, or as a selective inhibitor of transcription or translation of an inventive polynucleotide in a tissue, cell or cell extract.
  • GenBank Expressed Sequence Tags are specifically excluded as fragments of the invention.
  • ARSDRl related fragments AA 035790, AA 442517, AA 587226,
  • PN_10Dll_bd.rl Al 660243, Al 674580, AA 225818, AA 534046, D25996, AA 876896. Also excluded as a fragment of the invention is the 216 bp nucleic acid described in Paoloni-Giacobino et al , supra
  • a fragment of a polynucleotide molecule of the invention includes at least about 15 contiguous nucleotides from the reference polynucleotide or a complementary sequence thereto, can include at least about 16, 17, 18, 19, 20 or at least 25 nucleotides, often includes at least about 30, 40, 50, 100, 300 or 500 nucleotides, and can include up to the full length of the reference polynucleotide molecule minus one nucleotide Fragments of such lengths are able to selectively hybridize with the subject polynucleotide in a variety of detection formats described herein
  • the term "functional fragment,” when used in reference to a polynucleotide comprising the ARSDRl polynucleotide (SEQ ID NO 8), is intended to refer to any portion of the ARSDRl polynucleotide having at least one of the biological activities of the subject polynucleotides
  • a functional fragment can be a portion of the polynucleotide
  • a functional fragment when used in reference to the 5' promoter and regulatory region of TMPRSS2 (SEQ ID NO 9) is intended to refer to a portion of SEQ ID NO 10 having at least one of the activities of its parent polynucleotide molecule
  • a functional fragment of SEQ ID NO 9 may contain an ARE located at nucleotides 576-590 of SEQ ID NO 9 that exhibits increased expression upon androgen exposure
  • the term "functional fragment" when used in reference to a polypeptide of the invention is intended to refer to a peptide fragment that is a portion of a full length polypeptide, provided that the portion has a biological activity that is characteristic of the corresponding full length polypeptide
  • the term is also intended to include polypeptides that include, for example, modified forms of naturally occurring amino acids such as D-steroisomers, non-naturally occurring amino acids, amino acid analogues and mimetics so long as such polypeptides retain functional activity as defined below.
  • ARSDRl polypeptide functional fragment when used in reference to anARSDRl polypeptide, refers to any peptide sequence which can be identified using the binding and routine methods, such as bioassays described herein
  • An ARSDRl polypeptide functional fragment can be, for example, a NAD(H)/NADP(H) binding site referenced herein as amino acids 44 to 50 of SEQ ID NO 2 or a catalytic activity site referenced as amino acids 198 to 202 of SEQ ID NO 2
  • the term "functional fragment" when used in reference to a PART-1, or TMPRSS2 polypeptide is intended to refer to a portion of the polypeptide which retains some or all of prostate-specifity and androgen regulated expression of the full length polypeptides shown in SEQ ID NOS 4 and 6
  • nucleotide sequence as used herein in reference to a polynucleotide of the invention, is intended to mean one of the sequences shown as SEQ ID NOS. l, 3, 5, 7, 8, 9, 10, and 1 1 or a similar, non-identical sequence that is considered by those skilled in the art to be a functionally equivalent sequence.
  • a polynucleotide sequence that has one or more nucleotide additions, deletions or substitutions with respect to the subject polynucleotide is encompassed by the invention, so long as the polynucleotide sequence encodes the same amino acid sequence or retains its ability to selectively hybridize with the subject polynucleotide
  • a polynucleotide having substantially the sequence of one of the subject polynucleotides can encode, for example, an isotype variant or species homolog
  • a polynucleotide having substantially the nucleotide sequence of the reference polynucleotide has at least 60% identity with respect to the reference nucleotide sequence.
  • a polynucleotide having substantially the same nucleotide sequence of the reference polynucleotide can have at least 70%, at least 90%>, or at least 95% identity to the reference nucleotide sequence.
  • Sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing sequences of the two sequences over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window”, as used herein, refers to a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned-
  • Optimal alignment of sequences for comparison may be conducted by local identity or similarity algorithms such as those described in Smith and Waterman (Adv. Appl Math. 2:482 (1981)), by the homology alignment algorithm of Needleman and Wunsch, (J.
  • Gapped BLAST is utilized as described in Altschul et al., (Nucleic Acids Res.
  • percent similarity is a statistical measure of the degree of relatedness of two compared protein sequences
  • the percent similarity is calculated by a computer program that assigns a numerical value to each compared pair of amino acids based on chemical similarity (e.g., whether the compared amino acids are acidic, basic, hydrophobic, aromatic, etc.) and/or evolutionary distance as measured by the minimum number of base pair changes that would be required to convert a codon encoding one member of a pair of compared amino acids to a codon encoding the other member of the pair. Calculations are made after a best fit alignment of the two sequences have been made empirically by iterative comparison of all possible alignments- (Henikoff et al., 1992 Proc. Natl. Acad. Sci. USA 89: 10915-10919).
  • substantially identical of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 60%> sequence identity, preferably at least 70%, more preferably at least 80% and most preferably at least 90%, compared to a reference sequence using the programs described above (preferably BLAST) using standard parameters.
  • sequence identity preferably at least 70%, more preferably at least 80% and most preferably at least 90%
  • BLAST BLAST sequence identity
  • Polypeptides which are "substantially similar" share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes.
  • Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine
  • a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine
  • a group of amino acids having amide-containing side chains is asparagine and glutamine
  • a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan
  • a group of amino acids having basic side chains is lysine, arginine, and histidine
  • a group of amino acids having sulfur-containing side chains is cysteine and methionine-
  • Preferred conservative amino acids substitution groups are: valine-leucine- isoleucine, phenylalanine
  • substantially the amino acid sequence when used in reference to a TMPRSS2 polypeptide is intended to refer to any amino acid sequence having at least about 56% identity with respect to the reference amino acid sequence shown as SEQ ID NO:4.
  • a polypeptide having substantially the same amino acid sequence as the reference polypeptide can have, for example, 60%, 70%, 80%, 90% or more amino acid sequence identity to the reference amino acid sequence shown as SEQ ID NO:4.
  • Amino acid sequence identity can be determined, for example, in the following manner- The portion of the amino acid sequence of TMPRSS2 (SEQ ED NO:4) extending from amino acid 1 up to and including amino acid 492 is used to search a nucleic acid sequence database, such as the Genbank database, using the program BLASTP version 2.0.9 (Altschul et al., 1997 Nucleic Acids Res. 25:3389-3402).
  • the term "substantially the amino acid sequence" when used in reference to an ARSDRl polypeptide is intended to refer to any amino acid sequence having at least about 26% identity with respect to the reference amino acid sequence shown as SEQ ID NO:2.
  • a polypeptide having substantially the same amino acid sequence as the reference polypeptide can have, for example, 30%, 40%), 50%, 60%, 70%), 80%), 90%) or more amino acid sequence identity to the reference amino acid sequence shown as SEQ ID NO:2.
  • a polypeptide having substantially the amino acid sequence of the reference polypeptide retains comparable functional and biological activity characteristic of the reference polypeptide. It is recognized, however, that polypeptides, or encoding nucleic acids, containing less than the described levels of sequence identity arising as splice variants or that are modified by conservative amino acid substitutions are also encompassed within the scope of the present invention.
  • the term "probe” is intended to refer to a single-stranded polynucleotide, or analogs thereof, that has a sequence of nucleotides that includes at least 10, at least 20, at least 50, at least 100, at least 200, at least 300, at least 400, or at least 500 contiguous bases that are the same as or the complement of any contiguous bases set forth in any of SEQ ID NOS: l, 3, 5, 7, 8, 9, 10 and 11.
  • the entire sequence corresponding to SEQ ID NOS: l, 3, 5, 7, 8, 9, 10 and 1 1 can be used as a probe.
  • a probe has the ability to selectively hybridize to its subject polynucleotide molecule and can be labeled by methods well-known in the art, as described hereinafter, and used, for example, in various diagnostic kits.
  • antibody encompasses polyclonal and monoclonal antibody preparations, as well as preparations including hybrid antibodies, altered antibodies, F(ab') 2 fragments, F(ab) molecules, Fv fragments, single domain antibodies, chimeric antibodies and functional fragments thereof which exhibit immunological binding properties of the parent antibody molecule.
  • the term "monoclonal antibody” refers to an antibody composition having a homogeneous antibody population- The term is not limited by the manner in which it is made. The term encompasses whole immunoglobulin molecules, as well as Fab molecules, F(ab') 2 fragments, Fv fragments, and other molecules that exhibit immunological binding properties of the parent monoclonal antibody molecule. Methods of making polyclonal and monoclonal antibodies are known in the art and described more fully below.
  • the term "antigen” is defined herein to include any substance that may be specifically bound by an antibody molecule-
  • An "immunogen” is an antigen that is capable of initiating lymphocyte activation resulting in an antigen-specific immune response.
  • epitope is used herein to mean a site on an antigen to which specific B-cells and T-cells respond.
  • the term is also used interchangeably with "antigenic determinant” or "antigenic determinant site.”
  • a peptide epitope can comprise 3 or more amino acids in a spatial conformation unique to the epitope. Generally, an epitope consists of at least 5 such amino acids and, more usually, consists of at least 8-10 such amino acids. Methods of determining spatial conformation of amino acids are known in the art and include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance spectroscopy. Furthermore, the identification of epitopes in a given protein is readily accomplished using techniques well known in the art.
  • a "standard” as used herein is a quantitative or qualitative measurement of a compound at a known concentration used for comparing samples with unknown concentrations of the same or related compounds- Preferably, it is based on a statistically appropriate number of samples and is created to use as a basis of comparison when performing diagnostic assays Diagnostic assays may in turn be used for monitoring clinical trials, or following patient treatment profiles
  • proliferating condition is intended to refer to a benign or malignant and metastatic prostate lesion of proliferating cells
  • primary prostate tumors are classified into stages TX, TO, Tl, T2, T3, and T4
  • Metastatic prostate cancer is classified into stages Dl, D2, and D3
  • the term is also intended to include prostate neoplasma
  • sample is intended to mean any biological fluid, cell, tissue, organ or portion thereof, that includes or potentially includes nucleic acids and polypeptides of the invention
  • samples present in an individual can be a histologic section of a specimen obtained by biopsy, or cells that are placed in or adapted to tissue culture
  • a sample further can be a subcellular fraction or extract, or a crude or substantially pure nucleic acid or protein preparation
  • a sample can be prepared by methods known in the art suitable for the particular format of the detection method
  • detectable label refers to a molecule that renders a nucleic acid of the invention detectable by an analytical method
  • An appropriate detectable label depends on the particular assay format and are well known by those skilled in the art
  • a detectable label specific for a polynucleotide molecule can be a complementary polynucleotide molecule, such as a hybridization probe, that selectively hybridizes to the polynucleotide molecule
  • a hybridization probe can be labeled with a measurable moiety, such as a radioisotope, fluorochrome, chemiluminescent marker, biotin, or other moiety known in the art that is measurable by analytical methods
  • a detectable label also can be a polynucleotide molecule without a measurable moiety
  • PCR or RT-PCR primers can be used without conjugation to selectively amplify all or a desired portion of the polynucleotide molecule
  • binding agent when used in reference to ARSDRl, TMPRSS2, and PART-1 polypeptides is intended to mean a compound, a macromolecule, including polypeptide, DNA, RNA and carbohydrate that selectively binds a reference polypeptide or fragment thereof
  • a binding agent can be a polypeptide that selectively binds with high affinity or avidity to the polypeptides of the present invention, without substantial cross-reactivity with other polypeptides that are unrelated to the reference polypeptide
  • the affinity of a binding agent that selectively binds to a reference polypeptide will generally be greater than about 10 "5 M and more usually greater than about 10 "6 M High affinity interactions can be preferred, and will generally be greater than about 10 "8 M to 10 "9 M
  • Specific samples of such selective binding agents include a polyclonal or monoclonal antibody specific or selective for a polypeptide of the present invention or a peptide, polynucleotide, nucleic acid, nucleic acid
  • the term "expression level" when used in reference to ARSDRl, TMPRSS2, and PART-1 is intended to refer to the extent, amount or rate of synthesis of the nucleotide sequences shown as SEQ ID NOS 1, 3, 5, 8, 9, 10 and 1 1 or the polypeptides shown as SEQ ID NOS 2, 4 and 6
  • the extent, amount or rate of synthesis can be determined by measuring the accumulation or synthesis of the reference RNA, reference polypeptide or by measuring the reference polypeptide activity
  • the term "analog" when used in reference to a short-chain dehydrogenase/reductase substrate is intended to mean any agent which can be oxidized or reduced in the presence of ARSDRl
  • the short-chain dehydrogenase/reductase substrate analog can be a heterocyclic organic compound having minor modifications of the short-chain dehydrogenase/reductase substrate amino acid sequence
  • the term "about” when used in reference to a particular activity or measurement is intended to refer to the referenced activity or measurement as being within a range of values encompassing the referenced value and within accepted standards of a credible assay within the art, or within accepted statistical variance of a credible assay within the art
  • the term "analog" when used in reference to a serine protease substrate is intended to mean any agent which is cleaved at about the same rate in the presence of TMPRSS2 as the referenced polypeptide
  • the serine protease substrate analog can be a peptide having minor modifications of the serine protease substrate amino acid sequence
  • inhibitor when used in reference to ARSDRl is intended to refer to an agent effecting a decrease in the extent, amount or rate of ARSDRl expression or effecting a decrease in the activity of ARSDRl .
  • one group of inhibitors which decrease the activity of ARSDRl include short-chain dehydrogenase/reductase inhibitors
  • Specific examples of short-chain dehydrogenase/reductase inhibitors include, for example, steroids, steroid derivatives and analogs.
  • ARSDRl inhibitors which effect a decrease in ARSDRl expression include ARSDRl antisense polynucleotides and transcriptional inhibitors that bind to the ARSDRl 5' promoter and regulatory region
  • the term "inhibitor" when used in reference to TMPRSS2 is intended to refer to an agent effecting a decrease in the extent, amount or rate of TMPRSS2 expression or effecting a decrease in the activity of TMPRSS2 activity
  • one group of inhibitors which decrease the activity of TMPRSS2 include serine protease inhibitors
  • serine protease inhibitors include, for example, antitrypsin and antithrombin
  • TMPRSS2 inhibitors which effect a decrease in TMPRSS2 expression include TMPRSS2 antisense polynucleotides and transcriptional inhibitors that bind to the TMPRSS2 5' promoter/regulatory region
  • inhibitory amount is intended to refer to the amount of an inhibitor necessary to effect a reduction of at least about 2-fold in the extent, amount or rate of transcription and/or protein synthesis and/or activity
  • the term "reduced coenzyme” when used in reference to ARSDRl is intended to refer to a coenzyme that has been reduced during a dehydrogenation reaction mediated by ARSDRl During the dehydrogenation reaction the substrate is oxidized by the removal of two hydrogen atoms from the substrate One of the removed hydrogen atoms is directly transferred to the coenzyme, thereby reducing the coenzyme, for example, nicotinamide-adenine dinucleotide (NAD + ) to NADH or nicotinamide- adenine dinucleotide phosphate (NADP ⁇ ) to NADPH
  • NAD + nicotinamide-adenine dinucleotide
  • NADP ⁇ nicotinamide- adenine dinucleotide phosphate
  • the term “non- reduced coenzyme” is intended to refers to the ARSDRl coenzyme in its oxidized form, for example, NAD + or
  • the term "substrate” when used in reference to ARSDRl is intended to refer to the non-oxidized state of a reactant that is known to become oxidized in an -ARSDRl -catalyzed reaction
  • product when used in reference to ARSDRl as used herein is intended to refer to a reactant in an oxidized state that is the product of a dehydrogenation reaction catalyzed by ARSDRl Isolation of Prostate-Specific cDNA Clones
  • the androgen-regulated prostate specific polynucleotide molecules of the present invention were identified by hybridization screening of prostate mRNA against a diverse population of prostate derived probes which were immobilized in a two-dimensional array.
  • a complete description of the methods used for identification, cloning and sequencing of transcripts (SEQ ID NOs.l, 3, 5, and 7) are set forth in the Example sections corresponding to each of the referenced polynucleotides
  • two-dimensional microarrays containing a diverse set of prostate derived cDNAs were screened using RNA from a prostate cell line A non-redundant set of 1500 prostate-derived cDNA clones was identified from the Prostate Expression Database
  • the inserts of the cDNAs were amplified with primers BL_ml3F (5'-GTAAAACGACGGCCAGTGAATTG-3') (SEQ ID NO 12) and BL_ml3R (5'-ACACAGGAAACAGCTATGACCATG-3') (SEQ ID NO.13) utilizing PCR and spotted onto glass microscope slides to form a microarray
  • the microarrays of prostate derived cDNAs were screened using total RNA isolated from LNCap cells cultured for 72 hours either in the presence or absence of androgen Hybridized microarray slides were scanned with an Array Scanner Generation II
  • ARSDRl Microarray hybridization with androgen-stimulated and androgen-starved LNCaP cDNA probes revealed four cDNAs, designated ARSDRl, TMPRSS2, PART-1 and clone 8C3 whose expression was consistently up-regulated using the above criteria Sequence analysis and BLAST searches against the GenBank databases identified ARSDRl, PART-1 and 8C3 cDNAs as novel genes Sequence database analysis of the TMPRSS2 cDNA revealed it to be identical to a previously identified serine protease gene that had been mistakenly designated as by expressed in a small intestine-specific fashion (Paoloni-Giacobino et al , Geno ics 44 309-329 (1997)) The polynucleotide and polypeptide sequences of the present inventive are generally described in the following sections corresponding to each of the prostate-specific, androgen-regulated polynucleotides of the present invention ARSDRl ARSDRl is a multi
  • nucleotide sequence of the ARSDRl promoter and regulatory region was found to include two progesterone response elements (PREs) at nucleotides 2,175 to 2, 189 and 2,627 to 2,641 of SEQ ID NO 8, respectively
  • the PREs are about 15 nucleotides in length with substantial similarity to consensus PREs (Lieberman et al, Mol. Endorcrinol. 7:515-527 (1993)).
  • the promoter and regulatory region contains binding sites for various transcription and related regulatory factors-
  • the domains carrying these binding sites are functional as binding agents in a variety of methods known in the art to inhibit or identify factors which bind to one or more of these domains in a sequence specific manner.
  • These domains are also useful to construct expression vectors which confer, for example, tissue specificity and androgen regulation- That is the ARSDRl promoter and regulatory region (as well as the other polynucleotide promoter regions of the present invention) can be use to make prostate-specific, androgen regulated expression vectors that comprising the inventive promoter operably linked to a heterologous nucleotide sequenc.
  • Functional fragments of the ARSDRl promoter and regulatory region which independently exhibit one or more binding activities or other transcriptional activity of the full length sequence are therefore considered functional fragments of the promoter and regulatory region.
  • Specific examples are portions of the ARSDRl nucleotide sequence containing the ARE sequence, set forth as nucleotides 2,246 to 2,259 in SEQ ID NO: 8, and the PRE sequences set forth as nucleotides 2,175 to 2,189 and 2,627 to 2,641 in SEQ ID NO:8.
  • ARSDRl is a member of the short-chain dehydrogenase/reductase (SDR) family of proteins.
  • SDR are a large family of NAD(H) or NADP(H) dependent oxidoreductases.
  • SDR family of proteins include many enzymes involved in steroid metabolism including, for example, estradiol 17-beta-dehydrogenase, human 15- hydroxyprostaglandin dehydrogenase, and 11-beta-hydroxisteroid dehydrogenase (Jornvall et al., Biochemistry 34:6003-6013(1995)).
  • Proteins belonging to the SDR family share amino acid residue identities of only 15-30%, indicating early evolutionary divergence.
  • the ARSDRl polypeptide consists of about 318 amino acid residues having the sequence shown in SEQ ID NO:2. Two consensus sequences are conserved in the SDR family, the NAD(H) or NADP(H) binding domain, a N-terminal segment GlyXXXGLYXXGly (SEQ ID NO: 14), and the catalytic domain, a sequence TyrXXXLys (SEQ ID NO: 15). (Jornvall et al., supra, 1995; Ghosh et al., Structure 2:629-640(1994)). The ARSDRl polypeptide contains both of these motifs conserved in the SDR family.
  • ARSDRl also contains two Asn-glycosylation sites at amino acid positions 174 and 198 (SEQ ID NO:2) that are conserved among SDR family proteins. Another characteristic of ARSDRl is that it contains two protein kinase C pfaosphorylation sites at amino acid positions 57 and 106 (SEQ ID NO:2). TMPRSS2
  • TMPRSS2 is encoded by a transcript of about 4,650 nucleotides in length.
  • a complete description of the methods used for identification, cloning and sequencing of the full length transcript is set forth below in Examples 8-11
  • the complete nucleotide sequence of the TMPRSS2 encoding transcript is shown in SEQ ID NO 3 and the deduced amino acid sequence is shown in SEQ ID NO 4
  • the full length transcript contains a 5' untranslated region (UTR) of 56 nucleotides and a 3' UTR consisting of 3, 115 nucleotides
  • TMPRSS2 encoding polynucleotide sequence described herein extends the Paoloni-Giacobino et al sequence by about 2, 172 nucleotides at the 3' terminus
  • the new TMPRSS2 3 1 UTR sequence is shown as nucleotides 914 to 3,118 in SEQ ID NO 10
  • cDNA clone 10D11 which consisted of 2,681 nucleotides in length Clone 10D11 begins about 286 nucleotides 5' to the translation stop codon of TMPRSS2 and terminates about 723 nucleotides from the 3' end of the full length transcript (see SEQ ID NO 3) Therefore, clone 10D11 contains a region of 1,4
  • TMPRSS2 full length transcript and fragments described above
  • a polynucleotide corresponding to the 5' promoter and regulatory region was additionally isolated and sequenced The method used for identifying this genomic sequence is described further below in Example 11
  • the isolated promoter/regulatory region is about 869 nucleotides in length and is set forth in SEQ ID NO 9
  • TMPRSS2 was identified as a prostate specific and androgen regulated polynucleotide Consistent with these functional characteristics, the nucleotide sequence of the TMPRSS2 promoter/regulatory region was found to include an ARE The ARE is located at nucleotides 576 to 590 of SEQ ID NO 9 and is an about 15 nucleotide sequence with substantial similarity to consensus AREs
  • the promoter/regulatory region contains binding sites for various transcription and related regulatory factors
  • the domains carrying these binding sites are functional as binding agents in a variety of methods known in the art to inhibit or identify factors which bind to one or more of these domains in a sequence specific manner
  • These domains are also useful to construct expression vectors which confer, for example, tissue specificity and androgen regulation Fragments of the TMPRSS2 promoter/regulatory region which independently exhibit one or more binding activities or other transcriptional activity of the full length sequence are therefore considered functional fragments of the promoter/regulatory region
  • a specific example is a TMPRSS2 polynucleotide fragment containing the ARE sequence set forth as nucleotides 576 to 590 of SEQ ID NO 9
  • TMPRSS2 is a multidomain serine protease that is predominantly expressed in prostate tissue
  • the polypeptide consists of 492 amino acid residues in length and includes functional domains for serine protease activity, a scavenger receptor cysteine
  • TMPRSS2 active site residues have been identified as His296, Asp345, and Ser441 and cleavage specificity has been deduced to hydrolyze peptide bonds after Lys or Arg residues due to the presence of Asp435 at the base of the SI subsite which binds to the substrate (SEQ ID NO 4)
  • TMPRSS2 contains nine conserved cysteine residues with the intersubunit disulfide bond between Cys758-Cys912 (SEQ ID NO 4) joining the catalytic protease subunit with the non-protease domains of the polypeptide
  • the amino- terminal He residue of the protease domain is included within the peptide sequence Arg- Ile-Val-Gly-Gly (RIVGG), which is characteristic for the proteolytic activator site of many serine protease zymogens (Rawlings and Barrett, supra)
  • TMPRSS2 contains a hydrophobic sequence at amino acids 84-106 of SEQ ID NO 4 that is characteristic of a transmembrane domain (Hofmann, K , and Stoffel, W, Biol Chem. Hoppe-Seyler 847 166 (1993) The transmembrane is not preceded by a peptide leader sequence, indicating that TMPRSS2 is a type II integral membrane proteins in which the amino terminus is located on the cytoplasmic side of the membrane (Parks, G.D., and Lamb, R A , J. Biol. Chem. 268 19101-19109 (1993))
  • TMPRSS2 contains a third region characteristic of a low-density lipoprotein receptor A domain (LDLRA domain) This domain extends from Cysl 13 to Cysl48 in TMPRSS2 (SEQ ID NO 4)
  • LDLRA domain low-density lipoprotein receptor A domain
  • a characteristic LDLRA domain is about 40 amino acids long and contains 6 disulfide-bonded cysteines (S ⁇ dhoff et al , Science 228 815-822 (1985)) These cysteines have been identified in TMPRSS2 as amino acid residues 113, 120, 126, 133, 139, and 148 (SEQ ID NO 4)
  • TMPRSS2 also contains a scavenger receptor cysteine-rich domain (SRCR) SRCR domains characteristically are about 100 amino acids long and rich in cysteine (Resnick et al , Trends Bwchem. Sci. 19 5-8 (1994))
  • the SRCR domain of TMPRSS2 corresponds to amino acid residues Vail 49 to Leu242 (SEQ ID NO 4)
  • the SRCR domain of TMPRSS2 contains a consensus sequence characteristic of group A SRCR Proteins with SRCR domains are known to be expressed either on the cell surface or secreted into plasma or other body fluids PART-1
  • PART-1 is an androgen-regulated polypeptide that is predominantly expressed in prostate tissue
  • the PART-1 polypeptide consists of 60 amino acid residues and has two protein kinase phosphorylation sites as well as one tyrosine kinase phosphorylation site
  • the PART-1 polypeptide is encoded by an approximately 2 1 kb messenger RNA (SEQ ID NO 2)
  • SEQ ID NO 3 The PART-1 promoter region (SEQ ID NO 3) is approximately 2 0 kb in size and contains a binding site for the homeo-domain containing protein Pbx-la at nucleotides 536 to 544 of SEQ ID NO 12 as described by Van Dijk et al , Proc. Natl. Acad.
  • nucleotide sequence corresponding to the PART-1 cDNA combined with a portion of its 5' promoter and regulatory region has been described as clone 14D7
  • the nucleotide sequence of this clone therefore is a composite sequence of the about 2,106 nucleotide PART-1 cDNA and the about 603 nucleotide 5' promoter and regulatory region of the PART-1 transcription unit
  • PART-1 polynucleotide having the nucleotide sequence shown as SEQ ID NO.2
  • SEQ ID NO.2 a polynucleotide corresponding to the 5' promoter and regulatory region was isolated The method used for identifying the nucleotide sequence of the 5' promoter and regulatory region is described further below in the Example 15
  • the isolated promoter/regulatory region polynucleotide is about 1969 nucleotides in length and is set forth in SEQ ID NO 11
  • PART-1 was identified as a prostate-specific and androgen regulated transcript
  • the PART-1 Pbx-la binding site region is shown in SEQ ID NO 11 as nucleotides 536 to 544
  • the PART-1 promoter/regulatory region contains binding sites for various transcription and related regulatory factors
  • the domains carrying these binding sites are functional as binding agents in a variety of methods known in the art to inhibit or identify factors which bind to one or more of these domains in a sequence specific manner
  • 8C3 is an androgen-regulated transcript of about 4,500 nucleotides in length that is predominantly expressed in prostate tissue-
  • the 8C3 polynucleotide was identified by hybridization screening of prostate mRNA against a diverse population of prostate derived probes which were immobilized in a two-dimensional array. A complete description of the methods used for identification, cloning and sequencing of the 8C3 polynucleotide is set forth below in the Example 1.
  • the 8C3 cDNA has been mapped to 16Q24, a region of the human genome that has been shown to experience a high incidence of chromosomal loss in advanced prostate cancer- Therefore, it is likely that 8C3 is a tumor suppressor-
  • the nucleotide sequence of the 8C3 encoding transcript is shown in SEQ ID NO:7-
  • the -ARSDRl, TMPRSS2, 8C3 and PART-1 polynucleotide and polypeptide sequences of the invention are collectively referred to herein as the polynucleotides and polypeptides of the invention, respectively-
  • substantially pure polynucleotides are provided that are capable of hybridizing under stringent conditions to at least 15 contiguous nucleotides of the nucleotide sequences shown as SEQ ID NOS: l, 3, 5 and 7, or complementary sequences thereof-
  • substantially pure polynucleotides having substantially the nucleotide sequences shown as SEQ ID NOS: l, 3, 5, 7, 8, 9, 10 and 11, or functional fragments thereof are also provided-
  • Functional fragments of SEQ ID NOS:8, 9 and 11 may contain, for example, a 5' promoter or a transcription regulatory region, such, as for example, an androgen regulatory element-
  • the promoter and regulatory regions of the present invention contain binding sites for various transcription and related regulatory factors-
  • the domains carrying these binding sites are functional as binding agents in a variety of methods known in the art to inhibit or identify factors which bind to one or more of these domains in a sequence specific manner. These domains are also useful to construct expression vectors which confer, for example, tissue specificity and androgen regulation.
  • Functional fragments of the ARSDRl, TMPRSS2 and PART-1 promoter and regulatory regions which independently exhibit one or more binding activities or other transcriptional activity of the full length sequence are therefore considered functional fragments of the inventive promoter and regulatory regions.
  • the probes can be as long as the full length transcript or as short as about 10-15 nucleotides, and preferably about 15-18 nucleotides. They can correspond to coding region or untranslated region sequence. The particular application and degree of desired specificity will be one consideration well known to those skilled in the art in selecting a probe.
  • probes and low stringency hybridization conditions can be chosen Alternatively, using high stringency conditions with the same probe will select only polynucleotides that actually encode the referenced inventive polypeptide Untranslated region sequences are useful regions to construct probes since there is little evolutionary pressure to conserve non-coding domains
  • probes as small as 15 nucleotides are statistically unique sequences within the human genome Therefore, fragments of the inventive sequences that are generally of 15 nucleotides or more in length can be constructed from essentially any region of the transcript or promoter and regulatory region and be capable of uniquely hybridizing to ARSDRl, TMPRSS2, PART-1 and 8C3 polynucleotides
  • the probes can be produced recombinantly or chemically synthesized using methods well known in the art Additionally.
  • ARSDRl , TMPRSS2, PART- 1 and 8C3 hybridization probes can be labeled with a variety of detectable labels including, for example, radioisotopes, fluorescent tags, reporter enzymes, biotin and other ligands
  • detectable labels can additionally be coupled with, for example, colorimetric or photometric indicator substrate for spectrophotometric detection Methods for labeling and detecting such probes are well known in the art and can be found described in, for example, Sambrook et al , Molecular Cloning A Laboratory Manual. 2nd ed , Cold Spring Harbor Press, Plainsview, New York (1989), and Ausubel et al , Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999)
  • the invention further provides a substantially pure polynucleotide probe having substantially the nucleotide sequence of SEQ ID NOS 1, 3, 5, 7, 8, 9, 10 and 11, or fragment thereof
  • a fragment of the above referenced polynucleotide probes having substantially the sequence of SEQ ID NOS 1, 3, 5, 7, 8, 9, 10 and 11 can, for example, be an oligonucleotide of about 15-18 nucleotides in length.
  • the present invention is directed to isolated prostate-specific polypeptides (such as polypeptides encoded by the polynucleotide molecules of the present invention) that are androgen regulated
  • the polypeptides of the present invention can be isolated, for example, by incorporating a polynucleotide molecule of the invention (such as a cDNA molecule) into an expression vector, introducing the expression vector into a host cell and expressing the polynucleotide molecule to yield polypeptide.
  • proteinase inhibitors include serine proteinase inhibitors (such as phenylmethylsulfonyl fluoride (PMSF), benzamide, benzamidine HCI, ⁇ -Amino-/->-caproic acid and aprotinin (Trasylol)), cysteine proteinase inhibitors, such as sodium -hydroxymercuribenzoate, competitive proteinase inhibitors, such as antipain and leupeptin, covalent proteinase inhibitors, such as iodoacetate and N-ethylmaleimide, aspartate (acidic) proteinase inhibitors, such as pepstatin and diazoacetylnorleucine methyl ester (DAN), metalloproteinase inhibitors, such as EGTA [ethylene glycol) inhibitors, ethylene glycol, glycerin, glycerin, glycerin, cysteine proteinase inhibitors, such as sodium -hydroxymercuribenzoate
  • E. coli XLl-Blue harboring a Bluescript vector including a cD ⁇ A molecule of interest can be grown overnight at 37°C in LB medium containing 100 ⁇ g ampicillin/ml
  • a 50 ⁇ l aliquot of the overnight culture can be used to inoculate 5 ml of fresh LB medium containing ampicillin, and the culture grown at 37°C with vigorous agitation to A-- 00 - 0.5 before induction with 1 mM IPTG
  • the suspension is centrifuged (1000 x g, 15 min, 4°C), the media removed, and the pelleted cells resuspended in 1 ml of cold buffer that preferably contains 1 mM EDTA and one or more proteinase inhibitors, such as those described herein
  • polypeptide fragments of the inventive proteins can be prepared using peptide synthesis methods that are well known in the art
  • the synthetic polypeptide fragment can then be used to prepare antibodies that are specific to any one of the proteins of the present invention Direct peptide synthesis using solid-phase techniques (Stewart et al., Solid-Phase Peptide Synthesis, W H Freeman Co, San Francisco Calif (1969), Merrifield, J. Am. Chem. Soc.
  • polypeptide sequences of the present invention or any fragment thereof may be mutated during direct synthesis and, if desired, combined using chemical methods with other amino acid sequences
  • the polypeptides used to induce specific antibodies may have an amino acid sequence consisting of at least five amino acids and preferably at least 10 amino acids. Short stretches of amino acid sequence may be attached with those of another polypeptide, and the chimeric polypeptide used for antibody production- Alternatively, the polypeptide may be of sufficient length to contain an entire domain for antibody recognition-
  • Representative examples of art-recognized techniques for purifying, or partially purifying, polypeptides from biological material are exclusion chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, reversed-phase chromatography and immobilized metal affinity chromatography-
  • Hydrophobic interaction chromatography and reversed-phase chromatography are two separation methods based on the interactions between the hydrophobic moieties of a sample and an insoluble, immobilized hydrophobic group present on the chromatography matrix.
  • hydrophobic interaction chromatography the matrix is hydrophilic and is substituted with short-chain phenyl or octyl nonpolar groups-
  • the mobile phase is usually an aqueous salt solution.
  • the matrix is silica that has been substituted with longer /?-alkyl chains, usually C 8 (octylsilyl) or C ⁇ 8 (octadecylsilyl).
  • the matrix is less polar than the mobile phase.
  • the mobile phase is usually a mixture of water and a less polar organic modifier.
  • Separations on hydrophobic interaction chromatography matrices are usually done in aqueous salt solutions, which generally are nondenaturing conditions. Samples are loaded onto the matrix in a high-salt buffer and elution is by a descending salt gradient. Separations on reversed-phase media are usually done in mixtures of aqueous and organic solvents, which are often denaturing conditions.
  • hydrophobic interaction chromatography depends on surface hydrophobic groups and is carried out under conditions which maintain the integrity of the polypeptide molecule
  • Reversed-phase chromatography depends on the native hydrophobicity of the polypeptide and is carried out under conditions which expose nearly all hydrophobic groups to the matrix, i.e., denaturing conditions.
  • Ion-exchange chromatography is designed specifically for the separation of ionic or ionizable compounds.
  • the stationary phase (column matrix material) carries ionizable functional groups, fixed by chemical bonding to the stationary phase. These fixed charges carry a counterion of opposite sign. This counterion is not fixed and can be displaced.
  • Ion-exchange chromatography is named on the basis of the sign of the displaceable charges. Thus, in anion ion-exchange chromatography the fixed charges are positive and in cation ion-exchange chromatography the fixed charges are negative.
  • Retention of a molecule on an ion-exchange chromatography column involves an electrostatic interaction between the fixed charges and those of the molecule, binding involves replacement of the nonfixed ions by the molecule.
  • Elution in turn, involves displacement of the molecule from the fixed charges by a new counterion with a greater affinity for the fixed charges than the molecule, and which then becomes the new, nonfixed ion
  • Solid-phase packings used in ion-exchange chromatography include cellulose, dextrans, agarose, and polystyrene
  • the exchange groups used include DEAE (diethylaminoethyl), a weak base, that will have a net positive charge when ionized and will therefore bind and exchange anions, and CM (carboxymethyl), a weak acid, with a negative charge when ionized that will bind and exchange cations
  • Another form of weak anion exchanger contains the PEI (polyethyleneimine) functional group.
  • polystyrene matrix can be obtained with quaternary ammonium functional groups for strong base anion exchange or with sulfonic acid functional groups for strong acid cation exchange
  • Intermediate and weak ion-exchange materials are also available- Ion-exchange chromatography need not be performed using a column, and can be performed as batch ion-exchange chromatography with the slurry of the stationary phase in a vessel such as a beaker
  • Gel filtration is performed using porous beads as the chromatographic support.
  • a column constructed from such beads will have two measurable liquid volumes, the external volume, consisting of the liquid between the beads, and the internal volume, consisting of the liquid within the pores of the beads. Large molecules will equilibrate only with the external volume while small molecules will equilibrate with both the external and internal volumes.
  • a mixture of molecules (such as proteins) is applied in a discrete volume or zone at the top of a gel filtration column and allowed to percolate through the column. The large molecules are excluded from the internal volume and therefore emerge first from the column while the smaller molecules, which can access the internal volume, emerge later.
  • the volume of a conventional matrix used for protein purification is typically 30 to 100 times the volume of the sample to be fractionated.
  • the absorbance of the column effluent can be continuously monitored at a desired wavelength using a flow monitor A technique that is often applied to the purification of polypeptides is High
  • HPLC Performance Liquid Chromatography
  • polyclonal antibodies specific for a purified protein can be raised in a New Zealand rabbit implanted with a whiffle ball One ⁇ g of protein is injected at intervals directly into the whiffle ball granuloma A representative injection regime is injections (each of 1 ⁇ g protein) at day 1, day 14 and day 35. Granuloma fluid is withdrawn one week prior to the first injection (preimmune serum), and forty days after the final injection (postimmune serum)
  • An antibody is specific for one of the inventive proteins if it is produced against an epitope of the polypeptide and binds to at least part of the natural or recombinant polypeptide
  • Antibody production includes not only the stimulation of an immune response by injection into animals, but also analogous processes such as the production of synthetic antibodies, the screening of recombinant immunoglobulin libraries for specific-binding molecules (Orlandi et al , Proc. Natl. Acad. Sci.
  • the invention also provides a method of diagnosing or predicting the susceptibility of a prostate neoplastic condition in an individual suspected of having a neoplastic condition of the prostate
  • the method comprises (a) obtaining a fluid sample from an individual, (b) determining the expression level of a polypeptide selected from the group of polypeptides whose amino acid sequences are shown in SEQ ID NOS 2, 4, and 6, or a polynucleotide selected from the group of polynucleotides whose nucleotide sequences are shown in SEQ ID NOS 1, 3, 5, 7, 8, 9, 10, and 11, and
  • a method of diagnosing or predicting the susceptibility of a prostate neoplastic condition in an individual suspected of having a neoplastic condition of the prostate is provided The method is performed by
  • a prostate neoplastic condition is a benign or malignant prostate lesion of proliferating cells
  • Prostate neoplastic conditions include, for example, prostate intraepithelial neoplasia (PIN) and prostate cancer
  • Prostate cancer is an uncontrolled proliferation of prostate cells which can invade and destroy adjacent tissues as well as metastasize.
  • Primary prostate tumors can be classified into stages TX, TO, Tl, T2, T3, and T4 and metastate tumors can be classified into stages Dl, D2 and D3 Similarly, there are classifications known by those skilled in the art for the progressive stages of precancerous lesions or PIN The methods herein are applicable for the diagnosis or treatment of any or all stages of prostate neoplastic conditions
  • the methods of the invention are also applicable to prostate pathologies other than neoplastic conditions
  • Such other pathologies include, for example, benign prostatic hyperplasia (BPH) and prostatitis
  • BPH benign prostatic hyperplasia
  • prostatitis BPH is one of the most common diseases in adult males Histological evidence of BPH has been found in more than 40% of men in their fifties and almost 90% of men in their eighties The disease results from the accumulation of non-malignant nodules arising in a small region around the proximal segment of the prostatic urethra which leads to an increase in prostate volume If left untreated, BPH can result in acute and chronic retention of urine, renal failure secondary to obstructive uropathy, serious urinary tract infection and irreversible bladder decompensation
  • Prostatitis is an infection of the prostate
  • Other prostate pathologies known to those skilled in the art exist as well and are similarly applicable for diagnosis or treatment using the methods of the invention
  • the methods of the invention are applicable to both prostate neoplastic conditions and prostate pathologies
  • the invention provides a method of diagnosing or predicting prostate neoplastic conditions based on the finding of a positive correlation between -ARSDRl, TMPRSS2, PART-1 and 8C3 polypeptide or polynucleotide expression in neoplastic cells of the prostate and the degree or extent of the neoplastic condition or pathology
  • the diagnostic methods of the invention are applicable to numerous prostate neoplastic conditions and pathologies as described above
  • One consequence of progression into these neoplastic and pathological conditions is an increased expression of at least one of the -ARSDRl, TMPRSS2, PART-1 and 8C3 polypeptides or polynucleotides in prostate tissue as well as secretion into the circulatory system and urine
  • the polypeptide or polynucleotide of the present invention can be used qualitatively to positively identify a prostate neoplastic condition or pathology as described above
  • the inventive reagents also can be used quantitiatively to determine the degree or susceptibility of a prostate neoplastic condition or pathology
  • successive increases in the expression levels of at least one of ARSDRl, TMPRSS2, PART-1 or 8C3, including levels of secreted polypeptide in circulating fluids and urine can be used as a predictive indicator of the degree or severity of a prostate neoplastic condition or pathology because increased expression, leading to a rise in accumulated levels, for example, also positively correlates with increased severity of a neoplastic condition of the prostate
  • the higher the level of expression of any one of ARSDRl, TMPRSS2, PART-1 or 8C3, the later the stage of the prostate neoplastic condition or pathology For example, increases in expression levels of two-fold or more compared to a
  • Correlative increases can be determined by comparison of expression of at least one of ARSDRl, TMPRSS2, PART-1 or 8C3 from the individual having, or suspected of having a neoplastic condition of the prostate to expression levels of the corresponding polypeptide or polynucleotide from known samples determined to exhibit a prostate neoplastic condition
  • correlative increases also can be determined by comparison of expression of at least one of ARSDRl, TMPRSS2, PART-1 or 8C3 from the test individual to expression levels of other known markers of prostate cancer such as prostate specific antigen (PSA), glandular kallikrein 2 (hK2) and prostase/PRSS18
  • PSA prostate specific antigen
  • hK2 glandular kallikrein 2
  • prostase/PRSS18 these other known markers can be used, for example, as an internal or external standard for correlation of stage-specific expression with increases in expression of any one of ARSDRl, TMPRSS2, PART-1 or 8C3 and severity of the
  • a regression in the severity of a prostate neoplastic condition or pathology is followed by a corresponding decrease in expression levels of at least one of ARSDRl, TMPRSS2, PART-1 or 8C3 and can similarly be assessed using the methods described above
  • the invention provides a method for both diagnosing and prognosing a prostate neoplastic condition including prostate cancer and prostate interepithelial neoplasia as well as other prostate pathologies such as BPH and prostatitis
  • ARSDRl TMPRSS2, PART-1 or 8C3 expression levels in prostate cells, the circulatory system and urine as a diagnostic indicator of a prostate pathology
  • the methods are applicable to any males, generally those over age 50, African-American males and males with familial history of prostate neoplastic conditions or pathologies
  • the diagnostic methods of the invention also are applicable to individuals predicted to be at risk for prostate neoplastic conditions or pathologies by reliable prognostic indicators prior to onset of overt clinical symptoms.
  • individuals suspected of having a prostate neoplastic condition or pathology can be identified by exhibiting presenting signs of prostate cancer which include, for example, a palpable nodule (which generally occurs in greater than 50% of the cases), dysuria, cystitis and prostatitis, frequency, urinary retention, or decreased urine stream Signs of advanced disease include pain, uremia, weight loss and systemic bleeding.
  • Prognostic methods of this invention are applicable to individuals after diagnosis of a prostate neoplastic condition, for example, to monitor improvements or identify residual neoplastic prostate cells using, for example, imaging methods known in the art and which targets at least one of ARSDRl, TMPRSS2, PART-1 or 8C3 polypeptides or polynucleotides
  • the invention provides a method of predicting the onset of a prostate neoplastic condition or pathology
  • the method consists of determining increased expression levels of at least one of ARSDRl, TMPRSS2, PART-1 and 8C3 in a prostate cell sample or in fluids from an individual having or suspected of having a prostate neoplastic condition or pathology compared to a sample isolated from a normal individual, where increased expression in the sample indicates the onset of the prostate neoplastic condition or pathology
  • the diagnostic methods of the invention are applicable for use with a variety of different types of samples isolated or obtained from an individual having, or suspected of having a prostate neoplastic condition or prostate pathology-
  • samples applicable for use in one or more diagnostic formats of the invention include tissue and cell samples- A tissue or cell sample can be obtained, for example, by biopsy or surgery.
  • the tissue can be used whole or subjected to various methods known in the art to disassociate the sample into smaller pieces, cell aggregrates or individual cells- Additionally, when combined with amplification methods such as polymerase chain reaction (PCR), a single prostate cell sample is sufficient for use in diagnostic assays of the invention which employ hybridization detection methods- Similarly, when measuring levels of any one of ARSDRl, TMPRSS2, and PART-1 polypeptide or activity levels, amplification of the signal with enzymatic coupling or photometric enhancement can be employed using only a few or a small number of cells. Whole tissue obtained from a prostate biopsy or surgery is one example of a prostate cell sample.
  • amplification methods such as polymerase chain reaction (PCR)
  • amplification of the signal with enzymatic coupling or photometric enhancement can be employed using only a few or a small number of cells.
  • Whole tissue obtained from a prostate biopsy or surgery is one example of a prostate cell sample.
  • Whole tissue prostate cell samples can be assayed employing any of the formats described below.
  • the prostate tissue sample can be mounted and hybridized in situ with a polynucleotide probe of the present invention- Similar histological formats employing protein detection methods and in situ activity assays also can be used to detect polypeptides of the invention in whole tissue prostate cell samples.
  • Polypeptide detection methods include, for example, staining with antibodies specific for at least one of the inventive polypeptides and activity assays which result in the deposition of an ARSDRl, TMPRSS2, or PART-1 end product at the site of enzyme activity in the sample.
  • Such histological methods as well as others are well known to those skilled in the art and are applicable for use in the diagnostic methods of the invention using whole tissue as the source of a prostate cell sample. Methods for preparing and mounting the samples are similarly well known in the art.
  • prostate cells and cell aggregates from an individual having, or suspected of having a prostate neoplastic condition or pathology is another example of a prostate cell sample which can be analyzed for increased expression of ARSDRl, TMPRSS2, PART-1 or 8C3, polypeptide or polynucleotide or activity.
  • the cells can be grown in culture and analyzed in situ using procedures such as those described above.
  • the expression level can be determined by, for example, binding agents specific for ARSDRl, TMPRSS2, or PART-1 polypeptides, or by hybridization to a probe specific to at least one of ARSDRl, TMPRSS2, PART-1 and 8C3 polynucleotides.
  • Other methods for measuring the expression level of the inventive polypeptides or polynucleotides in whole cell samples are known in the art and are similarly applicable in any of the diagnostic formats described below.
  • tissue or whole cell prostate cell sample obtained from an individual also can be analyzed for increased expression of at least one of ARSDRl, TMPRSS2, PART-1 or 8C3 by lysing the cell and measuring the expression levels of an inventive polypeptide or polynucleotide in the lysate, a fractionated portion thereof or a purified component thereof using any of diagnostic formats described below
  • RNA from one or more of the inventive polynucleotides can be amplified directly from the lysate using PCR, or other amplification procedures well known in the art such as RT-PCR, 5' or 3' RACE to directly measure the expression levels of at least one of ARSDRl, TMPRSS2, PART-1 or 8C3 RNA also can be isolated and probed directly such as by solution hybridization or indirectly by hybridization to immobilized RNA.
  • lysates can be assayed directly, or they can be further fractionated to enrich for the inventive polypeptides and their corresponding activities
  • Numerous other methods applicable for use with various cell fractions are well known to those skilled in the art and can accordingly be used in the methods of the invention.
  • the prostate tissue or cell sample can be obtained directly from the individual or, alternatively, it can be obtained form other sources for testing Similarly, the cell sample can be tested when it is freshly isolated or it can be tested following short or prolonged periods of cryopreservation without substantial loss in accuracy or sensitivity If the sample is to be tested following an indeterminate period of time, it can be obtained and then cryopreserved, or stored at 4°C for short periods of time, for example
  • An advantage of the diagnostic methods of the invention is that they do not require histological analysis of the sample As such, the sample can be initially disaggregated, lysed, fractionated or purified and the active component stored for later diagnosis
  • the diagnostic methods of the invention are applicable for use with a variety of different types of samples other than prostate cell samples
  • intracellular polynucleotides and polypeptides of the invention may leak into the extracellular space when a neoplastic prostate condition causes a disruption of the normal prostate architecture. Therefore, the diagnostic methods of the invention are applicable with fluid samples collected from an individual having, or suspected of having a neoplastic condition of the prostate or a prostate pathology
  • Fluid samples which can be measured for ARSDRl, TMPRSS2, PART-1 or 8C3 expression levels include, for example, blood, serum, lymph, urine and semen
  • Other bodily fluids are known to those skilled in the art and are similarly applicable for use as a sample in the diagnostic methods of the invention
  • One advantage of analyzing fluid samples is that they are readily obtainable, in sufficient quantity, without invasive procedures as required by biopsy and surgery Analysis of fluid samples such as blood, serum and urine will generally be in the diagnostic formats described above and below which measure ARSDRl, TMPRSS2, or PART-1 polypeptide levels or activity
  • the inventive polypeptides are circulating in bodily fluids, the methods will be similar to those which measure expression lev els from cell lysates, fractionated portions thereof or purified components
  • Prostate neoplastic conditions and prostate pathologies can be diagnosed, predicted or prognosed by measuring the expression levels of the polynucleotides and polypeptides of the present invention in a prostate cell sample, circulating fluid or other bodily fluid obtained from the individual
  • expression levels can be measured by a variety methods known in the art
  • the expression level of a nucleic acid of the invention can be determined by measuring the amount of an RNA or polypeptide of the invention in a sample from the individual
  • the expression level of the inventive polypeptides can be determined by measuring the amount of enzyme activity in the sample, the amount of activity being indicative of the expression level of the inventive polynucleotide
  • RNA, polypeptide or activity will be that of the user Considerations such as the sample type, availability and amount will also influence selection of a particular diagnostic format
  • diagnostic formats which measure the amount of RNA by, for example, PCR amplification, can be an appropriate choice for determining the expression level of a polynucleotide of the invention
  • a multi sample format such as an Enzyme Linked Immunoabsorbant Assay (ELISA)
  • ELISA Enzyme Linked Immunoabsorbant Assay
  • polynucleotides of the invention released into bodily fluids from the neoplastic or pathological prostate cells can also be analyzed by, for example, PCR or RT-PCR Those skilled in the
  • Hybridization methods are applicable for measuring the amount of inventive RNA as an indicator of expression levels
  • methods well known in the art for detecting polynucleotides by specific or selective hybridization with a complementary probe Such methods include both solution hybridization procedures and solid-phase hybridization procedures where the probe or sample is immobilized to a solid support.
  • PCR or RT-PCR can be used with isolated RNA or crude cell lysate preparations. As described previously, PCR is advantageous when there is little starting material. A further description of PCR methods can be found in, for example, Dieffenbach, C.W., and Dveksler, G.S., PCR Primer: A Laboratory Manual, Cold Spring Harbor Press, Plainsview, New York (1995). Multi sample formats such as an ELISA or two- dimensional array offer the advantage of analyzing numerous, different samples in a single assay. A particular example of a two-dimensional array used in a hybridization format is described further below in the Examples- In contrast, solid-phase dip stick- based methods offer the advantage of being able to rapidly analyze a patient's fluid sample and obtain an immediate result.
  • Polynucleotide probes useful for measuring the expression level of the polynucleotides of the invention by hybridization include, for example, all of the polynucleotides probes described previously- More specifically, ARSDRl probes include, for example, polynucleotides corresponding to the entire transcribed region of SEQ ID NOT and fragments thereof. Similarly, TMPRSS2, PART-1, and 8C3 probes include, for example, polynucleotides corresponding to the entire polynucleotide sequences designated as SEQ ID NOS: 1, 3, 5, 7 and fragments thereof, respectively.
  • the polynucleotides probes of the invention having detectable labels are added to a prostate cell sample or a fluid sample obtained from the individual having, or suspected of having a prostate neoplastic condition or pathology under conditions which allow annealing of the probe to RNA.
  • conditions are well known in the art for both solution and solid phase hybridization procedures.
  • optimization of hybridization conditions can be performed, if desired, by hybridization of an aliquot of the sample at different temperatures, durations and in different buffer conditions. Such procedures are routine and well known to those skilled. Following annealing, the sample is washed and the signal is measured and compared with a suitable control or standard value.
  • the magnitude of the hybridization signal is directly proportional to the expression levels of the polynucleotide of the invention for which the probe was specific.
  • a suitable control for comparison can be, for example, the expression level of a polynucleotide of the invention from a prostate cell or a fluid sample obtained from a normal individual.
  • Another suitable control for comparison is a prostate cell line that is androgen-dependent.
  • ARSDRl, TMPRSS2, PART-1, and 8C3 expression levels in cell lines generally should be determined under androgen depleted growth conditions, as their response to androgen stimulation will be indicative of their respective expression levels in neoplastic cells.
  • control sample for comparison can be measured simultaneously with one or more test samples or, alternatively, expression levels can be established for a particular type of sample and standardized to internal or external parameters such as polypeptide or polynucleotide content, cell number or mass of tissue. Such standardized control samples can then be directly compared with results obtained from the test sample. An increase of two-fold or more of expression levels of a polynucleotide of the invention indicates the presence of a prostate neoplastic condition or pathology in the tested individual.
  • the diagnostic procedures described above and below using ANSDRl, TMPRSS2, PART-1, and 8C3 polynucleotide and polypeptide probes can additionally be used in conjunction with other prostate markers, such as prostate specific antigen (PSA), human glandular kallikrein 2 (hk2) and prostase/PRSS18 for simultaneous or independent corroboration of a sample.
  • PSA prostate specific antigen
  • hk2 human glandular kallikrein 2
  • prostase/PRSS18 for simultaneous or independent corroboration of a sample.
  • PSA prostate specific antigen
  • hk2 human glandular kallikrein 2
  • prostase/PRSS18 for simultaneous or independent corroboration of a sample.
  • these markers can also be used in combination- Those skilled in the art will know which markers are applicable for use in conjunction with a polynucleotide or polypeptide of the invention to delineate more specific diagnostic information such as that described above.
  • the invention provides a method of diagnosing or predicting the susceptibility of a prostate neoplastic condition in an individual suspected of having a neoplastic condition of the prostate where the expression level of a polynucleotide of the invention is determined by measuring the amount of its respective RNA-
  • the amount of ANSDRl, TMPRSS2, PART-1, and 8C3 RNA can be determined by hybridization with a polynucleotide probe having substantially the nucleotide sequence of SEQ ID NOS: l, 3, 5, 7, or functional fragment thereof, respectively, and wherein the fragment consists of an oligonucleotide of about 15-18 nucleotides in length-
  • the invention additionally provides a method of diagnosing or predicting the susceptibility of a prostate neoplastic condition in an individual suspected of having a neoplastic condition of the prostate where the expression level of an inventive polypeptide is determined by measuring the amount of polypeptide-
  • the method comprises contacting a cell, a cell lysate, or fractionated sample thereof, from an individual suspected of having a neoplastic condition with a binding agent selective for one of the inventive polypeptides, and determining the amount of selective binding of the agent.
  • the fractionated sample can be a cell lysate or lipid membranes and the binding agent can be an antibody or a non-hydrolizable substrate analog depending upon which inventive polypeptide is being assayed.
  • the fraction when the assay is directed to PART-1 the fraction can be lipid membranes and the selective binding agent can be an antibody
  • the fractionated sample when the assayed polypeptide is ARSDRl, the fractionated sample can be a cell lysate and the binding agent can be an antibody or non-hydrolizable short-chain dehydrogenase/reductase substrate analog
  • affinity binding methods are simple and can be adjusted to be performed in a variety of clinical settings and under conditions to suit a variety of particular needs.
  • Affinity binding assays which are known and can be used in the methods of the invention include both soluble and solid phase formats
  • a specific example of a soluble phase affinity binding assay is immunoprecipitation using an antibody selective for a polypeptide of the invention or other binding agent, such as, for example a steroid or steroid derivative for ARSDRl
  • Solid phase formats are advantageous for the methods of the invention since they are rapid and can be performed more easily on multiple different samples simultaneously without losing sensitivity or accuracy.
  • solid phase affinity binding assays are further amenable to high throughput screening and automation.
  • solid phase affinity binding assays include immunoaffinity binding assays such as an ELISA and radioimmune assay (RIA)
  • RIA radioimmune assay
  • Other solid phase affinity binding assays are known to those skilled in the art and are applicable to the methods of the invention.
  • affinity binding assays are generally formatted for use with an antibody that is selective for the analyte or ligand of interest, essentially any binding agent can be alternatively substituted for selectively binding the antibody.
  • binding agents include, for example, steroids, steroid derivatives, macromolecules such as polypeptides, peptides, nucleic acids, lipids and sugars as well as small molecule compounds.
  • binding agents selective for ARSDRl and TMPRSS2 include, for example, non-hydrolizable short-chain dehydrogenase/reductase substrate analogs and non-hydrolizable serine protease substrate analogs, respectively.
  • Methods are known in the art for identifying such molecules which bind selectively to a particular analyte or ligand and include, for example, combinatorial libraries
  • the binding agent for a molecule other than an antibody to be used in an affinity binding assay, all that is necessary is for the binding agent to exhibit selective binding activity for the inventive polypeptide.
  • affinity binding assays include for example, solid phase ELISA and RIA as well as modifications thereof. Such modifications thereof include, for example, capture ⁇ -
  • the diagnostic formats employing affinity binding can be used in conjunction with a variety of detection labels and systems known in the art to quantitate amounts of a polypeptide of the invention in the analyzed sample
  • Detection systems include the detection of bound polypeptide of the invention by both direct and indirect means
  • Direct detection methods include labeling of an antibody or binding agent that binds specifically to a polypeptide of the invention
  • Indirect detection systems include, for example, the use of labeled secondary antibodies and binding agents
  • detectable labels can be essentially any label that can be quantitated or measured by analytical methods
  • labels include, for example, enzymes, radioisotopes, fluorochromes as well as chemi- and bioluminescent compounds
  • enzyme labels include horseradish peroxidase (HRP), alkaline phosphatase (AP), ⁇ -galactosidase, urease and luciferase
  • a horseradish-peroxidase detection system can be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB), which yields a soluble product in the presence of hydrogen peroxide that is detectable by measuring absorbance at 450 nm
  • TMB chromogenic substrate tetramethylbenzidine
  • An alkaline phosphatase detection system can be used with the chromogenic substrate /-Miitrophenyl phosphate, for example, which yields a soluble product readily detectable by measuring absorbance at 405 nm
  • a ⁇ -galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl- ⁇ -D-galactopyranoside (ONPG), which yields a soluble product detectable by measuring absorbance at 410 nm
  • a urease detection system can be used with a substrate such as urea-bromocresol purple (Sigma Immunochemicals, St Louis, MO)
  • Fluorochrome detection labels are rendered detectable through the emission of light of ultraviolet or visible wavelength after excitation by light or another energy source DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin,
  • R-phycoerythrin, rhodamine, Texas red and lissamine are specific examples of fluorochrome detection labels that can be utilized in the affinity binding formats of the invention
  • Particularly useful fluorochromes include fluorescein and rhodamine Chemiluminescent as well as bioluminescent detection labels are convenient for sensitive, non-radioactive detection of the inventive polynucleotides and polypeptides and can be obtained commercially from various sources such as Amersham Lifesciences, Inc (Arlington Heights, IL)
  • Radioisotopes can alternatively, be used as detectable labels for use in the binding assays of the invention
  • Iodine-125 is a specific example of a radioisotope useful for a detectable label
  • Signals from detectable labels can be analyzed, for example, using a spectrophotometer to detect color from a chromogenic substrate, a fluorometer to detect fluorescence in the presence of light of a certain wavelength, or a radiation counter to detect radiation, such as a gamma counter for detection of iodine-125
  • a quantitative analysis of the amount of bound agent can be made using a spectrophotometer such as an EMAX Microplate Reader (Molecular Devices, Menlo Park, CA) in accordance with the manufacturer's instructions
  • the assays of the invention can be automated or performed robotically, and the signal from multiple samples can be detected simultaneously
  • the diagnostic formats of the present invention can be forward, reverse or simultaneous as described in U S Patent No 4,376,1 10 and No 4,778,751
  • Separation steps for the various assay formats described herein, including the removal of unbound secondary antibody can be performed by methods known in the art (Harlow and Lane, supra) For example, washing with a suitable buffer can be followed by filtration, aspiration, vacuum or magnetic separation as well as by centrifugation
  • a binding agent selective for a polypeptide of the invention also can be utilized in imaging methods that are targeted at prostate cells expressing the nucleic acids of the invention These imaging techniques will have utility in identification of residual neoplastic cells at the primary site following standard treatments including, for example, radical prostatectomy, radiation or hormone therapy In addition, imaging techniques that detect neoplastic prostate cells have utility in detecting secondary sites of metastasis
  • a binding agent specific for one of the polypeptides of the invention can be radiolabeled with, for example, ⁇ ⁇ indium and infused intravenously as described by Kahn et al (Journal of Urology 152 1952-1955 (1994))
  • the binding agent selective for a polypeptide of the invention can be, for example, a monoclonal antibody selective for any one of the inventive polypeptides Imaging can be accomplished by, for example, radioimmunoscintigraphy as described by Kahn et al , supra
  • the invention additionally provides a method of diagnosing or predicting the
  • the inventive method comprises contacting a cell, a cell lysate, or fractionated sample thereof, from the individual with a serine protease substrate selective for TMPRSS2, and determining the amount of cleavage product produced by TMPRSS2.
  • TMPRSS2 activity is used in the method the fractionated sample can be lipid membranes.
  • Another diagnostic format which can be used for determining the expression levels of ARSDRl and TMPRSS2 is by measuring the activity of ARSDRl short-chain dehydrogenase/reductase activity and serine protease activity, respectively, in a sample.
  • activity assays can similarly be performed using essentially identical methods and modes of analysis- Therefore, solution and solid phase modes, including multi sample ELISA, RIA and two-dimensional array procedures are applicable for use in measuring the short-chain dehydrogenase/reductase activity of ARSDRl and the serine protease activity of TMPRSS2.
  • activity can be measured by, for example, incubating a short-chain dehydrogenase/reductase substrate with the sample and determining the amount of product formation from the short-chain dehydrogenase/reductase substrate-
  • TMPRSS2 activity is being measured, a serine protease substrate is incubated with the sample and the amount of protein cleavage is determined.
  • the enzyme products can be measured using, for example, any of the detectable labels and detection systems described previously.
  • the amount of product formed or rate of product formation can be measured either indirectly by measuring the appearance of reduced coenzyme or disappearance of non-reduced coenzyme or, can be measured directly by measuring the appearance of product or disappearance of substrate
  • the amount of product formation can be measured indirectly by measuring the appearance of reduced coenzyme, for example, NADH or NADPH, indicating that the substrate has been oxidized in the ARSDRl -catalyzed reaction.
  • the amount of product formed or rate of product formation can be measured indirectly measuring the disappearance of non-reduced coenzyme, for example, NAD T and NADP + , indicating that the coenzyme has been reduced in the ARSDRl catalyzed reaction.
  • the appearance of product and disappearance of substrate can also be used to measure the activity of ARSDRl
  • the magnitude of product formed will directly correlate with the ARSDRl activity in the sample and therefore, with the expression levels of ARSDRl in the sample
  • Methods applicable for determining the activity of ARSDRl in a sample include, for example, determining the presence of short-chain dehydrogenase/reductase substrates such as steroids or steroid derivatives containing hydroxyl groups and short-chain dehydrogenase/reductase coenzymes such as pyridine nucleotides NAD * and NADP + or derivatives thereof.
  • Derivatives can further exhibit the capability of releasing a dye or fluorochrome, for example, upon chemical modification by ARSDRl such as the oxidation of the substrate or reduction of the coenzyme
  • ARSDRl chemical modification by ARSDRl
  • the difference in light absorbance between the oxidized and reduced forms of coenzyme is routinely distinguished by spectral measurements well known in the art For example, NADH and NADPH are characterized by maximal absortion at about 340nm, while the non-reduced forms, NAD + and NADP + , absorb maximally at about 260mm.
  • Methods useful for the detection of changes in polarity are useful for measuring the disappearance of substrate and appearance of product and can include, for example, thin layer chromatography (TLC), nuclear magnetic resonance spectroscopy (NMR) and infrared spectroscopy.
  • TLC thin layer chromatography
  • NMR nuclear magnetic resonance spectroscopy
  • Short-chain dehydrogenase/reductase substrates, coenzymes and their respective derivatives are well known in the art and are similarly applicable in the methods of the invention for determining ARSDRl activity in a sample
  • Substrates applicable for determining the activity of TMPRSS2 in a sample include, for example, serine protease substrates such as Lys and Arg containing polypeptides and peptides.
  • TMPRSS2 substrates include PSA, hk2, semenogelin, hemoglobin, glucagon, and casein, all of which can be obtained from commercial sources- Peptides of these polypeptides can additionally be used as TMPRSS2 substrates so long as they contain a Lys or Arg residue.
  • serine protease substrate analogs also can be used for determining the amount of TMPRSS2 activity in a sample.
  • Such analogs can further exhibit the capability of releasing a dye or fluorochrome, for example, upon cleavage by TMPRSS2.
  • a serine protease analog capable of releasing dye is azo dye-impregnated collagen, which is also available commercially.
  • Other serine protease substrates and analogs are well known to those skilled in the art and are similarly applicable in the methods of the invention for determining TMPRSS2 activity in a sample.
  • the invention further provides a method of identifying a compound that inhibits the activity of an inventive polypeptide
  • the method consists of contacting a sample containing the inventive polypeptide and an appropriate substrate, with a test compound under conditions that allow product formation from the substrate, and measuring the amount of the product formation from the substrate- A decrease in the amount of product formation from the inventive polypeptide substrate in the presence of the test compound compared to the absence of the test compound indicates that the compound has inhibitory activity towards the inventive polypeptide activity-
  • compounds that increase the activity of an inventive polypeptide also can be identified- A test compound added to a sample containing an inventive polypeptide and an appropriate substrate which increases the amount of product or rate of product formation chemical modification of the substrate compared to the absence of the test compound indicates that the compound increases the activity of the inventive polypeptide- Therefore, the invention provides a method of identifying compounds that modulate the activity of the polypeptides of the present invention-
  • the polypeptide containing sample used for such a method can be serum, prostate tissue, a prostate cell population or
  • the methods for determining the activity of an inventive polypeptide in a sample described above can also be adapted for screening test compounds to determine their ability to inhibit or increase product formation catalyzed by an inventive polypeptide from its substrates.
  • a test compound is added to a reaction system and the effect of the test compound on production of product is observed.
  • Those compounds which inhibit the product formation or rate of product formation are considered as potential antagonists of the inventive polypeptides and further as potential therapeutic agents for treatment of neoplastic conditions of the prostate.
  • those compounds which increase the product or rate of product formation are considered as potential agonists of the inventive polypeptides and further as potential therapeutic agents for the treatment of neoplastic conditions of the prostate.
  • a reaction system for identifying a compound that inhibits or enhances the activity of the inventive polypeptides can be performed using essentially any source of inventive polypeptide activity.
  • sources include, for example, a prostate cell sample, lysate or fractionated portion thereof; a bodily fluid such as blood, serum or urine from an individual with a prostate neoplastic condition; a recombinant cell or soluble recombinant source, and an in vitro translated source.
  • the source of inventive polypeptide is combined with an appropriate substrate as described above and incubated in the presence or absence of a test inhibitory compound. The reaction rate or extent of the usage of the substrate in the presence of the test compound is compared with that in the absence of the test compound.
  • test compounds which provide inhibition of the reaction activity of at least about 50% are considered to be inhibitors of the inventive polypeptides.
  • those compounds which increase the reaction activity of twofold for more are considered to be enhancers of the activity of the inventive polypeptides.
  • Such inhibitors of the inventive polypeptides can then be subjected to further in vitro or m vivo testing to confirm that they inhibit the production of substrates of the inventive polypeptides in cellular and animal models
  • test compounds for the inhibition or enhancement assays can be any substance, molecule, compound, mixture of molecules or compounds, or any other composition which is suspected of being capable of inhibiting inventive polypeptide activity in vivo or m vitro
  • the test compounds can be heterocyclic organic compounds such as steroids or steroid derivatives, macromolecules, such as biological polymers, including proteins, polysacchrides and nucleic acids
  • Sources of test compounds which can be screened for inhibitory activity against the inventive polypeptides include, for example, libraries of peptides, polypeptides, DNA, RNA and small organic compounds
  • test compounds can be preselected based on a variety of criteria
  • suitable test compounds for ANSDRl can be selected as having known short- chain dehydrogenase/reductase inhibition or enhancement activity
  • Suitable test compounds for TMPRSS2 can be selected as having known serine protease inhibition or enhancement activity Specific examples of such serine protease inhibitory test compounds include chymostatin, Aprotinin, Pro
  • the invention provides a method of identifying a compound that inhibits or enhances the activity of an inventive polypeptide where the sample further consists of a prostate cell lysate, a recombinant cell lysate expressing one of the inventive polypeptides, an m vitro translation lysate containing mRNA encoding one of the inventive polypeptides, a fractionated sample of a prostate cell lysate, a fractionated sample of a recombinant cell lysate expressing one of the inventive polypeptides, a fractionated sample of an in vitro translation lysate containing mRNA encoding one of the inventive polypeptides or an isolated inventive polypeptide
  • the method can be in single or multiple sample format
  • polypeptides and peptides of the invention can be used as vaccines to prophylactically treat individuals for the occurrence of a prostate neoplastic condition or pathology
  • Such vaccines can be used to induce B or T cell immune responses or both aspects of the individuals endogenous immune mechanisms
  • the mode of administration and formulations to induce either or both of these immune responses are well known to those skilled in the art
  • polypeptides and peptides of the invention can be administered in many possible formulations, including pharmaceutically acceptable mediums They can be administered alone or, for example, in the case of a peptide, the peptide can be conjugated to a carrier, such as KLH, in order to increase its immunogenicity
  • the vaccine can include or be administered in conjunction with an adjuvant, various of which are known to those skilled in the art After initial immunization with the vaccine, further boosters can be provided if desired Therefore, the vaccines are administered by conventional methods in dosages which are sufficient to elicit an immunological response, which can be easily determined by those skilled in the
  • the vaccines can comprise anti-idiotypic antibodies which are internal images of the inventive polypeptides and peptides described above.
  • anti-idiotypic antibodies which are internal images of the inventive polypeptides and peptides described above
  • the invention additionally provides a method of treating or reducing the progression of a prostate neoplastic condition
  • the method consists of administering to an individual having a neoplastic condition of the prostate an inhibitory amount of an inhibitor specific for a polypeptide of the invention, wherein said inhibitory amount causes a reduction of at least about 2-fold in the amount or activity of the targeted polypeptide.
  • a specific example of a ARSDRl specific inhibitor is a short-chain dehydrogenase/reductase inhibitor or an ARSDRl antisense nucleic acid
  • a specific example of a TMPRSS2 inhibitor is a serine protease inhibitor or a TMPRSS2 antisense nucleic acid
  • a specific example of an 8C3 specific inhibitor is an 8C3 antisense nucleic acid
  • a specific example of PART-1 specific inhibitor is a PART-1 antisene nucleic acid.
  • inhibitors may be produced using methods which are generally known in the art, and include the use of purified inventive polypeptide to produce antibodies or to screen libraries of compounds, as described previously, for those which specifically bind to one of the inventive polypeptides
  • inventive polypeptides for example, known inhibitors of oxidoreductases belonging to the short-chain dehydrogenase/reductase family that inhibit -ARSDRl can be used.
  • Lipophilic compounds able to cross the lipid bilayer that makes up cell membranes are especially useful inhibitors for practicing the methods of the invention
  • Antibodies specific to the polypeptides of the present invention can be used, for example, directly as an antagonist, or indirectly as a targeting or delivery mechanism for bringing a cytotoxic or cytostatic agent to neoplastic prostate cells
  • Such agents can be, for example, radioisotopes.
  • the antibodies can be generated using methods that are well known in the art and include, for example, polyclonal, monoclonal, chimeric, humanized single chain, Fab fragments, and fragments produced by a Fab expression library
  • the polynucleotides encoding the inventive polypeptides, or any fragment thereof, or antisense molecules can be used for therapeutic purposes
  • antisense molecules to the polynucleotides encoding the polypeptides of the invention can be used to block the transcription or translation of a mRNA homologous to the antisense molecule
  • cells can be transformed with sequences complementary to mRNA transcripts encoding the inventive polypeptides
  • sense or antisense ohgonucleotides or larger polynucleotide fragments can be designed from various locations along the coding or control regions of sequences encoding the inventive polypeptides
  • antisense molecules may be used to modulate the activity of the inventive polypeptides, or to achieve regulation of gene function Expression vectors derived from retroviruses, adenovirus, adeno-associated virus
  • Retroviral vectors and adenoviruses offer an efficient, useful, and well characterized means of introducing and expressing foreign genes efficiently in mammalian cells These vectors are well known in the art and have very broad host and cell type ranges, express genes stably and efficiently Methods which are well known to those skilled in the art can be used to construct such recombinant vectors and are described in Sambrook et al (supra) Even in the absence of integration into the DNA, such vectors can continue to transcribe RNA molecules for a substantial period of time. Transient expression can last for a month or more with a non-replicating vector and even longer if appropriate replication elements are part of the vector system
  • Ribozymes enzymatic RNA molecules
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to a complementary target RNA, followed by endonucleolytic cleavage
  • Specific ribozyme cleavage sites within any potential RNA target are identified by scanning the a target RNA for ribozyme cleavage sites which include, for example, the following sequences GUN GUU, and GUC
  • short R ⁇ A sequences of between 10 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for secondary structural features which can render the oligonucleotide inoperable
  • the suitability of candidate targets can also be evaluated by testing accessibility to hybridization with complementary ohgonucleotides using ribonuclease protection assays Antisense molecules and rib
  • the -ARSDRl, TMPRSS2 and PART-1 promoter and regulatory regions can be used for constructing vectors for prostate cancer gene therapy
  • the promoter and regulatory region can be operably fused to a therapeutic polynucleotide for prostate specific expression
  • This method can include the addition of one or more enhancer elements which amplify expression of the heterologous therapeutic polynucleotide without compromising tissue specificity
  • Examples of therapeutic polynucleotides that are candidates for prostate gene therapy utilizing the ARSDRl, TMPRSS2 and PART-1 promoters include suicide genes
  • the expression of suicide genes produces a polypeptide or agent that directly or indirectly inhibits neoplastic prostate cell growth or promotes neoplastic prostate cell death.
  • Suicide genes include genes encoding enzymes such as thymidine kinase, oncogenes, tumor suppressor genes, genes encoding toxins, genes encoding cytokines, or a gene encoding oncostatin
  • the therapeutic polynucleotides of the present invention can be expressed using the vectors described previously for antisense expression as well as others well known in the art
  • EXAMPLE 1 Identification of ARSDRl, an Androgen-Regulated Polynucleotide This example shows identification of ARSDRl, TMPRSS2, PART-1 and 8C3 as genes that are transcriptionally-regulated by androgens in human prostate cancer cells
  • RNAs showing increased expression levels in response to androgen stimulation were identified and characterized further Specifically, a non-redundant set of 1500 prostate-derived cDNA clones was identified from the Prostate Expression Database, a public sequence repository of expressed sequence tag (EST) data derived from human prostate cDNA libraries (Hawkins et al , Nucleic Acids Res ⁇ _ 27 204-208 (1999)) These 1500 unique cDNAs were sequence verified and the clones were stored in 96 well microtiter plates.
  • EST expressed sequence tag
  • the inserts of the cDNAs were amplified with primers BL_ml3F (5'-GTAAAACGACGGCCAGTGAATTG-3') (SEQ ID NO.12) and BL_ml3R (5'-ACACAGGAAACAGCTATGACCATG-3') (SEQ ID NO: 13)
  • Two ⁇ l of bacteria culture were used as PCR templates PCR was performed with an initial incubation at 94°C for 5 minutes, followed by 35 cycles of94°C for 30 seconds, 57°C for 30 seconds, 72 °C for 5 minutes, and a final extension at 72 °C for 7 minutes PCR products were purified with Sephacryl S-500 (Pharmacia, Kalamazoo, MI) on 96-well silent screen filter plates (Nunc, Rochester, NY) The DNA concentration was 200-400 ng/ ⁇ l
  • the purified PCR products were mixed with an equal volume of DMSO and spotted twice onto Type IV glass microscope slides (Amersham, Piscataway, NJ) using
  • the microarrays of prostate derived cDNAs were screened using total RNA isolated from LNCap cells cultured for 72 hours either in the presence or absence of a synthetic androgen R1881 (NEN Life Science Products, Boston, MA
  • TRlzol Gibco-BRL, Germantown, MD
  • LNCaP cells were cultured as follows.
  • the culture medium for LNCap cells was RPMI 1640 with 5%> FBS (Gibco-BRL, Germantown, MD)
  • FBS Gibco-BRL, Germantown, MD
  • 6 flask (175 cm2) of LNCaP cells were starved for androgens by culturing in CS media (RPMI 1640 with 5%> of charcoal filtered FBS). After 72 hours of incubation, three flasks were incubated with CS media and the other three were incubated with CS media plus 1 nM of synthetic androgen R1881. All LNCaP cells were incubated for additional 72 hours and then harvested.
  • Fluorescence-labeled probes were constructed from the above-isolated mRNA as follows. One ⁇ g of mRNA or 30 ⁇ g of total RNA was mixed with l ⁇ l of anchored oligo dT primer (Amersham, Piscataway, NJ), incubated at 70 °C for 10 minutes and then chilled on ice.
  • One ⁇ l of 5M HC1 and 5 ⁇ l of 1M Tris-HCl (pH7 5) were added after the incubation to neutralize the reaction mixture.
  • the mixture was then purified by the Qiagen PCR purification kit (Qiagen, Valencia, CA) following the manufacturer's protocol except washing twice with PE buffer. Following the purification, DNA was eluted with 30 ⁇ l of distilled H2O.
  • Microarray hybridization was performed as follows One ⁇ l of dA/dT (12-18) (l ⁇ g/ ⁇ l) (Pharmacia, Kalamazoo, MI) and l ⁇ l of human Cotl DNA (l ⁇ g/ ⁇ l) (Gibco-BRL, Germantown, MD) were added to the probe The reaction mixture was then heat denatured at 94 °C for 5 minutes An equal volume of 2X Microarray Hybridization Solution (Amersham, Piscataway, NJ) was added and the mixture was prehybridized at 50°C for 1 hour After prehybridization, the probe mixture was placed onto a microarray slide with a covershp The hybridization was carried out in a humid chamber at 52 °C for 16 hours After hybridization, the slides were washed once with IX SSC, 0 2% SDS at room temperature for 5 minutes on a shaker, then twice washed with 0 IX SSC, 0 2% SDS at room temperature for 10 minutes After washing, the
  • ARSDRl is a novel polynucleotide A portion of ARSDRl matches to Est AA657851 (IMAGE ID 1207405), but shows no matches to any known genes in the non-redundant subdivision of the GenBank databases cDNA lODl l was found to be homologous to a serine protease termed TMPRSS2
  • TMPRSS2 Full-length sequencing of the microarray cDNA confirmed the identity of 1OD11 as TMPRSS2 and added additional 3' sequence information to the mRNA sequence available
  • RNA blot containing the same RNAs used for the microarray hybridization was hybridized with ARSDRl cDNA and control G3PDH cDNA
  • the RNA blots were made by fractionating 10 ⁇ g total RNA on a 1 2% formaldehyde gel and blotting onto nylon filters (Sambrook et al., T Molecular Cloning.
  • RNA hybridization confirmed the microarray hybridization results that ARSDRl is up-regulated by androgens Quantification by ImageQuant program (Molecular Dynamics, Sunnyvale, CA) revealed that ARSDRl expression levels in androgen stimulated LNCaP cells is about 15 times higher than in androgen starved LNCaP cells.
  • This example shows the prostate predominant expression and androgen- regulation of ARSDRl
  • ARSDRl The expression profile of ARSDRl in normal human tissues was determined by RNA analysis to determine whether ARSDRl exhibits tissue specific expression
  • a multiple tissue Nothern (MTN) blot (Clontech, Palo Alto, CA) containing RNAs from 8 human tissues and an RNA master blot (Clontech, Palo Alto, CA) containing RNAs from 50 human tissues were hybridized with ARSDRl cDNA probe
  • the 50 human tissues are whole brain, amygdala, caudate nucleus, cerebellum, cerebral cortex, frontal lobe, hippocampus, medulla oblongata, occipital lobe, putamen, substantia nigra, temporal lobe, thalamus, acumens, spinal cord, heart, aorta, skeletal muscle, colon, bladder, uterus, prostate, stomach, testis, ovary, pancreas, pituitary gland, adrenal gland, thyroid gland,
  • RNA hybridization was carried out in ExpressHyb hybdridization solution (Clontech, Palo Alto, CA) RNA blots were exposed to a phosphor screen (Molecular Dynamics, Sunnyvale, CA) and the images were scanned into a computer with a Phosphorimager Quantification was done using ImageQuant program (Molecular Dynamics, Sunnyvale) Overall, not double-counting the six tissues that appeared in both, the MTN blot and the RNA master blot, ARSDRl expression
  • RNA blot containing RNAs from cancer cell lines LNCaP, DU145 and PC3 was made and hybridized with ARSDRl cDNA probe and G3PDH cDNA control probe DUI145 and PC3 are androgen-unresponsive cell lines
  • the ARSDRl cDNA probe was labeled with [ - 32 P] dCTP (Amersham, Piscataway, NJ) using a rediprime II random primer labeling system (Amersham, Piscatawaaay, NJ) and purified with Sephadex G50 Nick column (Pharmacia, Kalamazoo, Ml)
  • the RNA blot was made by fractionating 10 ⁇ g total RNAs on a 1 2% formaldehyde gel and blotting (Sambrook et al , T Molecular Cloning, Cold Spring Harbor, NY Cold Spring Harbor Laboratory Press (1989)) Interestingly, ARSDRl is expressed both in the androgen- dependent (AD), AR-containing cell line LNCaP and in the
  • EXAMPLE 4 Isolation of the ARSDRl Full Length cDNA This example shows the isolation and deduced determination of the nucleotide and deduced amino acid sequence of the ARSDRl polynucleotide, which contains 2539 base pairs and encodes a 318 aa polypeptide
  • ARSDRl encodes a polypeptide of 318 amino acids (SEQ ID NO.2)
  • SEQ ID NO.2 The -ARSDRl start codon, has a strong translation start context according to similarity to the Kozak translation initiation consensus sequence (Kozak, Mamm.
  • IMAGE clone 1703429 has a poly-A stretch that uses the AATAAA polyadenylation signal at 2419, while ARSDRl uses the AATAAA signal at 2481 PCR primers flanking the start and stop codons were designed and an expected size band encompassing the coding region was amplified from human prostate Marathon-Ready cDNA (Clontech, Palo Alto, CA)
  • ARSDRl is a Novel Member of the Short-Chain Dehydrogenases/Reductases (SDR) This example shows that homology searches showed that ARSDRl is a novel member of the family of short-chain dehydrogenases/reductases (SDR)
  • BLAST searches were performed and established sequence homology between ARSDRl and many oxidoreductases from bacteria and plant sources Subsequently, the polypeptide sequence of ARSDRl was compared to sequences contained in the BLOCKS database (http //www blocks fhcrc org) (Henikoff and Henikoff, Nucleic Acids Res.
  • Blocks are multiply aligned ungapped segments corresponding to the most highly conserved regions of proteins
  • the BLOCKS database aids in the detection and verification of polypeptide sequence homology by comparing a polypeptide or DNA sequence to a database of polypeptide blocks
  • the BLOCKS search revealed that the ARSDRl polypeptide has three blocks that match to short-chain dehydrogenases/reductases (SDR) family protein signature BLOCK (BL00061) with a significant combined E-value of 2 6e-06 (Jornvall et al , Biochemistry 34 6003-6013 (1995))
  • SDRs are a large family of NAD(H)- or NADP(H)-dependent oxidoreductases, whose members include many enzymes involved in steroid metabolism such as estradiol 17-beta-dehydrogenase (also called 17-beta-hydroxysteroid dehydrogenase) (EC 1 1 1 62), human 15-hydroxypro
  • GenBank accession numbers for the SDR family members used in the alignment are as follows 20-beta HSD_Strex, Streptomyces 3 ⁇ /20 ⁇ -hydroxysteroid dehydrogenase, P 19992, 11 -beta HSDl man, P28845, 11-beta HSD2-human, U14631 , 17-beta_HSDl_human, P14061, 17-beta_HSSD2-human, LI 1708, 17-beta_HSD3_human, P37058 Only two polypeptide motifs were identified as being conserved in the SDR family The first is a common GlyXXXGlyXGly (SEQ ID NO 14) pattern where the coenzyme NAD(H) or NADP(H) binds
  • ARSDRl shows about 25% amino acid sequence identity with other members of the SDR family and was thus determined to be a novel member of the SDR family Because the polypeptide is androgen regulated and most predominantly expressed in the prostate, it was named androgen regulated short-chain dehydrogenase/reductase 1 (ARSDRl) Prosite pattern searches revealed that ARSDRl contains two Asn-glycosylation sites at amino acid positions 174 and 198
  • EXAMPLE 6 Genomic Organization of ARSDRl This example shows the determination of the ARSDRl promoter and regulatory as well as coding regions
  • ARSDRl cDNA sequences were aligned against genomic sequences originating from a 197 kb chromosome 14 BAC clone R-1012A1 recently sequenced by the National Sequencing Center-Genoscope in France and deposited to GenBank under accession number AL049779 BAC clone R-1012A1 contains the whole genomic sequence of the ARSDRl cDNA
  • the ARSDRl polynucleotide has 7 exons and 6 introns The sizes of exons, the sizes of introns, and the exon/intron junctional sequences are listed in Table 1 All the intron/exon junctions conform to the 5'-gt 3'-ag consensus except intron 2, which has a 5'-gc 3'-ag splicing signal (Breathnach and Chambon, Ann. Rev Biochem 50 349- 383 (1981)) The 5'-gc 3'-ag splicing signals have
  • ARSDRl 5' genomic sequences were examined for potential transcriptional start sites using a neural network promoter prediction program (http //www-hgc lbl gov/projects/promoter html, Reese et al , Large Scale Sequencing Specific Neural Networks for Promoter and Splice Site Recognition, Biocomputing Proceedings of the 1996 Pacific Symposium, ed Lawrence Hunter and Terri E Klein, World Scientific Publishing Company, Singapore (1996))
  • a TATA box (TATAAT) was found 30 base pairs 3' of the putative transcriptional initiation site
  • TESS Transcription Element Search Software
  • the medium-resolution Stanford G3 radiation hybrid panel was used to map the chromosome localization of -ARSDRl using primers
  • 6A4F (5'-GGGGCATTTCCTTACATTGTCCTTG-3') (SEQ ID NO 22)
  • 6A4R (5'-CACTCCAAACAAGTGATGGGAACAC-3')(SEQ ID NO 23) PCR was performed with 35 cycles of 94°C for 30 seconds, followed by 35 cycles at 54°C for 30 seconds and, finally, 35 cycles at 72°C for 30 seconds
  • the reaction products were separated on a 1 2%o agarose gel and the resulting product pattern was analyzed through the Stanford genome web server (www shgc Stanford edu) to determine the probable chromosomal location ARSDRl was determined to be localized to SHGC-
  • BAC clone R-1012A1 (GenBank accession number AL049779) containing ARSDRl comes from chromosome 14q
  • EXAMPLE 8 Expression of ARSDRl in Sections of Normal and Adenocarcinoma Prostate Specimen This example shows that ARSDRl is expressed in both normal prostate and prostate carcinoma
  • ARSDRl was expressed in both the luminal secretary cells and the basal cells of the epithelia of normal prostate Little to no hybridization was seen in stromal cells No background hybridization to normal prostate tissue was seen with the sense ARSDRl probe.
  • EXAMPLE 9 Determination of Androgen-Regulated and Prostate-Localized Expression of TMPRSS2 This example confirms that expression of TMPRSS2 is androgen-regulated and that TMPRSS2 is highly expressed in normal and neoplastic prostate epithelium relative to other human tissues
  • TMPRSS2 is a prostate-specific and androgen-regulated polynucleotide that encodes a 492 amino acid serine protease Androgen-regulated expression of TMPRSS2 was confirmed by Northern analysis using the same LNCaP RNA that was used to construct the probes for microarray hybridization
  • the LNCaP RNA was isolated using TRIzol (Life Technologies, Germantown, MD) according to the manufacturer's directions Ten ⁇ g of total RNA were fractionated on 1 2% agarose denaturing gels and transferred to nylon membranes by capillary method (Sambrook et al , T Molecular Cloning.
  • TMPRSS2 expression was also studied in the prostate carcinoma cell lines LNCaP, DUI 145, and PC3 as well as in androgen-dependent (PXe-AD) and androgen-independent (PXe-AI) prostate cancer xenografts, and prostate stroma (PS).
  • the prostate carcinoma cell lines LNCaP, DU145, and PC3 were cultured in RPMI 1640 medium supplemented with 10%) fetal calf serum (FCS) (Life Technologies, Germantown, MD).
  • LNCaP cells were transferred into RPMI 1640 media with 10% charcoal-stripped FCS (CS-FCS) (Life Technologies, Germantown, MD) This media was replaced with fresh CS-FCS media or CS-FCS supplemented with 1 nM of the synthetic androgen R1881 (NEN Life Science Products Inc., Boston, MA) Cells were harvested for RNA isolation at 0-, 1-, 2-, 4-, 8-, 24-, 48-, and 72-hour time points. Northern analysis was performed with total RNA isolated from cell lines, normal prostate tissue, and prostate cancer xenografts as described in Example 2.
  • TMPRSS2 expression could be detected in the normal prostate tissue and the steady-state LNCaP cells grown in FCS, but was not detectable after 24 hours of androgen depletion.
  • TMPRSS2 expression was not detectable in the androgen-unresponsive PC-3 and DU-145 cell lines, or in a short-term
  • TMPRSS2 normal secretory prostate epithelial cells and early-stage prostate carcinomas depend on androgens for growth-
  • Al androgen-independent
  • Northern analysis was further utilized to examine the expression of TMPRSS2 in human prostate cancers propagated in a xenograft system that recapitulates the androgen-dependent (AD) and subsequent Al characteristics of human prostate cancer growth (Bladou et al , Int. J. Cancer 67 785-790 (1996))
  • TMPRSS2 was expressed in both the AD and Al tumors, a finding that parallels PSA expression in this system, indicating a possible dysregulation of TMPRSS2 control
  • TMPRSS2 transcripts in normal human tissues were also determined by Northern analysis performed as described in Example 2 Northern blot analysis of TMPRSS2 expression was performed using RNA from 16 human tissues The human multiple tissue blots were obtained from Clontech (Palo Alto, CA) and contained 2 ⁇ g of (poly)A+ RNA in each lane A beta-actin control probe was used to verify equivalent loading of RNA Of 16 adult tissues examined, TMPRSS2 message was predominantly expressed in prostate tissues, with very low expression levels in colon, lung, liver, kidney, and pancreas, and no detectable expression in spleen, thymus, testes, ovary, peripheral leukocytes, heart, brain, placenta, or skeletal muscle
  • TMPRSS2 Expression in Prostate Basal Cells and Prostate Carcinoma This example shows that TMPRSS2 is expressed in prostate basal cells and prostate carcinoma
  • TMPRSS2 normal prostate contains two major epithelial cell populations, the luminal secretory cells and the basal cells
  • m situ hybridizations were performed on sections of normal prostate by using an antisense RNA probe specific for TMPRSS2
  • recombinant plasmid pCRII-TOPO Invitrogen, Carlsbad, CA
  • plasmid pCRII-TOPO containing a 489 bp TMPRSS2 fragment (nt 513-1002 of the published TMPRSS2 sequence (Paoloni-Giacobino et al , Genomics 44- 309-20 (1997)
  • pCRII-TOPO plasmid pCRII-TOPO
  • TMPRSS2 fragment nt 513-1002 of the published TMPRSS2 sequence (Paoloni-Giacobino et al , Genomics 44- 309-20 (1997)
  • TMPRSS2 was expressed exclusively in the normal basal cell population
  • In situ hybridization with an antisense RNA probe for TMPRSS2 was done to assay TMPRSS2 expression in normal and malignant prostate tissue
  • TMPRSS2 expression was observed in basal cells of normal prostate tissue, but not in secretory luminal epithelium
  • the /// sun images were digitally acquired and the staining intensity was enhanced to show contrast Little to no staining was seen in stroma, secretory cells, or infiltrating lymphocytes
  • In situ hybridizations with TMPRSS2 antisense and sense probes were also performed on sections of primary prostate adenocarcinoma obtained from radical prostatectomy specimens Adenocarcinoma cells were uniformly positive for TMPRSS2 expression
  • TMPRSS2 expression was observed in primary prostate carcinoma cells
  • the sense strand control TMPRSS2 probe exhibited no background
  • EXAMPLE 11 Sequence Analysis of the Putative TMPRSS2 Promoter This example shows that the TMPRSS2 polynucleotide contains an androgen response element (ARE) in the 5' promoter region at nucleotides 576 to 590 of SEQ ID NO:9.
  • ARE androgen response element
  • TMPRSS2 coding region was cloned by genome-walking in order An 1100 base pair
  • DNA fragment overlapping the TMPRSS2 cDNA by 100 nucleotides that contained 870 base pairs of sequence 5' to the putative transcriptional start site was obtained using the
  • DNA fragments were used as template for PCR reactions with the TMPRSS2 gene-specific primer U75329-71R 5'-TGAGTTCAAAGCCATCTTGCTGTTATCAAC-3'
  • AP2 5'-ACTATAGGGCACGCGTGGT-3' (SEQ ID NO.29) was performed PCR products were cloned into the pCR2 1 vector (Invitrogen, Carlsbad, CA) and sequenced using M13 forward and M13 reverse primers Nucleotide sequences were submitted for homology comparisons against the nonredundant public sequence databases using the
  • the BLAST search parameter prompts utilized are the default prompts located at the NCBI BLAST website
  • Sequences examined for promoter and potential transcriptional start sites using a neural network promoter prediction program (http //www-hgc lbl gov/projects/promoter html) identified a 51 base pair sequence beginning 250 nucleotides 5' of the putative translational start site that correlates highly (score of 0 97 indicating a 0 1 % false-positive prediction rate) with consensus promoter elements
  • TMPRSS2 peptide sequences were selected by direct primary structure comparison between the members of the serine protease gene family and computer-aided antigenicity, surface probability and hydrophobicity analyses
  • the TMPRSS2 peptides for antibody production were selected based on the following criteria 1) the peptide sequence should be on the protein surface and preferably it is in flexible loops, 2) the peptide sequence is at least 15 residues long, and 3) the number of cysteine and proline residues in the selected peptide sequence should kept to a minimum
  • the three-dimensional structure of trypsin was evaluated and its loop regions (which are also on the protein surface) were identified
  • the primary sequence of trypsin was aligned with that of TMPRSS2, and the corresponding loop regions in TMPRSS2 was deduced. Table 2 sets forth the TMPRSS2 peptide sequences that were selected using the these criteria
  • the peptides shown in Table 2 were synthesized and then conjugated with keyhole limpet hemocyanin (KLH) for immunizing rabbits Conjugated peptides and whole proteins were used for the production of rabbit polyclonal antibodies These procedures were contracted to the biotechnology company Research Genetics, Inc (Huntsville, AL)
  • the rabbit anti-TMPRSS2 sera designated in Table 2 were obtained one week following the second boost with each referenced TMPRSS2 peptide antigen Western blot analysis on lysates from LNCaP cells starved or stimulated with androgens was performed with anti-TMPRSS2 antibody
  • An induction of TMPRSS2 polypeptide was observed using the anti-TMPRSS2 antibody upon androgen administration
  • No TMPRSS2 polypeptide was detected in DU145 or PC3 cells which are non-responsive to androgen Immunohistochemichal analysis of TMPRSS2 polypeptide expression using polyclonal antibodies raised against the protease domain of the TMPRSS
  • TMPRSS2 is expressed in mammalian cells in order to produce soluble proteins with suitable post-translational modifications that closely resembles the form of the protein in physiologic sources
  • the TMPRSS2 full length cDNA sequence shown as SEQ ID NO 3 is cloned into the plasmid pGT-d (Berg et al , Bwtechniques 14 972-978 (1993)) and transfected into the AV12 hamster cell line (ATCC CRL 9595) as described previously for the expression of recombinant hK2 protein (Charlesworth et al , Urology 49 487-493 (1997))
  • the TMPRSS2 cDNAs is cloned into the pLNSX and pLNCX retroviral expression vectors (Miller et al , Bwtechniques 7 980-2, 984-6, 989- 90, (1989)) Stable transfectants are isolated under drug resistance Individual clones are isolated, expanded, and checked
  • mice (Jackson Laboratories) Briefly, six-week-old A/J mice (Jackson Laboratories) are immunized with two intraperitoneal injections of selected immunogen, titers are checked and the mice are boosted with intravenous administration of the TMPRSS2 polypeptide Hybridomas are produced by fusion of mouse splenocytes with P3 653 myeloma cells (Kohler et al, Nature 256 495-497 (1975)) Monoclonal antibodies are selected based upon reactivity with TMPRSS2 as well as the failure to react with PSA and hK2 serine proteases using ELISA Hybridomas are then expanded and antibodies are produced in vitro by mass culture or hollow fibers ELISA Analysis An ELISA method for the quantitative screening of patient sera for TMPRSS2 polypeptide is developed using a sandwich ELISA assays as previously described for prostate-specific antigen (PSA) (Corey et al , hit J Cancer 71 10
  • RNA or Northern blot containing the same RNAs used for the microarray hybridization was hybridized to PART-1 cDNA
  • the RNA blots were made by fractionating 10 ⁇ g total RNA on a 1 2% formaldehyde gel and blotting (Sambrook et al , T Molecular Cloning, Cold Spring Harbor, NY Cold Spring Harbor Laboratory Press (1989))
  • the PART-1 cDNA probe was labeled with [ ⁇ - 32 P] dCTP (Amersham, Piscataway, NJ) using a rediprime II random primer labeling system (Amersham, Piscataway, NJ) and the probes were purified with Sephadex G50 Nick column (Pharmacia, Kalamazoo, MI)
  • Northern hybridization confirmed the microarray hybridization results that PART-1 is up-regulated by androgens
  • the same blot was also hybridized to PSA and G3PDH PSA was shown to be strongly stimulated by androgens, consistent with previous observation
  • RNA Master Blot was purchased from Clontech (Palo Alto, CA) and Northern hybridization was carried out in ExpressHyb hybridization solution (Clontech) The Northern blot was exposed to a phosphor screen (Molecular Dynamics) and the images were scanned into a computer with a Phosphorimager Quantification was done using ImageQuant program (Molecular Dynamics) Hybridization of PART-1 cDNA probes to a Clonetech RNA Master Blot revealed that PART-1 is expressed most abundantly in prostate with little or no expression detected in colon, lung, liver, kidney, pancreas, spleen, thymus, testes, ovary, peripheral leukocytes, heart, brain, placenta, and skeletal muscle
  • This example shows cloning of the full length cDNA for PART-1 and determination of its nucleotide sequence
  • PART-1 cDNA encodes a 60 amino acid polypeptide (SEQ ID NO 6)
  • the translational start site conforms to the Kozak consensus motif for translational start site in an adequate context (Kozak, Mammalian Genome 1 563-574 (1996))
  • the PART-1 polypeptide has no homology to any known proteins in the database by BLAST and FASTA searches BLOCKS searches (httpV/www blocks fhcrc org) (Henikoff et al., Nucleic Acids Research, 27 204-208 (1999)) revealed that the PART-1 polypeptide has a XPG_1 BLOCK XPG_1 BLOCK as found in the DNA-damage inducible gene Din7 from yeast (Mieczkowski et al , Molecular and General Genetics 253 655-665 (1997)) and in the XPG DNA repair endonuclease (O'Donovan et al , Journal of Biological Chemistry 269 15965-15968(
  • EXAMPLE 15 Isolation of the PART-1 Promoter Region by Genomic Walking This example shows cloning and sequence analysis of the PART-1 promoter region.
  • the Human GenomeWalker kit (Clontech) was used to clone the promoter region of the PART-1 cDNA with primers 14D7RC3 and API (5'-GTAATACGACTCACTATAGGGC)(SEQ ID NO 27)(Clontech)
  • Each Genome Walker kit contains five premade "libraries" constructed by digesting human genomic DNA with 5 enzymes EcoR V, Sea I, Dra I, Pvu II and Ssp 1, and hgating the restriction fragments to specific adaptors PCR was performed with an initial incubation at 94°C for
  • This genomic walk produced a 1 3 kilobases (kb), a 2 3 kb and 0 8 kb band respectively from the Dra I, Pvu II and Ssp I human GenomeWalking libraries
  • the 2 3 kb band obtained from the Pvu II library was cloned into a PCR2 1-TOPO vector (Invitrogen) and 2325 base pairs of sequence were obtained The sequences were examined to identify a potential transcriptional start site using a neural network promoter prediction program
  • Dra I and a Ssp I site were found in the sequences corresponding to the respective 1 3 and 0 8 kb genomic walking PCR bands from the Dra I and Ssp I libraries
  • the PART-1 genomic walking sequence extends 2024 bps 5' of the start of the PART-1 cDNA
  • TATAAAA TATA box
  • TGTCTTCAAT putative transcriptional start site
  • a binding site for the homeo-domain containing protein Pbx-la Van Dijk et al , Prod. Nat. Acad.
  • the PART-1 promoter region also contains a binding site for NFAT-1 (nuclear factor of activated T cells) at nucleotides 926 to 935 of SEQ ID NO 9 (Rao , Immunol Today 15 274-281 (1994))
  • NFAT-1 nuclear factor of activated T cells
  • This example shows the chromosomal localization of PART-1 by both, polymerase chain reaction (PCR) typing and fluorescence in situ hybridization (FISH)
  • the medium-resolution Stanford G3 radiation hybrid panel was used to map the chromosomal localization of PART-1 with primers 14D7mapR
  • the chromosomal localization of 8C3 was mapped utilizing primers 8C3mapR (5'-TGGCTTCCTCCCTCCATTTTAGAG-3')(SEQ ID NO 39) and API (Clontech, Palo Alto, CA) in the first round, and primers 8C3mapF (5'-GGTGTCAAAAAACTGGCACATCAG-3')(SEQ ID NO 40) and AP2 (Clontech, Palo Alto, CA) in the second round
  • the PCR was performed with an initial incubation at 94°C for 30 seconds, followed by one cycle at 54°C for 30 seconds and 35 cycles at 72°C for 30 seconds

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Abstract

L'invention concerne des polynucléotides isolés codant des polypeptides endrogéno-régulés spécifiques à la prostate. L'invention concerne également des polynucléotides sensiblement purs, correspondant aux régions régulatrices génomiques de polynucléotides endrogéno-régulés, spécifiques à la prostate. Sont également décrits des fragments et des sondes de polynucléotides. De plus, l'invention concerne un procédé permettant de diagnostiquer ou de prévoir un état néoplasique de la prostate chez une personne présentant un risque de souffrir d'un tel état pathologique. Le procédé comprend les étapes suivantes: (a) obtention d'un échantillon de fluide ou de la prostate de la personne; (b) détermination du niveau d'expression du polynucléotide ou du polypeptide spécifique à la prostate, endrogéno-régulé; et (c) comparaison des niveaux d'expression du polynucléotide ou du polypeptide spécifique à la prostate endrogéno-régulé avec des niveaux d'expression présentés par un échantillon de fluide normal, par des cellules de prostate normales ou par des lignées cellulaires endrogéno-dépendantes, un doublement du niveau d'expression du polynucléotide ou polypeptide spécifique à la prostate, endrogéno-régulé dans l'échantillon de fluide ou de prostate de la personne, comparé au niveau d'expression du fluide normal ou des cellules de prostate normales ou d'une lignée cellulaire endrogéno-dépendante, indiquant la présence d'un état néoplasique de la prostate. Sont également décrits des procédés d'identification de composés inhibant ou augmentant de façon sélective le niveau d'expression des polypeptides spécifiques à la prostate selon l'invention, ainsi qu'un procédé permettant de traiter ou de réduire la progression d'un état néoplasique de la prostate.
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US6551778B1 (en) 1999-01-28 2003-04-22 Gen-Probe Incorporated Nucleic acid sequences for detecting genetic markers for cancer in a biological sample
US7037667B1 (en) 1998-06-01 2006-05-02 Agensys, Inc. Tumor antigen useful in diagnosis and therapy of prostate and colon cancer
US7071303B2 (en) 2001-03-28 2006-07-04 Institute For Systems Biology Androgen regulated prostate specific nucleic acids
EP1774959A1 (fr) * 2005-09-21 2007-04-18 L'Oréal Oligonucléotide d'ARN double brin inhibant l'expression de la tyrosinase
US7393921B2 (en) 2000-12-04 2008-07-01 Institute For Systems Biology Prostate-specific polypeptide pamp and encoding nucleic acid molecules
US7718369B2 (en) 2005-09-12 2010-05-18 The Regents Of The University Of Michigan Recurrent gene fusions in prostate cancer
EP2295606A1 (fr) * 2000-11-28 2011-03-16 Wyeth LLC Analyse d'expression d'acides nucléiques KIAA et polypeptides utilisés dans le diagnostic et le traitement du cancer de la prostate
US8106037B2 (en) 2007-08-03 2012-01-31 The Brigham And Women's Hospital, Inc. Identification and treatment of estrogen responsive prostate tumors
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US8945556B2 (en) 2010-11-19 2015-02-03 The Regents Of The University Of Michigan RAF gene fusions
US9303291B2 (en) 2007-07-06 2016-04-05 The Regents Of The University Of Michigan MIPOL1-ETV1 gene rearrangements
US9926602B2 (en) 2009-09-17 2018-03-27 The Regents Of The University Of Michigan Recurrent gene fusions in prostate cancer
US9957569B2 (en) 2005-09-12 2018-05-01 The Regents Of The University Of Michigan Recurrent gene fusions in prostate cancer

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