JP2004521602A - Novel prostate-specific or testis-specific nucleic acid molecules, polypeptides, and diagnostic and therapeutic methods - Google Patents

Abstract

The present invention provides novel prostate-specific or testis-specific nucleic acid molecules, polypeptides, antibodies, and modulators for use in the diagnosis, treatment, and prevention of prostate and testicular diseases and conditions, such as cancer. Compounds are provided.

Description

[0001]
Field of the invention
The present invention relates generally to the treatment of diseases associated with prostate and testicular dysfunction and cell proliferation, and more particularly, to the identification and use of novel genes for the diagnosis and treatment of such diseases.
[0002]
Background of the Invention
Genitourinary disorders are often difficult to diagnose and treat effectively. This is because genitourinary disorders exist non-specifically. Two causes of urogenital disease are prostate and testicular disease.
[0003]
The prostate is a gland of various sizes within the male pelvis, consisting of a wide variety of cells, including epithelial cells and stromal cells. Diseases involving the prostate include prostate cancer, benign prostatic hyperplasia, and prostatitis. The male hormone testosterone and other androgen-related hormones play a major role in prostate growth and function. The testis is also the organ where many diseases occur, including abnormalities, inflammation, and cancer.
[0004]
Prostate cancer is the most commonly diagnosed cancer in men and is the second leading cause of cancer death after skin cancer. In the early stages, prostate cancer relies on androgens for its growth, and this dependence is the basis for androgen ablation therapy. However, in many cases, prostate cancer progresses to an androgen-independent phenotype, in which case there is currently no effective treatment.
[0005]
Currently, there is limited information on the molecular details of prostate cancer progression. Several independent methods have identified several genes that are highly expressed in the prostate that may play a unique role in this process. The first example of such a gene is the Prostate Specific Antigen (PSA), the detection of which is currently a diagnostic tool, despite significant limitations, as well as a marker for the progression of prostate cancer. It is used as More recently, a group of genes that are highly expressed in several other prostates have been identified. These include prostate specific membrane antigen (PSMA), prostate cancer tumor antigen 1 (PCTA-1), NKX3.1, prostate stem cell antigen (PSCA), DD3, PCGEM1, and the like.
[0006]
It would be beneficial to provide reagents useful for diagnosing and treating diseases involving the prostate and testis, and other tissues.
[0007]
Summary of the Invention
The present invention generally relates to novel prostate-specific or testis-specific nucleic acid molecules, polypeptides, antibodies, and regulatory compounds for use in the diagnosis, treatment, and prevention of prostate and testicular diseases and conditions, such as cancer. I will provide a.
[0008]
In a first aspect, the invention provides a substantially pure prostate-specific or testis comprising a sequence substantially identical to the sequence of any of SEQ ID NOs: 14, 29, 32, 34, 36, 41, or 53. Provide a specific polypeptide. In a preferred embodiment of the first aspect, the substantially pure, prostate-specific or testis-specific polypeptide has the sequence of any of SEQ ID NOs: 14, 29, 32, 34, 36, 41, or 53. including. In another preferred embodiment, the invention relates to an isolated nucleic acid molecule encoding a polypeptide of the first aspect, for example the sequence of any of SEQ ID NO: 23, 28, 31, 33, 35, 40 or 52. A nucleic acid molecule comprising: Preferably, the polypeptide is from a mammal (eg, a human).
[0009]
In a second aspect, the invention provides a prostate-specific or testis-specific isolated nucleic acid molecule comprising a sequence substantially identical to SEQ ID NOs: 1-12, 22, 27, 30, and 51. .
[0010]
In a third aspect, the invention provides a prostate-specific and testis-specific isolated nucleic acid molecule consisting essentially of SEQ ID NOs: 15-21, 24-26, 42-50, and 54-70. .
[0011]
In preferred embodiments of some of the above aspects, the invention provides vectors, cells, cells containing the vectors, and non-human transgenic animals comprising the isolated nucleic acid molecules.
[0012]
In a fourth aspect, the present invention provides any of the sequences set forth in SEQ ID NOs: 1-12, 15-28, 30, 31, 33, 35, 40, 42-50, 51, 52, or 54-70. Provided herein that encodes a prostate-specific or testis-specific polypeptide that hybridizes under high stringency conditions to the complement of the nucleic acid molecule.
[0013]
In a fifth aspect, the present invention is described in SEQ ID NOs: 1-12, 15-28, 30, 31, 33, 35, 40, 42-50, 51, 52, or 54-70, or fragments thereof. An isolated nucleic acid molecule comprising an antisense sequence to the coding strand of either a prostate-specific or testis-specific nucleic acid molecule.
[0014]
In a sixth aspect, the present invention provides a method for analyzing prostate-specific or testis-specific genes or homologs or fragments thereof, comprising SEQ ID NOs: 1-12, 15-28, 30, 31, 33, 35, Probes having greater than 55% nucleotide sequence identity to the sequence encoding any of 40, 42-50, 51, 52, or 54-70, or fragments thereof. In this case, the fragment consists of at least 6 amino acids, and the probe hybridizes under high stringency conditions to at least a part of a prostate-specific or testis-specific nucleic acid molecule. In a preferred embodiment of this aspect, the probe comprises SEQ ID NOS: 1-12, 15-28, 30, 31, 33, 35, 40, 42-50, 51, 52, or 54-70, or a fragment thereof. It has 100% complementarity with a nucleic acid molecule encoding either. In this case, the fragment consists of at least 6 amino acids, and the probe hybridizes under high stringency conditions to at least a part of a prostate-specific or testis-specific nucleic acid molecule.
[0015]
In a seventh aspect, the invention provides a prostate-specific or testis-specific comprising an amino acid sequence substantially identical to the amino acid sequence of any of SEQ ID NOs: 14, 29, 32, 34, 36, 41, or 53. Antibodies that specifically bind to specific polypeptides.
[0016]
In an eighth aspect, the present invention provides SEQ ID NOS: 1 to 12, 15 to 28, 30, 31, 33, 35, 40, 42 to 50, 51, 52, or 54 to 70 or fragments thereof (fragment length is Contacting any nucleic acid molecule (greater than about 18 nucleotides) with a genomic DNA preparation from the cell under highly stringent hybridization conditions; and SEQ ID NOs: 1-12, 15-28, 30, 31. , 33, 35, 40, 42-50, 51, 52, or 54-70, by detecting a DNA sequence that has about 55% or more nucleotide sequence identity to either prostate or testis specific A method for detecting a prostate-specific or testis-specific gene or a fragment thereof in a cell, comprising the step of identifying a specific gene or a fragment thereof. Nucleotides encoding the polypeptides of SEQ ID NOs: 38, 39, or 71-73 can also be used in embodiments of this aspect. In a preferred embodiment of this aspect, the method comprises detecting neoplastic or cancer cells in a patient susceptible to cancer, such as, for example, prostate cancer, or a patient at high risk for cancer.
[0017]
In a ninth aspect, the invention relates to the steps of contacting a prostate-specific or testis-specific polypeptide with a test compound and determining the effect of the test compound on the expression or activity of the prostate-specific or testis-specific polypeptide. And a method for identifying a test compound that modulates the expression or activity of a prostate-specific or testis-specific polypeptide. In a preferred embodiment of this aspect, the prostate-specific or testis-specific polypeptide is SEQ ID NO: 14, 29, 32, 34, 36, 38, 39, 41, 53, or 71-73, and fragments thereof. And amino acid sequences that are substantially identical to the amino acid sequences of the analogs.
[0018]
In a tenth aspect, the present invention provides a method for administering to a mammal a therapeutically effective amount of a compound that modulates the activity or expression of a prostate-specific or testis-specific polypeptide and that has a beneficial effect on a disease in the mammal. There is provided a method of treating a mammal having a prostate or testicular disease, comprising: In a preferred embodiment of this aspect, the disease is prostate cancer, the mammal is human, or the prostate-specific or testis-specific polypeptide is SEQ ID NO: 14, 29, 32, 34, 36, 38, 39, 41, 53, or 71-73, and fragments and analogs thereof.
[0019]
In an eleventh aspect, the present invention provides a pharmaceutical composition comprising at least a single dose of a therapeutically effective amount of a prostate-specific or testis-specific polypeptide, or fragment thereof, contained in a pharmaceutically acceptable carrier. provide. In this case, the composition is formulated for the treatment of prostate or testicular disease.
[0020]
In a twelfth aspect, the present invention provides a kit for analyzing a prostate-specific or testis-specific nucleic acid molecule, the kit comprising a nucleic acid for analyzing a prostate-specific or testis-specific nucleic acid molecule present in a subject. Includes molecular probes.
[0021]
In a thirteenth aspect, the present invention provides a kit for analyzing a prostate-specific or testis-specific polypeptide, the kit comprising an antibody for analyzing a prostate-specific or testis-specific polypeptide present in a subject. including.
[0022]
As used herein, "polypeptide", "protein", or "polypeptide fragment" refers to a native or unnatural polypeptide, whether or not any post-translational modifications (eg, glycosylation or phosphorylation) have been made. Means a chain of two or more amino acids that constitutes all or part of By "post-translational modification" is meant any change that occurs to a polypeptide or polypeptide fragment during or after synthesis. Post-translational modifications can occur naturally (eg, during synthesis in a cell) or can be artificially created (eg, by recombinant or chemical methods). A protein may be made from one or more polypeptides.
[0023]
By "substantially pure polypeptide" or "substantially pure and isolated polypeptide" is meant a polypeptide (or fragment thereof) that has been separated from components that accompany it in its natural state. Typically, such polypeptides are substantially pure if they are at least 60% (wt%) free of proteins and naturally occurring organic molecules that are naturally associated. Preferably, such a polypeptide is at least 75%, more preferably at least 90%, most preferably at least 99% (wt%) pure prostate-specific or testis-specific polypeptide. Substantially pure prostate-specific or testis-specific polypeptides can be prepared using standard techniques, for example, by extraction from natural sources (eg, prostate or testis tissue or cell lines). Obtained by expressing a recombinant nucleic acid encoding a novel polypeptide, or by chemically synthesizing the polypeptide. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
[0024]
A protein or polypeptide, when separated from contaminants that naturally accompany it, is substantially free of components that naturally associate. Thus, a chemically synthesized protein, or a protein made in a cell line different from cells of natural origin, is substantially free of components that bind in its natural state. Thus, a substantially pure polypeptide includes not only polypeptides derived from eukaryotes, but also polypeptides synthesized in E. coli or other prokaryotes.
[0025]
The term "identity" as used herein, means that the sequence of a particular nucleic acid molecule or polypeptide is related to the sequence of a reference molecule of the same type. For example, if a polypeptide or nucleic acid molecule has the same amino acid residue or nucleotide residue at any position relative to an aligned reference molecule, then there is said to be "identity" at the position of interest.
[0026]
The level of sequence identity of a nucleic acid molecule or polypeptide to a reference molecule is typically determined using software for sequence analysis and default parameters specified in the software, such as the introduction of gaps to achieve optimal alignment. Measured. Thus, the term "identity" of two or more nucleic acid or polypeptide sequences is referred to as "Computational Molecular Biology", Lesk, A .; M. Ed., Oxford University Press, New York, 1988; "Biocomputing: Informatics and Genome Projects", Smith, D.M. W. Ed., Academic Press, New York, 1993; "Computer Analysis of Sequence Data", Vol. 1, Griffin, A. et al. M. And Griffin, H .; G. FIG. Ed., Humana Press, New Jersey, 1994; "Sequence Analysis in Molecular Biology", von Heinje, Academic Press, 1987; and "Sequence analysis primers (Analysis Press). Primer) ", edited by Gribskov and Devereux, M.P. Stockton Press, New York, 1991; and Carillo and Lipman, SIAM J. Clin. Applied Math. 48: 1073, 1988, and can be readily calculated by known methods, including, but not limited to, those described in US Pat.
[0027]
Methods for determining identity can utilize publicly available computer programs. Computer program methods for determining the identity between two sequences include the GCG program package (Deverex et al., Nucleic Acids Research 12 (1): 387, 1984), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215; 403 (1990)). Identity can also be determined using the well-known Smith-Waterman algorithm. The BLAST program is publicly available from NCBI and other sources (BLAST Manual, Altschul et al., NCBI NLM NIH, Bethesda, MD, 20894). Search for http: // www. ncbi. nlm. nih. gov / BLAST / unfinishedgenome. html; or http: // www. tigr. org / cgi-bin / BlastSearch / blast. It can be executed with a URL such as cgi. These software programs match similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine; Tyrosine.
[0028]
A nucleic acid molecule or polypeptide, over its entire length, is at least 50%, 60%, or 70%, preferably at least 80%, or 90%, more preferably at least 95%, most preferably at least 95%, relative to the sequence of the reference molecule. If it shows 99% identity, it is said to be "substantially identical" to the reference molecule. For polypeptides, the length of the comparison sequence is at least 16 amino acids, preferably at least 20 amino acids, or at least 25 amino acids, more preferably at least 35 amino acids, and most preferably a full-length polypeptide. For nucleic acid molecules, the comparison sequence is at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, or at least 110 nucleotides, most preferably a full length nucleic acid molecule. Alternatively or additionally, two nucleic acid sequences are "substantially identical" when they hybridize under highly stringent conditions.
[0029]
"Isolated nucleic acid molecule", "substantially pure nucleic acid molecule", or "substantially pure and isolated nucleic acid molecule" refers to the natural genome of the organism from which the nucleic acid molecule of the invention is derived. Means a nucleic acid molecule (eg, DNA) that does not contain a gene adjacent to the target nucleic acid. The term includes, for example, vectors; autonomously replicating plasmids or viruses; or recombinant DNA that is integrated into prokaryotic or eukaryotic genomic DNA, or another molecule that is independent of other sequences (eg, , CDNA or genomic or cDNA fragments produced by PCR or restriction enzyme digestion). Also included are recombinant DNAs that are part of a hybrid gene encoding additional polypeptide sequence.
[0030]
“Antisense” as used herein with respect to a nucleic acid molecule refers to at least 75 nucleotides, preferably at least 75, of the coding strand of a nucleic acid molecule encoding a prostate-specific or testis-specific polypeptide described herein. A molecule having a nucleic acid sequence (any length) complementary to 100, 150, or 200 nucleotides is meant. Antisense molecules include regulation of transcription enhancers, hormone response elements, ribosomes and binding sites for receptor polymerases, which can be located tens to tens of thousands of base pairs upstream or downstream of the coding region. An array can be included. An antisense nucleic acid molecule can have, for example, the ability to selectively reduce the production or expression of a prostate-specific or testis-specific polypeptide encoded by a prostate-specific or testis-specific nucleic acid molecule.
[0031]
A “prostate-specific” or “testis-specific” nucleic acid molecule is at least 50%, 60%, or 75% relative to the nucleic acids described herein, eg, in FIGS. 4, 11, and 14. , More preferably at least 80%, 85%, or 95%, most preferably 99%, nucleic acid molecules such as genomic DNA, cDNA, or RNA (eg, mRNA) molecules having an amino acid identity. Furthermore, at least 50%, 60%, or at least a nucleotide sequence encoding amino acids 1 to 200 of STMP1 (SEQ ID NO: 14), preferably a nucleotide sequence encoding amino acids 40 to 150 of STMP1. A nucleic acid molecule having 75%, more preferably at least 80%, 85%, or 95%, and most preferably 99% nucleotide identity can be considered a nucleic acid molecule specific to prostate or testis. Nucleic acids specifically excluded from this definition are STEAP (AF186249) (Hubert, RS et al., Proc Natl Acad Sci USA 96, 14523-14528, 1999), as well as EST AF 1322025, AF177622, BAB23615, BAA91839, BAB15559, and NP_032190 is a nucleic acid molecule sequence described or encoding the same.
[0032]
Preferred prostate-specific nucleic acid molecules are at least 5-fold, preferably at least 10-fold, more preferably at least 5 times higher than the level of the same nucleic acid molecule in at least one non-prostate tissue, preferably all other non-prostate tissues. It can be selectively expressed 15 times higher, most preferably at least 20 times higher. Prostate-specific nucleic acid molecules can also be expressed at high levels in non-prostate tissue, but generally the expression levels are highest in the prostate. Occasionally, as described herein, prostate-specific nucleic acid molecules are expressed at higher levels in non-prostate tissue (eg, placenta, lung, or liver) than in the prostate.
[0033]
Preferred testis-specific nucleic acid molecules are at least 5 times, preferably at least 10 times, more preferably at least 5 times higher than the level of the same nucleic acid molecule in testis tissue, preferably in at least one non-testis tissue, preferably in all other non-testis tissues. It can be selectively expressed 15 times higher, most preferably at least 20 times higher. Testis-specific nucleic acid molecules can also be expressed at high levels in non-testis tissue, but generally the expression level is highest in testis. Occasionally, as described herein, testis-specific nucleic acid molecules are expressed at higher levels in non-testis tissue (eg, placenta, lung, or liver) than testis.
[0034]
A “prostate-specific” or “testis-specific” polypeptide, or “prostate-specific” or “testis-specific” protein is a polypeptide encoded by a prostate-specific or testis-specific nucleic acid molecule Means A prostate-specific or testis-specific polypeptide also has at least 50%, 60%, or 75%, more preferably, a polypeptide described herein (eg, FIGS. 4, 11, and 14). May be defined as polypeptides having 80%, 85%, or 95%, most preferably at least 99%, amino acid identity. Polypeptides specifically excluded from this definition include STEAP (AF186249) (Hubert, RS, et al., Proc Natl Acad Sci USA 96, 14523-14528, 1999), as well as ESTs AF132020, AF177622, BAB23615, BAA91839, BAB15559, And the polypeptide sequence described or encoded in NP_032190. Also, at least 50%, 60%, or 75%, more preferably at least 80%, of amino acids 1 to 200 of STMP1 (SEQ ID NO: 14), preferably amino acids 40 to 150 of STMP1; Polypeptides having 85%, or 95%, most preferably at least 99%, amino acid identity can be considered polypeptides specific to prostate or testis.
[0035]
Preferred prostate-specific polypeptides are at least 5-fold higher, preferably at least 10-fold higher, more preferably at least 10 times higher than the level of the same polypeptide in at least one non-prostate tissue, preferably all other non-prostate tissues. It is selectively expressed 15 times higher, most preferably at least 20 times higher. Prostate-specific polypeptides can also be expressed at high levels in non-prostate tissue, but generally the expression levels are highest in the prostate. Sometimes, as described herein, prostate-specific polypeptides are expressed at higher levels in the non-prostate (eg, placenta, lung, liver) than the prostate.
[0036]
Preferred testis-specific polypeptides are at least 5 times, preferably at least 10 times, more preferably at least 10 times higher than the level of the same polypeptide in testis tissue, preferably in at least one non-testis tissue, preferably in all other non-testis tissues. It is selectively expressed 15 times higher, most preferably at least 20 times higher. Testis-specific polypeptides can also be expressed at high levels in non-testis tissues, but generally the expression level is highest in testis. Sometimes, as described herein, testis-specific polypeptides are expressed at higher levels in non-testis (eg, placenta, lung, liver) than testis.
[0037]
The term prostate-specific or testis-specific polypeptide includes homologs, analogs, fragments, and isoforms (eg, alternative splicing isoforms) of the sequences described herein. A “biologically active fragment” is, for example, at least 30%, preferably at least 30%, compared to the properties of a full-length prostate-specific or testis-specific polypeptide, eg, extracellular transport, intracellular signaling, A polypeptide fragment of a prostate or testis specific polypeptide is meant that exhibits at least 50%, more preferably at least 75%, most preferably at least 100% of other properties. "Analog" refers to at least 30%, preferably at least 50%, more preferably at least 75%, most preferably at least 100% of the extracellular transport or intracellular signaling properties of the polypeptide of origin. It refers to any substitution, addition, or deletion of the amino acid sequence of a prostate or testis-specific polypeptide that exhibits properties. Fragments, homologs, and analogs can be made by standard techniques, for example, solid phase peptide synthesis, or the polymerase chain reaction. For example, point mutations can occur at any position in the sequence from aplins, apyrimidines, or structurally impaired sites on the cDNA. Alternatively, point mutations can be introduced by random or site-specific mutagenesis (eg, PCR using oligonucleotides, or error-prone PCR). Similarly, deletions and / or insertions can be introduced into the sequence, with preferred insertions including fusions at 5 ′ and / or 3 ′ with a polynucleotide encoding a reporter moiety or affinity moiety. included. Other preferred insertions include nucleic acids that further comprise functional elements such as promoters, enhancers, hormone responsive elements, origins of replication, transcriptional and translational start sites. Where the insertion involves one or more functional elements, the resulting nucleic acid will be understood to be linear or circular (eg, a transcription or expression cassette, plasmid, etc.).
[0038]
In the use of the method of the present invention, the expression "prostate-specific" or "testis-specific" polypeptide is further described or described in EST AF1322025, AF177622, BAB23615, BAA91839, BAB15559, and NP_032190. A prostate-specific or testis-specific nucleic acid molecule comprising a polypeptide sequence but not a STEAP and a nucleotide sequence described or encoded in EST AF1322025, AF177622, BAB23615, BAA91839, BAB15559, and NP-032190. Includes but does not include STEAP.
[0039]
By "prostate-specific or testis-specific gene or homologue or fragment thereof" is meant a gene encoding a prostate-specific or testis-specific polypeptide, or a homologue of the gene.
[0040]
"Specifically binds" means that a compound, eg, an antibody, recognizes and binds to a protein or polypeptide (eg, a prostate-specific or testis-specific polypeptide), and is labeled for detection. It means that the compound cannot bind to the target protein or polypeptide due to an excess of an unlabeled compound for detection. Compounds that bind non-specifically do not become unbinding due to excess detectable labeling compound. Preferred antibodies are polypeptide sequences set forth in FIGS. 4, 11, and 14, or polypeptide sequences encoded by any of the nucleotide sequences set forth in FIGS. 3, 4, 11, and 14, Or a prostate-specific or testis-specific polypeptide sequence substantially identical to a portion thereof.
[0041]
"Compound," "test compound," or "candidate compound" means a naturally occurring molecule or an artificially created molecule, such as a peptide, protein, synthetic organic molecule, natural organic molecule, nucleic acid molecule, and Contains these components.
[0042]
"High stringency conditions" refers to 0.5 M NaHPO₄PH 7.2 in a buffer containing 7% SDS, 1 mM EDTA, and 1% BSA (fraction V) at a temperature of 65 ° C. or 48% formamide, 4.8 × SSC, 0.2 M In a buffer containing Tris-Cl, pH 7.6, 1 × Denhardt's solution, 10% dextran sulfate, and 0.1% SDS at a temperature of 42 ° C. (these are highly stringent Northern or Southern hybridizations). Typical conditions for hybridization), which means hybridization that is equivalent to hybridization that occurs when a DNA probe having a length of at least 500 nucleotides is used. High stringency hybridization also involves a number of techniques routinely performed by molecular biologists, such as high stringency PCR, DNA sequencing, single-strand conformation polymorphism (SSCP) analysis, and in situ hybridization. Depends on whether or not succeeds. For Northern and Southern hybridizations, the above techniques typically employ relatively short probes (typically 16 or more nucleotides for PCR or sequencing and 40 or more nucleotides for in situ hybridization). Done. The high stringency conditions used in such procedures are well known to those skilled in the field of molecular biology, and are described, for example, in Ausubel et al., "The Latest Protocols in Molecular Biology," which is incorporated herein by reference. Current Protocols in Molecular Biology), John Wiley & Sons, New York, 1998.
[0043]
“Probe” or “primer” means a single-stranded DNA or RNA molecule having a constant sequence that base pairs with a second DNA or RNA molecule that contains a complementary sequence (“target”). I do. The stability of the resulting hybrid depends on the degree of base pairing that occurs. This stability is affected by parameters such as the degree of complementarity between the probe and the target molecule and the degree of stringency of the hybridization conditions. The degree of stringency of hybridization is affected by parameters such as temperature, salt concentration, and organic molecule (eg, formamide) concentration, and is determined by methods well known in the art. Probes or primers specific for a prostate-specific or testis-specific nucleic acid molecule preferably have greater than 45% sequence identity to the nucleic acid sequence encoding the amino acid sequences described herein, more preferably 55% to 75% sequence identity, even more preferably at least 75% to 85% sequence identity, even more preferably at least 85% to 99% sequence identity, most preferably 100% sequence identity. Have. Probes can be labeled for detection with radioactive or non-radioactive materials using methods well known in the art. Probes include nucleic acid sequencing, nucleic acid amplification by polymerase chain reaction, single-stranded conformational polymorphism (SSCP) analysis, restriction fragment length polymorphism (RFLP) analysis, Southern hybridization, Northern hybridization, in situ hybridization, It can be used in methods involving nucleic acid hybridization, such as electrophoretic mobility shift assay (EMSA), and others well known in the art.
[0044]
For example, an oligonucleotide probe or primer molecule, gene or fragment thereof, cDNA molecule, polypeptide, or antibody is "labeled for detection" if it is marked so that its presence in a sample can be directly identified. It is said that. Methods for labeling molecules for detection are well known in the art and include radioactive labels (eg,³²P or³⁵But not limited to, a method using an isotope such as S) and a non-radioactive label (for example, a method using a fluorescent label such as fluorescein, or a method for producing a construct containing green fluorescent protein (GFP)).
[0045]
By "transgenic" is meant any cell that contains a DNA sequence or a transgene that is artificially inserted into a cell and becomes part of the genome of the organism that develops from the cell. As used herein, a transgenic organism is generally a transgenic animal (eg, mouse, rat, and goat), and the DNA (transgene) is artificially inserted into the nuclear genome. By "transgene" is meant any piece of DNA that becomes part of the genome of an organism that develops from a cell when inserted artificially into the cell. Such transgenes may include genes that are partially or wholly heterologous (ie, foreign) to the transgenic organism, and may be homologous to endogenous genes of the subject organism. A “knockout mutation” is an artificially induced change on a nucleic acid sequence (recombinant DNA technology, which usually reduces the biological activity of an encoded polypeptide by at least 80% relative to a non-mutant gene). Or a change made upon deliberate exposure to a mutagen). Such mutations include, but are not limited to, insertions, deletions, frameshift mutations, or missense mutations. Knockout mutations can be present ex vivo (eg, in tissue culture cells, or primary cells), or in vivo. A “knockout animal” is a mammal, preferably a mouse containing a knockout mutation as defined above.
[0046]
By "sample" is meant a tissue biopsy, cells, blood, serum, urine, stool, or other specimen obtained from a patient or subject. The sample is analyzed to determine mutations in the gene encoding the prostate-specific or testis-specific polypeptide, or the level of expression of the gene encoding the prostate-specific or testis-specific polypeptide, as is well known in the art. The method, or a method described herein, is detected, for example, as indicative of cancer progression. For example, prostate-specific or testis-specific polymorphisms are determined using methods such as sequencing of PCR products from patient samples, single-stranded conformational polymorphism (SSCP) analysis, or restriction fragment length polymorphism (RFLP) analysis. It is possible to detect mutations in the gene encoding the peptide, to measure the level of prostate-specific or testis-specific polypeptide by ELISA, and to detect prostate-specific or testis-specific by PCR. It is possible to measure the level of a nucleic acid encoding a specific polypeptide.
[0047]
"Pharmaceutically acceptable carrier" means a carrier that is physiologically acceptable to the mammal to be treated while retaining the therapeutic properties of the compound to be administered. One exemplary pharmaceutically acceptable carrier is physiological saline. Other pharmaceutically acceptable carriers and their dosage forms are well known in the art and are described, for example, in Remington's "The Science and Practice of Pharmacy" (19th Edition), A. . Gennaro, ed., 1995, Mack Publishing Company, Easton, PA.
[0048]
A "therapeutically effective amount", as described herein, with respect to a dose of a drug, refers to a specific amount of a pharmaceutically active agent tailored to an individual patient exhibiting symptoms characteristic of a particular disease. (Eg, a prostate-specific or testis-specific polypeptide, nucleic acid molecule, or regulatory compound). For example, a patient receiving the treatment of the present invention may have prostate cancer. One skilled in the art will recognize that the optimal dose of the administered drug will vary from individual to individual. The dosage for an individual patient should take into account the patient's height, weight, rate of absorption, and rate of metabolism of the drug of interest, the stage of the disease being treated, and other co-administered drugs.
[0049]
“Treating” or “treatment” means the medical management of a patient intended to cure, reduce, stabilize, or prevent the disease, pathological condition, or disease. The term refers to aggressive therapy, i.e., treatment specifically intended to improve or cure the disease, pathological condition, or cure of the disease, as well as causative therapy, i.e. Includes treatments intended for removal. The term also refers to palliative therapy, i.e., a treatment intended to alleviate the disease, pathological condition, or disease rather than cure it, as well as prophylactic therapy, i.e., the occurrence of a related disease, pathological condition, or disease. To help minimize, or partially or completely suppress, or even supportive therapy, i.e., support the associated disease, pathological condition, or other specific treatment intended to ameliorate the disease Treatments used for the treatment. The term "treatment" also includes symptomatic treatment, i.e., treatment of the associated disease, pathological condition, or systemic symptom of the disease.
[0050]
"Prostate or testicular disease" means a disease of prostate or testis function and / or structure in an organism that results from an external factor, genetic predisposition, physical or chemical trauma, or a combination thereof. I do. Such diseases include prostate or testis cell proliferation. The term "proliferation of cells" means the growth or regeneration of similar cells, and the present invention provides a reagent for suppressing proliferation and a reagent for promoting proliferation. By "inhibiting proliferation" is meant reducing the number of similar cells by at least 10%, more preferably at least 20%, and most preferably at least 50%. By "promoting proliferation" is meant increasing the number of similar cells by at least 10%, more preferably at least 20%, and most preferably at least 50%.
[0051]
Using the reagents described herein, e.g., antisense-expressing vectors, antagonists, or prostate-specific or testis-specific polypeptides, or inhibitors of nucleic acid molecules, e.g., in excess of prostate or testis cells Proliferation can be suppressed. Blocking the expression or activity of prostate-specific or testis-specific polypeptides or nucleic acid molecules in prostate or testis cells alters the molecular pathways in cancer cells to better understand apoptotic, or dying, cells A process of cell death can occur that exhibits the biochemical characteristics (such as cytolemmal vesicle formation, cell body shrinkage, chromatin condensation, and DNA ladder formation).
[0052]
Diseases of the prostate or testis include prostate cancer, benign prostatic hyperplasia, acute prostatitis, testicular cancer, abnormal prostate or testis development (such as stagnated testes, and traction or disappearance testes).
[0053]
By "proliferative disease" is meant a disease that results from inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. For example, cancers such as prostate cancer, testicular cancer, lymphoma, leukemia, melanoma, ovarian cancer, breast cancer, pancreatic cancer, liver cancer, and lung cancer are all examples of proliferative diseases.
[0054]
"Modulate" or "regulatory" refers to altering by reducing or increasing the expression or biological activity of a prostate-specific or testis-specific nucleic acid molecule or polypeptide described herein. Means Although the degree of modulation provided by a modulatory compound in any assay will vary, one of skill in the art will identify the level of biological activity that identifies the compound that modulates a prostate-specific or testis-specific nucleic acid molecule or polypeptide. It would be possible to determine a statistically significant change in.
[0055]
The present invention offers several advantages. For example, the invention relates to the diagnosis and treatment of prostate and testis related diseases, and other diseases and conditions that are sensitive to the biological activity of the reagents (eg, polypeptides, nucleic acids, antibodies) described herein. The present invention provides methods and reagents that can be used for: Since the prostate-specific or testis-specific polypeptides of the present invention have been found to be strongly expressed in the prostate and testis, these polypeptides may also be used in therapeutic methods to treat prostate and testis related disorders. It can also be used for screening. Such polypeptides are also expressed in other tissues and can be used as therapeutics and diagnostics for cell proliferative disorders.
[0056]
Other features and advantages of the invention will be apparent from the description and drawings, and from the claims.
[0057]
DETAILED DESCRIPTION OF THE INVENTION
The basic biology of normal prostate and testis, and the initiation and progression of prostate and testicular cancer is poorly understood. Thus, there is a need to clarify the molecular mechanisms underlying these processes. To this end, we have identified, cloned and characterized genes that are highly expressed in prostate and testis, where the gene product plays a key role in both the normal and pathophysiology of the prostate and testis. Analysis was performed. Such gene products may also have varying degrees of low and sometimes very high expression of a particular gene product in tissues where Northern analysis is detectable, such as in the heart, brain, liver, pancreas, kidney, and colon. It also plays an important role in diseases.
[0058]
The present invention provides prostate-specific or testis-specific polypeptides and nucleic acid molecules (see below), and diagnostic and therapeutic methods using these polypeptides and nucleic acid molecules. The present invention also provides methods for identifying compounds that modulate the biological activity of prostate-specific or testis-specific polypeptides and nucleic acid molecules, as well as methods of treatment using these compounds. After an initial description of the diagnostic, therapeutic, and screening methods of the present invention, a general approach used to implement these methods will be described. Finally, experimental results supporting the method of the present invention will be described.
[0059]
Bioassay method
Prostate-specific and testis-specific polypeptides are expressed in the prostate and testis, as well as in other tissues such as kidney, pancreas, liver, lung, and colon. To identify cell targets on which prostate-specific and testis-specific polypeptides act, based on the expression pattern of prostate-specific and testis-specific polypeptides in specific cells and tissues, and Biological activities associated with related diseases such as prostate cancer, testicular cancer, benign prostatic hyperplasia, acute prostatitis, and testicular abnormalities can be identified.
[0060]
The utility of the prostate-specific and testis-specific polypeptides in therapy and diagnosis is identified, for example, by performing bioassays in vitro. Culture systems are selected that reflect the expression pattern of the prostate-specific and testis-specific polypeptides along with the distribution of specific receptors, such as the androgen receptor. For example, LNCaP cells express androgen receptor and respond to one or more isoforms of a prostate and testis specific polypeptide in various bioassays. The activity of prostate and testis-specific polypeptides (eg, STMP1, SSH9, PSL22) includes inhibition of proliferation, apoptosis, signal transduction (eg, altered kinase activity), transcription factor activity (eg, androgen receptor transcription factor activity) ) Are compared using sister cultures in a variety of dose-response assays, including, but not limited to, alterations, intracellular trafficking, or intracellular signaling. The relative titers of prostate-specific and testis-specific polypeptides are determined, for example, based on protein concentration.
[0061]
Diagnostic methods using prostate-specific or testis-specific nucleic acid molecules, polypeptides, and antibodies
Diagnosis of a variety of diseases and conditions, including those in which prostate- or testis-specific nucleic acid molecules, polypeptides, and antibodies are associated with mutations or inappropriate expression of prostate- or testis-specific genes Method or monitoring method. Prostate-specific or testis-specific expression has been demonstrated in various tissues as described above. Thus, detection of prostate-specific or testis-specific genes, or abnormal expression thereof, is used in diagnostics, therapeutics, or methods of monitoring disease development in such tissues.
[0062]
The diagnostic methods of the present invention are used, for example, in patients with prostate or testis-related disorders, such as prostate or testicular cancer, for the purpose of determining the pathology and thereby facilitating the selection of an appropriate treatment route. The diagnostic method is also used for patients who have not yet developed a disease associated with the prostate or testis, but who are at risk of developing such a disease, or who are in the early stages of developing such a disease. Because many diseases associated with the prostate or testis occur during development, the diagnostic methods of the invention may be performed on a fetus or developing embryo. The diagnostic method of the present invention is used, for example, in a prenatal genetic test for identifying a parent who is a carrier of a recessive mutation associated with the prostate or testis.
[0063]
Prostate-specific or testis-specific abnormalities detected by the diagnostic methods of the invention include, for example, (i) abnormalities in prostate-specific or testis-specific polypeptides, (ii) production of such polypeptides. Prostate-specific or testis-specific genes, including mutations, and (iii) abnormalities characterized by mutations that result in the production of abnormal amounts of prostate-specific or testis-specific polypeptides. .
[0064]
The level of prostate-specific or testis-specific expression in a patient sample is determined by any of several standard techniques well known in the art. For example, prostate-specific or testis-specific expression in a biological sample from a patient (eg, blood, prostate or testis tissue sample, or amniotic fluid) is monitored by standard Northern blot analysis or quantitative PCR ( See, eg, Ausubel et al., "Current Protocols in Molecular Biology", John Wiley & Sons, New York, NY, 1998; DNA Amplification), edited by HA Ehrlich, Stockton Press, NY; Yap et al., Nucl. Ac. See 4294,1991):. Ds Res, 19.
[0065]
Biological samples taken from the patient can be analyzed by mismatch detection for the presence or absence of one or more mutations in the prostate-specific or testis-specific nucleic acid molecule. In general, this method involves detecting a change in hybridization, an abnormality in mobility on an electrophoretic gel, binding or cleavage by a mismatch binding protein, or performing nucleic acid amplification after PCR amplification of a nucleic acid molecule from a patient sample. Mutations (ie, mismatches) are identified by directly determining the molecular sequence. Any of these techniques can be used to facilitate the detection of mutant prostate-specific or testis-specific genes and are well known in the art. Examples of such techniques are described, for example, by Orita et al. (Proc. Natl. Acad. Sci. USA 86: 2766-2770, 1989), and by Sheffield et al. (Proc. Natl. Acad. Sci. USA). 86: 232-236, 1989).
[0066]
The mismatch detection method also provides an opportunity to diagnose a predisposition to develop a disease involving a prostate-specific or testis-specific gene before the onset of symptoms. For example, patients heterozygous for a prostate-specific or testis-specific mutation that suppresses normal prostate-specific or testis-specific biological activity or expression are associated with a prostate-specific or testis-specific gene There may be no clinical symptoms of the disease, but the probability of developing prostate or testicular disease may be higher than normal. In the case of such a diagnosis, precautions may be taken to minimize the exposure of the patient to adverse environmental factors and to carefully monitor medical symptoms (eg, perform frequent physical examinations). As described above, this type of diagnostic method can also be used to detect prostate-specific or testis-specific mutations in prenatal screening.
[0067]
The prostate-specific or testis-specific diagnostic assays described above may be performed on any biological sample in which a prostate-specific or testis-specific polypeptide or nucleic acid molecule is normally expressed, such as a blood, prostate or testis tissue sample, Or amniotic fluid). Mutant prostate-specific or testis-specific genes can also be identified using such sources as test samples. Alternatively, prostate-specific or testis-specific mutations may be detected as part of a diagnosis of a predisposition to a disease involving a prostate-specific or testis-specific gene using DNA samples derived from any cell, for example, mismatch detection. Can be considered by law. Preferably, the DNA sample is analyzed after performing PCR amplification.
[0068]
In yet another diagnostic method of the invention, an immunoassay is used to detect or monitor the expression of a prostate-specific or testis-specific protein in a biological sample. Polyclonal or monoclonal antibodies against prostate-specific or testis-specific polypeptides (described below) can be linked to any standard immunoassay format (eg, ELISA, Western blot, or RIA; see, eg, Ausubel et al., Supra). It can be used to measure prostate-specific or testis-specific polypeptide levels. These levels are compared to wild-type prostate-specific or testis-specific levels. For example, increased production of a prostate-specific or testis-specific polypeptide is indicative of a condition or predisposition to a condition involving overexpression of the biological activity of a prostate-specific or testis-specific polypeptide, such as late stage prostate cancer. There are cases.
[0069]
Prostate-specific or testis-specific polypeptides can also be detected using immunohistochemical techniques. For example, a tissue sample is taken from a patient, sectioned, and contains a prostate-specific or testis-specific antibody (described below), and any standard detection system (eg, a horseradish peroxidase-conjugated secondary antibody, including Staining is performed to determine the presence or absence of prostate-specific or testis-specific polypeptides. General guidance on such techniques can be found, for example, in Bancroft et al., "Theory and Practice of Histologic Technologies", Churchill Livingstone, 1982, and Ausubell. Ausubel et al. (Supra).
[0070]
In a preferred example, evaluation of prostate-specific or testis-specific protein production (eg, immunological techniques or protein cleavage tests (PTT) (Hogerlorst et al., Nature Genetics 10: 208-212, 1995), and finer Hybrid diagnostic methods can be used that include the evaluation of nucleic acid molecule-based detection methods intended to identify prostate-specific or testis-specific mutations (eg, point mutations). Assays are available to those of skill in the art, and any preferred technique can be used.Prostate-specific or testis-specific mutations in the gene can be expressed in prostate-specific or testis-specific polypeptides. Or loss or gain of expression of a nucleic acid molecule, or normal prostate-specific or It is possible to detect the result of loss or acquisition of the biological activities of nests specific polypeptide or nucleic acid molecule.
[0071]
Use prostate-specific or testis-specific polypeptides or nucleic acids to correlate prostate cancer progress with markers other than PSA, monitor the progress of anti-cancer therapy, or detect tumor cells contained in the system can do. For example, a predetermined amount for RNA encoding a prostate-specific or testis-specific polypeptide correlates with the presence of tumor cells obtained, for example, by biopsy. Total RNA was extracted from biopsy specimens and real-time quantitative rt-PCR using individual reactions with primer pairs specific for prostate-specific or testis-specific sequences was found to be cancer-free. Performed in parallel with biopsy specimen. A biopsy specimen is determined to contain cancer cells if the amount of detected prostate-specific or testis-specific mRNA is at least 5 times greater than that of a control specimen. Exemplary extraction of total RNA uses a Qiagen BioRobot kit equipped with a BioRobot 9600 system, and real-time rtPCR is performed on a Perkin Elmer ABI Prism 7700.
[0072]
In another aspect of the present subject matter, the method of detecting tumor cells need not be limited to biopsy tissue from prostate or testis tissue, but includes lymphoma tumor cells, and various solid tumor cells. A variety of other tissues can be used, as long as such tumor cells overproduce prostate-specific or testis-specific polypeptide mRNA. Other suitable tumor cells can be easily identified by the methods described above. Similarly, the system need not be limited to mammals, but may also include cultures of cells and tissues grown in vitro, as well as tumor cells and samples from non-mammalian animals. For example, tumor cells and tissues grown in vitro can be advantageously used to study drug action on such cells, and sequences encoding prostate-specific or testis-specific polypeptides can be used as tumor markers. Can be used conveniently. Alternatively, body fluids (eg, serum and saliva) that may or may not contain tumor cells, as long as they contain some mRNA that encodes a prostate-specific or testis-specific polypeptide. A suitable substrate used in the method described in (1).
[0073]
In yet another aspect of the subject method, the amount of the polypeptide is not necessarily limited to at least 5 times greater than the subject specimen to determine that the tissue contains cancer cells. For example, if the polypeptide concentration is hormone dependent, then 3- to 8-fold and higher amounts may be appropriate. In contrast, if the concentration of cancer cells in the biopsy specimen is relatively low, less than 5-fold (including 1.5-4.9 and less) is of interest.
[0074]
The detection process can include fluorescence detection, luminescence detection, scintigraphy, autoradiography, and dye formation. For example, in microscopic analysis of biopsy specimens, it is particularly advantageous to couple a luciferase-labeled probe to a luminescent substrate (eg luciferin). The quantification of luminescence can then be performed using a CCD camera and an image analysis system. Similarly, radioactivity can be detected by autoradiographic or scintigraphic techniques on tissue sections in liquid or on solid supports. Where the probe is a natural or synthetic ligand of a prostate-specific or testis-specific polypeptide, such ligands may be provided with increased affinity for the polypeptide and / or irreversible to the polypeptide. Molecules that have been chemically modified to induce binding can be included. For example, transition state analogs or cell death inhibitors for particular reactions catalyzed by such polypeptides are of particular interest. The labeling of antibodies, antibody fragments, small molecules, and conjugation of labels is a technique well known in the art, and all known methods are generally suitable for use with the methods of interest herein. Also, the probes need not be limited to antibodies labeled with fluorescein, and other probes include antibody fragments (e.g., Fab, Fab ', scFab, etc.).
[0075]
Yet another contemplated variation involves the replacement of one or more atoms or functional groups on the sequence with a radioactive atom or functional group. For example, when cDNA is used as a probe specific for hybridization, the position of the complementary sequence and / or Or the amount can be identified. Alternatively, if the cDNA of interest is used in an affinity isolation procedure, the cDNA may be converted to a high affinity (ie, K_d<10^-4 mol^-1) Can be attached to a molecule known to have a partner. In another example, one or more phosphate groups are³²P or³³By exchanging with a radioactive phosphate group containing a P isotope, detection and quantification can be assisted by using radiolabeled cDNA as a probe for hybridization.
[0076]
Prostate- or testis-specific nucleic acid molecules, polypeptides, and therapeutic methods using antibodies
The present invention includes a method for treating or preventing a prostate-specific or testis-specific disease. Therapy involves bypassing or overcoming prostate-specific or testis-specific gene deficiency, or inappropriate or excessive prostate-specific or testis-specific gene expression, to produce a prostate-specific or testis-specific gene. Alternatively, it is designed to regulate, and if possible alleviate, conditions involving protein deficiency. In considering various therapies, it is clear that such therapies preferably target the affected or likely affected organs (eg, prostate or testis). Reagents used in modulating prostate-specific or testis-specific biological activity include full-length prostate-specific or testis-specific polypeptide; prostate-specific or testis-specific cDNA, mRNA, or Antisense RNA; Prostate-specific or testis-specific antibodies; and any compound that modulates the biological activity, expression, or stability of a prostate-specific or testis-specific polypeptide or nucleic acid molecule can be included. Is not limited to these.
[0077]
Treatment or prevention of a disease caused by a mutant prostate-specific or testis-specific gene, for example, replacing a mutant prostate-specific or testis-specific gene with a normal prostate-specific or testis-specific gene By controlling the function of the mutant prostate-specific or testis-specific protein by administering a normal prostate-specific or testis-specific gene, the normal prostate-specific or testis-specific protein By altering the amount of normal or mutant prostate-specific or testis-specific protein. It is also possible to correct prostate-specific or testis-specific gene deficiencies to modify physiological pathways involving prostate-specific or testis-specific proteins (eg, intracellular transport pathways).
[0078]
Large amounts of pure prostate-specific to replace the mutant protein with normal protein, or to add protein to cells that do not express sufficient or normal prostate-specific or testis-specific genetic protein It may be necessary to obtain target or testis specific protein from a cultured cell line in which the protein of interest is expressed (see, eg, below). Delivery of the protein to the diseased tissue can then be achieved using a suitable packaging or administration system. Alternatively, the desired physiological effect can be obtained by administering a small molecule analog that acts as an agonist or antagonist of a prostate-specific or testis-specific molecule (see below).
[0079]
Gene therapy is another treatment that prevents or alleviates diseases caused by prostate or testis-specific genetic defects. Nucleic acid molecules encoding wild-type prostate-specific or testis-specific proteins can be used in cells that lack sufficiently normal prostate-specific or testis-specific biological activity (eg, prostate-specific or testis-specific genes). (Mutated cells). Nucleic acid molecules must be delivered to these cells in such a way that they are taken up by the cells and sufficient protein is produced to provide effective prostate-specific or testis-specific function. Alternatively, in the case of some prostate-specific or testis-specific mutations, the introduction of another copy of the homologous gene with a second mutation on the gene slows the progression of the disease or prostate-specific or testis-specific It may be possible to modulate specific activity, change the mutation of interest, or block another negative effect with another gene.
[0080]
Introduction of vectors of retrovirus, adenovirus, and adeno-associated virus can be used for gene therapy targeting somatic cells. This is particularly due to the high efficiency of infection and the efficiency of stable integration and expression (eg, Cayouette et al., Human Gene Therapy 8: 423-430, 1997; Kido et al., Current Eye Research 15: 833-844, 1996; See Bloomer et al., Journal of Virology 71: 6641-6649, 1997; Naldini et al., Science 272: 263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. For example, a full-length prostate-specific or testis-specific gene, or a portion thereof, is cloned into a retroviral vector and its endogenous promoter, from the retroviral long terminal repeat (LTR), or from the target cell of interest ( Expression can be initiated from any of the promoters specific to aortic cells or other vascular cells). Other viral vectors that can be used include, for example, vaccinia virus, bovine papilloma virus, or herpes virus (such as Epstein-Barr virus) (eg, Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244). Eglitis et al., BioTechniques 6: 608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1: 55-61, 1990; Acid Research and Molecular Biology 3 : 31-322, 1987; Anderson, Science 226: 401-409, 1984; Moen, Blood Cells 17: 407-416, 1991; or also see the vector described in Miller et al., Biotechnology 7: 980-990, 1989). . Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N Engl. J. Med 323: 370, 1990; Anderson et al., US Pat. No. 5,399,346).
[0081]
Gene transfer can be achieved by non-viral techniques including in vitro transfection by any standard technique, including, but not limited to, the calcium phosphate method, the DEAE dextran method, the electroporation method, the protoplast fusion method, and the liposome method. Can also be achieved. Transplantation of a normal gene into a patient's diseased tissue involves transferring a normal prostate-specific or testis-specific gene ex vivo into culturable cells and then injecting the cells (or their progeny) into the target tissue. It can also be achieved. Other methods of using gene therapy to suppress prostate-specific or testis-specific function include prostate-specific or testis-specific antibodies, or partial prostate-specific or testis-specific antibodies in cells. And the like. For example, a gene (or gene fragment) encoding a monoclonal antibody that specifically binds to a prostate-specific or testis-specific polypeptide and inhibits its biological activity can be transcribed from a tissue-specific gene regulatory sequence. Place under control. In other treatments, the prostate-specific or testis-specific recombinant polypeptide is administered directly (eg, by injection) to a potentially affected or actually affected tissue site, or Administered systemically (by conventional recombinant protein administration methods). The dosage of the prostate-specific or testis-specific polypeptide will be affected by a number of factors, including the size and health of the individual patient, but will generally range from about 0.006 mg / kg to about 0.006 mg / kg per day. 0.6 mg / kg is administered to an adult in any pharmaceutically acceptable dosage form.
[0082]
Therapeutic DNA can also be introduced into cells predicted to develop a disease involving a prostate-specific or testis-specific disease using non-viral methods. For example, a prostate-specific or testis-specific nucleic acid molecule, or an antisense nucleic acid molecule can be prepared by lipofection (Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413, 1987; Ono et al., Neuroscience Letters 17: 259). Sci. 298: 278, 1989; Staubinger et al., Methods in Enzymology 101: 512, 1983), Asialo orosomucoid-polylysine junction method (Wu et al. 14621, 1988; Wu et al., Journal of Biological Chemistry 264: 16985, 1989). Alternatively, but less preferably, the cells can be introduced by microinjection under surgical conditions (Wolff et al., Science 247: 1465, 1990).
[0083]
Expression of prostate-specific or testis-specific cDNA used in gene therapy is derived from any suitable promoter, such as the promoter of human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein. And can be regulated by any suitable mammalian regulatory sequence. For example, if desired, prostate-specific or testis-specific expression can be induced using enhancers known to selectively induce gene expression in particular cells. Enhancers used may include, but are not limited to, enhancers characterized as tissue or cell specific enhancers. Alternatively, if a prostate-specific or testis-specific genomic clone could be used as a therapeutic construct (such clones could be identified by hybridization with a prostate-specific or testis-specific cDNA, as described above), Can be mediated by homologous regulatory sequences or, if desired, by regulatory sequences from heterologous sources, including any of the promoters or regulatory sequences described above.
[0084]
Antisense-based strategies can be used to examine prostate-specific or testis-specific gene function and as a basis for designing therapeutics. These strategies are based on the principle that sequence-specific suppression of gene expression (via transcription or translation) can be achieved by intracellular hybridization of genomic DNA or mRNA with complementary antisense species. Upon formation of the hybrid RNA duplex, transcription of a targeted prostate-specific or testis-specific genomic DNA molecule or processing, transport, translation, or stabilization of a targeted prostate-specific or testis-specific mRNA molecule Sex is disturbed.
[0085]
Antisense strategies can be implemented by various approaches. For example, an antisense oligonucleotide or an antisense RNA can be directly introduced into a subject (for example, intravenous injection) while being taken up by cells. Alternatively, a viral or plasmid vector encoding the antisense RNA (or a fragment of the antisense RNA) can be introduced into cells in vivo or ex vivo. Antisense effects can be induced by control (sense) sequences, but the degree of phenotypic change is highly variable. The effect on the phenotype induced by the effect of antisense is based on changes in reference values such as the amount of protein, the measured activity of the protein, and the amount of target mRNA.
[0086]
For example, prostate or testis-specific gene therapy can adversely affect prostate-specific or testis-specific antisense mRNA by expression of wild-type or mutant prostate-specific or testis-specific polypeptides It can also be achieved by direct administration to the expected cells. Antisense prostate-specific or testis-specific mRNA can be made and isolated by any standard technique, but with the antisense prostate-specific mRNA placed under the control of a high efficiency promoter (eg, the T7 promoter). It can be most easily prepared by an in vitro transcription method using target- or testis-specific cDNA. Administration of antisense prostate-specific or testis-specific mRNA to cells can be performed by any of the direct nucleic acid molecule transfer methods described above.
[0087]
Other strategies to suppress prostate or testis-specific function using gene therapy include prostate-specific or testis-specific antibodies, or intracellular expression of some prostate-specific or testis-specific antibodies There is. For example, a gene (or gene fragment) encoding a monoclonal antibody that specifically binds to a prostate-specific or testis-specific polypeptide and suppresses its biological activity can be transcribed by a tissue-specific gene regulatory sequence. Can be placed below.
[0088]
In another therapy encompassed by the present invention, a recombinant prostate-specific or testis-specific polypeptide is directly (eg, by injection) to a potentially affected or actually affected tissue site. Administered or administered systemically (eg, by any conventional recombinant protein administration method). The dosage of a prostate-specific or testis-specific polypeptide will be affected by a number of factors, including the size and health of the individual patient, but will generally be from 0.1 mg to 100 mg per day, It is administered to the adult in any pharmaceutically acceptable dosage form.
[0089]
Patients diagnosed with a prostate-specific or testis-specific mutant gene, or a prostate-specific or testis-specific disease, or a prostate-specific or testis-specific gene mutation, prostate-specific or testis-specific Aberrant expression of a polypeptide or nucleic acid molecule (even if the mutation or expression pattern does not yet result in an abnormality in prostate-specific or testis-specific expression or biological activity), or a prostate-specific or testis-specific disease In patients diagnosed as susceptible to, any of the treatments described above are administered before the onset of the disease phenotype. Compounds that may modulate the expression of a prostate-specific or testis-specific polypeptide or nucleic acid molecule, or the biological activity of a prostate-specific or testis-specific polypeptide or nucleic acid molecule, may also be affected. A patient or a patient who is actually affected is administered at any standard dosage and route of administration. Alternatively, gene therapy using a prostate-specific or testis-specific antisense mRNA expression construct can reverse or prevent gene deficiency before the onset of complete disease onset.
[0090]
The treatments of the invention are targeted in some cases to prenatal treatment. For example, administering a gene therapy vector containing a prostate-specific or testis-specific normal gene to a fetus known to have a prostate-specific or testis-specific mutation, or a normal prostate-specific or testis-specific A good protein. Such treatment may require only a small amount of time or, in some circumstances, throughout the life of the patient. However, whether continuation of treatment is required is determined, for example, using the diagnostic methods described above. Also, as noted above, abnormalities in prostate-specific or testis-specific polypeptides or nucleic acid molecules can be associated with disease in adults, and adults are also targeted by the treatment methods of the invention.
[0091]
In addition, prostate-specific or testis-specific polypeptides can be used to enhance the immune system, for example, to increase immunity to prostate cancer cells.
[0092]
The methods of the present invention can be used to diagnose or treat a disease that occurs in any of the mammals described herein (eg, humans, pets, or livestock). When treating or diagnosing a non-human mammal, the prostate-specific or testis-specific polypeptide, nucleic acid molecule or antibody used is preferably specific to the species of interest.
[0093]
Identification of molecules that modulate the biological activity of a prostate-specific or testis-specific polypeptide or nucleic acid molecule that is modulated by the prostate-specific or testis-specific polypeptide or nucleic acid molecule
Isolation of prostate-specific or testis-specific cDNA (as described herein) is also directed to a group of molecules that increase or decrease the biological activity of a prostate-specific or testis-specific polypeptide or nucleic acid molecule. Promote identification. Similarly, a group of molecules whose activity is modulated by the biological activity of a prostate-specific or testis-specific polypeptide or nucleic acid molecule can be identified. In one method, varying concentrations of a candidate molecule are added to a culture of cells expressing prostate-specific or testis-specific mRNA. The prostate-specific or testis-specific biological activity is then measured by standard techniques. Measuring biological activity includes, but is not limited to, measuring expression levels of prostate- or testis-specific protein and nucleic acid molecules, measuring response to androgens, or measuring localization and transport in cells Not done.
[0094]
If desired, the effect of candidate modulators on expression can also be determined by the same general methods and standard immunological detection methods (Western blotting using prostate-specific or testis-specific antibodies (see below), or immunoprecipitation). Method, etc.) at the level of prostate-specific or testis-specific protein production.
[0095]
The test compound to be screened by the above-described method may be a chemical substance, a natural substance, or an artificially produced one. Such compounds include, but are not limited to, for example, polypeptides, synthetic organic molecules, natural organic molecules, nucleic acid molecules, and components thereof. Candidate prostate-specific or testis-specific modulators include peptide molecules as well as non-peptide molecules (eg, cell extracts, mammalian serum, or peptides found in the growth medium in which mammalian cells are cultured). Molecules or non-peptide molecules).
[0096]
Prostate-specific or testis-specific polypeptide, prostate-specific or testis-specific Of novel nucleic acid molecules and modulators of the synthesis and function of prostate-specific or testis-specific polypeptides or nucleic acid molecules
Administer a unit dose of a prostate-specific or testis-specific protein, nucleic acid molecule, or modulator to a patient or experimental animal in a pharmaceutically acceptable diluent, carrier, or excipient . Also, using conventional pharmaceutical treatments, prostate-specific or testis-specific neutralizing antibodies, or prostate-specific or testis-specific inhibitory compounds (eg, prostate-specific or testis-specific antisense molecules, Or a prostate-specific or testis-specific dominantly negative variant) with a prostate-specific or testis-specific disease, such as prostate cancer, testicular cancer, benign prostatic hyperplasia, or a prostate or testis developmental disease Provide a suitable formulation or composition for administration to the patient. Administration can begin before or after the patient exhibits symptoms.
[0097]
Any suitable route of administration may be employed, for example, parenteral, intravenous, intraarterial, subcutaneous, intramuscular, intracranial, intraorbital, intraocular, intraventricular, intraarticular, intramedullary Intracavitary, intracisternal, intraperitoneal, intranasal, deep lung inhalation, aerosol, suppository use, oral or topical (eg, application of an adhesive patch containing a formulation that penetrates the skin and enters the bloodstream) ). Preferably, administration is local to the diseased tissue, such as prostate or testis tissue. Therapeutic dosage forms can be in liquid or suspension form; for oral administration, the dosage form can be tablets or capsules; and for intranasal dosage forms, powders , Nasal drops, or aerosols. Any of the above dosage forms may be a sustained release formulation.
[0098]
Methods well known in the art for making formulations are described, for example, in "Remington's Pharmaceutical Sciences", 18th Edition, A.M. Gennaro, 1990, Mack Publishing Company, Easton, PA. Dosage forms for parenteral administration may include, for example, sterile water or saline; polyalkylene glycols such as polyethylene glycol; vegetable oils; or excipients such as hydrogenated naphthalene. Controlled release can be controlled using sustained release, biocompatible, biodegradable lactide polymers, lactide / glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers. Other potentially useful parenteral delivery systems for prostate-specific or testis-specific modulatory compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. And so on. Formulations for inhalation can include excipients such as lactose, and can be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate, and deoxycholic acid. Alternatively, it can be an oily solution to be administered as a gel.
[0099]
Prostate-specific or testis-specific fragments
Polypeptide fragments containing various portions of a prostate-specific or testis-specific protein are useful for identifying domains that play important roles in biological activities such as protein-protein interactions and transcription. Methods for making such fragments using the nucleotide sequences described herein are well known in the art (see, eg, Ausubel et al., Supra). For example, a fragment of a prostate-specific or testis-specific protein can be obtained by using an oligonucleotide primer designed based on a prostate-specific or testis-specific nucleic acid sequence to produce a desired nucleic acid molecule fragment specific to the prostate or testis. It can be prepared by PCR amplification. The oligonucleotide primer preferably contains a unique restriction enzyme cleavage site that allows the fragment after amplification to be inserted into the cloning site of an expression vector (eg, a mammalian expression vector, see above). This vector is then artificially introduced into a cell (eg, a mammalian cell; see above) by any of a variety of methods known in the art described herein, and may be prostate-specific or testis-specific. A polypeptide fragment is produced in a cell containing the expression vector.
[0100]
Prostate-specific or testis-specific polypeptide fragments (eg, chimeric fusion proteins) can also be used to produce antibodies specific to various regions of the prostate-specific or testis-specific polypeptide. Preferred prostate-specific or testis-specific fragments include, but are not limited to, the N-terminal domain of STMP1 (amino acids 1-200), the P5CR domain, and fragments comprising those fragments.
[0101]
Synthesis of prostate-specific or testis-specific proteins, polypeptides, and polypeptide fragments
Those skilled in the field of molecular biology will understand that a variety of expression systems can be used to produce recombinant prostate-specific or testis-specific protein. The type of host cell used is not critical to the invention. Prostate-specific or testis-specific proteins may be prokaryotic hosts (eg, E. coli) or eukaryotic hosts (eg, S. cerevisiae, insect cells such as Sf9 cells, COS, NIH 3T3, CHO, Or mammalian cells such as HeLa cells). These cells are described, for example, in the American Type Culture Collection, Rockville, Md., MD (Ausubel et al., "Current Protocols in Molecular Biology & Wyogy." See also New York, NY, 1998). The method of transformation and the choice of transporter for expression (eg, expression vector) will depend on the host system chosen. Methods for transformation and transfection are described, for example, in Ausubel et al., "Current Protocols in Molecular Biology", John Wiley & Sons, New York, 1998, 1998. Transporters can be selected, for example, from those described in Powells et al., "Cloning Vectors: A Laboratory Manual", 1985, Supplement, 1987.
[0102]
The characteristics of prostate-specific or testis-specific nucleic acid molecules are analyzed by introducing such genes into various cells or using an in vitro extracellular system. The function of prostate-specific or testis-specific proteins made in such cells or systems is then examined under various physiological conditions. It also overexpresses prostate-specific or testis-specific gene products that enable the purification of prostate-specific or testis-specific proteins for biochemical characterization, large-scale production, antibody production, and treatment of patients Cell lines can be created.
[0103]
The polypeptides of the present invention can be made in vivo or in vitro, and can be modified chemically and / or using enzymes. Such polypeptides can be isolated from prostate tissue or prostate cancer cells, which may or may not be in a hormone-dependent state. Alternatively, and especially where large amounts (ie,> 10 mg) are desired, the production of recombinants (eg, in bacterial, yeast, insect cells, or mammalian cell lines) is advantageously used to produce significant amounts of prostate-specific Alternatively, testis-specific polypeptides can be produced.
[0104]
In addition, the production of recombinants not only provides a more economical way of making polypeptides of the invention, but also amino acid sequences tailored to specific biochemical, catalytic, and physical properties. It also allows for specific modifications of the composition. For example, if high solubility is desired, one or more hydrophobic amino acids may be replaced with hydrophilic amino acids. Alternatively, if it is necessary to increase or decrease the catalytic activity, one or more amino acids may be substituted or removed.
[0105]
In yet another example, the polypeptides of the invention can be fused to, for example, enzymatically active partners (eg, for pigmentation or substrate conversion) and fluorescent partners such as GFP, EGFB, BFP. Can be synthesized as a fusion protein.
[0106]
For chemical and enzymatic modifications of the polypeptides of interest, the addition of monofunctional and bifunctional linkers, binding to proteinaceous and non-proteinaceous macromolecules, and glycosyl Many modifications are appropriate, including the modification. For example, monofunctional and bifunctional linkers are used when the polypeptide is immobilized on a solid support or when covalently linked to a molecule that increases the immunogenicity of the polypeptide of interest (eg, binding to KLH or BSA). Is particularly advantageous. Alternatively, by binding such a polypeptide to an antibody or antibody fragment, the polypeptide can be rapidly recovered from the molecular mixture. Other linkages include the formation of covalent and non-covalent bonds between the polypeptide and a molecule that increases serum half-life and / or reduces immunogenicity (such as cyclodextrin and polyethylene glycol). is there.
[0107]
Use of prostate-specific or testis-specific antibodies
Antibodies to prostate-specific or testis-specific proteins can be used to suppress the biological activity of prostate-specific or testis-specific proteins, which detect prostate-specific or testis-specific proteins. For example, a nucleic acid molecule encoding an antibody or a portion of an antibody can be expressed in cells to inhibit prostate-specific or testis-specific function. A compound such as a radionuclide or a liposome can be bound to such an antibody for diagnosis or therapy. Antibodies that suppress the activity of prostate-specific or testis-specific polypeptides are also useful for preventing or delaying the onset of disease due to inappropriate expression of wild-type or mutant prostate-specific or testis-specific genes But also. For example, the antibodies of the invention can be used to localize and localize disease-specific markers (eg, in prostate or testicular cancer) in prostate or testis tissue sections.
[0108]
Detection of prostate-specific or testis-specific gene expression
As described above, prostate-specific or testis-specific protein expression can be monitored using the antibodies described above. By performing in situ hybridization of RNA, expression of a prostate-specific or testis-specific gene group can be detected. RNA in situ hybridization techniques are based on the hybridization of specifically labeled nucleic acid probes to cellular RNA contained in individual cells or tissues. Thus, RNA in situ hybridization is a powerful method for examining tissue or time-specific gene expression. This method uses oligonucleotides, cloned DNA fragments, or antisense RNA transcripts of cloned DNA fragments that correspond to unique parts of a group of prostate-specific or testis-specific genes, for example, in mice or the like. Specific mRNA species contained in animal tissues are detected at various stages of development or monitoring of tumor progression. Other gene expression detection methods are well known in the art and can be used to detect prostate-specific or testis-specific gene expression.
[0109]
Identification of additional prostate-specific or testis-specific genes
Prostate-specific or testis-specific homologs in other species are identified using standard techniques such as the polymerase chain reaction (PCR) and DNA hybridization, and SSH and other techniques described herein. And other prostate-specific or testis-specific genes in humans can be cloned. Prostate-specific or testis-specific genes and homologues can be easily obtained by performing stringency-reduced DNA hybridization using a human prostate-specific or testis-specific probe or primer, or by performing stringency-reduced PCR. Can be identified. Additional prostate-specific or testis-specific genes and homologs can be cloned by RT-PCR using degenerate primers encoding human prostate-specific or testis-specific amino acids.
[0110]
Other groups of prostate-specific or testis-specific genes include those expressed at various stages of growth and development involving pathological prostate or testis (eg, prostate cancer, benign prostatic hyperplasia, or testicular cancer). , And genes expressed as a result of drug therapy.
[0111]
Construction of transgenic and knockout animals
Analysis of the properties of prostate-specific or testis-specific genes provides information that enables the development of prostate-specific or testis-specific knockout animal models by homologous recombination. Preferably, the prostate-specific or testis-specific knockout animal is a mammal, most preferably a mouse. Similarly, animal models of prostate-specific or testis-specific overexpression can be created by incorporating one or more prostate-specific or testis-specific sequences into the animal genome using standard transgenic techniques. it can. In addition, the effect of a prostate-specific or testis-specific gene mutation (eg, a dominant gene mutation) can be determined by using a transgenic mouse having a mutant prostate-specific or testis-specific transgene, or by using such a mutation. It can be determined by introducing the gene into an endogenous prostate-specific or testis-specific gene using standard homologous recombination methods.
[0112]
A substitution type target vector that can be used for the production of a knockout model can be constructed using an isogenic genomic clone derived from a mouse strain such as 129 / Sv (Stratagene Inc., LaJolla, CA). By introducing the target vector into an appropriately extracted embryonic stem (ES) cell line by electroporation, an ES cell line having a large truncated form of the prostate-specific or testis-specific gene can be generated. it can. To generate chimeric primary mice, target cell lines are injected into blastula embryos of mice. Heterozygous offspring can be crossed to produce homozygous individuals. Prostate-specific or testis-specific knockout mice provide a tool for examining the role of prostate-specific or testis-specific polypeptides and nucleic acid molecules in embryonic development and disease. In addition, such mice may have in vivo pathways that rely on prostate-specific or testis-specific polypeptides or nucleic acid molecules, or pathways that are affected by prostate-specific or testis-specific polypeptides or nucleic acid molecules. It is an approach to study therapeutic compounds that alleviate the disease or condition involved.
[0113]
Animal model
By using the prostate-specific or testis-specific polypeptide or antisense compound of the present invention in an animal model of prostate or testicular disease, the therapeutic method, diagnostic method, and screening method of the present invention can be examined. it can. An exemplary prostate cancer model for transgenic mice is called TRAMP, in which the large T antigen of SV40 is targeted to the prostate (Greenberg, et al., PNAS 92, 3439-3443, 1995). Another test system includes CWR22 (androgen-independent) and CWR22R (androgen-independent) xenografts, which are well known in the art and described herein. Such xenograft growth, PSA secretion, metastasis, and the like can be monitored for the presence or absence of the prostate-specific or testis-specific polypeptides, nucleic acid molecules, and other compounds of the present invention. Other animal models can be used, for example, animal models of other types of cancer or immunodeficient animals (eg, nude mice).
[0114]
The following examples are provided to help those skilled in the art to better understand the present invention and its principles and advantages. These examples illustrate the invention and do not limit the scope of the invention.
[0115]
Example 1
Suppression subtraction of prostate-specific and testis-specific genes ( Subtraction )and Pzero Subcloning into
Poly A from 10 different normal human tissues⁺ CDNA derived from RNA was subtracted from normal human prostate cDNA by inhibitory subtraction hybridization (SSH) (Diatchenko, L. et al., Proc. Natl. Acad. Sci. USA 93, 6025-6030, 1996). The obtained cDNA fragment was cloned into an appropriate vector. SSH is obtained from normal human tissues (heart, brain, placenta, lung, liver, skeletal muscle, kidney, spleen, thymus, and ovary).⁺ Prostate poly A against a pool of RNA⁺ RNA was used in the procedure described in the literature (Clontech PCR-Select Cloning Kit). When performing secondary PCR amplification (12 cycles), the reaction was extracted with phenol / chloroform, the DNA was precipitated with ethanol, and the pellet was washed with 70% ethanol. After drying, the DNA pellet is 0.2 × TE or MQ dH₂It was dissolved in O, and cut with RsaI in a 20 μl reaction solution at 37 ° C. for 2 hours to cut out the adapter. After cleavage, the reaction was run on a 1.5% agarose gel with a molecular weight marker applied to another lane of the same gel at 5 V / cm for 40 minutes. At this time, care was taken not to expose the gel to short wavelength ultraviolet light. The band of the adapter was cut out, and the gel was electrophoresed at 5 V / cm for 15 minutes in a reverse electric field to concentrate the cDNA band.
[0116]
This gel was visualized (ultraviolet light with a long wavelength), and the amplified cDNA of 100 bp to 1 kb was cut out. This DNA was purified using a QAIEX gel DNA purification kit. The purified DNA was cloned into pZERO (Invitrogen) which had been cut with EcoRV and dephosphorylated. The ligation reaction was performed at 37 ° C. overnight in a final volume of 10 μl in the presence of 5% PEG, 1 × T4 ligase buffer, and a 1/5 dilution of 1 μl ligation mixture (PSL) was electrophoresed in DH10B. Competent cells (> 10 efficiency)¹⁰) Or equivalent. A colony was selected to confirm that there was a cDNA insert. For this, PCR was performed directly from the colonies using T7 and SP6 as primers. The 10% reaction was run on a 1.5% agarose gel to visualize the amplification products. Glycerol stocks (15%) were made by growing colonies with inserts and stored at -80 ° C.
[0117]
Example 2
Reverse snow blot and sequence analysis
The River Snowzan technique was used to clone the androgen responsive gene in PSL (Hedrick, SM et al., Nature 308, 149-153, 1984; Sakaguchi, N. et al., EMBO J 5: 2139-2147, 1986). ). In this method, a cDNA probe was prepared using RNA prepared from two cell populations to be compared, and then hybridized with two identical clone groups. For this, the PSL clone was amplified by PCR and spotted on a nylon filter in a 96-well format to generate two identical blots, each set consisting of 92 clones (the remaining four spots were used for positive and negative controls). Using). To make the probe, the prostate cancer cell line LNCaP, which responds to androgen, was used (Horoszewicz, JS, et al., Cancer Res, 43, 1809-1818, 1983), the rest untreated ((-) probe). Either left or treated with synthetic androgen R1881 for 24 hours ((+) probe). Poly A⁺ RNA is isolated from this cell,³²It was used for preparing a P-labeled probe. After hybridization with the (-) and (+) probes, clones that showed different hybridizations were selected for further analysis (confirmed by secondary reverse snow and northern blots).
[0118]
Reverse snowzan screening of cDNA clones was performed basically by partially modifying the procedure described in the literature (Hedrick, SM et al., supra; Sakaguchi, N. et al., supra). DNA (about 400 ng) from the PCR amplification of step 6 was diluted with 200 μl of 0.4 M NaOH, 10 mM EDTA and mixed well by pipetting. The tubes were cooled in ice after 5-10 minutes incubation at 95 ° C. The denatured DNA was blotted onto two other Zeta Probe GT + membranes (Bio-Rad) using a dot-blot apparatus (Bio-Rad). Two colonies of a positive control (cDNA for prostate specific antigen (PSA)) and a negative control (cDNA for glyceraldehyde triphosphate dehydrogenase (G3PDH)) were included on each blot (lower right). The membrane was washed with 2 × SSC, air-dried, and then heated at 80 ° C. for 30 minutes. An exemplary river snowzan analysis is shown in FIG. Here, there was a substantial increase in PSA hybridization in the (+) blot (probe prepared from cells stimulated with androgen) compared to the (-) blot (probe prepared from unstimulated cells), Note that hybridization of G3PDH shows no significant change between the two blots. Arrowheads indicate positive clones identified in the same experiment.
[0119]
To verify the tissue-specific nature of the isolated sequences, positive clones were examined on a standard Northern blot against RNA preparations of multiple non-prostate tissue samples. FIG. 2 shows a Northern blot of multiple tissues using NKX3A as a probe, showing an exemplary tissue expression pattern found in positive clones. Lanes 1-10 and 12-16 are RNA preparations from non-prostate tissue, lane 11 is RNA preparation from prostate, and lane 12 is RNA preparation from testis.
[0120]
Twelve clones without significant homology to the known sequence (by BLAST analysis) were isolated from prostate tissue and LNCaP cells. SEQ ID NOs: 1-9 were identified as a group of androgen-responsive genes that are differentially expressed in prostate, and SEQ ID NOs: 10-12 were identified as a group of androgen-responsive genes that are differentially expressed in LNCaP cells.
[0121]
Example 3
STMP1 Genes and mRNA Isolation and characterization
The normal prostate cDNA library was subjected to screening by 5'RACE analysis and 3'RACE analysis to obtain a full-length cDNA for L74. Computer-assisted secondary structure prediction of the deduced amino acid sequence of L74 suggested the presence of a six transmembrane domain on the C-terminal side, so L74 was named prostate six transmembrane protein 1 (STMP1).
[0122]
BLAST analysis was performed using the full-length STMP1 cDNA, which was found to match the BAC clone (GenBank Accession No. AC002064) except for the 313 bp repeat unit in the 3′UTR region. It was identified as the STMP1 gene, and was located on the long arm 21 of chromosome 7 (Chr7q21). Repeat regions are likely to be artefacts generated during the cloning or sequencing of BAC clones. Computer-based exon / intron boundary analysis and alignment of the full-length cDNA sequence with the BAC clone revealed that the STMP1 gene was composed of six exons and five introns (FIG. 4A). The transcription start site, exon and intron locations and sizes, and the location of the partial cDNA clone L74 (black squares) are shown. The start codon (atg) and stop codon (tga), as well as the putative polyadenylation signal (pA) are also shown. The first two exons are short non-coding exons (83 bp and 61 bp), and exons 3-6 encode reading frames (ORFs), 525 bp, 528 bp, 165 bp, and 3281 bp (FIG. 4C). The entire STMP1 gene is 26 kb, partially due to the presence of intron 2 (12713 bp) of extremely large size. Three different promoters are predicted within 4 kb upstream of the start codon of STMP1, but no clear TATA box or CAAT box consensus sequence is observed in any of them. It is suggested to be transcribed.
[0123]
The STMP1 cDNA (GenBank Accession No. AY008445) has a predicted putative 5 ′ untranslated region (5′UTR) of approximately 1 kb (estimated by RACE analysis) and an approximately 4 kb abnormality that accounts for ７７77% of the total cDNA sequence. It has a long 3'UTR. The ORF starts from within the third exon and is predicted to encode a 490 amino acid protein (FIG. 4B). As a result of searching for a protein motif, six putative transmembrane domains were found on the C-terminal side of STMP1, starting from F209 (FIGS. 4B and 4E). The figure shows only the cDNA sequence around the ORF. The exon-intron boundaries are indicated and the location of the putative transmembrane domain is highlighted (TM1-6) (FIG. 4B). The stop codon is indicated by an asterisk. As shown in FIGS. 4F to 4K, STMP1 is presumed to result in two types of isoforms as a result of two alternative splicings.
[0124]
Example 4
STMP1 Is 6 A new subfamily of proteins with a transmembrane domain
GenBank BLAST analysis of the deduced amino acid sequence of STMP1 identified two independent ESTs and STEAPs, recently discovered cell membrane proteins that are highly expressed in the prostate. An alignment of these sequences obtained with the Clustal and GenDoc programs is shown in FIG. Fully conserved residues are shown with black shadows. Residues conserved in two or three sequences are shown shaded in light gray and dark gray, respectively. This alignment suggested that the EST BAA91839 cDNA was near full length, while the BAB15559 cDNA was a partial sequence.
[0125]
The sequences of two proteins related to STMP1 were determined (FIGS. 4L and 4M show STMP2 and STMP3, respectively). The sequences of STMP2 and STMP3 include EST sequences. The GFP fusion of STMP2 shows a similar localization as STMP1. Both STMP2 and STMP3 are widely distributed and are present at higher levels in some tissues other than the prostate. For example, STMP2 has the highest expression in placenta and lung and strong expression in heart, liver, prostate, and testis, whereas STMP3 has highest expression in liver and also has high expression in heart, placenta, lung, kidney, It is also strongly expressed in the pancreas, prostate, testis, small intestine, and colon.
[0126]
The sequence similarity between STMP1 and STMP2 is low and not significant before the residue at position 210 of STMP1 where the putative six transmembrane coding domain begins. This suggests that the N-terminal region is structurally and functionally related between STMP proteins that form a six-transmembrane protein subfamily distinct from STEAP.
[0127]
Example 5
STMP1 Expression is highly concentrated in the prostate
The expression profile of STMP1 was then determined in a variety of human tissues by Northern analysis of multiple tissues with a Northern blot hybridizing the STMP1 probe (see "Materials and Methods" section). As shown in FIG. 6A, STMP1 hybridized with 6.5 kb major mRNA and three non-major mRNAs of 2.2 kb, 4.0 kb, and 4.5 kb in prostate tissue. The stronger hybridization observed between G3PDH in heart and skeletal muscle samples is due to strong expression in these tissues. Each lane represents the following subjects: 1 = heart, 2 = brain, 3 = placenta, 4 = lung, 5 = liver, 6 = skeletal muscle, 7 = kidney, 8 = pancreas, 9 = spleen, 10 = thymus, 11 = prostate, 12 = testis, 13 = ovary, 14 = small intestine, 15 = colon, 16 = peripheral blood leukocytes. The position of the full-length 6.5 kb mRNA, as well as the position of low molecular weight STMP1, are indicated by arrows on the left side of the figure. The 6.5 kb band showed 15-20 fold lower mRNA expression in heart, brain, kidney, pancreas, and ovaries compared to prostate, and no expression was detected in other tissues. In contrast, three low molecular weight mRNAs encoded by alternative splicing of STMP1 were detected only in the prostate. Hybridization of glyceraldehyde triphosphate dehydrogenase (G3PDH) with a cDNA probe yielded approximately equivalent signals in lanes except for heart and skeletal muscle, which were known to contain more G3PDH than other tissues. . These data indicate that STMP1 expression is also found in other tissues but is strong in the prostate, and that STMP1 has an isoform with limited expression in the prostate.
[0128]
Example 6
STMP1 Characterization of expression
Since androgen is a major hormonal stimulator for normal prostate and early prostate cancer, androgen regulation of STMP1 was assessed by Northern analysis in the androgen-responsive prostate cancer cell line LNCaP. The cells were untreated and the synthetic androgen R1881 (1010) increased over time as shown in FIG.^-8 M), recovered, and total RNA was isolated and subjected to Northern analysis using STMP1 cDNA as a probe. Northern analysis was also performed on the same membrane using the androgen-dependent gene PSA as a probe. For relative induction of mRNA accumulation, the values determined by phosphoimager analysis (Molecular Dynamics) are shown below the lanes. CWR22 xenografts were grown in nude mice and tumor samples were collected before castration (t = 0), or at 1, 2, and 4 weeks after gonadectomy. Total RNA was isolated and used for Northern analysis with the same probe. 18S RNA stained with ethidium bromide is shown as a control for RNA integrity and loading. At 6 hours, STMP1 expression had increased by about 25%, but by 24 hours it had been lost, and finally had decreased by 20% at 48 hours compared to basal levels. . In contrast, mRNA accumulation of the androgen-regulated gene PSA increased significantly over time after stimulation with androgen, and eventually reached an approximately 22-fold higher level at 48 hours. The relative induction of STMP1 mRNA accumulation determined by phosphoimager analysis is shown below the lanes. As shown in FIG. 6B, STMP1 showed similar expression levels in untreated LNCaP cells and R1881 treated LNCaP cells. This result indicates that STMP1 expression is not greatly regulated by androgens in LNCaP cells.
[0129]
To determine the potential for androgen regulation of STMP1 expression in vivo, an androgen-dependent xenograft model CWR22 derived from a primary human prostate tumor was used (Wainstein, MA et al., Cancer Res 54, 6049). ６０6052, 1994). Because growth is dependent on androgens, CWR22 tumors in nude mice show significant regression after gonadectomy and may completely regress. CWR22 xenografts were grown in nude mice with sustained release of testosterone pellets. After the tumors grew, the gonads of the mice were excised, the testosterone pellet was removed, and the regressing tumors were collected at 1, 2, and 4 weeks after gonadectomy. Total RNA was prepared from this tumor sample and used for Northern analysis. As shown in FIG. 6B, similar to the results seen with LNCaP cells, STMP1 mRNA accumulation in CWR22 tumors was not significantly altered after castration and was not affected by the presence of androgens ( Note the low loading of RNA to the sample at 2 weeks after CWR22). In contrast, mRNA accumulation of the androgen-regulated gene PSA was significantly reduced after castration and fell to about 16% of the pre-excision level by 2 weeks after castration. The above results are consistent with the findings obtained with LNCaP cells, indicating that the expression of STMP1 in prostate cancer cells is not significantly regulated by androgens. STMP1 expression was substantially lower in CWR22 tumors compared to LNCaP cells.
[0130]
The expression profile of STMP1 was also analyzed in the androgen-independent prostate cancer cell lines PC3 and DU145, and a recurrent derivative of four independent CWR22 tumors (named CWR22R) representative of advanced prostate cancer (FIG. 6C). (Nagabushhan, M. et al., Cancer Res 56, 3042-3046, 1996). LNCaP cells (R1881 (10^-8 In the presence (+) or absence (-) of M), PC-3 cells or DU-145 cells were grown and total RNA was isolated. Four independent lines of the androgen-independent human prostate cancer xenograft CWR22R were grown in nude mice, tumors were harvested and total RNA was isolated and probed with STMP1 or the cDNA for the androgen target gene NKX3.1. Used for analysis. 18S RNA stained with ethidium bromide is shown as a control for RNA integrity and loading. The relative induction of STMP1 and NKX3J mRNA accumulation as determined by phosphoimager analysis (Molecular Dynamics) is shown below the lanes. As shown in FIG. 6C, although STMP1 expression was strong in LNCaP cells, the change in response to R1881 treatment was not significant compared to the approximately 9-fold induction of the androgen target gene NKX3.1. In the prostate cancer cell lines PC-3 or DU-145 independent of androgen, STMP1 expression was not observed as in the case of NKX3.1. In contrast, STMP1 expression was significant in tumors from all four independent CWR22R xenograft lines examined, ranging from about 30% to 60% of the values found in LNCaP cells. Met. A similar overexpression pattern was also observed for NKX3.1 (FIG. 6C), which was consistent with previous findings (Korkmaz, KS, et al., Gene 260, 25-36, 2000).
[0131]
An interesting feature of the expression profile of STMP1 is the recurrent CWR22R, which is positive for the androgen receptor (AR) but does not respond to androgens, even though it is lowly expressed in androgen-dependent xenografts of CWR22. The point is that it is strongly expressed. This means that STMP1 expression is deregulated as prostate tumors progress from an androgen-dependent phase to an androgen-independent phase. STMP1 is also not expressed on the AR-negative prostate cancer cell lines PC-3 and DU-145, but is strongly expressed on the AR-positive cell line LNCaP and xenografts of CWR22 and CWR22R. Thus, STMP1 expression correlates with the presence or absence of functional AR in cells.
[0132]
It has been known for over 50 years that androgens play an important role in both the development and maintenance of normal prostate and the initiation and progression of prostate cancer. Withdrawal of androgens results in regression of normal prostate as well as prostate tumors in the early stages of androgen-dependent disease. Therefore, androgen elimination is commonly used as a therapy to reverse tumor growth. However, in the case of prostate tumors, in months and years later, in almost all cases, the tumors recur in an androgen-independent state. No effective treatment is available at this time, and the prognosis for survival is extremely poor. STMP1 is overexpressed in these late androgen-insensitive states, making it a useful tool for prostate cancer diagnostic and therapeutic applications.
[0133]
These data indicate that STMP1 expression is deregulated when prostate cancer progresses from an androgen-dependent state to an androgen-independent state.
[0134]
Example 7
STMP1 Localization in cells
To elucidate the intracellular localization pattern of STMP1, a green fluorescent protein (GFP) -STMP1 fusion protein was prepared. The use of such GFP chimeric proteins has recently become a standard method for assessing the localization and dynamics of proteins within cells. COS-1 cells were transiently transfected with GFP-STMP1 and fixed and processed for observation by confocal microscopy as described in Materials and Methods.
[0135]
A series of 11 confocal sections along the z-axis were obtained from one cell, nominally 100 nm apart. FIG. 7A shows projected images obtained by observing three continuous sections and a total of 11 sections. Arrows indicate tubular-vesicular structures (VTS) of different size, shape, and location (bar length 5 μm). Out of all 11 z-plane sections, GFP-STMP1 showed a bright perinuclear distribution pattern characteristic of the Golgi complex. GFP-STMP1 was dispersed in spots of various sizes in the entire cytoplasm and in the periphery of the cell (z-7, projected image). Some of these bright fluorescent spots are tubular (z-6, arrow and FIG. 8) or vesicular (z-5, arrow).
[0136]
To more directly determine whether GFP-STMP1 is localized to the Golgi complex, we determined its subcellular distribution by the two well-characterized Golgi markers, the Golgi marker. The enzyme mannosidase II (ManII) inside the device (Rabouille, C. et al., J Cell Sci 108, 1617-1627, 1995), and the coat protein β-COP (Pepperkok, R. et al., Cell 74, 71-82, 1993). COS-1 cells were transfected with GFP-STMP1, fixed, labeled with appropriate primary and secondary antibodies, and imaged with a confocal laser scanning microscope. The fluorescence of green GFP-STMP1 and the fluorescence of red (secondary antiserum labeled with Texas Red) β-COP and ManII were detected with a confocal laser microscope. The right panel shows a yellow / orange stained overlay image showing the area of co-localization. The bar length indicates 5 μm. As shown in FIG. 7B, the distribution of GFP-STMP1 was spread throughout the Golgi complex, as was evident from significant co-localization with both ManII and β-COP. However, there is some non-overlapping region between GFP-STMP1 and both Golgi markers, which indicates that STMP1 is differentially localized within the Golgi complex, at least in part, compared to the two markers. Suggest that.
[0137]
Because GFP-STMP1 is associated with the VTS (FIGS. 7A and 8), the trans-Golgi network (TGN), which is an important site for sorting proteins into GFP-STMP1 towards the plasma membrane, secretory vesicles, or lysosomes. Were evaluated (Farquhar, MG and Palade, GE Trends Cell Biol 8, 2-10, 1998; Mellman, I. and Warren, G., Cell 100). Lemmon, SK and Traub, LM, Curr Opin Cell Biol 12, 457-466, 2000). Antibodies against TGN46, a protein present in TGN that shuttles between TGN and the plasma membrane (Prescott AR et al., Eur J Cell Biol 72, 238-246, 1997; Ponnambalam, S. et al., J Cell Sci. 109, 675-675). 685, 1996) were used in the above experiments using immunofluorescence microscopy. As shown in FIG. 7B, GFP-STMP1 is extensively co-localized with TGN46, which is greater than the results observed with ManII and β-COP, so that in the Golgi complex STMP1 is mainly localized to TGN. You can see that it is doing. Also note that the image for TGN 46 was obtained with a low magnification objective.
[0138]
Example 8
STMP1 Shuttles between the Golgi and the plasma membrane and co-localizes with early endosomes
The dynamic properties and intracellular transport of GFP-STMP1 were examined by confocal time-lapse imaging of living cells. COS-1 cells were transiently transfected with GFP-STMP1 and 16 hours later, 12 consecutive images were taken of living cells at 37 ° C. every 20 seconds using a confocal laser scanning microscope. (FIG. 8). The upper panel shows the expansion of the VTS and return to the Golgi (white arrows). In the first image (160s) of the middle and lower panels, a red arrow indicates that the VTS has moved from the Golgi to the cell periphery. In the lower panel, yellow arrows indicate VTS movement from the cell edge to the Golgi. It should be noted that the above results are representative of multiple low speed analyses, and that changes in the image are not due to movement from the focal plane. The bar length indicates 5 μm.
[0139]
As shown in FIG. 8, a part of the VTS was dissociated or bound to the Golgi complex. Such VTSs were extremely dynamic and varied in size. Some of the VTS followed a linear or curvilinear path, some moved a little further and stopped, and some showed jumping movements. The VTS (white arrow) shown in the upper panel has returned again after leaving the Golgi. The VTS (red arrow) in the first image of the middle and lower panels left the Golgi complex and, after pausing, moved toward the perimeter of the cell until it disappeared at the edge of the cell. This suggests that STMP1 is involved in the secretory pathway. The VTS (yellow arrow) in the lower panel is moving from the cell periphery to the Golgi, suggesting that STMP1 is located in the phagocytic pathway.
[0140]
Example 9
GFP-STMP1 And early endosomal markers EEA1 Co-localization with
To determine whether GFP-STMP1 binds to the phagocytic pathway, the subcellular distribution of GFP-STMP1 was determined by examining the early endosomal protein EEA1 (Stenmark, H. et al., J Biol Chem 271, 204048-204054, 1996). It was compared with the intracellular distribution. COS-1 cells were transiently transfected with GFP-STMP1, fixed, immunostained with EEA1 antibody, and observed with a confocal laser scanning microscope. The fluorescence of green GFP-STMP1 and the fluorescence of red (secondary antiserum labeled with Texas Red) EEA1 were detected with a confocal laser microscope. The right panel shows an overlay image with yellow / orange staining showing the co-localized area. Arrows show examples of VTS in the cell periphery containing both EEA1 and STMP1. The bar length indicates 5 μm. As shown in FIG. 9, EEA1 showed a similar intracellular distribution in both transfected and untransfected cells. In addition, GFP-STMP1 was significantly co-localized with EEA1 in both the pericellular region and further in the perinuclear region (arrows in FIG. 9), indicating that STMP1 is linked to the early endosome and the phagocytic pathway. Is suggested.
[0141]
Example 10
SSH9 Genes and mRNA Isolation and characterization
The SSH9 gene was identified and mapped (FIG. 10). The location and size of the putative promoter site, transcription start site, and exons and introns are indicated. The start and stop codons and the two polyadenylation signals resulting in the two alternative splicing transcripts are also shown. FIGS. 11A-C show the nucleotide and predicted amino acid sequences of SSH9, as well as the putative promoter sequence and exon-intron boundaries.
[0142]
From the expression profile of SSH9 determined by Northern analysis (FIG. 12C) in various human tissues, a 0.7 kb splice variant of SSH9 was highly testis-specific and a 1.4 kb transcript was found. It was found to be expressed in both prostate and testis.
[0143]
Whether androgen regulates SSH9 was examined in LNCaP cells and CWR22 xenografts (FIG. 12A) and found that SSH9 is not regulated in LNCaP cells but is regulated in CWR22 xenografts. . The expression profile of SSH9 was also examined in the androgen-independent prostate cancer cell lines PC3 and DU145, and CWR22R cells (FIG. 12A).
[0144]
Example 11
PSL22 Genes and mRNA Isolation and characterization
The PSL22 gene was identified and a genetic map was prepared (FIG. 13). The location and size of exons and introns, the location of partial cDNA clones (closed squares), and alignment of full-length cDNA clones with GenBank accession numbers AC008551 and AC011449 are shown. 14A-C show the nucleotide sequence, cDNA and deduced amino acid sequence of the PSL22 ORF, as well as the deduced promoter, exon, and UTR sequences.
[0145]
The deduced amino acid sequence of PSL22 and BLAST analysis of GenBank indicated that PSL22 was a Rho binding protein. FIG. 15 shows multiple sequence alignments between PSL22 and related proteins. Fully conserved residues are shown in black, and residues found in the three sequences are shaded.
[0146]
The expression profile of PSL22 determined by Northern analysis (FIG. 16B) in various human tissues showed that the strongest expression was found in the prostate and the strong expression was found in the kidney, pancreas and colon.
[0147]
Regulation of PSL22 by androgens was examined in LNCaP cells, the androgen-independent prostate cancer cell lines PC3 and DU145, and CWR22R cells (FIG. 16A). The results showed that PSL22 was regulated by androgen in LNCaP cells that highly express it, but was not regulated by androgen in PC3 and DU145 cells.
[0148]
Example 12
Materials and methods
The following experiments and examples were performed using the following materials and methods. It will be appreciated by those skilled in the art that these materials and methods may be varied without changing the nature of the invention.
[0149]
probe
Poly A⁺ RNA 1 μg [(-) or (+)]
200 ng of random primer (N7)
Make up to 20 μl with RNAse-free sterile water.
Heat at 70 ° C. for 10 minutes and cool on ice.
[0150]
Prepare the following solutions while heating the RNA sample:
5x first strand buffer 10 μl
5 mM 0.1 mM DTT
2 μl each of 10 mM dTTP + dGTP
³²Pα dATP 5 μl
³²Pα dCTP 5 μl
2 μl of Superscript II (200 U / μl, BRL)
[0151]
The solution was mixed by pipetting, lightly centrifuged, incubated at 25 ° C for 5 minutes, and then further incubated at 37 ° C for 1 hour. 2 μl of 10 mM dCTP + dATP was added and the mixture was incubated at 37 ° C. for 30 minutes and then inactivated by heating at 70 ° C. for 10 minutes. Unincorporated nucleotides were removed using a G25 column (Bio-Rad). The specific activity (5 × 10⁸ cpm / μg).
[0152]
Hybridization
Immediately after preparation, 25 ml of the hybridization mixture (7% SDS, 0.5 M NaHPO₄, 1 mM EDTA) was pre-incubated at 65 ° C, and 12.5 ml of it was used for prehybridization of each membrane (5-10 minutes at 65 ° C). The probe was denatured by heating at 95 ° C for 3-5 minutes and transferred to the 65 ° C prehybridization mixture. Hybridization was performed at 65 ° C. overnight.
[0153]
Washing
Wash solution I (2 × SSC and 1% SDS) and wash solution II (0.1 × SSC and 0.5% SDS) are pre-incubated, and the membrane is washed once with solution I, and then washed with solution II for 30 minutes. Washed at 65 ° C for minutes. The membrane was covered with plastic wrap and a phosphorimager screen was exposed.
[0154]
Choice
Clones that showed a difference between the (-) and (+) blots were selected (usually one to eight for each pair of blots). A second round of reverse riverzan analysis for confirmation was performed (in this case, individual clones were spotted twice on each blot). After performing the phosphoimager analysis, the blot was detached from the blot at 95 ° C. in 0.1 × SSC and 0.5% SDS for 2 × 15 minutes and hybridized with the PSA probe (or used). Depending on the hormone, any amount of probe for the target gene in the tissue under study was used). For clones confirmed to be different from PSA, Northern analysis was performed according to established protocols for expression differences in secondary river snowzan analysis. LNCaP cells by R1881, and CWR22 xenograft model upon androgen deprivation (Wainstein, MA et al., Cancer Res. 54, 6049-6052, 1994), and androgen independent CWR22R recurrent xenografts (Nagabushhan, M. et al., Cancer Res. 56, 3042-6, 1996).
[0155]
Sequence analysis
Sequence analysis was performed by the dideoxy chain termination method using an ABI automatic sequencer. Homology searches were performed with the basic BLAST algorithm. FIG. 3 shows the results obtained by BLAST analysis of the isolated clones, and a table of homology to known genes (significant homology cutoff value was 50% identity).
[0156]
LNCaP Isolation of prostate cancer-related genes from cells
The prostate cancer cell line LNCaP was cultured in two batches under culture conditions similar to those reported in the literature (Horoszewicz JS et al., Cancer Res, 43: 1809-1818, 1983). The first batch was left untreated and the second batch was treated with the synthetic androgen R1881 for 24 hours. Cells from both batches were harvested and total RNA was isolated from each batch. From total RNA, poly A⁺ RNA was recovered by standard techniques and used in suppressive subtraction hybridization (SSH; Diatchenko et al., Supra) to identify a group of hormone-regulated genes. The tester for the SSH method was cDNA derived from untreated cells, and the driver was cDNA derived from cells treated with R1881. The protocol of suppression subtraction was performed according to the method described in the original (Diatchenko et al., Supra).
[0157]
Cell culture
LNCaP, PC-3, and DU-145 cells were maintained in a routine manner and treated as described in the literature (Korkmaz, KS, et al., DNA Cell Biol 19, 499-506, 2000). Korkmaz, KS, et al., Gene 260, 25-36, 2000).
[0158]
Investigation of xenografts
Implantation, growth, and recovery of tumors in CWR22 xenografts and mice bearing CWR22R xenografts were performed as described in the literature (Wainstein, MA, supra, Nagabushhan, M., supra).
[0159]
Cloning and plasmid construction
A 262 bp cDNA fragment was first obtained from a prostate-specific library screen (Ausubel, FM et al., (1997) "Current Protocols in Molecular Biology", John Wiley and. Sons, New York), L74. cDNA end 5 'rapid amplification (5'RACE) was performed using normal prostate tissue (Clontech) and / or Marathon-Ready prepared from the SMART-RACE LNCaP cDNA library (Clontech) generated according to the manufacturer's recommendations. Performed using cDNA (oligonucleotide sequences disclosed on request). The RACE product was cloned into pCRII-TOPO (Invitrogen), positive clones were confirmed by Southern analysis and sequenced. In parallel, screening of a λgt10 cDNA library generated from a pool of normal human prostates (Clontech) was performed in an established manner to obtain additional clones. The overlapping clones were used to deduce the full-length STMP1 cDNA sequence.
[0160]
The full-length STMP1 ORF was amplified using primers near the center of the start and stop codons (sequences disclosed on request) and the vector pcDNA3.1-NT-GFP-TOPO (Invitrogen) was used. In the same frame, GFP-STMP1 was prepared by fusing to the C-terminal of green fluorescent protein (GFP).
[0161]
Northern analysis
Total RNA was prepared in a one-step guanidine thiocyanate procedure and used for Northern analysis (18). 15 μg of total RNA was used per lane. Probes were made by random priming and> 3 × 10⁸ It had a specific activity of dpm / μg. A 145 bp to 2202 bp STMP1 cDNA fragment was used as a probe. Bands were visualized and quantified by phosphor imager analysis (Molecular Dynamics).
[0162]
Observation with a confocal microscope
COS-1 cells were transiently transfected by electroporation using a BTX square wave pulse generator at 150 V at a setting of 1 ms. Cells were plated on coverslips placed on 6-well tissue culture plates for indirect immunofluorescence, or on Lab-Tek Chambered Coverglass (Nalge Nunc International) for live cell microscopy. Grew. Transiently transfected cells were observed 16 hours after transfection with a Leica TCS-SP confocal microscope. All operations on living cells were performed at 37 ° C.
[0163]
Indirect immunofluorescence
Indirect immunofluorescence was performed as described in the literature (Mistelli, T. and Spector, DL MoI Cell 3, 697-705, 1999). The following antibodies were used: anti-β-coat protein (β-COP) antiserum (provided by J. Lippcott-Schwartz), anti-mannosidase II (provided by T. Mistelli), anti-TGN46 (Serotec, J.S. Bonifacino), and anti-EEA1 (Affinity Biotechnologies). Secondary antibodies conjugated to mouse and rabbit specific Texas Red were purchased from ICN Biomedicals (CostaMesa, CA).
[0164]
Other aspects
All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual independent publication or patent application was specifically and individually incorporated by reference. Although the invention has been described with respect to particular embodiments of the invention, it will be understood that further modifications are possible and this application is generally intended to cover any variations, uses, or modifications of the invention which are in accordance with the principles of the invention. , Or intended to include applications, and departures from the disclosure of the invention, which are included in known practice or practice in the art to which the invention pertains, and to essential features described above. It is understood that it can be applied and is subject to the appended claims.
[Brief description of the drawings]
FIG. 1 shows an exemplary reverse snowzan analysis of multiple clones of a prostate-specific cDNA library.
FIG. 2 shows an exemplary Northern blot of multiple tissues.
FIG. 3 is a table showing nucleotide sequences (SEQ ID NOs: 1 to 12) of 12 clones isolated from prostate tissue and LNCaP cells.
FIG. 4A is a schematic diagram showing the structure of the STMP1 gene.
FIG. 4B shows the nucleotide sequence containing the intron boundary sequence of STMP1 (SEQ ID NO: 13) and the deduced amino acid sequence (SEQ ID NO: 14).
FIG. 4C shows the nucleotide sequence of exons and 3 ′ UTRs of STMP1 (SEQ ID NOs: 15-21).
FIG. 4D shows the nucleotide sequence of the ORF of STMP1 (SEQ ID NO: 22).
FIG. 4E shows the cDNA sequence of STMP1 (SEQ ID NO: 23) and the deduced amino acid sequence (SEQ ID NO: 14).
FIG. 4F shows the nucleotide sequence of the exons and 3′UTR of STMP1 ORF2 (SEQ ID NOs: 17-20 and 24-26).
FIG. 4G shows the nucleotide sequence of the ORF of STMP1 ORF2 (SEQ ID NO: 27).
FIG. 4H shows the cDNA sequence of STMP1 ORF2 (SEQ ID NO: 28) and the deduced amino acid sequence (SEQ ID NO: 29).
FIG. 41 shows the nucleotide sequences of the exons and 3 ′ UTRs of STMP1 ORF3 (SEQ ID NOs: 17-19 and 24-26).
FIG. 4J shows the nucleotide sequence of the ORF of STMP1 ORF3 (SEQ ID NO: 30).
FIG. 4K shows the cDNA sequence of STMP1 ORF3 (SEQ ID NO: 31) and the deduced amino acid sequence (SEQ ID NO: 32).
FIG. 4L shows the cDNA sequence of STMP2 (SEQ ID NO: 33) and the deduced amino acid sequence (SEQ ID NO: 34).
FIG. 4M shows the cDNA sequence of STMP3 (SEQ ID NO: 35) and the deduced amino acid sequence (SEQ ID NO: 36).
FIG. 5: Sequence of STMP1 (SEQ ID NO: 14) with STEAP (SEQ ID NO: 37, Accession No. AF186249), and two ESTs (Accession Nos. BAA91839 and BAB15559; SEQ ID NOs: 38 and 39, respectively). Shows alignment.
FIG. 6A shows a Northern blot of multiple tissues using STMP1 or G3PDH cDNA as a probe. FIG. 6B is a Northern blot probed with STMP1 and PSA in the androgen-responsive prostate cancer cell line LNCaP and the CWR22 human prostate cancer xenograft model. FIG. 6C is a Northern blot probed with STMP1 and NKX3A in LNCaP, PC-3, and DU-145 cell lines, and a CWR22R human prostate cancer xenograft model.
FIG. 7A shows a fluorescence microscopy image of COS-1 cells transiently transfected with GFP-STMP1.
FIG. 7B shows fluorescence microscopy images of COS-1 cells transiently transfected with GFP-STMP1 and labeled with an antibody to the Golgi marker.
FIG. 8 shows a fluorescence microscope image of COS-1 cells transiently transfected with GFP-STMP1 and live cells observed with a confocal microscope.
FIG. 9 shows fluorescence microscopy images of COS-1 cells transiently transfected with GFP-STMP1 and labeled with an antibody to an early endosomal marker.
FIG. 10 is a schematic diagram showing the structure of the SSH9 gene and two mRNAs transcribed from the SSH9 gene.
FIG. 11A shows the cDNA of SSH9 (SEQ ID NO: 40) and the deduced amino acid sequence (SEQ ID NO: 41).
FIG. 11B shows the putative promoter sequence of SSH9 (SEQ ID NO: 42).
FIG. 11C shows the putative intron-exon boundaries of SSH9 (SEQ ID NOs: 43-50).
FIG. 12A is a Northern blot probed with SSH9 in the androgen-responsive prostate cancer cell line LNCaP cells and in the CWR22 human prostate cancer xenograft model. FIG. 12B is a Northern blot probed with SSH9 in the LNCaP, PC-3, and DU-145 cell lines, and the CWR22R human prostate cancer xenograft model. FIG. 12C is a northern blot of a plurality of tissues using the cDNA of SSH9 or GAPDH as a probe.
FIG. 13 is a schematic diagram showing the PSL22 gene structure.
FIG. 14A shows the nucleotide sequence of the ORF of PSL22 (SEQ ID NO: 51).
FIG. 14B shows the cDNA sequence of PSL22 (SEQ ID NO: 52) and the deduced amino acid sequence (SEQ ID NO: 53).
FIG. 14C shows the nucleotide sequence of the TATA promoter and transcription start site of PSL22, exons, and 5 'and 3' UTRs (SEQ ID NOs: 54-70).
FIG. 15 shows a sequence alignment of PSL22 (RhoBP) (SEQ ID NO: 53) with EST NP032190 (mRhoph), AF1322025 (dRhoph), and BAB23615 (SEQ ID NOs: 71-73).
FIG. 16A is a PSL22 probed Northern blot in the LNCaP, PC-3, and DU-145 cell lines, and the CWR22R human prostate cancer xenograft model. FIG. 16B is a Northern blot of multiple tissues using PSL22 cDNA as a probe.

Claims (26)

A substantially pure prostate-specific or testis-specific polypeptide, wherein the polypeptide sequence comprises a sequence substantially identical to any of SEQ ID NOs: 14, 29, 32, 34, 36, 41, or 53. peptide. 2. The substantially pure prostate-specific or testis-specific polypeptide of claim 1, wherein the polypeptide sequence comprises the sequence of any of SEQ ID NOs: 14, 29, 32, 34, 36, 41, or 53. An isolated nucleic acid molecule encoding the polypeptide of claim 1. 4. The isolated nucleic acid of claim 3, wherein the nucleic acid molecule comprises the sequence of any of SEQ ID NOs: 23, 28, 31, 33, 35, 40, or 52. A prostate-specific or testis-specific isolated nucleic acid molecule comprising a sequence substantially identical to SEQ ID NOs: 1-12, 22, 27, 30, and 51. A prostate-specific or testis-specific isolated nucleic acid molecule consisting essentially of SEQ ID NOs: 15-21, 24-26, 42-50, and 54-70. The polypeptide according to claim 1, which is derived from a mammal. The polypeptide according to claim 7, wherein the mammal is a human. A vector comprising the isolated nucleic acid molecule according to claim 3, 5, or 6. A cell comprising the isolated nucleic acid molecule according to claim 3, 5, or 6. A cell comprising the vector according to claim 9. A non-human transgenic animal comprising the isolated nucleic acid molecule according to claim 3, 5, or 6. SEQ ID NOS: 1 to 12, 15 to 28, 30, 31, 33, 35, 40, 42 to 50, 51, 52, or the complement of any of the sequences set forth in 54 to 70 under high stringency conditions An isolated nucleic acid molecule that encodes a hybridizing, prostate-specific or testis-specific polypeptide. Any prostate specific or testis specific set forth in SEQ ID NOs: 1-12, 15-28, 30, 31, 33, 35, 40, 42-50, 51, 52, or 54-70, or fragments thereof An isolated nucleic acid molecule comprising an antisense sequence to the coding strand of a specific nucleic acid molecule. SEQ ID NOS: 1-12, 15-28, wherein the fragment comprises at least six amino acids and the probe hybridizes under high stringency conditions to at least a portion of a prostate-specific or testis-specific nucleic acid molecule. Prostate-specific having greater than 55% nucleotide sequence identity to the sequence encoding any of 30, 31, 33, 35, 40, 42-50, 51, 52, or 54-70, or any of their fragments A probe for analyzing a target- or testis-specific gene or a homolog or fragment thereof. SEQ ID NOs: 1-12, 15-28, wherein the fragment comprises at least 6 amino acids and the probe hybridizes under high stringency conditions to at least a portion of a prostate-specific and testis-specific nucleic acid molecule. 16. The protein of claim 15, having 100% complementarity to a nucleic acid molecule encoding any of 30, 31, 33, 35, 40, 42-50, 51, 52, or 54-70, or a fragment thereof. Probe. Specific for a prostate-specific or testis-specific polypeptide, wherein the polypeptide comprises an amino acid sequence substantially identical to the amino acid sequence of any of SEQ ID NOs: 14, 29, 32, 34, 36, 41, or 53 Antibodies that bind to A method for detecting a prostate-specific or testis-specific gene, or a fragment thereof, in a cell, comprising the following steps:
SEQ ID NOS: 1-12, 15-28, 30, 31, 33, 35, 40, 42-50, 51, 52, or 54-70, or any of their fragments, wherein the fragment length is greater than about 18 nucleotides Contacting with a preparation of genomic DNA from said cells under highly stringent hybridization conditions; and SEQ ID NOs: 1-12, 15-28, 30, 31, 33, 35, 40, A DNA sequence having about 55% or more nucleotide sequence identity to any of 42-50, 51, 52, or 54-70 is detected, thereby prostate-specific or testis-specific gene or fragment thereof. Identifying. A method for identifying a test compound that modulates the expression or activity of a prostate-specific or testis-specific polypeptide, comprising the steps of:
Contacting a test compound with a prostate-specific or testis-specific polypeptide;
And determining the effect of the test compound on the expression or activity of the prostate-specific or testis-specific polypeptide. A method of treating a mammal having a disease of the prostate or testis, comprising the following steps:
Administering to the mammal a therapeutically effective amount of a compound that modulates the activity or expression of a prostate-specific or testis-specific polypeptide, wherein the compound has an effective effect on the disease in the mammal. 21. The method according to claim 20, wherein the disease is prostate cancer. A pharmaceutical comprising at least a single dose of a therapeutically effective amount of a prostate-specific or testis-specific polypeptide or fragment thereof in a pharmaceutically acceptable carrier formulated for the treatment of prostate or testicular disease. Composition. The prostate-specific or testis-specific polypeptide substantially differs from the amino acid sequence of SEQ ID NO: 14, 29, 32, 34, 36, 38, 39, 41, 53, or 71-73, and fragments and analogs thereof. 21. The method of claim 20, comprising substantially identical amino acid sequences. 21. The method according to claim 20, wherein the mammal is a human. A kit for analyzing a prostate-specific or testis-specific nucleic acid molecule, comprising a nucleic acid molecule probe for analyzing a prostate-specific or testis-specific nucleic acid molecule present in a subject. A kit for analysis of a prostate-specific or testis-specific polypeptide, comprising an antibody for analyzing a prostate-specific or testis-specific polypeptide present in a subject.

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