JPWO2005080562A1 - Prostate cancer determination method - Google Patents

Abstract

The present invention is a method for determining a subject who is at risk of developing prostate cancer or at risk of developing prostate cancer, and comprises the following steps: a) mutation of PCA-1 gene derived from the subject A step of analyzing the presence or absence or degree of the disease; and b) a step of determining whether or not the subject is at risk of developing prostate cancer or at risk of developing it by the presence or absence of the mutation of the PCA-1 gene; Including a method. By using the method for determining prostate cancer of the present invention, it is possible to easily and highly sensitively determine a subject who is at risk of developing prostate cancer or is at risk of developing prostate cancer. It is effective for observation, prognosis prediction, pre-onset diagnosis, carrier diagnosis, etc.

Description

The present invention relates to a method for determining prostate cancer. More particularly, the present invention is a method for determining a subject at risk of developing or at risk of developing prostate cancer, comprising the following steps:
a) a step of analyzing the presence or degree of mutation of the PCA-1 gene derived from the subject; and b) the subject developing prostate cancer depending on the presence or degree of mutation of the PCA-1 gene Determining the presence or degree of risk or risk of developing;
Including a method.

Prostate cancer is a malignant tumor that occupies the top male morbidity and mortality in the West. In Japan, the morbidity rate and mortality rate of prostate cancer are rapidly increasing with the westernization and aging of eating habits in recent years.
At present, the diagnosis of prostate cancer depends on histopathological determination such as cancer grade and stage, and biochemical parameters such as PSA value. In particular, the measurement of blood prostate specific antigen (PSA) concentration has rapidly spread in mass screening and medical checkups, and prostate cancer can be found relatively early. However, an increase in blood PSA concentration is observed after the onset of prostate cancer, and diagnosis by blood PSA concentration is difficult to distinguish from benign prostatic hyperplasia and prostatitis In some cases, the development of more specific and sensitive new markers for prostate cancer detection, diagnosis, and follow-up is desired.
The inventors of the present application have separated histopathologically the prostatic tissue extracted from the prostate cancer patient into a tumor part and a non-tumor part for the purpose of searching for a treatment target molecule for prostate cancer. Genes that differed in expression level from the part were analyzed. As a result, a gene called PCA-1 (also called human AlkB homolog 3 (hABH3)) was successfully cloned as a gene that is specifically highly expressed in the tumor part compared to the non-tumor part. (Abstracts of the 123rd Annual Meeting of the Pharmaceutical Society of Japan 4, p15, 2003). In recent years, it has been confirmed that PCA-1 is a DNA and RNA alkylating damage repair enzyme, and its function has attracted attention (Nature, 421, p859-863, 2003).
In view of the above circumstances, an object of the present invention is to provide a new method for determining a subject who is at risk of developing prostate cancer or at risk of developing prostate cancer.

As a result of intensive research aimed at achieving the above object, the present inventors have found that the expression of PCA-1 gene is increased in prostate cancer tissue, and that the increased function of PCA-1 has some causal relationship with the onset or progression of prostate cancer. In contrast to what has been thought to be the case, the present inventors have found that mutations in the PCA-1 gene that can cause PCA-1 dysfunction are frequently observed in prostate cancer patients, thereby completing the present invention. It came to.
That is, the present invention relates to the following (1) to (19).
(1) A method for determining a subject who is at risk of developing prostate cancer or at risk of developing prostate cancer, the following steps:
a) a step of analyzing the presence or degree of mutation of the PCA-1 gene derived from the subject; and b) the subject developing prostate cancer depending on the presence or degree of mutation of the PCA-1 gene Determining the presence or degree of risk or risk of developing;
Including the method.
(2) The method according to (1) above, wherein the mutation is selected from the group consisting of a missense mutation, a nonsense mutation, a silent mutation, and a frameshift mutation caused by deletion.
(3) The missense mutation is the mutation of the 7th arginine to leucine, the 8th alanine to valine, the 30th alanine to threonine of the PCA-1 polypeptide, the 41st Threonine to isoleucine, 73rd aspartate to asparagine, 137th glycine to arginine, 144th serine to proline, 228th aspartate to glutamate The method according to (2) above, wherein the mutation results in a mutation of an amino acid selected from the group consisting of a mutation of 233, glutamic acid to aspartic acid, and a mutation of 261 lysine to asparagine. .
(4) The missense mutation consists of a mutation of the 228th aspartic acid to glutamic acid, a 233rd glutamic acid to aspartic acid, and a 261nd lysine mutation to asparagine of the PCA-1 polypeptide. The method according to (3) above, wherein the mutation causes an amino acid mutation selected from the group.
(5) The method according to (4) above, wherein the missense mutation is a mutation that causes a mutation of the 228th aspartic acid to glutamic acid of the PCA-1 polypeptide.
(6) Missense mutation is mutation of PCA-1 gene from 426th guanine to thymine, 429th cytosine to thymine, 494th guanine to adenine, 528th cytosine Mutation to thymine, 623rd guanine to adenine, 815th guanine to adenine, 836th thymine to cytosine, 1090th cytosine to guanine, The method according to (3) above, wherein the mutation is selected from the group consisting of a mutation of the 1105th adenine to thymine and a mutation of the 1189th adenine to thymine.
(7) The missense mutation is selected from the group consisting of a 1090th cytosine mutation to guanine, a 1105th adenine mutation to thymine, and a 1189th adenine mutation to thymine in the PCA-1 gene. The method according to (6) above, wherein
(8) The method according to (7) above, wherein the missense mutation is a mutation of the 1090th cytosine of the PCA-1 gene to guanine.
(9) The method according to (2) above, wherein the nonsense mutation is a mutation that causes a mutation of the 261st lysine of the PCA-1 polypeptide to a stop codon.
(10) The method according to (9) above, wherein the nonsense mutation is a mutation of the 1187th adenine of the PCA-1 gene to thymine.
(11) In the above (2), the silent mutation is a mutation selected from the group consisting of a mutation from the 568th thymine to cytosine of the PCA-1 gene and a mutation from the 1132th guanine to thymine The method described.
(12) The method according to (11) above, wherein the silent mutation is a mutation of the 1132th guanine of the PCA-1 gene to thymine.
(13) The method according to (2) above, wherein the deletion is a deletion of residues from the 777th adenine to the 865th cytosine of the PCA-1 gene.
(14) The method according to (1) above, comprising the following steps:
a) isolating or purifying the polynucleotide from the subject;
b) amplifying the PCA-1 gene from the polynucleotide;
c) determining the base sequence of the amplified PCA-1 gene;
d) analyzing the presence or absence or degree of mutation of the PCA-1 gene derived from the subject;
e) A step of determining whether or not the subject has a risk of developing prostate cancer or a risk of developing it based on the presence or absence or degree of mutation of the PCA-1 gene.
(15) Compared with SEQ ID NO: 1, 426th guanine to thymine, 429th cytosine to thymine, 494th guanine to adenine, 528th cytosine Substitution of thymine, 623rd guanine to adenine, 815th guanine to adenine, 836th thymine to cytosine, 1090th cytosine to guanine, 1105th adenine to thymine, 1189th adenine to thymine, 1187th adenine to thymine, 568th thymine to cytosine, 1132th guanine to thymine One or more selected from the group consisting of substitution to, and deletion of the sequence from the 777th adenine to the 865th cytosine A polynucleotide comprising a nucleotide sequence having a substitution or deletion.
(16) Compared to SEQ ID NO: 1, substitution of 426th guanine with thymine, substitution of 429th cytosine with thymine, substitution of 494th guanine with adenine, substitution of 528th cytosine Substitution of thymine, 623rd guanine to adenine, 815th guanine to adenine, 836th thymine to cytosine, 1090th cytosine to guanine, Substitution of 1105th adenine to thymine, 1189th adenine to thymine, 1187th adenine to thymine, and deletion of sequence from 777th adenine to 865th cytosine A polypeptide comprising an amino acid sequence encoded by a base sequence having one or more substitutions or deletions selected from the group consisting of
(17) An antibody that immunospecifically recognizes the polypeptide of (16) above.
(18) A kit for determining a subject who is at risk of developing prostate cancer or is at risk of developing prostate cancer, comprising a reagent for analyzing PCA-1 gene mutation.
(19) The reagent for analyzing PCA-1 gene mutation can be used to determine a subject who is at risk of developing prostate cancer or at risk of developing prostate cancer. The kit according to (17), further comprising a description.
By using the method for determining prostate cancer of the present invention, it is possible to easily and highly sensitively determine a subject who is at risk of developing prostate cancer or is at risk of developing prostate cancer. It is effective for observation, prognosis prediction, pre-onset diagnosis, carrier diagnosis, etc.

FIG. 1 is a graph showing the relationship between MMS concentration and the number of adherent cells. The vertical axis shows the number of adherent cells as a relative value when the control group is 100%. The horizontal axis indicates MMS concentration (mM). The black column represents the control group, the shaded column represents the PCA-1WT transfection group, and the white column represents the PCA-1Δ777-865 transfection group.
Detailed Description of the Invention
In the present invention, “prostate cancer” is a concept that broadly encompasses cancers that have occurred in the prostate, and not only adenocarcinoma that has occurred in the prostate, but also squamous cell carcinoma, transitional cell carcinoma, neuroendocrine cancer, undifferentiated cancer, etc. Is also included. Preferably, the prostate cancer is an adenocarcinoma that has developed in the prostate.
In humans, PCA-1 is a molecule called human AlkB homolog 3 (hABH3) or DEPC-1.
Examples of the normal human PCA-1 gene base sequence (cDNA base sequence) include, for example, SEQ ID NO: 1 and the like, of which the 407th to 1267th residues (SEQ ID NO: 3) correspond to the protein coding region. And encoding a PCA-1 polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. In the present invention, the positions of the residues in the base sequence and amino acid sequence of the PCA-1 gene are based on the sequences of SEQ ID NOs: 1 and 2, respectively. Here, the 923rd to 1243rd residues in SEQ ID NO: 1 and the 172nd to 279th amino acid residues in SEQ ID NO: 2 correspond to the dealkylase active site of PCA-1.
The human PCA-1 gene is encoded by the locus of p11 of chromosome 11, and the PCA-1 gene and its surrounding chromosomal DNA base sequences are disclosed in, for example, Genebank Accession No. NT_009237 (NCBI website). ing.
The human PCA-1 gene contains 10 exons. In SEQ ID NO: 1, exon 1 is the first to 334th residue, exon 2 is the 335th to 485th residue, exon 3 Is the 486th-592nd residue, exon 4 is the 593rd-624th residue, exon 5 is the 625th-670th residue, exon 6 is the 671th residue -777th residue, exon 7 is 777th-865th residue, exon 8 is 866th-1075th residue, exon 9 is 1076th-1172th The residue, exon 10, corresponds to the 1173rd to 1520th residues, respectively.
The method for determining prostate cancer of the present invention comprises at least the following steps:
a) analyzing the presence or absence or degree of mutation in the PCA-1 gene derived from the subject; and
b) a step of determining the risk of the subject developing prostate cancer or the risk of developing prostate cancer according to the presence or absence or degree of mutation of the PCA-1 gene;
including.
In the step of analyzing the presence or degree of mutation of the PCA-1 gene derived from the subject, the PCA-1 gene derived from the subject is compared with the normal PCA-1 gene.
The comparison between the subject-derived PCA-1 gene and the normal PCA-1 gene is not particularly limited. For example, the comparison is with a base sequence of normal PCA-1 gene such as mRNA, cDNA, and chromosomal DNA.
Here, “comparison with the base sequence of normal PCA-1 gene” means, in addition to comparison with the full-length base sequence of normal PCA-1 gene, a complementary sequence of the sequence, a partial sequence, a complementary sequence of the partial sequence, It may be a comparison with a sequence of specific residues.
Here, the length of the partial sequence is at least 10 bases or more, preferably 30 bases or more. Preferred embodiments of the partial sequence include the base sequence of the protein coding region of PCA-1 and the base sequence of the exon region of the PCA-1 gene.
Any method known per se can be used to analyze the presence or absence or degree of mutation in the PCA-1 gene derived from the subject.
For example, you may determine the base sequence of PCA-1 gene in the polynucleotide derived from a subject. Also, to any method currently used to detect base changes in the field, such as RFLP method, TaqMan test, oligonucleotide ligase method, single strand conformation polymorphism, oligonucleotide array of template nucleic acids Test methods based on hybridization, denaturing gradient gel electrophoresis, PCR-SSPC method, Southern blot method, Northern blot method, ASO method, ARMS method using mismatch primers, and the like can also be used. Or the method developed in the future can also be used for the analysis method of the presence or absence of the mutation by the said comparison. Preferably, the base sequence of the PCA-1 gene in the polynucleotide derived from the subject is determined.
In determining the base sequence of the PCA-1 gene in a polynucleotide derived from a subject, the polynucleotide is first isolated or purified from the subject. The polynucleotide preferably contains a PCA-1 gene derived from the subject.
The polynucleotide is isolated or purified from a sample derived from the subject, for example, a tissue, a cell, a liquid component, or the like.
Although it does not specifically limit as a structure | tissue, For example, a prostate, hair, skin etc. are mentioned, Preferably it is a prostate.
The cell includes both a cell that has been separated from a subject and not cultured, and a cell that has been separated and cultured from a living body. Examples thereof include blood cells, prostate-derived cells, etc., preferably prostate-derived cells.
Examples of the liquid component include blood, semen, saliva, urine, sweat, and the like.
Examples of the polynucleotide include DNA (chromosomal DNA, cDNA, etc.) and RNA (total RNA, mRNA, cRNA, etc.), preferably chromosomal DNA, total RNA, or mRNA.
The polynucleotide can be isolated or purified by a method known per se.
For the isolation or purification of chromosomal DNA, for example, proteinase K / phenol extraction method, proteinase K / phenol / chloroform extraction method, alkali dissolution method, boiling method and the like can be used.
For isolation or purification of total RNA, for example, guanidine-cesium chloride ultracentrifugation, AGPC (Acid guanidinium-phenol-chloroform method) and the like can be used. Alternatively, commercially available kits such as TRIzol (manufactured by Life Technologies, Inc.) and Isogen (manufactured by Nippon Gene) can also be used.
Isolation or purification of mRNA can be achieved by subjecting the purified total RNA to an oligo dT column or the like.
When the polynucleotide is RNA, particularly total RNA or mRNA, it is preferable to synthesize cDNA by a method known per se using reverse transcriptase or the like.
In determining the base sequence of the PCA-1 gene in the polynucleotide, the PCA-1 gene in the polynucleotide is preferably amplified using the polynucleotide as a template. The polynucleotide used as a template is not particularly limited, but is preferably chromosomal DNA or cDNA.
The method of gene amplification is not particularly limited, however, polymerase chain reaction (PCR method), LAMP (Loop-mediated thermal amplification) (see, for example, WO 00/28082), ICAN (Isothermal and Chimeric-Pepper). Amplification of Nucleic acids) (see, for example, WO 00/56877), self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcription amplification system (Kwoh et al. (1989). Proc. Natl. Acad. Sci. USA 86: 1173-1177), Q- Replicase (Lizardi et al. (1988) Bio / Technology 6: 1197) or the like may be exemplified, but are both possible to use, PCR method is simple preferred. References for general use of PCR technology can be found in Mullis et al. , Cold Spring Harbor Symp. Quant. Biol. 51: 263, (1987), Ehrlich (ed), PCR technology, Stockton Press, NY, 1989, Ehrlich et al. , Science, 252: 1643-1650 (1991), PCR protocols; A Guide to Methods and Applications, Eds. Innis et al. , Academic Press, New York, (1990).
As the polymerase used in the amplification of the PCA-1 gene by the PCR method, any Taq polymerase or the like commonly used by those skilled in the art can be used, but a relatively high fidelity polymerase such as KOD Plus (TOYOBO) Etc.) are used.
In the amplification of the PCA-1 gene by the PCR method, an oligonucleotide primer pair that enables amplification of the gene is used. The primer pair includes, for example, information in the present specification such as the base sequence of SEQ ID NO: 1, the base sequence of the p11 region of human chromosome 11 disclosed in Genebank Accession No. NT_009237 (NCBI website), etc. Based on the above, it is possible for those skilled in the art to appropriately design using an appropriate program such as Oligo 4.0.6 (manufactured by National Bioscience, Plymouth MN).
When the template in PCR is cDNA, the region where the primer pair is set is not particularly limited, but the primer pair is preferably the 426th guanine and 429th cytosine in SEQ ID NO: 1. 494th guanine, 528th cytosine, 568th thymine, 623th guanine, 815th guanine, 836th thymine, 1090th cytosine, 1105th adenine, 1132st guanine, 1187th adenine, 1189th adenine, and at least one selected from the group consisting of residues from 777th adenine to 865th cytosine, more preferably 2 or more Most preferably, it is set so that all residues can be amplified.
In addition, when the template in PCR is chromosomal DNA, the region in which the primer pair is set is not particularly limited, but the primer pair is preferably set so that the exon region can be amplified, more preferably , It is set so that at least one, more preferably two or more, and most preferably all of the residues on the chromosomal DNA corresponding to the above-mentioned residues can be amplified. A plurality of primer pairs may be set.
The size of the oligonucleotide used as the primer can be appropriately selected, but is usually 10 to 100 bp, preferably 12 to 50 bp, more preferably 15 to 30 bp.
The GC content of the oligonucleotide used as a primer can be set as appropriate, but is usually in the range of 20 to 80%, preferably 30 to 70%, more preferably 40 to 60%.
The Tm value of the oligonucleotide used as a primer is usually set in the range of 60 ± 20 ° C. The Tm value differs depending on the salt concentration in the buffer, but can be calculated by a method known per se, such as the nearest neighbor method (see, for example, “Biochemistry, 35, 3555-3562, 1996”).
Oligonucleotides may be either naturally occurring or synthesized, but can be synthesized by a commonly used automatic nucleic acid synthesizer by the phosphotriethyl method, phosphodiester method or the like.
The primer is set so that the size of the amplification product is usually about 50 to 30000 bp. Considering the amplification efficiency and the like, the size of the amplification product is preferably set to about 50 to 3000 bp, more preferably about 100 to 2000 bp.
Preferred primer pairs include, for example,
Primer 1: CTGAAAGCTCCGAGCAGAAGC (SEQ ID NO: 4)
Primer 2: GGTCTACTGTGGGAACAG (SEQ ID NO: 5)
A primer pair consisting of
The base sequence of the amplified PCA-1 gene is determined by a well-known method. Preferred methods include those based on incorporation of terminal nucleotides into a template polymerase-generated copy using the method of Sanger et al. Alternatively, for example, adapter sequencing (PCT / US95 / 12678), sequencing based on ligation (PCT / US96 / 05245), sequencing by hybridization (AD Mirzabekov, TIBTech 12: 27-32). , 1994).
Based on the determined base sequence, the presence or absence or degree of mutation in the PCA-1 gene derived from the subject is analyzed.
When the template used in the amplification is chromosomal DNA, preferably, the base sequence contained in the exon region of the PCA-1 gene in the determined base sequence is the base sequence contained in the cDNA base sequence of the normal PCA-1 gene. Compare with the corresponding base sequence. When the template used in the amplification is cDNA, the determined base sequence is compared with the corresponding base sequence in the cDNA base sequence of the normal PCA-1 gene.
When a mutation was detected in the PCA-1 gene derived from the subject, that is, one or more residues different from the normal PCA-1 gene were detected in the analyzed PCA-1 gene If so, the subject is determined to be at risk (or high) for developing prostate cancer or at risk (or high) for developing prostate cancer. Conversely, if no mutation is detected in the PCA-1 gene derived from the subject, it is determined that the subject is not at risk (or low) or at risk (or low) of developing prostate cancer. To do.
In the method of the present invention, it can be determined that the greater the degree of mutation, the higher the risk that the subject will develop or develop prostate cancer. The “degree of mutation” refers to, for example, the number of mutation sites, the length of bases involved in mutation, and the like.
Further, if the PCA-1 gene mutation is present in either one of the alleles of the PCA-1 gene derived from the subject, the subject is at risk of developing prostate cancer or at risk of developing it. However, when there is a mutation in both alleles, it can be determined that there is a risk (or higher) than when there is a mutation in only one of the alleles.
Further, when there is a superiority or inferiority between alleles, if there is a mutation in the dominant gene, it can be determined that there is a risk (or higher) than in the case where there is a mutation in the inferior gene.
Further, the method of the present invention can improve the accuracy of determination by using it in combination with other methods for diagnosing prostate cancer. Other methods for diagnosing prostate cancer include palpation, method using serum-specific prostate specific antigen (PSA) concentration, pathological examination using prostate needle biopsy sample, ultrasonography, MRI, CT, bone scintigraphy, etc. It is done. In addition, the method of the present invention can be used in combination with a test method (see Non-Patent Document 1) using as an index that the expression of PCA-1 gene is improved in prostate cancer tissue.
The site of mutation of the PCA-1 gene is not particularly limited.
In the chromosomal DNA, it may be any region encoding the PCA-1 gene (for example, an enhancer region, a promoter region, an exon region, an intron region, etc.), preferably an exon region or an intron region, more preferably Exon region.
In cDNA and mRNA, the mutation site of the PCA-1 gene is preferably a protein coding region.
The type of mutation of the PCA-1 gene is not particularly limited, and examples include mutation by substitution such as missense mutation, nonsense mutation or silent mutation, frameshift mutation caused by deletion, insertion, etc., preferably mutation Is selected from the group consisting of missense mutations, nonsense mutations, silent mutations and frameshifts caused by deletions.
The aspect of the amino acid mutation in the missense mutation of the PCA-1 gene is not particularly limited. Preferably, the missense mutation is the 7th arginine, 8th alanine, 30th alanine, 41st threonine, 73rd aspartic acid, 137th glycine of PCA-1 polypeptide, It is a mutation that causes mutation of an amino acid residue selected from the group consisting of the 144th serine, the 228th aspartic acid, the 233rd glutamic acid, and the 261st lysine.
In this case, the type of amino acid after the substitution is not particularly limited. Preferably, the missense mutation is a mutation of the 7th arginine to leucine, a mutation of the 8th alanine to valine, a mutation of the 30th alanine to threonine, the 41st mutation of the PCA-1 polypeptide. Threonine to isoleucine, 73rd aspartate to asparagine, 137th glycine to arginine, 144th serine to proline, 228th aspartate to glutamate A mutation resulting in a mutation of an amino acid selected from the group consisting of a mutation of 233, glutamic acid to aspartic acid, and a mutation of 261 lysine to asparagine.
More preferably, the missense mutation is an amino acid mutation in the dealkylase active site of the PCA-1 polypeptide (corresponding to amino acid residues 172 to 279 of the PCA-1 polypeptide), ie, the 228th position. Aspartic acid to glutamic acid, 233rd glutamic acid to aspartic acid, and 261nd lysine to aspartic acid mutation.
More preferably, the missense mutation is a mutation resulting in a mutation of the 228th aspartic acid to glutamic acid of the PCA-1 polypeptide.
In the missense mutation, the base sequence encoding the amino acid after the mutation is not particularly limited. Preferably, the missense mutation is a mutation of the 426th guanine to thymine, a mutation of the 429th cytosine to thymine, a mutation of the 494th guanine to adenine, and the 528th mutation. Mutation of cytosine to thymine, mutation of 623rd guanine to adenine, mutation of 815th guanine to adenine, mutation of 836th thymine to cytosine, mutation of 1090th cytosine to guanine A mutation selected from the group consisting of a mutation, a mutation of the 1105th adenine to thymine, and a mutation of the 1189th adenine to thymine.
The aspect of the amino acid mutation in the nonsense mutation of the PCA-1 gene is not particularly limited, but the nonsense mutation is preferably a mutation that causes a mutation of the PCA-1 polypeptide to the 261st lysine stop codon.
In the nonsense mutation, the base sequence encoding the stop codon after the mutation is not particularly limited. Preferably, the nonsense mutation is a mutation of the 1187th adenine of the PCA-1 gene base sequence to thymine.
The mutation site in the silent mutation of PCA-1 gene is not particularly limited, but the silent mutation is preferably a residue selected from the group consisting of 568th thymine and 1132th guanine of PCA-1 gene. More preferably, the mutation at the 1132rd guanine.
In the silent mutation, the base after the mutation is not particularly limited. Preferably, the silent mutation is a mutation selected from the group consisting of a mutation of the 568th thymine to cytosine and a mutation of the 1132th guanine to thymine of the PCA-1 gene.
In the deletion of the PCA-1 gene, the number and position of bases to be deleted are not particularly limited. Preferably, the deletion is a deletion of one or more exon regions, more preferably the deletion is a deletion of one exon region.
The exon region to be deleted is not particularly limited, but is preferably exon 7 (the 777th to 865th residues in SEQ ID NO: 1) in the case of the human PCA-1 gene.
The deletion results in a substitution of the amino acid from position 124 of the PCA-1 polypeptide and a substitution of the 136th amino acid to a stop codon, so that protein translation is terminated up to the 135th amino acid. Is done.
Further, the present invention relates to the substitution of 426th guanine with thymine, the substitution of 429th cytosine with thymine, the substitution of 494th guanine with adenine, and the 528nd compared with SEQ ID NO: 1. Of cytosine to thymine, 623rd guanine to adenine, 815th guanine to adenine, 836th thymine to cytosine, 1090th cytosine to guanine 1105th adenine to thymine, 1189th adenine to thymine, 1187th adenine to thymine, 568th thymine to cytosine, 1132th Selected from the group consisting of substitution of guanine with thymine and deletion of the sequence from 777th adenine to 865th cytosine A polynucleotide comprising a nucleotide sequence having one or more substitutions or deletions. Preferably, the polynucleotide is a polynucleotide comprising a base sequence having one or more substitutions selected from the above group as compared with SEQ ID NO: 1.
In addition, a polynucleotide that is a part of the polynucleotide and has one or more substitutions or deletions selected from the group as compared with SEQ ID NO: 1 is also encompassed in the present invention. The length of a part of the polynucleotide is at least 10 bp, preferably 30 bp or more.
The above-described polynucleotide of the present invention has already been revealed to contain a PCA-1 gene having a mutation, for example, as a reagent such as a mismatch primer or a mismatch probe in the analysis of mutations in the human PCA-1 gene Useful as a control.
In addition, an appropriate expression vector into which the polynucleotide of the present invention is functionally inserted is introduced into a host cell such as a prostate cancer cell, and after the PCA-1 polypeptide having a mutation is expressed, the host cell is analyzed. Thus, the association between PCA-1 gene mutation and prostate cancer can be studied. Therefore, the polynucleotide of the present invention is useful as a reagent for prostate cancer research.
The polynucleotide of the present invention is preferably isolated or purified.
Further, the present invention relates to the substitution of 426th guanine with thymine, the substitution of 429th cytosine with thymine, the substitution of 494th guanine with adenine, and the 528nd compared with SEQ ID NO: 1. Of cytosine to thymine, 623rd guanine to adenine, 815th guanine to adenine, 836th thymine to cytosine, 1090th cytosine to guanine Substitution, substitution of 1105th adenine to thymine, substitution of 1189th adenine to thymine, substitution of 1187th adenine to thymine, and sequence from 777th adenine to 865th cytosine A polypeptide comprising an amino acid sequence encoded by a base sequence having one or more substitutions or deletions selected from the group consisting of deletions About. Preferably, the polypeptide is a polypeptide consisting of an amino acid sequence encoded by a base sequence having one or more substitutions or deletions selected from the above group as compared to SEQ ID NO: 1.
In addition, a polypeptide which is a part of the polypeptide and includes at least one amino acid substituted by substitution or deletion of the group is also encompassed in the present invention. The length of the partial polypeptide is at least 6 amino acids or more, preferably 8 amino acids or more.
The polypeptides of the present invention are preferably isolated or purified.
The polypeptide of the present invention is useful as an immunogen for the production of an antibody that immunospecifically recognizes the polypeptide of human PCA-1 having the above mutation. The present invention also relates to the antibody.
“Immunospecific” means that, under the conditions in which a person skilled in the art normally uses an antibody, the antibody exhibits a substantially higher affinity for a particular polypeptide than its affinity for other polypeptides. means. In the present invention, examples of the “other polypeptides” include normal human PCA-1 polypeptides.
Examples of antibodies include natural antibodies such as polyclonal antibodies and monoclonal antibodies (mAb), chimeric antibodies that can be produced using gene recombination techniques, humanized antibodies, single chain antibodies, human antibody-producing transgenic animals, and the like. Examples include, but are not limited to, human antibodies that can be produced, antibody fragments produced by Fab expression libraries, and binding fragments thereof. Preferably, the antibody is a polyclonal antibody, a monoclonal antibody and binding fragments thereof.
The binding fragment means a partial region of the aforementioned antibody, specifically, for example, F (ab ′) ₂ , Fab ′, Fab, Fv (variable fragment of antibody), sFv, dsFv (disulphide stabilized Fv), dAb (single domain antibody, etc.), Exp. V. Op. p.441-456, 1996).
The class of the antibody is not particularly limited, and includes antibodies having any isotype such as IgG, IgM, IgA, IgD, or IgE. IgG or IgM is preferable, and IgG is more preferable in view of ease of purification.
The antibody of the present invention can be obtained by administering the polypeptide or the like to mammals such as mice and rabbits (preferably mammals other than humans) using a conventional protocol.
The monoclonal antibody can be prepared according to the method of Köhler and Milstein et al. (Nature, Vol. 256, p. 495-497, 1975) and a modification method according thereto. In screening for a hybridoma that produces an antibody that specifically reacts with the polypeptide of the present invention, a corresponding control peptide in which the mutation site contained in the polypeptide of the present invention is replaced with an amino acid of normal PCA-1 is used. Clones that react with the inventive polypeptide but do not react with the corresponding control peptide can be selected.
The preparation of the polyclonal antibody is preferably purified by a column or the like to which the serum of an animal immunized with the polypeptide of the present invention is bound. More preferably, in order to further increase the specificity, a fraction capable of reacting with normal human PCA-1 is removed using a column to which the corresponding control peptide is bound.
The antibody of the present invention is preferably a saturated ammonium sulfate, euglobulin precipitation method, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52, etc.), anti-immunoglobulin column or protein A column, and immunogen. Isolated or purified by affinity column chromatography using a column or the like.
The antibody of the present invention immunospecifically recognizes a human PCA-1 polypeptide having a mutation. For example, the antibody can be examined by Western blotting, immunohistochemical staining, ELISA, RIA, surface plasmon resonance, protein chip, etc. It is possible to analyze whether a PCA-1 gene derived from a person has a mutation.
The method for determining a subject at risk of developing or at risk of developing prostate cancer of the present invention also includes a method using the above-mentioned antibody.
The present invention also relates to a kit for determining a subject who is at risk of developing prostate cancer or at risk of developing prostate cancer, and includes a reagent for analyzing PCA-1 gene mutation.
Preferably, in the kit, the PCA-1 gene mutation analysis reagent can or should be used to determine a subject who is or is at risk of developing prostate cancer. The description which described that is further included.
The reagent for PCA-1 gene mutation analysis may be any reagent used in the method of the present invention. For example, oligonucleotide primer pair used for amplification of PCA-1 gene, isolated or purified polynucleotide already known to contain normal PCA-1 gene, including PCA-1 gene having mutation Examples thereof include isolated or purified polynucleotides that have already been clarified, antibodies that immunospecifically bind to human PCA-1 polypeptides having mutations, and the like.
EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated more concretely, this invention is not limited at all by the Example shown below.

表１中、「Ａｌａ３０Ｔｈｒ」は「第３０番目のアラニンのスレオニンへの変異」を意味する。「Ｌｙｓ２６１＊」は「第２６１番目のリジンのストップコドンへの変異」を意味する。「Ｔ５６８Ｃ」は「第５６８番目のチミンのシトシンへの変異」を意味する。「ｄｅｌ（７７７−８６５）」は「第７７７番目−第８６５番目の残基の欠失」を意味する。
また検体番号１〜４は前立腺癌患者の癌部を、検体番号５はＬＮ−ＣａＰ細胞を、検体番号６はＰＣ−３細胞を、検体番号７はＤＵ１４５細胞をそれぞれ示す。
表１から明らかなように、全ての前立腺癌の検体において、ＰＣＡ−１遺伝子の変異が認められた（前立腺癌患者：４／４、前立腺癌細胞株：３／３）。変異の箇所、種類は多様であったが、Ｇ１１３２Ｔの変異が全ての前立腺癌患者において確認され（４／４）、またＡｓｐ２２８Ｇｌｕのアミノ酸置換を伴うＣ１０９０Ｇの変異が全ての前立腺癌細胞株（３／３）において確認された。第２６１番目のリジンにおける変異も比較的高頻度に認められた（２／７）。複数の変異を有する検体がほとんどであった（前立腺癌患者：４／４、前立腺癌細胞株：２／３）。
また、ＰＣＡ−１の脱アルキル化酵素活性部位のアミノ酸変異をもたらす変異が、極めて高頻度に確認された（前立腺癌患者：３／４、前立腺癌細胞株：３／３）。このことは、ＰＣＡ−１遺伝子の変異によるＰＣＡ−１の脱アルキル化酵素活性の異常と前立腺癌の発症、進行等との関連を示唆する。
一方、非癌部においては上記ＰＣＡ−１遺伝子の変異は認められなかった（３人中３人）。From the postoperative specimens of 4 prostate cancer patients who had never received chemotherapy or hormonal therapy, histopathologically, a tissue specimen was collected into a cancerous part and its surrounding non-cancerous part. A human prostate cancer cell line (PC-3, DU145, LN-CaP) was also used.
The collected tissues and cells were frozen and subjected to total RNA extraction. Frozen specimens were homogenized in TRIzol reagent (Life Technologies, Inc., Rockville, MD, USA) and total RNA was isolated according to the protocol provided by the manufacturer. DNA that may have been mixed was removed by RNase-free DNase I (manufactured by Wako Pure Chemical Industries, Ltd.) treatment. A reverse transcription reaction was performed using total RNA (5 μg), oligo dT primer, and AMV reverse transcriptase (manufactured by Life Technologies, Inc.). A part of the obtained cDNA was used to amplify the PCA-1 gene by PCR. The composition of the PCR reaction mixture used was the above cDNA (20 ng), KOD dash (manufactured by TOYOBO), dNTP (each 0.2 M), 10 × KOD dash buffer (2.5 μl), primer 1 in 25 μl PCR buffer. (SEQ ID NO: 4: 0.2 μM) and primer 2 (SEQ ID NO: 5: 0.2 μM). The reaction was carried out using a DNA thermal cycler (DNA Thermal Cycler 460, PerkinElmer Inc.) at 95 ° C. for 30 seconds, 60 ° C. for 30 seconds, 72 ° C. for 1 minute 30 seconds, and finally at 72 ° C. for 10 minutes. went. The reaction mixture was subjected to agarose gel electrophoresis to separate amplification products. A band containing the amplification product was isolated, and the amplification product was purified by Concert (Life Technologies, Inc.). The amplified product was subcloned into pT7-vector (manufactured by Novagen), and the base sequence was determined by PRISM 310 DNA sequencer (manufactured by PerinElmer).
The determined base sequence was compared with the base sequence of SEQ ID NO: 1, and the amino acid sequence predicted from the determined base sequence was compared with the sequence of SEQ ID NO: 2, and the presence or absence of mutation was analyzed. Table 1 shows the results.
Primer 1 (SEQ ID NO: 4): CTGAAAGCTCCGAGCAGAAGC (corresponding to residues 373 to 393 in SEQ ID NO: 1)
Primer 2 (SEQ ID NO: 5): GGTCTACTGTGGGAACAG (corresponding to residues 1442 to 1459 in SEQ ID NO: 1)

In Table 1, “Ala30Thr” means “mutation of 30th alanine to threonine”. “Lys261 *” means “mutation of 261st lysine to stop codon”. “T568C” means “mutation of 568th thymine to cytosine”. “Del (777-865)” means “deletion of residue at positions 777-865”.
Specimen Nos. 1 to 4 represent cancerous portions of prostate cancer patients, Specimen No. 5 represents LN-CaP cells, Specimen No. 6 represents PC-3 cells, and Specimen No. 7 represents DU145 cells.
As is clear from Table 1, mutations in the PCA-1 gene were observed in all prostate cancer specimens (prostate cancer patients: 4/4, prostate cancer cell lines: 3/3). Although the location and type of mutation were diverse, the G1132T mutation was confirmed in all prostate cancer patients (4/4), and the C1090G mutation with Asp228Glu amino acid substitution was found in all prostate cancer cell lines (3 / It was confirmed in 3). A mutation in the 261st lysine was also observed at a relatively high frequency (2/7). Most specimens had multiple mutations (prostate cancer patients: 4/4, prostate cancer cell lines: 2/3).
Moreover, the mutation which brings about the amino acid mutation of the dealkylase active site of PCA-1 was confirmed very frequently (prostate cancer patient: 3/4, prostate cancer cell line: 3/3). This suggests an association between abnormal PCA-1 dealkylase activity due to mutation in the PCA-1 gene and the onset, progression, etc. of prostate cancer.
On the other hand, no mutation of the PCA-1 gene was observed in the non-cancerous part (3 out of 3).

A normal PCA-1 (PCA-1WT) gene and a PCA-1 (PCA-1Δ777-865) gene from which the 777th to 865th bases were deleted were incorporated into a mammalian expression vector (pEGFP). These expression vectors (each 5 μg) were transfected into COS-7 cells (1 × 10 ⁶ ) by DEAE-Dextran method. As a control group, cells were transfected with an empty expression vector. At 48 hours after transfection, 0.25 mM methylmethanesulfonic acid (MMS) was added to induce nucleic acid alkylation injury, and the number of adherent cells was measured 24 hours later. The results are shown in FIG.
As apparent from FIG. 1, in the control group and the PCA-1Δ777-865 transfection group, about 40% cell death due to nucleic acid alkylation damage by 0.25 mM MMS was observed. On the other hand, in the PCA-1WT transfection group, MMS-induced alkylation injury was significantly rescued. From this result, it was shown that normal PCA-1 expresses dealkylation activity of alkylated DNA in vivo, while PCA-1Δ777-865 lacks that activity. It has also been shown that mutations in the active site of PCA-1 dealkylase can result in abnormal dealkylation activity.

By using the method for determining prostate cancer of the present invention, it is possible to easily and highly sensitively determine a subject who is at risk of developing prostate cancer or is at risk of developing prostate cancer. It is effective for observation, prognosis prediction, pre-onset diagnosis, carrier diagnosis, etc.
This application is based on patent application No. 2004-47036 filed in Japan, the contents of which are incorporated in full herein.

SEQ ID NO: 4: Oligonucleotide designed to act as a PCR primer for detecting the PCA-1 gene SEQ ID NO: 5: Oligonucleotide designed to act as a PCR primer for detecting the PCA-1 gene

Claims (19)

A method for determining a subject at risk of developing or at risk of developing prostate cancer comprising the following steps:
a) a step of analyzing the presence or degree of mutation of the PCA-1 gene derived from the subject; and b) the subject developing prostate cancer depending on the presence or degree of mutation of the PCA-1 gene Determining the presence or degree of risk or risk of developing;
Including the method. The method according to claim 1, wherein the mutation is selected from the group consisting of a missense mutation, a nonsense mutation, a silent mutation, and a frameshift mutation due to deletion. Missense mutations are PCA-1 polypeptide 7th arginine to leucine mutation, 8th alanine to valine mutation, 30th alanine to threonine mutation, 41st threonine isoleucine , Mutation of the 73rd aspartic acid to asparagine, mutation of the 137th glycine to arginine, mutation of the 144th serine to proline, mutation of the 228th aspartic acid to glutamic acid, The method according to claim 2, which is a mutation resulting in a mutation of an amino acid selected from the group consisting of a mutation of 233rd glutamic acid to aspartic acid and a mutation of 261st lysine to asparagine. The missense mutation is selected from the group consisting of the 228th aspartic acid to glutamic acid, 233rd glutamic acid to aspartic acid mutation, and the 261nd lysine to asparagine mutation of the PCA-1 polypeptide. The method according to claim 3, which is a mutation that results in a mutation of the amino acid. 5. The method of claim 4, wherein the missense mutation is a mutation that results in a mutation of the 228th aspartic acid to glutamic acid of the PCA-1 polypeptide. Missense mutations are mutations of 426th guanine to thymine, 429th cytosine to thymine, 494th guanine to adenine, and 528th cytosine to thymine. Mutation, 623rd guanine to adenine, 815th guanine to adenine, 836th thymine to cytosine, 1090th cytosine to guanine, 1105th The method according to claim 3, wherein the mutation is selected from the group consisting of a mutation of adenine to thymine and a mutation of the 1189th adenine to thymine. The missense mutation is selected from the group consisting of a mutation of the 1090th cytosine to guanine, a mutation of the 1105th adenine to thymine, and a mutation of the 1189th adenine to thymine of the PCA-1 gene The method of claim 6, wherein The method according to claim 7, wherein the missense mutation is a mutation of the 1090th cytosine of the PCA-1 gene to guanine. 3. The method of claim 2, wherein the nonsense mutation is a mutation that results in a mutation of the 261st lysine of the PCA-1 polypeptide to a stop codon. The method according to claim 9, wherein the nonsense mutation is a mutation of the 1187th adenine of the PCA-1 gene to thymine. The method according to claim 2, wherein the silent mutation is a mutation selected from the group consisting of a mutation from the 568th thymine to cytosine of the PCA-1 gene and a mutation from the 1132nd guanine to thymine. The method according to claim 11, wherein the silent mutation is a mutation of the 1132th guanine to thymine of the PCA-1 gene. The method according to claim 2, wherein the deletion is a deletion of residues from the 777th adenine to the 865th cytosine of the PCA-1 gene. The method of claim 1 comprising the following steps:
a) isolating or purifying the polynucleotide from the subject;
b) amplifying the PCA-1 gene from the polynucleotide;
c) determining the base sequence of the amplified PCA-1 gene;
d) analyzing the presence or absence or degree of mutation of the PCA-1 gene derived from the subject;
e) A step of determining whether or not the subject has a risk of developing prostate cancer or a risk of developing it based on the presence or absence or degree of mutation of the PCA-1 gene. Compared to SEQ ID NO: 1, substitution of 426th guanine to thymine, substitution of 429th cytosine to thymine, substitution of 494th guanine to adenine, substitution of 528th cytosine to thymine Substitution, 623rd guanine to adenine, 815th guanine to adenine, 836th thymine to cytosine, 1090th cytosine to guanine, 1105th Substitution of adenine with thymine, substitution of 1189th adenine with thymine, substitution of 1187th adenine with thymine, substitution of 568th thymine with cytosine, substitution of 1132th guanine with thymine And one or more substitutions selected from the group consisting of deletion of the sequence from the 777th adenine to the 865th cytosine A polynucleotide comprising a nucleotide sequence having a deletion. Compared to SEQ ID NO: 1, substitution of 426th guanine to thymine, substitution of 429th cytosine to thymine, substitution of 494th guanine to adenine, substitution of 528th cytosine to thymine Substitution, 623rd guanine to adenine, 815th guanine to adenine, 836th thymine to cytosine, 1090th cytosine to guanine, 1105th A group comprising substitution of adenine with thymine, substitution of 1189th adenine with thymine, substitution of 1187th adenine with thymine, and deletion of sequence from 777th adenine to 865th cytosine A polypeptide comprising an amino acid sequence encoded by a base sequence having one or more substitutions or deletions selected from An antibody that immunospecifically recognizes the polypeptide of claim 16. A kit for determining a subject who is at risk of developing prostate cancer or at risk of developing prostate cancer, comprising a reagent for analyzing PCA-1 gene mutation. A description describing that a reagent for analyzing PCA-1 gene mutation can or should be used to determine a subject at risk of developing or at risk of developing prostate cancer. The kit according to claim 18, further comprising:

Images