JP5354484B2 - Cancer detection method - Google Patents

Description

  The present invention relates to a cancer detection method characterized by detecting a deletion of a tumor suppressor gene present on the long arm q25.1 of human chromosome 6. The present invention further relates to the above gene, a protein encoded by the gene, and an antibody that recognizes the protein.

  Follicular lymphoma (hereinafter sometimes abbreviated as FL) is a lymphoma that progresses relatively slowly in the early stage, but is diffuse large B-cell lymphoma (hereinafter sometimes abbreviated as DLBCL). There are many cases that progress more rapidly by transformation of.

  A t (14; 18) (q32; 21) translocation has been reported as a feature of FL, whereby the BCL2 gene of 18q21 and the immunoglobulin heavy chain (IGH) of 14q32 are in close proximity (Non-patent Document 1). As a result, it is considered that BCL2 deregulation and subsequent inhibition of apoptosis occur and contribute to the onset of FL. Other chromosomal abnormalities such as +7, del (1) (p36), del (6) (q) and del (10) (q22-24) are clearly associated with the morphological course of FL (Non-Patent Document 2), however, their molecular significance has not yet been fully elucidated.

  The inventors have found that a translocation t (2; 6) (p12; q23) occurs in the process of transformation from FL to DLBCL, and that the translocation is extremely involved in the transformation from FL to DLBCL. This suggests that it plays an important role (Non-Patent Document 3). From this finding, it was deduced that the unknown gene of 6q23 may be deregulated as a result of gene translocation of the immunoglobulin κ locus (hereinafter sometimes abbreviated as IGK).

  It is estimated that the deletion of the long arm (6q) of chromosome 6 plays an important role in the development of tumors such as lymphoma, lung cancer, breast cancer and ovarian cancer (Non-patent Documents 4-6). It has been reported that FL having 6q23-26 deletion has a high risk of transformation to DLBCL and a poor prognostic factor (Non-patent Document 7).

Knutsen, T., “Cancer Surveys”, 1997, Vol. 30, p. 163-192. Horsman et al., “Jeans, Chromosomes and Cancer”, 2001, volume 30, p. 375-382. Yamamoto, K., et al., “Cancer Genetics and Cytogenetics”, 2003, vol. 147, p. 128-133. Offit, K., et al., “Blood”, 1993, 82, p. 2157-2162. Zhang et al. (Zhang, Y., et al.), “Human Genetics”, 1998, Vol. 103, p. 727-729. Bailey-Wilson et al., “American Journal of Human Genetics”, 2004, vol. 75, p. 460-474. Tilly, H., et al., “Blood”, 1994, 84, p. 1043-1049.

  To find a gene useful for detection of cancer, particularly malignant lymphoma (or various cancers including malignant lymphoma), and to provide a cancer detection method using the gene.

  The present inventors have made extensive studies to solve the above problems, cloned the t (2; 6) breakpoint found in malignant lymphoma, and the immunoglobulin κ locus located on chromosome 2q12 is located on chromosome 6q25. It was found that it fuses with a putative gene that is considered to be a tumor suppressor gene. And we estimated that the deletion of this gene, which is presumed to be a tumor suppressor gene, could contribute to the development, progression, progression and transformation of cancer. Moreover, the detection method of the deletion of this gene was established by utilizing FISH analysis, and the cancer detection method using the detection method of the deletion of this gene was found.

  Furthermore, the present inventors succeeded in isolating and identifying this gene, and obtained a protein encoded by the gene and an antibody that recognizes the protein.

  The present invention has been achieved based on these results.

  That is, the present invention comprises contacting a probe that hybridizes to human chromosome 6 long arm q25 region DNA with a chromosome in a test sample under conditions sufficient to hybridize the probe to the DNA, and The present invention relates to a method for detecting cancer comprising detecting a deletion of a tumor suppressor gene present in the long arm q25.1 region of human chromosome 6 by measuring a hybridization signal of the probe to DNA.

  The present invention also provides a sample derived from cancer when the test sample contains more chromosomes that exhibit a hybridization signal less than the number of hybridization signals observed in normal chromosomes compared to the normal sample. It is related with the said cancer detection method determined to be.

  Furthermore, the present invention relates to the cancer detection method, wherein the probe is a PAC probe RP1-281H8.

  Furthermore, the present invention relates to the cancer detection method, wherein the probe is a DNA represented by the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence listing, or a complementary DNA of the DNA.

  The present invention also relates to the cancer detection method, wherein the probe is a fluorescently labeled probe, and the hybridization signal of the probe to human chromosome 6 long arm q25 region DNA is fluorescence.

  Furthermore, the present invention relates to the cancer detection method, wherein the test sample is a sample containing lymphocytes, a lymph node biopsy sample, or a specimen derived from a tissue containing lymph nodes, tumor cells, or tumor cells.

Furthermore, the present invention provides (i) a DNA represented by the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence listing, or a complementary nucleotide sequence of the nucleotide sequence,
(Ii) DNA having 70% or more homology with the DNA described in (i) above, and (iii) DNA having 1 to several nucleotide mutations in the DNA base sequence described in (i) above,
Relates to DNA selected from

  The present invention also relates to a recombinant vector containing the DNA.

  Furthermore, the present invention relates to a transformant obtained by transformation with the above recombinant vector.

  Furthermore, this invention relates to the manufacturing method of protein including the process of culture | cultivating the said transformant.

  The present invention also relates to a protein encoded by the DNA.

  Furthermore, this invention relates to the said protein represented by the amino acid sequence shown to sequence number 2 or sequence number 4 of a sequence table.

  Furthermore, the present invention relates to an antibody that specifically recognizes the protein.

  The present invention also relates to a reagent kit comprising one or more of the DNA, the recombinant vector, the protein, and the antibody.

  The present invention can provide a method capable of easily detecting cancer, particularly malignant lymphoma (or various cancers including malignant lymphoma).

  In addition, the present invention can provide a tumor suppressor gene, a protein encoded by the gene, and an antibody that recognizes the protein.

  The gene according to the present invention is a gene present in human chromosome 6 long arm q25.1 (6q25.1), and is presumed to be a tumor suppressor gene. In the present specification, this gene is referred to as a follicular lymphoma transforming gene (hereinafter abbreviated as TFL gene).

  A gene is a structural unit that carries genetic information, is a factor that defines a genetic trait, and is defined as a genetic action unit that is defined by a base sequence of a certain region in high molecular DNA. A gene on a chromosome is composed of an exon that is a protein structure sequence portion and an intron that is a sequence portion that does not have amino acid sequence information interposed between exons. In eukaryotes, precursor mRNA is generated by transcription from a portion of DNA carrying genetic information. Subsequently, the part derived from the intron present in the precursor mRNA is excised (splicing), and only the exons are connected to produce a mature mRNA. The genetic information of the mature mRNA is read (translated) on the ribosome, and the protein is biosynthesized.

  In the present specification, “gene” means a nucleic acid having a structural unit carrying genetic information. The nucleic acid can be either DNA or RNA. For example, the gene includes DNA existing on a chromosome and composed of exons and introns, precursor mRNA, mature mRNA, and cDNA that is complementary to mature mRNA.

  “Tumor suppressor gene” refers to a gene that contributes to canceration due to its inactivation or dominant negative mutation. In highly carcinogenic families, there are frequent deletions of chromosomes and loss of heterozygosity (LOH). “Deletion” means that a part of a cell's chromosome or DNA is missing and lost. “Loss of heterozygosity (LOH)” refers to the loss of information on one of the alleles at a locus.

  Preferred examples of the TFL gene include DNA represented by the base sequence shown in SEQ ID NO: 1, DNA represented by the base sequence shown in SEQ ID NO: 3, and DNA represented by the complementary base sequence of the base sequence. These DNAs are splice variants of the TFL gene.

  “Splice variant” refers to two or more types of mature mRNAs generated from one gene by alternative splicing. Alternative splicing refers to the occurrence of two or more types of mature mRNA by changing the position and combination of splice sites between exons and excised introns during splicing in the precursor mRNA.

  It is expected that there are differences in the nucleotide sequence of the TFL gene that may occur due to natural mutation among individuals, but the TFL gene also includes DNA having such differences. The TFL gene includes, for example, the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 and usually 70% or more, preferably 80% or more, more preferably 90% or more, and still more preferably 95% or more of the entire nucleotide sequence. DNA represented by a base sequence having sequence homology or a complementary base sequence thereof is included. In addition, the TFL gene has one or more, for example, 1 to 100, preferably 1 to 30, more preferably 1 to 20, more preferably in the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. It is represented by a nucleotide sequence having a mutation such as deletion, substitution, addition or insertion of 1 to 10, even more preferably 1 to 5, particularly preferably 1 to 3 nucleotides, or a complementary nucleotide sequence thereof. DNA is included.

  The DNA represented by the base sequence shown in SEQ ID NO: 1 is exon 1 (1st to 305th), exon 2 (306th to 444th), exon 3 (445th to 680th), exon 4 (681st to 681). 787) and exon 5a (No. 788-1584), and encodes a protein represented by the amino acid sequence shown in SEQ ID NO: 2. Further, the C / T polymorphism exists at the 158th, 404th and 418th positions of the DNA represented by the nucleotide sequence shown in SEQ ID NO: 1, and thereby the protein encoded by the DNA ( Substitution of proline to leucine, serine to phenylalanine and proline to serine occurs at the 53rd, 135th and 140th amino acid residues of the amino acid sequence of SEQ ID NO: 2), respectively.

  The DNA represented by the nucleotide sequence shown in SEQ ID NO: 3 is exon 1 (1st to 305th position), exon 2 (306th to 444th position), exon 3 (position 445 to 680th position), exon 4 (681th position). 787), exon 5b (no. 788-886), exon 6 (no. 887-911) and exon 7 (no. 912-963), represented by the amino acid sequence shown in SEQ ID NO: 4 Encodes the protein. In addition, there are T / C polymorphisms at the 158th, 404th and 418th positions of the DNA represented by the nucleotide sequence shown in SEQ ID NO: 3, and the protein encoded by the DNA ( Substitution of leucine to proline, phenylalanine to serine and serine to proline occurs at the 53rd, 135th and 140th amino acid residues of the amino acid sequence of SEQ ID NO: 4), respectively.

  DNA containing the TFL gene can be obtained, for example, by the following method. Targeting mRNA extracted from normal human lymph nodes, performing RT-PCR using a part of the TFL gene as a primer, cloning the resulting cDNA into an appropriate plasmid, and including the TFL gene from the resulting cDNA clone It can be obtained by selecting one. Examples of the primer include a combination of a polynucleotide represented by the base sequence shown in SEQ ID NO: 7 and a polynucleotide represented by the base sequence shown in any one of SEQ ID NOs: 8 to 10. Alternatively, by screening a cDNA library prepared from an appropriate human-derived cell line according to a conventional method or a known cDNA library using a polynucleotide represented by the whole or a part of the base sequence of the TFL gene as a probe, TFL gene can be obtained.

  A protein encoded by the TFL gene (hereinafter sometimes referred to as TFL protein) has a zinc finger motif C-x8-C-x5-C-x3-H type. The motif usually plays a role in DNA binding, and sometimes binds to an AU rich element (hereinafter abbreviated as ARE) in the 3 ′ untranslated region of mRNA. Interestingly, many ARE binding proteins (hereinafter abbreviated as ABP) are involved in cell division or cell growth phase related regulation (Barreau et al., Barreau, C. et al., “Nucleic Acids Research”). ”, 2006, 33, p. 7138-71590).

  From these findings, the possibility that TFL protein controls cell proliferation as ABP emerged. In addition, it has been reported that the homologue ZC3H12D of the TFL protein caused cardiomyocyte apoptosis (Zhou, L., et al., “Circulation Research”, 2006, No. 1). 98, p. 1177-1185).

  Taken together, the TFL gene can be considered to play a crucial role in regulating lymphoma cell proliferation and / or survival.

  More recently, the association of the TFL homolog ZC3H12D to sporadic lung cancer has been shown by its loss of heterozygosity (Wang et al., “Cancer Research”, 2007). Year 67, p. 93-99). It has also been reported that the ZC3H12D gene has an A / G non-synonymous single nucleotide polymorphism at codon 106, and overexpression of the ZC3H12D A allele exerts tumor suppressor functions both in vitro and in vivo. ing.

  In the present invention, a translocation at 6q25 of the TFL gene and the IGK gene was identified in the transformed FL. The 5 ′ region containing exon 1 and exon 2 of the TFL gene was replaced with the Vκ and Cκ regions of the IGK gene. Genes shortened by translocations include one of the immunoglobulin loci and other B cell lymphomas with translocations associated with proto-oncogenes such as BCL2, BCL6 and MYC (Kuppers, R., “ Like the genes observed in “Nature Reviews Cancer”, 2005, Vol. 5, p.251-262), the possibility of expression cannot be denied. It is presumed that TFL gene dysfunction or loss of function due to gene translocation is associated with FL transformation.

  Thus, the TFL gene is presumed to be a tumor suppressor gene, and its dysfunction or loss of function is considered to be related to the occurrence, exacerbation / progression, and transformation of cancer. Therefore, by detecting the deletion of the TFL gene, it is possible to detect cancer in which the gene is deleted.

  The cancer detection method according to the present invention is characterized by detecting a deletion of a tumor suppressor gene present in the long arm q25.1 (6q25.1) region of human chromosome 6, ie, the TFL gene.

  Detection of the deletion of the TFL gene can be performed by measuring the expression of a nucleic acid having a structural unit carrying the genetic information of the TFL gene, for example, the TFL gene present on the chromosome, its mRNA, or the TFL protein.

  Examples of methods for measuring the TFL gene present on the chromosome include fluorescence in situ hybridization analysis (hereinafter sometimes abbreviated as FISH analysis) and Southern blotting. The FISH analysis method has an advantage that it is safe and simple and can obtain a result in a short time. On the other hand, the Southern blot method has an advantage of high accuracy, but lacks quantitativeness and is complicated.

  In the FISH analysis method, a cloned gene or DNA fragment is labeled with a non-radioactive compound and used as a probe, the probe and chromosomal DNA are hybridized, and a molecular hybrid site between the probe and chromosomal DNA is labeled. In this method, the compound is detected directly on the chromosome by detecting the compound. As the non-radioactive compound, for example, a fluorescent compound can be suitably used. When the probe is labeled with a fluorescent compound, the molecular hybrid site between the probe and chromosomal DNA can be detected by measuring the fluorescence of the fluorescent compound. An example of non-radioactive compounds other than fluorescent compounds is biotin. When biotin is used as a labeling compound, the probe is labeled with biotinylated deoxyuridine triphosphate (biotin-dUTP) or biotinylated deoxyadenosine triphosphate (biotin-dATP). In this case, the molecular hybrid site between the probe and chromosomal DNA is obtained by labeling avidin with high affinity for biotin with a fluorescent compound, binding it to biotin-dUTP or biotin-dATP, and measuring the fluorescent compound. It can be detected. Other non-radioactive compounds include digoxigenin. When digoxigenin is used as a labeling compound, the probe is labeled with digoxigenin-labeled dUTP (digoxigenin-dUTP) or digoxigenin-labeled deoxyadenosine triphosphate (digoxigenin-dATP). The labeling can be performed by a known labeling method such as a nick translation method, a random prime labeling method, or a PCR labeling method. The molecular hybrid site between the probe and chromosomal DNA can be detected by a fluorescently labeled anti-digoxigenin antibody or by a fluorescently labeled secondary antibody that recognizes the anti-digoxigenin antibody and the antibody. As fluorescent compounds, fluorescein-5-isothiocyanate (FITC), 5- (and -6) -carboxyfluorescein, 5- (and -6) -carboxy-X-rhodamine, 7-amino-4-methylcoumarin-3- Examples include, but are not limited to, acetic acid (AMCA), Texas Red ™ (Molecular Probes, OR, USA), 4 ′, 6-diamino-2-phenylindole (DAPI), Cy3, and Cy5.

  It is known that the FISH analysis method is not only a detection method targeting a metaphase or prometaphase chromosome, but can also detect an interphase nucleus as a target as an interface FISH. The interface FISH analysis method is useful because it can be applied even when growth of cells in a sample to be examined (hereinafter referred to as a test sample) is not observed or the degree of proliferation is low. The interface FISH analysis method can be implemented by the same method as other FISH analysis methods.

  Specifically, the detection of the TFL gene deletion is performed by, for example, hybridizing a probe that hybridizes to human chromosome 6 long arm q25.1 region DNA to a chromosome in the test sample and the probe to the DNA. To the DNA under sufficient conditions, and then detecting the hybridization signal of the probe to the DNA. When a chromosome showing a hybridization signal less than the number of hybridization signals observed on normal chromosomes is observed in the test sample, it can be determined that the test sample is a cancer-derived sample.

  An example of a probe that hybridizes to the long arm q25.1 (6q25.1) region DNA of human chromosome 6 is the PAC clone probe RP1-281H8. RP1-281H8 hybridizes to DNA on human chromosome 6 long arm q25.1-25.3. RP1-281H8 is registered with GenBank as accession number AL031133.1 [gi: 3676189] and can be obtained from Auckland Children's Hospital (Oakland, CA, USA) or RZPD German Resource Center for Genome Research, for example.

  Other examples of probes that hybridize to 6q25.1 region DNA include DNA represented by the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 and complementary DNA of the DNA.

  Examples of the test sample include cells, tissues, and samples containing cells. For example, lymphocytes, lymph node biopsy samples, lymph node specimens, blood and the like can be exemplified as test samples useful for detection of malignant lymphoma. Examples of test samples useful for detecting lung cancer include bronchial specimens, lung biopsy samples, and sputum samples. Examples of bronchial specimens include bronchial secretions, lavage fluid, lavage, aspiration and scraping. A lymph node biopsy sample and a biopsy sample of lung and other whole body tissues can be obtained by a sampling method such as surgery or needle puncture.

  In the FISH analysis method, cells are prepared from a test sample using techniques well known to those skilled in the art. For example, the cells can be prepared by collecting a lymph node biopsy sample by a known method, precipitating by centrifugation to form a cell pellet, and then resuspending in an appropriate buffer. A preferable example of the buffer is phosphate buffered saline (PBS). Alternatively, after centrifuging the cell suspension to obtain a cell pellet, the cells can be fixed with, for example, an acid alcohol solution, an acid acetone solution, or an aldehyde such as formaldehyde, paraformaldehyde and glutaraldehyde. A neutral buffered formalin solution can also be used as a fixative. When using fixed cells, most of the fixative is removed by centrifugation or the like and suspended in a buffer. The prepared cells or fixed cells can be used by being developed on the slide by dropping a suspension containing the cells onto the slide. At this time, the cell suspension is spread on the slide so that the cells do not overlap on the slide.

  Prior to in situ hybridization, the chromosomal probe and the chromosomal DNA contained in the cell are each denatured. In addition, when detecting chromosomal DNA contained in a cell, the cell can be pretreated by protease digestion. If the chromosomal probe is prepared as a single stranded nucleic acid, no denaturation of the probe is required. Denaturation is typically performed by incubation in the presence of high pH, heat (eg, temperatures of about 70 ° C. to about 95 ° C.), organic solvents such as formamide and tetraalkylammonium halides, or combinations thereof. . Chromosomal DNA denaturation is, for example, between a temperature above 70 ° C. (eg about 73 ° C.) and a denaturation buffer containing 70% formamide and 2 × SSC (0.3 M sodium chloride and 0.03 M sodium citrate). This can be done in combination. The denaturation of the chromosomal probe can be performed, for example, by heating at about 73 ° C. for about 5 minutes.

  Hybridization of the probe and chromosomal DNA is carried out under conditions sufficient for hybridization after removing the chemical substance or conditions used for denaturing the chromosomal probe and chromosomal DNA contained in the cell. “Conditions sufficient for hybridization” are conditions that promote annealing between the probe and the target chromosomal DNA. Conditions sufficient for hybridization will depend on probe concentration, base composition, complexity and length, and salt concentration, temperature and length of incubation. For example, in situ hybridization generally involves 1-2 × SSC, 50% -55% formamide, a hybridization promoter (eg, 10% dextran sulfate), and blocking DNA to inhibit non-specific hybridization. Perform in the contained hybridization buffer. In general, hybridization conditions as described above are performed at a temperature of about 25 ° C. to about 55 ° C. for about 0.5 hours to about 96 hours of incubation. More preferably, the hybridization is performed at about 32 ° C. to about 45 ° C. for about 2 hours to about 16 hours.

  Nonspecific binding of the chromosomal probe to other DNA in the target region can be eliminated by a series of washes. The temperature and salt concentration in each wash depends on the desired stringency. For example, at high stringency conditions, about 65 ° C. using 0.2-2 × SSC and a nonionic surfactant such as about 0.1% to about 1% Nonidet P-40 (NP40) Washing can be performed at ~ 80 ° C. Stringency can be lowered by reducing the temperature of the wash or by increasing the salt concentration of the wash.

  Detection of hybridization of the probe to chromosomal DNA is performed by measuring a hybridization signal. The hybridization signal means an index indicating that the probe and the chromosomal DNA are hybridized, and can be measured by a compound previously labeled on the probe. In the FISH analysis method, as described above, the hybridization signal is generally measured as a fluorescence signal. The method of measuring the fluorescence signal is well known to those skilled in the art, and a known technique can be selected and implemented according to the type of fluorescence used.

  Based on the measurement result of the hybridization signal of the probe to the chromosomal DNA, the deletion of the target chromosomal region DNA in the cell, in the present invention, the deletion of the 6q25 region DNA is evaluated. Since the chromosomes are paired, two hybridization signals of the probe to one target chromosomal DNA are observed in normal cells. On the other hand, when the target chromosomal DNA is deleted in one of the paired chromosomes, only one hybridization signal is observed. When the target chromosomal DNA is deleted in both chromosomes, the hybridization signal is Not observed. Therefore, it can be determined that the 6q25 region DNA is deleted when a chromosome showing a hybridization signal less than the number of hybridization signals observed in the normal chromosome is observed.

  Since the tumor suppressor gene according to the present invention is present in the 6q25 region, it is considered that the deletion of this region is related to the occurrence of cancer, exacerbation / progress and transformation. Therefore, it can be determined that the test sample containing the chromosome determined to have the 6q25 region DNA deleted is a cancer-derived sample. Specifically, when there are more chromosomes in the test sample showing hybridization signals less than the number of hybridization signals (two) observed in normal chromosomes compared to the normal sample, the test sample is peeled off. It can be determined that it is a sample derived from cancer. The determination can be made by using as an index the ratio of the cells in which chromosomes showing hybridization signals less than the number (two) of hybridization signals observed in normal chromosomes to the total cells evaluated. Since the detection sensitivity increases as the number of cells to be evaluated increases, the evaluation criterion can be set low by increasing the number of cells to be evaluated. The measurement of the hybridization signal is generally measured in a plurality of cells, for example about 100 or more, preferably about 200 or more, more preferably about 300 or more, more preferably about 400 or more, and even more preferably about Evaluate in over 500 cells. About 15% or more of the evaluated cells, preferably about 10% or more, more preferably 5% or more, more preferably about 3% or more, and even more preferably about 2% or more of high cells observed in normal chromosomes. When a chromosome showing a hybridization signal less than the number of hybridization signals (two) is observed, it can be determined that the 6q25 region DNA has been deleted. The evaluation criteria are set by measuring chromosome hybridization signals in a plurality of normal cells, and measuring the number of hybridization signals observed in normal chromosomes (two). This can be done by calculating the ratio and using that value as the evaluation criterion.

  Thus, in the present invention, a FISH analysis method for detecting a deletion of 6q25 region DNA is established, and further a cancer detection method characterized by detecting a deletion of 6q25 region DNA is provided. It is estimated that the deletion of the long arm (6q) of chromosome 6 plays an important role in the development of tumors such as lymphoma, lung cancer, breast cancer and ovarian cancer (Non-patent Documents 4-6). It has also been shown by the loss of heterozygosity that the TFL protein homolog ZC3H12D is associated with sporadic lung cancer (Wang, M., et al., “Cancer Research”, 2007). Year 67, p. 93-99). Therefore, the cancer detection method according to the present invention is extremely useful for diagnosis of malignant tumors associated with deletion of the TFL gene, for example, malignant lymphoma, lung cancer such as sporadic lung cancer, breast cancer and ovarian cancer.

  Detection of 6q25 region DNA deletion in the cancer detection method according to the present invention can be carried out by Southern blotting as well as FISH analysis. In Southern blotting, chromosomes are cleaved with restriction enzymes to be fragmented, electrophoresed on an agarose gel to make single-stranded DNA by alkaline denaturation, the gel is adsorbed to a nitrocellulose membrane, and then binds to a target gene. In this method, a double strand of the single-stranded DNA and the probe is formed using a probe or RNA probe, and the formed molecular hybrid is detected. Preferred examples of the probe include DNA represented by the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 or a complementary nucleotide sequence thereof. By this method, the presence or absence of the target gene can be detected. A molecular hybrid can be detected by detecting a labeled compound using a probe labeled with a fluorescent compound or a radioisotope. Southern blotting is well known to those skilled in the art and can be performed by known techniques.

  In addition, detection of TFL gene deletion can be carried out by measuring TFL mRNA expression or TFL protein expression. TFL mRNA expression can be measured by RNA blot analysis using a probe. Preferred examples of the probe include DNA represented by the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 or a complementary nucleotide sequence thereof. TFL protein expression can be measured by Western blot analysis using a specific antibody or immunostaining of tissues. It is also possible to examine the expression at the cell level by flow cytometry. RNA blot analysis, Western blot analysis, tissue immunostaining, and analysis methods by flow cytometry are all well known to those skilled in the art and can be performed by known techniques.

  A specific antibody against the TFL protein can be prepared using the protein as an antigen. The antigen may be a TFL protein or a fragment thereof. In order to produce an antibody specific to the TFL protein, it is preferable to use a region consisting of an amino acid sequence unique to the protein as an antigen. The antibody according to the present invention may be any antibody that specifically recognizes the TFL protein, and is not particularly limited. To specifically recognize the TFL protein means, for example, that it binds to the protein but does not bind or weakly binds proteins other than the protein. The presence or absence of recognition can be determined by a known antigen-antibody binding reaction.

  Antibody production methods are well known to those skilled in the art and can be performed by antibody production methods known per se. For example, an antibody can be obtained by administering an antigen to an animal, alone or in combination with a carrier, in the presence or absence of an adjuvant, and inducing immunity such as a humoral response and / or a cellular response. Carriers and adjuvants can be appropriately selected from known ones.

Polyclonal antibodies can be obtained from the sera of animals that have been immunized by known antibody recovery methods. As a preferred antibody recovery method, an immunoaffinity chromatography method can be exemplified.
Monoclonal antibodies include antibody-producing cells (for example, spleen or lymph node-derived lymphocytes) collected from animals that have been immunized and permanent proliferative cells known per se (for example, myeloma such as P3-X63-Ag8 strain). Can be produced using hybridomas produced by fusing the strain. For example, an antibody-producing cell and a permanently proliferating cell are fused by a method known per se to prepare a hybridoma, and then cloned. A hybridoma producing an antibody that specifically recognizes the protein of the present invention is selected from a number of cloned hybridomas, and the antibody is recovered from the culture medium of the hybridoma.

  A polyclonal antibody or monoclonal antibody capable of recognizing or binding to the TFL protein can be used as an antibody for purifying the protein, a reagent, a label marker, or the like.

  DNA containing the TFL gene, for example, (i) DNA represented by the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence listing, or a complementary nucleotide sequence of the nucleotide sequence, (ii) described in (i) above It is encoded by DNA having 70% or more homology with DNA, (iii) DNA having one to several nucleotide mutations in the base sequence of DNA described in (i) above, or DNA selected from the above DNA Any of the proteins, for example, the protein represented by the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, and the antibody recognizing the protein can be used alone or in combination as a reagent.

  In addition, a recombinant vector containing the DNA of the present invention and a transformant transformed with the recombinant vector can also be provided in the present invention, and these can be used as reagents and the like. A recombinant vector can be obtained by inserting the DNA according to the present invention into an appropriate vector DNA. The vector DNA is not particularly limited as long as it can replicate in the host, and is appropriately selected depending on the type of host and intended use. The vector DNA can be appropriately selected from vector DNAs well known to those skilled in the art, and any of expression vectors and cloning vectors can be used depending on the purpose. The recombinant expression vector containing the DNA according to the present invention is useful for producing a protein encoded by the DNA. As a method for incorporating the DNA according to the present invention into the vector DNA, a method known per se can be applied. For example, a method is used in which a gene containing the present DNA is treated with an appropriate restriction enzyme, cleaved at a specific site, then mixed with vector DNA treated in the same manner, and religated by ligase. Alternatively, a desired recombinant vector can be obtained by ligating a suitable linker to the DNA of the present invention and inserting it into a multicloning site of a vector suitable for the purpose. A transformant is obtained by transforming a host with the recombinant vector according to the present invention. The transformant introduced with the recombinant expression vector containing the DNA according to the present invention is useful for the production of the protein encoded by the DNA. Either prokaryotic or eukaryotic organisms can be used as the host. Prokaryotes, for example, Escherichia such as Escherichia coli, Bacillus such as Bacillus subtilis, Pseudomonas putida such as Pseudomonas genus, Rhizobium merilloti The bacteria to which it belongs. Examples of eukaryotes include animal cells such as yeast, insect cells and mammalian cells. Examples of the yeast include Saccharomyces cerevisiae and Schizosaccharomyces pombe. Examples of insect cells include Sf9 cells and Sf21 cells. Examples of mammalian cells include monkey kidney-derived cells (COS cells, Vero cells, etc.), Chinese hamster ovary cells (CHO cells), mouse L cells, rat GH3 cells, human FL cells, and 293EBNA cells. Transformation of host cells with a recombinant vector can be carried out by applying means known per se. For example, it can be carried out by a standard method described in a book (edited by Sambrook et al., “Molecular Cloning, Laboratory Manual, Second Edition”, 1989, Cold Spring Harbor Laboratory).

  When using the DNA, protein, antibody, recombinant vector, transformant, etc. according to the present invention as a reagent, in addition to at least one of these, a buffer solution, salt, stabilizer, and / or preservative, etc. Can be included. In the formulation, a known formulation method according to the properties of each substance may be introduced. The reagent can be used, for example, for cancer detection according to the present invention. In addition, the reagent is useful for basic research on diseases and the like based on abnormality of DNA or protein according to the present invention.

  The present invention further provides a reagent kit comprising one or more of the DNA, protein, antibody, recombinant vector, and transformant according to the present invention. In addition to the reagent kit, a labeling substance for detecting the DNA and protein according to the present invention, a labeling detection agent, a reaction diluent, a standard antibody, a buffer solution, a cleaning agent, a reaction stop solution, etc. Can be included. This reagent kit can be used for the cancer detection method according to the present invention. Furthermore, the reagent kit can be used as a test agent and a test kit in a test method using the cancer detection method according to the present invention, and also in a diagnostic method using the cancer detection method according to the present invention. It can be used as a diagnostic agent and a diagnostic kit.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited by this embodiment.

  In order to clone the t (2; 6) breakpoint found in patients with malignant lymphoma, the genome was first extracted from the lymph nodes of patients with malignant lymphoma who had t (2; 6) (p12; q23). Southern blot analysis of DNA was performed.

  Genomic DNA was extracted from lymph nodes by the method described previously (Non-Patent Document 3). Southern blot analysis was performed as follows. As a DNA probe, a DNA probe derived from a 1.8 kb genomic fragment containing the junction region (Jκ) of the IGK gene or a 323 bp fragment containing the constant region (Cκ) was synthesized using the polymerase chain reaction.

Forward primer: 5′-GAACTGTGGCTGCACCCATCT-3 ′ (SEQ ID NO: 5)
Reverse primer: 5'-CTAACACTCTCCCCTGTTTGA-3 '(SEQ ID NO: 6)

  As a result, no reconstructed band was detected with the Jκ probe, but two other bands were detected with the Cκ probe in addition to the germline band (FIG. 1-A).

  Subsequently, translocation breakpoints were cloned as follows using genomic DNA extracted from lymph nodes derived from malignant lymphoma patients.

  Genomic DNA extracted from lymph nodes from malignant lymphoma patients was completely digested with BamHI and electrophoresed on a 0.8% agarose gel. A fraction between 7 kb and 12 kb corresponding to the reconstituted band was collected. The extracted DNA was ligated to ZAP Express Pregested Vector (Stratagene, La Jolla, CA, USA) to construct a genomic library. The genomic library was screened by plaque hybridization using Cκ probe. Sequencing of the cloned DNA was performed by ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).

  As a result, 7 clones were isolated (FIG. 1-B). Nucleotide sequencing revealed that 5 clones were identical. That is, three separate clones were obtained (FIG. 2-A).

Clone I was a 7.6 kb fragment containing a normally rearranged IGK gene on chromosome 2. Clone II was a 6.7 kb fragment consisting of partially deleted clone I and an 865 bp fragment derived from 6q25. That is, this clone contained a translocation breakpoint of the IGK gene. Thus, the true breakpoint was located at 6q25, not 6q23. The nucleotide sequence of the 865 bp fragment was part of the second intron of the TFL gene located at 6q25. The TFL gene is referred to as ZC3H12D in the human genome database. The breakpoint of the IGK gene was located between Vκ _1-33 and Vκ _3-34 . The TFL gene was fused in a “head-to-head” fashion with the IGK gene and der (6) t (2; 6) (p12; q25). It was found that one nucleotide (T) was inserted between both genes (FIG. 2-B). Clone III was a 5.3 kb fragment consisting of a partially deleted clone II and a 1946 bp fragment derived from 5q34 bispecific phosphatase 1 (DUSP1).

From the above results, the breakpoint of t (2; 6) observed in patients with malignant lymphoma is between Vκ _1-33 and Vκ _3-34 of the 2p12 IGK gene on chromosome 2 and 6q25 on chromosome 6. It became clear that it is located in.

  Detection of t (2; 6) (p12; q25) was performed on lymph node samples from patients with malignant lymphoma by interface FISH analysis.

  As a probe for analysis, clone RP1-281H8 (obtained from Oakland Children's Hospital, Oakland, CA, USA), which is a PAC probe, was used. The FISH analysis protocol is slightly modified from the previously described method (Tibiletti, M., et al., “Cancer Research”, 1996, Vol. 56, p. 4493-4498). Used in addition.

  Specifically, the slide on which interphase cells were placed was subjected to pretreatment by protease digestion and denatured after fixation with formaldehyde. The probe was labeled with biotinylated 16-dUTP using a nick translation kit (Roche, Germany) and mixed with cot-1 DNA (Vysis, IL, USA) before hybridization with the sample on the slide I let you. Detection of the hybridized probe was performed using a Tyramide Signal Amplification Kit (Molecular Probes, OR, USA).

  At least 100 cells were examined for each sample. The case where the number of cells showing a single signal was 10% or more was set as a cut-off point for identifying the deletion.

  As a result, 30% of the total number of cells measured in a lymph node sample derived from a patient with malignant lymphoma showed a single signal (the right diagram in FIG. 3). On the other hand, in the normal control, only one of the positive cells was always 10% or less (the left figure in FIG. 3).

  Thus, by the FISH analysis, the TFL gene deletion was successfully detected in a lymph node sample derived from a malignant lymphoma patient. TFL gene deletion in patients with malignant lymphoma has not been detected by G-band analysis.

  Cloning of TFL cDNA was performed. MRNA was extracted from normal human lymph nodes according to a conventional method, and RT-PCR was performed on the mRNA using the following primers.

5'-ATGGAGCACCCCAGCAAGATGGGAATTC (SEQ ID NO: 7)
3'-TTAGGGCTTGCCCAGGGGGCCC (SEQ ID NO: 8)
3′-CTAGGGCGGTGTTCCCCCGCGCGG (SEQ ID NO: 9)

As a result, two separate cDNAs, TFL ^p58 and TFL ^p41, were isolated.

  The obtained cDNA was cloned into a mammalian expression plasmid based on pQBI25 (Takara, Japan) according to a conventional method. As the plasmid, a plasmid having a hemagglutinin (HA) tag or a green fluorescent protein (GFP) tag or a plasmid having no such tag was used. Each plasmid was then transiently transfected into BaF3 cells and the expression of cDNA in the cells was detected by immunoblot analysis using an anti-HA monoclonal antibody (Roche, Germany).

  In order to specify the 3 ′ untranslated region (UTR), cDNA was amplified in the same manner as described above using the above primer (SEQ ID NO: 7) and the following primer.

3′-ACTCTGTCTGTTTAAAAAAAAGAAAGAAG (SEQ ID NO: 10)

As a result, TFL ^p58 cDNA having a longer 3 ′ untranslated region (UTR) was isolated.

  Furthermore, to identify a splice variant of the TFL gene, the above human cDNA fragment was used to screen a human leukocyte large insert cDNA library (Clontech, CA, USA).

As a result, two different sizes of cDNA were isolated. The base sequences of all the isolated cDNAs were determined, and the base sequences of TFL ^p58 and TFL ^p41 cDNA (SEQ ID NO: 1 and SEQ ID NO: 3, respectively) were determined as at least two splice variants.

  An antibody that recognizes the protein encoded by the TFL gene was created (Operon Biotechnology, Tokyo, Japan).

As an antigen, the full-length protein encoded by ^{TFL p41} of cDNA clones isolated in Example 3 (amino acids 1-321: However amino acid 1-298 is a common sequence ^{TFL p58)} and the ^{TFL p58} and ^{TFL p41} All of the common N-terminal fragments (amino acids 1-88) were used fused with an N-terminal glutathione S-transferase (GST) tag.

^{Specifically,} the NdeI-BamHI fragment of plasmid containing the region encoding the N-terminal fragment of plasmid and ^{TFL p41} proteins contain an open reading frame of ^{TFL p41} protein, were each inserted into the same sites of pGEX2TL vector. GST-tagged fusion protein was expressed in BL21 transformed cells and separated by SDS-polyacrylamide gel electrophoresis. Two female rabbits were injected 6 times at 2-week intervals using a gel containing 4 mg of GST tag fusion protein. Seven days after the last injection, blood was collected from rabbits, and serum was collected by centrifugation. Antisera against TFL ^p41 full length protein was further affinity purified using human TFL ^p41 protein.

  The cancer detection method according to the present invention, particularly the malignant lymphoma detection method, contributes significantly to the pharmaceutical field. In addition, t (2; 6) (p12; q25) in malignant lymphoma found in the present invention is thought to play a crucial role in regulating the proliferation and / or survival of lymphoma cells, and thus malignant lymphoma and It also contributes to research to elucidate the various processes that cause cancer and the development of anticancer drugs.

It is a figure which shows the result of having performed the Southern blot analysis of the IGK gene about the genomic DNA extracted from the lymph node derived from the malignant lymphoma patient who has t (2; 6) (p12; q23). In the hybridization using the Jκ probe, only the germline band was observed, but in the hybridization using the Cκ probe, two reconstructed bands (arrows) were recognized in addition to the germline band (arrowhead). . In the figure, Patient refers to a lymphoma patient, and Normal control refers to a healthy person. HindIII and BamHI each indicate that the genomic DNA sample was treated with each restriction enzyme. It is a figure which shows the result of the cloning of the chimeric gene formed by t (2; 6) (p12; q25). Three different clones, clone # 1, clone # 2 and clone # 3, were isolated by screening a genomic library using a Cκ probe. Each extracted plasmid was digested with BamHI. Arrows indicate vector bands. It is a schematic diagram which shows the structure of a TFL chimera gene. From top, restriction map of germline IGK gene (Igκ gene) on chromosome 2, clone I (Clone I): rearranged IGK gene on chromosome 2, clone II (Clone II): der (6) Chimeric gene of IGK and TFL by t (2; 6) (p12; q25), clone III (Clone III): shows TGK, TFL and DUSP1 chimeric genes on chromosome 6, and TFL gene on chromosome 6 . The underline attached to the IGK gene indicates the position of the probe used for Southern blot analysis. The horizontal white arrow indicates the direction of ZC3H12D or IGK transcription, and the vertical black arrow indicates the breakpoint junction. The genomes for IGK, TFL and DUSP1 are indicated by black, white and gray horizontal thick lines, respectively. The reading frames of IGK and TFL are indicated by black frames and line-shadow frames, and the non-coding region of TFL is indicated by white frames. It is a figure which shows in parallel the nucleotide sequence including a breakpoint, and the related germline relative sequence of the said sequence and the 2nd chromosome (Chromosome 2) and the 6th chromosome (Chromosome 6). The analysis chromatogram which shows the base sequence of a breakpoint junction part is shown. It is a figure which shows the result of having detected the TFL gene deletion in the lymph node cell derived from a malignant lymphoma patient by interphase nuclear FISH analysis. The left figure (A) shows a normal control sample, and a double positive signal was observed for 90% or more of the nuclei. The right figure (B) has shown the lymphoma cell sample of a malignant lymphoma patient, and the number of single positive cells (white arrow) reached 30% or more. Although a chromosomal abnormality t (2; 5) was identified in this case, the 6q deletion was not identified by G-band analysis.

SEQ ID NO: 1: (158). . (158) C / T polymorphic site, thereby causing a substitution of proline to leucine at position 53 of the amino acid sequence shown in SEQ ID NO: 2.
SEQ ID NO: 1: (404). . (404) C / T polymorphic site, thereby causing a substitution of serine to phenylalanine at position 135 of the amino acid sequence shown in SEQ ID NO: 2.
SEQ ID NO: 1: (418). . (418) C / T polymorphic site, thereby causing a substitution of proline to serine at position 140 of the amino acid sequence shown in SEQ ID NO: 2.
Sequence number 1: (1). . (305) Exon 1
SEQ ID NO: 1: (306). . (444) Exon 2
SEQ ID NO: 1: (445). . (680) Exon 3
SEQ ID NO: 1: (681). . (787) Exon 4
SEQ ID NO: 1: (788). . (1584) Exon 5a
SEQ ID NO: 3: (158). . (158) T / C polymorphic site, thereby causing a substitution of leucine to proline at position 53 of the amino acid sequence shown in SEQ ID NO: 4.
SEQ ID NO: 3: (404). . (404) T / C polymorphic site, thereby causing a phenylalanine to serine substitution at position 135 of the amino acid sequence shown in SEQ ID NO: 4.
SEQ ID NO: 3: (418). . (418) T / C polymorphic site, thereby causing a serine to proline substitution at position 140 of the amino acid sequence shown in SEQ ID NO: 4.
Sequence number 3: (1). . (305) Exon 1
SEQ ID NO: 3: (306). . (444) Exon 2
SEQ ID NO: 3: (445). . (680) Exon 3
SEQ ID NO: 3: (681). . (787) Exon 4
SEQ ID NO: 3: (788). . (886) Exon 5b
SEQ ID NO: 3: (887). . (911) Exon 6
SEQ ID NO: 3: (912). . (963) Exon 7
SEQ ID NO: 5: Polynucleotide designed for primer SEQ ID NO: 6: Polynucleotide designed for primer SEQ ID NO: 7: Polynucleotide designed for primer SEQ ID NO: 8: Polynucleotide designed for primer 9: Polynucleotide designed for primer SEQ ID NO: 10: Polynucleotide designed for primer

Claims (13)

Of the long arm q25 region of human chromosome 6, a probe that hybridizes to DNA in the region encoding the mRNA of the follicular lymphoma transforming gene (TFL gene) is applied to the chromosome in the test sample, and the probe is added to the DNA. Deletion of the tumor suppressor gene present in the long arm q25.1 region of human chromosome 6 by contact under conditions sufficient for hybridization and then measuring the hybridization signal of the probe to the DNA Ri Na include detecting, wherein encoding mRNA of TFL gene region encoding a DNA represented by the complementary nucleotide sequence of the nucleotide sequence, or nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3 To check for malignant lymphoma, which is the area to be treated. A test sample is determined to be a sample derived from malignant lymphoma when there are more chromosomes showing a hybridization signal than the number of hybridization signals observed in normal chromosomes in the test sample. The method for examining malignant lymphoma according to claim 1. The method for examining malignant lymphoma according to claim 1, wherein the probe is a PAC probe RP1-281H8. The method for examining malignant lymphoma according to claim 1, wherein the probe is a DNA represented by the nucleotide sequence shown in SEQ ID NO: 1 or 3 in the sequence listing, or a complementary DNA of the DNA. The method for examining malignant lymphoma according to claim 1, wherein the probe is a fluorescently labeled probe, and the measurement of the hybridization signal is performed by measuring fluorescence . The method for examining malignant lymphoma according to claim 1, wherein the test sample is a sample containing lymphocytes, a lymph node biopsy sample, or a specimen derived from a tissue containing lymph nodes, tumor cells or tumor cells. A reagent for detecting malignant lymphoma, comprising a DNA represented by the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence listing, or a complementary nucleotide sequence of the nucleotide sequence. A reagent for detecting malignant lymphoma comprising a recombinant vector containing a DNA represented by the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence listing or a complementary nucleotide sequence of the nucleotide sequence . Detection of malignant lymphoma comprising a transformant transformed with a recombinant vector containing the DNA represented by the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence listing or a complementary nucleotide sequence of the nucleotide sequence Reagent . A reagent for detecting malignant lymphoma comprising a protein encoded by a DNA represented by the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence listing, or a complementary nucleotide sequence of the nucleotide sequence . A reagent for detecting malignant lymphoma comprising a protein represented by the amino acid sequence shown in SEQ ID NO: 2 or 4 in the sequence listing. A base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence listing, or a protein encoded by DNA represented by a complementary base sequence of the base sequence, or an amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 in the sequence listing A reagent for detecting malignant lymphoma, comprising an antibody that specifically recognizes the protein represented by DNA represented by the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence listing, or a complementary nucleotide sequence of the nucleotide sequence, a recombinant vector containing the DNA, a protein encoded by the DNA, or a sequence listing A reagent kit for detecting malignant lymphoma, comprising one or more of the protein represented by the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 and an antibody that specifically recognizes the protein .