WO2007043531A1 - Genetic marker for use in diagnosis of sensitivity to anti-tumor agent or concurrent chemoradiation therapy, and use thereof - Google Patents

Genetic marker for use in diagnosis of sensitivity to anti-tumor agent or concurrent chemoradiation therapy, and use thereof Download PDF

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Publication number
WO2007043531A1
WO2007043531A1 PCT/JP2006/320225 JP2006320225W WO2007043531A1 WO 2007043531 A1 WO2007043531 A1 WO 2007043531A1 JP 2006320225 W JP2006320225 W JP 2006320225W WO 2007043531 A1 WO2007043531 A1 WO 2007043531A1
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Prior art keywords
polynucleotide
seq
combination therapy
sensitivity
radiation
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PCT/JP2006/320225
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French (fr)
Japanese (ja)
Inventor
Hirofumi Doi
Kensaku Imai
Yasuo Uemura
Masakazu Fukusima
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Celestar Lexico-Sciences, Inc.
Taiho Pharmaceutical Co., Ltd.
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Priority to JP2007539949A priority Critical patent/JPWO2007043531A1/en
Publication of WO2007043531A1 publication Critical patent/WO2007043531A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a genetic marker for susceptibility diagnosis of an anti-tumor agent and radiation therapy, and more specifically, to examine the presence or absence of a specific polynucleotide in a biological sample collected from a subject such as a cancer patient. Whether or not it is sensitive to the combination therapy of tegafur, gimeracil and oteracil potassium combined antitumor agent and radiation
  • the present invention relates to a polynucleotide that functions as a genetic marker for rapidly diagnosing whether or not the combination of the above-described antitumor agent and radiation therapy is effective for treating cancer in a subject, and use thereof.
  • TS-1 tegafur 'gimeracil' oteracil potassium
  • S-1 S-1
  • Non-patent Document 1 an excellent cancer treatment method by combining the antitumor agent with radiation therapy is known.
  • Patent Document 1 Japanese Patent Publication No. 5-64123
  • Patent Document 2 Japanese Patent Publication No. 5-80451
  • Patent Document 3 Japanese Patent No. 2538422
  • Patent Document 4 Japanese Patent No. 2614164
  • the present invention has been made in view of the above-mentioned problems, and is a means for quickly and accurately diagnosing the sensitivity of an anti-tumor agent containing tegafur and gimeracil potassium terracil potassium and radiation therapy. It is intended to provide.
  • the present inventors provide a polynucleotide used as a primer or probe for the polynucleotide as the gene marker, a polypeptide encoded by the polynucleotide, and an antibody that recognizes the polypeptide.
  • the present inventors have found that it is useful for diagnosing the above-mentioned combination therapy sensitivity more quickly and accurately.
  • diagnostic agents and diagnostic kits containing the above primers, probes, and antibodies can more easily diagnose sensitivity.
  • the present invention has been completed based on such knowledge.
  • the present invention has the following [1] to [13] forces.
  • [1] A polynucleotide represented by the following (A) or (B).
  • (C) a nucleotide sequence that is hybridized under a highly stringent condition with the polynucleotide according to (A) or (B), and a tegafur / gimeracil / oteracil potassium-conjugated antitumor agent and radiation
  • one or a plurality of bases includes a base sequence in which deletion, substitution, insertion, and Z or addition are present, and tegafur 'gimeracil 'Polynucleotides that function as genetic markers for diagnosing sensitivity to combination therapy with anti-tumor agents containing oteracil potassium and radiation
  • (C) a nucleotide sequence that is hybridized under a highly stringent condition with the polynucleotide according to (A) or (B), and a tegafur / gimeracil / oteracil potassium-conjugated antitumor agent and radiation
  • one or a plurality of bases includes a base sequence in which deletion, substitution, insertion, and Z or addition are present, and tegafur 'gimeracil 'Polynucleotides that function as genetic markers for diagnosing sensitivity to combination therapy with anti-tumor agents containing oteracil potassium and radiation
  • a polynucleotide comprising at least 10 consecutive nucleotides in the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 70 in the Sequence Listing
  • polynucleotide according to (1) or (2) above which comprises a nucleotide sequence in which one or more bases are missing, substituted, inserted, and Z or added, and [1] A polynucleotide that functions as a probe or primer for the described polynucleotide
  • step (II) RNA or complementary polynucleotide bound to the polynucleotide in step (I) The step of measuring the presence or absence of the above-mentioned [3] to [5] using the polynucleotide described in any one of the above as an indicator
  • step (ii) a step of measuring the presence or absence of the polypeptide bound to the antibody or its partial peptide in step (i) using the antibody as an index
  • Step (iii) Based on the measurement results of step (ii), a diagnosis of the sensitivity of combination therapy with tegafur and gimeracil * oteracil potassium combined antitumor agent and radiation
  • FIG. 1 is a diagram showing an outline of the procedure up to the computer analysis after the creation of a cDNA library in the examples.
  • FIG. 2 is a diagram showing an outline of a procedure of array annotation processing in the embodiment.
  • FIG. 3 is a diagram showing an outline of a procedure for mapping genomes to clones and thereby clustering in Examples.
  • FIG. 4 is a graph showing the ratio of the amplification amount of the radiation treatment group and the combination treatment group in the example to the amplification amount of the control group.
  • the present invention relates to an antitumor agent containing tegafur, gimeracil, and oteracil potassium, and a genetic marker for diagnosing radiation therapy sensitivity.
  • the combination therapy of the antitumor agent and radiation means a cancer treatment method using a combination of radiotherapy with the antitumor agent.
  • the antitumor agent used in the combination therapy targeted by the present invention contains tegafur, gimeracil and oteracil potassium together with a pharmaceutically acceptable carrier, and tegafur: gimeracil: It is characterized by containing oteracil potassium in a molar ratio of 1: 4: 4: 1.
  • tegafur is a known drug that is known to release the active substance 5-fluorouracil (5-FU) upon receiving an in vivo activity.
  • the production method is not particularly limited, and a known method, for example, the method described in JP-B-49-10510 can be followed.
  • Gimeracil (2,4-dihydroxy-5-cloguchil pyridine) is a known compound that exerts an action to enhance the antitumor effect of 5-FU.
  • the production method is not particularly limited, and those produced according to known methods can be used.
  • Oteracil potassium (oxonic acid potassium) suppresses side effects such as inflammation caused by the above-mentioned tegafur.
  • Oteracil potassium itself is a known compound.
  • the pharmaceutically acceptable carrier include various types of commonly used drugs such as fillers, extenders, binders, disintegrants, surfactants, lubricants and the like. Can be illustrated.
  • an antitumor agent a commercially available product can be used. Specifically, it is manufactured and sold by Taiho Pharmaceutical Co., Ltd.! The commercially available products “TS1 capsule 20” and “TS1 capsule 25”.
  • TS-1 is a registered trademark of Taiho Pharmaceutical Co., Ltd., one of the applicants of the present application).
  • the dosage unit form of the antitumor agent is not particularly limited as long as it is a non-injectable form, and can be selected according to the therapeutic purpose. Specifically, tablets, coated tablets, pills, powders, granules And oral agents such as pushells, solutions, suspensions, emulsions, and parenterals such as suppositories, ointments, patches, etc., and these administration agents are conventional formulations commonly known in this field. Formulated by the method.
  • the administration method of the antitumor agent is not particularly limited as long as it is non-injection administration such as enteral administration, oral administration, rectal administration, buccal administration, transdermal administration, etc. It is determined according to gender and other conditions, the degree of symptoms of the patient, and the like. For example, tablets, pills, solutions, suspensions, emulsions, granules and capsules are administered orally. Suppositories are administered rectally. The ointment is applied to the skin, oral mucosa and the like.
  • the administration conditions of the antitumor agent can be appropriately selected depending on the usage, age, sex and other conditions, the degree of disease, and the like.
  • the amount of tegafur is about 0.1 to lOOmgZkgZ days, preferably about 1 to 30 mgZkgZ days, and the amount of gimeracil is about 0.1 to about LOOmgZkgZ days, preferably 1 to 50 mgZkgZ.
  • the amount of oteracil potassium is about 0.1 to about LOOmgZkgZ days, preferably about 1 to 40 mgZkgZ days.
  • These preparations of the present invention can be administered once a day or divided into about 2 to 4 times a day.
  • the amount of tegafur is about 1 to about LOOmgZkg, inserted once or twice a day at intervals of 6 to 12 hours and administered.
  • radiation therapy is cancer treatment by irradiating an affected area with radiation such as X-rays.
  • Radiation irradiation conditions can be appropriately selected depending on age, sex, degree of disease, and the like. For example, radiation therapy is performed 20-30 times (about 4-6 weeks) in one course. Can be set to 40-60 Gy for the entire network.
  • the irradiation site is not particularly limited as long as it contains a tumor tissue, but it is preferable to perform local irradiation on the tumor site.
  • the subject of the combination therapy of the antitumor agent and radiation is a cancer patient.
  • the types of cancer are not particularly limited, for example, head and neck cancer, stomach cancer, colon cancer, rectal cancer, liver cancer, gallbladder's bile duct cancer, spleen cancer, lung cancer, breast cancer, bladder cancer, prostate cancer, cervical cancer, etc. Is mentioned.
  • lung cancer is particularly preferred, with head and neck cancer, lung cancer and spleen cancer being preferred.
  • Conditions for performing the above-described combination therapy with an antitumor agent and radiation can be appropriately set depending on conditions such as age, sex, and degree of disease.
  • combination therapy sensitivity means individual differences in therapeutic effects in cancer patients who have undergone combination therapy. In other words, when a high therapeutic effect is obtained by combination therapy, the sensitivity is high. On the other hand, when a sufficient therapeutic effect is not obtained or no therapeutic effect is obtained, the sensitivity is low. .
  • Diagnosis of sensitivity to combination therapy means evaluation, identification, detection, judgment, and prediction of the presence / absence or level (level) of sensitivity to the above combination therapy based on some criteria.
  • the gene marker means a genetic index or predisposing factor for determining the sensitivity of a living body. That is, in the present invention, the gene marker for diagnosing susceptibility to a combination therapy of the above-described antitumor agent and radiation means that, as a result of the combination therapy, the expression level of the gene changes in response to sensitivity, etc. It indicates a genetic indicator or predisposing factor.
  • the present invention relates to a gene marker for diagnosing the sensitivity of such a combination therapy of a predetermined antitumor agent and radiation, and a polynucleotide (gene marker polynucleotide) functioning as the marker, Polynucleotides encoded by nucleotides, antibodies that recognize the polypeptides, polynucleotides that can serve as primers or probes for genetic marker polynucleotides (probes or polynucleotides for primers), and predetermined antitumors using these It provides a method for diagnosing susceptibility to combination therapy of drugs and radiation. These will be described in detail below. [0028] [1] Gene marker polynucleotide
  • the present invention first provides a novel polynucleotide.
  • the polynucleotide of the present invention is useful as a polynucleotide that functions as a genetic marker for diagnosing the sensitivity of a combination therapy of tegafur, gimeracil, and oteracil potassium, and a combination therapy with radiation (hereinafter referred to as a gene marker polynucleotide). .
  • the polynucleotide of the present invention can function as a genetic marker for diagnosing the sensitivity of a combination therapy of the antitumor agent and radiation. This is for patients whose expression level of each polynucleotide is sensitive to the above combination therapy of antitumor agents and radiation! This is due to the fact that it has a property that changes specifically before and after the combination therapy is performed. That is, the expression level of the polynucleotide of the present invention changes only in cancer patients having sensitivity to combination therapy and only when the combination therapy is applied.
  • the change in the expression level when the combination therapy is performed includes both an increase and a decrease in the expression level. That is, as a first example, if the expression level of the gene marker polynucleotide increases after the combination therapy compared to before the implementation, it is diagnosed that the combination therapy sensitivity is high when the expression level increases, and the expression level It is diagnosed that the combination therapy sensitivity is low (no change in the expression level). On the other hand, as a second example, when the expression level of the gene marker polynucleotide is decreased or not expressed at all after the combination therapy, it is diagnosed that the combination therapy sensitivity is high when it is decreased or not expressed at all. Conversely, the expression level will not decrease! / ⁇ (No change in expression level), low sensitivity to combination therapy! Diagnosed as sputum. Changes in the expression level after combination therapy can be confirmed by quantitative PCR.
  • Such changes in expression level differ depending on the base sequence constituting the marker, and even if the same marker is used, the expression level varies depending on the expression conditions, the target organism, the cancer type, etc. There are things to do.
  • RNA or DNA there is no particular limitation on whether it is RNA or DNA, its base length, whether it is single-stranded or double-stranded, circular or linear.
  • DNA it does not matter whether it is cDNA, genomic DNA, or synthetic DNA.
  • RNA it does not matter whether it is total RNA, mRNA, rRNA, or synthetic RNA.
  • a single strand it may be a sense strand or an antisense strand. It may be a double-stranded DNA, a double-stranded RNA, or a DNA: RNA hybrid.
  • the functional region is different, and may include, for example, an expression control region, a coding region, an exon, or an intron.
  • polynucleotide of the present invention include those represented by the following (A) to (D).
  • (C) a gene comprising a nucleotide sequence that can be hybridized under a highly stringent condition with the polynucleotide described in (i) or (ii) above, and a gene for diagnosing susceptibility to a combination therapy of the antitumor agent and radiation Polynucleotide that functions as a marker
  • the polynucleotide comprises one or more bases deleted, substituted, inserted, and inserted or added, and the antitumor agent and Polynucleotides that function as genetic markers for diagnosing susceptibility to combination therapy with radiation
  • the above (i) polynucleotide includes a base sequence described in any one of SEQ ID NOs: 1 to 70 in the sequence listing, and may comprise only the base sequence.
  • these polynucleotides are derived from humans by analyzing the sequence data from among those whose expression level specifically changes before and after the combination therapy with the antitumor agent and radiation. It has been confirmed that it is new.
  • SEQ ID NOs: 1 to 7, 9 to 15, 17 to 21, and 23 to 36 are preferable because they are easy to detect as markers because they are excellent in specificity in quantitative PCR.
  • polynucleotides containing the nucleotide sequences set forth in SEQ ID NOs: 1 to 33 in the Sequence Listing are selected as polynucleotides that may have the property of increasing the expression level when sensitivity to combination therapy is high (if present). It has been done.
  • a polynucleotide containing the nucleotide sequence of SEQ ID NOs: 34 to 36 may have a property that the expression level is reduced or not expressed at all when the sensitivity to combination therapy is high (existing). Selected as a nucleotide.
  • nucleotide sequences described in SEQ ID NOs: 37 to 70 are mRNA sequences predicted from the nucleotide sequences described in SEQ ID NOs: 1 to 9, 11 to 24, and 26 to 36, respectively, of the above. It is the base sequence expressed as NA.
  • the polynucleotide (B) includes a base sequence complementary to the polynucleotide described in (A). That is, it may be included in a part of the base sequence complementary to the polynucleotide described in (A) above, or only the complementary base sequence that is strong may be used.
  • the polynucleotide (C) includes a base sequence that hybridizes with the polynucleotide described in (A) or (B) under a highly stringent condition, and diagnoses the sensitivity to the combined treatment of the antitumor agent and radiation. It functions as a genetic marker for this purpose.
  • Nobbreviation can be performed according to a conventional method. Moreover, when using a commercially available library, it can be performed according to the method described in the attached instruction manual.
  • the stringent conditions are, for example, conditions in which a so-called specific hybrid is formed and a non-specific hybrid is not formed.
  • 1 X SSC is a condition washing ⁇ of typical Southern hybrida Izeshiyon, 0. 1 0/0 SDS, preferably ⁇ or 0. 1 XS SC, salt equivalent to 0. 1% SDS concentration
  • the condition for maintaining the hybridized state even after washing with (1) is mentioned.
  • C polynucleotide examples include, for example, at least 70%, preferably 90%, more preferably 95%, particularly preferably, the polynucleotides described in (A) and (B) above.
  • the base sequence homology can be calculated by homology search, sequence alignment program, BLAST, FASTA, ClustalW, or the like.
  • the polynucleotide described in (A) or (B) above has a nucleotide sequence in which one or more bases are deleted, substituted, inserted, and Z or added. In addition, it functions as a gene marker for diagnosing the sensitivity of the above antitumor agent and radiation therapy.
  • the number of bases that may be deleted, substituted, inserted, and Z or added is one or more, for example, 1 to: about L00, preferably about 1 to 30, more preferably 1 to About 10, more preferably 1 to 5.
  • Deletions, substitutions, insertions, and Z or additions The position of the base is not particularly limited.
  • (D) polynucleotide specifically, for example, at least 70%, preferably 90%, more preferably 95%, particularly preferably 97% with the polynucleotide described in (A) or (B) above.
  • a polynucleotide comprising a base sequence having homology can be calculated in the same manner as described in (C) above.
  • Such a gene marker polynucleotide of the present invention has a function as a gene marker by being targeted for detection of a change in expression level before and after the combination therapy of the antitumor agent and radiation. Demonstrate. To determine the change in the expression level, the expression level in the subject before the combination therapy or the expression level in another patient who is insensitive or sensitive to the combination therapy is used as a standard. This can be done by calculating the statistical significance of these.
  • the present invention also provides a diagnostic method for susceptibility to the above-mentioned combination therapy, characterized by examining the presence or absence of such a gene marker polynucleotide, the details of which are as described in the following [5]. .
  • detection of the expression level of the gene marker polynucleotide of the present invention is performed by collecting a biological sample from a subject, and expressing the gene marker polynucleotide expression product in the biological sample, for example, RNA or complementary thereto. This can be done by measuring the amount of DNA. In such detection, it is convenient to use a primer or probe designed based on the gene marker polynucleotide.
  • the present invention also provides a polynucleotide useful as a primer or probe useful for such sensitivity diagnosis, and the contents thereof are as described in detail in the following [2].
  • the detection of the expression level of the gene marker polynucleotide of the present invention involves measuring the amount of the polypeptide encoded by the gene marker polynucleotide prepared from the biological sample collected from the subject. This is also feasible. In such detection, it is convenient to use an antibody that recognizes the polypeptide.
  • the present invention also provides polypeptides and antibodies useful for such sensitivity diagnosis, the contents of which are as described in detail in the following [3].
  • the gene marker polynucleotide of this invention can be obtained by a conventional method. For example, it can be amplified by PCR using the primers detailed in [2] below.
  • the present invention relates to a probe or primer of the gene marker polynucleotide of the present invention described in [1] above in diagnosing the sensitivity of a combination therapy of tegafur 'gimeracil' oteracil potassium-containing antitumor agent and radiation.
  • a polynucleotide (hereinafter, referred to as a probe or primer polynucleotide) to be used as a DNA.
  • the probe or primer polynucleotide of the present invention is not particularly limited as long as it can detect or amplify the gene marker polynucleotide of the present invention of [1] above.
  • Southern hybridization DNA Any probe that can serve as a probe for a chip or a primer for PCR such as RT-PCR may be used.
  • a polynucleotide that can specifically bind to all or part of the gene marker polynucleotide is preferable.
  • its form there is no particular limitation on whether it is RNA force DNA, its base length, single-stranded or double-stranded, circular or linear. In the case of DNA, it does not matter whether it is cDNA, genomic DNA, or synthetic DNA.
  • RNA it does not matter whether total RNA, mRNA, rRNA, or synthetic RNA. Furthermore, in the case of a single strand, it may be a sense strand or an antisense strand. good. Further, it does not ask whether the functional region is different, and may include, for example, an expression control region, a coding region, an exon, or an intron.
  • the probe or primer polynucleotide of the present invention can be designed from the gene marker polynucleotide of the present invention of [1] above.
  • a polynucleotide having at least 10 bases or more among target polynucleotides of the gene marker polynucleotide of the present invention [1] can be used.
  • the amplification product when used as a primer, for example, when PCR is used for gene amplification.
  • the amplification product may be designed as a pair of polynucleotides containing at least 10 base pairs of the gene marker polynucleotide of the present invention of [1] above.
  • the other primer is It is only necessary to select an arbitrary sequence force 5 ′ from the sequence complementary to the primer in the complementary strand of the target polynucleotide.
  • the base length of the primer is not particularly limited as long as it can specifically bind to the gene marker polynucleotide of the present invention of [1] above, but is preferably a nucleic acid fragment of 10 to 150 bases.
  • a polynucleotide comprising at least 10 consecutive nucleotides in the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 70 in the Sequence Listing
  • polynucleotide according to (1) or (2) above which comprises a nucleotide sequence in which one or more bases are missing, substituted, inserted, and Z or added, and in the above [1] Polynucleotides that function as probes or primers for the described genetic marker polynucleotides
  • the polynucleotide (1) includes at least 10 consecutive nucleotides in the nucleotide sequence set forth in any one of SEQ ID NOS: 1 to 70 in the Sequence Listing.
  • the above (1) polynucleotide when used as a primer, it preferably contains 10 to 150 bases, especially 10 to LOO bases, particularly 15 to 50 bases.
  • the above (1) polynucleotide when used as a probe, it preferably contains 10 bases or more, more preferably 20 to 300 bases.
  • the polynucleotide (2) includes a base sequence complementary to the polynucleotide described in (1). That is, it may be included in a part of the base sequence complementary to the polynucleotide described in the above (1), or only a powerful complementary base sequence may be used. There may be.
  • the polynucleotide (2) when used as a primer, it preferably contains 10 to 150 bases, especially 10 to LOO bases, particularly 15 to 50 bases.
  • when used as a probe it preferably contains 10 bases or more, more preferably 20 to 300 bases.
  • the polynucleotide (3) includes a nucleotide sequence that hybridizes with the polynucleotide described in (1) or (2) under high stringency conditions, and the gene marker polynucleotide described in [1] above It functions as a probe or primer.
  • polynucleotide specifically, for example, the polynucleotide described in (1) and (2) is at least 70%, preferably 90%, more preferably 95%, particularly preferably 97%.
  • examples thereof include polynucleotides consisting of nucleotide sequences having the same homology.
  • the base sequence homology can be calculated in the same manner as described in [1] above.
  • the polynucleotide (3) when used as a primer, it preferably contains 10 to 150 bases, especially 10 to L00 bases, particularly 15 to 50 bases.
  • it when used as a probe, it preferably contains 10 bases or more, more preferably 20 to 300 bases.
  • the polynucleotide includes the base sequence in which one or more bases are deleted, substituted, inserted, and Z or added in the polynucleotide according to (1) or (2), and It functions as a probe or primer of the gene marker polynucleotide described in [1] above.
  • the number of bases that may be deleted, substituted, inserted, and Z or added is preferably 1 or more, more preferably 1 to: L0, and further preferably 1 to 5.
  • Specific examples of such (4) polynucleotide include, for example, at least 70%, preferably 90%, more preferably 95%, and particularly preferably 97% of the polynucleotide described in (1) and (2). Examples thereof include a polynucleotide having homology and a base sequence ability. salt The base sequence homology can be calculated in the same manner as described in [1] above.
  • the polynucleotide (4) when used as a primer, it preferably contains 10 to 150 bases, especially 10 to LOO bases, particularly 15 to 50 bases.
  • the polynucleotide (4) when used as a probe, it preferably contains 10 bases or more, more preferably 20 to 300 bases.
  • primer or probe polynucleotide of the present invention include a polynucleotide comprising the base sequence set forth in any of SEQ ID NOs: 71 to 142.
  • polynucleotides having the nucleotide sequences shown in SEQ ID NOs: 71 to 142 as described below are useful as probes and primers for the above gene marker polynucleotides, particularly polynucleotides containing the nucleotide sequences described in SEQ ID NOs: 1 to 36. Yes, preferably useful as a primer.
  • Polynucleotide fOla (SEQ ID NO: 71), rOla (SEQ ID NO: 72); polynucleotide f 02a (SEQ ID NO: 73), r02a (SEQ ID NO: 74); polynucleotide f03a (SEQ ID NO: 75), r03a (SEQ ID NO: 76); Polynucleotide f04b (SEQ ID NO: 77), r04b (SEQ ID NO: 78); Polynucleotide f05a (SEQ ID NO: 79), r05a (SEQ ID NO: 80); Polynucleotide f 06a (SEQ ID NO: 81), r06a (SEQ ID NO: 82); Poly Nucleotides f 07a (SEQ ID NO: 83), r07a (SEQ ID NO: 84); Polynucleotides f08b (SEQ ID NO: 85), r08b (SEQ ID
  • polynucleotide f 13a (SEQ ID No. 95), r 13a (SEQ ID No.) 96); polynucleotide f 14a (SEQ ID NO: 97), r 14a (SEQ ID NO: 98); fl5 b (SEQ ID NO: 99), rl5b (SEQ ID NO: 100); polynucleotide f 16a (SEQ ID NO: 101), rl6 a (sequence) No.
  • polynucleotide fl7a (SEQ ID NO: 103), rl7a (SEQ ID NO: 104); Polynucleotide f 18a (SEQ ID NO: 105), rl8a (SEQ ID NO: 106); Polynucleotide f 19a (SEQ ID NO: 107), r 19a (SEQ ID NO: 108); polynucleotide f 20a (SEQ ID NO: 109), r20 a (SEQ ID NO: 110); polynucleotide f21a (SEQ ID NO: 111), r21a (SEQ ID NO: 112); polynucleotide f 22b (SEQ ID NO: 113) R22b (SEQ ID NO: 114); polynucleotide f23b ( SEQ ID NO: 115), r23a (SEQ ID NO: 116); polynucleotide f 24a (SEQ ID NO: 117),
  • Each of these polynucleotides was designed from the gene marker polynucleotides of SEQ ID NOs corresponding to the numbers in the names of the polynucleotides among the gene-powered polynucleotides containing the nucleotide sequences set forth in SEQ ID NOS: 1-36. Is.
  • Each polynucleotide name containing “f” can be used as a forward primer, and “r” can be used as a reverse primer! That is, each polynucleotide can be used as a primer for a corresponding gene marker polynucleotide as a primer pair for each semicolon segment in the above list.
  • the primer was designed in the same manner as the nucleotide sequence set forth in SEQ ID NO: 71-142 from the nucleotide sequence set forth in SEQ ID NO: 1-36. Polynucleotides can be designed.
  • Such a primer or probe polynucleotide of the present invention is useful for the synthesis and detection of the expression level of the gene marker polynucleotide described in [1] above. It contributes to quick and accurate diagnosis of sensitivity.
  • the present invention provides such a method for susceptibility diagnosis, the contents of which will be described later. As described in [5].
  • primer or probe polynucleotide of the present invention in a sensitive diagnostic kit, it is possible to more easily diagnose the sensitivity of the combination therapy of the antitumor agent and radiation.
  • the present invention also provides diagnostic agents and kits for such sensitivity diagnosis, and diagnostic methods, the contents of which are as described in [4] below.
  • the present invention provides a polypeptide encoded by the gene marker polynucleotide described in [1] above, and an antibody that recognizes the polypeptide. As long as it is encoded by the gene marker polynucleotide described in [1] above, it is indicated by a specific amino acid sequence corresponding to the polynucleotide (A) to (D) described in [1] above. In addition to polypeptides, mutants, derivatives, mature forms and amino acid modified forms thereof are included on the condition that they exhibit substantially the same function as the polypeptide. Variants include naturally occurring allelic variants, non-naturally occurring variants, and variants having amino acid sequences modified by artificial deletion, addition, or substitution.
  • mutants include those that are at least 70%, preferably 80%, more preferably 95%, and even more preferably 97% homologous to a polypeptide having no mutation.
  • the amino acid modifications include naturally occurring amino acid modifications and non-naturally occurring amino acid modifications, and specifically include phosphorylated amino acids.
  • the polypeptide of the present invention can be obtained by a conventional method based on the gene marker polynucleotide described in [1] above.
  • the polynucleotide can be cloned, ligated into a vector plasmid, transformed into a host cell such as Escherichia coli, and the resulting transformed cell is cultured and recovered from the culture. .
  • human cells such as test subjects or tissue power can also be purified and produced by a conventional method. For example, after homogenizing a subject's tissue or cells, extraction with an acid or the like is performed, and the extract is chromatographed such as reverse phase chromatography or ion exchange chromatography. It can be purified and isolated by combining fees.
  • the polypeptide of the present invention is a polypeptide encoded by the genetic marker polynucleotide described in [1] above, the genetic marker polynucleotide prepared by subject test force is collected.
  • the expression level can be detected by measuring the amount of the polypeptide encoded by. In other words, if the expression level of the polypeptide encoded by the gene marker polynucleotide increases after the combination therapy than before the combination therapy, it is diagnosed that the sensitivity of the combination therapy is higher and the amount of the polypeptide increases. Diagnosis of low sensitivity to combination therapy when there is no increase (no change).
  • the expression level of the polypeptide encoded by the gene marker polynucleotide decreases or does not express at all after the combination therapy, it is diagnosed that the sensitivity of the combination therapy is high when it decreases or does not express at all, and conversely decreases. Is not confirmed (no change in the expression level), the sensitivity to combination therapy is low!
  • the form of the antibody of the present invention is not particularly limited, and may be V of the above-described polypeptide of the present invention, monoclonal antibody having any of the antigens as antigens, or polyclonal antibody! Further, it may be an antibody having an antigen binding property to a polypeptide having a partial amino acid sequence having at least 10 consecutive amino acids, particularly 15 amino acids, particularly 20 amino acids in the amino acid sequence of the polynucleotide of the present invention.
  • the antibody can be produced according to a conventional method.
  • a polyclonal antibody an experimental animal is immunized with the above-mentioned polypeptide expressed and purified in Escherichia coli according to a conventional method, or with a polypeptide synthesized with these partial amino acid sequences according to a conventional method.
  • it can be obtained from the serum of the immunized animal according to a conventional method.
  • the polypeptide can be used to immunize experimental animals, and spleen cells obtained from the experimental animals can be fused with osteomyeloma cells to synthesize hyperpridoma cells and obtained from the cells.
  • the polypeptide and antibody of the present invention contribute to rapid and accurate diagnosis of the combination therapy sensitivity of the above-described antitumor agent and radiation, and are used as diagnostic drugs and diagnostic kits for combination therapy sensitivity. It can be used widely.
  • the present invention provides a method for susceptibility diagnosis using such an antibody, the contents of which are as described in [5] below.
  • the antibody of the present invention in a sensitivity diagnostic agent or kit, it is possible to more easily diagnose the sensitivity of the above-described combination therapy with the antitumor agent and radiation.
  • the present invention also provides diagnostic agents and kits for such sensitivity diagnosis, and diagnostic methods, the contents of which are as described in [4] below.
  • the present invention provides a diagnostic agent characterized by comprising at least the polynucleotide for primer or probe described in [2] above, or the antibody described in [3] above, and a diagnostic kit. Is.
  • the diagnostic agent and diagnostic kit of the present invention contain at least the primer or probe polynucleotide described in [2] above, or the antibody described in [3] above, and are known diagnostics.
  • An appropriate dosage form can be obtained according to a conventional method which may contain reagents used for the preparation of biological samples from subjects and detection of amplification products.
  • the above combination therapy of the subject is detected by detecting the gene marker polynucleotide contained in the biological sample of the subject or the polypeptide encoded by the polynucleotide. Can be diagnosed.
  • the present invention is characterized by examining the presence or absence of the gene marker polynucleotide described in [1] above or the polypeptide described in [3] above in a biological sample collected from a subject.
  • the present invention also provides an antitumor agent containing tegafur, gimeracil and oteracil potassium, and a method for diagnosing radiation therapy sensitivity.
  • the subject may be a cancer patient who has already undergone the above-described combination therapy of an antitumor agent and radiation, or a cancer patient who has not been performed.
  • the type of cancer is not particularly limited as long as it is a cancer type that can be treated by the above-described combination therapy with an antitumor agent and radiation.
  • head and neck cancer, stomach cancer, colon cancer, rectal cancer, liver cancer, gallbladder, bile duct cancer examples include spleen cancer, lung cancer, breast cancer, bladder cancer, prostate cancer, cervical cancer, and the like, and lung cancer, in which head and neck cancer, lung cancer, and spleen cancer are preferred.
  • the biological sample is not particularly limited as long as it is a subject's own sample and may contain a tumor.
  • Body fluid blood, urine, etc.
  • tissue extract thereof, culture of collected tissue, etc.
  • blood, particularly serum is preferable.
  • a method for collecting a biological sample can be appropriately selected according to a method according to the biological sample or the cancer type.
  • the biological sample applied to the diagnostic method of the present invention may be a cancer sample that has already been collected from cancer patients who have already undergone the above-mentioned combination therapy of antitumor agent and radiation. What was collected from the patient's body may have been subjected to the combination therapy of the above-mentioned antitumor agent and radiation.
  • the time when the biological sample is collected when speaking is preferably the time when the effect of the combination therapy is expected to be exerted. Usually, it is 30 minutes to 5 hours, preferably 1 hour to 3 hours after the combination therapy.
  • the presence or absence of the gene marker polynucleotide described in [1] above or the presence or absence of the polypeptide described in [3] above is examined.
  • the presence or absence of the gene marker polynucleotide described in [1] above is set as the detection target will be described below.
  • step (II) Step of measuring the presence or absence of RNA or complementary polynucleotide bound to the polynucleotide in step (I) using the above-described probe or primer polynucleotide as an indicator.
  • step (I) RNA prepared from a biological sample collected from a subject or a complementary polynucleotide transcribed from the RNA and the above-described probe or primer polynucleotide are combined.
  • RNA from the biological sample and preparation of a complementary polynucleotide transcribed from RNA can be performed by conventional methods.
  • RNA to the probe or primer polynucleotide can be performed by a conventional method in consideration of the conditions where all or a part of both can be easily bound.
  • step (II) in step (I), the presence of the RNA or complementary polynucleotide bound to the probe or primer polynucleotide is determined using the above-described probe or primer polynucleotide. Measure as an indicator.
  • the probe or primer polynucleotide is labeled with a labeling substance such as a radioisotope or a fluorescent substance in advance and then bound to RNA or a complementary polynucleotide. Signals derived from substances can be measured with radiation detectors or fluorescence detectors.
  • Such a diagnostic method of the present invention can be performed using Northern blotting, RT-PCR, DNA chip analysis, in situ hybridization, and the like.
  • the above-described probe or primer polynucleotide is used as a probe, and the strength of the biological sample collected by the subject is determined whether the gene marker polynucleotide is expressed in the prepared RNA, Its expression level can be detected.
  • RNA prepared by a subject is transferred to a nylon membrane or the like, and labeled with the probe in advance and hybridized with the probe (step (1)). Subsequently, the presence or absence of a double-stranded polynucleotide and RNA is measured using the signal derived from the probe label as an index (step (ii)). In addition, it is also possible to carry out according to the protocol of the kit using a commercially available kit for Northern blotting.
  • the gene marker polynucleotide in cDNA prepared from R R prepared from a biological sample collected from a subject using the above-described probe or primer polynucleotide as a primer. The presence or absence of nucleotide expression and its expression level can be detected.
  • cDNA prepared from RNA derived from a subject is used as a cage, the above primer is hybridized with this, and PCR is performed according to a conventional method (corresponding to step (I)). Amplified double-stranded DNA can be detected (corresponding to step (ii)). It is also possible to use a commercially available RT-PCR kit according to the protocol of the kit.
  • RNA prepared by a subject is transferred to a nylon membrane or the like, and labeled with the probe in advance and hybridized with the probe (step (1)). Subsequently, the presence or absence of the double-stranded polynucleotide and RNA formed is measured using the signal derived from the probe label as an index (step (II)).
  • step (III) the antitumor agent and the radiation are obtained from the measurement result of the step (II). Diagnose sensitivity of combination therapy. As a result of the measurement, diagnosis of sensitivity to combination therapy
  • the measured RNA or complementary polynucleotide measured in (II) is the same as the RNA or complementary polynucleotide measured in the same manner in other subjects who are insensitive or sensitive to the combination therapy before or after the combination therapy. Nucleotide measurement values and similar measurement values can be used as a reference by calculating numerical values in advance and comparing them to calculating statistical significance.
  • the combination therapy sensitivity is high.
  • no increase in the expression level is confirmed! (No change in the expression level)
  • a gene marker polynucleotide whose expression level decreases when combined therapy is used as an indicator
  • the decrease in the expression level is not confirmed! In the case of (no change in the expression level), the sensitivity to combination therapy is diagnosed as low.
  • EIA enzyme immunoassay
  • RIA radioisotope Immunological detection methods
  • fluorescent immunization fluorescent immunization
  • the immune reaction between the antibody and the polypeptide or partial peptide may be either a competitive method or a sandwich method.
  • the sandwich method particularly an ELISA using a solid phase antigen or a solid phase antibody, is preferred.
  • Examples of the diagnostic method include (i) to (iii).
  • step (ii) Step of measuring the presence or absence of the polypeptide bound to the antibody or its partial peptide in step (i) using the antibody as an index
  • step (iii) Step of diagnosing the sensitivity of the above-mentioned antitumor agent and radiation combination therapy from the measurement result of step (ii)
  • the prepared biological sample force is collected from the subject force, and the prepared polypeptide and the antibody are combined.
  • the preparation of the biological sample strength polypeptide can be carried out by appropriately combining known fractionation and purification methods.
  • step (ii) in step (i), the presence or absence of a polypeptide bound to the antibody or a partial peptide thereof is measured using the antibody as an index.
  • the antibody is preliminarily labeled with a labeling substance such as a radioisotope or fluorescent substance, and the force is bound to the polypeptide to detect the signal derived from the labeling substance. This can be done by measuring with a detector or a fluorescence detector.
  • step (iii) the sensitivity of the combination therapy with the antitumor agent and radiation is diagnosed from the measurement result of step (ii). Measured results
  • the diagnosis of the sensitivity to combination therapy is as follows: @ ( ⁇ ) is used to determine the measured value of the polypeptide before subjecting the combination therapy to the subject or for other insensitivity or sensitivity to the combination therapy. It can be carried out by calculating a statistically significant difference by comparing a measured value of a polypeptide measured in the same manner or a similar measured value in advance with a numerical value as a reference.
  • the combination therapy is highly sensitive.
  • the increase in the expression level is not confirmed (no change in the expression level)
  • the polypeptide of [3] above whose expression level decreases when combined therapy is used as an index
  • the polypeptide of [3] above whose expression level decreases when combined therapy is used as an index
  • the expression level decreases or is not expressed at all.
  • the decrease in the expression level is not confirmed! ⁇ ⁇
  • the combination therapy sensitivity is low! Diagnosed as sputum.
  • the diagnostic method of the present invention By carrying out the diagnostic method of the present invention, the sensitivity of the combination therapy in a subject can be diagnosed, whereby the continuity and Z or feasibility of the combination therapy can be predicted.
  • the power to use the above combination therapy further depends on the sensitivity of the combination therapy obtained by the diagnostic method of the present invention. I can judge.
  • the above combination therapy when the above combination therapy is not performed in the subject, whether or not the above combination therapy should be performed can be predicted from the combination therapy sensitivity obtained by the diagnostic method of the present invention.
  • the patient By providing the above combination therapy only to a patient who is highly likely to continue and Z or feasible, the patient can receive appropriate treatment, and thus the treatment efficiency in the overall medical care and Economic power is also useful.
  • the S-1 drug solution used as the test solution for the following tests was prepared as follows. That is, first, tegafur, gimeracil, and potassium oxalate were suspended in a 0.5% hydroxypropyl methylcellulose (HPMC) solution at 0.83 mg / mU 0.25 mgZml and 0.82 mgZml, respectively, and at about room temperature. Stir for 10 minutes. Then, it was sonicated under ice-cooling to obtain a dose of 8.3 mg ZkgZday S-1 drug solution. This dose of S-1 is the maximum non-toxic dose when orally administered to mice for 14 days.
  • HPMC hydroxypropyl methylcellulose
  • irradiation position Using an MBR-1505R2 radiation irradiation device manufactured by Hitachi Medical Corporation, set the irradiation conditions (irradiation position) so that one irradiation per mouse is 2 Gy and 5 Gy, and transplanted to the right thigh of the mouse Local irradiation was performed on the tumor lines. Avoid whole-body irradiation during irradiation. In order to avoid this, the mouse was placed in a storage box made of lead so that only the right foot was exposed to radiation.
  • Human lung cancer strains (LC-11 and Lu-99) were isolated by transplanting to the right thigh of BALBZcA-nu mice aged 5-6 weeks under the skin of the back of mice of the same strain in advance. A piece of scissors approximately 2 mm square in physiological saline was transplanted subcutaneously using a transplant needle and bred for at least 1 to 2 weeks. After that, each group (6 mice per group) was divided into 4 groups so that the mean and standard deviation (SD) of the tumor volume were as uniform as possible, and then each was divided into the control group and the radiation alone group. (X ray), drug alone group (S-1) and drug and radiation combination group (S-1 + X my), and further divided into 3 groups or 4 groups depending on the treatment period. The group began drug administration and radiation. Table 1 shows the relationship between the strains derived from each of the 30 specimen groups, treatment details, and treatment periods in “ ⁇ ”.
  • the above-mentioned S-1 drug solution was used at a rate of 8.3 ml per 1 kg body weight, once a day, using an oral sonde for the treatment period shown in Table 1. Orally administered.
  • the method described under the above irradiation conditions within about 1 hour after administration of the S-1 drug. 2Gy was applied according to the above.
  • tumor tissues were collected from each group on the second, ninth, twelfth, and fifteenth days of the start of the study, and the origin strains, treatment details, and treatment periods shown in Table 1 were collected. Thirty kinds of specimens classified separately were used for the following processes.
  • RNA was extracted from each group to prepare polyA + RNA.
  • mixed groups were prepared from the 30 sample groups according to the treatment method, and the Control group (non-radiation irradiation, S-1 non-administration), S-1 treatment group (non-irradiation, S-1 administration), and radiation treatment
  • the Control group non-radiation irradiation, S-1 non-administration
  • S-1 treatment group non-irradiation, S-1 administration
  • radiation treatment Four cDNA libraries were prepared for the group (irradiation, S-1 non-administration) and the combination treatment group (irradiation, S-1 administration).
  • RNA sample total RNA
  • RNA sample was treated with Proteinase K. That is, proteinase K (l nvitrogen) was added to 1 mg of RNA sample and reacted at 37 ° C for 1 hour to inactivate RNase and then heated to 60 ° C for 10 minutes. Proteinase K was inactivated.
  • proteinase K l nvitrogen
  • RNA sample obtained by phenol treatment and ethanol precipitation was further subjected to the following steps.
  • the sample obtained in step 2.3 was treated with a DNA-degrading enzyme (DNase I: Takara Bio). That is, 1 unit of DNase I was added to 1 mg of the purified RNA sample and reacted at 37 ° C for 30 minutes. After completion of the reaction, RNA was recovered by phenol treatment and ethanol precipitation.
  • DNase I DNA-degrading enzyme
  • RNA thus recovered was subjected to the following steps as a DNase I-treated total RNA sample.
  • polyA + RNA was extracted from the DNase I-treated total RNA sample obtained in step 2.4.
  • Rigo dT used Oligotex—dT30 (Takara Bio) to absorb and extract mRNA. The operation procedure was performed according to the protocol of the product. The mRNA was purified by ethanol precipitation after extraction.
  • PolyA + RNA which has obtained the strength of 30 sample groups (see Table 1), is treated with Control, S-1 treatment, radiation treatment, S-1 and radiation treatment as shown in Table 2 “Mixed group”.
  • Control group S-1 treatment group
  • radiation treatment group S-1 and radiation treatment as shown in Table 2 “Mixed group”.
  • Table 2 shows the relationship between the 30 sample groups and the 4 mixed groups. Note that “ ⁇ ” in Table 2 indicates the correspondence of the derived strain, treatment content, and treatment period of each sample group. [0089] [Table 2]
  • First strand synthesis and cDNA library creation were performed using oligo dT primers using the polyA + RNA of each of the four mixed groups (Table 2) prepared in Step 2.5 as a template.
  • the following sequence of operations up to the creation of the cDNA library was performed using the cDNA Construction Kit (Takara Bio Inc.) according to the operation manual of the product.
  • the four cDNAs obtained from each of the four mixing groups were obtained.
  • the library was named as follows. That is, the Control group is called TH05C, the S-1 treatment group is TH05S, the radiation treatment group is TH05X, and the combination treatment group is TH05SX (see Figure 1).
  • This cDNA fragment was treated with restriction enzymes Not I (5 units) and EcoRI (5 units) at 37 ° C for 1 hour. After that, using a gel filtration column (Invitrogen, trade name: cDNA Size Fraction Column), size fractionation was performed according to the operation manual of the same product, and cDNA around 500 bp was extracted using electrophoresis and absorbance at 260 nm as indices. Further, ethanol precipitation was performed to purify a cDNA fragment having both ends cleaved with Not I and Eco RI (hereinafter referred to as a purified cDNA sample).
  • a gel filtration column Invitrogen, trade name: cDNA Size Fraction Column
  • pBlueScript II KS (—) (Stratagene) vector was treated with restriction enzymes Not I and EcoRI. After electrophoresis using 1% agarose, the vector was excised and purified.
  • Sequencing was performed on the clones derived from the four types of libraries obtained in Step 2 above. That is, after aligning E. coli clones from each library, PCR amplification is performed using a single vector primer to amplify the inserted cDNA fragment. did. Thereafter, the amplified cDNA fragment was purified, and a sequencing reaction was performed using this as a saddle shape, followed by sequencing.
  • agar plate obtained in step 2.9 colonies formed on the agar plate were colonized on the LB medium in advance using a colony pick device Q—PIX (Genetix). Inoculate and align. Shake overnight at 37 ° C. This is to reduce the difference between each well in the following work by suppressing the variation in growth among clones by arranging and re-culturing. Subsequent series of reactions were performed using 96 multiwell plates.
  • the culture solution in each well of the multiwell plate obtained in step 3.2 was heat-treated at 98 ° C. for 3 minutes to destroy the cell membrane of E. coli.
  • the plasmid DNA contained in the heat-treated solution (lysate solution) of the fungus is used as a saddle, and the primers (KS—F, KS-R) derived from the sequence near the multi-ring region of the pBlueScript II KS (—) vector are used.
  • PCR reaction was performed to amplify the inserted cDNA fragment.
  • PCR reaction solution composition fungus lysate solution; 10 ⁇ L, 10X PCR buffer; 5 L, primer; 10 pmol each, dNTPs; 20 nmol each, EX
  • Taq (Takara Bio Inc.); Sterilized water was added to 1 unit to make 50 ⁇ L.
  • the PCR thermal cycle was 94 ° C ⁇ 30 seconds ⁇ 60 ° C ⁇ 30 seconds ⁇ 70 ° C ⁇ 1 minute 30 times.
  • Sequencing reaction was performed using the cDNA fragment amplified and purified in step 3.3 as a cage.
  • BigDye terminator vl. 1 (Applied Biosystems) was used, and the reaction was carried out according to the operation manual of the product.
  • the sequence reaction product was purified using Sephadex G-50 (Amersham) according to the operation manual of the product.
  • TH05SX combination treatment group
  • TH05S S-1 treatment group
  • TH05C Control group
  • TH05X radiation treatment group
  • sequence data of each clone obtained in the above DNA sequencing section! First, a series of sequence cleaning consisting of quality trimming, vector trimming, cross-contamination confirmation (detection 'exclusion), etc. After processing, the inserted sequence was cut out (extracted). Next, sequence annotation is performed based on the obtained insertion sequence to determine whether each clone is a known gene or a new gene, and the derived gene species and the derived organism species (human or mouse). Of the difference).
  • Figure 2 shows the outline of the sequence annotation process.
  • a sequence cleaning process is performed, the sequence data output from the sequencer is processed for each clone obtained in the above DNA sequence section, and the cDNA fragment as the target of the sequence annotation process is extracted. It was. Since it is necessary to remove the cloning vector sequence in order to identify the cDN A fragment sequence, for example, Chou HH, Holmes MH .; DNA sequence quality trimming and vector removal. Bioinformatics. 2001 Dec; 17 (12): Refer to the description of 1093- 1104. Perform vector trimming or vector removal, quality trimming for removing low quality sequences, and detection and elimination of cross contamination. was also performed.
  • the unstable base sequences present near the beginning and end of the reading sequence were removed from both sides, and a highly accurate portion was extracted as a sequence.
  • sequence data that may contain a plurality of clones was detected and excluded at the stage of this processing, and excluded from the target of subsequent sequence annotation processing.
  • sequence annotation process was performed for the sequence data that was determined to have no quality problem by the following procedure for identifying the origin of genes and species.
  • human known gene mRNA sequence database and mouse known gene mRNA sequence database were used.
  • the known gene sequence databases used in this process are GenBank, RefSeq, and LocusLink of October 8, 2004.
  • cDNA fragment sequence data obtained in step 4.2 above a homology search using transcripts (mRNA) is performed on human known gene sequence data and mouse known gene sequence data available from NCBI. It was. The obtained search results were analyzed, and the gene corresponding to the sequence data of the cDNA fragment obtained in 4.2 was identified. Single gene at this stage It was judged that the sequence uniquely identified as a single species of the species could determine not only the gene but also the derived species, and the result was annotated.
  • mRNA transcripts
  • sequence data that was determined to be a clone of a human gene and not a known gene, that is, a clone as a candidate for a new gene, from each cDNA library.
  • step 4 Compared with mRNA registered in the public database in step 4 above, the ability to exclude sequence data of clones corresponding to known genes
  • the positions of known genes and clones in the public database at the genome level The position of the cluster of the sequence data is collated, the sequence data of the clone corresponding to the known gene is excluded, and new gene candidates are selected.
  • a clone having a homology of 98% or more between the clone sequence data and the genome sequence and mapped to only one locus site in one direction of one chromosome or mitochondria was extracted.
  • human genome data the database NCBI reference Build 35.1 (representative genome sequence of each chromosome and mitochondria) was used.
  • BLAST for analysis of mapping to genomic data, see McGinnis S., Madden T. L .; "BLAST: at the core of a powerful and diverse set of sequence analysis tools.
  • the position information on the genome of the obtained cluster can be predicted as the locus site of a new gene candidate.
  • the clone with the longest portion mapped to the genomic data is used as the representative sequence of the cluster.
  • Example 1 a confirmation experiment was conducted in which 36 clones (SEQ ID NOs: 1 to 36) obtained in practice could be actually amplified by the quantitative PCR method.
  • 5 g of DNase I-treated total RNA of Day 2 (second day from the start of the test) derived from human lung cancer cell line LC 11 used in Example 1 was used.
  • Table 1 On Day 2, no RNA preparation was performed on the xenograft sample in the S-1 treatment group, so the Dnase I treatment from each of the control group, radiation treatment group, and combination treatment group was completed. Total RNA 5 g was used.
  • Cells were cultured from the glycerol stock of each sample group obtained in Example 1, and plasmid DNA was extracted.
  • the plasmid DNA extraction reagent is Wizard plus Minpreps (Promega Was used.
  • the reagent was Oligo (dT) 12-18 primer (Invitrogen) 500 ng, Superscript II RT (Invitrogen) 200 U and the reaction was performed on a 20 ul scale.
  • primers fOla SEQ ID NO: 71
  • rOla SEQ ID NO: 72
  • primers f02a SEQ ID NO: 73
  • r02a SEQ ID NO: 74
  • Primers f03a SEQ ID NO: 75
  • r03a SEQ ID NO: 76
  • Primers f04b SEQ ID NO: 77
  • r04b SEQ ID NO: 78
  • Primer f 16a (SEQ ID NO: 101), rl6a (SEQ ID NO: 102) corresponding to the clone of SEQ ID NO: 16; Primer f 17a (SEQ ID NO: 103), rl7a (SEQ ID NO: 103) corresponding to the clone of SEQ ID NO: 17 104)
  • Primers f 18a (SEQ ID NO: 105), rl8a (SEQ ID NO: 106) corresponding to the clone of SEQ ID NO: 18; primers f 19a (SEQ ID NO: 107), rl9a (SEQ ID NO: 108) corresponding to the clone of SEQ ID NO: 19;
  • Primers f 20a (SEQ ID NO: 109), r20a (SEQ ID NO: 110) corresponding to the clone of SEQ ID NO: 20; primers f 21a (SEQ ID NO: 111), r21a (SEQ ID NO: 112) corresponding to the
  • the software parameters were set according to the protocol attached to TaKaRa Ex Taq R-PCR version 2.1 (Takarabio). From the candidates listed, primer pairs were selected in consideration of both the primer design guidelines recommended by Takara Neo and Applied Systems. The selected primers were subjected to a blast search and confirmed that no other gene sequences were amplified. Primer synthesis was requested from Invitrogen. The synthesized primer sequences are listed in Table 3. No. in Table 3 indicates the sequence number of the clone corresponding to each primer. [9 ⁇ 0]
  • the vertical type is a series of dilutions of plasmid DNA in 5 stages, ie, 10 pmolZul, lpmol / ul, 100 fmol / ul, 10 fmol / ul, lfmolZul, and 50 times the first strand cDNA (from the combination treatment group) Each diluted solution was used in lul. The reaction volume was 50 ul. The PCR program was 95 ° C, lmin ⁇ (95 ° C, 15sec ⁇ 60 ° Clmin) x 45cycles, and a default dissociation curve creation program was added. The primer pass / fail is determined by the dissociation curve.
  • SEQ ID NOs: 8, 16, and 22 had strong amplification of by-products and were unable to confirm specific amplification. This is because the clone of SEQ ID NO: 8 has a short sequence length of 197 bases obtained in the EST analysis of Example 1, and the clones of SEQ ID NO: 16 and 22 have many repeated sequences in the sequence. it is conceivable that. On the other hand, specific amplification was confirmed in 33 clones excluding SEQ ID NOs: 8, 16, and 22.
  • the reaction system was the same as the reaction system in the above-described primer specificity confirmation, except that the amount of the template DNA sample for the calibration curve was different from that of the first strand cDNA sample.
  • a calibration curve is prepared using samples obtained by serially diluting plasmid DNA in 6 stages, that is, samples containing 100 pmolZul, 10 pmol / ul, lpmol / ul, and 100 fmol / uU lOfmol / uU lfmol / ul.
  • the number of copies in the sample lul diluted 10-fold from the first strand cDNA was quantified.
  • GAPD Human GAPD
  • dalyceraldehyde 3-phosphate dehydrogenase was selected as a gene for the internal standard.
  • Primer for detecting this gene is Takarabio Used off-the-shelf products sold more.
  • the present inventors have confirmed in advance that DNA amplification does not occur when mouse first strand cDNA is subjected to PCR using this primer.
  • the rautoj setting was selected using the software supplied with the ABI7500 device.
  • the calibration curve was created and the number of copies in each sample was also calculated on the same software.
  • the copy number calculation formula is as follows, and the number of copies of double-stranded plasmid DNA is calculated.
  • the size of plasmid DNA actually varies from gene to gene, but was calculated as 3,500 bp (pbluescript II KS—: 3, OOObp, average length of insert: 500 bp).
  • GAPD [Calculation! Calculated as 4,700bp (pCR2.1: 4,200bp, insert: 500bp) only.
  • the number of moles of double-stranded DNA is the number of moles of double-stranded DNA.
  • Table 4 shows 33 clones (excluding SEQ ID Nos. 8, 16, and 22 of 36 clones) and internal standard of the control group, the radiation treatment group, and the combination treatment group of LC-11 on Day 2.
  • the double-stranded DNA copy number calculated for GAPD of the gene is shown.
  • Fig. 4 shows the ratio of the expression level in the control group to the expression level in the radiation treatment group and the combination treatment group.
  • Example 1 The clones selected in (1) were found to be useful as markers for sensitivity to combination therapy because their expression levels changed after combination therapy.
  • SEQ ID NOs: 37 to 70 these are sequences predicted from SEQ ID NOs: 1 to 9, 11 to 24, and 26 to 36. Therefore, if primers are designed in the same manner as the above clones, they are useful as markers. It can be fully analogized.
  • the present invention it is possible to quickly and accurately diagnose the sensitivity of combination therapy of tegafur / gimeracyl / oteracil potassium-containing antitumor agent and radiation. Therefore, early determination as to whether or not the combination therapy should be performed on cancer patients can be made early, and the efficiency of cancer treatment can be improved.

Abstract

It is directed to provide a means for rapid and accurate diagnosis of the sensitivity to a tegafur/gimeracil/oteracil potassium-containing anti-tumor agent or concurrent chemoradiation therapy. Thus, disclosed are: a polynucleotide which can serve as a genetic marker for diagnosis of the sensitivity to the anti-tumor agent or concurrent chemoradiation therapy; a polynucleotide which can serve as a probe or primer for the genetic marker polynucleotide; a polypeptide encoded by the genetic marker polynucleotide and an antibody capable of recognizing the polypeptide; and a diagnostic agent, a diagnosis kit and a method for diagnosis of the sensitivity to concurrent chemoradiation therapy which utilize the polynucleotide for the probe or primer or the antibody.

Description

抗腫瘍剤及び放射線併用療法感受性診断用遺伝子マーカー並びにそ の利用  Genetic markers for susceptibility diagnosis of antitumor agents and radiation therapy and their use
技術分野  Technical field
[0001] 本発明は、抗腫瘍剤及び放射線併用療法感受性診断用遺伝子マーカー並びに その利用に関し、詳しくは、がん患者等の被験者力 採取された生体試料中の特定 のポリヌクレオチドの有無を調べることを特徴とする、テガフール ·ギメラシル ·ォテラシ ルカリウム配合抗腫瘍剤及び放射線の併用療法に対して感受性を有するか否か、言 [0001] The present invention relates to a genetic marker for susceptibility diagnosis of an anti-tumor agent and radiation therapy, and more specifically, to examine the presence or absence of a specific polynucleotide in a biological sample collected from a subject such as a cancer patient. Whether or not it is sensitive to the combination therapy of tegafur, gimeracil and oteracil potassium combined antitumor agent and radiation
Vヽ換えれば、上記抗腫瘍剤及び放射線併用療法を施すことが被験者のがん治療に 有効か否かを迅速に診断するための遺伝子マーカーとして機能するポリヌクレオチド 、並びにその利用に関する。 In other words, the present invention relates to a polynucleotide that functions as a genetic marker for rapidly diagnosing whether or not the combination of the above-described antitumor agent and radiation therapy is effective for treating cancer in a subject, and use thereof.
背景技術  Background art
[0002] がん治療における化学療法においては、さまざまな抗腫瘍剤が開発されている。特 に、テガフール 'ギメラシル 'ォテラシルカリウム配合抗腫瘍剤 (特許文献 1〜4参照） は、いわゆるティーエスワン (TS— 1)、エスワン（S— 1)等として、癌、特に食道癌や 胃癌にぉ ヽて高 ヽ治癒率を誇り優れた抗腫瘍剤として広く利用されて!ヽる。  [0002] Various antitumor agents have been developed in chemotherapy for cancer treatment. In particular, anti-tumor agents containing tegafur 'gimeracil' oteracil potassium (see Patent Documents 1 to 4) are known as so-called TS-1 (TS-1), S-1 (S-1), etc. for cancer, particularly esophageal cancer and stomach cancer.ヽ It is widely used as an excellent antitumor agent with a high healing rate!
また、上記抗腫瘍剤を、放射線療法と組み合わせることによる優れた癌治療方法が 知られている (非特許文献 1)。  Further, an excellent cancer treatment method by combining the antitumor agent with radiation therapy is known (Non-patent Document 1).
[0003] 特許文献 1 :特公平 5— 64123号公報  [0003] Patent Document 1: Japanese Patent Publication No. 5-64123
特許文献 2：特公平 5 - 80451号公報  Patent Document 2: Japanese Patent Publication No. 5-80451
特許文献 3：特許第 2538422号公報  Patent Document 3: Japanese Patent No. 2538422
特許文献 4：特許第 2614164号公報  Patent Document 4: Japanese Patent No. 2614164
非特許文献 1 international Journal of Clinical Oncology, Vol. 9, No. 6 , 2004  Non-patent literature 1 international Journal of Clinical Oncology, Vol. 9, No. 6, 2004
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0004] このような上記抗腫瘍剤及び放射線併用療法は、癌の画期的な治療法として普及 しつつあるが、被験者によって治療の有効性に個人差があるという問題もあった。す なわち、根治する場合もあれば、ある程度効く場合もあり、一方、あまり効果がない場 合やまったく効かな 、場合もあった。 [0004] Such an anti-tumor agent and radiation therapy are widely used as a breakthrough treatment for cancer. However, there is also a problem that there are individual differences in the effectiveness of treatment depending on subjects. In other words, it may be cured or it may work to some extent, while it may or may not work at all.
[0005] このように、上記抗腫瘍剤及び放射線併用療法に対する感受性には、個人差があ ることが知られているものの、感受性を規定する因子は未だ具体的に解明されていな い。よって、感受性の相違の診断は、併用療法実施後の経過を長期間診察して行う しかなぐ感受性の低いまたは感受性のない被験者の場合は、治療効率および経済 的な面で不利であった。  [0005] Thus, although it is known that there are individual differences in sensitivity to the above-described anti-tumor agent and radiation therapy, the factors that define the sensitivity have not yet been clarified. Thus, the diagnosis of differences in susceptibility was disadvantageous in terms of treatment efficiency and economics in the case of insensitive subjects who could only be performed after long-term examination after the combination therapy.
[0006] 本発明は、上記問題点に鑑みてなされたものであって、テガフール ·ギメラシル'ォ テラシルカリウム配合抗腫瘍剤及び放射線併用療法の感受性を迅速に、かつ正確 に診断するための手段の提供を目的とするものである。  [0006] The present invention has been made in view of the above-mentioned problems, and is a means for quickly and accurately diagnosing the sensitivity of an anti-tumor agent containing tegafur and gimeracil potassium terracil potassium and radiation therapy. It is intended to provide.
課題を解決するための手段  Means for solving the problem
[0007] 本発明者らは、上記課題を解決するために鋭意研究を進めた。その過程で、腫瘍 細胞移植マウスにぉ ヽて上記抗腫瘍剤及び放射線併用療法を実施した場合、治療 を行わな力つた場合、抗腫瘍剤のみ投与した場合、及び放射線療法のみ実施した 場合のそれぞれにつ!ヽて遺伝子の発現量を比較したところ、併用療法を実施した場 合のみ特異的に発現量が変化するポリヌクレオチドが存在することに着目した。そし て、これらのポリヌクレオチドの中からの解析を重ねた結果得られた新規なヒト由来ポ リヌクレオチドが、上記抗腫瘍剤及び放射線併用療法感受性を迅速且つ正確に診 断するための遺伝子マーカーとして有用であることを見出した。  [0007] The inventors of the present invention have made extensive studies to solve the above problems. In the process, when the above-mentioned anti-tumor agent and radiation combination therapy is performed on tumor cell transplanted mice, when the treatment is not effective, only when the anti-tumor agent is administered, and when only radiation therapy is performed As a result, when the expression levels of the genes were compared, it was noted that there are polynucleotides whose expression levels specifically change only when combined therapy is performed. A novel human-derived polynucleotide obtained as a result of repeated analysis from these polynucleotides is used as a genetic marker for rapidly and accurately diagnosing the above-mentioned anti-tumor agent and radiation combination therapy sensitivity. I found it useful.
[0008] そして、本発明者らは、上記遺伝子マーカーとしてのポリヌクレオチドに対しプライ マーやプローブとして用いられるポリヌクレオチドや、該ポリヌクレオチドによりコードさ れるポリペプチド、並びに該ポリペプチドを認識する抗体は、上記併用療法感受性を より迅速かつ正確に診断するために有用であることを見出した。  [0008] Then, the present inventors provide a polynucleotide used as a primer or probe for the polynucleotide as the gene marker, a polypeptide encoded by the polynucleotide, and an antibody that recognizes the polypeptide. The present inventors have found that it is useful for diagnosing the above-mentioned combination therapy sensitivity more quickly and accurately.
さらに、本発明者らは、上記プライマーやプローブ、抗体を含む診断薬や診断用キ ットは、感受性の診断をより簡便に行うことができるものであることも見出した。  Furthermore, the present inventors have also found that diagnostic agents and diagnostic kits containing the above primers, probes, and antibodies can more easily diagnose sensitivity.
本発明は、係る知見に基づいて完成されたものである。  The present invention has been completed based on such knowledge.
[0009] 本発明は、以下の〔1〕〜〔13〕力もなる。 〔1〕下記の (A)または (B)で表されるポリヌクレオチド。 [0009] The present invention has the following [1] to [13] forces. [1] A polynucleotide represented by the following (A) or (B).
(A)配列表の配列番号 1〜70の!、ずれかに記載の塩基配列を含むポリヌクレオチド (A) a polynucleotide comprising any of the nucleotide sequences set forth in SEQ ID NOs: 1 to 70 in the sequence listing!
(B)前記 (A)記載のポリヌクレオチドと相補的な塩基配列を含むポリヌクレオチド(B) a polynucleotide comprising a base sequence complementary to the polynucleotide described in (A) above
(C)前記 (A)または（B)記載のポリヌクレオチドとハイストリンジェントな条件下でノ、ィ ブリダィズする塩基配列を含み、かつ、テガフール ·ギメラシル'ォテラシルカリウム配 合抗腫瘍剤及び放射線の併用療法感受性を診断するための遺伝子マーカーとして 機能するポリヌクレオチド (C) a nucleotide sequence that is hybridized under a highly stringent condition with the polynucleotide according to (A) or (B), and a tegafur / gimeracil / oteracil potassium-conjugated antitumor agent and radiation Polynucleotides that function as genetic markers for diagnosing combination therapy sensitivity
(D)前記 (A)または (B)記載のポリヌクレオチドにお 、て、 1または複数個の塩基が 欠失、置換、挿入、および Zまたは付加した塩基配列を含み、かつ、テガフール 'ギ メラシル 'ォテラシルカリウム配合抗腫瘍剤及び放射線の併用療法感受性を診断す るための遺伝子マーカーとして機能するポリヌクレオチド  (D) In the polynucleotide according to (A) or (B), one or a plurality of bases includes a base sequence in which deletion, substitution, insertion, and Z or addition are present, and tegafur 'gimeracil 'Polynucleotides that function as genetic markers for diagnosing sensitivity to combination therapy with anti-tumor agents containing oteracil potassium and radiation
〔2〕 テガフール ·ギメラシル ·ォテラシルカリウム配合抗腫瘍剤及び放射線の併用療 法感受性を診断するための遺伝子マーカーとして機能する、下記の (A)〜（D)で表 されるポリヌクレオチド。  [2] A polynucleotide represented by the following (A) to (D), which functions as a genetic marker for diagnosing susceptibility to combined treatment with tegafur, gimeracil and oteracil potassium combined antitumor agents and radiation.
(A)配列表の配列番号 1〜70の!、ずれかに記載の塩基配列を含むポリヌクレオチド (A) a polynucleotide comprising any of the nucleotide sequences set forth in SEQ ID NOs: 1 to 70 in the sequence listing!
(B)前記 (A)記載のポリヌクレオチドと相補的な塩基配列を含むポリヌクレオチド(B) a polynucleotide comprising a base sequence complementary to the polynucleotide described in (A) above
(C)前記 (A)または（B)記載のポリヌクレオチドとハイストリンジェントな条件下でノ、ィ ブリダィズする塩基配列を含み、かつ、テガフール ·ギメラシル'ォテラシルカリウム配 合抗腫瘍剤及び放射線の併用療法感受性を診断するための遺伝子マーカーとして 機能するポリヌクレオチド (C) a nucleotide sequence that is hybridized under a highly stringent condition with the polynucleotide according to (A) or (B), and a tegafur / gimeracil / oteracil potassium-conjugated antitumor agent and radiation Polynucleotides that function as genetic markers for diagnosing combination therapy sensitivity
(D)前記 (A)または (B)記載のポリヌクレオチドにお 、て、 1または複数個の塩基が 欠失、置換、挿入、および Zまたは付加した塩基配列を含み、かつ、テガフール 'ギ メラシル 'ォテラシルカリウム配合抗腫瘍剤及び放射線の併用療法感受性を診断す るための遺伝子マーカーとして機能するポリヌクレオチド  (D) In the polynucleotide according to (A) or (B), one or a plurality of bases includes a base sequence in which deletion, substitution, insertion, and Z or addition are present, and tegafur 'gimeracil 'Polynucleotides that function as genetic markers for diagnosing sensitivity to combination therapy with anti-tumor agents containing oteracil potassium and radiation
〔3〕 テガフール ·ギメラシル ·ォテラシルカリウム配合抗腫瘍剤及び放射線の併用療 法感受性を診断するにあたり、上記〔1〕または〔2〕記載のポリヌクレオチドのプローブ またはプライマーとして用いられるポリヌクレオチド。  [3] A polynucleotide used as a probe or primer for the polynucleotide according to [1] or [2] above in diagnosing the sensitivity of a combination therapy of tegafur, gimeracil, and oteracil potassium.
〔4〕 下記の（1)〜 (4)で表されるポリヌクレオチドである上記〔2〕記載のポリヌクレオ チド。 [4] The polynucleotide according to [2] above, which is a polynucleotide represented by the following (1) to (4): Chido.
(1)配列表の配列番号 1〜70のいずれかに記載の塩基配列内のすくなくとも連続す る 10塩基を含むポリヌクレオチド  (1) A polynucleotide comprising at least 10 consecutive nucleotides in the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 70 in the Sequence Listing
(2)前記（1)記載のポリヌクレオチドと相補的な塩基配列を含むポリヌクレオチド (2) a polynucleotide comprising a base sequence complementary to the polynucleotide of (1) above
(3)前記（1)または（2)記載のポリヌクレオチドとハイストリンジェントな条件下でハイ ブリダィズする塩基配列を含み、かつ、上記〔1〕記載のポリヌクレオチドのプローブま たはプライマーとして機能するポリヌクレオチド (3) A nucleotide sequence that hybridizes with the polynucleotide described in (1) or (2) under high stringency conditions, and functions as a probe or primer for the polynucleotide described in [1] above Polynucleotide
(4)前記（1)または（2)記載のポリヌクレオチドにお 、て、 1または複数個の塩基が欠 失、置換、挿入、および Zまたは付加した塩基配列を含み、かつ、上記〔1〕記載のポ リヌクレオチドのプローブまたはプライマーとして機能するポリヌクレオチド  (4) The polynucleotide according to (1) or (2) above, which comprises a nucleotide sequence in which one or more bases are missing, substituted, inserted, and Z or added, and [1] A polynucleotide that functions as a probe or primer for the described polynucleotide
[5] 配列番号 71〜142の 、ずれかに記載の塩基配列を含む上記〔2〕または〔3〕記 載のポリヌクレオチド。  [5] The polynucleotide according to [2] or [3] above, comprising the nucleotide sequence according to any one of SEQ ID NOs: 71 to 142.
〔6〕 上記〔1〕または〔2〕記載のポリヌクレオチドによりコードされるポリペプチド。 〔7〕 上記〔6〕記載のポリペプチドを認識する抗体。  [6] A polypeptide encoded by the polynucleotide described in [1] or [2] above. [7] An antibody that recognizes the polypeptide of [6] above.
〔8〕 上記〔3〕〜〔5〕のいずれかに記載のポリヌクレオチド、もしくは〔7〕記載の抗体 を少なくとも含む、テガフール ·ギメラシル ·ォテラシルカリウム配合抗腫瘍剤及び放 射線の併用療法感受性の診断薬。  [8] Susceptibility to combination therapy of tegafur, gimeracil, and oteracil potassium and radiotherapy including at least the polynucleotide according to any one of [3] to [5] above or the antibody according to [7]. Diagnostics.
〔9〕 上記〔3〕〜〔5〕のいずれかに記載のポリヌクレオチド、もしくは〔7〕記載の抗体 を少なくとも含む、テガフール ·ギメラシル ·ォテラシルカリウム配合抗腫瘍剤及び放 射線の併用療法感受性の診断用キット。  [9] Susceptibility to combination therapy of tegafur, gimeracil, and oteracil potassium and radiotherapy including at least the polynucleotide according to any one of [3] to [5] or the antibody according to [7] Diagnostic kit.
〔10〕 被験者から採取された生体試料中の上記〔1〕または〔2〕記載のポリヌクレオチ ドの有無を調べることを特徴とする、テガフール ·ギメラシル ·ォテラシルカリウム配合 抗腫瘍剤及び放射線の併用療法感受性の診断方法。  [10] Combination of anti-tumor agent and radiation combined with tegafur, gimeracil, and oteracil potassium, characterized in that the presence or absence of the polynucleotide described in [1] or [2] in a biological sample collected from a subject is examined A diagnostic method for therapy sensitivity.
〔11〕 下記の工程 (I)〜 (ΠΙ)を含む上記〔10〕記載の診断方法。 [11] The diagnostic method according to [10] above, comprising the following steps (I) to (i).
(I)被験者カゝら採取された生体試料カゝら調製された RNAまたは該 RNAカゝら転写され た相補的ポリヌクレオチドと上記〔3〕〜〔5〕の 、ずれかに記載のポリヌクレオチドとを 結合させる工程  (I) A biological sample collected from a subject, RNA prepared from a biological sample, or a complementary polynucleotide transcribed from the RNA, and the polynucleotide according to any one of [3] to [5] above The process of combining
(II)工程 (I)においてポリヌクレオチドに結合した RNAまたは相補的ポリヌクレオチド の有無を、上記〔3〕〜〔5〕の 、ずれかに記載のポリヌクレオチドを指標として測定す る工程 (II) RNA or complementary polynucleotide bound to the polynucleotide in step (I) The step of measuring the presence or absence of the above-mentioned [3] to [5] using the polynucleotide described in any one of the above as an indicator
(III)工程 (Π)の測定結果から、テガフール 'ギメラシル'ォテラシルカリウム配合抗腫 瘍剤及び放射線の併用療法感受性を診断する工程  (III) Process The process of diagnosing susceptibility to combination therapy with tegafur 'gimeracil' oteracil potassium-containing antitumor agent and radiation from the measurement results of (iii)
〔12〕 被験者力 採取された生体試料中の上記〔6〕記載のポリペプチドの有無を調 ベることを特徴とする、テガフール ·ギメラシル ·ォテラシルカリウム配合抗腫瘍剤及び 放射線の併用療法感受性の診断方法。  [12] Subject test power The anti-tumor agent containing tegafur / gimeracil / oteracil potassium, which is characterized by the presence or absence of the polypeptide according to [6] above in a collected biological sample, Diagnostic method.
〔13〕 下記の工程 (i)〜 (iii)を含む上記〔 12〕記載の診断方法。  [13] The diagnostic method according to the above [12], comprising the following steps (i) to (iii):
(i)被験者力 採取された生体試料力 調製されたポリペプチドと上記〔7〕記載の抗 体とを結合させる工程、  (i) subject force collected biological sample force a step of binding the prepared polypeptide and the antibody described in [7] above,
(ii)工程 (i)において抗体に結合したポリペプチドまたはその部分ペプチドの有無を 、該抗体を指標として測定する工程  (ii) a step of measuring the presence or absence of the polypeptide bound to the antibody or its partial peptide in step (i) using the antibody as an index
(iii)工程 (ii)の測定結果から、テガフール ·ギメラシル*ォテラシルカリウム配合抗腫 瘍剤及び放射線の併用療法感受性を診断する工程  (iii) Step (ii) Based on the measurement results of step (ii), a diagnosis of the sensitivity of combination therapy with tegafur and gimeracil * oteracil potassium combined antitumor agent and radiation
発明の効果  The invention's effect
[0010] 本発明によれば、テガフール 'ギメラシル'ォテラシルカリウム配合抗腫瘍剤及び放 射線の併用療法感受性を迅速かつ正確に診断することができる。よって、癌患者に 対し併用療法を実施すべきか否かの早期決定が可能であると共に、癌治療の効率 ィ匕を図ることができる。  [0010] According to the present invention, it is possible to rapidly and accurately diagnose the combination therapy sensitivity of tegafur 'gimeracil' oteracil potassium-containing antitumor agent and radiation. Therefore, it is possible to make an early decision as to whether or not combination therapy should be performed for cancer patients, and to improve the efficiency of cancer treatment.
図面の簡単な説明  Brief Description of Drawings
[0011] [図 1]図 1は、実施例における cDNAライブラリの作成カゝら計算機解析までの手順の 概略を示す図である。  FIG. 1 is a diagram showing an outline of the procedure up to the computer analysis after the creation of a cDNA library in the examples.
[図 2]図 2は、実施例における配列ァノテーシヨン処理の手順の概略を示す図である。  [Fig. 2] Fig. 2 is a diagram showing an outline of a procedure of array annotation processing in the embodiment.
[図 3]図 3は、実施例におけるクローンへのゲノムのマッピング及びそれによるクラスタ リングの手順の概略を示す図である。  [FIG. 3] FIG. 3 is a diagram showing an outline of a procedure for mapping genomes to clones and thereby clustering in Examples.
[図 4]図 4は、実施例における放射線処理群及び併用処理群の増幅量の、コントロー ル群の増幅量に対する比率を示す図である。  [FIG. 4] FIG. 4 is a graph showing the ratio of the amplification amount of the radiation treatment group and the combination treatment group in the example to the amplification amount of the control group.
発明を実施するための最良の形態 [0012] 以下に、本発明の実施形態について説明する。 BEST MODE FOR CARRYING OUT THE INVENTION [0012] Hereinafter, embodiments of the present invention will be described.
なお、本発明における生物化学的なあるいは遺伝子工学的な手法を実施するにあ たっては、例えば、 Molecular Cloning : A LABORATORY MANUAL,第 3版、 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Ne w York(2001)、新遺伝子工学ノヽンドブック (村松正實ら編、羊土社、実験医学別 冊、第 3版、 1999年)、タンパク質実験の進め方（岡田雅人、宫崎香編、羊土社、第 1版、 1998年)、タンパク質実験ノート（岡田雅人、宫崎香編、羊土社、第 2版、 1999 年)、タンパク質実験ハンドブック (竹縛忠臣編集、実験医学別冊、初版、 2003年 8 月 15日）、 PCR実験ノート (谷ロ武俊編集、羊土社、第 1版、 1997年)などの種々の 実験マニュアルの記載が参照される。  In carrying out the biochemical or genetic engineering method in the present invention, for example, Molecular Cloning: A LABORATORY MANUAL, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York ( 2001), New Genetic Engineering Nord Book (edited by Masaaki Muramatsu et al., Yodosha, experimental medicine separate volume, third edition, 1999), How to proceed with protein experiments (Masato Okada, Kaori Amagasaki, Yodosha, No. 1) Edition, 1998), Protein Experiment Notes (Masato Okada, Kasaki Kasaki, Yodosha, 2nd Edition, 1999), Protein Experiment Handbook (Edited by Tadaomi Takeshige, Experimental Medicine Separate Volume, First Edition, August 2003 15) ), PCR experiment notes (edited by Taketoshi Taniro, Yodosha, 1st edition, 1997), etc. are referred to.
[0013] 本発明は、テガフール ·ギメラシル ·ォテラシルカリウム配合抗腫瘍剤及び放射線の 併用療法感受性を診断するための遺伝子マーカーに関するものである。  The present invention relates to an antitumor agent containing tegafur, gimeracil, and oteracil potassium, and a genetic marker for diagnosing radiation therapy sensitivity.
[0014] ここで、上記抗腫瘍剤及び放射線の併用療法とは、上記抗腫瘍剤を用いたィ匕学療 法と放射線療法の併用による癌治療法を意味する。  [0014] Here, the combination therapy of the antitumor agent and radiation means a cancer treatment method using a combination of radiotherapy with the antitumor agent.
[0015] ここで、本発明が対象とする併用療法に用いられる抗腫瘍剤は、テガフール、ギメラ シルおよびォテラシルカリウムを、薬学的に許容される担体と共に含有し、且つ、テガ フール:ギメラシル:ォテラシルカリウム = 1 : 0. 4 : 1のモル比で含有することを特徴と するものである。  [0015] Here, the antitumor agent used in the combination therapy targeted by the present invention contains tegafur, gimeracil and oteracil potassium together with a pharmaceutically acceptable carrier, and tegafur: gimeracil: It is characterized by containing oteracil potassium in a molar ratio of 1: 4: 4: 1.
[0016] ここで、テガフール (tegafur)は、生体内で活性ィ匕を受けて活性本体である 5—フ ルォロウラシル（5— FU)を放出することが知られている公知の薬剤である。その製造 方法については特に限定されず、公知の方法、例えば特公昭 49— 10510号公報に 記載されて 、る方法に従うことができる。  [0016] Here, tegafur is a known drug that is known to release the active substance 5-fluorouracil (5-FU) upon receiving an in vivo activity. The production method is not particularly limited, and a known method, for example, the method described in JP-B-49-10510 can be followed.
[0017] ギメラシル（2, 4—ジヒドロキシ一 5—クロ口ピリジン）は、 5— FUの抗腫瘍効果を増 強させる作用を発揮する公知の化合物である。その製造方法については特に限定さ れず、公知の方法に従って製造されたものを用いることができる。  [0017] Gimeracil (2,4-dihydroxy-5-cloguchil pyridine) is a known compound that exerts an action to enhance the antitumor effect of 5-FU. The production method is not particularly limited, and those produced according to known methods can be used.
[0018] ォテラシルカリウム（ォキソン酸（oxonic acid)カリウム）は、上述のテガフールによ る炎症等の副作用を抑制するものである。ォテラシルカリウム自体は公知の化合物で ある。 [0019] 薬学的に許容される担体としては、通常の薬剤に汎用される各種のもの、例えば充 填剤、増量剤、結合剤、崩壊剤、表面活性剤、滑沢剤等の賦形剤を例示できる。 このような抗腫瘍剤としては、市販品を用いることができ、具体的には、大鵬薬品ェ 業株式会社から製造販売されて!、る市販品「ティーエスワン カプセル 20」「ティーェ スワン カプセル 25」（尚、「ティーエスワン」は、本願出願人の一人である大鵬薬品 工業株式会社の登録商標である。）を用いることができる。 [0018] Oteracil potassium (oxonic acid potassium) suppresses side effects such as inflammation caused by the above-mentioned tegafur. Oteracil potassium itself is a known compound. [0019] Examples of the pharmaceutically acceptable carrier include various types of commonly used drugs such as fillers, extenders, binders, disintegrants, surfactants, lubricants and the like. Can be illustrated. As such an antitumor agent, a commercially available product can be used. Specifically, it is manufactured and sold by Taiho Pharmaceutical Co., Ltd.! The commercially available products “TS1 capsule 20” and “TS1 capsule 25”. ("TS-1" is a registered trademark of Taiho Pharmaceutical Co., Ltd., one of the applicants of the present application).
[0020] 上記抗腫瘍剤の投与単位形態としては、非注射剤形態であれば特に限定されず、 治療目的に応じて選択でき、具体的には錠剤、被覆錠剤、丸剤、散剤、顆粒剤、力 プセル剤、液剤、懸濁剤、乳剤等の経口剤、坐剤、軟膏剤、貼付剤等の非経口剤を 例示でき、これら投与剤は、この分野で通常知られた慣用的な製剤方法により製剤 化される。  [0020] The dosage unit form of the antitumor agent is not particularly limited as long as it is a non-injectable form, and can be selected according to the therapeutic purpose. Specifically, tablets, coated tablets, pills, powders, granules And oral agents such as pushells, solutions, suspensions, emulsions, and parenterals such as suppositories, ointments, patches, etc., and these administration agents are conventional formulations commonly known in this field. Formulated by the method.
上記抗腫瘍剤の投与方法は、例えば、経腸投与、経口投与、直腸投与、口腔内投 与、経皮投与等の非注射投与であれば特に制限されず、各種製剤形態、患者の年 齢、性別その他の条件、患者の症状の程度等に応じて決定される。例えば錠剤、丸 剤、液剤、懸濁剤、乳剤、顆粒剤及びカプセル剤は、経口投与される。坐剤は直腸 内投与される。軟膏剤は、皮膚、口腔内粘膜等に塗布される。  The administration method of the antitumor agent is not particularly limited as long as it is non-injection administration such as enteral administration, oral administration, rectal administration, buccal administration, transdermal administration, etc. It is determined according to gender and other conditions, the degree of symptoms of the patient, and the like. For example, tablets, pills, solutions, suspensions, emulsions, granules and capsules are administered orally. Suppositories are administered rectally. The ointment is applied to the skin, oral mucosa and the like.
[0021] 上記抗腫瘍剤の投与条件は、用法、年齢、性別その他の条件、疾患の程度等によ り適宜選択できる。通常、経口投与の場合、成人に対して、テガフールの量が 0. 1〜 lOOmgZkgZ日程度、好ましくは l〜30mgZkgZ日程度、ギメラシルの量が 0. 1 〜： LOOmgZkgZ日程度、好ましくは l〜50mgZkgZ日程度、ォテラシルカリウム の量が 0. 1〜： LOOmgZkgZ日程度、好ましくは l〜40mgZkgZ日程度の範囲と なる量を目安とするのがよい。これら本発明製剤は 1日に 1回又は 2〜4回程度に分 けて投与することができる。坐剤の場合、通常、成人に対して、テガフール量として、 1〜： LOOmgZkg程度を、 1日 1〜2回、 6〜12時間の間隔をおいて、直腸内に挿入 して投与する。 [0021] The administration conditions of the antitumor agent can be appropriately selected depending on the usage, age, sex and other conditions, the degree of disease, and the like. In general, for oral administration, the amount of tegafur is about 0.1 to lOOmgZkgZ days, preferably about 1 to 30 mgZkgZ days, and the amount of gimeracil is about 0.1 to about LOOmgZkgZ days, preferably 1 to 50 mgZkgZ. The amount of oteracil potassium is about 0.1 to about LOOmgZkgZ days, preferably about 1 to 40 mgZkgZ days. These preparations of the present invention can be administered once a day or divided into about 2 to 4 times a day. In the case of a suppository, usually, for adults, the amount of tegafur is about 1 to about LOOmgZkg, inserted once or twice a day at intervals of 6 to 12 hours and administered.
[0022] 一方、放射線療法とは、 X線等の放射線を患部に対し照射することによるがんの治 療法である。放射線の照射条件は、年齢、性別、疾患の程度等により適宜選択でき る。例えば、放射線療法は 1コースで 20〜30回（約 4〜6週間）に分けて行われ、コ ース全体で 40〜60Gyとなるように設定できる。また、照射部位は腫瘍組織が含まれ る部位であれば特に制限されないが、腫瘍部位に対して局所照射を行うことが好まし い。 [0022] On the other hand, radiation therapy is cancer treatment by irradiating an affected area with radiation such as X-rays. Radiation irradiation conditions can be appropriately selected depending on age, sex, degree of disease, and the like. For example, radiation therapy is performed 20-30 times (about 4-6 weeks) in one course. Can be set to 40-60 Gy for the entire network. The irradiation site is not particularly limited as long as it contains a tumor tissue, but it is preferable to perform local irradiation on the tumor site.
[0023] 上記抗腫瘍剤及び放射線の併用療法の対象は癌患者である。癌の種類に関して は、特に限定はなぐ例えば、頭頸部癌、胃癌、結腸癌、直腸癌、肝臓癌、胆のう'胆 管癌、脾臓癌、肺癌、乳癌、膀胱癌、前立腺癌、子宮頸癌等が挙げられる。これらの うち、頭頸部癌、肺癌、脾臓癌が好ましぐ肺癌が特に好ましい。  [0023] The subject of the combination therapy of the antitumor agent and radiation is a cancer patient. The types of cancer are not particularly limited, for example, head and neck cancer, stomach cancer, colon cancer, rectal cancer, liver cancer, gallbladder's bile duct cancer, spleen cancer, lung cancer, breast cancer, bladder cancer, prostate cancer, cervical cancer, etc. Is mentioned. Of these, lung cancer is particularly preferred, with head and neck cancer, lung cancer and spleen cancer being preferred.
[0024] 上記抗腫瘍剤及び放射線の併用療法の実施条件は、年齢、性別、疾患の程度等 の条件により適宜設定することが可能である。  [0024] Conditions for performing the above-described combination therapy with an antitumor agent and radiation can be appropriately set depending on conditions such as age, sex, and degree of disease.
[0025] 一方、併用療法感受性とは、併用療法が施された癌患者における治療効果の個人 差を意味する。すなわち、併用療法により高い治療効果が得られる場合は、感受性 が高いとされ、一方、十分な治療効果が得られない場合やまったく治療効果が得ら れない場合には、感受性が低いとされる。  [0025] On the other hand, combination therapy sensitivity means individual differences in therapeutic effects in cancer patients who have undergone combination therapy. In other words, when a high therapeutic effect is obtained by combination therapy, the sensitivity is high. On the other hand, when a sufficient therapeutic effect is not obtained or no therapeutic effect is obtained, the sensitivity is low. .
また、併用療法感受性を診断するとは、上記併用療法に対する感受性の有無また は程度 (レベル)を何らかの基準に基づき評価、特定、検知、判断、予測することを意 味するものとする。  Diagnosis of sensitivity to combination therapy means evaluation, identification, detection, judgment, and prediction of the presence / absence or level (level) of sensitivity to the above combination therapy based on some criteria.
[0026] また、遺伝子マーカーとは、生体の感受性を判断するための遺伝的な指標または 素因を意味する。つまり、本発明において、上記抗腫瘍剤及び放射線の併用療法感 受性を診断するための遺伝子マーカーとは、該併用療法を施した結果、感受性に対 応して遺伝子の発現量が変化する等の遺伝的な指標または素因となるものを示すも のである。  [0026] The gene marker means a genetic index or predisposing factor for determining the sensitivity of a living body. That is, in the present invention, the gene marker for diagnosing susceptibility to a combination therapy of the above-described antitumor agent and radiation means that, as a result of the combination therapy, the expression level of the gene changes in response to sensitivity, etc. It indicates a genetic indicator or predisposing factor.
[0027] 本発明は、このような所定の抗腫瘍剤及び放射線の併用療法感受性を診断するた めの遺伝子マーカーに関するものであり、該マーカーとして機能するポリヌクレオチド (遺伝子マーカーポリヌクレオチド）、該ポリヌクレオチドによりコードされるポリべプチ ド、該ポリペプチドを認識する抗体、遺伝子マーカーポリヌクレオチドのプライマーま たはプローブとなりうるポリヌクレオチド（プローブまたはプライマー用ポリヌクレオチド )、並びにこれらを利用した所定の抗腫瘍剤及び放射線の併用療法感受性の診断 方法を提供するものである。これらに関し、以下詳細に説明する。 [0028] [1]遺伝子マーカーポリヌクレオチド [0027] The present invention relates to a gene marker for diagnosing the sensitivity of such a combination therapy of a predetermined antitumor agent and radiation, and a polynucleotide (gene marker polynucleotide) functioning as the marker, Polynucleotides encoded by nucleotides, antibodies that recognize the polypeptides, polynucleotides that can serve as primers or probes for genetic marker polynucleotides (probes or polynucleotides for primers), and predetermined antitumors using these It provides a method for diagnosing susceptibility to combination therapy of drugs and radiation. These will be described in detail below. [0028] [1] Gene marker polynucleotide
本発明は、第一に、新規なポリヌクレオチドを提供するものである。本発明のポリヌ クレオチドは、テガフール ·ギメラシル ·ォテラシルカリウム配合抗腫瘍剤及び放射線 の併用療法感受性を診断するための遺伝子マーカーとして機能するポリヌクレオチド (以下、遺伝子マーカーポリヌクレオチドという。）として有用である。  The present invention first provides a novel polynucleotide. The polynucleotide of the present invention is useful as a polynucleotide that functions as a genetic marker for diagnosing the sensitivity of a combination therapy of tegafur, gimeracil, and oteracil potassium, and a combination therapy with radiation (hereinafter referred to as a gene marker polynucleotide). .
[0029] 本発明のポリヌクレオチドは、上記抗腫瘍剤及び放射線の併用療法感受性を診断 するための遺伝子マーカーとして機能することができる。これは、各ポリヌクレオチド の発現量が、上記抗腫瘍剤及び放射線の併用療法感受性を有する患者にお！ヽては 、該併用療法を実施する前後で特異的に変化する性質を持つことに起因するもので ある。すなわち、本発明のポリヌクレオチドの発現量は、併用療法感受性を有する癌 患者において、かつ、上記併用療法が施された場合にのみに変化する。 [0029] The polynucleotide of the present invention can function as a genetic marker for diagnosing the sensitivity of a combination therapy of the antitumor agent and radiation. This is for patients whose expression level of each polynucleotide is sensitive to the above combination therapy of antitumor agents and radiation! This is due to the fact that it has a property that changes specifically before and after the combination therapy is performed. That is, the expression level of the polynucleotide of the present invention changes only in cancer patients having sensitivity to combination therapy and only when the combination therapy is applied.
ここで、併用療法が施された場合の発現量の変化は、発現量の増加と低下の両者 を含む。すなわち、第一の例として、遺伝子マーカーポリヌクレオチドの発現量が併 用療法実施後に実施前と比較して増加する場合、発現量が増加するときには併用療 法感受性が高 、と診断され、発現量の増加しな!、 (発現量に変化なし)ときに併用療 法感受性が低いと診断される。一方、第 2の例として、遺伝子マーカーポリヌクレオチ ドの発現量が、併用療法実施後に実施前よりも低下もしくは全く発現しない場合、低 下もしくは発現しない際には併用療法感受性が高いと診断され、逆に発現量が低下 しな！/ヽ (発現量に変化なし)際には併用療法感受性が低！ヽと診断される。併用療法 実施後の発現量の変化は、定量 PCRなどにより確認することができる。  Here, the change in the expression level when the combination therapy is performed includes both an increase and a decrease in the expression level. That is, as a first example, if the expression level of the gene marker polynucleotide increases after the combination therapy compared to before the implementation, it is diagnosed that the combination therapy sensitivity is high when the expression level increases, and the expression level It is diagnosed that the combination therapy sensitivity is low (no change in the expression level). On the other hand, as a second example, when the expression level of the gene marker polynucleotide is decreased or not expressed at all after the combination therapy, it is diagnosed that the combination therapy sensitivity is high when it is decreased or not expressed at all. Conversely, the expression level will not decrease! / ヽ (No change in expression level), low sensitivity to combination therapy! Diagnosed as sputum. Changes in the expression level after combination therapy can be confirmed by quantitative PCR.
尚、このような発現量の変化の態様は、マーカーを構成する塩基配列により相違す る他、同じマーカーであっても、発現させる際の条件、対象となる生体、癌種等により ネ目違することがある。  Such changes in expression level differ depending on the base sequence constituting the marker, and even if the same marker is used, the expression level varies depending on the expression conditions, the target organism, the cancer type, etc. There are things to do.
[0030] 本発明のポリヌクレオチドの構造に関し、 RNAか DNAかの別、その塩基長、一本 鎖か二本鎖の別、環状か線状の別、などについては特に限定されない。また、 DNA の場合、 cDNA、ゲノム DNA、および合成 DNAの別を問わない。さらに、 RNAの場 合、全 RNA、 mRNA、 rRNA、および合成 RNAの別も問わない。さらにまた、一本 鎖の場合は、センス鎖であっても、アンチセンス鎖であっても良ぐ二本鎖の場合は、 二本鎖 DNA、二本鎖 RNAまたは DNA:RNAのハイブリッドでも良い。そして、機能 領域の別を問うものでもなぐ例えば発現制御領域、コード領域、ェキソン、またはィ ントロンを含むものであっても良い。 [0030] Regarding the structure of the polynucleotide of the present invention, there is no particular limitation on whether it is RNA or DNA, its base length, whether it is single-stranded or double-stranded, circular or linear. In the case of DNA, it does not matter whether it is cDNA, genomic DNA, or synthetic DNA. Furthermore, in the case of RNA, it does not matter whether it is total RNA, mRNA, rRNA, or synthetic RNA. Furthermore, in the case of a single strand, it may be a sense strand or an antisense strand. It may be a double-stranded DNA, a double-stranded RNA, or a DNA: RNA hybrid. Further, it does not ask whether the functional region is different, and may include, for example, an expression control region, a coding region, an exon, or an intron.
[0031] このような本発明のポリヌクレオチドとして、具体的には、下記の (A)〜（D)で表さ れるものを ί列示することができる。 [0031] Specific examples of the polynucleotide of the present invention include those represented by the following (A) to (D).
(Α)配列表の配列番号 1〜70の!、ずれかに記載の塩基配列を含むポリヌクレオチド (Β)前記 (Α)記載のポリヌクレオチドと相補的な塩基配列を含むポリヌクレオチド (Ii) a polynucleotide comprising a base sequence described in any of SEQ ID NOs: 1 to 70 in the sequence listing (ii) a polynucleotide comprising a base sequence complementary to the polynucleotide described in (ii) above
(C)前記 (Α)または（Β)記載のポリヌクレオチドとハイストリンジェントな条件下でノ、ィ ブリダィズする塩基配列を含み、かつ上記抗腫瘍剤及び放射線の併用療法感受性 を診断するための遺伝子マーカーとして機能するポリヌクレオチド (C) a gene comprising a nucleotide sequence that can be hybridized under a highly stringent condition with the polynucleotide described in (i) or (ii) above, and a gene for diagnosing susceptibility to a combination therapy of the antitumor agent and radiation Polynucleotide that functions as a marker
(D)前記 (Α)または (Β)記載のポリヌクレオチドにお 、て、 1または複数個の塩基が 欠失、置換、挿入、および Ζまたは付加した塩基配列を含み、かつ上記抗腫瘍剤及 び放射線の併用療法感受性を診断するための遺伝子マーカーとして機能するポリヌ クレオチド  (D) In the polynucleotide described in (i) or (ii) above, the polynucleotide comprises one or more bases deleted, substituted, inserted, and inserted or added, and the antitumor agent and Polynucleotides that function as genetic markers for diagnosing susceptibility to combination therapy with radiation
[0032] 上記 (Α)ポリヌクレオチドは、配列表の配列番号 1〜70のいずれかに記載の塩基 配列を含むものであり、該塩基配列のみからなるものであってもよい。これらのポリヌ クレオチドは、後述の実施例に示すように、上記抗腫瘍剤及び放射線の併用療法を 実施する前後でその発現量が特異的に変化するものの中から配列データの解析に より、ヒト由来でありかつ新規であることが確認されたものである。このうち、配列番号 1 〜7、 9〜15、 17〜21、 23〜36は、定量 PCRにおける特異'性に優れるので、マー カーとしての検出が容易であり、好ましい。  [0032] The above (i) polynucleotide includes a base sequence described in any one of SEQ ID NOs: 1 to 70 in the sequence listing, and may comprise only the base sequence. As shown in the examples described later, these polynucleotides are derived from humans by analyzing the sequence data from among those whose expression level specifically changes before and after the combination therapy with the antitumor agent and radiation. It has been confirmed that it is new. Among these, SEQ ID NOs: 1 to 7, 9 to 15, 17 to 21, and 23 to 36 are preferable because they are easy to detect as markers because they are excellent in specificity in quantitative PCR.
このうち、配列表の配列番号 1〜33に記載の塩基配列を含むポリヌクレオチドは、 併用療法感受性が高 ヽ (有る)場合に発現量が増加する性質を持つ可能性があるポ リヌクレオチドとして選抜されたものである。また、配列番号 34〜36に記載の塩基配 列を含むポリヌクレオチドは、併用療法感受性が高 ヽ (有る）場合に発現量が低下す るもしくは全く発現しな 、性質を持つ可能性があるポリヌクレオチドとして選抜された ものである。一方、配列番号 37〜70記載の塩基配列は、上記のうち配列番号 1〜9 、 11〜24、 26〜36のそれぞれに記載の塩基配列から予測される mRNA配列を D NAとして表した塩基配列である。 Among these, the polynucleotides containing the nucleotide sequences set forth in SEQ ID NOs: 1 to 33 in the Sequence Listing are selected as polynucleotides that may have the property of increasing the expression level when sensitivity to combination therapy is high (if present). It has been done. In addition, a polynucleotide containing the nucleotide sequence of SEQ ID NOs: 34 to 36 may have a property that the expression level is reduced or not expressed at all when the sensitivity to combination therapy is high (existing). Selected as a nucleotide. On the other hand, the nucleotide sequences described in SEQ ID NOs: 37 to 70 are mRNA sequences predicted from the nucleotide sequences described in SEQ ID NOs: 1 to 9, 11 to 24, and 26 to 36, respectively, of the above. It is the base sequence expressed as NA.
[0033] 上記 (B)ポリヌクレオチドは、前記 (A)記載のポリヌクレオチドと相補的な塩基配列 を含むものである。すなわち、前記 (A)記載のポリヌクレオチドと相補的な塩基配列 のその一部に含むものであっても良いし、力かる相補的な塩基配列のみ力もなるもの であっても良い。 [0033] The polynucleotide (B) includes a base sequence complementary to the polynucleotide described in (A). That is, it may be included in a part of the base sequence complementary to the polynucleotide described in (A) above, or only the complementary base sequence that is strong may be used.
[0034] 上記 (C)ポリヌクレオチドは、前記 (A)または（B)記載のポリヌクレオチドとハイストリ ンジェントな条件下でハイブリダィズする塩基配列を含み、かつ上記抗腫瘍剤及び 放射線の併用療法感受性を診断するための遺伝子マーカーとして機能するものであ る。  [0034] The polynucleotide (C) includes a base sequence that hybridizes with the polynucleotide described in (A) or (B) under a highly stringent condition, and diagnoses the sensitivity to the combined treatment of the antitumor agent and radiation. It functions as a genetic marker for this purpose.
ノ、イブリダィゼーシヨンは、常法に従って行なうことができる。また、市販のライブラリ を使用する場合、添付の使用説明書に記載の方法に従って行うことができる。  Nobbreviation can be performed according to a conventional method. Moreover, when using a commercially available library, it can be performed according to the method described in the attached instruction manual.
[0035] ノ、イストリンジェントな条件とは、例えば、いわゆる特異的なハイブリッドが形成され、 非特異的なハイブリッドが形成されない条件を言う。例えば、通常のサザンハイブリダ ィゼーシヨンの洗 ヽの条件である 60°C、 1 X SSC、 0. 10/0SDS、好ましく ίま 0. 1 X S SC、 0. 1%SDSに相当する塩濃度で洗浄しても、ハイブリダィズ状態を維持する条 件が挙げられる。 [0035] The stringent conditions are, for example, conditions in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. For example, 60 ° C, 1 X SSC is a condition washingヽof typical Southern hybrida Izeshiyon, 0. 1 0/0 SDS, preferably ί or 0. 1 XS SC, salt equivalent to 0. 1% SDS concentration The condition for maintaining the hybridized state even after washing with (1) is mentioned.
このような (C)ポリヌクレオチドとして、具体的には、例えば、前記 (A)および (B)記 載のポリヌクレオチドと少なくとも 70%、好ましくは 90%、より好ましくは 95%、特に好 ましくは 97%の相同性 (homology)を有する塩基配列からなるポリヌクレオチドを挙 げることができる。塩基配列の相同性は、相同性検索、配列アラインメントプログラム、 BLAST, FASTA、 ClustalWなどにて計算することができる。  Specific examples of such (C) polynucleotide include, for example, at least 70%, preferably 90%, more preferably 95%, particularly preferably, the polynucleotides described in (A) and (B) above. Can include a polynucleotide having a base sequence having 97% homology. The base sequence homology can be calculated by homology search, sequence alignment program, BLAST, FASTA, ClustalW, or the like.
[0036] 上記 (D)ポリヌクレオチドは、前記 (A)または (B)記載のポリヌクレオチドにお 、て、 1または複数個の塩基が欠失、置換、挿入、および Zまたは付加した塩基配列を含 み、かつ上記抗腫瘍剤及び放射線の併用療法感受性を診断するための遺伝子マー カーとして機能するものである。 [0036] In the polynucleotide (D), the polynucleotide described in (A) or (B) above has a nucleotide sequence in which one or more bases are deleted, substituted, inserted, and Z or added. In addition, it functions as a gene marker for diagnosing the sensitivity of the above antitumor agent and radiation therapy.
ここで、欠失、置換、挿入、および Zまたは付加しても良い塩基の数は、 1または複 数個、例えば 1〜： L00個程度、好ましくは 1〜30個程度、より好ましくは 1〜10個程度 、さらに好ましくは 1〜5個である。欠失、置換、挿入、および Zまたは付加しても良い 塩基の位置は、とくに限定されない。このような (D)ポリヌクレオチドとして、具体的に は、例えば、前記 (A)または (B)記載のポリヌクレオチドと少なくとも 70%、好ましくは 90%、より好ましくは 95%、特に好ましくは 97%の相同性 (homology)を有する塩 基配列からなるポリヌクレオチドを挙げることができる。塩基配列の相同性の計算に ついては、上記 (C)にて説明した条件と同様とすることができる。 Here, the number of bases that may be deleted, substituted, inserted, and Z or added is one or more, for example, 1 to: about L00, preferably about 1 to 30, more preferably 1 to About 10, more preferably 1 to 5. Deletions, substitutions, insertions, and Z or additions The position of the base is not particularly limited. As such (D) polynucleotide, specifically, for example, at least 70%, preferably 90%, more preferably 95%, particularly preferably 97% with the polynucleotide described in (A) or (B) above. And a polynucleotide comprising a base sequence having homology. The base sequence homology can be calculated in the same manner as described in (C) above.
[0037] このような本発明の遺伝子マーカーポリヌクレオチドは、上記抗腫瘍剤及び放射線 の併用療法を実施する前後における発現量の変化の検出対象とされることにより、遺 伝子マーカーとしての機能を発揮する。発現量の変化を判断するには、被験者にお ける併用療法実施前の発現量や併用療法に対し非感受性または感受性の別の患 者における発現量を予め数値化するなどして基準としておき、これらとの統計学的有 意差を算出して行うことができる。  [0037] Such a gene marker polynucleotide of the present invention has a function as a gene marker by being targeted for detection of a change in expression level before and after the combination therapy of the antitumor agent and radiation. Demonstrate. To determine the change in the expression level, the expression level in the subject before the combination therapy or the expression level in another patient who is insensitive or sensitive to the combination therapy is used as a standard. This can be done by calculating the statistical significance of these.
本発明は、このような遺伝子マーカーポリヌクレオチドの有無を調べることを特徴と する、上記併用療法感受性の診断方法をも提供するものであり、その内容は下記 [5 ]において詳述するとおりである。  The present invention also provides a diagnostic method for susceptibility to the above-mentioned combination therapy, characterized by examining the presence or absence of such a gene marker polynucleotide, the details of which are as described in the following [5]. .
[0038] また、本発明の遺伝子マーカーポリヌクレオチドの発現量の検出は、被験者から生 体試料を採取し、該生体試料中における遺伝子マーカーポリヌクレオチドの発現産 物、例えば RNAやこれと相補的な DNAの量を測定することにより行うことができる。 係る検出においては、上記遺伝子マーカーポリヌクレオチドを基に設計されたプライ マーやプローブを用いることが簡便である。  [0038] In addition, detection of the expression level of the gene marker polynucleotide of the present invention is performed by collecting a biological sample from a subject, and expressing the gene marker polynucleotide expression product in the biological sample, for example, RNA or complementary thereto. This can be done by measuring the amount of DNA. In such detection, it is convenient to use a primer or probe designed based on the gene marker polynucleotide.
本発明は、このような感受性診断にあたり有用なプライマーやプローブとして有用な ポリヌクレオチドをも提供するものであり、その内容は下記 [2]において詳細に説明さ れる通りである。  The present invention also provides a polynucleotide useful as a primer or probe useful for such sensitivity diagnosis, and the contents thereof are as described in detail in the following [2].
[0039] また、本発明の遺伝子マーカーポリヌクレオチドの発現量の検出は、被験者から採 取された生体試料カゝら調製された遺伝子マーカーポリヌクレオチドによりコードされる ポリペプチドの量を測定することによつても実現可能である。係る検出においては、該 ポリペプチドを認識する抗体を用いることが簡便である。  [0039] Furthermore, the detection of the expression level of the gene marker polynucleotide of the present invention involves measuring the amount of the polypeptide encoded by the gene marker polynucleotide prepared from the biological sample collected from the subject. This is also feasible. In such detection, it is convenient to use an antibody that recognizes the polypeptide.
本発明は、このような感受性診断にあたり有用なポリペプチドや抗体をも提供するも のであり、その内容は下記 [3]において詳細に説明される通りである。 そして、本発明の遺伝子マーカーポリヌクレオチドは、常法により得ることができる。 例えば、下記 [2]において詳述されるプライマーを用いた PCR法により増幅すること ができる。 The present invention also provides polypeptides and antibodies useful for such sensitivity diagnosis, the contents of which are as described in detail in the following [3]. And the gene marker polynucleotide of this invention can be obtained by a conventional method. For example, it can be amplified by PCR using the primers detailed in [2] below.
[0040] [2]本発明のプローブまたはプライマー用ポリヌクレオチド  [0040] [2] Polynucleotide for probe or primer of the present invention
本発明は、第二に、テガフール 'ギメラシル 'ォテラシルカリウム配合抗腫瘍剤及び 放射線の併用療法感受性を診断するにあたり、上記 [1]で説明された本発明の遺伝 子マーカーポリヌクレオチドのプローブまたはプライマーとして用いられるポリヌクレオ チド (以下、プローブまたはプライマー用ポリヌクレオチドという。）を提供するものであ る。  Secondly, the present invention relates to a probe or primer of the gene marker polynucleotide of the present invention described in [1] above in diagnosing the sensitivity of a combination therapy of tegafur 'gimeracil' oteracil potassium-containing antitumor agent and radiation. A polynucleotide (hereinafter, referred to as a probe or primer polynucleotide) to be used as a DNA.
[0041] 本発明のプローブまたはプライマー用ポリヌクレオチドは、上記 [1]の本発明の遺 伝子マーカーポリヌクレオチドを検出又は増幅できるものであれば良ぐ例えば、サ ザンハイブリダィゼーシヨン、 DNAチップ用等のプローブまたは RT— PCR等の PCR 用のプライマーとなりうるものであれば良い。特に、遺伝子マーカーポリヌクレオチド の全部または一部に特異的に結合することのできるポリヌクレオチドであることが好ま しい。その形態に関し、 RNA力 DNAかの別、その塩基長、一本鎖か二本鎖の別、 環状か線状の別については特に限定されない。また、 DNAの場合、 cDNA、ゲノム DNA、および合成 DNAの別を問わない。さらに、 RNAの場合、全 RNA、 mRNA、 rRNA、および合成 RNAの別も問わない。さらにまた、一本鎖の場合は、センス鎖で あっても、アンチセンス鎖であっても良ぐ二本鎖の場合は、二本鎖 DNA、二本鎖 R NAまたは DNA:RNAのハイブリッドでも良い。そして、機能領域の別を問うものでも なぐ例えば発現制御領域、コード領域、ェキソン、またはイントロンを含むものであつ ても良い。  [0041] The probe or primer polynucleotide of the present invention is not particularly limited as long as it can detect or amplify the gene marker polynucleotide of the present invention of [1] above. For example, Southern hybridization, DNA Any probe that can serve as a probe for a chip or a primer for PCR such as RT-PCR may be used. In particular, a polynucleotide that can specifically bind to all or part of the gene marker polynucleotide is preferable. Regarding its form, there is no particular limitation on whether it is RNA force DNA, its base length, single-stranded or double-stranded, circular or linear. In the case of DNA, it does not matter whether it is cDNA, genomic DNA, or synthetic DNA. Furthermore, in the case of RNA, it does not matter whether total RNA, mRNA, rRNA, or synthetic RNA. Furthermore, in the case of a single strand, it may be a sense strand or an antisense strand. good. Further, it does not ask whether the functional region is different, and may include, for example, an expression control region, a coding region, an exon, or an intron.
[0042] 本発明のプローブまたはプライマー用ポリヌクレオチドは、上記 [1]の本発明の遺 伝子マーカーポリヌクレオチドから設計することができる。  [0042] The probe or primer polynucleotide of the present invention can be designed from the gene marker polynucleotide of the present invention of [1] above.
例えばプローブとして用いる場合には、上記 [1]の本発明の遺伝子マーカーポリヌ クレオチドのうちの標的とするポリヌクレオチドのうち少なくとも 10塩基以上を有するポ リヌクレオチドを用いることができる。  For example, when used as a probe, a polynucleotide having at least 10 bases or more among target polynucleotides of the gene marker polynucleotide of the present invention [1] can be used.
一方、プライマーとして用いる場合には、例えば PCR法で遺伝子増幅を行った場 合、増幅産物が上記 [1]の本発明の遺伝子マーカーポリヌクレオチドの、少なくとも 1 0塩基対以上を含むような 1対のポリヌクレオチドとして設計すればよい。具体的には 、一方のプライマーが上記 [1]の本発明の遺伝子マーカーポリヌクレオチドのうちの 標的とするポリヌクレオチドのうち少なくとも 10塩基以上を含むように設計すれば、も う一方のプライマーは、その標的となるポリヌクレオチドの相補鎖において前記プライ マーと相補的な配列より、 5'側の任意の配列力 選べばよい。プライマーの塩基長 は上記 [1]の本発明の遺伝子マーカーポリヌクレオチドと特異的に結合可能なもの であれば特に限定されないが、好ましくは 10塩基から 150塩基の核酸断片である。 On the other hand, when used as a primer, for example, when PCR is used for gene amplification. In other words, the amplification product may be designed as a pair of polynucleotides containing at least 10 base pairs of the gene marker polynucleotide of the present invention of [1] above. Specifically, if one primer is designed to contain at least 10 bases of the target polynucleotide of the gene marker polynucleotide of the present invention of [1] above, the other primer is It is only necessary to select an arbitrary sequence force 5 ′ from the sequence complementary to the primer in the complementary strand of the target polynucleotide. The base length of the primer is not particularly limited as long as it can specifically bind to the gene marker polynucleotide of the present invention of [1] above, but is preferably a nucleic acid fragment of 10 to 150 bases.
[0043] 本発明のプローブまたはプライマー用ポリヌクレオチドとして、具体的には、下記の [0043] As the probe or primer polynucleotide of the present invention, specifically, the following
(1)〜 (4)で表されるものを例示することができる。  Examples represented by (1) to (4) can be given.
(1)配列表の配列番号 1〜70のいずれかに記載の塩基配列内のすくなくとも連続す る 10塩基を含むポリヌクレオチド  (1) A polynucleotide comprising at least 10 consecutive nucleotides in the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 70 in the Sequence Listing
(2)前記（1)記載のポリヌクレオチドと相補的な塩基配列を含むポリヌクレオチド (2) a polynucleotide comprising a base sequence complementary to the polynucleotide of (1) above
(3)前記（1)または（2)記載のポリヌクレオチドとハイストリンジェントな条件下でハイ ブリダィズする塩基配列を含み、かつ上記 [1]で説明された遺伝子マーカーポリヌク レオチドのプローブまたはプライマーとして機能するポリヌクレオチド (3) As a probe or primer for the gene marker polynucleotide described in [1] above, comprising a nucleotide sequence that hybridizes with the polynucleotide described in (1) or (2) above under highly stringent conditions Functional polynucleotide
(4)前記（1)または（2)記載のポリヌクレオチドにお 、て、 1または複数個の塩基が欠 失、置換、挿入、および Zまたは付加した塩基配列を含み、かつ上記 [1]で説明され た遺伝子マーカーポリヌクレオチドのプローブまたはプライマーとして機能するポリヌ クレオチド  (4) The polynucleotide according to (1) or (2) above, which comprises a nucleotide sequence in which one or more bases are missing, substituted, inserted, and Z or added, and in the above [1] Polynucleotides that function as probes or primers for the described genetic marker polynucleotides
[0044] 上記（1)ポリヌクレオチドは、配列表の配列番号 1〜70のいずれかに記載の塩基 配列内のすくなくとも連続する 10塩基を含むものである。ここで、上記（1)ポリヌクレ ォチドがプライマーとして用いられる場合は、 10〜150塩基、中でも 10〜： LOO塩基、 特に 15〜50塩基を含むことが好ましい。一方、プローブとして用いられる場合は、 1 0塩基以上、より好ましくは 20〜300塩基を含むことが好まし 、。  [0044] The polynucleotide (1) includes at least 10 consecutive nucleotides in the nucleotide sequence set forth in any one of SEQ ID NOS: 1 to 70 in the Sequence Listing. Here, when the above (1) polynucleotide is used as a primer, it preferably contains 10 to 150 bases, especially 10 to LOO bases, particularly 15 to 50 bases. On the other hand, when used as a probe, it preferably contains 10 bases or more, more preferably 20 to 300 bases.
[0045] 上記（2)ポリヌクレオチドは、前記（1)記載のポリヌクレオチドと相補的な塩基配列 を含むものである。すなわち、前記（1)記載のポリヌクレオチドと相補的な塩基配列の その一部に含むものであっても良いし、力かる相補的な塩基配列のみ力もなるもので あっても良い。ここで、当該（2)ポリヌクレオチドがプライマーとして用いられる場合は 、 10〜150塩基、中でも 10〜： LOO塩基、特に 15〜50塩基を含むことが好ましい。一 方、プローブとして用いられる場合は、 10塩基以上、より好ましくは 20〜300塩基を 含むことが好ましい。 [0045] The polynucleotide (2) includes a base sequence complementary to the polynucleotide described in (1). That is, it may be included in a part of the base sequence complementary to the polynucleotide described in the above (1), or only a powerful complementary base sequence may be used. There may be. Here, when the polynucleotide (2) is used as a primer, it preferably contains 10 to 150 bases, especially 10 to LOO bases, particularly 15 to 50 bases. On the other hand, when used as a probe, it preferably contains 10 bases or more, more preferably 20 to 300 bases.
[0046] 上記（3)ポリヌクレオチドは、前記（1)または（2)記載のポリヌクレオチドとハイストリ ンジェントな条件下でハイブリダィズする塩基配列を含み、かつ上記 [1]で説明され た遺伝子マーカーポリヌクレオチドのプローブまたはプライマーとして機能するもので ある。  [0046] The polynucleotide (3) includes a nucleotide sequence that hybridizes with the polynucleotide described in (1) or (2) under high stringency conditions, and the gene marker polynucleotide described in [1] above It functions as a probe or primer.
ノ、イブリダィゼーシヨンの方法およびノヽイストリンジェントな条件は前記 [1]の（C)に ぉ 、て説明したのと同様である。  The method of the hybridization and the noisy stringent conditions are the same as described in (C) of [1] above.
このような（3)ポリヌクレオチドとして、具体的には、例えば、（1)および（2)記載のポ リヌクレオチドと少なくとも 70%、好ましくは 90%、より好ましくは 95%、特に好ましく は 97%の相同性 (homology)を有する塩基配列からなるポリヌクレオチドを挙げるこ とができる。塩基配列の相同性の計算については、上記 [1]にて説明した条件と同 様とすることができる。  As such (3) polynucleotide, specifically, for example, the polynucleotide described in (1) and (2) is at least 70%, preferably 90%, more preferably 95%, particularly preferably 97%. Examples thereof include polynucleotides consisting of nucleotide sequences having the same homology. The base sequence homology can be calculated in the same manner as described in [1] above.
ここで、当該（3)ポリヌクレオチドがプライマーとして用いられる場合は、 10〜150塩 基、中でも 10〜： L00塩基、特に 15〜50塩基を含むことが好ましい。一方、プローブ として用いられる場合は、 10塩基以上、より好ましくは 20〜300塩基を含むことが好 ましい。  Here, when the polynucleotide (3) is used as a primer, it preferably contains 10 to 150 bases, especially 10 to L00 bases, particularly 15 to 50 bases. On the other hand, when used as a probe, it preferably contains 10 bases or more, more preferably 20 to 300 bases.
[0047] 上記 (4)ポリヌクレオチドは、前記（1)または（2)記載のポリヌクレオチドにおいて、 1 または複数個の塩基が欠失、置換、挿入、および Zまたは付加した塩基配列を含み 、かつ上記 [1]で説明された遺伝子マーカーポリヌクレオチドのプローブまたはプライ マーとして機能するものである。  [0047] (4) The polynucleotide includes the base sequence in which one or more bases are deleted, substituted, inserted, and Z or added in the polynucleotide according to (1) or (2), and It functions as a probe or primer of the gene marker polynucleotide described in [1] above.
ここで、欠失、置換、挿入、および Zまたは付加しても良い塩基の数は、好ましくは 1または複数個、より好ましくは 1〜： L0個、更に好ましくは 1〜5個である。このような (4 )ポリヌクレオチドとして、具体的には、例えば、（1)および（2)記載のポリヌクレオチド と少なくとも 70%、好ましくは 90%、より好ましくは 95%、特に好ましくは 97%の相同 性 (homology)を有する塩基配列力もなるポリヌクレオチドを挙げることができる。塩 基配列の相同性の計算については、上記 [1]にて説明した条件と同様とすることが できる。 Here, the number of bases that may be deleted, substituted, inserted, and Z or added is preferably 1 or more, more preferably 1 to: L0, and further preferably 1 to 5. Specific examples of such (4) polynucleotide include, for example, at least 70%, preferably 90%, more preferably 95%, and particularly preferably 97% of the polynucleotide described in (1) and (2). Examples thereof include a polynucleotide having homology and a base sequence ability. salt The base sequence homology can be calculated in the same manner as described in [1] above.
ここで、当該 (4)ポリヌクレオチドがプライマーとして用いられる場合は、 10〜150塩 基、中でも 10〜： LOO塩基、特に 15〜50塩基を含むことが好ましい。一方、プローブ として用いられる場合は、 10塩基以上、より好ましくは 20〜300塩基を含むことが好 ましい。  Here, when the polynucleotide (4) is used as a primer, it preferably contains 10 to 150 bases, especially 10 to LOO bases, particularly 15 to 50 bases. On the other hand, when used as a probe, it preferably contains 10 bases or more, more preferably 20 to 300 bases.
本発明のプライマーまたはプローブ用ポリヌクレオチドとして、具体的には配列番号 71〜142のいずれかに記載の塩基配列を含むポリヌクレオチドを挙げることができる 。特に、下記のように配列番号 71〜142記載の各塩基配列力もなるポリヌクレオチド は、上記遺伝子マーカーポリヌクレオチド、特に配列番号 1〜36に記載の塩基配列 を含むポリヌクレオチドのプローブ及びプライマーとして有用であり、好ましくはプライ マーとして有用である。  Specific examples of the primer or probe polynucleotide of the present invention include a polynucleotide comprising the base sequence set forth in any of SEQ ID NOs: 71 to 142. In particular, the polynucleotides having the nucleotide sequences shown in SEQ ID NOs: 71 to 142 as described below are useful as probes and primers for the above gene marker polynucleotides, particularly polynucleotides containing the nucleotide sequences described in SEQ ID NOs: 1 to 36. Yes, preferably useful as a primer.
ポリヌクレオチド fOla (配列番号 71)、 rOla (配列番号 72)；ポリヌクレオチド f 02a ( 配列番号 73)、 r02a (配列番号 74)；ポリヌクレオチド f03a (配列番号 75)、 r03a (配 列番号 76)；ポリヌクレオチド f04b (配列番号 77)、 r04b (配列番号 78)；ポリヌクレオ チド f05a (配列番号 79)、 r05a (配列番号 80)；ポリヌクレオチド f 06a (配列番号 81) 、 r06a (配列番号 82)；ポリヌクレオチド f 07a (配列番号 83)、 r07a (配列番号 84)； ポリヌクレオチド f08b (配列番号 85)、 r08b (配列番号 86)；ポリヌクレオチド f 09a (配 列番号 87)、 r09a (配列番号 88)；ポリヌクレオチド f 10a (配列番号 89)、 r 10a (配列 番号 90)；ポリヌクレオチド f 11a (配列番号 91)、 rl la (配列番号 92)；ポリヌクレオチ ド f 12a (配列番号 93)、 rl2a (配列番号 94)；ポリヌクレオチド f 13a (配列番号 95)、 r 13a (配列番号 96)；ポリヌクレオチド f 14a (配列番号 97)、 r 14a (配列番号 98)； fl5 b (配列番号 99)、 rl5b (配列番号 100)；ポリヌクレオチド f 16a (配列番号 101)、 rl6 a (配列番号 102)；ポリヌクレオチド fl7a (配列番号 103)、 rl7a (配列番号 104)；ポ リヌクレオチド f 18a (配列番号 105)、 rl8a (配列番号 106)；ポリヌクレオチド f 19a ( 配列番号 107)、 r 19a (配列番号 108)；ポリヌクレオチド f 20a (配列番号 109)、 r20 a (配列番号 110)；ポリヌクレオチド f21a (配列番号 111)、 r21a (配列番号 112)；ポ リヌクレオチド f 22b (配列番号 113)、 r22b (配列番号 114)；ポリヌクレオチド f 23b ( 配列番号 115)、 r23a (配列番号 116)；ポリヌクレオチド f 24a (配列番号 117)、 r24 a (配列番号 118)；ポリヌクレオチド f25a (配列番号 119)、 r25a (配列番号 120)；ポ リヌクレオチド f26a (配列番号 121)、 r26a (配列番号 122)；ポリヌクレオチド f 27a ( 配列番号 123)、 r27a (配列番号 124)；ポリヌクレオチド f 28a (配列番号 125)、 r28 a (配列番号 126)；ポリヌクレオチド f29a (配列番号 127)、 r29a (配列番号 128)；ポ リヌクレオチド f 30a (配列番号 129)、 r30a (配列番号 130)；ポリヌクレオチド f 31b ( 配列番号 131)、 r3 lb (配列番号 132)；ポリヌクレオチド f 32a (配列番号 133)、 r32 a (配列番号 134)；ポリヌクレオチド f 33a (配列番号 135)、 r33a (配列番号 136)；ポ リヌクレオチド f34a (配列番号 137)、 r34a (配列番号 138)；ポリヌクレオチド f 35a ( 配列番号 139)、 r35a (配列番号 140)；ポリヌクレオチド f36a (配列番号 141)、 r36 a (配列番号 142)。 Polynucleotide fOla (SEQ ID NO: 71), rOla (SEQ ID NO: 72); polynucleotide f 02a (SEQ ID NO: 73), r02a (SEQ ID NO: 74); polynucleotide f03a (SEQ ID NO: 75), r03a (SEQ ID NO: 76); Polynucleotide f04b (SEQ ID NO: 77), r04b (SEQ ID NO: 78); Polynucleotide f05a (SEQ ID NO: 79), r05a (SEQ ID NO: 80); Polynucleotide f 06a (SEQ ID NO: 81), r06a (SEQ ID NO: 82); Poly Nucleotides f 07a (SEQ ID NO: 83), r07a (SEQ ID NO: 84); Polynucleotides f08b (SEQ ID NO: 85), r08b (SEQ ID NO: 86); Polynucleotides f 09a (SEQ ID NO: 87), r09a (SEQ ID NO: 88); Polynucleotide f 10a (SEQ ID NO: 89), r 10a (SEQ ID NO: 90); Polynucleotide f 11a (SEQ ID NO: 91), rl la (SEQ ID NO: 92); Polynucleotide f 12a (SEQ ID NO: 93), rl2a (SEQ ID NO: 93) No. 94); polynucleotide f 13a (SEQ ID No. 95), r 13a (SEQ ID No.) 96); polynucleotide f 14a (SEQ ID NO: 97), r 14a (SEQ ID NO: 98); fl5 b (SEQ ID NO: 99), rl5b (SEQ ID NO: 100); polynucleotide f 16a (SEQ ID NO: 101), rl6 a (sequence) No. 102); polynucleotide fl7a (SEQ ID NO: 103), rl7a (SEQ ID NO: 104); Polynucleotide f 18a (SEQ ID NO: 105), rl8a (SEQ ID NO: 106); Polynucleotide f 19a (SEQ ID NO: 107), r 19a (SEQ ID NO: 108); polynucleotide f 20a (SEQ ID NO: 109), r20 a (SEQ ID NO: 110); polynucleotide f21a (SEQ ID NO: 111), r21a (SEQ ID NO: 112); polynucleotide f 22b (SEQ ID NO: 113) R22b (SEQ ID NO: 114); polynucleotide f23b ( SEQ ID NO: 115), r23a (SEQ ID NO: 116); polynucleotide f 24a (SEQ ID NO: 117), r24 a (SEQ ID NO: 118); polynucleotide f25a (SEQ ID NO: 119), r25a (SEQ ID NO: 120); polynucleotide f26a (SEQ ID NO: 121), r26a (SEQ ID NO: 122); polynucleotide f 27a (SEQ ID NO: 123), r27a (SEQ ID NO: 124); polynucleotide f 28a (SEQ ID NO: 125), r28 a (SEQ ID NO: 126); polynucleotide f29a (SEQ ID NO: 127), r29a (SEQ ID NO: 128); polynucleotide f 30a (SEQ ID NO: 129), r30a (SEQ ID NO: 130); polynucleotide f 31b (SEQ ID NO: 131), r3 lb (SEQ ID NO: 132); Polynucleotide f 32a (SEQ ID NO: 133), r32 a (SEQ ID NO: 134); Polynucleotide f 33a (SEQ ID NO: 135), r33a (SEQ ID NO: 136); Polynucleotide f34a (SEQ ID NO: 137), r34a (SEQ ID NO: 138) ); Polynucleotide f 35a (SEQ ID NO: 139), r35a (SEQ ID NO: 140) Polynucleotide F36a (SEQ ID NO: 141), r36 a (SEQ ID NO: 142).
これらの各ポリヌクレオチドは、配列番号 1〜36に記載の塩基配列を含む遺伝子マ 一力一ポリヌクレオチドのうち、各ポリヌクレオチド名中の番号に対応する配列番号の 遺伝子マーカーポリヌクレオチドから設計されたものである。各ポリヌクレオチド名中「 f」が含まれて 、るものはフォワードプライマーとして、「r」が含まれて!/、るものはリバ一 スプライマーとして用いることができる。すなわち、各ポリヌクレオチドは、上記列挙中 のセミコロンによる区切りごとに、プライマー対として、対応する遺伝子マーカーポリヌ クレオチドのためのプライマーとして用いることができる。これらのうち、配列番号 71〜 84、 87〜100、 103〜112、 115〜 142の!ヽずれ力の塩基酉己歹 IJを含む遺伝子マー カーポリヌクレオチドの中力も適宜選択して用いることが好ましい。また、配列番号 37 〜70に記載の塩基配列を含む遺伝子マーカーポリヌクレオチドからも、上記配列番 号 1〜36記載の塩基配列から配列番号 71〜142の塩基配列を設計したのと同様に 、プライマーポリヌクレオチドを設計することができる。  Each of these polynucleotides was designed from the gene marker polynucleotides of SEQ ID NOs corresponding to the numbers in the names of the polynucleotides among the gene-powered polynucleotides containing the nucleotide sequences set forth in SEQ ID NOS: 1-36. Is. Each polynucleotide name containing “f” can be used as a forward primer, and “r” can be used as a reverse primer! That is, each polynucleotide can be used as a primer for a corresponding gene marker polynucleotide as a primer pair for each semicolon segment in the above list. Among these, it is preferable to select and use the intermediate force of the gene marker polynucleotide of SEQ ID NOs: 71 to 84, 87 to 100, 103 to 112, 115 to 142 as well as the gene marker polynucleotides including the misalignment base IJI. . In addition, from the gene marker polynucleotide containing the nucleotide sequence set forth in SEQ ID NOs: 37-70, the primer was designed in the same manner as the nucleotide sequence set forth in SEQ ID NO: 71-142 from the nucleotide sequence set forth in SEQ ID NO: 1-36. Polynucleotides can be designed.
このような本発明のプライマーまたはプローブ用ポリヌクレオチドは、上記 [1]で説 明された遺伝子マーカーポリヌクレオチドの合成及び発現量の検出に有用であるの で、上記抗腫瘍剤及び放射線の併用療法感受性の迅速かつ正確な診断に寄与す るものである。  Such a primer or probe polynucleotide of the present invention is useful for the synthesis and detection of the expression level of the gene marker polynucleotide described in [1] above. It contributes to quick and accurate diagnosis of sensitivity.
本発明は、このような感受性診断の方法を提供するものであり、その内容は、後述 の [5]において説明される通りである。 The present invention provides such a method for susceptibility diagnosis, the contents of which will be described later. As described in [5].
また、本発明のプライマーまたはプローブ用ポリヌクレオチドを感受性診断薬ゃキッ トに含めることにより、上記抗腫瘍剤及び放射線の併用療法感受性の診断をより簡便 に行うことができる。  Further, by including the primer or probe polynucleotide of the present invention in a sensitive diagnostic kit, it is possible to more easily diagnose the sensitivity of the combination therapy of the antitumor agent and radiation.
本発明は、このような感受性診断のための診断薬およびキット、ならびに診断方法 をも提供するものであり、その内容は、後述の [4]において説明される通りである。  The present invention also provides diagnostic agents and kits for such sensitivity diagnosis, and diagnostic methods, the contents of which are as described in [4] below.
[0050] [3]本発明のポリペプチドおよび抗体 [0050] [3] Polypeptide and antibody of the present invention
本発明は、第三に、前記 [1]で説明された遺伝子マーカーポリヌクレオチドによりコ ードされるポリペプチド、および該ポリペプチドを認識する抗体を提供するものである 本発明のポリペプチドは、前記 [1]で説明された遺伝子マーカーポリヌクレオチドに よりコードされるものであれば良ぐ前記 [1]で説明された (A)〜（D)ポリヌクレオチド に対応する特定のアミノ酸配列で示されるポリペプチドのほか、該ポリペプチドと実質 的に同等の機能を示すことを条件として、その変異体、誘導体、成熟体およびアミノ 酸修飾体などを包含する。変異体としては、天然に存在するアレル変異体、天然に 存在しない変異体、および人為的に欠失、付加、置換されることによって改変された アミノ酸配列を有する変異体が包含される。なお、上記変異体としては、変異のない ポリペプチドと少なくとも 70%、好ましくは 80%、より好ましくは 95%、さらにより好まし くは 97%相同なものを挙げることができる。また、アミノ酸修飾体には、天然に存在す るアミノ酸修飾体、天然に存在しないアミノ酸修飾体が包含され、具体的にはアミノ酸 のリン酸化体が挙げられる。  Third, the present invention provides a polypeptide encoded by the gene marker polynucleotide described in [1] above, and an antibody that recognizes the polypeptide. As long as it is encoded by the gene marker polynucleotide described in [1] above, it is indicated by a specific amino acid sequence corresponding to the polynucleotide (A) to (D) described in [1] above. In addition to polypeptides, mutants, derivatives, mature forms and amino acid modified forms thereof are included on the condition that they exhibit substantially the same function as the polypeptide. Variants include naturally occurring allelic variants, non-naturally occurring variants, and variants having amino acid sequences modified by artificial deletion, addition, or substitution. Examples of the mutant include those that are at least 70%, preferably 80%, more preferably 95%, and even more preferably 97% homologous to a polypeptide having no mutation. The amino acid modifications include naturally occurring amino acid modifications and non-naturally occurring amino acid modifications, and specifically include phosphorylated amino acids.
[0051] 本発明のポリペプチドは、前記 [1]で説明された遺伝子マーカーポリヌクレオチドを 基にして常法により得ることができる。例えば、該ポリヌクレオチドをクローユングし、ベ クタ一プラスミドにライゲーシヨンした後、大腸菌等の宿主細胞へ形質転換し、得られ る形質転換細胞を培養し、培養物中から回収することにより得ることができる。 [0051] The polypeptide of the present invention can be obtained by a conventional method based on the gene marker polynucleotide described in [1] above. For example, the polynucleotide can be cloned, ligated into a vector plasmid, transformed into a host cell such as Escherichia coli, and the resulting transformed cell is cultured and recovered from the culture. .
また、被験者などのヒトの細胞または組織力も常法により精製して製造することもで きる。例えば、被験者の組織または細胞をホモジナイズした後、酸などで抽出を行い 、該抽出液を逆相クロマトグラフィー、イオン交換クロマトグラフィーなどのクロマトダラ フィーを組み合わせることにより精製単離することができる。 In addition, human cells such as test subjects or tissue power can also be purified and produced by a conventional method. For example, after homogenizing a subject's tissue or cells, extraction with an acid or the like is performed, and the extract is chromatographed such as reverse phase chromatography or ion exchange chromatography. It can be purified and isolated by combining fees.
さら〖こ、固相合成法、液相合成法等のペプチド合成法に準じて製造することもでき る。  It can also be produced according to peptide synthesis methods such as Sarako, solid phase synthesis, and liquid phase synthesis.
[0052] 本発明のポリペプチドは、前記 [1]で説明された遺伝子マーカーポリヌクレオチドに よりコードされるポリペプチドであることから、被験者力 採取された生体試料力 調 製された遺伝子マーカーポリヌクレオチドによりコードされるポリペプチドの量を測定 することにより発現量の検出が可能である。すなわち、遺伝子マーカーポリヌクレオチ ドによりコードされるポリペプチドの発現量が、併用療法実施後に実施前よりも増加す る場合には、増加すれば併用療法感受性が高いと診断され、ポリペプチドの量が増 加しない (変化なし)ときに併用療法感受性が低いと診断される。一方、遺伝子マー カーポリヌクレオチドによりコードされるポリペプチドの発現量が併用療法実施後に低 下もしくは全く発現しない場合には、低下もしくはまったく発現しないときに併用療法 感受性が高いと診断され、逆に低下が確認されない (発現量に変化なし)ときに併用 療法感受性が低!ヽと診断される。  [0052] Since the polypeptide of the present invention is a polypeptide encoded by the genetic marker polynucleotide described in [1] above, the genetic marker polynucleotide prepared by subject test force is collected. The expression level can be detected by measuring the amount of the polypeptide encoded by. In other words, if the expression level of the polypeptide encoded by the gene marker polynucleotide increases after the combination therapy than before the combination therapy, it is diagnosed that the sensitivity of the combination therapy is higher and the amount of the polypeptide increases. Diagnosis of low sensitivity to combination therapy when there is no increase (no change). On the other hand, if the expression level of the polypeptide encoded by the gene marker polynucleotide decreases or does not express at all after the combination therapy, it is diagnosed that the sensitivity of the combination therapy is high when it decreases or does not express at all, and conversely decreases. Is not confirmed (no change in the expression level), the sensitivity to combination therapy is low!
[0053] このような本発明のポリペプチドの量の検出においては、該ポリペプチドを認識する 抗体を用いることが簡便であり、本発明は、このようなポリペプチドを認識する抗体を も提供する。 [0053] In detecting the amount of the polypeptide of the present invention, it is convenient to use an antibody that recognizes the polypeptide, and the present invention also provides an antibody that recognizes such a polypeptide. .
本発明の抗体としては、その形態に特に制限はなぐ上記本発明のポリペプチドの V、ずれかを抗原とするモノクローナル抗体、およびポリクローナル抗体の!/、ずれであ つても良い。また、本発明のポリヌクレオチドのアミノ酸配列のうち少なくとも連続する 10アミノ酸、中でも 15アミノ酸、特に 20アミノ酸力もなる部分アミノ酸配列を有するポ リペプチドに対して抗原結合性を有する抗体であっても良い。  The form of the antibody of the present invention is not particularly limited, and may be V of the above-described polypeptide of the present invention, monoclonal antibody having any of the antigens as antigens, or polyclonal antibody! Further, it may be an antibody having an antigen binding property to a polypeptide having a partial amino acid sequence having at least 10 consecutive amino acids, particularly 15 amino acids, particularly 20 amino acids in the amino acid sequence of the polynucleotide of the present invention.
[0054] 抗体は常法に従って製造することができる。例えば、ポリクローナル抗体の場合は、 常法に従って大腸菌で発現し精製した上記ポリペプチドを用いて、或いは常法に従 つてこれらの部分アミノ酸配列を有するよう合成したポリペプチドを用いて、実験動物 に免疫し、該免疫動物の血清から常法に従って得ることが可能である。一方、例えば モノクローナル抗体の場合は、常法に従って大腸菌等で発現し精製した上記ポリヌク レオチドを用いて、或いは常法に従ってこれらの部分アミノ酸配列を有するよう合成し たポリペプチドを用いて、実験動物に免疫し、該実験動物から得られた脾臓細胞と骨 髄腫細胞とを融合させてハイプリドーマ細胞を合成し、該細胞中から得ることができる [0054] The antibody can be produced according to a conventional method. For example, in the case of a polyclonal antibody, an experimental animal is immunized with the above-mentioned polypeptide expressed and purified in Escherichia coli according to a conventional method, or with a polypeptide synthesized with these partial amino acid sequences according to a conventional method. However, it can be obtained from the serum of the immunized animal according to a conventional method. On the other hand, for example, in the case of a monoclonal antibody, the above-mentioned polynucleotide expressed and purified in Escherichia coli or the like according to a conventional method, or synthesized to have these partial amino acid sequences according to a conventional method. The polypeptide can be used to immunize experimental animals, and spleen cells obtained from the experimental animals can be fused with osteomyeloma cells to synthesize hyperpridoma cells and obtained from the cells.
[0055] このように、本発明のポリペプチドおよび抗体は、上記抗腫瘍剤及び放射線の併用 療法感受性の迅速かつ正確な診断に寄与するものであり、併用療法感受性の診断 薬や診断用キットとして広く活用することができるものである。 [0055] Thus, the polypeptide and antibody of the present invention contribute to rapid and accurate diagnosis of the combination therapy sensitivity of the above-described antitumor agent and radiation, and are used as diagnostic drugs and diagnostic kits for combination therapy sensitivity. It can be used widely.
本発明は、このような抗体を用いた感受性診断の方法を提供するものであり、その 内容は、後述の [5]において説明される通りである。  The present invention provides a method for susceptibility diagnosis using such an antibody, the contents of which are as described in [5] below.
また、本発明の抗体を感受性診断薬やキットに含めることにより、上記抗腫瘍剤及 び放射線の併用療法感受性の診断をより簡便に行うことができる。  Further, by including the antibody of the present invention in a sensitivity diagnostic agent or kit, it is possible to more easily diagnose the sensitivity of the above-described combination therapy with the antitumor agent and radiation.
本発明は、このような感受性診断のための診断薬およびキット、ならびに診断方法 をも提供するものであり、その内容は、後述の [4]において説明される通りである。  The present invention also provides diagnostic agents and kits for such sensitivity diagnosis, and diagnostic methods, the contents of which are as described in [4] below.
[0056] [4]診断薬及び診断用キット  [0056] [4] Diagnostic agent and diagnostic kit
本発明は、第四に、上記 [2]で説明されたプライマー又はプローブ用ポリヌクレオ チド、もしくは上記 [3]で説明された抗体を少なくとも含むことを特徴とする診断薬、 並びに診断キットを提供するものである。  Fourth, the present invention provides a diagnostic agent characterized by comprising at least the polynucleotide for primer or probe described in [2] above, or the antibody described in [3] above, and a diagnostic kit. Is.
[0057] 本発明の診断薬並びに診断キットは、上記 [2]で説明されたプライマー又はプロ一 ブ用ポリヌクレオチド、もしくは上記 [3]で説明された抗体を少なくとも含むものであり 、公知の診断薬や診断用キットにおいて用いることのできる他の材料、例えば、上記 ポリヌクレオチドや抗体の生体試料に対する反応に用いるための緩衝液や酵素を含 むことができ、さらに簡便に診断を行うためには、被験者からの生体試料の調製や増 幅産物の検出に使用される試薬類を含んでいてもよぐ常法に従い、適宜の剤形と することができる。  [0057] The diagnostic agent and diagnostic kit of the present invention contain at least the primer or probe polynucleotide described in [2] above, or the antibody described in [3] above, and are known diagnostics. Other materials that can be used in drugs and diagnostic kits, such as buffers and enzymes for use in the reaction of the above polynucleotides and antibodies with biological samples, can be included. An appropriate dosage form can be obtained according to a conventional method which may contain reagents used for the preparation of biological samples from subjects and detection of amplification products.
従って、本発明の診断薬又は診断キットを用いることにより、被験者の生体試料中 に含まれる遺伝子マーカーポリヌクレオチド、又は該ポリヌクレオチドによりコードされ るポリペプチドを検出することによって、該被験者の上記併用療法に対する感受性を 診断することができる。  Therefore, by using the diagnostic agent or diagnostic kit of the present invention, the above combination therapy of the subject is detected by detecting the gene marker polynucleotide contained in the biological sample of the subject or the polypeptide encoded by the polynucleotide. Can be diagnosed.
[0058] [5]本発明の診断方法 本発明は、第五に、被験者から採取された生体試料中の上記 [1]で説明された遺 伝子マーカーポリヌクレオチド、或いは上記 [3]で説明されたポリペプチドの有無を 調べることを特徴とする、テガフール .ギメラシル .ォテラシルカリウム配合抗腫瘍剤及 び放射線の併用療法感受性の診断方法をも提供するものである。 [0058] [5] Diagnostic method of the present invention Fifth, the present invention is characterized by examining the presence or absence of the gene marker polynucleotide described in [1] above or the polypeptide described in [3] above in a biological sample collected from a subject. The present invention also provides an antitumor agent containing tegafur, gimeracil and oteracil potassium, and a method for diagnosing radiation therapy sensitivity.
[0059] 被験者は、既に上記抗腫瘍剤及び放射線の併用療法が施されている癌患者であ つても、未実施の癌患者であってもよい。 [0059] The subject may be a cancer patient who has already undergone the above-described combination therapy of an antitumor agent and radiation, or a cancer patient who has not been performed.
癌の種類については前記した抗腫瘍剤及び放射線の併用療法により治療できる癌 種であれば特に限定はなぐ例えば、頭頸部癌、胃癌、結腸癌、直腸癌、肝臓癌、胆 嚢，胆管癌、脾臓癌、肺癌、乳癌、膀胱癌、前立腺癌、子宮頸癌等が挙げられ、頭頸 部癌、肺癌、脾臓癌が好ましぐ肺癌が特に好ましい。  The type of cancer is not particularly limited as long as it is a cancer type that can be treated by the above-described combination therapy with an antitumor agent and radiation. For example, head and neck cancer, stomach cancer, colon cancer, rectal cancer, liver cancer, gallbladder, bile duct cancer, Examples include spleen cancer, lung cancer, breast cancer, bladder cancer, prostate cancer, cervical cancer, and the like, and lung cancer, in which head and neck cancer, lung cancer, and spleen cancer are preferred.
[0060] 生体試料は、被験者自身のものであり腫瘍を含む可能性がある試料であれば特に 限定されず、体液 (血液、尿等）、組織、その抽出物及び採取した組織の培養物など が例示できる力 採取の容易さから、血液、特に血清であることが好ましい。 [0060] The biological sample is not particularly limited as long as it is a subject's own sample and may contain a tumor. Body fluid (blood, urine, etc.), tissue, extract thereof, culture of collected tissue, etc. In view of ease of collection, blood, particularly serum, is preferable.
また、生体試料の採取方法は、生体試料や癌種に応じた方法により適宜選択する ことができる。  In addition, a method for collecting a biological sample can be appropriately selected according to a method according to the biological sample or the cancer type.
本発明の診断方法に適用される生体試料は、既に上記抗腫瘍剤及び放射線の併 用療法が施されている癌患者カゝら採取されたものであってもよぐ併用療法未実施の 癌患者カゝら採取されたものに上記抗腫瘍剤及び放射線の併用療法を施したもので あってもよい。  The biological sample applied to the diagnostic method of the present invention may be a cancer sample that has already been collected from cancer patients who have already undergone the above-mentioned combination therapy of antitumor agent and radiation. What was collected from the patient's body may have been subjected to the combination therapy of the above-mentioned antitumor agent and radiation.
被験者にお！ヽて既に上記抗腫瘍剤及び放射線の併用療法が施されて！/ヽる場合の 生体試料の採取を行う時期は、併用療法の効果が発揮されることが見込まれる時間 であることが好ましい。通常は、併用療法実施後 30分〜 5時間、好ましくは 1時間〜 3 時間である。  To the subject! A combination therapy of the antitumor agent and radiation has already been given! / The time when the biological sample is collected when speaking is preferably the time when the effect of the combination therapy is expected to be exerted. Usually, it is 30 minutes to 5 hours, preferably 1 hour to 3 hours after the combination therapy.
[0061] 本発明の診断方法においては、上記 [1]で説明された遺伝子マーカーポリヌクレオ チドの有無、或いは上記 [3]で説明されたポリペプチドの有無を調べる。まず、上記 [ 1]で説明された遺伝子マーカーポリヌクレオチドの有無を検出対象とする場合につ いて、以下に説明する。  [0061] In the diagnostic method of the present invention, the presence or absence of the gene marker polynucleotide described in [1] above or the presence or absence of the polypeptide described in [3] above is examined. First, the case where the presence or absence of the gene marker polynucleotide described in [1] above is set as the detection target will be described below.
[0062] 本発明の診断方法において上記 [1]で説明された遺伝子マーカーポリヌクレオチド の有無を調べるにあたり、前記 [2]で説明されたプローブまたはプライマー用のポリヌ クレオチドを利用して、ノーザンブロット法、 RT—PCR法、 DNAチップ解析法、 in s ituノヽイブリダィゼーシヨン法等の公知の方法を利用して、下記の工程 (I)〜（ΠΙ)を 含む方法により実施することができる。 [0062] The gene marker polynucleotide described in [1] above in the diagnostic method of the present invention When examining the presence or absence of DNA, using the probe or primer polynucleotide described in [2] above, Northern blot method, RT-PCR method, DNA chip analysis method, in situ hybridization method Etc. can be carried out by a method comprising the following steps (I) to (ii).
(I)被験者カゝら採取された生体試料カゝら調製された RNAまたは該 RNAカゝら転写され た相補的ポリヌクレオチドと上述したプローブまたはプライマー用ポリヌクレオチドとを 結合させる工程  (I) A step of binding the RNA prepared from the biological sample collected from the subject or the complementary polynucleotide transcribed from the RNA and the above-described probe or primer polynucleotide.
(II)工程 (I)においてポリヌクレオチドに結合した RNAまたは相補的ポリヌクレオチド の有無を、上述したプローブまたはプライマー用ポリヌクレオチドを指標として測定す る工程  (II) Step of measuring the presence or absence of RNA or complementary polynucleotide bound to the polynucleotide in step (I) using the above-described probe or primer polynucleotide as an indicator.
(III)工程 (Π)の測定結果から、上記抗腫瘍剤及び放射線の併用療法感受性を診断 する工程  (III) Step Diagnosing the combination therapy sensitivity of the antitumor agent and radiation from the measurement result of (iii)
[0063] 工程 (I)にお ヽては、被験者から採取された生体試料から調製された RNAまたは 該 RNAから転写された相補的ポリヌクレオチドと上述したプローブまたはプライマー 用ポリヌクレオチドとを結合させる。  [0063] In step (I), RNA prepared from a biological sample collected from a subject or a complementary polynucleotide transcribed from the RNA and the above-described probe or primer polynucleotide are combined.
該生体試料力ゝらの RNAの調製や、 RNAからの転写された相補的ポリヌクレオチド の調製は、常法により行うことができる。  Preparation of RNA from the biological sample and preparation of a complementary polynucleotide transcribed from RNA can be performed by conventional methods.
RNAとプローブまたはプライマー用ポリヌクレオチドとの結合は、常法により両者の 全部または一部が結合し易い条件を考慮して行うことができる。  The binding of RNA to the probe or primer polynucleotide can be performed by a conventional method in consideration of the conditions where all or a part of both can be easily bound.
[0064] 工程 (II)にお 、ては、工程 (I)にお 、てプローブまたはプライマー用ポリヌクレオチ ドに結合した RNAまたは相補的ポリヌクレオチドの有無を、上述したプローブまたは プライマー用ポリヌクレオチドを指標として測定する。測定は、工程 (I)において、あら かじめプローブまたはプライマー用ポリヌクレオチドを放射性同位元素や蛍光物質な どの標識物質で標識しておいてから RNAや相補的ポリヌクレオチドと結合させること により、該標識物質に由来するシグナルを放射線検出器や蛍光検出器で測定して 行うことができる。  [0064] In step (II), in step (I), the presence of the RNA or complementary polynucleotide bound to the probe or primer polynucleotide is determined using the above-described probe or primer polynucleotide. Measure as an indicator. In step (I), the probe or primer polynucleotide is labeled with a labeling substance such as a radioisotope or a fluorescent substance in advance and then bound to RNA or a complementary polynucleotide. Signals derived from substances can be measured with radiation detectors or fluorescence detectors.
[0065] このような本発明の診断方法は、ノーザンブロット法、 RT— PCR法、 DNAチップ解 析法、 in situハイブリダィゼーシヨンなどを利用して行うことができる。 [0066] ノーザンブロット法を利用する場合には、上述したプローブまたはプライマー用ポリ ヌクレオチドをプローブとして用いて、被験者力 採取された生体試料力 調製され た RNA中の遺伝子マーカーポリヌクレオチドの発現の有無やその発現レベルを検 出することができる。 [0065] Such a diagnostic method of the present invention can be performed using Northern blotting, RT-PCR, DNA chip analysis, in situ hybridization, and the like. [0066] When the Northern blot method is used, the above-described probe or primer polynucleotide is used as a probe, and the strength of the biological sample collected by the subject is determined whether the gene marker polynucleotide is expressed in the prepared RNA, Its expression level can be detected.
具体例を挙げると、まず、被験者カゝら調製された RNAをナイロンメンブレン等にトラ ンスファーしておき、これにあらかじめ標識してお!ヽたプローブとハイブリダィズさせる (工程 (1) )。続いて、形成されたポリヌクレオチドと RNAの二本鎖の有無を、該プロー ブの標識に由来するシグナルを指標として測定する（工程 (Π) )。なお、市販ノーザン ブロット法用キットを利用して、該キットのプロトコルにしたがって実施することも可能 である。  To give a specific example, first, RNA prepared by a subject is transferred to a nylon membrane or the like, and labeled with the probe in advance and hybridized with the probe (step (1)). Subsequently, the presence or absence of a double-stranded polynucleotide and RNA is measured using the signal derived from the probe label as an index (step (ii)). In addition, it is also possible to carry out according to the protocol of the kit using a commercially available kit for Northern blotting.
[0067] RT— PCR法を利用する場合には、上述したプローブまたはプライマー用ポリヌクレ ォチドをプライマーとして用いて、被験者から採取された生体試料から調製された R ΝΑから調製した cDNA中の遺伝子マーカーポリヌクレオチドの発現の有無やその 発現レベルを検出することができる。  [0067] When the RT-PCR method is used, the gene marker polynucleotide in cDNA prepared from R R prepared from a biological sample collected from a subject using the above-described probe or primer polynucleotide as a primer. The presence or absence of nucleotide expression and its expression level can be detected.
具体例を挙げるとまず、被験者由来の RNAから調製した cDNAを铸型として、上 記プライマーをこれとハイブリダィズさせて、常法に従 ヽ PCRを行 ヽ（工程 (I)に相当 )、得られた増幅二本鎖 DNAを検出する（工程 (Π)に相当）ことができる。なお、市販 RT—PCR用キットを利用して、該キットのプロトコルにしたがって実施することも可能 である。  As a specific example, first, cDNA prepared from RNA derived from a subject is used as a cage, the above primer is hybridized with this, and PCR is performed according to a conventional method (corresponding to step (I)). Amplified double-stranded DNA can be detected (corresponding to step (ii)). It is also possible to use a commercially available RT-PCR kit according to the protocol of the kit.
[0068] DNAチップ解析法を利用する場合は、上述したプローブまたはプライマー用ポリヌ クレオチドを 1本鎖または 2本鎖のプローブとして適当な担体に固定させてなる DNA チップを用いて、被験者から採取された生体試料から調製された RNA中の遺伝子 マーカーポリヌクレオチドの発現の有無やその発現レベルを検出することができる。 具体例を挙げると、まず、被験者カゝら調製された RNAをナイロンメンブレン等にトラ ンスファーしておき、これにあらかじめ標識してお!ヽたプローブとハイブリダィズさせる (工程 (1) )。続いて、形成されたポリヌクレオチドと RNAの二本鎖の有無を、該プロー ブの標識に由来するシグナルを指標として測定する（工程 (II) )。  [0068] When using the DNA chip analysis method, a DNA chip obtained by immobilizing the above-described probe or primer polynucleotide on a suitable carrier as a single-stranded or double-stranded probe is collected from a subject. The presence or absence of expression of a gene marker polynucleotide in RNA prepared from a biological sample can be detected. To give a specific example, first, RNA prepared by a subject is transferred to a nylon membrane or the like, and labeled with the probe in advance and hybridized with the probe (step (1)). Subsequently, the presence or absence of the double-stranded polynucleotide and RNA formed is measured using the signal derived from the probe label as an index (step (II)).
[0069] 工程 (III)にお ヽては、前記工程 (II)の測定結果から、上記抗腫瘍剤及び放射線 の併用療法感受性を診断する。測定の結果力ゝらの併用療法感受性の診断は、工程 ([0069] In the step (III), the antitumor agent and the radiation are obtained from the measurement result of the step (II). Diagnose sensitivity of combination therapy. As a result of the measurement, diagnosis of sensitivity to combination therapy
II)において測定された RNAまたは相補的ポリヌクレオチドの測定値を、被験者にお ける併用療法実施前や併用療法に対し非感受性または感受性の別の被験者におい て同様に測定された RNAまたは相補的ポリヌクレオチドの測定値や同様の測定値を 予め数値ィ匕するなどして基準としておき、これらと比較して統計学的有意差を算出す ること〖こより行うことができる。 The measured RNA or complementary polynucleotide measured in (II) is the same as the RNA or complementary polynucleotide measured in the same manner in other subjects who are insensitive or sensitive to the combination therapy before or after the combination therapy. Nucleotide measurement values and similar measurement values can be used as a reference by calculating numerical values in advance and comparing them to calculating statistical significance.
具体的には、上記したように、併用療法が施されると発現量が増加する遺伝子マー カーポリヌクレオチドを指標とした場合、発現量の増加が確認された際は併用療法感 受性が高!、と診断され、発現量の増加が確認されな!、 (発現量に変化なし)際には 併用療法感受性が低いと診断される。一方、併用療法が施されると発現量が低下す る遺伝子マーカーポリヌクレオチドを指標とした場合は、発現量の低下もしくは全く発 現しないことが確認された場合には併用療法感受性が高いと診断され、逆に発現量 の低下が確認されな！、 (発現量に変化なし)場合には併用療法感受性が低 ヽと診断 される。  Specifically, as described above, when gene marker polynucleotides whose expression level increases when combined therapy is used as an index, when the increase in expression level is confirmed, the combination therapy sensitivity is high. When no increase in the expression level is confirmed! (No change in the expression level), it is diagnosed that the sensitivity to combination therapy is low. On the other hand, when a gene marker polynucleotide whose expression level decreases when combined therapy is used as an indicator, it is diagnosed that the combination therapy is highly sensitive if it is confirmed that the expression level decreases or does not occur at all. On the contrary, the decrease in the expression level is not confirmed! In the case of (no change in the expression level), the sensitivity to combination therapy is diagnosed as low.
[0070] 次に、本発明の診断方法において前記 [3]で説明されたポリペプチドの有無を検 出対象とする場合について、以下に説明する。  [0070] Next, the case where the presence or absence of the polypeptide described in [3] above is to be detected in the diagnostic method of the present invention will be described below.
本発明の診断方法において上記 [3]で説明されたポリペプチドの有無を調べるに あたり、前記 [3]で説明された抗体を利用したウェスタンプロット法のほか、酵素免疫 法 (EIA)、ラジオアイソトープ免疫法 (RIA)、蛍光免疫法などの免疫学的検出法等 によることができる。また、抗体とポリペプチドまたは部分ペプチドとの免疫反応は、 競合法、サンドイッチ法のいずれでもよいが、このうちサンドイッチ法、特に固相抗原 または固相抗体を用いた ELISAが好まし 、。  In examining the presence or absence of the polypeptide described in [3] above in the diagnostic method of the present invention, in addition to the Western plot method using the antibody described in [3] above, enzyme immunoassay (EIA), radioisotope Immunological detection methods such as immunization (RIA) and fluorescent immunization can be used. In addition, the immune reaction between the antibody and the polypeptide or partial peptide may be either a competitive method or a sandwich method. Among these, the sandwich method, particularly an ELISA using a solid phase antigen or a solid phase antibody, is preferred.
[0071] このようなポリペプチドを検出対象とした場合の診断の具体例としては、下記の工程  [0071] Specific examples of diagnosis when such a polypeptide is a detection target include the following steps:
(i)〜 (iii)を含む診断方法を挙げることができる。  Examples of the diagnostic method include (i) to (iii).
(i)被験者力 採取された生体試料力 調製されたポリペプチドと上述した抗体とを 結合させる工程、  (i) subject force collected biological sample force a step of binding the prepared polypeptide and the above-mentioned antibody,
(ii)工程 (i)において抗体に結合したポリペプチドまたはその部分ペプチドの有無の 有無を、該抗体を指標として測定する工程 (iii)工程 (ii)の測定結果から、上記抗腫瘍剤及び放射線の併用療法感受性を診断 する工程 (ii) Step of measuring the presence or absence of the polypeptide bound to the antibody or its partial peptide in step (i) using the antibody as an index (iii) Step of diagnosing the sensitivity of the above-mentioned antitumor agent and radiation combination therapy from the measurement result of step (ii)
[0072] 工程 (i)にお 、ては、被験者力 採取された生体試料力 調製されたポリペプチドと 抗体とを結合させる。生体試料力 のポリペプチドの調製は、公知の分画、精製方法 を適宜組み合わせて行うことができる。  [0072] In the step (i), the prepared biological sample force is collected from the subject force, and the prepared polypeptide and the antibody are combined. The preparation of the biological sample strength polypeptide can be carried out by appropriately combining known fractionation and purification methods.
[0073] 工程 (ii)にお 、ては、工程 (i)にお 、て抗体に結合したポリペプチドまたはその部 分ペプチドの有無を、該抗体を指標として測定する。測定は、工程 (i)において、あら 力じめ抗体を放射性同位元素や蛍光物質などの標識物質で標識しておいて力もポリ ペプチドと結合させることにより、該標識物質に由来するシグナルを放射線検出器や 蛍光検出器で測定して行うことができる。  [0073] In step (ii), in step (i), the presence or absence of a polypeptide bound to the antibody or a partial peptide thereof is measured using the antibody as an index. In step (i), the antibody is preliminarily labeled with a labeling substance such as a radioisotope or fluorescent substance, and the force is bound to the polypeptide to detect the signal derived from the labeling substance. This can be done by measuring with a detector or a fluorescence detector.
[0074] 工程 (iii)にお ヽては、前記工程 (ii)の測定結果から、上記抗腫瘍剤及び放射線の 併用療法感受性を診断する。測定結果力ゝらの併用療法感受性の診断は、 @ (ϋ) にお 、て測定されたポリペプチドの測定値を、被験者における併用療法実施前や併 用療法に対し非感受性または感受性の別の被験者において同様に測定されたポリ ペプチドの測定値や同様の測定値を予め数値ィ匕するなどして基準としておき、これら と比較して統計学的有意差を算出することにより行うことができる。  [0074] In step (iii), the sensitivity of the combination therapy with the antitumor agent and radiation is diagnosed from the measurement result of step (ii). Measured results The diagnosis of the sensitivity to combination therapy is as follows: @ (ϋ) is used to determine the measured value of the polypeptide before subjecting the combination therapy to the subject or for other insensitivity or sensitivity to the combination therapy. It can be carried out by calculating a statistically significant difference by comparing a measured value of a polypeptide measured in the same manner or a similar measured value in advance with a numerical value as a reference.
具体的には、上記したように、併用療法が施されると発現量が増加する上記 [3]の ポリペプチドを指標とした場合、発現量の増加が確認された際は併用療法感受性が 高いと診断され、発現量の増加が確認されない (発現量に変化なし)際には併用療 法感受性が低いと診断される。一方、併用療法が施されると発現量が低下する上記 [ 3]のポリペプチドを指標とした場合は、発現量の低下もしくは全く発現しな 、ことが確 認された場合には併用療法感受性が高いと診断され、逆に発現量の低下が確認さ れな！ヽ (発現量に変化なし)場合には併用療法感受性が低！ヽと診断される。  Specifically, as described above, when the polypeptide of [3] above, whose expression level increases when combined therapy is used as an index, when the increase in expression level is confirmed, the combination therapy is highly sensitive. When the increase in the expression level is not confirmed (no change in the expression level), it is diagnosed that the combination therapy sensitivity is low. On the other hand, when the polypeptide of [3] above, whose expression level decreases when combined therapy is used as an index, is confirmed that the expression level decreases or is not expressed at all. However, the decrease in the expression level is not confirmed!併 用 In the case of no change in the expression level, the combination therapy sensitivity is low! Diagnosed as sputum.
[0075] 本発明の診断方法を実施することにより、被験者における上記併用療法の感受性 が診断でき、それにより上記併用療法の継続可能性及び Zまたは実施可能性を予 測できる。  [0075] By carrying out the diagnostic method of the present invention, the sensitivity of the combination therapy in a subject can be diagnosed, whereby the continuity and Z or feasibility of the combination therapy can be predicted.
すなわち、被験者において既に上記併用療法が施されている場合、本発明診断方 法により得られる併用療法感受性から、さらに上記併用療法を継続するべき力否力を 判断できる。 That is, when the above-mentioned combination therapy has already been given in the subject, the power to use the above combination therapy further depends on the sensitivity of the combination therapy obtained by the diagnostic method of the present invention. I can judge.
また、被験者において上記併用療法が未実施である場合、本発明診断方法により 得られる併用療法感受性から、上記併用療法を実施するべきか否カゝを予測できる。 継続可能性及び Zまたは実施可能性が高 、患者にっ 、てのみ上記併用療法を実 施することにより、該患者は適正な治療を受けることができ、ひいては、医療全体にお ける治療効率および経済的な面力 も有用である。  In addition, when the above combination therapy is not performed in the subject, whether or not the above combination therapy should be performed can be predicted from the combination therapy sensitivity obtained by the diagnostic method of the present invention. By providing the above combination therapy only to a patient who is highly likely to continue and Z or feasible, the patient can receive appropriate treatment, and thus the treatment efficiency in the overall medical care and Economic power is also useful.
実施例  Example
[0076] 以下、実施例に基づき、本発明についてさらに詳細に説明する。なお、本発明は下 記実施例に限定されるものではな 、。  Hereinafter, the present invention will be described in more detail based on examples. The present invention is not limited to the following examples.
[0077] 実施例 1 [0077] Example 1
以下の手順に従 、、遺伝子マーカーとして機能する可能性の高 、ポリヌクレオチド の調製、検索を行った。  According to the following procedure, a polynucleotide having a high possibility of functioning as a genetic marker was prepared and searched.
[0078] 1. 30種の検体群の調製 [0078] 1. Preparation of 30 sample groups
ヌードマウス皮下移植ヒト肺癌株から、 30種の検体群を、以下の条件で調製した。 尚、本実施例にぉ ヽて使用したヌードマウス皮下移植ヒト肺癌株は併用療法感受性 陽性であることが予め確認されて ヽる。  Thirty specimen groups were prepared from human lung cancer lines transplanted subcutaneously into nude mice under the following conditions. It should be noted that it has been confirmed in advance that the nude mouse subcutaneously transplanted human lung cancer strain used in the present example is positive for combination therapy sensitivity.
[0079] 〔被験液の調製〕 [0079] [Preparation of test solution]
下記の試験にぉ 、て被験液として用いた S— 1薬液は、以下のようにして調製した。 すなわち、まず、テガフール、ギメラシルおよびォキソン酸カリウムをそれぞれ 0. 83 mg/mU 0. 25mgZml、0. 82mgZmlになるように 0. 5%ヒドロキシプロピルメチ ルセルロース (HPMC)溶液に懸濁し、室温で約 10分間攪拌した。その後、氷冷下 で超音波処理して、用量 8. 3mgZkgZdayの S— 1薬液を得た。なお、この S— 1薬 液の用量は、マウスに 14日間経口投与したときの最大無毒性用量である。  The S-1 drug solution used as the test solution for the following tests was prepared as follows. That is, first, tegafur, gimeracil, and potassium oxalate were suspended in a 0.5% hydroxypropyl methylcellulose (HPMC) solution at 0.83 mg / mU 0.25 mgZml and 0.82 mgZml, respectively, and at about room temperature. Stir for 10 minutes. Then, it was sonicated under ice-cooling to obtain a dose of 8.3 mg ZkgZday S-1 drug solution. This dose of S-1 is the maximum non-toxic dose when orally administered to mice for 14 days.
[0080] 〔放射線の照射条件〕 [0080] [Radiation irradiation conditions]
下記の試験における放射線 (X線)の照射条件は、以下の通りとした。  Radiation (X-ray) irradiation conditions in the following tests were as follows.
日立メディコ社製の MBR— 1505R2型放射線照射装置を用いて、マウスあたり 1 回の照射が 2Gyおよび 5Gyとなるように照射条件 (照射位置)を設定し、マウスの右 大腿部に移植したヒト腫瘍株に対して局所照射を行った。照射の際、全身照射を避 けるため、鉛で作られた収納箱にマウスを入れて右足のみ放射線に暴露されるように した。 Using an MBR-1505R2 radiation irradiation device manufactured by Hitachi Medical Corporation, set the irradiation conditions (irradiation position) so that one irradiation per mouse is 2 Gy and 5 Gy, and transplanted to the right thigh of the mouse Local irradiation was performed on the tumor lines. Avoid whole-body irradiation during irradiation. In order to avoid this, the mouse was placed in a storage box made of lead so that only the right foot was exposed to radiation.
[0081] 〔投与試験〕  [0081] [Administration study]
生後 5〜6週齢の BALBZcA— nuマウスの右大腿部に、前もって同系統のマウス 背部皮下に移植して増殖させて 、たヒト肺癌株 (LC— 11および Lu— 99)を摘出して 生理食塩液中で約 2mm角にハサミで細片化したものを、移植針を使って皮下移植 して、少なくとも 1〜2週間飼育した。その後、各群（1群 6匹)の腫瘍体積の平均およ び標準偏差 (S. D. )ともできる限り均等になるように 4群に分けた後、それぞれを対 照群 (Control)、放射線単独群 (X ray)、薬剤単独群 (S— 1)および薬剤と放射線 併用群（S— 1 +X my)と位置づけ、さらに、処置期間により 3群又は 4群に分け計 3 0検体群として、各群に薬剤投与および放射線照射を開始した。表 1に 30検体群そ れぞれの由来株、処置内容及び処置期間との対応関係を「參」で示した。  Human lung cancer strains (LC-11 and Lu-99) were isolated by transplanting to the right thigh of BALBZcA-nu mice aged 5-6 weeks under the skin of the back of mice of the same strain in advance. A piece of scissors approximately 2 mm square in physiological saline was transplanted subcutaneously using a transplant needle and bred for at least 1 to 2 weeks. After that, each group (6 mice per group) was divided into 4 groups so that the mean and standard deviation (SD) of the tumor volume were as uniform as possible, and then each was divided into the control group and the radiation alone group. (X ray), drug alone group (S-1) and drug and radiation combination group (S-1 + X my), and further divided into 3 groups or 4 groups depending on the treatment period. The group began drug administration and radiation. Table 1 shows the relationship between the strains derived from each of the 30 specimen groups, treatment details, and treatment periods in “參”.
薬剤投与群、および薬剤と放射線併用群には、体重 lkgに対し上記の S—1薬液 をそれぞれ 8. 3mlの割合で、 1日 1回、表 1に示す処置期間経口投与用ゾンデを用 いて経口投与した。放射線照射群、および薬剤と放射線併用群には、試験開始の 1 日目と 8日目において、 S—1薬剤を投与した後の約 1時間以内に、上記放射線の照 射条件で説明した方法に従って 2Gyを照射した。対照群 (非放射線照射、非薬剤投 与群）および放射線照射単独群の担癌マウスには、 0. 5%HPMC液のみを、 S- 1 薬液の場合と同じ条件に従って表 1に示す処置期間経口投与した。  In the drug administration group and the drug and radiation combination group, the above-mentioned S-1 drug solution was used at a rate of 8.3 ml per 1 kg body weight, once a day, using an oral sonde for the treatment period shown in Table 1. Orally administered. In the irradiation group and the drug and radiation combination group, on the first and eighth days of the study, the method described under the above irradiation conditions within about 1 hour after administration of the S-1 drug. 2Gy was applied according to the above. For the tumor-bearing mice in the control group (non-irradiated, non-drug-administered group) and in the irradiation-only group, only 0.5% HPMC solution is treated according to the same conditions as in the S-1 drug solution. Orally administered.
[0082] [表 1] [0082] [Table 1]
Figure imgf000029_0001
Figure imgf000029_0001
[0083] そこで、各群から、試験開始の 2日目、 9日目、 12日目、および 15日目の腫瘍組織 を採取し、表 1に示す由来株別、処置内容別、および処置期間別に分類される 30種 類の検体として、以下の工程に供した。 [0083] Therefore, tumor tissues were collected from each group on the second, ninth, twelfth, and fifteenth days of the start of the study, and the origin strains, treatment details, and treatment periods shown in Table 1 were collected. Thirty kinds of specimens classified separately were used for the following processes.
[0084] 2. cDNAライブラリ作製部  [0084] 2. cDNA library preparation section
2. 1 概略  2.1 Overview
がん細胞株 ·処置方法及処置時間別に分類される 30種の検体群 (表 1参照）につ いて、各群から RNAを抽出し、 polyA+RNAを調製した。  For 30 sample groups (see Table 1) classified by cancer cell line, treatment method, and treatment time, RNA was extracted from each group to prepare polyA + RNA.
その後、 30検体群から処置方法別に混合グループを調製し、 Control群 (非放射 線照射、 S— 1非投与)、 S— 1処理群 (非放射線照射、 S— 1投与)、および放射線処 理群 (放射線照射、 S— 1非投与)、および併用処理群 (放射線照射、 S— 1投与)とし て 4つの cDNAライブラリを作成した。  Thereafter, mixed groups were prepared from the 30 sample groups according to the treatment method, and the Control group (non-radiation irradiation, S-1 non-administration), S-1 treatment group (non-irradiation, S-1 administration), and radiation treatment Four cDNA libraries were prepared for the group (irradiation, S-1 non-administration) and the combination treatment group (irradiation, S-1 administration).
[0085] 2. 2 Total RNA抽出 [0085] 2.2 Total RNA extraction
前述の工程 1で得られた 30種の検体群 (表 1)を、それぞれ、液体窒素中にて凍結 し、凍結状態のままで破砕し粉末化した。その後、 Trizol Reagent (Invitrogen社) を、粉末 50mgあたり lmL添カ卩してホモジェナイズし、 Total RNA (以下、 RNA試 料という。）を抽出した。抽出は、同製品のプロトコルに従って行った。 The 30 specimen groups (Table 1) obtained in Step 1 above were each frozen in liquid nitrogen, crushed and powdered in the frozen state. Then Trizol Reagent (Invitrogen) This was homogenized by adding 1 mL per 50 mg of powder, and total RNA (hereinafter referred to as RNA sample) was extracted. Extraction was performed according to the protocol of the product.
[0086] 2. 3 Proteinase K処理 [0086] 2.3 Proteinase K treatment
工程 2. 2で得られた RNA試料中の RNA分解酵素を不活ィ匕するために、 RNA試 料を Proteinase K処理した。すなわち、 RNA試料 lmgに対し、 Proteinase K(l nvitrogen社)を 0. カロえ、 37°Cで 1時間反応させることで RNA分解酵素を不活 化させた後、 60°Cに 10分間加熱して Proteinase Kを失活させた。  In order to inactivate the RNase in the RNA sample obtained in step 2.2, the RNA sample was treated with Proteinase K. That is, proteinase K (l nvitrogen) was added to 1 mg of RNA sample and reacted at 37 ° C for 1 hour to inactivate RNase and then heated to 60 ° C for 10 minutes. Proteinase K was inactivated.
その後、フエノール処理およびエタノール沈澱により得られた RNA試料をさらに以 下の工程に供した。  Thereafter, the RNA sample obtained by phenol treatment and ethanol precipitation was further subjected to the following steps.
[0087] 2. 4 DNase I処理 [0087] 2.4 DNase I treatment
精製 RNA試料に混入して ヽる可能性のあるゲノム DNAを除去するために、工程 2 . 3で得られた試料を DNA分解酵素（DNase I：タカラバィォ社)処理した。すなわ ち、精製 RNA試料 lmgに対し、 DNase Iを 1ユニット加え、 37°Cで 30分反応させた 。反応終了後、フエノール処理およびエタノール沈澱により RNAを回収した。  In order to remove genomic DNA that could be mixed into the purified RNA sample, the sample obtained in step 2.3 was treated with a DNA-degrading enzyme (DNase I: Takara Bio). That is, 1 unit of DNase I was added to 1 mg of the purified RNA sample and reacted at 37 ° C for 30 minutes. After completion of the reaction, RNA was recovered by phenol treatment and ethanol precipitation.
こうして回収された RNAを、 DNase I処理済の Total RNA試料として、以下のェ 程に供した。  The RNA thus recovered was subjected to the following steps as a DNase I-treated total RNA sample.
[0088] 2. 5 polyA+RNA抽出 [0088] 2.5 polyA + RNA extraction
mRNAの polyA配列とオリゴ dTとの親和性を利用して、工程 2. 4で得られた DNa se I処理済の Total RNA試料から polyA+RNAを抽出した。ォ  Using the affinity between mRNA polyA sequence and oligo dT, polyA + RNA was extracted from the DNase I-treated total RNA sample obtained in step 2.4. O
リゴ dTは Oligotex— dT30 (タカラバイオ社）を利用し、 mRNAの吸着及抽出を行な つた。なお操作手順は同製品のプロトコルに従って行った。 mRNAは抽出後、ェタノ ール沈澱により精製した。  Rigo dT used Oligotex—dT30 (Takara Bio) to absorb and extract mRNA. The operation procedure was performed according to the protocol of the product. The mRNA was purified by ethanol precipitation after extraction.
こうして 30種類の検体群（表 1参照）力も得られた polyA+RNAを、表 2の「混合グ ループ」に示すように、 Control, S— 1処理、放射線処理、 S— 1および放射線併用 処理の処置別に、各検体群について 2 gずつ混合調製し、 4つの混合グループ (C ontrol群、 S— 1処理群、放射線処理群、併用処理群）とした。 30種類の検体群と 4 つの混合グループとの関係を、表 2に示す。なお、表 2中の「參」は、各検体群の由来 株、処置内容及び処置期間の対応を示すものである。 [0089] [表 2] PolyA + RNA, which has obtained the strength of 30 sample groups (see Table 1), is treated with Control, S-1 treatment, radiation treatment, S-1 and radiation treatment as shown in Table 2 “Mixed group”. For each treatment, 2 g of each sample group was mixed and prepared, and four mixed groups (Control group, S-1 treatment group, radiation treatment group, combined treatment group) were prepared. Table 2 shows the relationship between the 30 sample groups and the 4 mixed groups. Note that “參” in Table 2 indicates the correspondence of the derived strain, treatment content, and treatment period of each sample group. [0089] [Table 2]
表 2  Table 2
Figure imgf000031_0001
Figure imgf000031_0001
[0090] 2. 6 First strand合成 ' cDN Aライブラリ作成の概略  [0090] 2.6 First strand synthesis '' Outline of cDN A library creation
工程 2. 5にて混合調製された 4つの各混合グループ（表 2)の polyA+RNAを铸 型として、オリゴ dTプライマーを使用し、 First strand合成および cDNAライブラリ 作成を行った。 cDNAライブラリ作成までの以下の一連の操作は、 cDNA Constru ction Kit (タカラバイオ社)を利用して、同製品の操作マニュアルに従って実施した こうして 4混合グループのそれぞれカゝら得られた 4種の cDNAライブラリを、以下の ように命名した。すなわち、 Control群は TH05C、 S— 1処理群は TH05S、放射線 処理群は TH05X、併用処理群は TH05SXと呼ぶ（図 1参照）。  First strand synthesis and cDNA library creation were performed using oligo dT primers using the polyA + RNA of each of the four mixed groups (Table 2) prepared in Step 2.5 as a template. The following sequence of operations up to the creation of the cDNA library was performed using the cDNA Construction Kit (Takara Bio Inc.) according to the operation manual of the product. Thus, the four cDNAs obtained from each of the four mixing groups were obtained. The library was named as follows. That is, the Control group is called TH05C, the S-1 treatment group is TH05S, the radiation treatment group is TH05X, and the combination treatment group is TH05SX (see Figure 1).
[0091] 2. 7 Not I + EcoRl切断 cDNAの作製 *精製 [0091] 2.7 Construction of Not I + EcoRl-cleaved cDNA * Purification
工程 2. 5にて混合調製された 4つの各混合グループの polyA+RNAそれぞれを 铸型に、 cDNA Construction Kit (タカラバイオ社）の操作手順に従い、制限酵 素 Not I認識配列を含むオリゴ dTプライマーと 5— methyl dCTPを使用した First strand合成、 DNA polymerase I、 E. coli RNase H、 E. coli DNA Ligas eを使用した Second strand合成、及び T4 DNA polymeraseを用いた末端構 造の平滑化を行なった。ついでこれに対し、 Kitに付属の EcoRI— Sma I Adapto rを T4 DNA Ligaseにより連結させた。こうして得られた cDNA断片の両末端には 制限酵素 NotI、 EcoRIの認識部位が存在する。この cDNA断片に対し制限酵素 No t I (5ユニット）と EcoRI (5ユニット）で 37°C、 1時間の処理を行なった。その後、ゲル ろ過カラム（インビトロジェン社、商品名： cDNA Size Fraction Column)を用い て、同製品操作マニュアルに従いサイズ分画を行い、電気泳動と 260nmの吸光度を 指標として 500bp付近の cDNAを抽出した。更に、エタノール沈澱を行って、その両 末端が Not Iおよび Eco RIで切断されている cDNA断片を精製した (以下、精製 c DNA試料）。 Oligo dT primer containing restriction enzyme Not I recognition sequence in the shape of each polyA + RNA of each of the four mixed groups prepared in Step 2.5 according to the procedure of cDNA Construction Kit (Takara Bio Inc.) And 5-strand first strand synthesis using methyl dCTP, second strand synthesis using DNA polymerase I, E. coli RNase H, E. coli DNA Ligase, and T4 DNA polymerase. The structure was smoothed. Subsequently, EcoRI—Sma I Adaptor included in the kit was ligated with T4 DNA Ligase. There are recognition sites for the restriction enzymes NotI and EcoRI at both ends of the cDNA fragment thus obtained. This cDNA fragment was treated with restriction enzymes Not I (5 units) and EcoRI (5 units) at 37 ° C for 1 hour. After that, using a gel filtration column (Invitrogen, trade name: cDNA Size Fraction Column), size fractionation was performed according to the operation manual of the same product, and cDNA around 500 bp was extracted using electrophoresis and absorbance at 260 nm as indices. Further, ethanol precipitation was performed to purify a cDNA fragment having both ends cleaved with Not I and Eco RI (hereinafter referred to as a purified cDNA sample).
[0092] 2. 8 ベクターの準備  [0092] 2.8 Vector preparation
pBlueScript II KS (—）（ストラタジーン社）ベクターを、制限酵素 Not Iおよび EcoRIで処理した。 1 %ァガロースを用いた電気泳動を実施後、ベクターを切り出し 、精製を行った。  pBlueScript II KS (—) (Stratagene) vector was treated with restriction enzymes Not I and EcoRI. After electrophoresis using 1% agarose, the vector was excised and purified.
[0093] 2. 9 ベクターと cDNAの Ligationと形質転換（図 1)  [0093] 2.9 Ligation and transformation of vector and cDNA (Figure 1)
工程 2. 7にて得られた 4種の精製 cDNA試料を各々、工程 2. 8にて準備したベタ ターに、 Ligation High (TOYOBO社）を用いて、同製品の操作マ-ユアノレに従 ヽ 、 Ligationし、挿入した。得られた 4種の Ligation産物はそれぞれ大腸菌 DH5 a (T OYOBO社）に形質転換し、寒天培地に塗沫し 37°Cで一晩培養後、大腸菌のコロ- 一を形成させた。培地組成は、 15gバタトァガー/ L、 10gトリプトン ZL、 5gイースト エキストラタト ZL、 100 /z gアンピシリン ZmLとした。個々のコロニー（大腸菌クロー ン）は、それぞれ 1種類の cDNA断片が挿入された (組み込まれた)ベクターを保持し ている。こうして、 4種 cDNAライブラリに対しそれぞれ形質転換と大腸菌コロニーの 形成を行なうことにより、 4種類のライブラリ由来のクローンが得られた。  Using the Ligation High (TOYOBO) for each of the 4 types of purified cDNA samples obtained in Step 2.7, follow the operation manual of the product. Ligation and inserted. Each of the four Ligation products obtained was transformed into Escherichia coli DH5a (T OYOBO), smeared on an agar medium, and cultured overnight at 37 ° C to form E. coli colonies. The medium composition was 15 g Batatoguar / L, 10 g Tryptone ZL, 5 g East Extraato ZL, 100 / z g Ampicillin ZmL. Each colony (E. coli clone) carries a vector with one kind of cDNA fragment inserted (incorporated). Thus, clones derived from four types of libraries were obtained by transforming and forming E. coli colonies respectively for the four types of cDNA libraries.
[0094] 3 DNAシーケンス  [0094] 3 DNA sequencing
3. 1 概略  3.1 Overview
前述の工程 2で得られた 4種のライブラリ由来のクローンを対象としてシーケンシン グを実施した。すなわち、各ライブラリ由来の大腸菌クローンを整列化した後、ベクタ 一プライマーを利用して PCR増幅を行なうことで、挿入されて!、る cDNA断片を増幅 した。その後、増幅 cDNA断片を精製し、これを铸型としたシーケンス反応を行い、 配列決定を行った。 Sequencing was performed on the clones derived from the four types of libraries obtained in Step 2 above. That is, after aligning E. coli clones from each library, PCR amplification is performed using a single vector primer to amplify the inserted cDNA fragment. did. Thereafter, the amplified cDNA fragment was purified, and a sequencing reaction was performed using this as a saddle shape, followed by sequencing.
[0095] 3. 2 コロニーピックアップ.培養（図 1) [0095] 3.2 Colony pickup culture (Fig. 1)
工程 2. 9にて得られた寒天プレート毎に、寒天プレート上に生じたコロニーを、コロ ニーピック装置 Q— PIX(Genetix社）を使用して、予め LB培地分注済みの 96マル チウエルプレートに植菌 ·整列化する。 37°Cにて一晩震とう培養した。これは整列化 して再培養することで各クローン間における増殖のばらつきを押さえ、以下の作業に おける各ゥエル間の差を低減させるためである。これ以降の一連の反応は、 96マル チウエルプレートを利用して行なった。  For each agar plate obtained in step 2.9, colonies formed on the agar plate were colonized on the LB medium in advance using a colony pick device Q—PIX (Genetix). Inoculate and align. Shake overnight at 37 ° C. This is to reduce the difference between each well in the following work by suppressing the variation in growth among clones by arranging and re-culturing. Subsequent series of reactions were performed using 96 multiwell plates.
[0096] 3. 3 铸型の直線化（図 1) [0096] 3.3 Rabbit linearization (Figure 1)
工程 3. 2で得られたマルチウエルプレートの各ゥエル内の培養液を、 98°C3分間加 熱処理し、大腸菌の細胞膜を破壊した。菌の加熱処理済み液 (ライゼート液）に含ま れるプラスミド DNAを铸型として、 pBlueScript II KS (—）ベクターのマルチクロ 一ユング部位近傍配列由来配列のプライマー (KS— F、 KS-R)を利用して PCR反 応を行い、挿入 cDNA断片を増幅した。 PCRの反応液組成は、菌ライゼート液； 10 μ L、 10XPCRバッファー；5 L、プライマー；各 10pmol、 dNTPs ;各 20nmol、 EX The culture solution in each well of the multiwell plate obtained in step 3.2 was heat-treated at 98 ° C. for 3 minutes to destroy the cell membrane of E. coli. The plasmid DNA contained in the heat-treated solution (lysate solution) of the fungus is used as a saddle, and the primers (KS—F, KS-R) derived from the sequence near the multi-ring region of the pBlueScript II KS (—) vector are used. PCR reaction was performed to amplify the inserted cDNA fragment. PCR reaction solution composition: fungus lysate solution; 10 μL, 10X PCR buffer; 5 L, primer; 10 pmol each, dNTPs; 20 nmol each, EX
Taq (タカラバイオ社）； 1ユニットに対し滅菌水を添加して 50 μ Lとした。 また、 PCRのサーマルサイクルは、 94°C · 30秒→60°C · 30秒→70°C · 1分を 30回 繰り返した。 Taq (Takara Bio Inc.); Sterilized water was added to 1 unit to make 50 μL. The PCR thermal cycle was 94 ° C · 30 seconds → 60 ° C · 30 seconds → 70 ° C · 1 minute 30 times.
[0097] 3. 4 シーケンス反応（図 1) [0097] 3.4 Sequence reaction (Figure 1)
工程 3. 3にて増幅'精製した cDNA断片を铸型としてシーケンス反応を行った。標 識には、 BigDyeターミネータ vl. 1 (アプライドバイオシステムズ社)を使用し、同製 品の操作マニュアルに従 、反応を行った。  Sequencing reaction was performed using the cDNA fragment amplified and purified in step 3.3 as a cage. For labeling, BigDye terminator vl. 1 (Applied Biosystems) was used, and the reaction was carried out according to the operation manual of the product.
シーケンス反応産物は、 SephadexG— 50 (Amersham社）を使用して、同製品の 操作マニュアルに従 、精製した。  The sequence reaction product was purified using Sephadex G-50 (Amersham) according to the operation manual of the product.
[0098] 3. 5 泳動 ·配列決定（図 1)  [0098] 3.5 Electrophoresis · Sequencing (Figure 1)
工程 3. 4において得られた精製シーケンス反応産物について、 ABI3730xl (ァプ ライドバイオシステムズ社)を使用して、同製品の操作マニュアルに従い、泳動'配列 決定を行なった。 Use the ABI3730xl (Applied Biosystems) for the purified sequencing reaction product obtained in step 3.4 according to the operation manual for the product. A decision was made.
その結果、 TH05SX (併用処理群）、 TH05S (S— 1処理群）、 TH05C (Control 群）および TH05X(放射線処理群）のそれぞれのライブラリ毎にクローンの配列デー タを得た。  As a result, clone sequence data was obtained for each library of TH05SX (combination treatment group), TH05S (S-1 treatment group), TH05C (Control group) and TH05X (radiation treatment group).
[0099] 4.配列ァノテーシヨン処理 [0099] 4. Sequence annotation processing
4. 1 概略  4.1 Overview
上記 DNAシーケンス部にお!、て得られた各クローンの配列データにつ!、て、まず 、品質トリミング、ベクタートリミング、クロスコンタミネーシヨンの確認 (検出'排除)等か らなる一連の配列クリーニング処理を行な 、、挿入配列の切り出し (抽出）を行なった 。次に、得られた挿入配列を基に配列ァノテーシヨン処理を行ない、各クローンが既 知遺伝子であるか、あるいは新規遺伝子であるかの判定を行うと共に、由来遺伝子 種および由来生物種 (ヒトかマウスの 、ずれか）の特定を行なった。  The sequence data of each clone obtained in the above DNA sequencing section! First, a series of sequence cleaning consisting of quality trimming, vector trimming, cross-contamination confirmation (detection 'exclusion), etc. After processing, the inserted sequence was cut out (extracted). Next, sequence annotation is performed based on the obtained insertion sequence to determine whether each clone is a known gene or a new gene, and the derived gene species and the derived organism species (human or mouse). Of the difference).
配列ァノテーシヨン処理の手順の概略を図 2に示す。  Figure 2 shows the outline of the sequence annotation process.
[0100] 4. 2 配列クリーニング処理 [0100] 4.2 Array cleaning process
4. 2. 1 概略  4.2.1 Overview
配列ァノテーシヨン処理の前に、配列クリーニング処理を行い、上記 DNAシーケン ス部において得られた各クローンについて、シーケンサーから出力された配列データ を処理し、配列ァノテーシヨン処理の対象としての cDNA断片の抽出を行った。 cDN A断片の配列を同定するにあたり、クローユングベクターの配列を取り除く必要がある ので、例えば、 Chou HH, Holmes MH. ； DNA sequence quality trimmin g and vector removal. Bioinformatics. 2001 Dec ; 17 (12) :1093- 1104 .の記載を参考にして、ベクタートリミング処理（vector trimmingあるいは vector removal)を行うと共に、低品質配列の除去のための品質トリミング処理（quality tri mming)、クロスコンタミネーシヨンの検出'排除をも併せて行なった。  Prior to the sequence annotation process, a sequence cleaning process is performed, the sequence data output from the sequencer is processed for each clone obtained in the above DNA sequence section, and the cDNA fragment as the target of the sequence annotation process is extracted. It was. Since it is necessary to remove the cloning vector sequence in order to identify the cDN A fragment sequence, for example, Chou HH, Holmes MH .; DNA sequence quality trimming and vector removal. Bioinformatics. 2001 Dec; 17 (12): Refer to the description of 1093- 1104. Perform vector trimming or vector removal, quality trimming for removing low quality sequences, and detection and elimination of cross contamination. Was also performed.
[0101] 4. 2. 2 品質トリミング処理  [0101] 4.2.2 Quality trimming process
各配列データについて、読み取り配列の先頭と終端付近に存在する品質の不安定 な塩基配列を、それぞれ両側から取り除き、配列として確度の高い部分を抽出した。  For each sequence data, the unstable base sequences present near the beginning and end of the reading sequence were removed from both sides, and a highly accurate portion was extracted as a sequence.
[0102] 4. 2. 3 ベクタートリミング処理 クロー-ングベクター（pBluescript II KS (—））の配列を取り除くことにより、 cD NA断片を抽出した。このとき、想定されるクローユング部位が見当たらない、あるい はクロー-ング部位に cDNA断片の配列が確認できな 、、等の不具合を持つ配列 データを、この処理段階で排除した。 [0102] 4.2.3 Vector trimming process The cDNA fragment was extracted by removing the sequence of the cloning vector (pBluescript II KS (—)). At this time, sequence data having defects such as the expected cloning site not being found or the sequence of the cDNA fragment not being confirmed at the cloning site were excluded at this processing stage.
[0103] 4. 2. 4 クロスコンタミネーシヨンの検出.排除  [0103] 4.2.4 Cross contamination detection and elimination
ベクタートリミング処理により抽出された cDNA断片の配列の品質を評価し、クロス コンタミネーシヨンの有無を判定した。すなわち、複数のクローンが混入している恐れ のある配列データについて、この処理の段階で検出'排除し、以後の配列ァノテーシ ヨン処理の対象から排除した。  The quality of the cDNA fragment sequence extracted by vector trimming was evaluated, and the presence or absence of cross-contamination was determined. That is, sequence data that may contain a plurality of clones was detected and excluded at the stage of this processing, and excluded from the target of subsequent sequence annotation processing.
[0104] 4. 3 配列ァノテーシヨン処理  [0104] 4.3 Array annotation processing
4. 3. 1 概略  4.3.1 Overview
配列クリーニング処理の結果、品質に問題がな 、と判定された cDNA断片力 なる 配列データについて、遺伝子および生物種の由来を特定すベぐ以下の手順で配 列ァノテーシヨン処理を行った。  As a result of the sequence cleaning process, the sequence annotation process was performed for the sequence data that was determined to have no quality problem by the following procedure for identifying the origin of genes and species.
ここで、出発材料として、ヌードマウス皮下移植ヒト肺がん株、いわゆる xenograft組 織を用いたため、ヒト由来のクローンの配列データだけでなくマウス由来のクローンの 配列データが混在しているおそれがあるため、ヒトおよびマウスのいずれに由来する 配列であるかの生物種判定も併せて行なった。  Here, since the human lung cancer strain transplanted subcutaneously into nude mice, the so-called xenograft organization, was used as a starting material, not only human clone sequence data but also mouse clone sequence data may be mixed. Biological species determination of whether the sequence was derived from human or mouse was also performed.
本処理にお!、ては、ヒト既知遺伝子 mRNA配列データベースおよびマウス既知遺 伝子 mRNA配列データベースを使用した。なお、本処理において使用した既知遺 伝子配列データベースは、 2004年 10月 8日版の GenBank, RefSeq, LocusLink である。  In this treatment, human known gene mRNA sequence database and mouse known gene mRNA sequence database were used. The known gene sequence databases used in this process are GenBank, RefSeq, and LocusLink of October 8, 2004.
[0105] 4. 3. 2 既知遺伝子配列データベースとの相同性 (homology)検索（遺伝子名の 特定）  [0105] 4.3.2 Homology search with known gene sequence database (specification of gene name)
前述の工程 4. 2で得られた cDNA断片の配列データについて、 NCBIより入手可 能なヒト既知遺伝子配列データよびマウス既知遺伝子配列データに対して、転写物（ mRNA)での相同性検索を行なった。得られた検索結果を分析して 4. 2で得られた cDNA断片の配列データが対応付く遺伝子を特定した。この段階で遺伝子が単一 生物種の単一遺伝子種に一意に特定された配列は、遺伝子だけでなく由来生物種 も決定できたものと判断され、その結果がァノテーシヨンされた。 For the cDNA fragment sequence data obtained in step 4.2 above, a homology search using transcripts (mRNA) is performed on human known gene sequence data and mouse known gene sequence data available from NCBI. It was. The obtained search results were analyzed, and the gene corresponding to the sequence data of the cDNA fragment obtained in 4.2 was identified. Single gene at this stage It was judged that the sequence uniquely identified as a single species of the species could determine not only the gene but also the derived species, and the result was annotated.
このァノテーシヨンを用いて、各 cDNAライブラリ由来のクローン力 ヒトの遺伝子で あり、かつ既知遺伝子ではないと判断できた配列データ、すなわち新規遺伝子候補 となるクローンの配列データが得られた。  Using this annotation, we obtained sequence data that was determined to be a clone of a human gene and not a known gene, that is, a clone as a candidate for a new gene, from each cDNA library.
[0106] 5.ゲノムデータをもちいた新規遺伝子の同定（図 3) [0106] 5. Identification of novel genes using genomic data (Figure 3)
5. 1 概略  5.1 Overview
上記工程 4において、公開データベースに登録された mRNAと比較し、既知の遺 伝子に対応するクローンの配列データを除いた力 当該工程では、ゲノムのレベル で公開データベースの既知遺伝子の位置とクローンの配列データのクラスターの位 置を照合し、既知遺伝子に対応するクローンの配列データを除き、新規遺伝子候補 を選別する。  Compared with mRNA registered in the public database in step 4 above, the ability to exclude sequence data of clones corresponding to known genes In this step, the positions of known genes and clones in the public database at the genome level The position of the cluster of the sequence data is collated, the sequence data of the clone corresponding to the known gene is excluded, and new gene candidates are selected.
[0107] 5. 2 ゲノムデータへのマッピング解析  [0107] 5.2 Analysis of mapping to genomic data
工程 4で得られた新規遺伝子候補となる各クローンの配列データの中から、ヒトゲノ ムのデータにユニークにマッピングされるクローンを抽出した。すなわち、各クローン の配列データをクエリーとして、 Eバリューを le— 100に設定した Blast処理を実施し From the sequence data of each clone that was a novel gene candidate obtained in step 4, clones that were uniquely mapped to human genome data were extracted. In other words, Blast processing with the E value set to le-100 was performed using the sequence data of each clone as a query.
、クローンの配列データとゲノム配列に相同性が 98%以上有り、且つ、 1つの染色体 又はミトコンドリアの 1方向の 1遺伝子座部位のみにマッピングされているクローンを抽 出した。ヒトゲノムデータとしては、データベース NCBI reference Build35. 1 (各 染色体とミトコンドリアの代表ゲノム配列）を用いた。ゲノムデータへのマッピング解析 については、 McGinnis S. , Madden T. L. ；" BLAST: at the core of a powerful and diverse set of sequence analysis tools. Nucleic AcidsA clone having a homology of 98% or more between the clone sequence data and the genome sequence and mapped to only one locus site in one direction of one chromosome or mitochondria was extracted. As human genome data, the database NCBI reference Build 35.1 (representative genome sequence of each chromosome and mitochondria) was used. For analysis of mapping to genomic data, see McGinnis S., Madden T. L .; "BLAST: at the core of a powerful and diverse set of sequence analysis tools. Nucleic Acids
Res. 2004 ; 32 :W20-W25.の記載力 S参照される。 Res. 2004; 32: Descriptive power of W20-W25.
[0108] 5. 3 新規遺伝子候補抽出  [0108] 5.3 New gene candidate extraction
上記工程 5. 2で得られたクローンの配列データについて、ゲノム上のクラスターを 計算する。得られたクラスターのゲノム上の位置情報は、新規遺伝子候補の遺伝子 座部位と予測できる。クラスターを構成するクローン (新規遺伝子候補)の中で、ゲノ ムのデータにマップされる部分が最も長いクローンをクラスターの代表配列とする。 [0109] これらの解析の結果、 TH05SXのライブラリのみから獲得されたが他の 3つのライ ブラリからは獲得されなかったクローンは、 33個（配列表の配列番号 1〜33参照）で あり、一方、 TH05SXのライブラリのみ力も獲得されな力つたが他の 3つのライブラリ のいずれからも獲得されたクローンは、 3個（配列表の配列番号 34〜36参照）であつ た。前者のクローン 33個は、 S—1及び放射線併用療法実施後に特異的に発現量が 増加する遺伝子マーカーとして有用であり、一方、後者のクローン 3個は、上記併用 療法実施後に発現量が低下する遺伝子マーカーとして有用性が高いものと考えられ る。 For the sequence data of the clone obtained in step 5.2 above, calculate the cluster on the genome. The position information on the genome of the obtained cluster can be predicted as the locus site of a new gene candidate. Among the clones (new gene candidates) constituting the cluster, the clone with the longest portion mapped to the genomic data is used as the representative sequence of the cluster. [0109] As a result of these analyses, there were 33 clones (see SEQ ID NOs: 1 to 33 in the Sequence Listing) that were acquired only from the TH05SX library but not from the other three libraries. There were three clones (see SEQ ID NOs: 34 to 36 in the Sequence Listing) that only the TH05SX library was able to acquire power but was acquired from any of the other three libraries. The former 33 clones are useful as genetic markers that specifically increase expression after S-1 and radiation therapy, while the latter 3 clones decrease expression after the above combination therapy. It is considered to be highly useful as a genetic marker.
更に、 mRNAの全長を予測するため、 Exon, Intronの部分の高精度解析を実施 し、 N端 trancateおよび C端 trancateを評価して、 mRNAの配列が取れていない部 分を予測した。予測された mRNAが全長遺伝子からの転写物であり得るか否かを判 断し、予測された mRNA配列中のアンビギュイティーコードを解消し、コンサーブド' ドメインの存在を確認し、さらに Genome上でのユニーク性を確認した上で、最終的 な予測アミノ酸配列を決定した。その結果得られた、各クローン (配列表の配列番号 1 〜9、 11〜24、 26〜36参照）から予測される全長 mRNA配列に対応する DNA配 列は、それぞれ、配列表の配列番号 37〜 70に示すとおりであった。尚、 mRNA配 列自体の配列は、配列番号 143〜176に示すとおりである。  Furthermore, in order to predict the total length of mRNA, high-precision analysis of the Exon and Intron parts was performed, and the N-terminal trancate and C-terminal trancate were evaluated to predict the part where the mRNA sequence was not taken. Determine whether the predicted mRNA can be a transcript from the full-length gene, eliminate the ambiguity code in the predicted mRNA sequence, confirm the presence of the conserved domain, and After confirming the uniqueness, the final predicted amino acid sequence was determined. The resulting DNA sequence corresponding to the full-length mRNA sequence predicted from each clone (see SEQ ID NOs: 1-9, 11-24, 26-36 in the sequence listing) is SEQ ID NO: 37 in the sequence listing, respectively. ~ 70 as shown. The sequence of the mRNA sequence itself is as shown in SEQ ID NOs: 143-176.
[0110] 実施例 2 [0110] Example 2
実施例 1にお 、て獲得された 36個のクローン (配列番号 1〜36)が実際に、定量 P CR法で増幅できるカゝ、確認実験を実施した。定量 PCRには、実施例 1で用いたヒト 肺癌細胞株 LC 11由来の Day2 (試験開始から 2日目）の DNase I処理済みのト 一タル RNA5 gを用いた。表 1にあるように、 Day2においては、 S— 1処理群のゼノ グラフトサンプルについて、 RNA調製を行わなかったので、 control群、放射線処理 群、および併用処理群のそれぞれ由来の Dnase I処理済みのトータル RNA5 gを 用いた。  In Example 1, a confirmation experiment was conducted in which 36 clones (SEQ ID NOs: 1 to 36) obtained in practice could be actually amplified by the quantitative PCR method. For quantitative PCR, 5 g of DNase I-treated total RNA of Day 2 (second day from the start of the test) derived from human lung cancer cell line LC 11 used in Example 1 was used. As shown in Table 1, on Day 2, no RNA preparation was performed on the xenograft sample in the S-1 treatment group, so the Dnase I treatment from each of the control group, radiation treatment group, and combination treatment group was completed. Total RNA 5 g was used.
[0111] 1.プラスミド DNA調製  [0111] 1. Plasmid DNA preparation
実施例 1で得られた各検体群のグリセロールストックより細胞を培養し、プラスミド D NAを抽出した。プラスミド DNA抽出用試薬は Wizard plus Minpreps (プロメガ 社)を用いた。 Cells were cultured from the glycerol stock of each sample group obtained in Example 1, and plasmid DNA was extracted. The plasmid DNA extraction reagent is Wizard plus Minpreps (Promega Was used.
[0112] 2. first strand cDNA合成  [0112] 2. First strand cDNA synthesis
実施例 1で得られた Day2における LC— 11株の control群、放射線処理群、およ び併用処理群のそれぞれ由来の DNase I処理済みトータル RNA 5ugを铸型にし てそれぞれ個別に first strand cDNA合成を行った。試薬は Oligo (dT) 12— 18 primer (インビトロジェン社） 500ng、 Superscript II RT (インビトロジェン社） 20 0Uを用い 20ulスケールにて反応を行った。  First-strand cDNA synthesis of Day 5 obtained in Example 1 by separately using 5 μg of DNase I-treated total RNA from the control group, radiation treatment group, and combination treatment group of the LC-11 strain. Went. The reagent was Oligo (dT) 12-18 primer (Invitrogen) 500 ng, Superscript II RT (Invitrogen) 200 U and the reaction was performed on a 20 ul scale.
[0113] 3.プライマー設計、合成 [0113] 3. Primer design and synthesis
実施例 1で得られた 36個のクローンの配列（配列番号 1〜36)をプライマー設計ソ フトウェアに適用してプライマーペアの候補 (配列番号 71〜 142)を得た。すなわち、 配列番号 1のクローンに対応するプライマー fOla (配列番号 71)、 rOla (配列番号 7 2)；配列番号 2のクローンに対応するプライマー f02a (配列番号 73)、 r02a (配列番 号 74)；配列番号 3のクローンに対応するプライマー f03a (配列番号 75)、 r03a (配 列番号 76)；配列番号 4のクローンに対応するプライマー f04b (配列番号 77)、 r04b (配列番号 78)；配列番号 5のクローンに対応するプライマー f05a (配列番号 79)、 r 05a (配列番号 80)；配列番号 6のクローンに対応するプライマー f 06a (配列番号 81 )、 r06a (配列番号 82)；配列番号 7のクローンに対応するプライマー f 07a (配列番号 83)、 r07a (配列番号 84)；配列番号 8のクローンに対応するプライマー f 08b (配列 番号 85)、 r08b (配列番号 86)；配列番号 9のクローンに対応するプライマー f09a ( 配列番号 87)、 r09a (配列番号 88)；配列番号 10のクローンに対応するプライマー f 10a (配列番号 89)、 rlOa (配列番号 90)；配列番号 11のクローンに対応するプライ マー f 11a (配列番号 91)、 rl la (配列番号 92)；配列番号 12のクローンに対応する プライマー f 12a (配列番号 93)、 rl2a (配列番号 94)；配列番号 13のクローンに対応 するプライマー f 13a (配列番号 95)、 rl3a (配列番号 96)；配列番号 14のクローンに 対応するプライマー f 14a (配列番号 97)、 rl4a (配列番号 98)；配列番号 15のクロー ンに対応するプライマー f 15b (配列番号 99)、 rl5b (配列番号 100)；配列番号 16の クローンに対応するプライマー f 16a (配列番号 101)、 rl6a (配列番号 102)；配列番 号 17のクローンに対応するプライマー f 17a (配列番号 103)、 rl7a (配列番号 104) ；配列番号 18のクローンに対応するプライマー f 18a (配列番号 105)、 rl8a (配列番 号 106)；配列番号 19のクローンに対応するプライマー f 19a (配列番号 107)、 rl9a (配列番号 108)；配列番号 20のクローンに対応するプライマー f 20a (配列番号 109 )、 r20a (配列番号 110)；配列番号 21のクローンに対応するプライマー f 21a (配列 番号 111)、 r21a (配列番号 112)；配列番号 22のクローンに対応するプライマー f 22 b (配列番号 113)、 r22b (配列番号 114)；配列番号 23のクローンに対応するプライ マー f23b (配列番号 115)、 r23a (配列番号 116)；配列番号 24のクローンに対応す るプライマー f24a (配列番号 117)、 r24a (配列番号 118)；配列番号 25のクローン に対応するプライマー f 25a (配列番号 119)、 r25a (配列番号 120)；配列番号 26の クローンに対応するプライマー f 26a (配列番号 121)、 r26a (配列番号 122)；配列番 号 27のクローンに対応するプライマー f 27a (配列番号 123)、 r27a (配列番号 124) ；配列番号 28のクローンに対応するプライマー f 28a (配列番号 125)、 r28a (配列番 号 126)；配列番号 29のクローンに対応するプライマー f 29a (配列番号 127)、 r29a (配列番号 128)；配列番号 30のクローンに対応するプライマー f 30a (配列番号 129 )、 r30a (配列番号 130)；配列番号 31のクローンに対応するプライマー f31b (配列 番号 131)、 r31b (配列番号 132)；配列番号 32のクローンに対応するプライマー f 3 2a (配列番号 133)、 r32a (配列番号 134)；配列番号 33のクローンに対応するプラ イマ一 f 33a (配列番号 135)、 r33a (配列番号 136)；配列番号 34のクローンに対応 するプライマー f34a (配列番号 137)、 r34a (配列番号 138)；配列番号 35のクロー ンに対応するプライマー f35a (配列番号 139)、 r35a (配列番号 140)；配列番号 36 のクローンに対応するプライマー f 36a (配列番号 141)、 r36a (配列番号 142)。 なお、ソフトウェアのパラメーター設定は TaKaRa Ex Taq R-PCR version 2. 1 (タカラバィォ社)付属のプロトコールに従った。挙げられた候補の中から、タカラ ノィォ社およびアプライドバイォシステムズ社が推奨するプライマー設計指針の両者 を考慮してプライマーペアを選択した。選んだプライマーは blast検索にかけて、他の 遺伝子配列が増幅しな 、ことを確認した。プライマー合成はインビトロジェン社に依 頼した。合成したプライマーの配列は表 3に記載した。表 3中の No.は、各プライマ 一に対応するクローンの配列番号を示す。 ム έ ' [9Π0] The sequence of 36 clones (SEQ ID NOs: 1 to 36) obtained in Example 1 was applied to primer design software to obtain candidate primer pairs (SEQ ID NOs: 71 to 142). That is, primers fOla (SEQ ID NO: 71), rOla (SEQ ID NO: 72) corresponding to the clone of SEQ ID NO: 1; primers f02a (SEQ ID NO: 73), r02a (SEQ ID NO: 74) corresponding to the clone of SEQ ID NO: 2; Primers f03a (SEQ ID NO: 75), r03a (SEQ ID NO: 76) corresponding to the clone of SEQ ID NO: 3; Primers f04b (SEQ ID NO: 77), r04b (SEQ ID NO: 78) corresponding to the clone of SEQ ID NO: 4; SEQ ID NO: 5 Primers f05a (SEQ ID NO: 79), r 05a (SEQ ID NO: 80) corresponding to clones of SEQ ID NO: 6; Primers f06a (SEQ ID NO: 81), r06a (SEQ ID NO: 82) corresponding to clones of SEQ ID NO: 6; clones of SEQ ID NO: 7 Primers f 07a (SEQ ID NO: 83), r07a (SEQ ID NO: 84) corresponding to the clones of primers f 08b (SEQ ID NO: 85), r08b (SEQ ID NO: 86) corresponding to the clone of SEQ ID NO: 8; corresponding to the clone of SEQ ID NO: 9 Primer f09a (SEQ ID NO: 87), r09a (SEQ ID NO: 88); primers corresponding to the clone of SEQ ID NO: 10 f 10a (SEQ ID NO: 89), rlOa (SEQ ID NO: 90); primers corresponding to the clone of SEQ ID NO: 11 f 11a (SEQ ID NO: 91), rl la (SEQ ID NO: 92); primers f 12a (SEQ ID NO: 93), rl2a (SEQ ID NO: 94) corresponding to the clone of SEQ ID NO: 12; primers f 13a (SEQ ID NO: 95), rl3a (corresponding to the clone of SEQ ID NO: 13 SEQ ID NO: 96); primers f 14a (SEQ ID NO: 97), rl4a (SEQ ID NO: 98) corresponding to the clone of SEQ ID NO: 14; primers f 15b (SEQ ID NO: 99), rl5b (sequence) corresponding to the clone of SEQ ID NO: 15. Primer f 16a (SEQ ID NO: 101), rl6a (SEQ ID NO: 102) corresponding to the clone of SEQ ID NO: 16; Primer f 17a (SEQ ID NO: 103), rl7a (SEQ ID NO: 103) corresponding to the clone of SEQ ID NO: 17 104) Primers f 18a (SEQ ID NO: 105), rl8a (SEQ ID NO: 106) corresponding to the clone of SEQ ID NO: 18; primers f 19a (SEQ ID NO: 107), rl9a (SEQ ID NO: 108) corresponding to the clone of SEQ ID NO: 19; Primers f 20a (SEQ ID NO: 109), r20a (SEQ ID NO: 110) corresponding to the clone of SEQ ID NO: 20; primers f 21a (SEQ ID NO: 111), r21a (SEQ ID NO: 112) corresponding to the clone of SEQ ID NO: 21; SEQ ID NO: Primers corresponding to 22 clones f 22 b (SEQ ID NO: 113), r22b (SEQ ID NO: 114); primers corresponding to clones of SEQ ID NO: 23 f23b (SEQ ID NO: 115), r23a (SEQ ID NO: 116); SEQ ID NO: 24 Primers f24a (SEQ ID NO: 117), r24a (SEQ ID NO: 118) corresponding to clones of SEQ ID NO: 25; Primers f25a (SEQ ID NO: 119), r25a (SEQ ID NO: 120) corresponding to clones of SEQ ID NO: 25; clones of SEQ ID NO: 26 Primer f 26a corresponding to (Column number 121), r26a (SEQ ID NO: 122); primer f 27a (SEQ ID NO: 123) corresponding to the clone of SEQ ID NO: 27; r27a (SEQ ID NO: 124); primer f 28a (sequence) corresponding to the clone of SEQ ID NO: 28 No. 125), r28a (SEQ ID NO: 126); primer f 29a (SEQ ID NO: 127) corresponding to the clone of SEQ ID NO: 29, r29a (SEQ ID NO: 128); primer f 30a (SEQ ID NO: 30) corresponding to the clone of SEQ ID NO: 30 129), r30a (SEQ ID NO: 130); primer f31b (SEQ ID NO: 131) corresponding to the clone of SEQ ID NO: 31; r31b (SEQ ID NO: 132); primer f 3 2a (SEQ ID NO: 133) corresponding to the clone of SEQ ID NO: 32 R32a (SEQ ID NO: 134); primers corresponding to the clone of SEQ ID NO: 33 f 33a (SEQ ID NO: 135), r33a (SEQ ID NO: 136); primer f34a (SEQ ID NO: 137) corresponding to the clone of SEQ ID NO: 34, r34a (SEQ ID NO: 138); claw of SEQ ID NO: 35 Corresponding primer f35a (SEQ ID NO: 139), r35a (SEQ ID NO: 140); primer f 36a (SEQ ID NO: 141) corresponding to clone SEQ ID NO: 36, R36A (SEQ ID NO: 142). The software parameters were set according to the protocol attached to TaKaRa Ex Taq R-PCR version 2.1 (Takarabio). From the candidates listed, primer pairs were selected in consideration of both the primer design guidelines recommended by Takara Neo and Applied Systems. The selected primers were subjected to a blast search and confirmed that no other gene sequences were amplified. Primer synthesis was requested from Invitrogen. The synthesized primer sequences are listed in Table 3. No. in Table 3 indicates the sequence number of the clone corresponding to each primer. [9έ0]
Figure imgf000040_0001
Figure imgf000040_0002
Figure imgf000040_0001
Figure imgf000040_0002
ε拏  ε 拏
[ε挲] [sno]  [ε 挲] [sno]
SZZ0if/900idf/X3d 6ε ι ε嫁 ooz ΟΛ\ 定量試験を実施し、上述のようにして作成した各プライマーカ、対応するクローンを 特異的に増幅させるかどうかを確認した。定量試験はアプライドバイォシステムズ社 の装置 (ABI7500)と試薬の組み合わせで行った。具体的な反応系は以下の通りと した。すなわち基本試薬に Power SYBR Green PCR Master Mix (アプライ ドバイオシステムズ社)を用い、プライマーはそれぞれ終濃度 200nMとした。铸型は 、プラスミド DNAを 5段階に系列希釈したもの、すなわち 10pmolZul、 lpmol/ul, 100fmol/ul, 10fmol/ul, lfmolZulに希釈したもの、および first strand cD NA (併用処理群由来）の 50倍希釈液をそれぞれ lul用いた。反応液量は 50ulとし た。 PCRプログラムは 95°C、 lmin→(95°C、 15sec→60°Clmin) x 45cycleの 後、 defaultの dissociation curve作成プログラムを追加した。プライマーの合否は dissociation curveにより半 [J定しに。 SZZ0if / 900idf / X3d 6ε ι ε bride ooz ΟΛ \ A quantitative test was performed to confirm whether each primer prepared as described above and the corresponding clone were specifically amplified. Quantitative tests were performed using a combination of Applied Systems (ABI7500) and reagents. The specific reaction system was as follows. That is, Power SYBR Green PCR Master Mix (Applied Biosystems) was used as the basic reagent, and the primers were each at a final concentration of 200 nM. The vertical type is a series of dilutions of plasmid DNA in 5 stages, ie, 10 pmolZul, lpmol / ul, 100 fmol / ul, 10 fmol / ul, lfmolZul, and 50 times the first strand cDNA (from the combination treatment group) Each diluted solution was used in lul. The reaction volume was 50 ul. The PCR program was 95 ° C, lmin → (95 ° C, 15sec → 60 ° Clmin) x 45cycles, and a default dissociation curve creation program was added. The primer pass / fail is determined by the dissociation curve.
[0117] その結果、 36個のクローンのうち、配列番号 8、 16、 22については、副産物の増幅 が多く特異的な増幅が確認できな力つた。これは、配列番号 8のクローンについては 実施例 1の EST解析で得られた配列長さが 197塩基と短いこと、配列番号 16、 22の クローンについては配列中に繰り返し配列が多いこと、が原因と考えられる。一方、 配列番号 8、 16、 22を除く 33個のクローンは特異的な増幅が確認できた。  [0117] As a result, among the 36 clones, SEQ ID NOs: 8, 16, and 22 had strong amplification of by-products and were unable to confirm specific amplification. This is because the clone of SEQ ID NO: 8 has a short sequence length of 197 bases obtained in the EST analysis of Example 1, and the clones of SEQ ID NO: 16 and 22 have many repeated sequences in the sequence. it is conceivable that. On the other hand, specific amplification was confirmed in 33 clones excluding SEQ ID NOs: 8, 16, and 22.
[0118] 5.定量 PCR  [0118] 5.Quantitative PCR
次に、定量 PCRにおける、各クローンの増幅量を定量した。反応系は検量線用プ ラスミド DNAサンプルと first strand cDNAサンプルの铸型量が異なるほかは、 上記プライマー特異性確認における反応系と同様とした。具体的にはプラスミド DN Aを 6段階に系列希釈したもの、すなわち 100pmolZul、 10pmol/ul, lpmol/ul 、 100fmol/uU lOfmol/uU lfmol/ulをそれぞれ lul添カ卩したサンプルにより検 量線を作成し、 first strand cDNAを 10倍希釈したサンプル lul中のコピー数を 定量した。なお 1枚の 96穴 PCRプレート中で、検量線用プラスミド DNAサンプルおよ び first strand cDNAサンプルについて各 2反応ずつ PCR反応を行い、これをも つて N = 2とした。  Next, the amount of amplification of each clone in quantitative PCR was quantified. The reaction system was the same as the reaction system in the above-described primer specificity confirmation, except that the amount of the template DNA sample for the calibration curve was different from that of the first strand cDNA sample. Specifically, a calibration curve is prepared using samples obtained by serially diluting plasmid DNA in 6 stages, that is, samples containing 100 pmolZul, 10 pmol / ul, lpmol / ul, and 100 fmol / uU lOfmol / uU lfmol / ul. The number of copies in the sample lul diluted 10-fold from the first strand cDNA was quantified. In each 96-well PCR plate, two PCR reactions were performed for each of the plasmid DNA sample for calibration curve and the first strand cDNA sample, and this was set to N = 2.
内部標準用の遺伝子としてヒト GAPD (GAPDH；ダリセルアルデヒド 3—リン酸デヒ ドロゲナーゼ）を選択した。この遺伝子を検出するためのプライマーはタカラバィォ社 より販売している既製品を用いた。尚、本発明者らは、このプライマーを用いてマウス first strand cDNAを铸型にして PCRを行った場合、 DNAの増幅が起こらない 事を事前に確認した。 Human GAPD (GAPDH; dalyceraldehyde 3-phosphate dehydrogenase) was selected as a gene for the internal standard. Primer for detecting this gene is Takarabio Used off-the-shelf products sold more. In addition, the present inventors have confirmed in advance that DNA amplification does not occur when mouse first strand cDNA is subjected to PCR using this primer.
[0119] また、 Ct値（Cycle Threshold)の設定は、装置 ABI7500に付属のソフトウェアで rautojの設定を選択した。検量線の作成と各サンプル中のコピー数の算出も同ソフ トウエア上で行った。コピー数の計算式は以下の通りで、 2本鎖プラスミド DNAのコピ 一数が算出される。プラスミド DNAのサイズは実際には遺伝子ごとにまちまちだが、 一律 3, 500bp (pbluescript II KS— : 3, OOObp, insert平均鎖長； 500bp)とし て計算した。 GAPD【こつ!ヽてのみ 4, 700bp (pCR2. 1 :4, 200bp, insert : 500bp )として計算した。  [0119] For the Ct value (Cycle Threshold) setting, the rautoj setting was selected using the software supplied with the ABI7500 device. The calibration curve was created and the number of copies in each sample was also calculated on the same software. The copy number calculation formula is as follows, and the number of copies of double-stranded plasmid DNA is calculated. The size of plasmid DNA actually varies from gene to gene, but was calculated as 3,500 bp (pbluescript II KS—: 3, OOObp, average length of insert: 500 bp). GAPD [Calculation! Calculated as 4,700bp (pCR2.1: 4,200bp, insert: 500bp) only.
[0120] <計算式 >  [0120] <Calculation formula>
2本鎖 DNAのモル数は  The number of moles of double-stranded DNA is
[X (mol) ] = [ 2本鎖 DNA (pg) ]xl0- 12/ [MW (Da) ]  [X (mol)] = [Double-stranded DNA (pg)] xl0-12 / [MW (Da)]
= [2本鎖DNA(pg) ]xlO—12Z[鎖長(bP) ]Z660 = [Double-stranded DNA (pg)] xlO-12Z [Strand length (b P )] Z660
であるから  Because
[2本鎖DNAコピー数] = [X]x6. 02x1023  [Double-stranded DNA copy number] = [X] x6. 02x1023
[0121] 表 4は、 Day2における LC— 11株の control群、放射線処理群、および併用処理 群の 33個のクローン（36個のクローンのうち配列番号 8、 16、 22を除く）および内部 標準遺伝子の GAPDについて算出された 2本鎖 DNAコピー数を示す。尚、表 4中に 示す数値は、 2回 (N = 2)の定量 PCR解析で得られたデータを平均した数値である [0121] Table 4 shows 33 clones (excluding SEQ ID Nos. 8, 16, and 22 of 36 clones) and internal standard of the control group, the radiation treatment group, and the combination treatment group of LC-11 on Day 2. The double-stranded DNA copy number calculated for GAPD of the gene is shown. The numerical values shown in Table 4 are the average values of the data obtained by two times of quantitative PCR analysis (N = 2).
[0122] [表 4] 表 4 [0122] [Table 4] Table 4
Figure imgf000043_0001
Figure imgf000043_0001
表 4に示す 2本鎖 DNAコピー数の結果を、各細胞当たりの GAPDHの発現量は同 じであるとの仮定のもとに、 GAPDHの発現量で補正した。放射線処理群と併用処理 群のそれぞれの発現量 control群における発現量に対し、比率をとつた結果を図 4に 示す。  The double-stranded DNA copy number results shown in Table 4 were corrected with the GAPDH expression level on the assumption that the GAPDH expression level per cell was the same. Fig. 4 shows the ratio of the expression level in the control group to the expression level in the radiation treatment group and the combination treatment group.
以上のように、 33個のクローンは特異的な増幅が確認できた。このように、実施例 1 で選抜された各クローンは、それらの発現量が併用療法実施後に変化することから、 併用療法感受性マーカーとして有用であることが明らかとなった。また、配列番号 37 〜70についても、これらは配列番号 1〜9、 11〜24、 26〜36から予測される配列で あることから、上記各クローンと同様にプライマーを設計すればマーカーとして有用で あることが充分に類推できる。 As described above, specific amplification was confirmed in 33 clones. Thus, Example 1 The clones selected in (1) were found to be useful as markers for sensitivity to combination therapy because their expression levels changed after combination therapy. In addition, regarding SEQ ID NOs: 37 to 70, these are sequences predicted from SEQ ID NOs: 1 to 9, 11 to 24, and 26 to 36. Therefore, if primers are designed in the same manner as the above clones, they are useful as markers. It can be fully analogized.
産業上の利用可能性 Industrial applicability
本発明によれば、テガフール ·ギメラシル'ォテラシルカリウム配合抗腫瘍剤及び放射 線の併用療法感受性を迅速かつ正確に診断することができる。よって、癌患者に対 し併用療法を実施すべき力否かの早期決定が可能であると共に、癌治療の効率ィ匕を 図ることができる。 According to the present invention, it is possible to quickly and accurately diagnose the sensitivity of combination therapy of tegafur / gimeracyl / oteracil potassium-containing antitumor agent and radiation. Therefore, early determination as to whether or not the combination therapy should be performed on cancer patients can be made early, and the efficiency of cancer treatment can be improved.

Claims

請求の範囲 [1] 下記の (A)または (B)で表されるポリヌクレオチド。 (A)配列表の配列番号 1〜70の!、ずれかに記載の塩基配列を含むポリヌクレオチド(B)前記 (A)記載のポリヌクレオチドと相補的な塩基配列を含むポリヌクレオチド(C)前記 (A)または（B)記載のポリヌクレオチドとハイストリンジェントな条件下でノ、ィ ブリダィズする塩基配列を含み、かつ、テガフール ·ギメラシル'ォテラシルカリウム配 合抗腫瘍剤及び放射線の併用療法感受性を診断するための遺伝子マーカーとして 機能するポリヌクレオチド (D)前記 (A)または (B)記載のポリヌクレオチドにお 、て、 1または複数個の塩基が 欠失、置換、挿入、および Zまたは付加した塩基配列を含み、かつ、テガフール 'ギ メラシル 'ォテラシルカリウム配合抗腫瘍剤及び放射線の併用療法感受性を診断す るための遺伝子マーカーとして機能するポリヌクレオチド [2] テガフール ·ギメラシル ·ォテラシルカリウム配合抗腫瘍剤及び放射線の併用療法 感受性を診断するための遺伝子マーカーとして機能する、下記の (A)〜（D)で表さ れるポリヌクレオチド。 (A)配列表の配列番号 1〜70の!、ずれかに記載の塩基配列を含むポリヌクレオチド(B)前記 (A)記載のポリヌクレオチドと相補的な塩基配列を含むポリヌクレオチド(C)前記 (A)または（B)記載のポリヌクレオチドとハイストリンジェントな条件下でノ、ィ ブリダィズする塩基配列を含み、かつ、テガフール ·ギメラシル'ォテラシルカリウム配 合抗腫瘍剤及び放射線の併用療法感受性を診断するための遺伝子マーカーとして 機能するポリヌクレオチド (D)前記 (A)または (B)記載のポリヌクレオチドにお 、て、 1または複数個の塩基が 欠失、置換、挿入、および Zまたは付加した塩基配列を含み、かつ、テガフール 'ギ メラシル 'ォテラシルカリウム配合抗腫瘍剤及び放射線の併用療法感受性を診断す るための遺伝子マーカーとして機能するポリヌクレオチド [3] テガフール ·ギメラシル ·ォテラシルカリウム配合抗腫瘍剤及び放射線の併用療法 感受性を診断するにあたり、請求項 1または 2記載のポリヌクレオチドのプローブまた はプライマーとして用いられるポリヌクレオチド。 [4] 下記の（1)〜 (4)で表されるポリヌクレオチドである請求項 3記載のポリヌクレオチド Claims [1] A polynucleotide represented by the following (A) or (B). (A) SEQ ID NO: 1 to 70 in the sequence listing !, a polynucleotide comprising a base sequence according to any one of the above (B) a polynucleotide comprising a base sequence complementary to the polynucleotide of (A) (C) (A) or (B) containing a nucleotide sequence that is hybridized under highly stringent conditions with the polynucleotide of (A) or (B), and is sensitive to combination therapy with tegafur and gimeracil potassium oteracil potassium and an anti-tumor agent Polynucleotide that functions as a genetic marker for diagnosis (D) In the polynucleotide according to (A) or (B), one or more bases are deleted, substituted, inserted, and Z or added. Polynucleotide that contains a base sequence and functions as a genetic marker for diagnosing the sensitivity of combination therapy with anti-tumor agents containing tegafur 'gimeracil' oteracil potassium and radiation Tide [2] Combination therapy of anti-tumor agent containing tegafur, gimeracil and oteracil potassium and radiation A polynucleotide represented by the following (A) to (D), which functions as a genetic marker for diagnosing sensitivity. (A) SEQ ID NO: 1 to 70 in the sequence listing !, a polynucleotide comprising a base sequence according to any one of the above (B) a polynucleotide comprising a base sequence complementary to the polynucleotide of (A) (C) (A) or (B) containing a nucleotide sequence that is hybridized under highly stringent conditions with the polynucleotide of (A) or (B), and is sensitive to combination therapy with tegafur and gimeracil potassium oteracil potassium and an anti-tumor agent Polynucleotide that functions as a genetic marker for diagnosis (D) In the polynucleotide according to (A) or (B), one or more bases are deleted, substituted, inserted, and Z or added. Polynucleotide that contains a base sequence and functions as a genetic marker for diagnosing the sensitivity of combination therapy with anti-tumor agents containing tegafur 'gimeracil' oteracil potassium and radiation Tide [3] Combination therapy with tegafur, gimeracil and oteracil potassium antitumor agent and radiation A polynucleotide used as a probe or primer of the polynucleotide according to claim 1 or 2 in diagnosing sensitivity. [4] The polynucleotide according to claim 3, which is a polynucleotide represented by the following (1) to (4):
(1)配列表の配列番号 1〜70のいずれかに記載の塩基配列内のすくなくとも連続す る 10塩基を含むポリヌクレオチド (1) A polynucleotide comprising at least 10 consecutive nucleotides in the nucleotide sequence set forth in any one of SEQ ID NOs: 1 to 70 in the Sequence Listing
(2)前記（1)記載のポリヌクレオチドと相補的な塩基配列を含むポリヌクレオチド (2) a polynucleotide comprising a base sequence complementary to the polynucleotide of (1) above
(3)前記（1)または（2)記載のポリヌクレオチドとハイストリンジェントな条件下でハイ ブリダィズする塩基配列を含み、かつ、請求項 1記載のポリヌクレオチドのプローブま たはプライマーとして機能するポリヌクレオチド (3) A polynucleotide comprising a base sequence that hybridizes with the polynucleotide according to (1) or (2) above under a highly stringent condition, and which functions as a probe or primer of the polynucleotide according to claim 1. Nucleotide
(4)前記（1)または（2)記載のポリヌクレオチドにお 、て、 1または複数個の塩基が欠 失、置換、挿入、および Zまたは付加した塩基配列を含み、かつ、請求項 1記載のポ リヌクレオチドのプローブまたはプライマーとして機能するポリヌクレオチド  (4) The polynucleotide according to (1) or (2) above, which comprises a nucleotide sequence in which one or more bases are missing, substituted, inserted, and Z or added, and A polynucleotide that functions as a probe or primer for polynucleotides
[5] 配列番号 71〜142の 、ずれかに記載の塩基配列を含む請求項 3または 4記載の ポリヌクレオチド。  [5] The polynucleotide according to claim 3 or 4, comprising the nucleotide sequence of any one of SEQ ID NOs: 71 to 142.
[6] 請求項 1または 2記載のポリヌクレオチドによりコードされるポリペプチド。  [6] A polypeptide encoded by the polynucleotide according to claim 1 or 2.
[7] 請求項 6記載のポリペプチドを認識する抗体。 [7] An antibody that recognizes the polypeptide according to claim 6.
[8] 請求項 3〜5のいずれかに記載のポリヌクレオチド、もしくは請求項 7記載の抗体を 少なくとも含む、テガフール ·ギメラシル ·ォテラシルカリウム配合抗腫瘍剤及び放射 線の併用療法感受性の診断薬。  [8] An antitumor agent containing tegafur, gimeracil, and oteracil potassium, and a diagnostic agent sensitive to radiation combination therapy, comprising at least the polynucleotide according to any one of claims 3 to 5 or the antibody according to claim 7.
[9] 請求項 3〜5のいずれかに記載のポリヌクレオチド、もしくは請求項 7記載の抗体を 少なくとも含む、テガフール ·ギメラシル ·ォテラシルカリウム配合抗腫瘍剤及び放射 線の併用療法感受性の診断用キット。 [9] A diagnostic kit for sensitivity to a combination therapy of tegafur, gimeracil, and oteracil potassium and radiation, comprising at least the polynucleotide according to any one of claims 3 to 5 or the antibody according to claim 7. .
[10] 被験者力 採取された生体試料中の請求項 1または 2記載のポリヌクレオチドの有 無を調べることを特徴とする、テガフール ·ギメラシル ·ォテラシルカリウム配合抗腫瘍 剤及び放射線の併用療法感受性の診断方法。 [10] Subject strength The sensitivity of the combination therapy with tegafur, gimeracil, and oteracil potassium, which is characterized by examining the presence or absence of the polynucleotide according to claim 1 or 2 in a collected biological sample. Diagnosis method.
[11] 下記の工程 (I)〜 (III)を含む請求項 10記載の診断方法。 [11] The diagnostic method according to claim 10, comprising the following steps (I) to (III):
(I)被験者カゝら採取された生体試料カゝら調製された RNAまたは該 RNAカゝら転写され た相補的ポリヌクレオチドと請求項 3〜5のいずれかに記載のポリヌクレオチドとを結 合させる工程 (II)工程 (I)においてポリヌクレオチドに結合した RNAまたは相補的ポリヌクレオチド の有無を、請求項 3〜5のいずれかに記載のポリヌクレオチドを指標として測定する 工程 (I) Binding RNA prepared from a biological sample collected from a subject or a complementary polynucleotide transcribed from the RNA and the polynucleotide according to any one of claims 3 to 5. Process (II) A step of measuring the presence or absence of RNA or complementary polynucleotide bound to the polynucleotide in step (I) using the polynucleotide according to any one of claims 3 to 5 as an index.
(III)工程 (Π)の測定結果から、テガフール 'ギメラシル'ォテラシルカリウム配合抗腫 瘍剤及び放射線の併用療法感受性を診断する工程  (III) Process The process of diagnosing susceptibility to combination therapy with tegafur 'gimeracil' oteracil potassium-containing antitumor agent and radiation from the measurement results of (iii)
[12] 被験者力 採取された生体試料中の請求項 6記載のポリペプチドの有無を調べる ことを特徴とする、テガフール ·ギメラシル ·ォテラシルカリウム配合抗腫瘍剤及び放 射線の併用療法感受性の診断方法。  [12] Test force A method for diagnosing susceptibility to combination therapy of tegafur, gimeracil, and oteracil potassium and radiotherapy, characterized by examining the presence or absence of the polypeptide according to claim 6 in the collected biological sample .
[13] 下記の工程 (i)〜 (iii)を含む請求項 12記載の診断方法。  [13] The diagnostic method according to claim 12, comprising the following steps (i) to (iii):
(i)被験者力 採取された生体試料力 調製されたポリペプチドと請求項 7記載の抗 体とを結合させる工程、  (i) Subject force Collected biological sample force The step of binding the prepared polypeptide and the antibody of claim 7,
(ii)工程 (i)において抗体に結合したポリペプチドまたはその部分ペプチドの有無を 、該抗体を指標として測定する工程  (ii) a step of measuring the presence or absence of the polypeptide bound to the antibody or its partial peptide in step (i) using the antibody as an index
(iii)工程 (ii)の測定結果から、テガフール ·ギメラシル*ォテラシルカリウム配合抗腫 瘍剤及び放射線の併用療法感受性を診断する工程  (iii) Step (ii) Based on the measurement results of step (ii), a diagnosis of the sensitivity of combination therapy with tegafur and gimeracil * oteracil potassium combined antitumor agent and radiation
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