WO2011129371A1 - GENE GROUP CAPABLE OF ENHANCING ANTI-TUMOR EFFECT OF 5-FU ALONE OR IFN-α/5-FU COMBINATION - Google Patents

GENE GROUP CAPABLE OF ENHANCING ANTI-TUMOR EFFECT OF 5-FU ALONE OR IFN-α/5-FU COMBINATION Download PDF

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WO2011129371A1
WO2011129371A1 PCT/JP2011/059192 JP2011059192W WO2011129371A1 WO 2011129371 A1 WO2011129371 A1 WO 2011129371A1 JP 2011059192 W JP2011059192 W JP 2011059192W WO 2011129371 A1 WO2011129371 A1 WO 2011129371A1
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gene
amino acid
fluorouracil
mutant
acid sequence
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汐田　剛史
博之 土谷
友彦 坂部
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国立大学法人鳥取大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • 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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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 method for screening a gene having an effect of enhancing the sensitivity of an anticancer agent, a 5-fluorouracil sensitive sensitizer, an anticancer agent kit containing the same, a diagnostic agent for determining 5-fluorouracil sensitivity, and a 5-fluorouracil sensitivity inhibitor.
  • Hepatocellular carcinoma is a type of malignant tumor that is increasing all over the world, accounting for the third largest number of cancer deaths in Japan.
  • treatment results have improved due to the recent development and progress of various diagnosis and treatment methods.
  • HCC patients are carcinogenic after chronic liver diseases such as hepatitis, there are many cases of recurrence after treatment, and an increase in highly advanced cases is a problem.
  • This advanced progressive HCC has a very low success rate of existing therapies, and the development of new therapies with higher therapeutic effects has been desired.
  • IFN- ⁇ / 5-FU combination therapy in which the cytokine interferon- ⁇ (IFN- ⁇ ) having antiviral activity and the anticancer agent 5-fluorouracil (5-FU) are used in combination is highly effective.
  • the results are reported from multiple institutions and are expected as a novel treatment for advanced HCC.
  • Non-Patent Document 1 is a paper that attempts to predict the effect of IFN- ⁇ / 5-FU combination therapy from genetic analysis.
  • Non-Patent Document 2 reports a susceptibility gene for 5-FU in liver cancer cells.
  • Patent Document 1 describes a system for predicting the malignancy of cancer patients and the response to chemotherapy. Furthermore, Patent Document 2 describes a marker for diagnosing cancer progression, outcome, and prognosis. Patent Document 3 describes cancer treatment using a combination of antisense RNA and 5FU.
  • Non-Patent Documents 1 and 2 it is difficult to comprehensively find genes related to the susceptibility of IFN- ⁇ / 5-FU combination therapy. It is assumed that there are many genes whose relationship is not yet elucidated. In Non-Patent Documents 1 and 2, the genes shown in the examples described later cannot actually be found.
  • Patent Document 3 EXT1 is used as a gene
  • 5-FU is used as a drug
  • liver cancer is used as an example of cancer.
  • EXT1 increases the sensitivity of 5-FU is not actually tested.
  • TGFBR2 is exemplified as a marker for diagnosing cancer progression, outcome, and prognosis
  • liver cancer is exemplified as cancer
  • 5FU is exemplified as adjuvant chemotherapy.
  • TGFBR2 is sensitive to 5-FU. I have not experimented to raise it.
  • Patent Document 5 does not even search for genes related to 5-FU susceptibility in the first place. Therefore, Patent Document 5 does not particularly touch the genes shown in the examples described later.
  • the present invention has been made in view of the above circumstances, and an object thereof is to find a gene group that enhances the antitumor effect of 5-fluorouracil.
  • a 5-fluorouracil sensitive sensitizer comprising a nucleotide fragment encoding one or more genes selected from the group consisting of PRAKG2, TGFBR2 and EXT1 genes.
  • the present invention also provides a 5-fluorouracil sensitive sensitizer comprising a vector that expresses one or more genes selected from the group consisting of PRAKG2, TGFBR2 and EXT1 genes.
  • the vector since the vector includes any one of the PRAK2 gene, the TGFBR2 gene, and the EXT1 gene, which has been demonstrated in Examples described later, to have an action of enhancing the antitumor effect of 5-fluorouracil, As the sensitizer itself, an effect of enhancing the antitumor effect of 5-fluorouracil can be obtained.
  • the present invention also provides a 5-fluorouracil sensitive sensitizer comprising a protein encoded by one or more genes selected from the group consisting of PRAKG2, TGFBR2 and EXT1 genes.
  • an anticancer agent kit comprising the above sensitizer and 5-fluorouracil.
  • a 5-fluorouracil sensitivity inhibitor comprising siRNA, shRNA, or antisense RNA that suppresses the expression of one or more genes selected from the group consisting of PRAK2 gene, TGFBR2 gene, and EXT1 gene An inhibitor is provided.
  • siRNA, shRNA, or the like that suppresses the expression of any of PRAK2 gene, TGFBR2 gene, or EXT1 gene, which has been demonstrated in Examples to be described later, has an action of enhancing the antitumor effect of 5-fluorouracil. Since the antisense RNA is contained, the inhibitor itself can suppress the antitumor effect of 5-fluorouracil.
  • a diagnostic agent for determining 5-fluorouracil sensitivity a reagent for measuring the expression level of one or more genes selected from the group consisting of PRAKG2, TGFBR2 and EXT1 genes
  • a diagnostic agent is provided.
  • a reagent for measuring the expression level of any of PRAK2 gene, TGFBR2 gene, or EXT1 gene which has been demonstrated in Examples described later, to have an action of enhancing the antitumor effect of 5-fluorouracil Therefore, by measuring the expression level of any one of the PRAK2 gene, TGFBR2 gene or EXT1 gene in the subject's living body, it is possible to determine whether or not the subject is likely to be effective in the anti-tumor effect of 5-fluorouracil. It can be used as a diagnostic agent.
  • a method for screening a gene having an effect of enhancing the sensitivity of an anticancer agent comprising (m) introducing a ribozyme library into a predetermined cancer cell line of a mammal, and (n) a ribozyme library.
  • ribozymes from viable cells among cancer cells treated with anticancer agents to obtain a novel ribozyme library, and comprising: Randomized ribozyme library, wherein the library includes a plurality of ribozymes that cleave mRNA specifically for a target recognition sequence and suppress gene expression, and the target recognition sequences recognized by each ribozyme are a plurality of randomized sequences
  • a ribozyme library obtained by repeating the steps (p) (m), (n) and (o) multiple times is recognized.
  • detecting the target recognition sequence, (q) genetic information corresponding to the detected target recognition sequence further comprising the steps of: extracting from the genome database, a screening method is provided.
  • the ribozyme library obtained by repeating the above steps a plurality of times includes: This means that many ribozymes that suppress the expression of genes having a sensitivity enhancing action of anticancer agents are concentrated and contained. Therefore, if the target recognition sequence recognized by the ribozyme library obtained in this way is detected and the gene information corresponding to the detected target recognition sequence is extracted from the genome database, genes having an anticancer agent sensitivity enhancing action can be detected with high probability. It is possible to find out.
  • the antitumor effect of 5-fluorouracil can be enhanced by using the RAKG2 gene, TGFBR2 gene, EXT1 gene or the protein encoded by those genes that enhance the antitumor effect of 5-fluorouracil.
  • the antitumor effect of 5-fluorouracil can be enhanced using siRNA, shRNA or antisense RNA that suppresses the expression of RAKG2 gene, TGFBR2 gene or EXT1 gene that enhances the antitumor effect of 5-fluorouracil. Can be suppressed.
  • the subject can use a reagent for measuring the expression level of any of PRAK2 gene, TGFBR2 gene, or EXT1 gene that enhances the antitumor effect of 5-fluorouracil, so that the subject It can be determined whether the constitution is effective.
  • a reagent for measuring the expression level of any of PRAK2 gene, TGFBR2 gene, or EXT1 gene that enhances the antitumor effect of 5-fluorouracil so that the subject It can be determined whether the constitution is effective.
  • FIG. 1 It is a conceptual diagram for demonstrating construction
  • FIG. 10 is a fluorescence micrograph (FIG. 10a) and a graph (FIG. 10b) showing enhancement of 5-FU and IFN- ⁇ / 5-FU-induced apoptosis by overexpression of TGFBR2 and EXT1.
  • FIG. 11 is a graph (FIGS. 11a and 11b) and an electrophoretic photograph (FIG. 11c) showing changes in TGF- ⁇ signal activation and apoptosis-related protein expression due to overexpression of TGFBR2. It is a graph (FIG. 12a, FIG. 12b) and an electrophoretic photograph (FIG. 12c) which show the involvement of ER stress with respect to the enhancement of apoptosis by EXT1 overexpression.
  • 15 is a graph showing mRNA expression of PRKAG2, TGFBR2 and EXT1 in liver cancer patients (FIGS. 14A, 14B, 14C, and 14D).
  • homology is the ratio of the number of identical amino acids in an amino acid sequence between two or more amino acids calculated according to a method known in the art. Before calculating the ratio, the amino acid sequences of the amino acid sequences to be compared are aligned, and a gap is introduced into a part of the amino acid sequence if necessary to maximize the same ratio. Nor do any conservative substitutions be considered identical. Further, it means the ratio of the same number of amino acids to all amino acid residues including overlapping amino acids in an optimally aligned state.
  • stringent conditions means, for example, (1) low ionic strength for washing and high temperature, for example, 0.015 M sodium chloride / 0.00% at 50 ° C. 0015M sodium citrate / 0.1% sodium dodecyl sulfate, (2) denaturing agents such as formamide during hybridization, eg 50% (vol / vol) formamide and 0.1% bovine serum at 42 ° C. Albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer (pH 6.5) and 750 mM sodium chloride, 75 mM sodium citrate, or (3) 50% formamide at 42 ° C.
  • moderately stringent conditions are 20% formamide, 5 ⁇ SSC, 50 mM sodium phosphate (pH 7.6), 5 ⁇ Denhart solution, 10% dextran sulfate, and 20 mg / ml denatured sheared salmon sperm DNA. Conditions such as overnight incubation at 37 ° C. in a solution containing, followed by washing the filter at 37-50 ° C. in 1 ⁇ SSC.
  • the stringency of the hybridization reaction can be easily determined by those skilled in the art and generally depends on the probe length, the washing temperature, and the salt concentration. In general, longer probes require higher temperatures for proper annealing, and shorter probes require lower temperatures. In general, stringency is inversely proportional to salt concentration.
  • Hybridization when applied to a polynucleotide means a property that allows pairing between nucleotides by hydrogen bonding between nucleotide bases. Base pairs can occur in Watson-Crick base pairs, Hoogsteen base pairs, or any other sequence specific form.
  • the 5-fluorouracil-sensitive sensitizer in the present embodiment is a sensitizer containing a nucleotide fragment encoding one or more genes selected from the group consisting of PRAK2 gene, TGFBR2 gene and EXT1 gene.
  • the 5-fluorouracil-sensitive sensitizer is not particularly limited as long as it improves the mortality of cancer cells when 5-fluorouracil is administered (decreases the survival rate).
  • Student's t-test which is a parametric test. That is, in the Student's t-test, one-sided test should be p ⁇ 0.05, more preferably one-sided test should be p ⁇ 0.03, and most preferably one-sided test should be p ⁇ 0.01. Good.
  • the student t-test is not limited to a one-sided test, but may be a two-sided test. Further, when it cannot be assumed that the population follows a normal distribution, the presence or absence of a significant difference may be tested by performing a Mann-Whitney U test as a nonparametric test.
  • the PRAKG2 gene encoded by the nucleotide fragment contained in the 5-fluorouracil-sensitive sensitizer of the present embodiment is a protein kinase, AMP-activated, AMP-activated protein kinase ⁇ 2 non-catalytic subunit.
  • the gene encodes gamma 2 non-catalytic subunit) and is not particularly limited.
  • human wild-type PRAK2 gene Entrez Gene ID: 51422 5'-AMP-activated protein kinase subunit gamma subunit subunit gamma isoform a [a gene encoding Homo sapiens]
  • human wild-type PRAK2 gene Entrez Gene ID: 51422 5'-AMP-activated protein kinase subunit gamma subunit subunit gamma isoform a [a gene encoding Homo sapiens]
  • the PRAK2 gene encoded by the nucleotide fragment contained in the 5-fluorouracil-sensitive sensitizer of the present embodiment may be a mutant gene of the human wild type PRAK2 gene. This is because a gene having high homology with the human wild-type PRAK2 gene has a high possibility of enhancing 5-fluorouracil sensitivity in mammalian cells in the same manner as the human wild-type PRAKA2 gene.
  • the mutant type includes those resulting from differences in DNA sequences between individuals.
  • the mutant gene of the human wild-type PRAK2 gene consists of a base sequence encoding the amino acid sequence of the protein encoded by the human wild-type PRAK2 gene (ie, a degenerate sequence of the base sequence of the human wild-type PRAK2 gene). It may be a mutant gene. Because the amino acid sequence encoded by the mutant gene comprising the degenerate sequence of the human wild-type PRAK2 gene is the same as the amino acid sequence encoded by the wild-type gene, the function of the protein expressed from this mutant gene is wild-type. This is because it is the same as a protein expressed from a gene. It should be noted that a person skilled in the art can easily understand which base sequence corresponds to a degenerate sequence using a universal codon correspondence table.
  • the mutant gene of the human wild-type PRAK2 gene encodes an amino acid sequence obtained by deleting, substituting, or adding one or several amino acid residues in the amino acid sequence of the protein encoded by the human wild-type PRAK2 gene. It may be a mutant gene consisting of a base sequence.
  • the “one or several” is preferably 30 or less, more preferably 20 or less, more preferably 15 or less, more preferably 10 or less, more preferably 5 or less. Preferably, it is 4 or less, more preferably 3 or less, more preferably 2 or less, and still more preferably 1. This is because, as the base sequence of the mutant gene has fewer base deletions, substitutions or additions to the base sequence of the wild-type gene, it has a property closer to the gene encoding the base sequence of the wild-type gene. Because it will be.
  • amino acid sequence encoded by the nucleotide sequence of the mutant gene has one or several substitutions with respect to the amino acid sequence encoded by the human wild type PRAK2 gene, the properties of the amino acid side chain are preserved. It is preferably substituted with another amino acid.
  • hydrophobic amino acids A, I, L, M, F, P, W, Y, V
  • hydrophilic amino acids R, D, N, C, E, Q, G, H, K, S, T
  • amino acids having aliphatic side chains G, A, V, L, I, P
  • amino acids having hydroxyl group-containing side chains S, T, Y
  • the mutant gene of the human wild-type PRAK2 gene is a mutant gene comprising a base sequence encoding an amino acid sequence having 80% or more homology with the amino acid sequence of the protein encoded by the human wild-type PRAK2 gene. May be.
  • “80% or more” is preferably 85% or more, more preferably 90% or more, further preferably 95% or more, and most preferably 98% or more. This is because the higher the homology between the base sequence of the atypical gene and the base sequence of the wild-type gene, the closer to the gene encoding the base sequence of the wild-type gene. is there.
  • the mutant gene of the human wild type PRAK2 gene may be a mutant gene comprising a base sequence that hybridizes under stringent conditions to a base sequence complementary to the base sequence of the human wild type PRAK2 gene. .
  • a mutant gene consisting of a base sequence that hybridizes under stringent conditions is highly homologous to the base sequence of the wild-type gene, and therefore has characteristics close to those of the gene encoding the base sequence of the wild-type gene. It is because it will have.
  • TGFBR2 gene encoded by the nucleotide fragment contained in the 5-fluorouracil-sensitive sensitizer of the present embodiment encodes a mammalian TGF- ⁇ 2 type receptor (transforming growth factor, beta receptor II).
  • the gene may be any gene, and is not particularly limited.
  • human wild-type TGFBR2 gene encoding gene growth factor, beta receptor II (70/80 kDa) [Homo sapiens] encoding Entrez Gene ID: 7048
  • GenBank is a database of National Center for Biotechnology Information (NCBI).
  • the TGFBR2 gene encoded by the nucleotide fragment contained in the 5-fluorouracil-sensitive sensitizer of the present embodiment may be a mutant gene of the human wild type TGFBR2 gene. This is because a gene having a high homology with the human wild-type TGFBR2 gene is likely to enhance 5-fluorouracil sensitivity in mammalian cells in the same manner as the human wild-type TGFBR2 gene.
  • the mutant type includes those resulting from differences in DNA sequences between individuals.
  • the mutant gene of the human wild type TGFBR2 gene consists of a base sequence encoding the amino acid sequence of the protein encoded by the human wild type TGFBR2 gene (that is, a degenerate sequence of the base sequence of the human wild type TGFBR2 gene). It may be a mutant gene. Because the amino acid sequence encoded by the mutant gene consisting of the degenerate sequence of the human wild type TGFBR2 gene is the same as the amino acid sequence encoded by the wild type gene, the function of the protein expressed from the mutant gene is wild type. This is because it is the same as a protein expressed from a gene. It should be noted that a person skilled in the art can easily understand which base sequence corresponds to a degenerate sequence using a universal codon correspondence table.
  • the mutant gene of the human wild type TGFBR2 gene encodes an amino acid sequence formed by deleting, substituting, or adding one or several amino acid residues in the amino acid sequence of the protein encoded by the human wild type TGFBR2 gene. It may be a mutant gene consisting of a base sequence.
  • the “one or several” is preferably 30 or less, more preferably 20 or less, more preferably 15 or less, more preferably 10 or less, more preferably 5 or less. Preferably, it is 4 or less, more preferably 3 or less, more preferably 2 or less, and still more preferably 1. This is because, as the base sequence of the mutant gene has fewer base deletions, substitutions or additions to the base sequence of the wild-type gene, it has a property closer to the gene encoding the base sequence of the wild-type gene. Because it will be.
  • the amino acid sequence encoded by the nucleotide sequence of the mutant gene has one or several substitutions with respect to the amino acid sequence encoded by the human wild type TGFBR2 gene, the properties of the amino acid side chain are preserved. It is preferably substituted with another amino acid. The description of the nature of the amino acid side chain has already been made and will not be repeated.
  • the mutant gene of the human wild type TGFBR2 gene is a mutant gene comprising a base sequence encoding an amino acid sequence having 80% or more homology with the amino acid sequence of the protein encoded by the human wild type TGFBR2 gene. May be.
  • “80% or more” is preferably 85% or more, more preferably 90% or more, further preferably 95% or more, and most preferably 98% or more. This is because the higher the homology between the base sequence of the atypical gene and the base sequence of the wild-type gene, the closer to the gene encoding the base sequence of the wild-type gene. is there.
  • the mutant gene of the human wild type TGFBR2 gene may be a mutant gene comprising a base sequence that hybridizes under stringent conditions to a base sequence complementary to the base sequence of the human wild type TGFBR2 gene. .
  • a mutant gene consisting of a base sequence that hybridizes under stringent conditions is highly homologous to the base sequence of the wild-type gene, and therefore has characteristics close to those of the gene encoding the base sequence of the wild-type gene. It is because it will have.
  • EXT1 gene encoded by the nucleotide fragment contained in the 5-fluorouracil-sensitive sensitizer of the present embodiment is an endoplasmic reticulum-resident type II in mammalian endoplasmic reticulum. Any gene may be used as long as it is a gene that encodes transmembrane glycosyltransferase (for example, human EXT1 gene (Entrez Gene ID: 2131 EXT1 genes (multiple) 1)). Can be suitably used. This is because this human wild EXT1 gene has an effect of enhancing the anti-tumor effect of 5-fluorouracil, which has been demonstrated in Examples described later. The details of the base sequence and the like related to the above gene can be confirmed in GenBank which is a database of National Center for Biotechnology Information (NCBI).
  • the mammalian endoplasmic reticulum type II transmembrane glycosyltransferase encoded by this EXT1 gene is related to a sugar chain elongation reaction in biosynthesis of heparan sulfate (involved in the chain elongation step of heparan). sulfate biosynthesis).
  • the EXT1 gene encoded by the nucleotide fragment contained in the 5-fluorouracil sensitive sensitizer of the present embodiment may be a mutant gene of the human wild type EXT1 gene. This is because a gene having high homology with the human wild-type EXT1 gene is likely to enhance 5-fluorouracil sensitivity in mammalian cells in the same manner as the human wild-type EXT1 gene.
  • the mutant type includes those resulting from differences in DNA sequences between individuals.
  • the mutant gene of the human wild type EXT1 gene consists of a base sequence encoding the amino acid sequence of the protein encoded by the human wild type EXT1 gene (that is, a degenerate sequence of the base sequence of the human wild type EXT1 gene). It may be a mutant gene. Because the amino acid sequence encoded by the mutant gene consisting of the degenerate sequence of the human wild type EXT1 gene is the same as the amino acid sequence encoded by the wild type gene, the function of the protein expressed from this mutant gene is wild type. This is because it is the same as a protein expressed from a gene. It should be noted that a person skilled in the art can easily understand which base sequence corresponds to a degenerate sequence using a universal codon correspondence table.
  • the mutant gene of the human wild type EXT1 gene encodes an amino acid sequence obtained by deleting, substituting, or adding one or several amino acid residues in the amino acid sequence of the protein encoded by the human wild type EXT1 gene. It may be a mutant gene consisting of a base sequence.
  • the “one or several” is preferably 30 or less, more preferably 20 or less, more preferably 15 or less, more preferably 10 or less, more preferably 5 or less. Preferably, it is 4 or less, more preferably 3 or less, more preferably 2 or less, and still more preferably 1. This is because, as the base sequence of the mutant gene has fewer base deletions, substitutions or additions to the base sequence of the wild-type gene, it has a property closer to the gene encoding the base sequence of the wild-type gene. Because it will be.
  • the amino acid sequence encoded by the base sequence of the mutant gene has one or several substitutions with respect to the amino acid sequence encoded by the human wild type EXT1 gene, the properties of the amino acid side chain are preserved. It is preferably substituted with another amino acid. The description of the nature of the amino acid side chain has already been made and will not be repeated.
  • the mutant gene of the human wild type EXT1 gene is a mutant gene comprising a base sequence encoding an amino acid sequence having 80% or more homology to the amino acid sequence of the protein encoded by the human wild type EXT1 gene. May be.
  • “80% or more” is preferably 85% or more, more preferably 90% or more, further preferably 95% or more, and most preferably 98% or more. This is because the higher the homology between the base sequence of the atypical gene and the base sequence of the wild-type gene, the closer to the gene encoding the base sequence of the wild-type gene. is there.
  • the mutant gene of the human wild type EXT1 gene may be a mutant gene comprising a base sequence that hybridizes under stringent conditions to a base sequence complementary to the base sequence of the human wild type EXT1 gene. .
  • a mutant gene consisting of a base sequence that hybridizes under stringent conditions is highly homologous to the base sequence of the wild-type gene, and therefore has characteristics close to those of the gene encoding the base sequence of the wild-type gene. It is because it will have.
  • the 5-fluorouracil-sensitive sensitizer is a sensitizer including a vector that expresses one or more genes selected from the group consisting of PRAK2 gene, TGFBR2 gene, and EXT1 gene.
  • PRAK2 gene the genetic marker of the chromosome
  • TGFBR2 gene the genetic marker of the chromosome
  • EXT1 gene the genetic marker of the chromosome
  • the sensitizer itself an effect of enhancing the antitumor effect of 5-fluorouracil can be obtained.
  • the expression vector used in this embodiment includes a vector and one or more genes selected from the group consisting of a PRAK2 gene, a TGFBR2 gene, and an EXT1 gene operably linked to the vector.
  • This vector can suitably express one or more genes selected from the group consisting of PRAK2 gene, TGFBR2 gene, and EXT1 gene (and the protein encoded by the gene), as shown in the Examples described later. .
  • Adenovirus vector is not particularly limited, and a vector that can be introduced into a general mammalian cell can be used.
  • an adenovirus used in Examples described later is used.
  • a vector can be preferably used.
  • Adenovirus is one of the most well-studied viruses, and since the currently widely used adenovirus vector lacks the E1 gene, special cells that continuously express the E1 gene (293 cells)
  • Adenovirus vector is a safe and highly efficient vector, and it is possible to infect normal cells. Widely used up to the field.
  • Use of an adenovirus vector provides the following advantages. 1) A high titer virus can be obtained. A virus solution of about 10 8 to 10 9 PFU / ml can be easily obtained.
  • any of the “full-length DNA introduction method” or the “COS-TPC method” may be used.
  • the full-length DNA introduction method is a method for obtaining a recombinant adenovirus by transfecting a restriction digested recombinant cosmid into 293 cells.
  • homologous recombination in cells is not required, so there is no contamination with the parent virus, and most of the resulting recombinant adenovirus is the target virus.
  • the “COS-TPC method” is a method for producing a recombinant adenovirus by co-transfecting a recombinant cosmid and an Adenovirus gene DNA-TPC into 293 cells and utilizing homologous recombination occurring in 293 cells. is there.
  • Adenovirus genome DNA-TPC is bound to a terminal protein (TP) that is originally bound to both ends of adenovirus genomic DNA. Therefore, a recombinant adenovirus can be produced with high efficiency, and the target virus can be obtained almost certainly.
  • TP terminal protein
  • the 5-fluorouracil-sensitive sensitizer according to the present embodiment is a sensitizer containing a protein encoded by one or more genes selected from the group consisting of PRAK2 gene, TGFBR2 gene and EXT1 gene. According to this configuration, since it includes a protein encoded by any one of PRKG2 gene, TGFBR2 gene or EXT1 gene, which has been demonstrated in Examples described later, to have an action of enhancing the antitumor effect of 5-fluorouracil, As the sensitizer itself, an effect of enhancing the antitumor effect of 5-fluorouracil can be obtained.
  • the protein When obtaining a protein encoded by any one of the PRAK2 gene, TGFBR2 gene, or EXT1 gene, the protein may be artificially chemically synthesized, but it is inexpensive and large-scale to obtain by a production method using cell culture. It is preferable because it can be produced.
  • the PRAKG2 gene, TGFBR2 gene, TGFBR2 gene, TGFBR2 gene, TGFBR2 gene, TGFBR2 gene, TGFBR2 gene, TGFBR2 gene, TGFBR2 gene, and EXT1 gene are mass-produced and separated and purified. A general method for separating and purifying a protein encoded by either the gene or the EXT1 gene can be used.
  • it can be carried out by disrupting large mammalian cells that have mass-produced a protein encoded by any of PRAK2 gene, TGFBR2 gene or EXT1 gene, and then using a commonly used separation and purification means.
  • a commonly used separation and purification means for destruction of mammalian cells, for example, ultrasonic treatment, high-pressure homogenizer treatment, osmotic shock method and the like are preferably used.
  • methods such as salting out, gel filtration and ion exchange chromatography can be used in appropriate combination.
  • AMP-activated protein kinase encoded by PRKG2 gene The protein encoded by the PRAK2 gene contained in the 5-fluorouracil-sensitive sensitizer of the present embodiment is a AMP-activated protein kinase ⁇ 2 non-catalytic subunit (protein) of mammals.
  • Kinase, AMP-activated, gamma 2 non-catalytic subunit) is not particularly limited.
  • human wild-type AMP-activated protein kinase ⁇ 2 non-catalytic subunit (5′-AMP-activated protein kinase)
  • Subunit gamma-2 isoform a [Homo sapiens]) can be preferably used.
  • the human wild-type AMP-activated protein kinase ⁇ 2 non-catalytic subunit has an effect of enhancing the antitumor effect of 5-fluorouracil, which is demonstrated in Examples described later. Details of the amino acid sequence and the like regarding the AMP-activated protein kinase ⁇ 2 non-catalytic subunit can be confirmed in GenBank, which is a database of National Center for Biotechnology Information (NCBI).
  • the AMP-activated protein kinase contained in the 5-fluorouracil-sensitive sensitizer of this embodiment may be a mutant protein of a human wild-type AMP-activated protein kinase ⁇ 2 non-catalytic subunit. This is because a protein having high homology with human wild-type AMP-activated protein kinase ⁇ 2 non-catalytic subunit is sensitive to 5-fluorouracil in mammalian cells in the same manner as human wild-type AMP-activated protein kinase ⁇ 2 non-catalytic subunit. This is because there is a high possibility of strengthening.
  • the mutant type includes those resulting from differences in amino acid sequences between individuals.
  • the mutant protein of the human wild-type AMP-activated protein kinase ⁇ 2 non-catalytic subunit is one or several amino acid residues in the amino acid sequence of the human wild-type AMP-activated protein kinase ⁇ 2 non-catalytic subunit. It may be a mutant protein consisting of an amino acid sequence obtained by deleting, substituting, or adding.
  • the “one or several” is preferably 30 or less, more preferably 20 or less, more preferably 15 or less, more preferably 10 or less, more preferably 5 or less. Preferably, it is 4 or less, more preferably 3 or less, more preferably 2 or less, and still more preferably 1.
  • the amino acid sequence of the mutant protein has fewer amino acid deletions, substitutions or additions to the amino acid sequence of the wild-type protein, it has a property closer to that of the wild-type AMP-activated protein kinase ⁇ 2 non-catalytic subunit. It is because it will have.
  • amino acid sequence of the mutant AMP-activated protein kinase ⁇ 2 non-catalytic subunit has one or several substitutions with respect to the amino acid sequence of the human wild-type AMP-activated protein kinase ⁇ 2 non-catalytic subunit
  • the amino acid side chain is preferably substituted with another amino acid having a conserved property.
  • it already demonstrated about the property of an amino acid side chain it does not repeat.
  • the mutant protein of the human wild-type AMP-activated protein kinase ⁇ 2 non-catalytic subunit is an amino acid sequence having 80% or more homology with the amino acid sequence of the human wild-type AMP-activated protein kinase ⁇ 2 non-catalytic subunit.
  • a mutant protein consisting of Here, “80% or more” is preferably 85% or more, more preferably 90% or more, further preferably 95% or more, and most preferably 98% or more.
  • the mutant protein of the human wild-type AMP-activated protein kinase ⁇ 2 non-catalytic subunit has a base sequence complementary to the base sequence of the gene encoding the human wild-type AMP-activated protein kinase ⁇ 2 non-catalytic subunit. It may be a mutant protein consisting of an amino acid sequence encoded by a base sequence that hybridizes under stringent conditions. This is because a mutant AMP-activated protein kinase ⁇ 2 non-catalytic subunit consisting of an amino acid sequence encoded by a base sequence that hybridizes under stringent conditions is highly homologous to the amino acid sequence of a wild-type protein. This is because it has characteristics close to those of the wild-type AMP-activated protein kinase ⁇ 2 non-catalytic subunit.
  • TGF- ⁇ 2 type receptor encoded by TGFBR2 gene The protein encoded by the TGFBR2 gene contained in the 5-fluorouracil-sensitive sensitizer of the present embodiment is a mammalian growth factor, transforming growth factor, beta receptor II), and is not particularly limited.
  • human wild type TGF- ⁇ 2 type receptor transforming growth factor, beta receptor II (70/80 kDa) [Homo sapiens]
  • GenBank is a database of National Center for Biotechnology Information (NCBI).
  • the TGF- ⁇ 2 type receptor contained in the 5-fluorouracil-sensitive sensitizer of the present embodiment may be a mutant protein of human wild type TGF- ⁇ 2 type receptor. This is because a protein having high homology with human wild-type TGF- ⁇ 2 type receptor is likely to enhance 5-fluorouracil sensitivity in mammalian cells in the same manner as human wild-type TGF- ⁇ 2 type receptor. It is.
  • the mutant type includes those resulting from differences in amino acid sequences between individuals.
  • the mutant protein of the human wild type TGF- ⁇ 2 type receptor deletes, substitutes, or adds one or several amino acid residues in the amino acid sequence of the human wild type TGF- ⁇ 2 type receptor.
  • a mutant protein consisting of the amino acid sequence may be used.
  • the “one or several” is preferably 30 or less, more preferably 20 or less, more preferably 15 or less, more preferably 10 or less, more preferably 5 or less. Preferably, it is 4 or less, more preferably 3 or less, more preferably 2 or less, and still more preferably 1.
  • the amino acid sequence of the mutant protein has fewer amino acid deletions, substitutions or additions to the amino acid sequence of the wild-type protein, it has a property closer to that of the wild-type TGF- ⁇ 2 type receptor. Because it will be.
  • the amino acid sequence of the mutant TGF- ⁇ 2 type receptor has one or several substitutions with respect to the amino acid sequence of the human wild type TGF- ⁇ 2 type receptor, the properties of the amino acid side chain are preserved. It is preferable to substitute another amino acid. In addition, since it already demonstrated about the property of an amino acid side chain, it does not repeat.
  • the human wild type TGF- ⁇ 2 type receptor mutant protein is a mutant protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence of human wild type TGF- ⁇ 2 type receptor. Also good.
  • “80% or more” is preferably 85% or more, more preferably 90% or more, further preferably 95% or more, and most preferably 98% or more. This is because the higher the homology of the amino acid sequence of the mutant TGF- ⁇ 2 type receptor with the amino acid sequence of the wild type protein, the closer to the wild type TGF- ⁇ 2 type receptor. Because it becomes.
  • the mutant protein of the human wild type TGF- ⁇ 2 type receptor hybridizes under stringent conditions to the base sequence complementary to the base sequence of the gene encoding the human wild type TGF- ⁇ 2 type receptor. It may be a mutant protein consisting of an amino acid sequence encoded by a base sequence. This is because a mutant TGF- ⁇ 2 type receptor consisting of an amino acid sequence encoded by a base sequence that hybridizes under stringent conditions has high homology to the amino acid sequence of a wild-type protein. This is because it has characteristics similar to those of - ⁇ 2 type receptors.
  • Endoplasmic reticulum type II transmembrane glycosyltransferase encoded by the EXT1 gene The protein encoded by the EXT1 gene contained in the 5-fluorouracil-sensitive sensitizer of the present embodiment is an endoplasmic reticulum type II in mammals. Any transmembrane glycosyltransferase (endoplasmic reticulum-resident type II transmembrane glycosyltransferase) may be used.
  • human wild-type endoplasmic reticulum transmembrane glycosyltransferase (EXT1) 1 [Homo sapiens]) or the like can be preferably used.
  • the mammalian endoplasmic reticulum type II transmembrane glycosyltransferase encoded by this EXT1 gene is related to a sugar chain elongation reaction in biosynthesis of heparan sulfate (involved in the chain elongation step of heparan). sulfate biosynthesis).
  • the endoplasmic reticulum type II transmembrane glycosyltransferase included in the 5-fluorouracil-sensitive sensitizer of the present embodiment is a mutant protein of human wild type endoplasmic reticulum type II transmembrane glycosyltransferase. Also good. This is because a protein having a high homology with human wild-type endoplasmic reticulum type II transmembrane glycosyltransferase is similar to human wild-type endoplasmic reticulum type II transmembrane glycosyltransferase in mammalian cells. This is because the possibility of enhancing sensitivity to fluorouracil is high.
  • the mutant type includes those resulting from differences in amino acid sequences between individuals.
  • the mutant protein of human wild type endoplasmic reticulum type II transmembrane glycosyltransferase is one or several amino acids of the amino acid sequence of human wild type endoplasmic reticulum type II transmembrane glycosyltransferase. It may be a mutant protein consisting of an amino acid sequence obtained by deleting, substituting, or adding residues.
  • the “one or several” is preferably 30 or less, more preferably 20 or less, more preferably 15 or less, more preferably 10 or less, more preferably 5 or less. Preferably, it is 4 or less, more preferably 3 or less, more preferably 2 or less, and still more preferably 1.
  • the amino acid sequence of the mutant protein has fewer amino acid deletions, substitutions or additions to the amino acid sequence of the wild-type protein, it is closer to the wild-type endoplasmic reticulum type II transmembrane glycosyltransferase. It is because it will have.
  • the amino acid side chain is preferably substituted with another amino acid in which the nature of the amino acid side chain is conserved.
  • it since it already demonstrated about the property of an amino acid side chain, it does not repeat.
  • the mutant protein of human wild-type endoplasmic reticulum type II transmembrane glycosyltransferase has 80% or more homology with the amino acid sequence of human wild-type endoplasmic reticulum type II transmembrane glycosyltransferase. It may be a mutant protein consisting of an amino acid sequence.
  • “80% or more” is preferably 85% or more, more preferably 90% or more, further preferably 95% or more, and most preferably 98% or more.
  • the mutant protein of human wild-type endoplasmic reticulum type II transmembrane glycosyltransferase has a nucleotide sequence complementary to the base sequence of the gene encoding human wild-type endoplasmic reticulum type II transmembrane glycosyltransferase.
  • it may be a mutant protein comprising an amino acid sequence encoded by a base sequence that hybridizes under stringent conditions. This is because a mutant endoplasmic reticulum type II transmembrane glycosyltransferase consisting of an amino acid sequence encoded by a base sequence that hybridizes under stringent conditions is highly homologous to the amino acid sequence of a wild-type protein. This is because it has characteristics similar to wild-type endoplasmic reticulum type II transmembrane glycosyltransferase.
  • ⁇ 5-fluorouracil / sensitizer for interferon ⁇ > The sensitizers of the above-described embodiments described so far can be used as sensitizers that enhance the sensitivity to 5-fluorouracil and interferon ⁇ when 5-fluorouracil and interferon ⁇ are used in combination.
  • the TGFBR2 gene and the EXT1 gene have an effect of enhancing the antitumor action when 5-fluorouracil / interferon ⁇ is used in combination. This is because it can be assumed that there is an effect of enhancing the antitumor action when 5-fluorouracil / interferon ⁇ is used in combination.
  • the TGF- ⁇ 2 type receptor encoded by the TGFBR2 gene and the endoplasmic reticulum type II transmembrane glycosyltransferase encoded by the EXT1 gene have anti-antibody activity in combination with 5-fluorouracil / interferon ⁇ . Since it has been demonstrated that there is an effect of enhancing the tumor action, those skilled in the art will be able to use anti-tumor in combination with 5-fluorouracil / interferon ⁇ in the AMP-activated protein kinase ⁇ 2 non-catalytic subunit encoded by the PRAKG2 gene. This is because it can be assumed that the effect is enhanced.
  • the most effective route for treatment is preferably used.
  • Oral administration, or intraoral, respiratory tract, intrarectal Parenteral administration such as subcutaneous, intramuscular, intraocular and intravenous, can be given and can be administered systemically or locally.
  • the administration route is preferably intravenous administration.
  • the route of administration need not be the same as the route of administration of 5-fluorouracil or interferon alpha.
  • Administration forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.
  • Suitable formulations for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.
  • Liquid preparations such as emulsions and syrups include sugars such as water, sucrose, sorbitol, fructose, glycols such as polyethylene glycol, propylene glycol, oils such as sesame oil, olive oil, soybean oil, p-hydroxybenzoic acid
  • Preservatives such as esters, and flavors such as strawberry flavor and peppermint can be used as additives.
  • capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl alcohol , Hydroxypropylcellulose, gelatin and other binders, surfactants such as fatty acid esters, plasticizers such as glycerin and the like can be used as additives.
  • excipients such as lactose, glucose, sucrose and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl alcohol , Hydroxypropylcellulose, gelatin and other binders, surfactants such as fatty acid esters, plasticizers such as glycerin and the like can be used as additives.
  • Preparations suitable for parenteral administration include injections, suppositories, sprays and the like.
  • Aqueous solutions for injection include, for example, isotonic solutions containing physiological saline, glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol and sodium chloride.
  • Suitable solubilizers such as Alcohols, specifically ethanol, polyalcohols such as propylene glycol, polyethylene glycol, nonionic surfactants such as polysorbate 80 (TM), HCO-50 may be used in combination.
  • Suppositories are prepared using a carrier such as cocoa butter, hydrogenated fat or carboxylic acid.
  • the spray is prepared using a carrier that does not irritate the recipient's oral cavity and airway mucosa, and that facilitates absorption by dispersing the sensitizer as fine particles.
  • a carrier that does not irritate the recipient's oral cavity and airway mucosa, and that facilitates absorption by dispersing the sensitizer as fine particles.
  • the carrier include lactose and glycerin.
  • aerosols, rye powders, and the like it is possible to formulate aerosols, rye powders, and the like.
  • the components exemplified as additives for oral preparations can also be added.
  • the sensitizer includes a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (eg, human serum albumin, Polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like.
  • a buffer eg, phosphate buffer, sodium acetate buffer
  • a soothing agent eg, benzalkonium chloride, procaine hydrochloride, etc.
  • a stabilizer eg, human serum albumin, Polyethylene glycol, etc.
  • preservatives eg, benzyl alcohol, phenol, etc.
  • antioxidants eg, benzyl alcohol, phenol, etc.
  • the administration method can be appropriately selected depending on the patient's age, symptoms, target organs, and the like.
  • the dose of this sensitizer can be selected, for example, in the range of 0.0001 mg to 1000 mg per kg of body weight at a time.
  • the dose can be selected in the range of 0.001 to 100,000 mg / body per patient, but is not necessarily limited to these values. It varies depending on the intended therapeutic effect, administration method, treatment period, age, body weight, type and amount of anticancer agent used in combination.
  • the dose and administration method vary depending on the weight, age, symptoms, etc. of the patient, but can be appropriately selected by those skilled in the art. It may also be administered in combination with another appropriate chemotherapeutic agent.
  • the anticancer agent kit of this embodiment is a kit comprising the sensitizer of the above-described embodiment described above and 5-fluorouracil.
  • the PRAK2 gene, the TGFBR2 gene, or the EXT1 gene (or a protein encoded by these genes), which has been demonstrated in Examples described later, to have an action of enhancing the antitumor effect of 5-fluorouracil was used. Since it contains a sensitizer, the antitumor effect of 5-fluorouracil is enhanced and an excellent antitumor effect can be obtained.
  • 5-fluorouracil is a fluoropyrimidine-based antimetabolite and includes a compound used as an antineoplastic agent (anticancer agent).
  • This compound has a structure in which the 5-position hydrogen atom of uracil is replaced with a fluorine atom.
  • This compound was synthesized in 1956 by Dushinsky et al., And was later established as an anti-neoplastic agent in a wide range of basic and clinical studies centered on Heidelberger et al.
  • “5-FU Kyowa” (Kyowa Hakko Kirin) series is commercially available.
  • the anticancer agent kit according to the present embodiment may be a kit further containing interferon ⁇ in addition to the sensitizer of the above-described embodiment described above and 5-fluorouracil.
  • interferon ⁇ in addition to the sensitizer of the above-described embodiment described above and 5-fluorouracil.
  • the TGF- ⁇ 2 type receptor encoded by the TGFBR2 gene and the endoplasmic reticulum type II transmembrane glycosyltransferase encoded by the EXT1 gene have anti-antibody activity in combination with 5-fluorouracil / interferon ⁇ . Since it has been demonstrated that there is an effect of enhancing the tumor action, those skilled in the art will be able to use anti-tumor in combination with 5-fluorouracil / interferon ⁇ in the AMP-activated protein kinase ⁇ 2 non-catalytic subunit encoded by the PRAKG2 gene. This is because it can be assumed that the effect is enhanced.
  • IFN interferon
  • cytokine a protein secreted by cells in response to the invasion of foreign substances such as pathogens (particularly viruses) and tumor cells. This protein acts to stop viral growth, inhibit cell growth, regulate the immune system and inflammation. This protein is a kind of cytokine. This protein is used as a pharmaceutical for the treatment of malignant tumors such as hepatitis C and multiple myeloma.
  • Interferon ⁇ means IFN- ⁇ : 13 types (1, 2, 4, 5, 6, 7, 8, 10, 13, 14, 16, 17, 21).
  • Examples of pharmaceuticals using IFN ⁇ include: Sumiferon (R): renal cancer, multiple myeloma, chronic myelogenous leukemia, hairy cell leukemia, subacute sclerosing panencephalitis, HTLV-1 myelopathy, hepatitis B, C Hepatitis B, OH (R): Chronic myeloid leukemia, hepatitis B, hepatitis C and the like are commercially available.
  • interferon ⁇ includes intron A (R), which is a commercially available drug using IFN ⁇ 2b: hepatitis B / hepatitis C, pegasis (R), which is a commercially available drug using PEG-IFN ⁇ 2a.
  • R intron A
  • IFN ⁇ 2b hepatitis B / hepatitis C
  • pegasis R
  • PEG-IFN ⁇ 2a a commercially available drug using PEG-IFN ⁇ 2a
  • PEG-IFN ⁇ 2a hepatitis C
  • Pegintron (R) Hepatitis C
  • Advaferon (R) a commercially available drug using C-IFN ⁇ : Hepatitis C and the like are also included.
  • the 5-fluorouracil sensitivity inhibitor according to this embodiment is an inhibitor comprising siRNA, shRNA, or antisense RNA that suppresses the expression of one or more genes selected from the group consisting of PRAK2 gene, TGFBR2 gene, and EXT1 gene .
  • siRNA, shRNA, or the like that suppresses the expression of any of PRAK2 gene, TGFBR2 gene, or EXT1 gene, which has been demonstrated in Examples to be described later, has an action of enhancing the antitumor effect of 5-fluorouracil. Since the antisense RNA is contained, the inhibitor itself can suppress the antitumor effect of 5-fluorouracil.
  • the 5-fluorouracil sensitivity inhibitor according to the present embodiment when used, for example, when 5-fluorouracil is administered systemically or extensively by oral or intravenous injection or the like, the 5-fluorouracil sensitivity is detected by an injection or the like at a site where no cancer focus exists. Inhibitors can be injected. In this way, it is possible to reduce the possibility of serious side effects due to 5-fluorouracil occurring at sites where cancerous lesions do not exist.
  • a 5-fluorouracil sensitivity inhibitor is injected into the site where there is no cancer nest with an injection or the like, while a 5-fluorouracil sensitive sensitizer is injected into the cancer nest with an injection or the like, no cancer nest exists. While reducing the possibility of serious side effects due to 5-fluorouracil at the site, it is possible to enhance the antitumor effect of 5-fluorouracil in the cancer nest.
  • the 5-fluorouracil sensitivity inhibitor is not particularly limited as long as it reduces the mortality of cancer cells (improves the survival rate) when 5-fluorouracil is administered.
  • Student's t-test which is a parametric test. That is, in the Student's t-test, one-sided test should be p ⁇ 0.05, more preferably one-sided test should be p ⁇ 0.03, and most preferably one-sided test should be p ⁇ 0.01. Good.
  • the student t-test is not limited to a one-sided test, but may be a two-sided test. Further, when it cannot be assumed that the population follows a normal distribution, the presence or absence of a significant difference may be tested by performing a Mann-Whitney U test as a nonparametric test.
  • any of the above inhibitors can also be used as an inhibitor that suppresses sensitivity to 5-fluorouracil and interferon ⁇ when 5-fluorouracil and interferon ⁇ are used in combination.
  • the TGFBR2 gene and the EXT1 gene have an effect of enhancing the antitumor effect when 5-fluorouracil / interferon ⁇ is used in combination. Therefore, those skilled in the art will recognize the TGFBR2 gene and the EXT1 gene. This is because it can be assumed that if the expression of the gene is suppressed, there is an effect of suppressing the antitumor action when 5-fluorouracil / interferon ⁇ is used together. Further, it is also possible for those skilled in the art to similarly assume that suppressing the expression of the PRAK2 gene has an effect of suppressing the antitumor action when 5-fluorouracil / interferon ⁇ is used in combination.
  • siRNA siRNA small interfering RNA is a small double-stranded RNA consisting of 21-23 base pairs. siRNA is involved in a phenomenon called RNA interference (RNAi), and suppresses gene expression in a sequence-specific manner by destroying mRNA.
  • RNAi RNA interference
  • the 5-fluorouracil sensitivity-suppressing agent includes two containing 21-23 base pairs corresponding to a part of the base sequence of one or more genes selected from the group consisting of PRAK2 gene, TGFBR2 gene and EXT1 gene. It is preferable that siRNA which consists of strand RNA is included. Such siRNA consisting of double-stranded RNA can effectively suppress the expression of one or more genes selected from the group consisting of PRAK2 gene, TGFBR2 gene and EXT1 gene. As a result, 5-fluorouracil An effect of suppressing the antitumor effect is obtained.
  • shRNA shRNA small hairpin RNA
  • shRNA is an RNA molecule in which a small double-stranded RNA consisting of 21-23 base pairs is connected as a single strand via a single loop structure.
  • shRNA is also involved in a phenomenon called RNA interference (RNAi), and suppresses gene expression in a sequence-specific manner by destroying mRNA.
  • RNAi RNA interference
  • the 5-fluorouracil sensitivity-suppressing agent includes two containing 21-23 base pairs corresponding to a part of the base sequence of one or more genes selected from the group consisting of PRAK2 gene, TGFBR2 gene and EXT1 gene.
  • the stranded RNA includes a form of shRNA connected as a single strand via a single loop structure.
  • shRNA having a hairpin structure can effectively suppress the expression of one or more genes selected from the group consisting of PRAK2 gene, TGFBR2 gene and EXT1 gene, and as a result, anti-tumor of 5-fluorouracil An effect of suppressing the effect is obtained.
  • Antisense RNA is antisense RNA having a sequence complementary to a target mRNA.
  • antisense DNA is antisense DNA having a sequence complementary to the target mRNA.
  • the 5-fluorouracil sensitivity inhibitor according to the present embodiment is an antisense RNA corresponding to at least a part (or all) of the base sequence of one or more genes selected from the group consisting of PRAK2 gene, TGFBR2 gene, and EXT1 gene. It is preferable to contain.
  • Such an antisense RNA can effectively suppress the expression of one or more genes selected from the group consisting of PRAK2 gene, TGFBR2 gene and EXT1 gene, and as a result, the antitumor effect of 5-fluorouracil can be reduced. The effect of suppressing is acquired.
  • An antisense DNA may be used instead of the antisense RNA.
  • the diagnostic agent for determining 5-fluorouracil sensitivity includes a reagent for measuring the expression level of one or more genes selected from the group consisting of PRAKG2 gene, TGFBR2 gene and EXT1 gene It is.
  • a reagent for measuring the expression level of any of PRAK2 gene, TGFBR2 gene, or EXT1 gene which has been demonstrated in Examples described later, to have an action of enhancing the antitumor effect of 5-fluorouracil Therefore, by measuring the expression level of any one of the PRAK2 gene, TGFBR2 gene or EXT1 gene in the subject's living body, it is possible to determine whether or not the subject is likely to be effective in the anti-tumor effect of 5-fluorouracil. It can be used as a diagnostic agent.
  • the method of using the diagnostic agent is not particularly limited.
  • the PRAK2 gene, the TGFBR2 gene, or the EXT1 gene in cells, blood, serum, body fluid, or pathological sections in a subject's living body, and standard cells. Diagnosis is possible by examining and comparing the expression level of any of the above. For example, if these expression levels exceed a predetermined threshold value, it can be determined that the subject has a constitution that allows the anti-tumor effect of 5-fluorouracil to be effective. On the other hand, if the expression level falls below a predetermined threshold, it can be determined that the subject has a constitution that makes it difficult for the antitumor effect of 5-fluorouracil to be effective.
  • this threshold value may be set to 1.5 times or more of the average expression level of healthy subjects, may be set to 2 times or more, may be set to 5 times or more, and may be set to 10 times or more. It may be set.
  • this threshold value may be set, for example, when the standard deviation is twice or more larger than the average value of the expression level of healthy subjects, or may be set when the standard deviation is five times or more larger than the standard deviation. You may set when it is 10 times or more larger.
  • the determination may be made based on whether the expression level of any of PRAK2 gene, TGFBR2 gene, or EXT1 gene in the subject's living body is so large that a significant difference is recognized, for example, compared to the expression level of a healthy person.
  • a significant difference for example, when it can be assumed that the population follows a normal distribution, it is preferable that there is a significant difference in Student's t-test, which is a parametric test. That is, in the Student's t-test, one-sided test should be p ⁇ 0.05, more preferably one-sided test should be p ⁇ 0.03, and most preferably one-sided test should be p ⁇ 0.01. Good.
  • the student t-test is not limited to a one-sided test, but may be a two-sided test. Further, when it cannot be assumed that the population follows a normal distribution, the presence or absence of a significant difference may be tested by performing a Mann-Whitney U test as a nonparametric test.
  • All of the above diagnostic agents can also be used as diagnostic agents for determining whether a subject has a predisposition to the antitumor effect of 5-fluorouracil and interferon when 5-fluorouracil and interferon ⁇ are used in combination. it can.
  • the TGFBR2 gene and the EXT1 gene have an effect of enhancing the antitumor effect when 5-fluorouracil / interferon ⁇ is used in combination. Therefore, those skilled in the art will recognize the TGFBR2 gene and the EXT1 gene.
  • the reagent for measuring the expression level of any one of the PRAK2 gene, TGFBR2 gene, or EXT1 gene in the subject's living body is not particularly limited.
  • it is complementary to the base sequences of the PRAKG2, TGFBR2 gene, and EXT1 gene. It can be measured using a probe containing a typical base sequence.
  • the expression level of any mRNA of the PRAKG2 gene, TGFBR2 gene or EXT1 gene in the subject's living body can be suitably measured.
  • These probes are preferably labeled with a fluorescent dye or a radioisotope. By labeling in this way, the expression level of these mRNAs can be measured with a simple method with high sensitivity.
  • the reagent for measuring the expression level of any of PRAKG2 gene, TGFBR2 gene or EXT1 gene in the subject's living body is encoded by one or more genes selected from the group consisting of PRAKG2, TGFBR2 gene and EXT1 gene It may be a reagent for measuring the expression level of protein.
  • an antibody that specifically binds to a protein encoded by one or more genes selected from the group consisting of PRAKG2 gene, TGFBR2 gene, and EXT1 gene can be preferably used.
  • the expression level of the protein encoded by any of PRAKG2 gene, TGFBR2 gene or EXT1 gene in the body of the subject can be suitably measured.
  • These antibodies are preferably labeled with a fluorescent dye or a radioisotope. By labeling in this way, the expression level of these proteins can be measured with a simple method with high sensitivity.
  • the method for screening a gene having an effect of enhancing the sensitivity of an anticancer drug comprises a step of introducing a ribozyme library into a predetermined cancer cell line of a mammal, and a cancer cell into which the ribozyme library has been introduced with a predetermined anticancer drug. And a step of collecting a ribozyme from a living cell among cancer cells treated with an anticancer agent to obtain a novel ribozyme library. That is, the gene screening method according to the present embodiment is a so-called “modified random ribozyme method”.
  • This “modified random ribozyme method” is a method (gene discovery system) in which useful genes can be searched in terms of function using a ribozyme library in which substrate recognition sites are randomized.
  • the outline of this “modified random ribozyme method” is that a ribozyme library is made to function in the cell, and a cell in which a phenotype of a cell of interest (such as suppression of sensitivity of an anticancer drug to a cancer cell) appears is collected. Then, by analyzing the target recognition sequence of the ribozyme expressed there, it is possible to know the relationship between the focused phenotype and the sequence of the gene cleaved into the ribozyme in the cell.
  • the ribozyme library used in the screening method according to this embodiment includes a plurality of ribozymes that specifically cleave mRNA and suppress gene expression, and target recognition sequences recognized by each ribozyme are randomized.
  • a randomized ribozyme library which is a plurality of sequences that have been designed, is used.
  • the ribozyme library that suppresses the sensitivity of the anticancer drug to cancer cells is concentrated.
  • the ribozyme library contains a target recognition sequence that recognizes a gene that enhances the sensitivity of the anticancer drug to cancer cells. Therefore, the gene corresponding to the target recognition sequence is exactly the anticancer drug for cancer cells. It means that it is a gene that enhances susceptibility.
  • each ribozyme may be detected by detecting a fluorescent dye having a wavelength specific to each target recognition sequence labeled on each ribozyme.
  • each target recognition sequence labeled on each ribozyme can be identified by simply detecting the wavelength of the fluorescent dye, and screening can be performed easily with high sensitivity.
  • the human wild-type PRAK2 gene, the human wild-type TGFBR2 gene, the human wild-type EXT1 gene, and their mutant genes have been mainly described, but the present invention is not particularly limited to human-derived genes. That is, the origin of the PRAK2 gene, TGFBR2 gene, and EXT1 gene is not particularly limited, and may be, for example, human, mouse, rat, rabbit, pig, sheep, cow, horse, cat, dog, monkey, or chimpanzee. Preferred are mouse, rat, monkey, chimpanzee and human, and particularly preferred is human. This is because humans can be used to treat human diseases and develop therapeutic drugs. In addition, mice, rats, monkeys, and chimpanzees are widely used as model animals for research all over the world, and many characteristics have been clarified. Therefore, by controlling the expression of the above genes in these organisms, therapeutic drugs Especially useful information for the development of
  • DMEM Dulbecco's Modified Eagle's Medium powder, 10 mL 10% NaOHCO 3 , 5 mL 100 ⁇ glucose, 10 mL 50 ⁇ glutamine PBS (-): 137 mM NaCl, 8.10 mM Na 2 HPO 4 ⁇ 12H 2 O, 2.68 mM KCl, 1.47 mM KH 2 PO 4 Trypsin / EDTA solution: Nacalai Tesque, 0.25% Trypsin, 1 mM EDTA IFN- ⁇ : 3 million U / mL Intron A (Schering-Plough Corporation) 5-FU: Kyowa Hakko Kogyo Co., Ltd.
  • HCC cell line is 5% CO 2 , 37 ° C., 100% using DMEM containing 10% fetal calf serum (EQUITECH-BIO, INC) on a 10 cm cell culture dish (FALCON). Cultured under humidity. In a state of 70-90% confluence, 200 ⁇ L of Trypsin / EDTA Solution was added to detach the cells, and the cells were collected by centrifugation at 1000 rpm, 5 min, 4 ° C., and 1 dish portion was divided into 4 dishes and subcultured.
  • DMEM containing 10% fetal calf serum
  • FALCON 10 cm cell culture dish
  • V Construction of random ribozyme library (v-1) Construction of attB1-tRNAVal-RRz-attB2 RRz was inserted downstream of the tRNAVal promoter by the following method (FIG. 1).
  • V-1-1) RRz gene was synthesized by annealing by PCR using 20 pmol of RRz1 and RRz2 primers each having 8 or 7 random base sequences (FIG. 1A).
  • KOD Plus TOYOBO
  • V-1-3 PCR was performed using 100 pg piGENE tRNA pur (iGEN Therapeutics, Inc.) as a template and 10 pmol each of RRz5 and RRz6 primers (Table 1).
  • KOD Plus TOYOBO
  • a tRNAVal promoter having an attB1 sequence upstream was synthesized (FIG. 1B).
  • (V-1-4) PCR was performed using the products of (v-1-2) and (v-1-3) as templates and 10 pmol of RRz4 and RRz5 primers (Table 1).
  • KOD Plus TOYOBO
  • tRNAVal-RRz having an attB1 sequence upstream and an attB2 downstream was synthesized (FIG. 1C).
  • E. coli in 2 mL total volume. I asked for a number. The remaining E. coli solution was centrifuged at 3,500 rpm for 15 minutes at 4 ° C., the pellet was suspended in 500 ⁇ L of LB medium containing 20% glycerol, and stored at ⁇ 80 ° C. as a glycerol stock. This operation was repeated until the total number of E. coli reached about 6 million. Thereafter, all the preserved glycerol stocks were added to 1 L of LB medium containing 30 ⁇ g / mL kanamycin, and cultured with shaking until sufficient turbidity was obtained for 12 hours or more. Plasmid DNA was extracted from this E. coli using QIAGEN Plasmid Maxi Kit (QIAGEN). The extracted plasmid DNA was dissolved in TE (10 mM Tris, 1 mM EDTA) buffer so as to be 1 ⁇ g / ⁇ L, and this was used as an RRz library.
  • TE 10 mM Tri
  • siRNA Cells were seeded in 96-well plates at 5.0 ⁇ 10 3 cells / well and incubated in a 37 ° C. CO 2 incubator for 24 hours. After the incubation, 4 pmol of siRNA was introduced using 0.2 ⁇ L Lipofectamine 2000 (Invitrogen).
  • RNA was extracted using TRIzol Reagent (Invitrogen).
  • SuperScript First-Stand Synthesis System for RT-PCR (Invitrogen) was used.
  • cDNA was synthesized by reacting for 1 cycle at 65 ° C. for 5 minutes, 1 cycle at 42 ° C. for 120 minutes, and 70 ° C. for 10 minutes.
  • the cDNA after the reaction was diluted 20 times and used for Real-Time PCR.
  • Example 1 Screening for sensitizing genes sensitive to 5-FU>
  • IFN- ⁇ / 5-FU combination therapy enhancement in IFN- ⁇ / 5-FU combination therapy in highly advanced HCC patients. Identification of genes contributing to susceptibility to - ⁇ / 5-FU combination therapy was performed. As a result, a plurality of susceptibility enhancing genes were identified.
  • FIG. 2A a library of ribozymes (FIG. 2A), an RNA enzyme that cleaves mRNA specifically for the target recognition sequence and suppresses gene expression, was used.
  • This library has 6 million ribozyme recognition sequences at random, and can comprehensively suppress gene expression (FIG. 2B).
  • the following screening was performed using the constructed library (FIG. 2C).
  • the human HepG2 cell line was treated with 5-FU alone after introduction of the prepared ribozyme library, and screening was performed using as an index the enhanced resistance to 5-FU at a 50% growth inhibitory concentration.
  • ribozymes were collected from viable cells and reintroduced into HepG2 cells.
  • Candidate genes were extracted from the finally obtained ribozyme recognition sequence using BLAST.
  • the identified gene was examined for 5-FU alone and IFN- ⁇ / 5-FU sensitivity enhancement using an adenovirus forced expression system. Ten screenings were performed and 5-FU resistance increased with each increase (FIG. 3).
  • POLR2J4 which is a pseudogene
  • FOXP2 whose expression could not be confirmed
  • Example 2 Confirmation of 5-FU sensitivity and examination of IFN- ⁇ / 5-FU sensitivity> SiRNAs of PRKAG2, TGFBR2, and EXT1 were prepared.
  • siRNAs When these siRNAs were used to suppress gene expression of PRKAG2, TGFBR2, and EXT1, the sensitivity to 5-FU was significantly reduced in all three genes (FIG. 4A). This is because administration of siRNA targeting PRKAG2, TGFBR2, and EXT1 suppresses gene expression of PRKAG2, TGFBR2, and EXT1, resulting in suppression of sensitivity to 5-FU (FIG. 4B).
  • an adenovirus vector and a control adenovirus vector for forced expression of PRKAG2, TGFBR2, and EXT1 were prepared, respectively (FIGS. 5 and 6).
  • proteins encoded by the PRKAG2, TGFBR2, and EXT1 genes were overexpressed in HepG2 cells, respectively (FIG. 7).
  • the sensitivity to 5-FU was significantly increased by forced expression of 3 genes using these vectors (FIG. 8A). This is because administration of an adenoviral vector that overexpresses PRKAG2, TGFBR2, and EXT1 results in overexpression of PRKAG2, TGFBR2, and EXT1, resulting in enhanced sensitivity to 5-FU (FIG. 4B). . Furthermore, two of these genes (TGFBR2, EXT1) significantly enhanced sensitivity to IFN- ⁇ / 5-FU even when IFN- ⁇ / 5-FU was used together (FIG. 8B).
  • Example 3 Confirmation of IFN- ⁇ / 5-FU sensitivity in vivo> Mice were administered by intraperitoneal injection of an adenoviral vector for forced expression of TGFBR2 under the following conditions, and changes in the sensitivity of IFN- ⁇ / 5-FU were observed (FIG. 9).
  • Example 4 Confirmation of enhancement of 5-FU and IFN- ⁇ / 5-FU-induced apoptosis by overexpression of TGFBR2, EXT1> (4-1) Hoechst staining
  • EXT1 4-1) Hoechst staining
  • LacZ, TGFBR2, and EXT1 genes were overexpressed by adenovirus infection of HepG2, and then treated with 5 ⁇ g / mL 5-FU and 200 U / mL IFN- ⁇ . After 48 hours, 10 ⁇ M Hoechst 33258 was added, and after 1 hour, observation with a fluorescence microscope was performed. As an observation result, a fluorescence micrograph is shown in FIG.
  • caspase 3/7 activation was carried out by the following procedure. Measurements were made. First, LacZ, TGFBR2, and EXT1-expressing cells (HepG2) were treated with 5 ⁇ g / mL 5-FU and 200 U / mL IFN- ⁇ . After 48 hours, caspase 3 and 7 activities were measured according to the manual attached to Capsapse-Glo3 / 7 assay kit (Promega). The measurement results are summarized in a graph and shown in FIG.
  • the meanings of *, +, and ++ are the same in FIGS.
  • RNA synthesis was performed according to the following procedure. First, LacZ and TGFBR2-expressing cells (HepG2) were treated with 5 ⁇ g / mL 5-FU and 200 U / mL IFN- ⁇ . Total RNA was extracted 48 hours after drug treatment using TRIzol Reagent (Invitrogen). In the RT reaction, SuperScript First-Stand Synthesis System for RT-PCR (Invitrogen) was used.
  • TGF ⁇ 1 mRNA expression level causing apoptosis was measured, and it was interpreted that if TGF ⁇ mRNA expression level increased, apoptosis was promoted via a signal transduction pathway involving TGF- ⁇ .
  • the amount of TGF ⁇ 1 mRNA expression was significantly increased when 5-Fu alone or IFN-a / 5-FU was added, compared to the measurement results of the corresponding controls. From this, it is clear that 5-Fu alone or drug treatment with IFN-a / 5-FU enhances apoptosis through a signal transduction pathway involving TGF- ⁇ .
  • TGFBR2 is overexpressed after adding 5-Fu alone or IFN-a / 5-FU
  • the expression level of TGF ⁇ 1 mRNA is not significantly increased. Therefore, whether 5-Fu alone or IFN-a / 5-FU is added and TGFBR2 is overexpressed is unclear whether apoptosis is further enhanced through a signaling pathway involving TGF- ⁇ It is.
  • Luciferase Assay was performed according to the following procedure. First, HepG2 cells into which reporter gene p3TP-Lux and phRL-TK as an internal control gene were transiently introduced by FuGENE6 were infected with 5FUSGs overexpression adenovirus. After 24 hours of infection, DMSO was treated with Negative Control (NC), 5 ⁇ g / mL 5-FU, and 200 U / mL IFN- ⁇ , and Luciferase fluorescence was detected 48 hours after drug treatment. Each numerical value in the graph is corrected by an internal control, and the value of the drug-untreated LacZ overexpression group is 1. The measurement results are summarized in a graph and shown in FIG.
  • the activity of firefly luciferase expressed from p3TP-Lux, a reporter vector of TGF- ⁇ causing apoptosis was measured, and if the activity of firefly luciferase increased, the transcriptional activity of TGF- ⁇ increased. It is interpreted that apoptosis is enhanced through a signal transduction pathway involving TGF- ⁇ .
  • the p3TP-Luc reporter plasmid is activated via three TRE elements (Smad) derived from the human collagenase gene in the -740 / -636 region of the PAI-1 promoter selectively induced by TGF- ⁇ .
  • a luciferase gene is connected downstream of a sequence to which an element specific to the activin pathway of the TGF- ⁇ family is bound. Therefore, when the transcriptional activity of TGF- ⁇ is increased, overexpression of firefly luciferase from the p3TP-Luc reporter plasmid is induced. From the experimental results, it is clear that when TGFBR2 is overexpressed after adding 5-Fu alone or IFN-a / 5-FU, the activity of firefly luciferase is significantly increased. It is clear that the transcriptional activity of TGF- ⁇ is increased. That is, it is clear that apoptosis is enhanced through a signal transduction pathway involving TGF- ⁇ .
  • HepG2 was seeded at 1 ⁇ 10 6 cells / 6 cm-dish, and TGFBR2 gene was introduced by adenovirus 5 hours later.
  • 5 ⁇ g / mL 5-FU and 200 U / mL IFN- ⁇ treatment was performed.
  • the cells were washed twice with 1 ⁇ PBS ( ⁇ ), RIPA Buffer was added, and the cells were collected with a cell scraper.
  • the collected cells were centrifuged at 15,000 rpm for 10 minutes at 4 ° C., and the supernatant was used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
  • Example 6 Confirmation of involvement of ER stress in enhancement of apoptosis due to EXT1 overexpression> (6-1) Real-Time RT-PCR In order to examine the involvement of ER stress in the enhancement of apoptosis due to EXT1 overexpression, Real-Time RT-PCR was performed in the same manner as in Example 5. The measurement results are summarized in a graph and shown in FIGS. 12a and 12b.
  • DDIT3 (CHOP) mRNA expression level and HSPA5 (BIP) mRNA expression level which are known to increase in response to ER stress causing apoptosis, were measured, and DDIT3 (CHOP) and It is interpreted that the ER stress that causes apoptosis is increased when the mRNA expression level of HSPA5 (BIP) is increased.
  • DDIT3 (CHOP) and HSPA5 (BIP) increased significantly. ing. Therefore, it is apparent that ER stress causing apoptosis is increased when EXT1 is overexpressed after adding 5-Fu alone or IFN-a / 5-FU.
  • Example 7 Effect on human liver cancer model mouse by overexpression of TGFBR2 and EXT1>
  • a human liver cancer cell line HepG2; 6.8 ⁇ 10 6 cells / mouse
  • LacZ, TGFBR2, and EXT1 genes were directly introduced into the tumor site using adenovirus.
  • 20,000 U / body / day IFN- ⁇ was subcutaneously injected and 30 mg / kg / day 5-FU was administered by intraperitoneal injection.
  • Tumor diameter (TV) major axis ⁇ (minor diameter) 2/2.
  • the experimental results are summarized and shown as a graph in FIG.
  • RNA expression of PRKAG2, TGFBR2, and EXT1 in a liver cancer patient was analyzed by the following procedure. First, RNA was collected from tissues collected from cancer cases and non-cancerous cases of non-B non-C, B-type, and C-type HCC patients, respectively. Next, using these RNA samples, mRNA expression of PRKAG2, TGFBR2, and EXT1 was analyzed by the Real-Time RT-PCR method in the same manner as in Examples 5 and 6 above.
  • the antitumor effect when TGFBR2 is overexpressed after adding 5-Fu alone or IFN-a / 5-FU in the cancerous part of a type B liver cancer patient, the antitumor effect may be particularly increased. It is suggested. In addition, in the cancerous part of a non-B non-C type liver cancer patient, when 5-FU alone or IFN-a / 5-FU is added and EXT1 is overexpressed, the antitumor effect is particularly increased. The possibility is suggested.
  • 5-FU sensitivity was enhanced by forced expression of 5-FU sensitivity contributing genes (5FUSGs: PRKAG2, TGFBR2, EXT1) identified by functional screening using a random ribozyme library. That is, it was shown that the three genes identified in this study (PRKAG2, TGFBR2, EXT1) enhance the sensitivity when 5-FU alone and IFN- ⁇ / 5-FU are used together.
  • PRKAG2, TGFBR2, EXT1 enhance the sensitivity when 5-FU alone and IFN- ⁇ / 5-FU are used together.
  • TGFBR2 and EXT1 with an adenoviral vector enhanced the sensitivity of HepG2 cells to IFN- ⁇ / 5-FU combination therapy in vitro.
  • overexpression of TGFBR2 by adenoviral vectors enhanced the sensitivity of mouse tumor tissues to IFN- ⁇ / 5-FU combination therapy in vivo. Therefore, the use of these genes is expected to improve the long-term prognosis of various malignant tumor patients to which 5-FU alone or IFN- ⁇ / 5-FU combination therapy is indicated.
  • 5-FU and IFN- ⁇ / 5-FU are anticancer agents used in various malignant tumors
  • the three types of genes PRKAG2, TGFBR2, and EXT1 are only for advanced hepatocellular carcinoma.
  • it is expected to be effective as a sensitizer for anticancer agents against other malignant tumors.

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Abstract

A group of genes which can enhance the anti-tumor effect of 5-fluorouracil are found. Disclosed is an enhancer of the sensitivity to 5-fluorouracil, which comprises a nucleotide fragment encoding at least one gene selected from the group consisting of PRAKG2 gene, TGFBR2 gene and EXT1 gene.

Description

５－ＦＵ単独及びＩＦＮ－α／５－ＦＵ併用時の抗腫瘍効果を増強する遺伝子群Genes that enhance antitumor effects when 5-FU alone and IFN-α / 5-FU are used together
　本発明は、抗癌剤の感受性増強作用を有する遺伝子のスクリーニング方法、５－フルオロウラシル感受性増感剤、それを含む抗癌剤キット、５－フルオロウラシル感受性を判定するための診断薬、５－フルオロウラシル感受性抑制剤に関する。 The present invention relates to a method for screening a gene having an effect of enhancing the sensitivity of an anticancer agent, a 5-fluorouracil sensitive sensitizer, an anticancer agent kit containing the same, a diagnostic agent for determining 5-fluorouracil sensitivity, and a 5-fluorouracil sensitivity inhibitor.
　肝細胞癌（ｈｅｐａｔｏｃｅｌｌｕｌａｒ　ｃａｒｃｉｎｏｍａ，ＨＣＣ）は世界中で増加している悪性腫瘍の一種であり、日本における癌死亡者数の第３位を占めている。このＨＣＣに対しては、近年の様々な診断・治療法の開発及び進歩によって、治療成績は向上している。しかし、その一方で、ＨＣＣ患者の多くが、肝炎等の慢性肝疾患を経た発癌であるため、治療後に再発する症例が極めて多く、高度進行型症例の増加が問題となっている。この高度進行型ＨＣＣは、既存の治療法の奏功率が極めて低く、より治療効果の高い新規治療法の開発が望まれていた。 Hepatocellular carcinoma (HCC) is a type of malignant tumor that is increasing all over the world, accounting for the third largest number of cancer deaths in Japan. With respect to this HCC, treatment results have improved due to the recent development and progress of various diagnosis and treatment methods. However, on the other hand, since many HCC patients are carcinogenic after chronic liver diseases such as hepatitis, there are many cases of recurrence after treatment, and an increase in highly advanced cases is a problem. This advanced progressive HCC has a very low success rate of existing therapies, and the development of new therapies with higher therapeutic effects has been desired.
　これに対して、近年、抗ウイルス活性を有するサイトカインｉｎｔｅｒｆｅｒｏｎ－α（ＩＦＮ－α）と抗癌剤５－ｆｌｕｏｒｏｕｒａｃｉｌ（５－ＦＵ）を併用するＩＦＮ－α／５－ＦＵ併用療法が、高い有効性を示すとの結果が複数の施設から報告され、進行型ＨＣＣに対する新規治療法として期待されている。 On the other hand, in recent years, IFN-α / 5-FU combination therapy in which the cytokine interferon-α (IFN-α) having antiviral activity and the anticancer agent 5-fluorouracil (5-FU) are used in combination is highly effective. The results are reported from multiple institutions and are expected as a novel treatment for advanced HCC.
　ＩＦＮ－α／５－ＦＵ併用療法の感受性に関する遺伝子についての研究は幾つか行われている。例えば、非特許文献１は、遺伝子解析からＩＦＮ－α／５－ＦＵ併用療法の効果予測を行おうとする論文である。また、非特許文献２は、肝癌細胞の５－ＦＵに対する感受性遺伝子を報告している。 Several studies have been conducted on genes related to susceptibility to IFN-α / 5-FU combination therapy. For example, Non-Patent Document 1 is a paper that attempts to predict the effect of IFN-α / 5-FU combination therapy from genetic analysis. Non-Patent Document 2 reports a susceptibility gene for 5-FU in liver cancer cells.
　また、特許文献１には、癌患者の悪性度や化学療法に対する応答を予測するシステムが説明されている。さらに、特許文献２には、癌の進行、転帰および予後を診察するマーカーが説明されている。そして、特許文献３には、アンチセンスＲＮＡと５ＦＵとの併用による癌治療について説明されている。 Patent Document 1 describes a system for predicting the malignancy of cancer patients and the response to chemotherapy. Furthermore, Patent Document 2 describes a marker for diagnosing cancer progression, outcome, and prognosis. Patent Document 3 describes cancer treatment using a combination of antisense RNA and 5FU.
特表２００９－５０６７７７号公報Special table 2009-506777 gazette 特開２００９－５１５１４８号公報JP 2009-515148 A 特表２００５－５０４５２２号公報JP 2005-504522 A
　しかしながら、上記文献記載の従来技術は、以下の点で改善の余地を有していた。
　第一に、進行型ＨＣＣ治療法として、注目されているＩＦＮ－α／５－ＦＵ併用療法であるが、高度進行型ＨＣＣに対する奏功率は約５０％である（S obi, et al. Cancer. 2006. 106, 1990-7.）。そのため、この治療法を用いても、約半数の患者においては治療効果が得られず、全てのＨＣＣ患者に対して好ましい効果を期待できるわけではなく、より治療効果の高い新規治療法の開発が望まれる。 However, the prior art described in the above literature has room for improvement in the following points.
First, IFN-α / 5-FU combination therapy is gaining attention as a treatment for advanced HCC, but the response rate for highly advanced HCC is about 50% (Sobi, et al. Cancer. 2006. 106, 1990-7.). Therefore, even if this treatment method is used, a therapeutic effect cannot be obtained in about half of the patients, and a favorable effect cannot be expected for all HCC patients. desired.
　第二に、非特許文献１～２では、ＩＦＮ－α／５－ＦＵ併用療法の感受性に関する遺伝子を網羅的に見つけることは困難であり、未だＩＦＮ－α／５－ＦＵ併用療法の感受性との関係が未解明の遺伝子が多く存在すると想定される。なお、非特許文献１～２では、実際に後述する実施例で示す遺伝子を発見できていない。 Second, in Non-Patent Documents 1 and 2, it is difficult to comprehensively find genes related to the susceptibility of IFN-α / 5-FU combination therapy. It is assumed that there are many genes whose relationship is not yet elucidated. In Non-Patent Documents 1 and 2, the genes shown in the examples described later cannot actually be found.
　第三に、特許文献３～４でも、ＩＦＮ－α／５－ＦＵ併用療法の感受性に関する遺伝子を網羅的に見つけることは困難であり、未だＩＦＮ－α／５－ＦＵ併用療法の感受性との関係が未解明の遺伝子が多く存在すると想定される。なお、特許文献３では、遺伝子としてＥＸＴ１、薬剤として５－ＦＵ、癌として肝癌を例に挙げているが、実際にはＥＸＴ１が５－ＦＵの感受性を高めるかどうか実験していない。なお、特許文献４では、癌の進行、転帰および予後を診断するマーカーとしてＴＧＦＢＲ２、癌として肝臓癌、アジュバント化学療法として５ＦＵを例に挙げているが、実際にはＴＧＦＢＲ２が５－ＦＵの感受性を高めるかどうか実験していない。 Thirdly, even in Patent Documents 3 to 4, it is difficult to comprehensively find genes related to the sensitivity of the IFN-α / 5-FU combination therapy, and the relationship with the sensitivity of the IFN-α / 5-FU combination therapy is still difficult. However, there are many unexplained genes. In Patent Document 3, EXT1 is used as a gene, 5-FU is used as a drug, and liver cancer is used as an example of cancer. However, whether EXT1 increases the sensitivity of 5-FU is not actually tested. In Patent Document 4, TGFBR2 is exemplified as a marker for diagnosing cancer progression, outcome, and prognosis, liver cancer is exemplified as cancer, and 5FU is exemplified as adjuvant chemotherapy. In fact, TGFBR2 is sensitive to 5-FU. I have not experimented to raise it.
　第四に、特許文献５では、５－ＦＵの感受性に関する遺伝子をそもそも探索すらしていない。そのため、特許文献５では、後述する実施例で示す遺伝子には特に触れていない。 Fourth, Patent Document 5 does not even search for genes related to 5-FU susceptibility in the first place. Therefore, Patent Document 5 does not particularly touch the genes shown in the examples described later.
　本発明は上記事情に鑑みてなされたものであり、５－フルオロウラシルの抗腫瘍効果を増強する遺伝子群を見出すことを目的とする。 The present invention has been made in view of the above circumstances, and an object thereof is to find a gene group that enhances the antitumor effect of 5-fluorouracil.
　本発明によれば、５－フルオロウラシル感受性増感剤であって、ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子からなる群から選ばれる１以上の遺伝子をコードするヌクレオチド断片を含む、増感剤が提供される。 According to the present invention, there is provided a 5-fluorouracil sensitive sensitizer comprising a nucleotide fragment encoding one or more genes selected from the group consisting of PRAKG2, TGFBR2 and EXT1 genes. .
　この構成によれば、５－フルオロウラシルの抗腫瘍効果を増強する作用を有することが後述する実施例において実証されているＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子またはＥＸＴ１遺伝子のいずれかをコードするヌクレオチド断片を含むため、増感剤自体としても５－フルオロウラシルの抗腫瘍効果を増強する効果が得られる。 According to this configuration, since it contains a nucleotide fragment encoding any of PRAK2 gene, TGFBR2 gene or EXT1 gene, which has been demonstrated in Examples described later, to have an action of enhancing the antitumor effect of 5-fluorouracil, As a sensitizer itself, an effect of enhancing the antitumor effect of 5-fluorouracil can be obtained.
　また、本発明によれば、５－フルオロウラシル感受性増感剤であって、ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子からなる群から選ばれる１以上の遺伝子を発現するベクターを含む、増感剤が提供される。 The present invention also provides a 5-fluorouracil sensitive sensitizer comprising a vector that expresses one or more genes selected from the group consisting of PRAKG2, TGFBR2 and EXT1 genes. The
　この構成によれば、５－フルオロウラシルの抗腫瘍効果を増強する作用を有することが後述する実施例において実証されているＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子またはＥＸＴ１遺伝子のいずれかを発現するベクターを含むため、増感剤自体としても５－フルオロウラシルの抗腫瘍効果を増強する効果が得られる。 According to this configuration, since the vector includes any one of the PRAK2 gene, the TGFBR2 gene, and the EXT1 gene, which has been demonstrated in Examples described later, to have an action of enhancing the antitumor effect of 5-fluorouracil, As the sensitizer itself, an effect of enhancing the antitumor effect of 5-fluorouracil can be obtained.
　また、本発明によれば、５－フルオロウラシル感受性増感剤であって、ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子からなる群から選ばれる１以上の遺伝子のコードする蛋白質を含む、増感剤が提供される。 The present invention also provides a 5-fluorouracil sensitive sensitizer comprising a protein encoded by one or more genes selected from the group consisting of PRAKG2, TGFBR2 and EXT1 genes. The
　この構成によれば、５－フルオロウラシルの抗腫瘍効果を増強する作用を有することが後述する実施例において実証されているＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子またはＥＸＴ１遺伝子のコードする蛋白質を含むため、増感剤自体としても５－フルオロウラシルの抗腫瘍効果を増強する効果が得られる。 According to this configuration, since the PRAK2 gene, TGFBR2 gene, or EXT1 gene-encoded protein that has been demonstrated in Examples described later to have an action of enhancing the antitumor effect of 5-fluorouracil is contained, the sensitizer itself However, the effect of enhancing the antitumor effect of 5-fluorouracil can be obtained.
　また、本発明によれば、抗癌剤キットであって、上記の増感剤と、５－フルオロウラシルと、を含む、キットが提供される。 In addition, according to the present invention, there is provided an anticancer agent kit comprising the above sensitizer and 5-fluorouracil.
　この構成によれば、５－フルオロウラシルの抗腫瘍効果を増強する作用を有することが後述する実施例において実証されているＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子またはＥＸＴ１遺伝子を用いた増感剤を含むため、５－フルオロウラシルの抗腫瘍効果が増強されて優れた抗腫瘍効果が得られる。 According to this configuration, since it contains a sensitizer using PRAKG2 gene, TGFBR2 gene or EXT1 gene, which has been demonstrated in Examples described later, to have an action of enhancing the antitumor effect of 5-fluorouracil, The antitumor effect of fluorouracil is enhanced and an excellent antitumor effect is obtained.
　また、本発明によれば、５－フルオロウラシル感受性抑制剤であって、ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子からなる群から選ばれる１以上の遺伝子の発現を抑制するｓｉＲＮＡ、ｓｈＲＮＡまたはアンチセンスＲＮＡを含む、抑制剤が提供される。 Further, according to the present invention, a 5-fluorouracil sensitivity inhibitor, comprising siRNA, shRNA, or antisense RNA that suppresses the expression of one or more genes selected from the group consisting of PRAK2 gene, TGFBR2 gene, and EXT1 gene An inhibitor is provided.
　この構成によれば、５－フルオロウラシルの抗腫瘍効果を増強する作用を有することが後述する実施例において実証されているＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子またはＥＸＴ１遺伝子のいずれかの発現を抑制するｓｉＲＮＡ、ｓｈＲＮＡまたはアンチセンスＲＮＡを含むため、抑制剤自体としても５－フルオロウラシルの抗腫瘍効果を抑制する効果が得られる。 According to this configuration, siRNA, shRNA, or the like that suppresses the expression of any of PRAK2 gene, TGFBR2 gene, or EXT1 gene, which has been demonstrated in Examples to be described later, has an action of enhancing the antitumor effect of 5-fluorouracil. Since the antisense RNA is contained, the inhibitor itself can suppress the antitumor effect of 5-fluorouracil.
　また、本発明によれば、５－フルオロウラシル感受性を判定するための診断薬であって、ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子からなる群から選ばれる１以上の遺伝子の発現量を測定するための試薬を含む、診断薬が提供される。 Further, according to the present invention, a diagnostic agent for determining 5-fluorouracil sensitivity, a reagent for measuring the expression level of one or more genes selected from the group consisting of PRAKG2, TGFBR2 and EXT1 genes A diagnostic agent is provided.
　この構成によれば、５－フルオロウラシルの抗腫瘍効果を増強する作用を有することが後述する実施例において実証されているＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子またはＥＸＴ１遺伝子のいずれかの発現量を測定するための試薬を含むため、被験者の生体内におけるＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子またはＥＸＴ１遺伝子のいずれかの発現量を測定することで、被験者が５－フルオロウラシルの抗腫瘍効果が効きやすい体質であるかどうか判定するための診断薬として利用できる。 According to this configuration, a reagent for measuring the expression level of any of PRAK2 gene, TGFBR2 gene, or EXT1 gene, which has been demonstrated in Examples described later, to have an action of enhancing the antitumor effect of 5-fluorouracil Therefore, by measuring the expression level of any one of the PRAK2 gene, TGFBR2 gene or EXT1 gene in the subject's living body, it is possible to determine whether or not the subject is likely to be effective in the anti-tumor effect of 5-fluorouracil. It can be used as a diagnostic agent.
　また、本発明によれば、抗癌剤の感受性増強作用を有する遺伝子のスクリーニング方法であって、（ｍ）哺乳動物の所定の癌細胞株にリボザイムライブラリーを導入する工程と、（ｎ）リボザイムライブラリーが導入された癌細胞を所定の抗癌剤で処理する工程と、（ｏ）抗癌剤で処理された癌細胞のうち生存細胞からリボザイムを回収して新規なリボザイムライブラリーを得る工程と、を含み、リボザイムライブラリーが、標的認識配列特異的にｍＲＮＡを切断し、遺伝子発現を抑制する複数のリボザイムを含み、各リボザイムが認識する標的認識配列がランダム化された複数の配列である、ランダム化リボザイムライブラリーであり、（ｐ）（ｍ）、（ｎ）および（ｏ）の工程を複数回繰り返して得られるリボザイムライブラリーが認識する標的認識配列を検出する工程と、（ｑ）検出された標的認識配列に対応する遺伝子情報をゲノムデータベースから抽出する工程と、　をさらに含む、スクリーニング方法が提供される。 According to the present invention, there is also provided a method for screening a gene having an effect of enhancing the sensitivity of an anticancer agent, the method comprising (m) introducing a ribozyme library into a predetermined cancer cell line of a mammal, and (n) a ribozyme library. And (o) recovering ribozymes from viable cells among cancer cells treated with anticancer agents to obtain a novel ribozyme library, and comprising: Randomized ribozyme library, wherein the library includes a plurality of ribozymes that cleave mRNA specifically for a target recognition sequence and suppress gene expression, and the target recognition sequences recognized by each ribozyme are a plurality of randomized sequences A ribozyme library obtained by repeating the steps (p) (m), (n) and (o) multiple times is recognized. And detecting the target recognition sequence, (q) genetic information corresponding to the detected target recognition sequence further comprising the steps of: extracting from the genome database, a screening method is provided.
　この方法によれば、リボザイムライブラリーが、標的認識配列特異的にｍＲＮＡを切断し、遺伝子発現を抑制する複数のリボザイムを含むため、上記の工程を複数回繰り返して得られるリボザイムライブラリーには、抗癌剤の感受性増強作用を有する遺伝子の発現を抑制するリボザイムが濃縮されて多く含まれていることになる。そのため、こうして得られるリボザイムライブラリーが認識する標的認識配列を検出して、検出された標的認識配列に対応する遺伝子情報をゲノムデータベースから抽出すれば、抗癌剤の感受性増強作用を有する遺伝子を高確率で見出すことが可能である。 According to this method, since the ribozyme library includes a plurality of ribozymes that cleave mRNA specifically for the target recognition sequence and suppress gene expression, the ribozyme library obtained by repeating the above steps a plurality of times includes: This means that many ribozymes that suppress the expression of genes having a sensitivity enhancing action of anticancer agents are concentrated and contained. Therefore, if the target recognition sequence recognized by the ribozyme library obtained in this way is detected and the gene information corresponding to the detected target recognition sequence is extracted from the genome database, genes having an anticancer agent sensitivity enhancing action can be detected with high probability. It is possible to find out.
　本発明によれば、５－フルオロウラシルの抗腫瘍効果を増強するＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子またはＥＸＴ１遺伝子またはそれらの遺伝子がコードする蛋白質を用いて、５－フルオロウラシルの抗腫瘍効果を増強することができる。また、本発明によれば、５－フルオロウラシルの抗腫瘍効果を増強するＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子またはＥＸＴ１遺伝子の発現を抑制するｓｉＲＮＡ、ｓｈＲＮＡまたはアンチセンスＲＮＡを用いて、５－フルオロウラシルの抗腫瘍効果を抑制することができる。 According to the present invention, the antitumor effect of 5-fluorouracil can be enhanced by using the RAKG2 gene, TGFBR2 gene, EXT1 gene or the protein encoded by those genes that enhance the antitumor effect of 5-fluorouracil. In addition, according to the present invention, the antitumor effect of 5-fluorouracil can be enhanced using siRNA, shRNA or antisense RNA that suppresses the expression of RAKG2 gene, TGFBR2 gene or EXT1 gene that enhances the antitumor effect of 5-fluorouracil. Can be suppressed.
　また、本発明によれば、５－フルオロウラシルの抗腫瘍効果を増強するＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子またはＥＸＴ１遺伝子のいずれかの発現量を測定するための試薬を用いて、被験者が５－フルオロウラシルの抗腫瘍効果が効きやすい体質であるかどうか判定することができる。また、本発明によれば、標的認識配列特異的にｍＲＮＡを切断し、遺伝子発現を抑制する複数のリボザイムを用いて、抗癌剤の感受性増強作用を有する遺伝子を高確率で見出すことが可能である。 In addition, according to the present invention, the subject can use a reagent for measuring the expression level of any of PRAK2 gene, TGFBR2 gene, or EXT1 gene that enhances the antitumor effect of 5-fluorouracil, so that the subject It can be determined whether the constitution is effective. In addition, according to the present invention, it is possible to find a gene having an anti-cancer drug sensitivity enhancing action with a high probability by using a plurality of ribozymes that cleave mRNA specifically for a target recognition sequence and suppress gene expression.
実施例のスクリーニング法で用いるプラスミドの構築について説明するための概念図である。It is a conceptual diagram for demonstrating construction | assembly of the plasmid used with the screening method of an Example. 実施例のスクリーニング法で用いるリボザイムの構造について説明するための概念図である。It is a conceptual diagram for demonstrating the structure of the ribozyme used by the screening method of an Example. 実施例のスクリーニング法で用いるランダムリボザイムライブラリーについて説明するための概念図である。It is a conceptual diagram for demonstrating the random ribozyme library used with the screening method of an Example. 実施例のスクリーニング法について説明するための概念図である。It is a conceptual diagram for demonstrating the screening method of an Example. 実施例のスクリーニング法による５－ＦＵ抵抗性の亢進を示すグラフである。It is a graph which shows the enhancement of 5-FU resistance by the screening method of an Example. 実施例のスクリーニング法で見出した５－ＦＵ感受性増強遺伝子の発現抑制による５－ＦＵ抵抗性の亢進を示すグラフである。It is a graph which shows the enhancement of 5-FU resistance by suppression of the expression of 5-FU sensitivity enhancement gene discovered by the screening method of an Example. ５－ＦＵ感受性増強遺伝子のｓｉＲＮＡおよびアデノウイルス発現ベクターの投与による５－ＦＵ感受性の増強・抑制のメカニズムを説明するための概念図である。It is a conceptual diagram for explaining the mechanism of enhancement / suppression of 5-FU sensitivity by administration of siRNA of 5-FU sensitivity enhancement gene and adenovirus expression vector. ５ＦＵＳＧｓ発現アデノウイルスプラスミドの構築について説明するための概念図である。It is a conceptual diagram for demonstrating construction | assembly of 5FUSGs expression adenovirus plasmid. コントロールアデノウイルスプラスミドの構築について説明するための概念図である。It is a conceptual diagram for demonstrating construction | assembly of a control adenovirus plasmid. Ａｄ－５ＦＵＳＧｓによる遺伝子過剰発現確認を示す電気泳動写真である。It is an electrophoresis photograph showing confirmation of gene overexpression by Ad-5FUSGs. 実施例のスクリーニング法で見出した５－ＦＵ感受性増強遺伝子の強制発現による５－ＦＵ感受性の亢進を示すグラフである。It is a graph which shows the enhancement of 5-FU sensitivity by forced expression of the 5-FU sensitivity enhancement gene discovered by the screening method of an Example. 実施例のスクリーニング法で見出した５－ＦＵ感受性増強遺伝子の強制発現によるＩＮＦ－α／５－ＦＵ感受性の亢進を示すグラフである。It is a graph which shows the enhancement of INF-α / 5-FU sensitivity by forced expression of a 5-FU sensitivity enhancing gene found by the screening method of the example. Ａｄ／ＴＧＦＢＲ２－ｍＣｈｅｒｒｙによるＩＦＮ－α／５－ＦＵ感受性の亢進（ｉｎ　ｖｉｖｏ）を示すグラフである。2 is a graph showing the enhancement (in vivo) of IFN-α / 5-FU sensitivity by Ad / TGFBR2-mCherry. ＴＧＦＢＲ２，ＥＸＴ１過剰発現による５－ＦＵ及びＩＦＮ－α／５－ＦＵ誘導性アポトーシスの増強を示す蛍光顕微鏡写真（図１０ａ）およびグラフ（図１０ｂ）である。FIG. 10 is a fluorescence micrograph (FIG. 10a) and a graph (FIG. 10b) showing enhancement of 5-FU and IFN-α / 5-FU-induced apoptosis by overexpression of TGFBR2 and EXT1. ＴＧＦＢＲ２過剰発現によるＴＧＦ－βシグナル活性化及びアポトーシス関連タンパク発現の変化を示すグラフ（図１１ａ、図１１ｂ）および電気泳動写真（図１１ｃ）である。FIG. 11 is a graph (FIGS. 11a and 11b) and an electrophoretic photograph (FIG. 11c) showing changes in TGF-β signal activation and apoptosis-related protein expression due to overexpression of TGFBR2. ＥＸＴ１過剰発現によるアポトーシス亢進に対するＥＲストレスの関与を示すグラフ（図１２ａ、図１２ｂ）および電気泳動写真（図１２ｃ）である。It is a graph (FIG. 12a, FIG. 12b) and an electrophoretic photograph (FIG. 12c) which show the involvement of ER stress with respect to the enhancement of apoptosis by EXT1 overexpression. ＴＧＦＢＲ２、ＥＸＴ１過剰発現によるヒト肝癌モデルマウスへの効果を示すグラフである。It is a graph which shows the effect to a human liver cancer model mouse by TGFBR2 and EXT1 overexpression. 肝癌患者におけるＰＲＫＡＧ２、ＴＧＦＢＲ２、ＥＸＴ１のｍＲＮＡ発現を示すグラフ（図１４Ａ、図１４Ｂ、図１４Ｃ、図１４Ｄ）である。15 is a graph showing mRNA expression of PRKAG2, TGFBR2 and EXT1 in liver cancer patients (FIGS. 14A, 14B, 14C, and 14D).
　＜用語の説明＞
　（１）相同性
　本明細書において、「相同性」とは、２つもしくは複数間のアミノ酸配列の同一のアミノ酸数の割合を、当該技術分野で公知の方法に従って算定したものである。割合を算定する前には、比較するアミノ酸配列群のアミノ酸配列を整列させ、同一の割合を最大にするために必要である場合はアミノ酸配列の一部に間隙を導入する。また、いかなる保存的置換も同一と考えない。また、最適に整列した状態において、オーバーラップするアミノ酸を含めた全アミノ酸残基に対する、同一のアミノ酸数の割合を意味する。整列のための方法、割合の算定方法、およびそれらに関連するコンピュータプログラムは、当該技術分野で従来からよく知られており、一般的な配列分析プログラム（例えば、ＧＥＮＥＴＹＸ、ＧｅｎｅＣｈｉｐ　Ｓｅｑｕｅｎｃｅ　Ａｎａｌｙｓｉｓなど）を使用して測定することができる。また「相同性」は、２つもしくは複数間のＤＮＡ鎖、または２つもしくは複数間のＲＮＡ鎖において、同一の塩基の割合を、上記と同様に当該技術分野で公知の方法に従って算定したものである。 <Explanation of terms>
(1) Homology In this specification, “homology” is the ratio of the number of identical amino acids in an amino acid sequence between two or more amino acids calculated according to a method known in the art. Before calculating the ratio, the amino acid sequences of the amino acid sequences to be compared are aligned, and a gap is introduced into a part of the amino acid sequence if necessary to maximize the same ratio. Nor do any conservative substitutions be considered identical. Further, it means the ratio of the same number of amino acids to all amino acid residues including overlapping amino acids in an optimally aligned state. Methods for alignment, percentage calculation methods, and related computer programs are well known in the art and use common sequence analysis programs (eg, GENETYX, GeneChip Sequence Analysis, etc.) Can be measured. In addition, “homology” is obtained by calculating the ratio of the same base in two or more DNA strands or two or more RNA strands according to a method known in the art as described above. is there.
　（２）ストリンジェントな条件
　本明細書において、「ストリンジェントな条件」とは、例えば（１）洗浄のための低イオン強度と高温度、例えば、５０℃において０．０１５Ｍの塩化ナトリウム／０．００１５Ｍのクエン酸ナトリウム／０．１％のドデシル硫酸ナトリウムを用いる、（２）ハイブリダイゼーション中にホルムアミド等の変性剤、例えば、４２℃において５０％（ｖｏｌ／ｖｏｌ）ホルムアミドと０．１％ウシ血清アルブミン／０．１％フィコール／０．１％ポリビニルピロリドン／５０ｍＭリン酸ナトリウムバッファー（ｐＨ６．５）、および７５０ｍＭの塩化ナトリウム、７５ｍＭクエン酸ナトリウムを用いる、または（３）４２℃において５０％ホルムアミド、５×ＳＳＣ（０．７５ＭのＮａＣｌ、０．０７５Ｍのクエン酸ナトリウム）、５０ｍＭのリン酸ナトリウム（ｐＨ６．８）、０．１％のピロリン酸ナトリウム、５×デンハート液、超音波処理サケ精子ＤＮＡ（５０μｇ／ｍｌ）、０．１％ＳＤＳ、および１０％のデキストラン硫酸と、４２℃において０．２×ＳＳＣ（塩化ナトリウム／クエン酸ナトリウム）中での洗浄および５５℃のホルムアミド、次いで５５℃におけるＥＤＴＡを含む０．１×ＳＳＣにてストリンジェントな洗浄を含む条件であっても良い。また中程度にストリンジェントな条件の例は、２０％ホルムアミド、５×ＳＳＣ、５０ｍＭリン酸ナトリウム（ｐＨ７．６）、５×デンハート液、１０％デキストラン硫酸、および２０ｍｇ／ｍｌの変性剪断サケ精子ＤＮＡを含む溶液中において、３７℃で一晩インキュベーション、次いで１×ＳＳＣ中３７～５０℃でのフィルターの洗浄のような条件である。なお、ハイブリダイゼーション反応のストリンジェンシーは、当業者によって容易に決定でき、一般的にプローブ長、洗浄温度、および塩濃度に依存する。一般に、長いプローブは適当なアニーリングのために高温を必要とし、短いプローブは低温を必要とする。また一般に、ストリンジェンシーは塩濃度に逆比例する。 (2) Stringent conditions In this specification, “stringent conditions” means, for example, (1) low ionic strength for washing and high temperature, for example, 0.015 M sodium chloride / 0.00% at 50 ° C. 0015M sodium citrate / 0.1% sodium dodecyl sulfate, (2) denaturing agents such as formamide during hybridization, eg 50% (vol / vol) formamide and 0.1% bovine serum at 42 ° C. Albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer (pH 6.5) and 750 mM sodium chloride, 75 mM sodium citrate, or (3) 50% formamide at 42 ° C. 5 × SSC (0.75M NaCl, 0.075M Que Acid sodium), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 × Denhart's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% Of dextran sulfate at 42 ° C. in 0.2 × SSC (sodium chloride / sodium citrate) and 55 ° C. formamide followed by 0.1 × SSC containing EDTA at 55 ° C. The conditions may be included. Examples of moderately stringent conditions are 20% formamide, 5 × SSC, 50 mM sodium phosphate (pH 7.6), 5 × Denhart solution, 10% dextran sulfate, and 20 mg / ml denatured sheared salmon sperm DNA. Conditions such as overnight incubation at 37 ° C. in a solution containing, followed by washing the filter at 37-50 ° C. in 1 × SSC. The stringency of the hybridization reaction can be easily determined by those skilled in the art and generally depends on the probe length, the washing temperature, and the salt concentration. In general, longer probes require higher temperatures for proper annealing, and shorter probes require lower temperatures. In general, stringency is inversely proportional to salt concentration.
　（３）ハイブリダイズ
　また、本明細書において、ポリヌクレオチドに適用される場合の「ハイブリダイズ」とは、ヌクレオチドの塩基間の水素結合等によってヌクレオチド間の対ができる性質のことを表す。塩基対はワトソン・クリック型塩基対、フーグスティーン型塩基対、または任意の他の配列特異的な形で生じうる。 (3) Hybridization In addition, in this specification, “hybridization” when applied to a polynucleotide means a property that allows pairing between nucleotides by hydrogen bonding between nucleotide bases. Base pairs can occur in Watson-Crick base pairs, Hoogsteen base pairs, or any other sequence specific form.
　以下、本発明の実施の形態について説明する。
　＜ヌクレオチド断片を含む５－フルオロウラシル感受性増感剤＞
　本実施形態における５－フルオロウラシル感受性増感剤は、ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子からなる群から選ばれる１以上の遺伝子をコードするヌクレオチド断片を含む、増感剤である。この構成によれば、５－フルオロウラシルの抗腫瘍効果を増強する作用を有することが後述する実施例において実証されているＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子またはＥＸＴ１遺伝子のいずれかをコードするヌクレオチド断片を含むため、増感剤自体としても５－フルオロウラシルの抗腫瘍効果を増強する効果が得られる。 Embodiments of the present invention will be described below.
<5-Fluorouracil-sensitive sensitizer containing nucleotide fragment>
The 5-fluorouracil-sensitive sensitizer in the present embodiment is a sensitizer containing a nucleotide fragment encoding one or more genes selected from the group consisting of PRAK2 gene, TGFBR2 gene and EXT1 gene. According to this configuration, since it contains a nucleotide fragment encoding any of PRAK2 gene, TGFBR2 gene or EXT1 gene, which has been demonstrated in Examples described later, to have an action of enhancing the antitumor effect of 5-fluorouracil, As a sensitizer itself, an effect of enhancing the antitumor effect of 5-fluorouracil can be obtained.
　ここで、この５－フルオロウラシル感受性増感剤は、５－フルオロウラシルを投与した場合の癌細胞の死亡率を向上させる（生存率を低下させる）ものであればよく、特に限定するものではないが、例えば母集団が正規分布に従うと仮定できる場合にはパラメトリック検定であるスチューデントのｔ検定（Ｓｔｕｄｅｎｔ'ｓ　ｔ－ｔｅｓｔ）において有意差があれば好ましい。すなわち、スチューデントのｔ検定において片側検定でｐ＜０．０５となればよく、より好ましくは片側検定でｐ＜０．０３となればよく、最も好ましくは片側検定でｐ＜０．０１となればよい。なお、スチューデントのｔ検定は特に片側検定に限定するわけではなく、両側検定で行っても良い。さらに、母集団が正規分布に従うと仮定できない場合には、ノンパラメトリック検定として、マン・ホイットニーのＵ検定などを行って有意差の有無を検定しても良い。 Here, the 5-fluorouracil-sensitive sensitizer is not particularly limited as long as it improves the mortality of cancer cells when 5-fluorouracil is administered (decreases the survival rate). For example, when it can be assumed that the population follows a normal distribution, it is preferable if there is a significant difference in Student's t-test, which is a parametric test. That is, in the Student's t-test, one-sided test should be p <0.05, more preferably one-sided test should be p <0.03, and most preferably one-sided test should be p <0.01. Good. The student t-test is not limited to a one-sided test, but may be a two-sided test. Further, when it cannot be assumed that the population follows a normal distribution, the presence or absence of a significant difference may be tested by performing a Mann-Whitney U test as a nonparametric test.
　（ｉ）ＰＲＡＫＧ２遺伝子
　本実施形態の５－フルオロウラシル感受性増感剤に含まれるヌクレオチド断片にコードされるＰＲＡＫＧ２遺伝子は、哺乳動物のＡＭＰ活性化蛋白質キナーゼγ２非触媒サブユニット（ｐｒｏｔｅｉｎ　ｋｉｎａｓｅ，ＡＭＰ－ａｃｔｉｖａｔｅｄ，ｇａｍｍａ　２　ｎｏｎ－ｃａｔａｌｙｔｉｃ　ｓｕｂｕｎｉｔ）をコードする遺伝子であればよく、特に限定するものではないが、例えば、ヒト野生型ＰＲＡＫＧ２遺伝子（Ｅｎｔｒｅｚ　Ｇｅｎｅ　ＩＤ：５１４２２の５'－ＡＭＰ－ａｃｔｉｖａｔｅｄ　ｐｒｏｔｅｉｎ　ｋｉｎａｓｅ　ｓｕｂｕｎｉｔ　ｇａｍｍａ－２　ｉｓｏｆｏｒｍ　ａ［Ｈｏｍｏ　ｓａｐｉｅｎｓ］をコードする遺伝子）などを好適に用いることができる。なぜなら、このヒト野生型ＰＲＡＫＧ２遺伝子に５－フルオロウラシルの抗腫瘍効果を増強する効果があることは、後述の実施例で実証されているからである。なお、上記遺伝子に関する塩基配列等の詳細は、Ｎａｔｉｏｎａｌ　Ｃｅｎｔｅｒ　ｆｏｒ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ　Ｉｎｆｏｒｍａｔｉｏｎ（ＮＣＢＩ）のデータベースであるＧｅｎＢａｎｋで確認することができる。 (I) PRKG2 gene The PRAKG2 gene encoded by the nucleotide fragment contained in the 5-fluorouracil-sensitive sensitizer of the present embodiment is a protein kinase, AMP-activated, AMP-activated protein kinase γ2 non-catalytic subunit. The gene encodes gamma 2 non-catalytic subunit) and is not particularly limited. For example, human wild-type PRAK2 gene (Entrez Gene ID: 51422 5'-AMP-activated protein kinase subunit gamma subunit subunit gamma isoform a [a gene encoding Homo sapiens]) and the like can be preferably used. This is because this human wild-type PRAK2 gene has an effect of enhancing the antitumor effect of 5-fluorouracil, which has been demonstrated in Examples described later. The details of the base sequence and the like related to the above gene can be confirmed in GenBank which is a database of National Center for Biotechnology Information (NCBI).
　もっとも、本実施形態の５－フルオロウラシル感受性増感剤に含まれるヌクレオチド断片にコードされるＰＲＡＫＧ２遺伝子は、ヒト野生型ＰＲＡＫＧ２遺伝子の変異型遺伝子であってもよい。なぜなら、ヒト野生型ＰＲＡＫＧ２遺伝子との相同性の高い遺伝子であれば、ヒト野生型ＰＲＡＫＧ２遺伝子と同様に哺乳動物細胞における５－フルオロウラシル感受性を増強させる可能性が高いからである。ここで、変異型とは、個体間のＤＮＡ配列の差異に起因するものを含む。 However, the PRAK2 gene encoded by the nucleotide fragment contained in the 5-fluorouracil-sensitive sensitizer of the present embodiment may be a mutant gene of the human wild type PRAK2 gene. This is because a gene having high homology with the human wild-type PRAK2 gene has a high possibility of enhancing 5-fluorouracil sensitivity in mammalian cells in the same manner as the human wild-type PRAKA2 gene. Here, the mutant type includes those resulting from differences in DNA sequences between individuals.
　具体的には、ヒト野生型ＰＲＡＫＧ２遺伝子の変異型遺伝子は、ヒト野生型ＰＲＡＫＧ２遺伝子がコードする蛋白質のアミノ酸配列をコードする塩基配列（すなわちヒト野生型ＰＲＡＫＧ２遺伝子の塩基配列の縮重配列）からなる変異型遺伝子であってもよい。なぜなら、ヒト野生型ＰＲＡＫＧ２遺伝子の縮重配列からなる変異型遺伝子がコードするアミノ酸配列は野生型遺伝子がコードするアミノ酸配列と同じであるため、この変異型遺伝子から発現される蛋白質の機能は野生型遺伝子から発現される蛋白質と同じだからである。なお、どのような塩基配列が縮重配列にあたるかは、ユニバーサルコドンの対応表を用いて当業者であれば容易に理解することが可能である。 Specifically, the mutant gene of the human wild-type PRAK2 gene consists of a base sequence encoding the amino acid sequence of the protein encoded by the human wild-type PRAK2 gene (ie, a degenerate sequence of the base sequence of the human wild-type PRAK2 gene). It may be a mutant gene. Because the amino acid sequence encoded by the mutant gene comprising the degenerate sequence of the human wild-type PRAK2 gene is the same as the amino acid sequence encoded by the wild-type gene, the function of the protein expressed from this mutant gene is wild-type. This is because it is the same as a protein expressed from a gene. It should be noted that a person skilled in the art can easily understand which base sequence corresponds to a degenerate sequence using a universal codon correspondence table.
　また、ヒト野生型ＰＲＡＫＧ２遺伝子の変異型遺伝子は、ヒト野生型ＰＲＡＫＧ２遺伝子がコードする蛋白質のアミノ酸配列のうち１若しくは数個のアミノ酸残基を欠失・置換・付加してなるアミノ酸配列をコードする塩基配列からなる変異型遺伝子であってもよい。また上記「１若しくは数個」は好ましくは３０個以下であり、より好ましくは２０個以下であり、より好ましくは１５個以下であり、より好ましくは１０個以下であり、より好ましくは５個以下であり、好ましくは４個以下であり、より好ましくは３個以下であり、より好ましくは２個以下であり、さらに好ましくは１個である。なぜならば、変異型遺伝子の塩基配列が、野生型の遺伝子の塩基配列に対して塩基の欠失、置換若しくは付加が少ないほど、野生型の遺伝子の塩基配列をコードする遺伝子に近い特性を有していることになるからである。 The mutant gene of the human wild-type PRAK2 gene encodes an amino acid sequence obtained by deleting, substituting, or adding one or several amino acid residues in the amino acid sequence of the protein encoded by the human wild-type PRAK2 gene. It may be a mutant gene consisting of a base sequence. The “one or several” is preferably 30 or less, more preferably 20 or less, more preferably 15 or less, more preferably 10 or less, more preferably 5 or less. Preferably, it is 4 or less, more preferably 3 or less, more preferably 2 or less, and still more preferably 1. This is because, as the base sequence of the mutant gene has fewer base deletions, substitutions or additions to the base sequence of the wild-type gene, it has a property closer to the gene encoding the base sequence of the wild-type gene. Because it will be.
　なお、この変異型遺伝子の塩基配列のコードするアミノ酸配列が、ヒト野生型ＰＲＡＫＧ２遺伝子のコードするアミノ酸配列に対して１または数個の置換がある場合には、アミノ酸側鎖の性質が保存されている別のアミノ酸に置換していることが好ましい。例えばアミノ酸側鎖の性質としては、疎水性アミノ酸（Ａ、Ｉ、Ｌ、Ｍ、Ｆ、Ｐ、Ｗ、Ｙ、Ｖ）、親水性アミノ酸（Ｒ、Ｄ、Ｎ、Ｃ、Ｅ、Ｑ、Ｇ、Ｈ、Ｋ、Ｓ、Ｔ）、脂肪族側鎖を有するアミノ酸（Ｇ、Ａ、Ｖ、Ｌ、Ｉ、Ｐ）、水酸基含有側鎖を有するアミノ酸（Ｓ、Ｔ、Ｙ）、硫黄原子含有側鎖を有するアミノ酸（Ｃ、Ｍ）、カルボン酸及びアミド含有側鎖を有するアミノ酸（Ｄ、Ｎ、Ｅ、Ｑ）、塩基含有側鎖を有するアミノ離（Ｒ、Ｋ、Ｈ）、および、芳香族含有側鎖を有するアミノ酸（Ｈ、Ｆ、Ｙ、Ｗ）を挙げることができる（括弧内はいずれもアミノ酸の一文字標記を表す）。これらの各グループ内のアミノ酸同士の置換は保存的置換と総称される。あるアミノ酸配列に対する１または複数個のアミノ酸残基の欠失、付加、または他のアミノ酸による置換により修飾されたアミノ酸配列を有するポリペプチドがその生物学的活性を維持することはすでに知られている（Mark et al., Proc Natl Acad Sci U SA. 1984Sep; 81(18): 5662-5666., Zoller et al., Nucleic Acids Res. 1982 Oct 25; 10(20): 6487-6500., Wang et al., Science. 1984 Jun 29; 224 (4656): 1431-1433.）。 In addition, when the amino acid sequence encoded by the nucleotide sequence of the mutant gene has one or several substitutions with respect to the amino acid sequence encoded by the human wild type PRAK2 gene, the properties of the amino acid side chain are preserved. It is preferably substituted with another amino acid. For example, as the properties of amino acid side chains, hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids having aliphatic side chains (G, A, V, L, I, P), amino acids having hydroxyl group-containing side chains (S, T, Y), sulfur atom-containing side chains Amino acids (C, M) having carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids having base-containing side chains (R, K, H), and aromatic-containing Amino acids having side chains (H, F, Y, W) can be mentioned (the parentheses indicate single letter symbols of amino acids). Substitutions between amino acids within each of these groups are collectively referred to as conservative substitutions. It is already known that a polypeptide having an amino acid sequence modified by deletion, addition or substitution by one or more amino acid residues with respect to a certain amino acid sequence maintains its biological activity. (Mark et al., Proc Natl Acad Sci U SA. 1984Sep; 81 (18): 5662-5666., Zoller et al., Nucleic Acids Res. 1982 Oct 25; 10 (20): 6487-6500., Wang et al., Science. 1984 Jun 29; 224 (4656): 1431-1433.).
　また、ヒト野生型ＰＲＡＫＧ２遺伝子の変異型遺伝子は、ヒト野生型ＰＲＡＫＧ２遺伝子がコードする蛋白質のアミノ酸配列に対して８０％以上の相同性を有するアミノ酸配列をコードする基配列からなる変異型遺伝子であってもよい。ここで上記「８０％以上」は好ましくは８５％以上であり、より好ましくは９０％以上であり、さらに好ましくは９５％以上であり、最も好ましくは９８％以上である。なぜならば、異型遺伝子の塩基配列が、野生型の遺伝子の塩基配列に対して相同性が高いほど、野生型の遺伝子の塩基配列をコードする遺伝子に近い特性を有していることになるからである。 The mutant gene of the human wild-type PRAK2 gene is a mutant gene comprising a base sequence encoding an amino acid sequence having 80% or more homology with the amino acid sequence of the protein encoded by the human wild-type PRAK2 gene. May be. Here, “80% or more” is preferably 85% or more, more preferably 90% or more, further preferably 95% or more, and most preferably 98% or more. This is because the higher the homology between the base sequence of the atypical gene and the base sequence of the wild-type gene, the closer to the gene encoding the base sequence of the wild-type gene. is there.
　また、ヒト野生型ＰＲＡＫＧ２遺伝子の変異型遺伝子は、ヒト野生型ＰＲＡＫＧ２遺伝子の塩基配列に相補的な塩基配列に対してストリンジェントな条件でハイブリダイズする塩基配列からなる変異型遺伝子であってもよい。なぜなら、ストリンジェントな条件でハイブリダイズする塩基配列からなる変異型遺伝子は、野生型の遺伝子の塩基配列に対して相同性が高いため、野生型の遺伝子の塩基配列をコードする遺伝子に近い特性を有していることになるからである。 Further, the mutant gene of the human wild type PRAK2 gene may be a mutant gene comprising a base sequence that hybridizes under stringent conditions to a base sequence complementary to the base sequence of the human wild type PRAK2 gene. . This is because a mutant gene consisting of a base sequence that hybridizes under stringent conditions is highly homologous to the base sequence of the wild-type gene, and therefore has characteristics close to those of the gene encoding the base sequence of the wild-type gene. It is because it will have.
　（ｉｉ）ＴＧＦＢＲ２遺伝子
　本実施形態の５－フルオロウラシル感受性増感剤に含まれるヌクレオチド断片にコードされるＴＧＦＢＲ２遺伝子は、哺乳動物のＴＧＦ－β２型受容体（ｔｒａｎｓｆｏｒｍｉｎｇ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ，ｂｅｔａ　ｒｅｃｅｐｔｏｒ　ＩＩ）をコードする遺伝子であればよく、特に限定するものではないが、例えば、ヒト野生型ＴＧＦＢＲ２遺伝子（Ｅｎｔｒｅｚ　Ｇｅｎｅ　ＩＤ：７０４８のｔｒａｎｓｆｏｒｍｉｎｇ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ，　ｂｅｔａ　ｒｅｃｅｐｔｏｒ　ＩＩ（７０／８０ｋＤａ）［Ｈｏｍｏ　ｓａｐｉｅｎｓ］をコードする遺伝子）などを好適に用いることができる。なぜなら、このヒト野生型ＴＧＦＢＲ２遺伝子に５－フルオロウラシルの抗腫瘍効果を増強する効果があることは、後述の実施例で実証されているからである。なお、上記遺伝子に関する塩基配列等の詳細は、Ｎａｔｉｏｎａｌ　Ｃｅｎｔｅｒ　ｆｏｒ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ　Ｉｎｆｏｒｍａｔｉｏｎ（ＮＣＢＩ）のデータベースであるＧｅｎＢａｎｋで確認することができる。 (Ii) TGFBR2 gene The TGFBR2 gene encoded by the nucleotide fragment contained in the 5-fluorouracil-sensitive sensitizer of the present embodiment encodes a mammalian TGF-β2 type receptor (transforming growth factor, beta receptor II). The gene may be any gene, and is not particularly limited. For example, human wild-type TGFBR2 gene (encoding gene growth factor, beta receptor II (70/80 kDa) [Homo sapiens] encoding Entrez Gene ID: 7048) Can be suitably used. This is because this human wild-type TGFBR2 gene has an effect of enhancing the antitumor effect of 5-fluorouracil, which has been demonstrated in Examples described later. The details of the base sequence and the like related to the above gene can be confirmed in GenBank which is a database of National Center for Biotechnology Information (NCBI).
　もっとも、本実施形態の５－フルオロウラシル感受性増感剤に含まれるヌクレオチド断片にコードされるＴＧＦＢＲ２遺伝子は、ヒト野生型ＴＧＦＢＲ２遺伝子の変異型遺伝子であってもよい。なぜなら、ヒト野生型ＴＧＦＢＲ２遺伝子との相同性の高い遺伝子であれば、ヒト野生型ＴＧＦＢＲ２遺伝子と同様に哺乳動物細胞における５－フルオロウラシル感受性を増強させる可能性が高いからである。ここで、変異型とは、個体間のＤＮＡ配列の差異に起因するものを含む。 However, the TGFBR2 gene encoded by the nucleotide fragment contained in the 5-fluorouracil-sensitive sensitizer of the present embodiment may be a mutant gene of the human wild type TGFBR2 gene. This is because a gene having a high homology with the human wild-type TGFBR2 gene is likely to enhance 5-fluorouracil sensitivity in mammalian cells in the same manner as the human wild-type TGFBR2 gene. Here, the mutant type includes those resulting from differences in DNA sequences between individuals.
　具体的には、ヒト野生型ＴＧＦＢＲ２遺伝子の変異型遺伝子は、ヒト野生型ＴＧＦＢＲ２遺伝子がコードする蛋白質のアミノ酸配列をコードする塩基配列（すなわちヒト野生型ＴＧＦＢＲ２遺伝子の塩基配列の縮重配列）からなる変異型遺伝子であってもよい。なぜなら、ヒト野生型ＴＧＦＢＲ２遺伝子の縮重配列からなる変異型遺伝子がコードするアミノ酸配列は野生型遺伝子がコードするアミノ酸配列と同じであるため、この変異型遺伝子から発現される蛋白質の機能は野生型遺伝子から発現される蛋白質と同じだからである。なお、どのような塩基配列が縮重配列にあたるかは、ユニバーサルコドンの対応表を用いて当業者であれば容易に理解することが可能である。 Specifically, the mutant gene of the human wild type TGFBR2 gene consists of a base sequence encoding the amino acid sequence of the protein encoded by the human wild type TGFBR2 gene (that is, a degenerate sequence of the base sequence of the human wild type TGFBR2 gene). It may be a mutant gene. Because the amino acid sequence encoded by the mutant gene consisting of the degenerate sequence of the human wild type TGFBR2 gene is the same as the amino acid sequence encoded by the wild type gene, the function of the protein expressed from the mutant gene is wild type. This is because it is the same as a protein expressed from a gene. It should be noted that a person skilled in the art can easily understand which base sequence corresponds to a degenerate sequence using a universal codon correspondence table.
　また、ヒト野生型ＴＧＦＢＲ２遺伝子の変異型遺伝子は、ヒト野生型ＴＧＦＢＲ２遺伝子がコードする蛋白質のアミノ酸配列のうち１若しくは数個のアミノ酸残基を欠失・置換・付加してなるアミノ酸配列をコードする塩基配列からなる変異型遺伝子であってもよい。また上記「１若しくは数個」は好ましくは３０個以下であり、より好ましくは２０個以下であり、より好ましくは１５個以下であり、より好ましくは１０個以下であり、より好ましくは５個以下であり、好ましくは４個以下であり、より好ましくは３個以下であり、より好ましくは２個以下であり、さらに好ましくは１個である。なぜならば、変異型遺伝子の塩基配列が、野生型の遺伝子の塩基配列に対して塩基の欠失、置換若しくは付加が少ないほど、野生型の遺伝子の塩基配列をコードする遺伝子に近い特性を有していることになるからである。 The mutant gene of the human wild type TGFBR2 gene encodes an amino acid sequence formed by deleting, substituting, or adding one or several amino acid residues in the amino acid sequence of the protein encoded by the human wild type TGFBR2 gene. It may be a mutant gene consisting of a base sequence. The “one or several” is preferably 30 or less, more preferably 20 or less, more preferably 15 or less, more preferably 10 or less, more preferably 5 or less. Preferably, it is 4 or less, more preferably 3 or less, more preferably 2 or less, and still more preferably 1. This is because, as the base sequence of the mutant gene has fewer base deletions, substitutions or additions to the base sequence of the wild-type gene, it has a property closer to the gene encoding the base sequence of the wild-type gene. Because it will be.
　なお、この変異型遺伝子の塩基配列のコードするアミノ酸配列が、ヒト野生型ＴＧＦＢＲ２遺伝子のコードするアミノ酸配列に対して１または数個の置換がある場合には、アミノ酸側鎖の性質が保存されている別のアミノ酸に置換していることが好ましい。アミノ酸側鎖の性質についての説明は既に行ったので繰り返さない。 In addition, when the amino acid sequence encoded by the nucleotide sequence of the mutant gene has one or several substitutions with respect to the amino acid sequence encoded by the human wild type TGFBR2 gene, the properties of the amino acid side chain are preserved. It is preferably substituted with another amino acid. The description of the nature of the amino acid side chain has already been made and will not be repeated.
　また、ヒト野生型ＴＧＦＢＲ２遺伝子の変異型遺伝子は、ヒト野生型ＴＧＦＢＲ２遺伝子がコードする蛋白質のアミノ酸配列に対して８０％以上の相同性を有するアミノ酸配列をコードする基配列からなる変異型遺伝子であってもよい。ここで上記「８０％以上」は好ましくは８５％以上であり、より好ましくは９０％以上であり、さらに好ましくは９５％以上であり、最も好ましくは９８％以上である。なぜならば、異型遺伝子の塩基配列が、野生型の遺伝子の塩基配列に対して相同性が高いほど、野生型の遺伝子の塩基配列をコードする遺伝子に近い特性を有していることになるからである。 The mutant gene of the human wild type TGFBR2 gene is a mutant gene comprising a base sequence encoding an amino acid sequence having 80% or more homology with the amino acid sequence of the protein encoded by the human wild type TGFBR2 gene. May be. Here, “80% or more” is preferably 85% or more, more preferably 90% or more, further preferably 95% or more, and most preferably 98% or more. This is because the higher the homology between the base sequence of the atypical gene and the base sequence of the wild-type gene, the closer to the gene encoding the base sequence of the wild-type gene. is there.
　また、ヒト野生型ＴＧＦＢＲ２遺伝子の変異型遺伝子は、ヒト野生型ＴＧＦＢＲ２遺伝子の塩基配列に相補的な塩基配列に対してストリンジェントな条件でハイブリダイズする塩基配列からなる変異型遺伝子であってもよい。なぜなら、ストリンジェントな条件でハイブリダイズする塩基配列からなる変異型遺伝子は、野生型の遺伝子の塩基配列に対して相同性が高いため、野生型の遺伝子の塩基配列をコードする遺伝子に近い特性を有していることになるからである。 Further, the mutant gene of the human wild type TGFBR2 gene may be a mutant gene comprising a base sequence that hybridizes under stringent conditions to a base sequence complementary to the base sequence of the human wild type TGFBR2 gene. . This is because a mutant gene consisting of a base sequence that hybridizes under stringent conditions is highly homologous to the base sequence of the wild-type gene, and therefore has characteristics close to those of the gene encoding the base sequence of the wild-type gene. It is because it will have.
　（ｉｉｉ）ＥＸＴ１遺伝子
　本実施形態の５－フルオロウラシル感受性増感剤に含まれるヌクレオチド断片にコードされるＥＸＴ１遺伝子は、哺乳動物の小胞体内在性ＩＩ型膜貫通糖転移酵素（ｅｎｄｏｐｌａｓｍｉｃ　ｒｅｔｉｃｕｌｕｍ－ｒｅｓｉｄｅｎｔ　ｔｙｐｅ　ＩＩ　ｔｒａｎｓｍｅｍｂｒａｎｅ　ｇｌｙｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅ）をコードする遺伝子であればよく、特に限定するものではないが、例えば、ヒト野生型ＥＸＴ１遺伝子（Ｅｎｔｒｅｚ　Ｇｅｎｅ　ＩＤ：２１３１のＥＸＴ１　ｅｘｏｓｔｏｓｅｓ　（ｍｕｌｔｉｐｌｅ）１［Ｈｏｍｏ　ｓａｐｉｅｎｓ］をコードする遺伝子）などを好適に用いることができる。なぜなら、このヒト野生ＥＸＴ１遺伝子に５－フルオロウラシルの抗腫瘍効果を増強する効果があることは、後述の実施例で実証されているからである。なお、上記遺伝子に関する塩基配列等の詳細は、Ｎａｔｉｏｎａｌ　Ｃｅｎｔｅｒ　ｆｏｒ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ　Ｉｎｆｏｒｍａｔｉｏｎ（ＮＣＢＩ）のデータベースであるＧｅｎＢａｎｋで確認することができる。 (Iii) EXT1 gene The EXT1 gene encoded by the nucleotide fragment contained in the 5-fluorouracil-sensitive sensitizer of the present embodiment is an endoplasmic reticulum-resident type II in mammalian endoplasmic reticulum. Any gene may be used as long as it is a gene that encodes transmembrane glycosyltransferase (for example, human EXT1 gene (Entrez Gene ID: 2131 EXT1 genes (multiple) 1)). Can be suitably used. This is because this human wild EXT1 gene has an effect of enhancing the anti-tumor effect of 5-fluorouracil, which has been demonstrated in Examples described later. The details of the base sequence and the like related to the above gene can be confirmed in GenBank which is a database of National Center for Biotechnology Information (NCBI).
　なお、このＥＸＴ１遺伝子にコードされている哺乳動物の小胞体内在性ＩＩ型膜貫通糖転移酵素は、ヘパラン硫酸の生合成における糖鎖延長反応に関係している（ｉｎｖｏｌｖｅｄ　ｉｎ　ｔｈｅ　ｃｈａｉｎ　ｅｌｏｎｇａｔｉｏｎ　ｓｔｅｐ　ｏｆ　ｈｅｐａｒａｎ　ｓｕｌｆａｔｅ　ｂｉｏｓｙｎｔｈｅｓｉｓ）。 In addition, the mammalian endoplasmic reticulum type II transmembrane glycosyltransferase encoded by this EXT1 gene is related to a sugar chain elongation reaction in biosynthesis of heparan sulfate (involved in the chain elongation step of heparan). sulfate biosynthesis).
　もっとも、本実施形態の５－フルオロウラシル感受性増感剤に含まれるヌクレオチド断片にコードされるＥＸＴ１遺伝子は、ヒト野生型ＥＸＴ１遺伝子の変異型遺伝子であってもよい。なぜなら、ヒト野生型ＥＸＴ１遺伝子との相同性の高い遺伝子であれば、ヒト野生型ＥＸＴ１遺伝子と同様に哺乳動物細胞における５－フルオロウラシル感受性を増強させる可能性が高いからである。ここで、変異型とは、個体間のＤＮＡ配列の差異に起因するものを含む。 However, the EXT1 gene encoded by the nucleotide fragment contained in the 5-fluorouracil sensitive sensitizer of the present embodiment may be a mutant gene of the human wild type EXT1 gene. This is because a gene having high homology with the human wild-type EXT1 gene is likely to enhance 5-fluorouracil sensitivity in mammalian cells in the same manner as the human wild-type EXT1 gene. Here, the mutant type includes those resulting from differences in DNA sequences between individuals.
　具体的には、ヒト野生型ＥＸＴ１遺伝子の変異型遺伝子は、ヒト野生型ＥＸＴ１遺伝子がコードする蛋白質のアミノ酸配列をコードする塩基配列（すなわちヒト野生型ＥＸＴ１遺伝子の塩基配列の縮重配列）からなる変異型遺伝子であってもよい。なぜなら、ヒト野生型ＥＸＴ１遺伝子の縮重配列からなる変異型遺伝子がコードするアミノ酸配列は野生型遺伝子がコードするアミノ酸配列と同じであるため、この変異型遺伝子から発現される蛋白質の機能は野生型遺伝子から発現される蛋白質と同じだからである。なお、どのような塩基配列が縮重配列にあたるかは、ユニバーサルコドンの対応表を用いて当業者であれば容易に理解することが可能である。 Specifically, the mutant gene of the human wild type EXT1 gene consists of a base sequence encoding the amino acid sequence of the protein encoded by the human wild type EXT1 gene (that is, a degenerate sequence of the base sequence of the human wild type EXT1 gene). It may be a mutant gene. Because the amino acid sequence encoded by the mutant gene consisting of the degenerate sequence of the human wild type EXT1 gene is the same as the amino acid sequence encoded by the wild type gene, the function of the protein expressed from this mutant gene is wild type. This is because it is the same as a protein expressed from a gene. It should be noted that a person skilled in the art can easily understand which base sequence corresponds to a degenerate sequence using a universal codon correspondence table.
　また、ヒト野生型ＥＸＴ１遺伝子の変異型遺伝子は、ヒト野生型ＥＸＴ１遺伝子がコードする蛋白質のアミノ酸配列のうち１若しくは数個のアミノ酸残基を欠失・置換・付加してなるアミノ酸配列をコードする塩基配列からなる変異型遺伝子であってもよい。また上記「１若しくは数個」は好ましくは３０個以下であり、より好ましくは２０個以下であり、より好ましくは１５個以下であり、より好ましくは１０個以下であり、より好ましくは５個以下であり、好ましくは４個以下であり、より好ましくは３個以下であり、より好ましくは２個以下であり、さらに好ましくは１個である。なぜならば、変異型遺伝子の塩基配列が、野生型の遺伝子の塩基配列に対して塩基の欠失、置換若しくは付加が少ないほど、野生型の遺伝子の塩基配列をコードする遺伝子に近い特性を有していることになるからである。 The mutant gene of the human wild type EXT1 gene encodes an amino acid sequence obtained by deleting, substituting, or adding one or several amino acid residues in the amino acid sequence of the protein encoded by the human wild type EXT1 gene. It may be a mutant gene consisting of a base sequence. The “one or several” is preferably 30 or less, more preferably 20 or less, more preferably 15 or less, more preferably 10 or less, more preferably 5 or less. Preferably, it is 4 or less, more preferably 3 or less, more preferably 2 or less, and still more preferably 1. This is because, as the base sequence of the mutant gene has fewer base deletions, substitutions or additions to the base sequence of the wild-type gene, it has a property closer to the gene encoding the base sequence of the wild-type gene. Because it will be.
　なお、この変異型遺伝子の塩基配列のコードするアミノ酸配列が、ヒト野生型ＥＸＴ１遺伝子のコードするアミノ酸配列に対して１または数個の置換がある場合には、アミノ酸側鎖の性質が保存されている別のアミノ酸に置換していることが好ましい。アミノ酸側鎖の性質についての説明は既に行ったので繰り返さない。 In addition, when the amino acid sequence encoded by the base sequence of the mutant gene has one or several substitutions with respect to the amino acid sequence encoded by the human wild type EXT1 gene, the properties of the amino acid side chain are preserved. It is preferably substituted with another amino acid. The description of the nature of the amino acid side chain has already been made and will not be repeated.
　また、ヒト野生型ＥＸＴ１遺伝子の変異型遺伝子は、ヒト野生型ＥＸＴ１遺伝子がコードする蛋白質のアミノ酸配列に対して８０％以上の相同性を有するアミノ酸配列をコードする基配列からなる変異型遺伝子であってもよい。ここで上記「８０％以上」は好ましくは８５％以上であり、より好ましくは９０％以上であり、さらに好ましくは９５％以上であり、最も好ましくは９８％以上である。なぜならば、異型遺伝子の塩基配列が、野生型の遺伝子の塩基配列に対して相同性が高いほど、野生型の遺伝子の塩基配列をコードする遺伝子に近い特性を有していることになるからである。 The mutant gene of the human wild type EXT1 gene is a mutant gene comprising a base sequence encoding an amino acid sequence having 80% or more homology to the amino acid sequence of the protein encoded by the human wild type EXT1 gene. May be. Here, “80% or more” is preferably 85% or more, more preferably 90% or more, further preferably 95% or more, and most preferably 98% or more. This is because the higher the homology between the base sequence of the atypical gene and the base sequence of the wild-type gene, the closer to the gene encoding the base sequence of the wild-type gene. is there.
　また、ヒト野生型ＥＸＴ１遺伝子の変異型遺伝子は、ヒト野生型ＥＸＴ１遺伝子の塩基配列に相補的な塩基配列に対してストリンジェントな条件でハイブリダイズする塩基配列からなる変異型遺伝子であってもよい。なぜなら、ストリンジェントな条件でハイブリダイズする塩基配列からなる変異型遺伝子は、野生型の遺伝子の塩基配列に対して相同性が高いため、野生型の遺伝子の塩基配列をコードする遺伝子に近い特性を有していることになるからである。 Moreover, the mutant gene of the human wild type EXT1 gene may be a mutant gene comprising a base sequence that hybridizes under stringent conditions to a base sequence complementary to the base sequence of the human wild type EXT1 gene. . This is because a mutant gene consisting of a base sequence that hybridizes under stringent conditions is highly homologous to the base sequence of the wild-type gene, and therefore has characteristics close to those of the gene encoding the base sequence of the wild-type gene. It is because it will have.
　＜ベクターを含む５－フルオロウラシル感受性増感剤＞
　本実施形態に係る５－フルオロウラシル感受性増感剤は、ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子からなる群から選ばれる１以上の遺伝子を発現するベクターを含む、増感剤である。この構成によれば、５－フルオロウラシルの抗腫瘍効果を増強する作用を有することが後述する実施例において実証されているＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子またはＥＸＴ１遺伝子のいずれかを発現するベクターを含むため、増感剤自体としても５－フルオロウラシルの抗腫瘍効果を増強する効果が得られる。 <5-Fluorouracil-sensitive sensitizer including vector>
The 5-fluorouracil-sensitive sensitizer according to this embodiment is a sensitizer including a vector that expresses one or more genes selected from the group consisting of PRAK2 gene, TGFBR2 gene, and EXT1 gene. According to this configuration, since the vector includes any one of the PRAK2 gene, the TGFBR2 gene, and the EXT1 gene, which has been demonstrated in Examples described later, to have an action of enhancing the antitumor effect of 5-fluorouracil, As the sensitizer itself, an effect of enhancing the antitumor effect of 5-fluorouracil can be obtained.
　具体的には、本実施形態に用いる発現ベクターは、ベクターと、そのベクターに作動可能に連結されてなるＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子からなる群から選ばれる１以上の遺伝子と、を備える。このベクターは、後述する実施例で示すように、ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子からなる群から選ばれる１以上の遺伝子（およびその遺伝子のコードする蛋白質）を好適に発現することが可能である。 Specifically, the expression vector used in this embodiment includes a vector and one or more genes selected from the group consisting of a PRAK2 gene, a TGFBR2 gene, and an EXT1 gene operably linked to the vector. This vector can suitably express one or more genes selected from the group consisting of PRAK2 gene, TGFBR2 gene, and EXT1 gene (and the protein encoded by the gene), as shown in the Examples described later. .
　（ｉ）アデノウイルスベクター
　ここで、このベクターとしては、特に限定されず、一般的な哺乳動物細胞に導入可能なベクターを利用可能であるが、例えば、後述する実施例で用いられているアデノウイルスベクターを好適に用いることができる。アデノウイルスは最もよく研究されているウイルスのひとつであり、現在汎用されているアデノウイルスベクターはＥ１遺伝子を欠損しているため、Ｅ１遺伝子を持続的に発現している特殊な細胞（２９３細胞）でのみ増殖することができ、通常の細胞には感染することは可能であるが増殖することはないため、アデノウイルスベクターは安全かつ高効率なベクターとして、遺伝子機能解析などの基礎研究から遺伝子治療の分野まで広く利用されている。アデノウイルスベクターを用いれば、以下のような利点が得られる。１）高力価のウイルスを得ることができる。１０８～１０９ＰＦＵ／ｍｌ程度のウイルス液を容易に得ることができる。さらに１０１１ＰＦＵ／ｍｌ程度まで濃縮することも可能である。そのため、接着性の細胞ではほぼ１００％の細胞に遺伝子導入することが可能である。２）広い動物種に用いることができる。３）増殖細胞だけではなく静止期の細胞にも感染・発現できる。４）神経系を含む多くの分化・未分化動物培養細胞をターゲットにすることができ、さらに動物個体への直接注入・投与による遺伝子発現が可能である。 (I) Adenovirus vector Here, the vector is not particularly limited, and a vector that can be introduced into a general mammalian cell can be used. For example, an adenovirus used in Examples described later is used. A vector can be preferably used. Adenovirus is one of the most well-studied viruses, and since the currently widely used adenovirus vector lacks the E1 gene, special cells that continuously express the E1 gene (293 cells) Adenovirus vector is a safe and highly efficient vector, and it is possible to infect normal cells. Widely used up to the field. Use of an adenovirus vector provides the following advantages. 1) A high titer virus can be obtained. A virus solution of about 10 8 to 10 9 PFU / ml can be easily obtained. Further, it can be concentrated to about 10 11 PFU / ml. Therefore, it is possible to introduce a gene into almost 100% of adherent cells. 2) Can be used for a wide range of animal species. 3) It can infect and express not only proliferating cells but also stationary cells. 4) Many differentiated and undifferentiated animal cultured cells including the nervous system can be targeted, and gene expression by direct injection / administration to individual animals is possible.
　組換えアデノウイルスを作成するには、「完全長ＤＮＡ導入法」あるいは「ＣＯＳ－ＴＰＣ法」のいずれの方法を用いても良い。完全長ＤＮＡ導入法とは、制限消化処理した組換えコスミドを２９３細胞にｔｒａｎｓｆｅｃｔｉｏｎすることにより、組換えアデノウイルスを取得する方法である。完全長ＤＮＡ導入法では、細胞内での相同組換えが必要ないため、親ウイルスの混入はなく、得られる組換えアデノウイルスのほとんどが目的ウイルスである。「ＣＯＳ－ＴＰＣ法」とは、組換えコスミド　およびＡｄｅｎｏｖｉｒｕｓ　ｇｅｎｏｍｅ　ＤＮＡ－ＴＰＣを２９３細胞にｃｏ－ｔｒａｎｓｆｅｃｔｉｏｎし、２９３細胞内でおこる相同組換えを利用することにより、組換えアデノウイルスを作製する方法である。Ａｄｅｎｏｖｉｒｕｓ　ｇｅｎｏｍｅ　ＤＮＡ－ＴＰＣには、本来アデノウイルスゲノムＤＮＡの両末端に結合している末端タンパク質（ＴＰ；　Ｔｅｒｍｉｎａｌ　Ｐｒｏｔｅｉｎ）が結合している。そのため、高効率に組換えアデノウイルスを作製することができ、ほぼ確実に目的ウイルスを得ることができる。 In order to prepare a recombinant adenovirus, any of the “full-length DNA introduction method” or the “COS-TPC method” may be used. The full-length DNA introduction method is a method for obtaining a recombinant adenovirus by transfecting a restriction digested recombinant cosmid into 293 cells. In the full-length DNA introduction method, homologous recombination in cells is not required, so there is no contamination with the parent virus, and most of the resulting recombinant adenovirus is the target virus. The “COS-TPC method” is a method for producing a recombinant adenovirus by co-transfecting a recombinant cosmid and an Adenovirus gene DNA-TPC into 293 cells and utilizing homologous recombination occurring in 293 cells. is there. Adenovirus genome DNA-TPC is bound to a terminal protein (TP) that is originally bound to both ends of adenovirus genomic DNA. Therefore, a recombinant adenovirus can be produced with high efficiency, and the target virus can be obtained almost certainly.
　＜蛋白質を含む増感剤＞
　本実施形態に係る５－フルオロウラシル感受性増感剤は、ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子からなる群から選ばれる１以上の遺伝子のコードする蛋白質を含む、増感剤である。この構成によれば、５－フルオロウラシルの抗腫瘍効果を増強する作用を有することが後述する実施例において実証されているＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子またはＥＸＴ１遺伝子のいずれかがコードする蛋白質を含むため、増感剤自体としても５－フルオロウラシルの抗腫瘍効果を増強する効果が得られる。 <Sensitizer containing protein>
The 5-fluorouracil-sensitive sensitizer according to the present embodiment is a sensitizer containing a protein encoded by one or more genes selected from the group consisting of PRAK2 gene, TGFBR2 gene and EXT1 gene. According to this configuration, since it includes a protein encoded by any one of PRKG2 gene, TGFBR2 gene or EXT1 gene, which has been demonstrated in Examples described later, to have an action of enhancing the antitumor effect of 5-fluorouracil, As the sensitizer itself, an effect of enhancing the antitumor effect of 5-fluorouracil can be obtained.
　このＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子またはＥＸＴ１遺伝子のいずれかがコードする蛋白質を得る場合には、その蛋白質は人工的に化学合成されてもよいが、細胞培養を用いる生産方法によって得ることが安価かつ大量に生産可能であるため好ましい。具体的には、ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子またはＥＸＴ１遺伝子のいずれかがコードする蛋白質を大量生産した上で分離・精製するために、上記のアデノウイルスベクターを感染させた哺乳動物細胞からＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子またはＥＸＴ１遺伝子のいずれかがコードする蛋白質を分離・精製するための一般的な方法を用いることができる。具体的には、ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子またはＥＸＴ１遺伝子のいずれかがコードする蛋白質を大量生産した大哺乳動物細胞を破壊後、通常用いられる分離精製手段を用いることにより行うことができる。哺乳動物細胞の破壊には、例えば、超音波処理、高圧ホモジナイザー処理、浸透圧ショック法などが好適に用いられる。ＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子またはＥＸＴ１遺伝子のいずれかがコードする蛋白質の分離精製手段には、例えば塩析、ゲルろ過法、イオン交換クロマトグラフィーなどの方法を適宜組み合わせて用いることができる。 When obtaining a protein encoded by any one of the PRAK2 gene, TGFBR2 gene, or EXT1 gene, the protein may be artificially chemically synthesized, but it is inexpensive and large-scale to obtain by a production method using cell culture. It is preferable because it can be produced. Specifically, the PRAKG2 gene, TGFBR2 gene, TGFBR2 gene, TGFBR2 gene, TGFBR2 gene, TGFBR2 gene, TGFBR2 gene, and EXT1 gene are mass-produced and separated and purified. A general method for separating and purifying a protein encoded by either the gene or the EXT1 gene can be used. Specifically, it can be carried out by disrupting large mammalian cells that have mass-produced a protein encoded by any of PRAK2 gene, TGFBR2 gene or EXT1 gene, and then using a commonly used separation and purification means. For destruction of mammalian cells, for example, ultrasonic treatment, high-pressure homogenizer treatment, osmotic shock method and the like are preferably used. As a means for separating and purifying a protein encoded by any of the RAKG2 gene, the TGFBR2 gene and the EXT1 gene, for example, methods such as salting out, gel filtration and ion exchange chromatography can be used in appropriate combination.
　（ｉ）ＰＲＡＫＧ２遺伝子のコードするＡＭＰ活性化蛋白質キナーゼ
　本実施形態の５－フルオロウラシル感受性増感剤に含まれるＰＲＡＫＧ２遺伝子のコードする蛋白質は、哺乳動物のＡＭＰ活性化蛋白質キナーゼγ２非触媒サブユニット（ｐｒｏｔｅｉｎ　ｋｉｎａｓｅ，ＡＭＰ－ａｃｔｉｖａｔｅｄ，ｇａｍｍａ　２　ｎｏｎ－ｃａｔａｌｙｔｉｃ　ｓｕｂｕｎｉｔ）であればよく、特に限定するものではないが、例えば、ヒト野生型ＡＭＰ活性化蛋白質キナーゼγ２非触媒サブユニット（５'－ＡＭＰ－ａｃｔｉｖａｔｅｄ　ｐｒｏｔｅｉｎ　ｋｉｎａｓｅ　ｓｕｂｕｎｉｔ　ｇａｍｍａ－２　ｉｓｏｆｏｒｍ　ａ［Ｈｏｍｏ　ｓａｐｉｅｎｓ］）などを好適に用いることができる。なぜなら、このヒト野生型ＡＭＰ活性化蛋白質キナーゼγ２非触媒サブユニットに５－フルオロウラシルの抗腫瘍効果を増強する効果があることは、後述の実施例で実証されているからである。なお、上記ＡＭＰ活性化蛋白質キナーゼγ２非触媒サブユニットに関するアミノ酸配列等の詳細は、Ｎａｔｉｏｎａｌ　Ｃｅｎｔｅｒ　ｆｏｒ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ　Ｉｎｆｏｒｍａｔｉｏｎ（ＮＣＢＩ）のデータベースであるＧｅｎＢａｎｋで確認することができる。 (I) AMP-activated protein kinase encoded by PRKG2 gene The protein encoded by the PRAK2 gene contained in the 5-fluorouracil-sensitive sensitizer of the present embodiment is a AMP-activated protein kinase γ2 non-catalytic subunit (protein) of mammals. Kinase, AMP-activated, gamma 2 non-catalytic subunit) is not particularly limited. For example, human wild-type AMP-activated protein kinase γ2 non-catalytic subunit (5′-AMP-activated protein kinase) Subunit gamma-2 isoform a [Homo sapiens]) can be preferably used. This is because the human wild-type AMP-activated protein kinase γ2 non-catalytic subunit has an effect of enhancing the antitumor effect of 5-fluorouracil, which is demonstrated in Examples described later. Details of the amino acid sequence and the like regarding the AMP-activated protein kinase γ2 non-catalytic subunit can be confirmed in GenBank, which is a database of National Center for Biotechnology Information (NCBI).
　もっとも、本実施形態の５－フルオロウラシル感受性増感剤に含まれるＡＭＰ活性化蛋白質キナーゼは、ヒト野生型ＡＭＰ活性化蛋白質キナーゼγ２非触媒サブユニットの変異型蛋白質であってもよい。なぜなら、ヒト野生型ＡＭＰ活性化蛋白質キナーゼγ２非触媒サブユニットとの相同性の高い蛋白質であれば、ヒト野生型ＡＭＰ活性化蛋白質キナーゼγ２非触媒サブユニットと同様に哺乳動物細胞における５－フルオロウラシル感受性を増強させる可能性が高いからである。ここで、変異型とは、個体間のアミノ酸配列の差異に起因するものを含む。 However, the AMP-activated protein kinase contained in the 5-fluorouracil-sensitive sensitizer of this embodiment may be a mutant protein of a human wild-type AMP-activated protein kinase γ2 non-catalytic subunit. This is because a protein having high homology with human wild-type AMP-activated protein kinase γ2 non-catalytic subunit is sensitive to 5-fluorouracil in mammalian cells in the same manner as human wild-type AMP-activated protein kinase γ2 non-catalytic subunit. This is because there is a high possibility of strengthening. Here, the mutant type includes those resulting from differences in amino acid sequences between individuals.
　具体的には、ヒト野生型ＡＭＰ活性化蛋白質キナーゼγ２非触媒サブユニットの変異型蛋白質は、ヒト野生型ＡＭＰ活性化蛋白質キナーゼγ２非触媒サブユニットのアミノ酸配列のうち１若しくは数個のアミノ酸残基を欠失・置換・付加してなるアミノ酸配列からなる変異型蛋白質であってもよい。また上記「１若しくは数個」は好ましくは３０個以下であり、より好ましくは２０個以下であり、より好ましくは１５個以下であり、より好ましくは１０個以下であり、より好ましくは５個以下であり、好ましくは４個以下であり、より好ましくは３個以下であり、より好ましくは２個以下であり、さらに好ましくは１個である。なぜならば、変異型蛋白質のアミノ酸配列が、野生型の蛋白質のアミノ酸配列に対してアミノ酸の欠失、置換若しくは付加が少ないほど、野生型のＡＭＰ活性化蛋白質キナーゼγ２非触媒サブユニットに近い特性を有していることになるからである。 Specifically, the mutant protein of the human wild-type AMP-activated protein kinase γ2 non-catalytic subunit is one or several amino acid residues in the amino acid sequence of the human wild-type AMP-activated protein kinase γ2 non-catalytic subunit. It may be a mutant protein consisting of an amino acid sequence obtained by deleting, substituting, or adding. The “one or several” is preferably 30 or less, more preferably 20 or less, more preferably 15 or less, more preferably 10 or less, more preferably 5 or less. Preferably, it is 4 or less, more preferably 3 or less, more preferably 2 or less, and still more preferably 1. Because the amino acid sequence of the mutant protein has fewer amino acid deletions, substitutions or additions to the amino acid sequence of the wild-type protein, it has a property closer to that of the wild-type AMP-activated protein kinase γ2 non-catalytic subunit. It is because it will have.
　なお、この変異型ＡＭＰ活性化蛋白質キナーゼγ２非触媒サブユニットのアミノ酸配列が、ヒト野生型ＡＭＰ活性化蛋白質キナーゼγ２非触媒サブユニットのアミノ酸配列に対して１または数個の置換がある場合には、アミノ酸側鎖の性質が保存されている別のアミノ酸に置換していることが好ましい。なお、アミノ酸側鎖の性質について既に説明したので繰り返さない。 When the amino acid sequence of the mutant AMP-activated protein kinase γ2 non-catalytic subunit has one or several substitutions with respect to the amino acid sequence of the human wild-type AMP-activated protein kinase γ2 non-catalytic subunit The amino acid side chain is preferably substituted with another amino acid having a conserved property. In addition, since it already demonstrated about the property of an amino acid side chain, it does not repeat.
　また、ヒト野生型ＡＭＰ活性化蛋白質キナーゼγ２非触媒サブユニットの変異型蛋白質は、ヒト野生型ＡＭＰ活性化蛋白質キナーゼγ２非触媒サブユニットのアミノ酸配列に対して８０％以上の相同性を有するアミノ酸配列からなる変異型蛋白質であってもよい。ここで上記「８０％以上」は好ましくは８５％以上であり、より好ましくは９０％以上であり、さらに好ましくは９５％以上であり、最も好ましくは９８％以上である。なぜならば、変異型ＡＭＰ活性化蛋白質キナーゼγ２非触媒サブユニットのアミノ酸配列が、野生型の蛋白質のアミノ酸配列に対して相同性が高いほど、野生型のＡＭＰ活性化蛋白質キナーゼγ２非触媒サブユニットに近い特性を有していることになるからである。 The mutant protein of the human wild-type AMP-activated protein kinase γ2 non-catalytic subunit is an amino acid sequence having 80% or more homology with the amino acid sequence of the human wild-type AMP-activated protein kinase γ2 non-catalytic subunit. A mutant protein consisting of Here, “80% or more” is preferably 85% or more, more preferably 90% or more, further preferably 95% or more, and most preferably 98% or more. This is because the higher the homology of the amino acid sequence of the mutant AMP-activated protein kinase γ2 non-catalytic subunit with the amino acid sequence of the wild-type protein, the more the wild-type AMP-activated protein kinase γ2 non-catalytic subunit becomes. This is because they have close characteristics.
　また、ヒト野生型ＡＭＰ活性化蛋白質キナーゼγ２非触媒サブユニットの変異型蛋白質は、ヒト野生型ＡＭＰ活性化蛋白質キナーゼγ２非触媒サブユニットをコードする遺伝子の塩基配列に相補的な塩基配列に対してストリンジェントな条件でハイブリダイズする塩基配列にコードされるアミノ酸配列からなる変異型蛋白質であってもよい。なぜなら、ストリンジェントな条件でハイブリダイズする塩基配列にコードされるアミノ酸配列からなる変異型ＡＭＰ活性化蛋白質キナーゼγ２非触媒サブユニットは、野生型の蛋白質のアミノ酸配列に対して相同性が高いため、野生型のＡＭＰ活性化蛋白質キナーゼγ２非触媒サブユニットに近い特性を有していることになるからである。 In addition, the mutant protein of the human wild-type AMP-activated protein kinase γ2 non-catalytic subunit has a base sequence complementary to the base sequence of the gene encoding the human wild-type AMP-activated protein kinase γ2 non-catalytic subunit. It may be a mutant protein consisting of an amino acid sequence encoded by a base sequence that hybridizes under stringent conditions. This is because a mutant AMP-activated protein kinase γ2 non-catalytic subunit consisting of an amino acid sequence encoded by a base sequence that hybridizes under stringent conditions is highly homologous to the amino acid sequence of a wild-type protein. This is because it has characteristics close to those of the wild-type AMP-activated protein kinase γ2 non-catalytic subunit.
　（ｉｉ）ＴＧＦＢＲ２遺伝子のコードするＴＧＦ－β２型受容体
　本実施形態の５－フルオロウラシル感受性増感剤に含まれるＴＧＦＢＲ２遺伝子のコードする蛋白質は、哺乳動物のＴＧＦ－β２型受容体（ｔｒａｎｓｆｏｒｍｉｎｇ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ，ｂｅｔａ　ｒｅｃｅｐｔｏｒ　ＩＩ）であればよく、特に限定するものではないが、例えば、ヒト野生型ＴＧＦ－β２型受容体（ｔｒａｎｓｆｏｒｍｉｎｇ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ，　ｂｅｔａ　ｒｅｃｅｐｔｏｒ　ＩＩ（７０／８０ｋＤａ）［Ｈｏｍｏ　ｓａｐｉｅｎｓ］）などを好適に用いることができる。なぜなら、このヒト野生型ＴＧＦ－β２型受容体に５－フルオロウラシルの抗腫瘍効果を増強する効果があることは、後述の実施例で実証されているからである。なお、上記ＴＧＦ－β２型受容体に関するアミノ酸配列等の詳細は、Ｎａｔｉｏｎａｌ　Ｃｅｎｔｅｒ　ｆｏｒ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ　Ｉｎｆｏｒｍａｔｉｏｎ（ＮＣＢＩ）のデータベースであるＧｅｎＢａｎｋで確認することができる。 (Ii) TGF-β2 type receptor encoded by TGFBR2 gene The protein encoded by the TGFBR2 gene contained in the 5-fluorouracil-sensitive sensitizer of the present embodiment is a mammalian growth factor, transforming growth factor, beta receptor II), and is not particularly limited. For example, human wild type TGF-β2 type receptor (transforming growth factor, beta receptor II (70/80 kDa) [Homo sapiens]) is preferably used. Can be used. This is because the human wild-type TGF-β2 type receptor has an effect of enhancing the antitumor effect of 5-fluorouracil, which has been demonstrated in Examples described later. The details of the amino acid sequence and the like regarding the TGF-β2 type receptor can be confirmed in GenBank which is a database of National Center for Biotechnology Information (NCBI).
　もっとも、本実施形態の５－フルオロウラシル感受性増感剤に含まれＴＧＦ－β２型受容体は、ヒト野生型ＴＧＦ－β２型受容体の変異型蛋白質であってもよい。なぜなら、ヒト野生型ＴＧＦ－β２型受容体との相同性の高い蛋白質であれば、ヒト野生型ＴＧＦ－β２型受容体と同様に哺乳動物細胞における５－フルオロウラシル感受性を増強させる可能性が高いからである。ここで、変異型とは、個体間のアミノ酸配列の差異に起因するものを含む。 However, the TGF-β2 type receptor contained in the 5-fluorouracil-sensitive sensitizer of the present embodiment may be a mutant protein of human wild type TGF-β2 type receptor. This is because a protein having high homology with human wild-type TGF-β2 type receptor is likely to enhance 5-fluorouracil sensitivity in mammalian cells in the same manner as human wild-type TGF-β2 type receptor. It is. Here, the mutant type includes those resulting from differences in amino acid sequences between individuals.
　具体的には、ヒト野生型ＴＧＦ－β２型受容体の変異型蛋白質は、ヒト野生型ＴＧＦ－β２型受容体のアミノ酸配列のうち１若しくは数個のアミノ酸残基を欠失・置換・付加してなるアミノ酸配列からなる変異型蛋白質であってもよい。また上記「１若しくは数個」は好ましくは３０個以下であり、より好ましくは２０個以下であり、より好ましくは１５個以下であり、より好ましくは１０個以下であり、より好ましくは５個以下であり、好ましくは４個以下であり、より好ましくは３個以下であり、より好ましくは２個以下であり、さらに好ましくは１個である。なぜならば、変異型蛋白質のアミノ酸配列が、野生型の蛋白質のアミノ酸配列に対してアミノ酸の欠失、置換若しくは付加が少ないほど、野生型のＴＧＦ－β２型受容体に近い特性を有していることになるからである。 Specifically, the mutant protein of the human wild type TGF-β2 type receptor deletes, substitutes, or adds one or several amino acid residues in the amino acid sequence of the human wild type TGF-β2 type receptor. A mutant protein consisting of the amino acid sequence may be used. The “one or several” is preferably 30 or less, more preferably 20 or less, more preferably 15 or less, more preferably 10 or less, more preferably 5 or less. Preferably, it is 4 or less, more preferably 3 or less, more preferably 2 or less, and still more preferably 1. This is because, as the amino acid sequence of the mutant protein has fewer amino acid deletions, substitutions or additions to the amino acid sequence of the wild-type protein, it has a property closer to that of the wild-type TGF-β2 type receptor. Because it will be.
　なお、この変異型ＴＧＦ－β２型受容体のアミノ酸配列が、ヒト野生型ＴＧＦ－β２型受容体のアミノ酸配列に対して１または数個の置換がある場合には、アミノ酸側鎖の性質が保存されている別のアミノ酸に置換していることが好ましい。なお、アミノ酸側鎖の性質について既に説明したので繰り返さない。 When the amino acid sequence of the mutant TGF-β2 type receptor has one or several substitutions with respect to the amino acid sequence of the human wild type TGF-β2 type receptor, the properties of the amino acid side chain are preserved. It is preferable to substitute another amino acid. In addition, since it already demonstrated about the property of an amino acid side chain, it does not repeat.
　また、ヒト野生型ＴＧＦ－β２型受容体の変異型蛋白質は、ヒト野生型ＴＧＦ－β２型受容体のアミノ酸配列に対して８０％以上の相同性を有するアミノ酸配列からなる変異型蛋白質であってもよい。ここで上記「８０％以上」は好ましくは８５％以上であり、より好ましくは９０％以上であり、さらに好ましくは９５％以上であり、最も好ましくは９８％以上である。なぜならば、変異型ＴＧＦ－β２型受容体のアミノ酸配列が、野生型の蛋白質のアミノ酸配列に対して相同性が高いほど、野生型のＴＧＦ－β２型受容体に近い特性を有していることになるからである。 The human wild type TGF-β2 type receptor mutant protein is a mutant protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence of human wild type TGF-β2 type receptor. Also good. Here, “80% or more” is preferably 85% or more, more preferably 90% or more, further preferably 95% or more, and most preferably 98% or more. This is because the higher the homology of the amino acid sequence of the mutant TGF-β2 type receptor with the amino acid sequence of the wild type protein, the closer to the wild type TGF-β2 type receptor. Because it becomes.
　また、ヒト野生型ＴＧＦ－β２型受容体の変異型蛋白質は、ヒト野生型ＴＧＦ－β２型受容体をコードする遺伝子の塩基配列に相補的な塩基配列に対してストリンジェントな条件でハイブリダイズする塩基配列にコードされるアミノ酸配列からなる変異型蛋白質であってもよい。なぜなら、ストリンジェントな条件でハイブリダイズする塩基配列にコードされるアミノ酸配列からなる変異型ＴＧＦ－β２型受容体は、野生型の蛋白質のアミノ酸配列に対して相同性が高いため、野生型のＴＧＦ－β２型受容体に近い特性を有していることになるからである。 The mutant protein of the human wild type TGF-β2 type receptor hybridizes under stringent conditions to the base sequence complementary to the base sequence of the gene encoding the human wild type TGF-β2 type receptor. It may be a mutant protein consisting of an amino acid sequence encoded by a base sequence. This is because a mutant TGF-β2 type receptor consisting of an amino acid sequence encoded by a base sequence that hybridizes under stringent conditions has high homology to the amino acid sequence of a wild-type protein. This is because it has characteristics similar to those of -β2 type receptors.
　（ｉｉｉ）ＥＸＴ１遺伝子のコードする小胞体内在性ＩＩ型膜貫通糖転移酵素
　本実施形態の５－フルオロウラシル感受性増感剤に含まれるＥＸＴ１遺伝子のコードする蛋白質は、哺乳動物の小胞体内在性ＩＩ型膜貫通糖転移酵素（ｅｎｄｏｐｌａｓｍｉｃ　ｒｅｔｉｃｕｌｕｍ－ｒｅｓｉｄｅｎｔ　ｔｙｐｅ　ＩＩ　ｔｒａｎｓｍｅｍｂｒａｎｅ　ｇｌｙｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅ）であればよく、特に限定するものではないが、例えば、ヒト野生型小胞体内在性ＩＩ型膜貫通糖転移酵素（ＥＸＴ１　ｅｘｏｓｔｏｓｅｓ　（ｍｕｌｔｉｐｌｅ）１［Ｈｏｍｏ　ｓａｐｉｅｎｓ］）などを好適に用いることができる。なぜなら、このヒト野生型小胞体内在性ＩＩ型膜貫通糖転移酵素に５－フルオロウラシルの抗腫瘍効果を増強する効果があることは、後述の実施例で実証されているからである。なお、上記小胞体内在性ＩＩ型膜貫通糖転移酵素に関するアミノ酸配列等の詳細は、Ｎａｔｉｏｎａｌ　Ｃｅｎｔｅｒ　ｆｏｒ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ　Ｉｎｆｏｒｍａｔｉｏｎ（ＮＣＢＩ）のデータベースであるＧｅｎＢａｎｋで確認することができる。 (Iii) Endoplasmic reticulum type II transmembrane glycosyltransferase encoded by the EXT1 gene The protein encoded by the EXT1 gene contained in the 5-fluorouracil-sensitive sensitizer of the present embodiment is an endoplasmic reticulum type II in mammals. Any transmembrane glycosyltransferase (endoplasmic reticulum-resident type II transmembrane glycosyltransferase) may be used. For example, human wild-type endoplasmic reticulum transmembrane glycosyltransferase (EXT1) 1 [Homo sapiens]) or the like can be preferably used. This is because the human wild-type endoplasmic reticulum type II transmembrane glycosyltransferase has an effect of enhancing the antitumor effect of 5-fluorouracil as demonstrated in the examples described later. The details of the amino acid sequence and the like regarding the endoplasmic reticulum type II transmembrane glycosyltransferase can be confirmed in GenBank which is a database of National Center for Biotechnology Information (NCBI).
　なお、このＥＸＴ１遺伝子にコードされている哺乳動物の小胞体内在性ＩＩ型膜貫通糖転移酵素は、ヘパラン硫酸の生合成における糖鎖延長反応に関係している（ｉｎｖｏｌｖｅｄ　ｉｎ　ｔｈｅ　ｃｈａｉｎ　ｅｌｏｎｇａｔｉｏｎ　ｓｔｅｐ　ｏｆ　ｈｅｐａｒａｎ　ｓｕｌｆａｔｅ　ｂｉｏｓｙｎｔｈｅｓｉｓ）。 In addition, the mammalian endoplasmic reticulum type II transmembrane glycosyltransferase encoded by this EXT1 gene is related to a sugar chain elongation reaction in biosynthesis of heparan sulfate (involved in the chain elongation step of heparan). sulfate biosynthesis).
　もっとも、本実施形態の５－フルオロウラシル感受性増感剤に含まれる小胞体内在性ＩＩ型膜貫通糖転移酵素は、ヒト野生型小胞体内在性ＩＩ型膜貫通糖転移酵素の変異型蛋白質であってもよい。なぜなら、ヒト野生型小胞体内在性ＩＩ型膜貫通糖転移酵素との相同性の高い蛋白質であれば、ヒト野生型小胞体内在性ＩＩ型膜貫通糖転移酵素と同様に哺乳動物細胞における５－フルオロウラシル感受性を増強させる可能性が高いからである。ここで、変異型とは、個体間のアミノ酸配列の差異に起因するものを含む。 However, the endoplasmic reticulum type II transmembrane glycosyltransferase included in the 5-fluorouracil-sensitive sensitizer of the present embodiment is a mutant protein of human wild type endoplasmic reticulum type II transmembrane glycosyltransferase. Also good. This is because a protein having a high homology with human wild-type endoplasmic reticulum type II transmembrane glycosyltransferase is similar to human wild-type endoplasmic reticulum type II transmembrane glycosyltransferase in mammalian cells. This is because the possibility of enhancing sensitivity to fluorouracil is high. Here, the mutant type includes those resulting from differences in amino acid sequences between individuals.
　具体的には、ヒト野生型小胞体内在性ＩＩ型膜貫通糖転移酵素の変異型蛋白質は、ヒト野生型小胞体内在性ＩＩ型膜貫通糖転移酵素のアミノ酸配列のうち１若しくは数個のアミノ酸残基を欠失・置換・付加してなるアミノ酸配列からなる変異型蛋白質であってもよい。また上記「１若しくは数個」は好ましくは３０個以下であり、より好ましくは２０個以下であり、より好ましくは１５個以下であり、より好ましくは１０個以下であり、より好ましくは５個以下であり、好ましくは４個以下であり、より好ましくは３個以下であり、より好ましくは２個以下であり、さらに好ましくは１個である。なぜならば、変異型蛋白質のアミノ酸配列が、野生型の蛋白質のアミノ酸配列に対してアミノ酸の欠失、置換若しくは付加が少ないほど、野生型の小胞体内在性ＩＩ型膜貫通糖転移酵素に近い特性を有していることになるからである。 Specifically, the mutant protein of human wild type endoplasmic reticulum type II transmembrane glycosyltransferase is one or several amino acids of the amino acid sequence of human wild type endoplasmic reticulum type II transmembrane glycosyltransferase. It may be a mutant protein consisting of an amino acid sequence obtained by deleting, substituting, or adding residues. The “one or several” is preferably 30 or less, more preferably 20 or less, more preferably 15 or less, more preferably 10 or less, more preferably 5 or less. Preferably, it is 4 or less, more preferably 3 or less, more preferably 2 or less, and still more preferably 1. Because the amino acid sequence of the mutant protein has fewer amino acid deletions, substitutions or additions to the amino acid sequence of the wild-type protein, it is closer to the wild-type endoplasmic reticulum type II transmembrane glycosyltransferase. It is because it will have.
　なお、この変異型小胞体内在性ＩＩ型膜貫通糖転移酵素のアミノ酸配列が、ヒト野生型小胞体内在性ＩＩ型膜貫通糖転移酵素のアミノ酸配列に対して１または数個の置換がある場合には、アミノ酸側鎖の性質が保存されている別のアミノ酸に置換していることが好ましい。なお、アミノ酸側鎖の性質について既に説明したので繰り返さない。 When the amino acid sequence of the mutant endoplasmic reticulum type II transmembrane glycosyltransferase has one or several substitutions with respect to the amino acid sequence of the human wild type endoplasmic reticulum type II transmembrane glycosyltransferase In this case, the amino acid side chain is preferably substituted with another amino acid in which the nature of the amino acid side chain is conserved. In addition, since it already demonstrated about the property of an amino acid side chain, it does not repeat.
　また、ヒト野生型小胞体内在性ＩＩ型膜貫通糖転移酵素の変異型蛋白質は、ヒト野生型小胞体内在性ＩＩ型膜貫通糖転移酵素のアミノ酸配列に対して８０％以上の相同性を有するアミノ酸配列からなる変異型蛋白質であってもよい。ここで上記「８０％以上」は好ましくは８５％以上であり、より好ましくは９０％以上であり、さらに好ましくは９５％以上であり、最も好ましくは９８％以上である。なぜならば、変異型小胞体内在性ＩＩ型膜貫通糖転移酵素のアミノ酸配列が、野生型の蛋白質のアミノ酸配列に対して相同性が高いほど、野生型の小胞体内在性ＩＩ型膜貫通糖転移酵素に近い特性を有していることになるからである。 Moreover, the mutant protein of human wild-type endoplasmic reticulum type II transmembrane glycosyltransferase has 80% or more homology with the amino acid sequence of human wild-type endoplasmic reticulum type II transmembrane glycosyltransferase. It may be a mutant protein consisting of an amino acid sequence. Here, “80% or more” is preferably 85% or more, more preferably 90% or more, further preferably 95% or more, and most preferably 98% or more. This is because the higher the homology of the amino acid sequence of the mutant endoplasmic reticulum type II transmembrane glycosyltransferase with the amino acid sequence of the wild type protein, the wild type endoplasmic reticulum type II transmembrane glycosyltransferase This is because it has characteristics close to those of enzymes.
　また、ヒト野生型小胞体内在性ＩＩ型膜貫通糖転移酵素の変異型蛋白質は、ヒト野生型小胞体内在性ＩＩ型膜貫通糖転移酵素をコードする遺伝子の塩基配列に相補的な塩基配列に対してストリンジェントな条件でハイブリダイズする塩基配列にコードされるアミノ酸配列からなる変異型蛋白質であってもよい。なぜなら、ストリンジェントな条件でハイブリダイズする塩基配列にコードされるアミノ酸配列からなる変異型小胞体内在性ＩＩ型膜貫通糖転移酵素は、野生型の蛋白質のアミノ酸配列に対して相同性が高いため、野生型の小胞体内在性ＩＩ型膜貫通糖転移酵素に近い特性を有していることになるからである。 The mutant protein of human wild-type endoplasmic reticulum type II transmembrane glycosyltransferase has a nucleotide sequence complementary to the base sequence of the gene encoding human wild-type endoplasmic reticulum type II transmembrane glycosyltransferase. Alternatively, it may be a mutant protein comprising an amino acid sequence encoded by a base sequence that hybridizes under stringent conditions. This is because a mutant endoplasmic reticulum type II transmembrane glycosyltransferase consisting of an amino acid sequence encoded by a base sequence that hybridizes under stringent conditions is highly homologous to the amino acid sequence of a wild-type protein. This is because it has characteristics similar to wild-type endoplasmic reticulum type II transmembrane glycosyltransferase.
　＜５－フルオロウラシル／インターフェロンαへの感受性増感剤＞
　これまで説明してきた上記の実施形態の増感剤は、いずれも５－フルオロウラシルおよびインターフェロンαの併用時における５－フルオロウラシルおよびインターフェロンαへの感受性を増強する、増感剤としても活用できる。後述する実施例で、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子には、５－フルオロウラシル／インターフェロンαの併用時における抗腫瘍作用を増強する効果があることが実証されているため、当業者であればＰＲＡＫＧ２遺伝子にも５－フルオロウラシル／インターフェロンαの併用時における抗腫瘍作用を増強する効果があると想定できるためである。 <5-fluorouracil / sensitizer for interferon α>
The sensitizers of the above-described embodiments described so far can be used as sensitizers that enhance the sensitivity to 5-fluorouracil and interferon α when 5-fluorouracil and interferon α are used in combination. In the examples described below, it has been demonstrated that the TGFBR2 gene and the EXT1 gene have an effect of enhancing the antitumor action when 5-fluorouracil / interferon α is used in combination. This is because it can be assumed that there is an effect of enhancing the antitumor action when 5-fluorouracil / interferon α is used in combination.
　また、後述する実施例で、ＴＧＦＢＲ２遺伝子のコードするＴＧＦ－β２型受容体およびＥＸＴ１遺伝子のコードする小胞体内在性ＩＩ型膜貫通糖転移酵素には、５－フルオロウラシル／インターフェロンαの併用時における抗腫瘍作用を増強する効果があることが実証されているため、当業者であればＰＲＡＫＧ２遺伝子のコードするＡＭＰ活性化蛋白質キナーゼγ２非触媒サブユニットにも５－フルオロウラシル／インターフェロンαの併用時における抗腫瘍作用を増強する効果があると想定できるためである。 In the examples described later, the TGF-β2 type receptor encoded by the TGFBR2 gene and the endoplasmic reticulum type II transmembrane glycosyltransferase encoded by the EXT1 gene have anti-antibody activity in combination with 5-fluorouracil / interferon α. Since it has been demonstrated that there is an effect of enhancing the tumor action, those skilled in the art will be able to use anti-tumor in combination with 5-fluorouracil / interferon α in the AMP-activated protein kinase γ2 non-catalytic subunit encoded by the PRAKG2 gene. This is because it can be assumed that the effect is enhanced.
　＜投与形態および剤形＞
　これまで説明してきた上記の実施形態の増感剤を生体に投与する際の投与経路は、治療に際して最も効果的なものを使用するのが好ましく、経口投与、または口腔内、気道内、直腸内、皮下、筋肉内、眼内および静脈内などの非経口投与をあげることができ、全身または局部的に投与することができる。投与経路は、好ましくは静脈内投与をあげることができる。また、投与経路は、５－フルオロウラシルまたはインターフェロンαの投与経路と同一である必要はない。 <Dosage form and dosage form>
As the administration route when administering the sensitizer of the above-described embodiment described above to the living body, the most effective route for treatment is preferably used. Oral administration, or intraoral, respiratory tract, intrarectal Parenteral administration, such as subcutaneous, intramuscular, intraocular and intravenous, can be given and can be administered systemically or locally. The administration route is preferably intravenous administration. Also, the route of administration need not be the same as the route of administration of 5-fluorouracil or interferon alpha.
　投与形態としては、噴霧剤、カプセル剤、錠剤、顆粒剤、シロップ剤、乳剤、座剤、注射剤、軟膏、テープ剤などがあげられる。経口投与に適当な製剤としては、乳剤、シロップ剤、カプセル剤、錠剤、散剤、顆粒剤などがあげられる。乳剤およびシロップ剤のような液体調製物は、水、ショ糖、ソルビトール、果糖などの糖類、ポリエチレングリコール、プロピレングリコールなどのグリコール類、ゴマ油、オリーブ油、大豆油などの油類、ｐ－ヒドロキシ安息香酸エステル類などの防腐剤、ストロベリーフレーバー、ペパーミントなどのフレーバー類などを添加剤として用いて製造できる。さらに、カプセル剤、錠剤、散剤、顆粒剤などは、乳糖、ブドウ糖、ショ糖、マンニトールなどの賦形剤、デンプン、アルギン酸ナトリウムなどの崩壊剤、ステアリン酸マグネシウム、タルクなどの滑沢剤、ポリビニルアルコール、ヒドロキシプロピルセルロース、ゼラチンなどの結合剤、脂肪酸エステルなどの界面活性剤、グリセリンなどの可塑剤などを添加剤として用いて製造できる。 Administration forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like. Suitable formulations for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like. Liquid preparations such as emulsions and syrups include sugars such as water, sucrose, sorbitol, fructose, glycols such as polyethylene glycol, propylene glycol, oils such as sesame oil, olive oil, soybean oil, p-hydroxybenzoic acid Preservatives such as esters, and flavors such as strawberry flavor and peppermint can be used as additives. In addition, capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl alcohol , Hydroxypropylcellulose, gelatin and other binders, surfactants such as fatty acid esters, plasticizers such as glycerin and the like can be used as additives.
　非経口投与に適当な製剤としては、注射剤、座剤、噴霧剤などがあげられる。注射用の水溶液としては、例えば生理食塩水、ブドウ糖やその他の補助薬を含む等張液、例えばＤ－ソルビトール、Ｄ－マンノース、Ｄ－マンニトール、塩化ナトリウムが挙げられ、適当な溶解補助剤、例えばアルコール、具体的にはエタノール、ポリアルコール、例えばプロピレングリコール、ポリエチレングリコール、非イオン性界面活性剤、例えばポリソルベート８０（ＴＭ）、ＨＣＯ－５０と併用してもよい。座剤はカカオ脂、水素化脂肪またはカルボン酸などの担体を用いて調製される。また、噴霧剤は、受容者の口腔および気道粘膜を刺激せず、かつこの増感剤を微細な粒子として分散させ吸収を容易にさせる担体などを用いて調製される。この担体としては具体的には乳糖、グリセリンなどが例示できる。このインテグリンα８β１およびリガンドの結合阻害剤と、用いる担体の性質により、エアロゾル、ライパウダーなどの製剤化が可能である。また、これらの非経口剤においても経口剤で添加剤として例示した成分を添加することもできる。 Preparations suitable for parenteral administration include injections, suppositories, sprays and the like. Aqueous solutions for injection include, for example, isotonic solutions containing physiological saline, glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol and sodium chloride. Suitable solubilizers such as Alcohols, specifically ethanol, polyalcohols such as propylene glycol, polyethylene glycol, nonionic surfactants such as polysorbate 80 (TM), HCO-50 may be used in combination. Suppositories are prepared using a carrier such as cocoa butter, hydrogenated fat or carboxylic acid. The spray is prepared using a carrier that does not irritate the recipient's oral cavity and airway mucosa, and that facilitates absorption by dispersing the sensitizer as fine particles. Specific examples of the carrier include lactose and glycerin. Depending on the properties of the integrin α8β1 and ligand binding inhibitor and the carrier used, it is possible to formulate aerosols, rye powders, and the like. In these parenteral preparations, the components exemplified as additives for oral preparations can also be added.
　また、上記増感剤は、緩衝剤（例えば、リン酸塩緩衝液、酢酸ナトリウム緩衝液）、無痛化剤（例えば、塩化ベンザルコニウム、塩酸プロカインなど）、安定剤（例えば、ヒト血清アルブミン、ポリエチレングリコールなど）、保存剤（例えば、ベンジルアルコール、フェノールなど）、酸化防止剤などと配合してもよい。調製された注射液は通常、適当なアンプルに充填される。このようにして得られる製剤は安全で低毒性であるので、例えば、ヒトや哺乳動物（例えば、ラット、マウス、ウサギ、ヒツジ、ブタ、ウシ、ネコ、イヌ、サルなど）に対して投与することができる。 The sensitizer includes a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (eg, human serum albumin, Polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants and the like. The prepared injection solution is usually filled in a suitable ampoule. Since the preparation thus obtained is safe and has low toxicity, it can be administered to, for example, humans and mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). Can do.
　また、投与方法は患者の年齢、症状、対象臓器等などにより適宜選択することができる。この増感剤の投与量としては、例えば、一回につき体重１ｋｇあたり０．０００１ｍｇから１０００ｍｇの範囲で選ぶことが可能である。あるいは、例えば、患者あたり０．００１～１０００００ｍｇ／ｂｏｄｙの範囲で投与量を選ぶことができるが、これらの数値に必ずしも制限されるものではない。目的とする治療効果、投与方法、治療期間、年齢、体重、併用する抗癌剤の種類および量などにより異なる。投与量、投与方法は、患者の体重や年齢、症状などにより変動するが、当業者であれば適宜選択することが可能である。また、別の適切な化学療法薬と併用で投与してもよい。 In addition, the administration method can be appropriately selected depending on the patient's age, symptoms, target organs, and the like. The dose of this sensitizer can be selected, for example, in the range of 0.0001 mg to 1000 mg per kg of body weight at a time. Alternatively, for example, the dose can be selected in the range of 0.001 to 100,000 mg / body per patient, but is not necessarily limited to these values. It varies depending on the intended therapeutic effect, administration method, treatment period, age, body weight, type and amount of anticancer agent used in combination. The dose and administration method vary depending on the weight, age, symptoms, etc. of the patient, but can be appropriately selected by those skilled in the art. It may also be administered in combination with another appropriate chemotherapeutic agent.
　＜抗癌剤キット＞
　本実施形態の抗癌剤キットは、これまで説明してきた上記の実施形態の増感剤と、５－フルオロウラシルと、を含む、キットである。この構成によれば、５－フルオロウラシルの抗腫瘍効果を増強する作用を有することが後述する実施例において実証されているＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子またはＥＸＴ１遺伝子（またはこれら遺伝子のコードする蛋白質）を用いた増感剤を含むため、５－フルオロウラシルの抗腫瘍効果が増強されて優れた抗腫瘍効果が得られる。 <Anticancer agent kit>
The anticancer agent kit of this embodiment is a kit comprising the sensitizer of the above-described embodiment described above and 5-fluorouracil. According to this configuration, the PRAK2 gene, the TGFBR2 gene, or the EXT1 gene (or a protein encoded by these genes), which has been demonstrated in Examples described later, to have an action of enhancing the antitumor effect of 5-fluorouracil, was used. Since it contains a sensitizer, the antitumor effect of 5-fluorouracil is enhanced and an excellent antitumor effect can be obtained.
　ここで、本明細書において、「５－フルオロウラシル」（５－ｆｌｕｏｒｏｕｒａｃｉｌ）とは、フッ化ピリミジン系の代謝拮抗剤であり、抗悪性腫瘍薬（抗がん剤）として用いられる化合物を含む。この化合物は、ウラシルの５位水素原子がフッ素原子に置き換わった構造をしている。この化合物は、１９５６年にＤｕｓｈｉｎｓｋｙらによって合成され、その後Ｈｅｉｄｅｌｂｅｒｇｅｒらを中心として基礎および臨床にわたる広範な研究で抗悪性腫瘍剤としての評価が確立された。代表商品としては、「５－ＦＵ協和」（協和発酵キリン）シリーズが市販されている。 Here, in this specification, “5-fluorouracil” is a fluoropyrimidine-based antimetabolite and includes a compound used as an antineoplastic agent (anticancer agent). This compound has a structure in which the 5-position hydrogen atom of uracil is replaced with a fluorine atom. This compound was synthesized in 1956 by Dushinsky et al., And was later established as an anti-neoplastic agent in a wide range of basic and clinical studies centered on Heidelberger et al. As a representative product, “5-FU Kyowa” (Kyowa Hakko Kirin) series is commercially available.
　また、本実施形態に係る抗癌剤キットは、これまで説明してきた上記の実施形態の増感剤と、５－フルオロウラシルと、にくわえて、さらにインターフェロンαを含む、キットであってもよい。後述する実施例で、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子には、５－フルオロウラシル／インターフェロンαの併用時における抗腫瘍作用を増強する効果があることが実証されているため、当業者であればＰＲＡＫＧ２遺伝子にも５－フルオロウラシル／インターフェロンαの併用時における抗腫瘍作用を増強する効果があると想定できるためである。 In addition, the anticancer agent kit according to the present embodiment may be a kit further containing interferon α in addition to the sensitizer of the above-described embodiment described above and 5-fluorouracil. In the examples described below, it has been demonstrated that the TGFBR2 gene and the EXT1 gene have an effect of enhancing the antitumor action when 5-fluorouracil / interferon α is used in combination. This is because it can be assumed that there is an effect of enhancing the antitumor action when 5-fluorouracil / interferon α is used in combination.
　また、後述する実施例で、ＴＧＦＢＲ２遺伝子のコードするＴＧＦ－β２型受容体およびＥＸＴ１遺伝子のコードする小胞体内在性ＩＩ型膜貫通糖転移酵素には、５－フルオロウラシル／インターフェロンαの併用時における抗腫瘍作用を増強する効果があることが実証されているため、当業者であればＰＲＡＫＧ２遺伝子のコードするＡＭＰ活性化蛋白質キナーゼγ２非触媒サブユニットにも５－フルオロウラシル／インターフェロンαの併用時における抗腫瘍作用を増強する効果があると想定できるためである。 In the examples described later, the TGF-β2 type receptor encoded by the TGFBR2 gene and the endoplasmic reticulum type II transmembrane glycosyltransferase encoded by the EXT1 gene have anti-antibody activity in combination with 5-fluorouracil / interferon α. Since it has been demonstrated that there is an effect of enhancing the tumor action, those skilled in the art will be able to use anti-tumor in combination with 5-fluorouracil / interferon α in the AMP-activated protein kinase γ2 non-catalytic subunit encoded by the PRAKG2 gene. This is because it can be assumed that the effect is enhanced.
　ここで、本明細書において、「インターフェロン」（Ｉｎｔｅｒｆｅｒｏｎ、略号：ＩＦＮ）とは、動物体内で病原体（特にウイルス）や腫瘍細胞などの異物の侵入に反応して細胞が分泌する蛋白質を含む。この蛋白質は、ウイルス増殖の阻止や細胞増殖の抑制、免疫系および炎症の調節などの働きをする。また、この蛋白質は、サイトカインの一種である。この蛋白質は、医薬品としてはＣ型肝炎・多発性骨髄腫等の悪性腫瘍の治療に用いられている。 Here, in this specification, “interferon” (abbreviation: IFN) includes a protein secreted by cells in response to the invasion of foreign substances such as pathogens (particularly viruses) and tumor cells. This protein acts to stop viral growth, inhibit cell growth, regulate the immune system and inflammation. This protein is a kind of cytokine. This protein is used as a pharmaceutical for the treatment of malignant tumors such as hepatitis C and multiple myeloma.
　また、本明細書において、「インターフェロンα」（Ｉｎｔｅｒｆｅｒｏｎ－α、略号：ＩＦＮ－α）とは、ＩＦＮ－α：１３種類（１，２，４，５，６，７，８，１０，１３，１４，１６，１７，２１）を含む。ＩＦＮαを用いた医薬品としては、例えば、スミフェロン（Ｒ）：腎癌・多発性骨髄腫・慢性骨髄性白血病・ヘアリー細胞白血病・亜急性硬化性全脳炎・ＨＴＬＶ－１脊髄症・Ｂ型肝炎・Ｃ型肝炎、オーアイエフ（Ｒ）：慢性骨髄性白血病・Ｂ型肝炎・Ｃ型肝炎などが市販されている。なお、本明細書において、「インターフェロンα」には、ＩＦＮα２ｂを用いた市販薬であるイントロンＡ（Ｒ）：Ｂ型肝炎・Ｃ型肝炎、ＰＥＧ－ＩＦＮα２ａを用いた市販薬であるペガシス（Ｒ）：Ｃ型肝炎、ＰＥＧ－ＩＦＮα２ｂを用いた市販薬であるペグイントロン（Ｒ）：Ｃ型肝炎、Ｃ－ＩＦＮαを用いた市販薬であるアドバフェロン（Ｒ）：Ｃ型肝炎なども含まれる。 In the present specification, “Interferon α” (abbreviation: IFN-α) means IFN-α: 13 types (1, 2, 4, 5, 6, 7, 8, 10, 13, 14, 16, 17, 21). Examples of pharmaceuticals using IFNα include: Sumiferon (R): renal cancer, multiple myeloma, chronic myelogenous leukemia, hairy cell leukemia, subacute sclerosing panencephalitis, HTLV-1 myelopathy, hepatitis B, C Hepatitis B, OH (R): Chronic myeloid leukemia, hepatitis B, hepatitis C and the like are commercially available. In this specification, “interferon α” includes intron A (R), which is a commercially available drug using IFNα2b: hepatitis B / hepatitis C, pegasis (R), which is a commercially available drug using PEG-IFNα2a. : Hepatitis C, a commercially available drug using PEG-IFNα2b, Pegintron (R): Hepatitis C, Advaferon (R), a commercially available drug using C-IFNα: Hepatitis C and the like are also included.
　＜５－フルオロウラシル感受性抑制剤＞
　本実施形態に係る５－フルオロウラシル感受性抑制剤は、ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子からなる群から選ばれる１以上の遺伝子の発現を抑制するｓｉＲＮＡ、ｓｈＲＮＡまたはアンチセンスＲＮＡを含む、抑制剤である。この構成によれば、５－フルオロウラシルの抗腫瘍効果を増強する作用を有することが後述する実施例において実証されているＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子またはＥＸＴ１遺伝子のいずれかの発現を抑制するｓｉＲＮＡ、ｓｈＲＮＡまたはアンチセンスＲＮＡを含むため、抑制剤自体としても５－フルオロウラシルの抗腫瘍効果を抑制する効果が得られる。 <5-Fluorouracil sensitivity inhibitor>
The 5-fluorouracil sensitivity inhibitor according to this embodiment is an inhibitor comprising siRNA, shRNA, or antisense RNA that suppresses the expression of one or more genes selected from the group consisting of PRAK2 gene, TGFBR2 gene, and EXT1 gene . According to this configuration, siRNA, shRNA, or the like that suppresses the expression of any of PRAK2 gene, TGFBR2 gene, or EXT1 gene, which has been demonstrated in Examples to be described later, has an action of enhancing the antitumor effect of 5-fluorouracil. Since the antisense RNA is contained, the inhibitor itself can suppress the antitumor effect of 5-fluorouracil.
　本実施形態に係る５－フルオロウラシル感受性抑制剤を用いれば、例えば経口または静脈注射などで全身または広範囲に５－フルオロウラシルを投与した場合に、癌巣が存在しない部位に注射剤などで５－フルオロウラシル感受性抑制剤を注入することができる。このようにすれば、癌巣が存在しない部位において５－フルオロウラシルによる深刻な副作用が発生する可能性を低減することができる。また、癌巣が存在しない部位に注射剤などで５－フルオロウラシル感受性抑制剤を注入し、一方で癌巣には注射剤などで５－フルオロウラシル感受性増感剤を注入すれば、癌巣が存在しない部位において５－フルオロウラシルによる深刻な副作用が発生する可能性を低減しつつも、癌巣では５－フルオロウラシルによる抗腫瘍作用を増強することができる。 When the 5-fluorouracil sensitivity inhibitor according to the present embodiment is used, for example, when 5-fluorouracil is administered systemically or extensively by oral or intravenous injection or the like, the 5-fluorouracil sensitivity is detected by an injection or the like at a site where no cancer focus exists. Inhibitors can be injected. In this way, it is possible to reduce the possibility of serious side effects due to 5-fluorouracil occurring at sites where cancerous lesions do not exist. In addition, if a 5-fluorouracil sensitivity inhibitor is injected into the site where there is no cancer nest with an injection or the like, while a 5-fluorouracil sensitive sensitizer is injected into the cancer nest with an injection or the like, no cancer nest exists. While reducing the possibility of serious side effects due to 5-fluorouracil at the site, it is possible to enhance the antitumor effect of 5-fluorouracil in the cancer nest.
　ここで、この５－フルオロウラシル感受性抑制剤は、５－フルオロウラシルを投与した場合の癌細胞の死亡率を低下させる（生存率を向上させる）ものであればよく、特に限定するものではないが、例えば母集団が正規分布に従うと仮定できる場合にはパラメトリック検定であるスチューデントのｔ検定（Ｓｔｕｄｅｎｔ'ｓ　ｔ－ｔｅｓｔ）において有意差があれば好ましい。すなわち、スチューデントのｔ検定において片側検定でｐ＜０．０５となればよく、より好ましくは片側検定でｐ＜０．０３となればよく、最も好ましくは片側検定でｐ＜０．０１となればよい。なお、スチューデントのｔ検定は特に片側検定に限定するわけではなく、両側検定で行っても良い。さらに、母集団が正規分布に従うと仮定できない場合には、ノンパラメトリック検定として、マン・ホイットニーのＵ検定などを行って有意差の有無を検定しても良い。 Here, the 5-fluorouracil sensitivity inhibitor is not particularly limited as long as it reduces the mortality of cancer cells (improves the survival rate) when 5-fluorouracil is administered. When the population can be assumed to follow a normal distribution, it is preferable if there is a significant difference in Student's t-test, which is a parametric test. That is, in the Student's t-test, one-sided test should be p <0.05, more preferably one-sided test should be p <0.03, and most preferably one-sided test should be p <0.01. Good. The student t-test is not limited to a one-sided test, but may be a two-sided test. Further, when it cannot be assumed that the population follows a normal distribution, the presence or absence of a significant difference may be tested by performing a Mann-Whitney U test as a nonparametric test.
　なお、上記の抑制剤は、いずれも５－フルオロウラシルおよびインターフェロンαの併用時における５－フルオロウラシルおよびインターフェロンαへの感受性を抑制する、抑制剤としても活用できる。後述する実施例で、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子には、５－フルオロウラシル／インターフェロンαの併用時における抗腫瘍作用を増強する効果があることが実証されているため、当業者であればＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子の発現を抑制すれば、５－フルオロウラシル／インターフェロンαの併用時における抗腫瘍作用を抑制する効果があると想定できるためである。また、当業者であればＰＲＡＫＧ２遺伝子の発現を抑制すれば、５－フルオロウラシル／インターフェロンαの併用時における抗腫瘍作用を抑制する効果があることも同様に想定できるためである。 Note that any of the above inhibitors can also be used as an inhibitor that suppresses sensitivity to 5-fluorouracil and interferon α when 5-fluorouracil and interferon α are used in combination. In the examples described below, it has been demonstrated that the TGFBR2 gene and the EXT1 gene have an effect of enhancing the antitumor effect when 5-fluorouracil / interferon α is used in combination. Therefore, those skilled in the art will recognize the TGFBR2 gene and the EXT1 gene. This is because it can be assumed that if the expression of the gene is suppressed, there is an effect of suppressing the antitumor action when 5-fluorouracil / interferon α is used together. Further, it is also possible for those skilled in the art to similarly assume that suppressing the expression of the PRAK2 gene has an effect of suppressing the antitumor action when 5-fluorouracil / interferon α is used in combination.
　（ｉ）ｓｉＲＮＡ
　ｓｉＲＮＡ（ｓｍａｌｌ　ｉｎｔｅｒｆｅｒｉｎｇ　ＲＮＡ）とは２１－２３塩基対から成る低分子二本鎖ＲＮＡである。ｓｉＲＮＡはＲＮＡ干渉（ＲＮＡｉ）と呼ばれる現象に関与しており、ｍＲＮＡの破壊によって配列特異的に遺伝子の発現を抑制する。 (I) siRNA
siRNA (small interfering RNA) is a small double-stranded RNA consisting of 21-23 base pairs. siRNA is involved in a phenomenon called RNA interference (RNAi), and suppresses gene expression in a sequence-specific manner by destroying mRNA.
　そのため、本実施形態に係る５－フルオロウラシル感受性抑制剤は、ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子からなる群から選ばれる１以上の遺伝子の塩基配列の一部分に対応する２１－２３塩基対を含む二本鎖ＲＮＡからなるｓｉＲＮＡを含むことが好ましい。このような二本鎖ＲＮＡからなるｓｉＲＮＡであれば、ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子からなる群から選ばれる１以上の遺伝子の発現を効果的に抑制することができ、その結果５－フルオロウラシルの抗腫瘍効果を抑制する効果が得られる。 Therefore, the 5-fluorouracil sensitivity-suppressing agent according to the present embodiment includes two containing 21-23 base pairs corresponding to a part of the base sequence of one or more genes selected from the group consisting of PRAK2 gene, TGFBR2 gene and EXT1 gene. It is preferable that siRNA which consists of strand RNA is included. Such siRNA consisting of double-stranded RNA can effectively suppress the expression of one or more genes selected from the group consisting of PRAK2 gene, TGFBR2 gene and EXT1 gene. As a result, 5-fluorouracil An effect of suppressing the antitumor effect is obtained.
　（ｉｉ）ｓｈＲＮＡ
　ｓｈＲＮＡ（ｓｍａｌｌ　ｈａｉｒｐｉｎ　ＲＮＡ）とは２１－２３塩基対から成る低分子二本鎖ＲＮＡが１つのループ構造を介して一本鎖としてつながった形のＲＮＡ分子である。ｓｈＲＮＡもＲＮＡ干渉（ＲＮＡｉ）と呼ばれる現象に関与しており、ｍＲＮＡの破壊によって配列特異的に遺伝子の発現を抑制する。 (Ii) shRNA
shRNA (small hairpin RNA) is an RNA molecule in which a small double-stranded RNA consisting of 21-23 base pairs is connected as a single strand via a single loop structure. shRNA is also involved in a phenomenon called RNA interference (RNAi), and suppresses gene expression in a sequence-specific manner by destroying mRNA.
　そのため、本実施形態に係る５－フルオロウラシル感受性抑制剤は、ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子からなる群から選ばれる１以上の遺伝子の塩基配列の一部分に対応する２１－２３塩基対を含む二本鎖ＲＮＡが１つのループ構造を介して一本鎖としてつながった形のｓｈＲＮＡを含むことが好ましい。このようなヘアピン構造を有するｓｈＲＮＡであれば、ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子からなる群から選ばれる１以上の遺伝子の発現を効果的に抑制することができ、その結果５－フルオロウラシルの抗腫瘍効果を抑制する効果が得られる。 Therefore, the 5-fluorouracil sensitivity-suppressing agent according to the present embodiment includes two containing 21-23 base pairs corresponding to a part of the base sequence of one or more genes selected from the group consisting of PRAK2 gene, TGFBR2 gene and EXT1 gene. It is preferable that the stranded RNA includes a form of shRNA connected as a single strand via a single loop structure. Such a shRNA having a hairpin structure can effectively suppress the expression of one or more genes selected from the group consisting of PRAK2 gene, TGFBR2 gene and EXT1 gene, and as a result, anti-tumor of 5-fluorouracil An effect of suppressing the effect is obtained.
　（ｉｉｉ）アンチセンスＲＮＡ（アンチセンスＤＮＡ）
　アンチセンスＲＮＡとは、標的ｍＲＮＡに相補的な配列を持つアンチセンスＲＮＡである。このように標的ｍＲＮＡに相補的な配列を持つアンチセンスＲＮＡを、標的ｍＲＮＡにハイブリダイズさせると、標的ｍＲＮＡが翻訳されることを妨げるので、標的ｍＲＮＡの発現を抑制することになる。なお、同様の現象は、標的ｍＲＮＡに相補的な配列を持つアンチセンスＤＮＡでも実現可能である。 (Iii) Antisense RNA (antisense DNA)
Antisense RNA is antisense RNA having a sequence complementary to a target mRNA. Thus, when antisense RNA having a sequence complementary to the target mRNA is hybridized to the target mRNA, the target mRNA is prevented from being translated, and thus the expression of the target mRNA is suppressed. The same phenomenon can be realized with antisense DNA having a sequence complementary to the target mRNA.
　そのため、本実施形態に係る５－フルオロウラシル感受性抑制剤は、ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子からなる群から選ばれる１以上の遺伝子の塩基配列の少なくとも一部分（全部でもよい）に対応するアンチセンスＲＮＡを含むことが好ましい。このようなアンチセンスＲＮＡであれば、ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子からなる群から選ばれる１以上の遺伝子の発現を効果的に抑制することができ、その結果５－フルオロウラシルの抗腫瘍効果を抑制する効果が得られる。なお、アンチセンスＲＮＡの代わりにアンチセンスＤＮＡを用いても良い。 Therefore, the 5-fluorouracil sensitivity inhibitor according to the present embodiment is an antisense RNA corresponding to at least a part (or all) of the base sequence of one or more genes selected from the group consisting of PRAK2 gene, TGFBR2 gene, and EXT1 gene. It is preferable to contain. Such an antisense RNA can effectively suppress the expression of one or more genes selected from the group consisting of PRAK2 gene, TGFBR2 gene and EXT1 gene, and as a result, the antitumor effect of 5-fluorouracil can be reduced. The effect of suppressing is acquired. An antisense DNA may be used instead of the antisense RNA.
　＜診断薬＞
　本実施形態に係る５－フルオロウラシル感受性を判定するための診断薬は、ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子からなる群から選ばれる１以上の遺伝子の発現量を測定するための試薬を含む、診断薬である。この構成によれば、５－フルオロウラシルの抗腫瘍効果を増強する作用を有することが後述する実施例において実証されているＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子またはＥＸＴ１遺伝子のいずれかの発現量を測定するための試薬を含むため、被験者の生体内におけるＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子またはＥＸＴ１遺伝子のいずれかの発現量を測定することで、被験者が５－フルオロウラシルの抗腫瘍効果が効きやすい体質であるかどうか判定するための診断薬として利用できる。 <Diagnostics>
The diagnostic agent for determining 5-fluorouracil sensitivity according to the present embodiment includes a reagent for measuring the expression level of one or more genes selected from the group consisting of PRAKG2 gene, TGFBR2 gene and EXT1 gene It is. According to this configuration, a reagent for measuring the expression level of any of PRAK2 gene, TGFBR2 gene, or EXT1 gene, which has been demonstrated in Examples described later, to have an action of enhancing the antitumor effect of 5-fluorouracil Therefore, by measuring the expression level of any one of the PRAK2 gene, TGFBR2 gene or EXT1 gene in the subject's living body, it is possible to determine whether or not the subject is likely to be effective in the anti-tumor effect of 5-fluorouracil. It can be used as a diagnostic agent.
　ここで、診断薬の使用方法は特に限定しないが、例えば、被験者の生体内における細胞、血液、血清、体液、または病理切片等と、標準的な細胞等において、ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子またはＥＸＴ１遺伝子のいずれかの発現量を検査、比較することで診断が可能である。例えば、これらの発現量が所定の閾値を超えていれば、被験者が５－フルオロウラシルの抗腫瘍効果が効きやすい体質であると判定することができる。一方、これらの発現量が所定の閾値を下回れば、被験者が５－フルオロウラシルの抗腫瘍効果が効きにくい体質であると判定することができる。この閾値は、例えば健常者の発現量の平均値の１．５倍以上に設定してもよく、２倍以上に設定してもよく、５倍以上に設定してもよく、１０倍以上に設定してもよい。また、この閾値は、例えば健常者の発現量の平均値よりも標準偏差の２倍以上大きい場合に設定してもよく、標準偏差の５倍以上大きい場合に設定してもよく、標準偏差の１０倍以上大きい場合に設定してもよい。 Here, the method of using the diagnostic agent is not particularly limited. For example, the PRAK2 gene, the TGFBR2 gene, or the EXT1 gene in cells, blood, serum, body fluid, or pathological sections in a subject's living body, and standard cells. Diagnosis is possible by examining and comparing the expression level of any of the above. For example, if these expression levels exceed a predetermined threshold value, it can be determined that the subject has a constitution that allows the anti-tumor effect of 5-fluorouracil to be effective. On the other hand, if the expression level falls below a predetermined threshold, it can be determined that the subject has a constitution that makes it difficult for the antitumor effect of 5-fluorouracil to be effective. For example, this threshold value may be set to 1.5 times or more of the average expression level of healthy subjects, may be set to 2 times or more, may be set to 5 times or more, and may be set to 10 times or more. It may be set. In addition, this threshold value may be set, for example, when the standard deviation is twice or more larger than the average value of the expression level of healthy subjects, or may be set when the standard deviation is five times or more larger than the standard deviation. You may set when it is 10 times or more larger.
　あるいは、被験者の生体内におけるＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子またはＥＸＴ１遺伝子のいずれかの発現量が、例えば健常者の発現量に比べて有意差が認められるほど大きいかどうかで判定してもよい。有意差があるかどうかの判定としては、例えば母集団が正規分布に従うと仮定できる場合にはパラメトリック検定であるスチューデントのｔ検定（Ｓｔｕｄｅｎｔ'ｓ　ｔ－ｔｅｓｔ）において有意差があれば好ましい。すなわち、スチューデントのｔ検定において片側検定でｐ＜０．０５となればよく、より好ましくは片側検定でｐ＜０．０３となればよく、最も好ましくは片側検定でｐ＜０．０１となればよい。なお、スチューデントのｔ検定は特に片側検定に限定するわけではなく、両側検定で行っても良い。さらに、母集団が正規分布に従うと仮定できない場合には、ノンパラメトリック検定として、マン・ホイットニーのＵ検定などを行って有意差の有無を検定しても良い。 Alternatively, the determination may be made based on whether the expression level of any of PRAK2 gene, TGFBR2 gene, or EXT1 gene in the subject's living body is so large that a significant difference is recognized, for example, compared to the expression level of a healthy person. For the determination of whether there is a significant difference, for example, when it can be assumed that the population follows a normal distribution, it is preferable that there is a significant difference in Student's t-test, which is a parametric test. That is, in the Student's t-test, one-sided test should be p <0.05, more preferably one-sided test should be p <0.03, and most preferably one-sided test should be p <0.01. Good. The student t-test is not limited to a one-sided test, but may be a two-sided test. Further, when it cannot be assumed that the population follows a normal distribution, the presence or absence of a significant difference may be tested by performing a Mann-Whitney U test as a nonparametric test.
　なお、上記の診断薬は、いずれも被験者が５－フルオロウラシルおよびインターフェロンαの併用時における５－フルオロウラシルおよびインターフェロンの抗腫瘍効果が効きやすい体質であるかどうか判定するためのする、診断薬としても活用できる。後述する実施例で、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子には、５－フルオロウラシル／インターフェロンαの併用時における抗腫瘍作用を増強する効果があることが実証されているため、当業者であればＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子の発現量を測定すれば、５－フルオロウラシル／インターフェロンαの併用時における被験者の感受性を判定するために活用できると想定できるためである。また、当業者であればＰＲＡＫＧ２遺伝子の発現量を測定すれば、５－フルオロウラシル／インターフェロンαの併用時における被験者の感受性を判定するために活用できると想定できるためである。 All of the above diagnostic agents can also be used as diagnostic agents for determining whether a subject has a predisposition to the antitumor effect of 5-fluorouracil and interferon when 5-fluorouracil and interferon α are used in combination. it can. In the examples described below, it has been demonstrated that the TGFBR2 gene and the EXT1 gene have an effect of enhancing the antitumor effect when 5-fluorouracil / interferon α is used in combination. Therefore, those skilled in the art will recognize the TGFBR2 gene and the EXT1 gene. This is because it can be assumed that if the expression level of the gene is measured, it can be used to determine the sensitivity of the subject during the combined use of 5-fluorouracil / interferon α. Moreover, it is because a person skilled in the art can assume that if the expression level of the PRAK2 gene is measured, it can be used to determine the sensitivity of the subject at the time of the combined use of 5-fluorouracil / interferon α.
　被験者の生体内におけるＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子またはＥＸＴ１遺伝子のいずれかの発現量の測定のための試薬は、特に限定するものではないが、例えば、ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子の塩基配列に相補的な塩基配列を含むプローブを用いて測定することができる。この方法を用いれば、被験者の生体内におけるＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子またはＥＸＴ１遺伝子のいずれかのｍＲＮＡの発現量を好適に測定することができる。また、これらのプローブは、蛍光色素または放射性同位元素で標識されていることが好ましい。このように標識すれば簡便な方法で感度良くこれらのｍＲＮＡの発現量を測定することができる。 The reagent for measuring the expression level of any one of the PRAK2 gene, TGFBR2 gene, or EXT1 gene in the subject's living body is not particularly limited. For example, it is complementary to the base sequences of the PRAKG2, TGFBR2 gene, and EXT1 gene. It can be measured using a probe containing a typical base sequence. By using this method, the expression level of any mRNA of the PRAKG2 gene, TGFBR2 gene or EXT1 gene in the subject's living body can be suitably measured. These probes are preferably labeled with a fluorescent dye or a radioisotope. By labeling in this way, the expression level of these mRNAs can be measured with a simple method with high sensitivity.
　もっとも、被験者の生体内におけるＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子またはＥＸＴ１遺伝子のいずれかの発現量の測定のための試薬は、ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子からなる群から選ばれる１以上の遺伝子がコードする蛋白質の発現量を測定するための試薬であってもよい。具体的には、ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子からなる群から選ばれる１以上の遺伝子のコードする蛋白質に特異的に結合する抗体を好適に用いることができる。この方法を用いれば、被験者の生体内におけるＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子またはＥＸＴ１遺伝子のいずれかがコードする蛋白質の発現量を好適に測定することができる。また、これらの抗体は、蛍光色素または放射性同位元素で標識されていることが好ましい。このように標識すれば簡便な方法で感度良くこれらの蛋白質の発現量を測定することができる。 However, the reagent for measuring the expression level of any of PRAKG2 gene, TGFBR2 gene or EXT1 gene in the subject's living body is encoded by one or more genes selected from the group consisting of PRAKG2, TGFBR2 gene and EXT1 gene It may be a reagent for measuring the expression level of protein. Specifically, an antibody that specifically binds to a protein encoded by one or more genes selected from the group consisting of PRAKG2 gene, TGFBR2 gene, and EXT1 gene can be preferably used. By using this method, the expression level of the protein encoded by any of PRAKG2 gene, TGFBR2 gene or EXT1 gene in the body of the subject can be suitably measured. These antibodies are preferably labeled with a fluorescent dye or a radioisotope. By labeling in this way, the expression level of these proteins can be measured with a simple method with high sensitivity.
　＜スクリーニング方法＞
　本実施形態に係る抗癌剤の感受性増強作用を有する遺伝子のスクリーニング方法は、哺乳動物の所定の癌細胞株にリボザイムライブラリーを導入する工程と、リボザイムライブラリーが導入された癌細胞を所定の抗癌剤で処理する工程と、抗癌剤で処理された癌細胞のうち生存細胞からリボザイムを回収して新規なリボザイムライブラリーを得る工程と、を含む。すなわち、本実施形態に係る遺伝子のスクリーニング方法は、いわば「改変型ランダムリボザイム法」と言えるものである。 <Screening method>
The method for screening a gene having an effect of enhancing the sensitivity of an anticancer drug according to this embodiment comprises a step of introducing a ribozyme library into a predetermined cancer cell line of a mammal, and a cancer cell into which the ribozyme library has been introduced with a predetermined anticancer drug. And a step of collecting a ribozyme from a living cell among cancer cells treated with an anticancer agent to obtain a novel ribozyme library. That is, the gene screening method according to the present embodiment is a so-called “modified random ribozyme method”.
　この「改変型ランダムリボザイム法」では、基質認識部位をランダマイズしたリボザイムライブラリーを用いて、有用な遺伝子を機能の面から探索できる方法(ジーンディスカバリーシステム)である。この「改変型ランダムリボザイム法」の概要としては、リボザイムライブラリーを細胞内で機能させ、ある着目した細胞の表現型(癌細胞への抗癌剤の感受性が抑制されるなど)が現れた細胞を回収して、そこで発現しているリボザイムの標的認識配列を解析すると、その着目した表現型と細胞内でリボザイムに切断された遺伝子の配列との関係を知ることができる。 This “modified random ribozyme method” is a method (gene discovery system) in which useful genes can be searched in terms of function using a ribozyme library in which substrate recognition sites are randomized. The outline of this “modified random ribozyme method” is that a ribozyme library is made to function in the cell, and a cell in which a phenotype of a cell of interest (such as suppression of sensitivity of an anticancer drug to a cancer cell) appears is collected. Then, by analyzing the target recognition sequence of the ribozyme expressed there, it is possible to know the relationship between the focused phenotype and the sequence of the gene cleaved into the ribozyme in the cell.
　そのため、本実施形態に係るスクリーニング方法で用いるリボザイムライブラリーとしては、標的認識配列特異的にｍＲＮＡを切断し、遺伝子発現を抑制する複数のリボザイムを含み、各リボザイムが認識する標的認識配列がランダム化された複数の配列である、ランダム化リボザイムライブラリーを用いている。本実施形態に係るスクリーニング方法では、このようなランダム化リボザイムライブラリーを用いて、上記の工程を複数回繰り返して得られるリボザイムライブラリーが認識する標的認識配列を検出する工程と、こうして検出された標的認識配列に対応する遺伝子情報をゲノムデータベースから抽出する工程と、をさらに含むため、癌細胞への抗癌剤の感受性を抑制するリボザイムライブラリーが濃縮されることになる。そして、そのリボザイムライブラリーには、癌細胞への抗癌剤の感受性を増強する遺伝子を認識する標的認識配列が含まれているので、その標的認識配列に対応する遺伝子は、まさに、癌細胞への抗癌剤の感受性を増強する遺伝子であるということになるわけである。 Therefore, the ribozyme library used in the screening method according to this embodiment includes a plurality of ribozymes that specifically cleave mRNA and suppress gene expression, and target recognition sequences recognized by each ribozyme are randomized. A randomized ribozyme library, which is a plurality of sequences that have been designed, is used. In the screening method according to the present embodiment, using such a randomized ribozyme library, a step of detecting a target recognition sequence recognized by a ribozyme library obtained by repeating the above steps a plurality of times, and thus detected And a step of extracting gene information corresponding to the target recognition sequence from the genome database. Therefore, the ribozyme library that suppresses the sensitivity of the anticancer drug to cancer cells is concentrated. The ribozyme library contains a target recognition sequence that recognizes a gene that enhances the sensitivity of the anticancer drug to cancer cells. Therefore, the gene corresponding to the target recognition sequence is exactly the anticancer drug for cancer cells. It means that it is a gene that enhances susceptibility.
　なお、この「改変型ランダムリボザイム法」では、各リボザイムに標識された各標的認識配列に特異的な波長を有する蛍光色素を検出する形で各リボザイムを検出してもよい。このようにすれば、蛍光色素の波長を検出するだけで、各リボザイムに標識された各標的認識配列を同定することができ、簡単に感度良くスクリーニングを行うことができる。 In this “modified random ribozyme method”, each ribozyme may be detected by detecting a fluorescent dye having a wavelength specific to each target recognition sequence labeled on each ribozyme. In this way, each target recognition sequence labeled on each ribozyme can be identified by simply detecting the wavelength of the fluorescent dye, and screening can be performed easily with high sensitivity.
　以上、図面を参照して本発明の実施形態について述べたが、これらは本発明の例示であり、上記以外の様々な構成を採用することもできる。 As described above, the embodiments of the present invention have been described with reference to the drawings. However, these are exemplifications of the present invention, and various configurations other than the above can be adopted.
　例えば、上記実施の形態では、ヒト野生型ＰＲＡＫＧ２遺伝子、ヒト野生型ＴＧＦＢＲ２遺伝子およびヒト野生型ＥＸＴ１遺伝子、そしてそれらの変異型遺伝子を中心に説明したが、特にヒト由来遺伝子に限定する趣旨ではない。すなわち、ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子の由来は特に限定せず、例えば、ヒト、マウス、ラット、ウサギ、ブタ、ヒツジ、ウシ、ウマ、ネコ、イヌ、サル、またはチンパンジーであっても良い。好ましくは、マウス、ラット、サル、チンパンジー、およびヒトであり、特に好ましくはヒトである。なぜならば、ヒトであればヒトの疾患の治療や、治療薬等の開発に利用できるためである。また、マウス、ラット、サル、およびチンパンジーは、世界中で研究用のモデル動物として汎用され多くの特性が明らかになっているため、これらの生物の上記遺伝子の発現を制御することで、治療薬等の開発のために特に有用な情報が得られる。 For example, in the above embodiment, the human wild-type PRAK2 gene, the human wild-type TGFBR2 gene, the human wild-type EXT1 gene, and their mutant genes have been mainly described, but the present invention is not particularly limited to human-derived genes. That is, the origin of the PRAK2 gene, TGFBR2 gene, and EXT1 gene is not particularly limited, and may be, for example, human, mouse, rat, rabbit, pig, sheep, cow, horse, cat, dog, monkey, or chimpanzee. Preferred are mouse, rat, monkey, chimpanzee and human, and particularly preferred is human. This is because humans can be used to treat human diseases and develop therapeutic drugs. In addition, mice, rats, monkeys, and chimpanzees are widely used as model animals for research all over the world, and many characteristics have been clarified. Therefore, by controlling the expression of the above genes in these organisms, therapeutic drugs Especially useful information for the development of
　以下、本発明を実施例によりさらに説明するが、本発明はこれらに限定されるものではない。 Hereinafter, the present invention will be further described with reference to examples, but the present invention is not limited thereto.
　＜材料と方法＞
　（ｉ）使用した試薬
DMEM: Dulbecco's Modified Eagle's Medium粉末、10 mL 10% NaOHCO3、
5 mL 100×glucose、10 mL 50×glutamine
PBS（-）: 137 mM NaCl、8.10 mM Na2HPO4・12H2O、2.68 mM KCl、1.47 mM KH2PO4
Trypsin/EDTA solution: Nacalai Tesque、0.25% Trypsin、1 mM EDTA
IFN-α: 300万U/mL イントロンA（Schering-Plough Corporation）
5-FU: 協和発酵工業株式会社
TetraColor ONE: TetraColor ONE（SEIKAGAKU CORPORATION）
Solution I: 50 mM glucose、25 mM Tris pH8.0、10 mM EDTA
Solution II: 0.2 N NaOH、1% SDS
Solution III: 5 M acetic acid:5 M potassium acetate=2:3
フェノール/クロロホルム: TE飽和フェノール:クロロホルム:イソアミルアルコール
=25:24:1
LB培地: Nacalai Tesque
LB寒天培地: Lennox、Becton Dickinson
SOB（Mg-）培地: 10 g Bacto Tryptone、2.5 g Yeast Extract、5 mL 5% NaCl、5 mL 250 mM KCl
SOC培地: SOB（Mg-）50 mL、1 M MgCl2 500 mL、2 M MgSO4 250 μL、
1 M glucose 1 mL
RIPA Buffer: 50mM Tris-HCl（pH7.9）、150mM NaCl、1%NP-40、
0.5% Sodium Deoxycholate、0.1% SDS、
protease inhibitor（Complete: Roche）、
phosphatase inhibitor（Phos stop: Roche）
1次抗体: rabbit anti-V5 antibody（MBL）
2次抗体: horse radish peroxidase（HRP）標識 donkey anti-Rabbit IgG
（GEヘルスケア）
ウエスタン検出試薬: ECL Western Blotting Detection Reagents（GEヘルスケア） <Materials and methods>
(I) Reagent used
DMEM: Dulbecco's Modified Eagle's Medium powder, 10 mL 10% NaOHCO 3 ,
5 mL 100 × glucose, 10 mL 50 × glutamine
PBS (-): 137 mM NaCl, 8.10 mM Na 2 HPO 4 · 12H 2 O, 2.68 mM KCl, 1.47 mM KH 2 PO 4
Trypsin / EDTA solution: Nacalai Tesque, 0.25% Trypsin, 1 mM EDTA
IFN-α: 3 million U / mL Intron A (Schering-Plough Corporation)
5-FU: Kyowa Hakko Kogyo Co., Ltd.
TetraColor ONE: TetraColor ONE (SEIKAGAKU CORPORATION)
Solution I: 50 mM glucose, 25 mM Tris pH 8.0, 10 mM EDTA
Solution II: 0.2 N NaOH, 1% SDS
Solution III: 5 M acetic acid: 5 M potassium acetate = 2: 3
Phenol / chloroform: TE saturated phenol: chloroform: isoamyl alcohol
= 25: 24: 1
LB medium: Nacalai Tesque
LB agar: Lennox, Becton Dickinson
SOB (Mg-) medium: 10 g Bacto Tryptone, 2.5 g Yeast Extract, 5 mL 5% NaCl, 5 mL 250 mM KCl
SOC medium: SOB (Mg-) 50 mL, 1 M MgCl 2 500 mL, 2 M MgSO 4 250 μL,
1 M glucose 1 mL
RIPA Buffer: 50 mM Tris-HCl (pH 7.9), 150 mM NaCl, 1% NP-40,
0.5% Sodium Deoxycholate, 0.1% SDS,
protease inhibitor (Complete: Roche),
phosphatase inhibitor (Phos stop: Roche)
Primary antibody: rabbit anti-V5 antibody (MBL)
Secondary antibody: horse radish peroxidase (HRP) -labeled donkey anti-Rabbit IgG
(GE Healthcare)
Western Detection Reagents: ECL Western Blotting Detection Reagents (GE Healthcare)
　（ｉｉ）細胞培養
　ＨＣＣ細胞株は、１０ｃｍ細胞培養皿（ＦＡＬＣＯＮ）上にて、１０％ウシ胎児血清（ＥＱＵＩＴＥＣＨ－ＢＩＯ，ＩＮＣ）を含むＤＭＥＭを用いて、５％ＣＯ２、３７℃、１００％湿度下において培養した。７０～９０％コンフルエントになった状態でＴｒｙｐｓｉｎ／ＥＤＴＡ　Ｓｏｌｕｔｉｏｎ　２００μＬを加えて細胞を剥がし、１０００ｒｐｍ、５ｍｉｎ、４℃で遠心し細胞を回収、１ｄｉｓｈ分を４ｄｉｓｈに分けて継代した。 (Ii) Cell Culture The HCC cell line is 5% CO 2 , 37 ° C., 100% using DMEM containing 10% fetal calf serum (EQUITECH-BIO, INC) on a 10 cm cell culture dish (FALCON). Cultured under humidity. In a state of 70-90% confluence, 200 μL of Trypsin / EDTA Solution was added to detach the cells, and the cells were collected by centrifugation at 1000 rpm, 5 min, 4 ° C., and 1 dish portion was divided into 4 dishes and subcultured.
　（ｉｉｉ）ＷＳＴ　ａｓｓａｙ
　「細胞培養」に示した方法によって、７０～９０％コンフルエントになったＨＣＣ細胞株を回収し、９６穴プレート（ＦＡＬＣＯＮ）の各ｗｅｌｌに１００μＬずつ捲いた。２４時間後に５－ＦＵ及びＩＦＮ－αを加えて３７℃でインキュベートした。薬剤処理３日後に１０％　ＴｅｔｒａＣｏｌｏｒ　ＯＮＥ　１００μＬを加え、３７℃、２時間インキュベートし、９６穴用Ｍｉｃｒｏ　Ｐｌａｔｅ　Ｒｅａｄｅｒ（ＭＲＰ－Ａ４ｉ、ＴＯＳＯＨ）を用いて吸光度（測定波長４５０ｎｍ／対照波長６００ｎｍ）を測定した。測定結果は、試薬のみの吸光度との差をとり、細胞のみの吸光度とし、これを生細胞数とした。 (Iii) WST assay
The HCC cell line which became 70-90% confluent was collected by the method shown in “Cell culture”, and 100 μL was plated on each well of a 96-well plate (FALCON). After 24 hours, 5-FU and IFN-α were added and incubated at 37 ° C. Three days after drug treatment, 100 μL of 10% TetraColor ONE was added, incubated at 37 ° C. for 2 hours, and absorbance (measuring wavelength: 450 nm / control wavelength: 600 nm) was measured using a 96-well microplate reader (MRP-A4i, TOSOH). . The measurement result was the difference between the absorbance of the reagent alone and the absorbance of the cells alone, which was the number of living cells.
　（ｉｖ）アルカリ溶菌法
　３７℃、オーバーナイトで振盪培養した大腸菌液（ＤＨ５α）１．５ｍＬを１５，０００ｒｐｍ、５分、室温で遠心分離した。上清を除去し、ペレットに１００μＬ　Ｓｏｌｕｔｉｏｎ　Ｉを加えてボルテックスで攪拌した後、１５０μＬ　Ｓｏｌｕｔｉｏｎ　ＩＩを加えて転倒混和した。次に、２００μＬ　Ｓｏｌｕｔｉｏｎ　ＩＩＩを加えて転倒混和後、１５，０００ｒｐｍ、５分、４℃で遠心分離した。上清回収後、３００μＬフェノール／クロロホルムを加えてボルテックスで攪拌し、１５，０００ｒｐｍ、５分、４℃で遠心分離した。上清に１ｍＬ　１００％エタノールを加えて転倒混和した後、１５，０００ｒｐｍ、５分、４℃で遠心した。上清除去後１ｍＬ　７０％エタノールを加えて洗い、１５，０００ｒｐｍ、５分、４℃で遠心した。最後に上清を除き、風乾後に滅菌水５０μＬに溶かした。 (Iv) Alkaline lysis method 1.5 mL of Escherichia coli solution (DH5α) cultured with shaking at 37 ° C. overnight was centrifuged at 15,000 rpm for 5 minutes at room temperature. The supernatant was removed, 100 μL Solution I was added to the pellet and stirred by vortexing, and then 150 μL Solution II was added and mixed by inversion. Next, 200 μL Solution III was added and mixed by inversion, followed by centrifugation at 15,000 rpm for 5 minutes at 4 ° C. After recovering the supernatant, 300 μL phenol / chloroform was added, and the mixture was vortexed and centrifuged at 15,000 rpm for 5 minutes at 4 ° C. 1 mL 100% ethanol was added to the supernatant and mixed by inversion, followed by centrifugation at 15,000 rpm for 5 minutes at 4 ° C. After removing the supernatant, 1 mL 70% ethanol was added and washed, followed by centrifugation at 15,000 rpm for 5 minutes at 4 ° C. Finally, the supernatant was removed, air-dried and dissolved in 50 μL of sterilized water.
　（ｖ）ランダムリボザイムライブラリーの構築
　（ｖ－１）ａｔｔＢ１－ｔＲＮＡＶａｌ－ＲＲｚ－ａｔｔＢ２の構築
　以下の方法でｔＲＮＡＶａｌプロモーター下流にＲＲｚを挿入した（図１）。 (V) Construction of random ribozyme library (v-1) Construction of attB1-tRNAVal-RRz-attB2 RRz was inserted downstream of the tRNAVal promoter by the following method (FIG. 1).
　（ｖ－１－１）
　ランダムな塩基配列をそれぞれ８、７塩基有したＲＲｚ１、ＲＲｚ２プライマーを２０ｐｍｏｌずつ用いてＰＣＲによってアニーリングし、ＲＲｚ遺伝子を合成した（図１Ａ）。ＰＣＲにはＫＯＤ　Ｐｌｕｓ（ＴＯＹＯＢＯ）を使用し、９４℃２分で１サイクル、９４℃１５秒、６０℃３０秒、６８℃１０秒で２０サイクル行った。 (V-1-1)
RRz gene was synthesized by annealing by PCR using 20 pmol of RRz1 and RRz2 primers each having 8 or 7 random base sequences (FIG. 1A). For PCR, KOD Plus (TOYOBO) was used, and 1 cycle at 94 ° C. for 2 minutes, 20 cycles at 94 ° C. for 15 seconds, 60 ° C. for 30 seconds, and 68 ° C. for 10 seconds.
　（ｖ－１－２）
　合成したＲＲｚ遺伝子をテンプレートとして、ＲＲｚ３、ＲＲｚ４プライマー（表１）を３０ｐｍｏｌずつ用いてＰＣＲを行った。ＰＣＲにはＫＯＤ　Ｐｌｕｓ（ＴＯＹＯＢＯ）を使用し、９４℃２分で１サイクル、９４℃１５秒、６１℃３０秒、６８℃１０秒で２０サイクル行った。この反応によって下流にａｔｔＢ２配列を有するＲＲｚ遺伝子を合成した（図１Ａ）。 (V-1-2)
Using the synthesized RRz gene as a template, PCR was performed using 30 pmol of RRz3 and RRz4 primers (Table 1). For PCR, KOD Plus (TOYOBO) was used, and one cycle at 94 ° C. for 2 minutes, 20 cycles at 94 ° C. for 15 seconds, 61 ° C. for 30 seconds, and 68 ° C. for 10 seconds. By this reaction, an RRz gene having an attB2 sequence was synthesized downstream (FIG. 1A).
　（ｖ－１－３）
　１００ｐｇ　ｐｉＧＥＮＥ　ｔＲＮＡ　ｐｕｒ（ｉＧＥＮＥ　Ｔｈｅｒａｐｅｕｔｉｃｓ，　Ｉｎｃ．）をテンプレートとして、ＲＲｚ５、ＲＲｚ６プライマー（表１）を１０ｐｍｏｌずつ用いてＰＣＲを行った。ＰＣＲにはＫＯＤ　Ｐｌｕｓ（ＴＯＹＯＢＯ）を使用し、９４℃２分で１サイクル、９４℃１５秒、６０℃３０秒、６８℃１０秒で２６サイクル行った。この反応によって上流にａｔｔＢ１配列を有するｔＲＮＡＶａｌプロモーターを合成した（図１Ｂ）。 (V-1-3)
PCR was performed using 100 pg piGENE tRNA pur (iGEN Therapeutics, Inc.) as a template and 10 pmol each of RRz5 and RRz6 primers (Table 1). For PCR, KOD Plus (TOYOBO) was used, and 1 cycle at 94 ° C. for 2 minutes, 26 cycles at 94 ° C. for 15 seconds, 60 ° C. for 30 seconds, and 68 ° C. for 10 seconds. By this reaction, a tRNAVal promoter having an attB1 sequence upstream was synthesized (FIG. 1B).
　（ｖ－１－４）
　（ｖ－１－２）、（ｖ－１－３）の生成物をテンプレートに、ＲＲｚ４、ＲＲｚ５プライマー（表１）を１０ｐｍｏｌ用いてＰＣＲを行った。ＰＣＲにはＫＯＤ　Ｐｌｕｓ（ＴＯＹＯＢＯ）を使用し、９４℃２分で１サイクル、９４℃１５秒、６０℃３０秒、６８℃１０秒で２５サイクル行った。この反応によって上流にａｔｔＢ１配列、下流にａｔｔＢ２を有するｔＲＮＡＶａｌ－ＲＲｚを合成した（図１Ｃ）。 (V-1-4)
PCR was performed using the products of (v-1-2) and (v-1-3) as templates and 10 pmol of RRz4 and RRz5 primers (Table 1). For PCR, KOD Plus (TOYOBO) was used, and one cycle at 94 ° C. for 2 minutes, 25 cycles at 94 ° C. for 15 seconds, 60 ° C. for 30 seconds, and 68 ° C. for 10 seconds. By this reaction, tRNAVal-RRz having an attB1 sequence upstream and an attB2 downstream was synthesized (FIG. 1C).
　（ｖ－１－５）
　（ｖ－１－４）のＰＣＲ終了後、ＰＣＲ反応液を電気泳動し、ＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（ＱＩＡＧＥＮ）を用いて、ゲルからＤＮＡを抽出した。その後、ＮＡＰ－５　Ｃｏｌｕｍｎ（Ａｍｅｒｓｈａｍ　Ｂｉｏｓｃｉｅｎｃｅｓ）を用いて再度ＤＮＡを生成し、これをａｔｔＢ１－ｔＲＮＡＶａｌ－ＲＲｚ－ａｔｔＢ２とした。 (V-1-5)
After completion of PCR in (v-1-4), the PCR reaction solution was electrophoresed, and DNA was extracted from the gel using a QIAquick Gel Extraction Kit (QIAGEN). Thereafter, DNA was again generated using NAP-5 Column (Amersham Biosciences), and this was designated as attB1-tRNAVal-RRz-attB2.
　（ｖｉ）ｐＥＮＴＲ－ＲＲｚの構築
３０ｎｇ　ａｔｔＢ１－ｔＲＮＡＶａｌ－ＲＲｚ－ａｔｔＢ２、３００ｎｇ　ｐＤＯＮＲ２２１（Ｉｎｖｉｔｒｏｇｅｎ）、２μＬ　ＢＰ　Ｃｌｏｎａｓｅ　ＩＩ　Ｅｎｚｙｍｅ　Ｍｉｘ（Ｉｎｖｉｔｒｏｇｅｎ）を混合し、２５℃、１８時間反応させた。これによって、ａｔｔＢ１－ｔＲＮＡＶａｌ－ＲＲｚ－ａｔｔＢ２に含まれるａｔｔＢ１及びａｔｔＢ２とｐＤＯＮＲ２２１に含まれるａｔｔＰ１及びａｔｔＰ２との配列間で組換え反応を行った（図１Ｄ）。その後、２μｇ　ｐｒｏｔｅｉｎａｓｅ　Ｋを添加し、３７℃で１０分反応させた。この反応によって作成したａｔｔＢ１－ｔＲＮＡＶａｌ－ＲＲｚ－ａｔｔＢ２を有するプラスミドをｐＥＮＴＲ－ＲＲｚとした。 (Vi) Construction of pENTR-RRz 30 ng attB1-tRNAVal-RRz-attB2, 300 ng pDONR221 (Invitrogen), 2 μL BP Clonase II Enzyme Mix (Invitrogen) were mixed and reacted at 25 ° C. for 18 hours. Thereby, a recombination reaction was performed between the sequences of attB1 and attB2 contained in attB1-tRNAVal-RRz-attB2 and attP1 and attP2 contained in pDONR221 (FIG. 1D). Thereafter, 2 μg proteinase K was added and reacted at 37 ° C. for 10 minutes. A plasmid having attB1-tRNAVal-RRz-attB2 prepared by this reaction was designated as pENTR-RRz.
　（ｖｉｉ）ＲＲｚライブラリーの構築
　構築したｐＥＮＴＲ－ＲＲｚ　２μＬを２００μＬのｅｌｅｃｔｒｏｃｏｍｐｉｔｅｎｔ　Ｅ．　ｃｏｌｉに、エレクトロポレーション（２，５００Ｖ、２５μＦ、２０１ｋΩ）によって導入した。遺伝子導入後、ＳＯＣ培地を加え、２ｍＬの大腸菌液を３７℃で１時間振盪培養した。この大腸菌液のうち４μＬは５０倍希釈して、３０μｇ／ｍＬカナマイシンを加えたＬＢ寒天培地（Ｌｅｎｎｏｘ、Ｂｅｃｔｏｎ　Ｄｉｃｋｉｎｓｏｎ）にまき、３７℃で２４時間インキュベートし、コロニー数を数え、総量２ｍＬ中の大腸菌数を求めた。残りの大腸菌液は３，５００ｒｐｍ、１５分、４℃で遠心し、ペレットを２０％グリセロールを含む５００μＬのＬＢ培地に懸濁し、グリセロールストックとして－８０℃で保存した。この作業を大腸菌数の総計が約６００万個に達するまで繰り返し行った。その後、保存していた全てのグリセロールストックを３０μｇ／ｍＬカナマイシンを含むＬＢ培地１Ｌに加え、１２時間以上十分な濁度が得られるまで振盪培養した。この大腸菌よりＱＩＡＧＥＮ　Ｐｌａｓｍｉｄ　Ｍａｘｉ　Ｋｉｔ（ＱＩＡＧＥＮ）を用いてプラスミドＤＮＡを抽出した。抽出したプラスミドＤＮＡは１μｇ／μＬとなるようにＴＥ（１０ｍＭ　Ｔｒｉｓ、１ｍＭ　ＥＤＴＡ）バッファーに溶解し、これをＲＲｚライブラリーとした。 (Vii) Construction of RRz library 2 μL of the constructed pENTR-RRz was replaced with 200 μL of electrocompetent E. coli. E. coli was introduced by electroporation (2,500 V, 25 μF, 201 kΩ). After the gene introduction, SOC medium was added and 2 mL of E. coli solution was cultured with shaking at 37 ° C. for 1 hour. 4 μL of this E. coli solution was diluted 50-fold, seeded on LB agar medium (Lennox, Becton Dickinson) supplemented with 30 μg / mL kanamycin, incubated at 37 ° C. for 24 hours, counted for colony, and E. coli in 2 mL total volume. I asked for a number. The remaining E. coli solution was centrifuged at 3,500 rpm for 15 minutes at 4 ° C., the pellet was suspended in 500 μL of LB medium containing 20% glycerol, and stored at −80 ° C. as a glycerol stock. This operation was repeated until the total number of E. coli reached about 6 million. Thereafter, all the preserved glycerol stocks were added to 1 L of LB medium containing 30 μg / mL kanamycin, and cultured with shaking until sufficient turbidity was obtained for 12 hours or more. Plasmid DNA was extracted from this E. coli using QIAGEN Plasmid Maxi Kit (QIAGEN). The extracted plasmid DNA was dissolved in TE (10 mM Tris, 1 mM EDTA) buffer so as to be 1 μg / μL, and this was used as an RRz library.
　（ｖｉｉｉ）シークエンス解析
　構築したＲＲｚライブラリーから１００個のコロニーを選択し、アルカリ溶菌法によって、リボザイムプラスミドを回収した後、ＲＲｚ４、ＲＲｚ５プライマー（表１）を用いてリボザイム配列を調べた。解析には、Ｔｅｒｍｉｎａｔｏｒ　Ｒｅａｄｙ　Ｍｉｘを使用し、９５℃　１０秒、５０℃　５秒、６０℃　２分３０秒で４０サイクル反応させた。 (Viii) Sequence analysis After selecting 100 colonies from the constructed RRz library and recovering the ribozyme plasmid by the alkaline lysis method, the ribozyme sequence was examined using RRz4 and RRz5 primers (Table 1). For the analysis, Terminator Ready Mix was used, and 40 cycles were reacted at 95 ° C. for 10 seconds, 50 ° C. for 5 seconds, and 60 ° C. for 2 minutes 30 seconds.
　（ｉｘ）スクリーニング
　ＲＲｚライブラリーを、ＨｅｐＧ２に一過性に導入し、５μｇ／ｍＬ　５－ＦＵ処理を３日間行った。その後、細胞からプラスミドを回収し、再度ＨｅｐＧ２へと導入した。これを１サイクルとして１０回のサイクルを繰り返した（図２Ｂ）。 (Ix) Screening The RRz library was transiently introduced into HepG2 and treated with 5 μg / mL 5-FU for 3 days. Thereafter, the plasmid was recovered from the cells and again introduced into HepG2. This was regarded as one cycle and 10 cycles were repeated (FIG. 2B).
　（ｘ）プラスミドの導入
　６穴プレートに細胞を３．０×１０５ｃｅｌｌｓ／ｗｅｌｌずつ捲き、３７℃　ＣＯ２インキュベーター内で、２４時間インキュベートした。インキュベート後、５４μＬ　ＦｕＧＥＮＥ６（Ｒｏｃｈｅ）を使用し、２μｇのプラスミドＤＮＡを導入した。また、６ｃｍ細胞培養皿、９６穴プレートの場合は細胞数（５．０×１０３ｃｅｌｌｓ／ｗｅｌｌ、３．５×１０５ｃｅｌｌｓ／ｗｅｌｌ）、ＦｕＧＥＮＥ６量（０．０５μＬ、７μＬ）、プラスミドＤＮＡ量（０．１５μＬ、２μＬ）を変更して行った。 (X) Introduction of plasmid Cells were seeded in 6-well plates at 3.0 × 10 5 cells / well and incubated in a 37 ° C. CO 2 incubator for 24 hours. After incubation, 2 μg of plasmid DNA was introduced using 54 μL FuGENE6 (Roche). In the case of a 6 cm cell culture dish or a 96-well plate, the number of cells (5.0 × 10 3 cells / well, 3.5 × 10 5 cells / well), FuGENE6 amount (0.05 μL, 7 μL), plasmid DNA amount (0.15 μL, 2 μL) was changed.
　（ｘｉ）ｓｉＲＮＡの導入
　９６穴プレートに細胞を５．０×１０３ｃｅｌｌｓ／ｗｅｌｌずつ捲き、３７℃ＣＯ２インキュベーター内で、２４時間インキュベートした。インキュベート後、０．２μＬ　Ｌｉｐｏｆｅｃｔａｍｉｎ　２０００（Ｉｎｖｉｔｒｏｇｅｎ）を使用し、４ｐｍｏｌのｓｉＲＮＡを導入した。 (Xi) Introduction of siRNA Cells were seeded in 96-well plates at 5.0 × 10 3 cells / well and incubated in a 37 ° C. CO 2 incubator for 24 hours. After the incubation, 4 pmol of siRNA was introduced using 0.2 μL Lipofectamine 2000 (Invitrogen).
　（ｘｉｉ）Ｒｅｖｅｒｓｅ　Ｔｒａｎｓｃｒｉｐｔｉｏｎ－Ｐｏｌｙｍｅｒａｓｅ　Ｃｈａｉｎ　Ｒｅａｃｔｉｏｎ（ＲＴ－ＰＣＲ）
　ＴＲＩｚｏｌ　Ｒｅａｇｅｎｔ（Ｉｎｖｉｔｒｏｇｅｎ）を用いてＴｏｔａｌ　ＲＮＡを抽出した。ＲＴ反応には、ＳｕｐｅｒＳｃｒｉｐｔ　Ｆｉｒｓｔ－Ｓｔａｎｄ　Ｓｙｎｔｈｅｓｉｓ　Ｓｙｓｔｅｍ　ｆｏｒ　ＲＴ－ＰＣＲ（Ｉｎｖｉｔｒｏｇｅｎ）を使用した。０．５μｇのｔｏｔａｌ　ＲＮＡをテンプレートとして使用し、ｏｌｉｇｏ　ｄＴをプライマーとして、６５℃　５分で１サイクル、４２℃　１２０分、７０℃　１０分で１サイクル反応させ、ｃＤＮＡを合成した。反応後のｃＤＮＡは２０倍希釈してＲｅａｌ－Ｔｉｍｅ　ＰＣＲに用いた。 (Xii) Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total RNA was extracted using TRIzol Reagent (Invitrogen). In the RT reaction, SuperScript First-Stand Synthesis System for RT-PCR (Invitrogen) was used. Using 0.5 μg of total RNA as a template and oligo dT as a primer, cDNA was synthesized by reacting for 1 cycle at 65 ° C. for 5 minutes, 1 cycle at 42 ° C. for 120 minutes, and 70 ° C. for 10 minutes. The cDNA after the reaction was diluted 20 times and used for Real-Time PCR.
　（ｘｉｉｉ）Ｒｅａｌ　Ｔｉｍｅ　ＰＣＲ
　Ｒｅａｌ－Ｔｉｍｅ　ＰＣＲ反応には、ＬｉｇｈｔＣｙｃｌｅｒ　ＦａｓｔＳｔａｒｔ　ＤＮＡ　Ｍａｓｔｅｒ　ＳＹＢＲ　Ｇｒｅｅｎ　Ｉ（Ｒｏｃｈｅ）を使用した。ＲＴ－ＰＣＲによって合成したｃＤＮＡ　５μＬをテンプレートとして使用し、各遺伝子に対するプライマーを用いて反応させた。 (Xiii) Real Time PCR
For the Real-Time PCR reaction, LightCycler FastStart DNA Master SYBR Green I (Roche) was used. Using 5 μL of cDNA synthesized by RT-PCR as a template, the reaction was carried out using primers for each gene.
　（ｘｖ）Ｗｅｓｔｅｒｎ　Ｂｌｏｔ
　（ｘｖ－１）タンパク質回収
　ＨＣＣ細胞株（ＨＬＦ、ＨｕＨ７、ＨｅｐＧ２）を３．５×１０５ｃｅｌｌｓ／６ｃｍ－ｄｉｓｈ（Ｂｅｃｔｏｎ　Ｄｉｃｋｉｎｓｏｎ）ずつ捲き、２４時間後に遺伝子導入を行った。遺伝子導入４８時間後に１×ＰＢＳ（－）で２回ｗａｓｈし、ＲＩＰＡ　Ｂｕｆｆｅｒを加え、セルスクレイパー（住友ベークライト）で細胞を回収した。回収した細胞を１５，０００ｒｐｍ、１０分、４℃で遠心し、上清をｓｏｄｉｕｍ　ｄｏｄｅｃｙｌ　ｓｕｌｆａｔｅ－ｐｏｌｙａｃｒｙｌａｍｉｄｅ　ｇｅｌ　ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ（ＳＤＳ－ＰＡＧＥ）に使用した。 (Xv) Western Blot
(Xv-1) Protein recovery HCC cell lines (HLF, HuH7, HepG2) were seeded at 3.5 × 10 5 cells / 6 cm-dish (Becton Dickinson), and gene transfer was performed 24 hours later. 48 hours after gene introduction, the cells were washed twice with 1 × PBS (−), RIPA Buffer was added, and the cells were collected with a cell scraper (Sumitomo Bakelite). The collected cells were centrifuged at 15,000 rpm for 10 minutes at 4 ° C., and the supernatant was used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
　（ｘｖ－２）ＳＤＳ－ＰＡＧＥ
　回収した上清に含まれるタンパク質をＳＤＳ－ＰＡＧＥ（７．５％　ａｃｒｙｌａｍｉｄｅ）により分離した。泳動条件は、２０ｍＡで２時間行った。泳動後のゲルは、セミドライ式トランスファー装置を用いて、１００ｍＡ、１時間反応させ、ｐｏｌｙｖｉｎｙｌｉｄｅｎｅ　ｄｉｆｌｕｏｒｉｄｅメンブレンに転写した。 (Xv-2) SDS-PAGE
Proteins contained in the collected supernatant were separated by SDS-PAGE (7.5% acrylamide). The electrophoresis conditions were 20 mA for 2 hours. The gel after electrophoresis was reacted at 100 mA for 1 hour using a semi-dry transfer device and transferred to a polyvinylidene fluoride membrane.
　（ｘｖ－３）Ｗｅｓｔｅｒｎ　Ｂｌｏｔ
　５％スキムミルク（雪印）／ＴＢＳ－０．１％Ｔでブロッキングを行った後、抗Ｖ５抗体（１：３，０００）を用いて室温で１時間、１次抗体反応を行った。反応終了後、メンブレンをＴＢＳ－０．１％Ｔで３回ｗａｓｈした。次に、ＨＲＰ標識　ａｎｔｉ－ｒａｂｂｉｔ　ＩｇＧ抗体（１：５，０００）を使用して室温で１時間、２次抗体反応を行った。反応終了後、ＴＢＳ－０．１％Ｔで３回ｗａｓｈした。その後、ＥＣＬ　Ｗｅｓｔｅｒｎ　Ｂｌｏｔｔｉｎｇ　Ｄｅｔｅｃｔｉｏｎ　Ｒｅａｇｅｎｔｓ及びＬＡＳ－１０００　ｓｙｓｔｅｍ（ＦＵＪＩ　ＦＩＬＭ）を用いて、バンドを検出した。 (Xv-3) Western Blot
After blocking with 5% skim milk (snow mark) /TBS-0.1% T, a primary antibody reaction was performed with an anti-V5 antibody (1: 3,000) for 1 hour at room temperature. After completion of the reaction, the membrane was washed 3 times with TBS-0.1% T. Next, a secondary antibody reaction was performed for 1 hour at room temperature using an HRP-labeled anti-rabbit IgG antibody (1: 5,000). After completion of the reaction, washing was performed 3 times with TBS-0.1% T. Subsequently, bands were detected using ECL Western Blotting Detection Reagents and LAS-1000 system (FUJI FILM).
　＜実施例１：５－ＦＵ感受性の増感遺伝子のスクリーニング＞
　本実施例では、より奏功率の高い新規治療法開発によるＨＣＣ患者の長期予後改善を目的とし、高度進行型ＨＣＣ患者におけるＩＦＮ－α／５－ＦＵ併用療法感受性増強による治療効果増強を目指し、ＩＦＮ－α／５－ＦＵ併用療法感受性に寄与する遺伝子の同定を行った。その結果、複数の感受性増強遺伝子を同定した。 <Example 1: Screening for sensitizing genes sensitive to 5-FU>
In this example, with the aim of improving the long-term prognosis of HCC patients by developing a new treatment method with higher response rate, IFN-α / 5-FU combination therapy enhancement in IFN-α / 5-FU combination therapy in highly advanced HCC patients Identification of genes contributing to susceptibility to -α / 5-FU combination therapy was performed. As a result, a plurality of susceptibility enhancing genes were identified.
　感受性増強遺伝子のスクリーニングには、標的認識配列特異的にｍＲＮＡを切断し、遺伝子発現を抑制するＲＮＡ酵素であるリボザイム（図２Ａ）によるライブラリーを使用した。このライブラリーはリボザイムの認識配列をランダムに６００万通り有しており、網羅的な遺伝子発現抑制が可能である（図２Ｂ）。 In the screening for sensitivity-enhancing genes, a library of ribozymes (FIG. 2A), an RNA enzyme that cleaves mRNA specifically for the target recognition sequence and suppresses gene expression, was used. This library has 6 million ribozyme recognition sequences at random, and can comprehensively suppress gene expression (FIG. 2B).
　構築したライブラリーを用いて以下のスクリーニングを行った（図２Ｃ）。ヒトＨｅｐＧ２細胞株において、作製したリボザイムライブラリー導入後に５－ＦＵ単剤で処理を行い、５０％増殖抑制濃度での５－ＦＵへの抵抗性の増強を指標にスクリーニングを行った。３日間の薬剤処理後、生存細胞からリボザイムを回収し、ＨｅｐＧ２細胞への再導入を繰り返した。最終的に得られたリボザイムの認識配列から、ＢＬＡＳＴを用いて候補遺伝子を抽出した。同定した遺伝子については、アデノウイルスによる強制発現系を用いて、５－ＦＵ単独、及びＩＦＮ－α／５－ＦＵ感受性増強作用を検討した。１０回のスクリーニングを行い、５－ＦＵへの抵抗性は回を増す毎に増加した（図３）。 The following screening was performed using the constructed library (FIG. 2C). The human HepG2 cell line was treated with 5-FU alone after introduction of the prepared ribozyme library, and screening was performed using as an index the enhanced resistance to 5-FU at a 50% growth inhibitory concentration. After 3 days of drug treatment, ribozymes were collected from viable cells and reintroduced into HepG2 cells. Candidate genes were extracted from the finally obtained ribozyme recognition sequence using BLAST. The identified gene was examined for 5-FU alone and IFN-α / 5-FU sensitivity enhancement using an adenovirus forced expression system. Ten screenings were performed and 5-FU resistance increased with each increase (FIG. 3).
　このライブラリー中のリボザイムが有する標的認識配列をもとに、ＢＬＡＳＴによって５－ＦＵ感受性に寄与する遺伝子を５遺伝子（表１：ＰＲＫＡＧ２、ＴＧＦＢＲ２、ＥＸＴ１、ＰＯＬＲ４Ｊ２、ＦＯＸＰ２）に絞り込んだ。 Based on the target recognition sequence possessed by the ribozyme in this library, the genes contributing to 5-FU sensitivity were narrowed down to 5 genes (Table 1: PRKAG2, TGFBR2, EXT1, POLR4J2, FOXP2) by BLAST.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　このうち、偽遺伝子であるＰＯＬＲ２Ｊ４、および発現が確認できなかったＦＯＸＰ２を除外し、残りの３遺伝子（ＰＲＫＡＧ２、ＴＧＦＢＲ２、ＥＸＴ１）についてさらに検討した。 Of these, POLR2J4, which is a pseudogene, and FOXP2, whose expression could not be confirmed, were excluded, and the remaining three genes (PRKAG2, TGFBR2, EXT1) were further examined.
　＜実施例２：５－ＦＵ感受性の確認およびＩＦＮ－α／５－ＦＵ感受性の検討＞
　ＰＲＫＡＧ２、ＴＧＦＢＲ２、ＥＸＴ１のｓｉＲＮＡを作成した。 <Example 2: Confirmation of 5-FU sensitivity and examination of IFN-α / 5-FU sensitivity>
SiRNAs of PRKAG2, TGFBR2, and EXT1 were prepared.
　これらのｓｉＲＮＡを用いてＰＲＫＡＧ２、ＴＧＦＢＲ２、ＥＸＴ１の遺伝子発現を抑制すると、５－ＦＵに対する感受性が３遺伝子全てで有意に減少した（図４Ａ）。なぜなら、ＰＲＫＡＧ２、ＴＧＦＢＲ２、ＥＸＴ１を標的とするｓｉＲＮＡを投与すると、ＰＲＫＡＧ２、ＴＧＦＢＲ２、ＥＸＴ１の遺伝子発現が抑制されて、その結果５－ＦＵに対する感受性が抑制されるためである（図４Ｂ）。 When these siRNAs were used to suppress gene expression of PRKAG2, TGFBR2, and EXT1, the sensitivity to 5-FU was significantly reduced in all three genes (FIG. 4A). This is because administration of siRNA targeting PRKAG2, TGFBR2, and EXT1 suppresses gene expression of PRKAG2, TGFBR2, and EXT1, resulting in suppression of sensitivity to 5-FU (FIG. 4B).
　また、ＰＲＫＡＧ２、ＴＧＦＢＲ２、ＥＸＴ１の強制発現用のアデノウイルスベクターおよびコントロールアデノウイルスベクターをそれぞれ作成した（図５、図６）。これらのベクターを用いて３遺伝子の強制発現を行うことにより、ＨｅｐＧ２細胞中にＰＲＫＡＧ２、ＴＧＦＢＲ２、ＥＸＴ１の遺伝子がコードする蛋白質がそれぞれ過剰発現した（図７）。  Moreover, an adenovirus vector and a control adenovirus vector for forced expression of PRKAG2, TGFBR2, and EXT1 were prepared, respectively (FIGS. 5 and 6). By forcibly expressing 3 genes using these vectors, proteins encoded by the PRKAG2, TGFBR2, and EXT1 genes were overexpressed in HepG2 cells, respectively (FIG. 7).
　また、これらのベクターを用いて３遺伝子の強制発現を行うことにより、５－ＦＵへの感受性は有意に増加した（図８Ａ）。なぜなら、ＰＲＫＡＧ２、ＴＧＦＢＲ２、ＥＸＴ１を過剰発現するアデノウイルスベクターを投与すると、ＰＲＫＡＧ２、ＴＧＦＢＲ２、ＥＸＴ１の遺伝子発現が過剰発現されて、その結果５－ＦＵに対する感受性が増強されるためである（図４Ｂ）。さらに、これらのうち２遺伝子（ＴＧＦＢＲ２、ＥＸＴ１）はＩＦＮ－α／５－ＦＵ併用時においても、ＩＦＮ－α／５－ＦＵへの感受性を有意に増強した（図８Ｂ）。 Moreover, the sensitivity to 5-FU was significantly increased by forced expression of 3 genes using these vectors (FIG. 8A). This is because administration of an adenoviral vector that overexpresses PRKAG2, TGFBR2, and EXT1 results in overexpression of PRKAG2, TGFBR2, and EXT1, resulting in enhanced sensitivity to 5-FU (FIG. 4B). . Furthermore, two of these genes (TGFBR2, EXT1) significantly enhanced sensitivity to IFN-α / 5-FU even when IFN-α / 5-FU was used together (FIG. 8B).
　＜実施例３：ＩＦＮ－α／５－ＦＵ感受性のｉｎ　ｖｉｖｏでの確認＞
　以下の条件でマウスにＴＧＦＢＲ２の強制発現用のアデノウイルスベクターを腹腔内注射することによって投与し、ＩＦＮ－α／５－ＦＵの感受性の変化を観察した（図９）。 <Example 3: Confirmation of IFN-α / 5-FU sensitivity in vivo>
Mice were administered by intraperitoneal injection of an adenoviral vector for forced expression of TGFBR2 under the following conditions, and changes in the sensitivity of IFN-α / 5-FU were observed (FIG. 9).
Mouse: male BALB/cAJcl-nu/nu
Xenograft cell line: HepG2 (5×106 cells)
Drug concentration: 15 mg/kg/day 5-FU (i.p.)
10,000 units/body IFN-α (s.c.)
Administration time: 1 time/3 days
Tumor volume (TV) calculation: measure 1/week using calipers
(TV=Length×(Width)2/2)
Adenovirus: 2×108 pfu 1 time/5 days Mouse: male BALB / cAJcl-nu / nu
Xenograft cell line: HepG2 (5 × 106 cells)
Drug concentration: 15 mg / kg / day 5-FU (ip)
10,000 units / body IFN-α (sc)
Administration time: 1 time / 3 days
Tumor volume (TV) calculation: measure 1 / week using calipers
(TV = Length × (Width) 2/2)
Adenovirus: 2 × 108 pfu 1 time / 5 days
　＜実施例４：ＴＧＦＢＲ２，ＥＸＴ１過剰発現による５－ＦＵ及びＩＦＮ－α／５－ＦＵ誘導性アポトーシスの増強の確認＞
　（４－１）ヘキスト染色
　ＴＧＦＢＲ２，ＥＸＴ１過剰発現による５－ＦＵ及びＩＦＮ－α／５－ＦＵ誘導性アポトーシスの増強を確認するために、以下の手順でヘキスト染色を行った。まず、ＨｅｐＧ２へのアデノウイルス感染によるＬａｃＺ、ＴＧＦＢＲ２、ＥＸＴ１遺伝子過剰発現後、５μｇ／ｍＬ　５－ＦＵ及び２００Ｕ／ｍＬ　ＩＦＮ－α処理を行った。４８時間後、１０μＭ　Ｈｏｅｃｈｓｔ３３２５８を添加し、１時間後に蛍光顕微鏡による観察を行った。観察結果として、蛍光顕微鏡写真を図１０ａに示す。 <Example 4: Confirmation of enhancement of 5-FU and IFN-α / 5-FU-induced apoptosis by overexpression of TGFBR2, EXT1>
(4-1) Hoechst staining In order to confirm the enhancement of 5-FU and IFN-α / 5-FU-induced apoptosis by overexpression of TGFBR2 and EXT1, Hoechst staining was performed according to the following procedure. First, LacZ, TGFBR2, and EXT1 genes were overexpressed by adenovirus infection of HepG2, and then treated with 5 μg / mL 5-FU and 200 U / mL IFN-α. After 48 hours, 10 μM Hoechst 33258 was added, and after 1 hour, observation with a fluorescence microscope was performed. As an observation result, a fluorescence micrograph is shown in FIG.
　この実験では、ヘキスト染色でクロマチンの凝集の様子を観察し、クロマチンの凝集が見られればアポトーシスが亢進していると解釈する。図１０ａの実験結果を見ると、明らかに、５－Ｆｕ単独又はＩＦＮ－ａ／５－ＦＵを添加した上で、ＴＧＦＢＲ２又はＥＸＴ１を過剰発現させた場合には、クロマチンの凝集が見られるためアポトーシスが亢進していることが明らかである。 In this experiment, the state of chromatin aggregation is observed by Hoechst staining, and if chromatin aggregation is observed, it is interpreted that apoptosis is enhanced. From the experimental results shown in FIG. 10a, it is apparent that when TGFBR2 or EXT1 is overexpressed with addition of 5-Fu alone or IFN-a / 5-FU, aggregation of chromatin is observed and apoptosis occurs. It is clear that is enhanced.
　（４－２）カスパーゼ３／７活性化測定
　ＴＧＦＢＲ２，ＥＸＴ１過剰発現による５－ＦＵ及びＩＦＮ－α／５－ＦＵ誘導性アポトーシスの増強を確認するために、以下の手順でカスパーゼ３／７活性化測定を行った。まず、ＬａｃＺ、ＴＧＦＢＲ２、ＥＸＴ１発現細胞（ＨｅｐＧ２）に対して５μｇ／ｍＬ　５－ＦＵ及び２００Ｕ／ｍＬ　ＩＦＮ－α処理を行った。４８時間後にＣａｐｓａｐｓｅ－Ｇｌｏ３／７　ａｓｓａｙ　ｋｉｔ　（Ｐｒｏｍｅｇａ）付属のマニュアルに従い、カスパーゼ３及び７活性を測定した。測定結果をグラフにまとめて図１０ｂに示す。 (4-2) Measurement of caspase 3/7 activation To confirm the enhancement of 5-FU and IFN-α / 5-FU-induced apoptosis by overexpression of TGFBR2 and EXT1, caspase 3/7 activation was carried out by the following procedure. Measurements were made. First, LacZ, TGFBR2, and EXT1-expressing cells (HepG2) were treated with 5 μg / mL 5-FU and 200 U / mL IFN-α. After 48 hours, caspase 3 and 7 activities were measured according to the manual attached to Capsapse-Glo3 / 7 assay kit (Promega). The measurement results are summarized in a graph and shown in FIG.
　この実験では、アポトーシスを起こすカスパーゼカスケードにおけるエフェクターであるカスパーゼ３とカスパーゼ７の活性を測定し、カスパーゼ３とカスパーゼ７の活性が上昇すればアポトーシスが亢進していると解釈する。図１０ｂの実験結果を見ると、明らかに、５－Ｆｕ単独又はＩＦＮ－ａ／５－ＦＵを添加した上で、ＴＧＦＢＲ２又はＥＸＴ１を過剰発現させた場合には、カスパーゼ３とカスパーゼ７の活性が有意に上昇しているためアポトーシスが亢進していることが明らかである。 In this experiment, the activities of caspase 3 and caspase 7, which are effectors in the caspase cascade causing apoptosis, are measured, and if the activities of caspase 3 and caspase 7 increase, it is interpreted that apoptosis is enhanced. From the experimental results shown in FIG. 10b, it is clear that when TGFBR2 or EXT1 is overexpressed with addition of 5-Fu alone or IFN-a / 5-FU, the activities of caspase 3 and caspase 7 are increased. It is clear that apoptosis is enhanced due to the significant increase.
　なお、図１０の＊印は、ｔ検定（ｎ＝３）においてｐ＜０．０５（両側検定）であることを意味している。また、図１０の＋印は、対応するコントロールの測定結果に対してｔ検定（ｎ＝３）においてｐ＜０．０５（両側検定）であることを意味している。さらに、図１０の＋＋印は、対応するコントロールの測定結果に対してｔ検定（ｎ＝３）においてｐ＜０．０１（両側検定）であることを意味している。これらの＊、＋、＋＋の意味は、図１１～図１３でも同様である。また、ｎ＝３であることは、図１１～図１３でも同様である。 In addition, * mark of FIG. 10 means that it is p <0.05 (two-sided test) in t test (n = 3). Further, the + mark in FIG. 10 means that p <0.05 (two-sided test) in the t test (n = 3) with respect to the measurement result of the corresponding control. Furthermore, the ++ mark in FIG. 10 means that p <0.01 (two-sided test) in the t test (n = 3) with respect to the measurement result of the corresponding control. The meanings of *, +, and ++ are the same in FIGS. Also, n = 3 is the same in FIGS. 11 to 13.
　＜実施例５：ＴＧＦＢＲ２過剰発現によるＴＧＦ－βシグナル活性化及びアポトーシス関連タンパク発現の変化の確認＞
　（５－１）Ｒｅｖｅｒｓｅ　Ｔｒａｎｓｃｒｉｐｔｉｏｎ（ＲＴ）　Ｒｅａｃｔｉｏｎ
　ＴＧＦＢＲ２過剰発現によるＴＧＦ－βシグナル活性化について検討するために、以下の手順でｃＤＮＡ合成を行った。まず、ＬａｃＺ、ＴＧＦＢＲ２発現細胞（ＨｅｐＧ２）に対して５μｇ／ｍＬ　５－ＦＵ及び２００Ｕ／ｍＬ　ＩＦＮ－α処理を行った。薬剤処理４８時間後にＴＲＩｚｏｌ　Ｒｅａｇｅｎｔ（Ｉｎｖｉｔｒｏｇｅｎ）を用いてＴｏｔａｌ　ＲＮＡを抽出した。ＲＴ反応には、ＳｕｐｅｒＳｃｒｉｐｔ　Ｆｉｒｓｔ－Ｓｔａｎｄ　Ｓｙｎｔｈｅｓｉｓ　Ｓｙｓｔｅｍ　ｆｏｒ　ＲＴ－ＰＣＲ（Ｉｎｖｉｔｒｏｇｅｎ）を使用した。０．５μｇのｔｏｔａｌ　ＲＮＡをテンプレートとして使用し、ｏｌｉｇｏ　ｄＴをプライマーとして、６５℃５分で１サイクル、４２℃１２０分、７０℃１０分で１サイクル反応させ、ｃＤＮＡを合成した。反応後のｃＤＮＡは２０倍希釈してＲｅａｌ－Ｔｉｍｅ　ＰＣＲに用いた。 <Example 5: Confirmation of changes in TGF-β signal activation and apoptosis-related protein expression by overexpression of TGFBR2>
(5-1) Reverse Transcription (RT) Reaction
In order to examine TGF-β signal activation by overexpression of TGFBR2, cDNA synthesis was performed according to the following procedure. First, LacZ and TGFBR2-expressing cells (HepG2) were treated with 5 μg / mL 5-FU and 200 U / mL IFN-α. Total RNA was extracted 48 hours after drug treatment using TRIzol Reagent (Invitrogen). In the RT reaction, SuperScript First-Stand Synthesis System for RT-PCR (Invitrogen) was used. Using 0.5 μg of total RNA as a template and oligo dT as a primer, 1 cycle reaction was performed at 65 ° C. for 5 minutes, 42 ° C. for 120 minutes, and 70 ° C. for 10 minutes to synthesize cDNA. The cDNA after the reaction was diluted 20 times and used for Real-Time PCR.
　（５－２）Ｒｅａｌ　Ｔｉｍｅ　ＰＣＲ
　次いで、Ｒｅａｌ－Ｔｉｍｅ　ＰＣＲ反応には、ＬｉｇｈｔＣｙｃｌｅｒ　ＦａｓｔＳｔａｒｔ　ＤＮＡ　Ｍａｓｔｅｒ　ＳＹＢＲ　Ｇｒｅｅｎ　Ｉ（Ｒｏｃｈｅ）を使用した。ＲＴ－ＰＣＲによって合成したｃＤＮＡ　５μＬをテンプレートとして使用し、ＴＧＦβ１の遺伝子に対するプライマーを用いて、反応を行った。測定結果をグラフにまとめて図１１ａに示す。 (5-2) Real Time PCR
Next, for the Real-Time PCR reaction, LightCycler FastStart DNA Master SYBR Green I (Roche) was used. The reaction was carried out using 5 μL of cDNA synthesized by RT-PCR as a template and using a primer for the TGFβ1 gene. The measurement results are summarized in a graph and shown in FIG. 11a.
　この実験では、アポトーシスを起こすＴＧＦβ１のｍＲＮＡ発現量を測定し、ＴＧＦβのｍＲＮＡ発現量が上昇すればＴＧＦ－βが関係するシグナル伝達経路を介してアポトーシスが亢進していると解釈している。実験結果を見ると、対応するコントロールの測定結果に比べて、５－Ｆｕ単独又はＩＦＮ－ａ／５－ＦＵを添加した場合にはＴＧＦβ１のｍＲＮＡ発現量が有意に上昇している。このことから、５－Ｆｕ単独又はＩＦＮ－ａ／５－ＦＵによる薬剤処理によって、ＴＧＦ－βが関係するシグナル伝達経路を介してアポトーシスが亢進していることが明らかである。しかしながら、５－Ｆｕ単独又はＩＦＮ－ａ／５－ＦＵを添加した上で、ＴＧＦＢＲ２を過剰発現させた場合にも特に、ＴＧＦβ１のｍＲＮＡ発現量が有意には上昇していない。そのため、５－Ｆｕ単独又はＩＦＮ－ａ／５－ＦＵを添加した上で、ＴＧＦＢＲ２を過剰発現させた場合にＴＧＦ－βが関係するシグナル伝達経路を介してアポトーシスがさらに亢進されるかどうかは不明である。 In this experiment, TGFβ1 mRNA expression level causing apoptosis was measured, and it was interpreted that if TGFβ mRNA expression level increased, apoptosis was promoted via a signal transduction pathway involving TGF-β. Looking at the experimental results, the amount of TGFβ1 mRNA expression was significantly increased when 5-Fu alone or IFN-a / 5-FU was added, compared to the measurement results of the corresponding controls. From this, it is clear that 5-Fu alone or drug treatment with IFN-a / 5-FU enhances apoptosis through a signal transduction pathway involving TGF-β. However, especially when TGFBR2 is overexpressed after adding 5-Fu alone or IFN-a / 5-FU, the expression level of TGFβ1 mRNA is not significantly increased. Therefore, whether 5-Fu alone or IFN-a / 5-FU is added and TGFBR2 is overexpressed is unclear whether apoptosis is further enhanced through a signaling pathway involving TGF-β It is.
　なお、図１１の＊印は、ｔ検定（ｎ＝３）においてｐ＜０．０５（両側検定）であることを意味している。また、図１１のＮ．Ｓ．印は、ｔ検定（ｎ＝３）において有意差なし（ｎｏｔ　ｓｉｇｎｉｆｉｃａｎｔの略）であることを意味している。この＊、Ｎ．Ｓ．の意味は、図１２～図１３でも同様である。 In addition, the * mark of FIG. 11 means that p <0.05 (two-sided test) in the t test (n = 3). N. of FIG. S. The symbol means that there is no significant difference (not abbreviation for notsignificant) in the t-test (n = 3). This *, N.I. S. The meaning of is also the same in FIGS.
　（５－３）Ｌｕｃｉｆｅｒａｓｅ　Ａｓｓａｙ
　ＴＧＦＢＲ２過剰発現によるＴＧＦ－βシグナル活性化について検討するために、以下の手順でＬｕｃｉｆｅｒａｓｅ　Ａｓｓａｙを行った。まず、レポーター遺伝子ｐ３ＴＰ－Ｌｕｘ及び内部コントロール遺伝子としてｐｈＲＬ－ＴＫをＦｕＧＥＮＥ６により一過性に導入したＨｅｐＧ２細胞に対し、５ＦＵＳＧｓ過剰発現アデノウイルス感染させた。感染２４時間後にＤＭＳＯ；　Ｎｅｇａｔｉｖｅ　Ｃｏｎｔｒｏｌ（ＮＣ）、５μｇ／ｍＬ　５－ＦＵ、２００Ｕ／ｍＬ　ＩＦＮ－αでそれぞれ処理し、薬剤処理４８時間後にＬｕｃｉｆｅｒａｓｅ蛍光を検出した。グラフの各数値は、内部コントロールで補正した上で、薬剤未処理ＬａｃＺ過剰発現群の値を１としている。測定結果をグラフにまとめて図１１ｂに示す。 (5-3) Luciferase Assay
In order to examine TGF-β signal activation by overexpression of TGFBR2, Luciferase Assay was performed according to the following procedure. First, HepG2 cells into which reporter gene p3TP-Lux and phRL-TK as an internal control gene were transiently introduced by FuGENE6 were infected with 5FUSGs overexpression adenovirus. After 24 hours of infection, DMSO was treated with Negative Control (NC), 5 μg / mL 5-FU, and 200 U / mL IFN-α, and Luciferase fluorescence was detected 48 hours after drug treatment. Each numerical value in the graph is corrected by an internal control, and the value of the drug-untreated LacZ overexpression group is 1. The measurement results are summarized in a graph and shown in FIG.
　この実験では、アポトーシスを起こすＴＧＦ－βのレポーターベクターであるｐ３ＴＰ－Ｌｕｘから発現するホタルルシフェラーゼの活性を測定し、ホタルルシフェラーゼの活性が上昇すれば、ＴＧＦ－βの転写活性が上昇しており、ＴＧＦ－βが関係するシグナル伝達経路を介してアポトーシスが亢進していると解釈している。ここで、ｐ３ＴＰ－Ｌｕｃレポータープラスミドは、ＴＧＦ－βによって選択的に誘導されるＰＡＩ－１プロモーターの－７４０／－６３６領域にヒトコラゲナーゼ遺伝子由来の三つのＴＲＥエレメント（Ｓｍａｄを介して活性化されるＴＧＦ－βファミリーの一つａｃｔｉｖｉｎ経路に特異的なエレメント）を結合させた配列の下流にルシフェラーゼ遺伝子が接続されている。そのため、ＴＧＦ－βの転写活性が上昇すると、ｐ３ＴＰ－Ｌｕｃレポータープラスミドからのホタルルシフェラーゼの過剰発現が誘導されるためである。実験結果を見ると、明らかに、５－Ｆｕ単独又はＩＦＮ－ａ／５－ＦＵを添加した上で、ＴＧＦＢＲ２を過剰発現させた場合には、ホタルルシフェラーゼの活性が有意に上昇しているため、ＴＧＦ－βの転写活性が上昇していることが明らかである。すなわち、ＴＧＦ－βが関係するシグナル伝達経路を介してアポトーシスが亢進していることが明らかである。 In this experiment, the activity of firefly luciferase expressed from p3TP-Lux, a reporter vector of TGF-β causing apoptosis, was measured, and if the activity of firefly luciferase increased, the transcriptional activity of TGF-β increased. It is interpreted that apoptosis is enhanced through a signal transduction pathway involving TGF-β. Here, the p3TP-Luc reporter plasmid is activated via three TRE elements (Smad) derived from the human collagenase gene in the -740 / -636 region of the PAI-1 promoter selectively induced by TGF-β. A luciferase gene is connected downstream of a sequence to which an element specific to the activin pathway of the TGF-β family is bound. Therefore, when the transcriptional activity of TGF-β is increased, overexpression of firefly luciferase from the p3TP-Luc reporter plasmid is induced. From the experimental results, it is clear that when TGFBR2 is overexpressed after adding 5-Fu alone or IFN-a / 5-FU, the activity of firefly luciferase is significantly increased. It is clear that the transcriptional activity of TGF-β is increased. That is, it is clear that apoptosis is enhanced through a signal transduction pathway involving TGF-β.
　（５－４）Ｗｅｓｔｅｒｎ　Ｂｌｏｔ
　ＴＧＦＢＲ２過剰発現によるアポトーシス関連タンパク質の発現の変化について検討するために、以下の手順でＷｅｓｔｅｒｎ　Ｂｌｏｔを行った。 (5-4) Western Blot
In order to examine changes in the expression of apoptosis-related proteins due to overexpression of TGFBR2, Western Blot was performed according to the following procedure.
　＜タンパク質回収＞
　ＨｅｐＧ２を１×１０６ｃｅｌｌｓ／６ｃｍ－ｄｉｓｈずつ捲き、５時間後にアデノウイルスによるＴＧＦＢＲ２の遺伝子導入を行った。遺伝子導入１９時間後に５μｇ／ｍＬ　５－ＦＵ及び２００Ｕ／ｍＬ　ＩＦＮ－α処理を行った。４８時間後、１×ＰＢＳ（－）で２回ｗａｓｈし、ＲＩＰＡ　Ｂｕｆｆｅｒを加え、セルスクレイパーで細胞を回収した。回収した細胞を１５，０００ｒｐｍ、１０分、４℃で遠心し、上清をｓｏｄｉｕｍ　ｄｏｄｅｃｙｌ　ｓｕｌｆａｔｅ－ｐｏｌｙａｃｒｙｌａｍｉｄｅ　ｇｅｌ　ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ（ＳＤＳ－ＰＡＧＥ）に使用した。 <Protein recovery>
HepG2 was seeded at 1 × 10 6 cells / 6 cm-dish, and TGFBR2 gene was introduced by adenovirus 5 hours later. Nineteen hours after gene introduction, 5 μg / mL 5-FU and 200 U / mL IFN-α treatment was performed. After 48 hours, the cells were washed twice with 1 × PBS (−), RIPA Buffer was added, and the cells were collected with a cell scraper. The collected cells were centrifuged at 15,000 rpm for 10 minutes at 4 ° C., and the supernatant was used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
　＜ＳＤＳ－ＰＡＧＥ＞
　回収した上清に含まれるタンパク質をＳＤＳ－ＰＡＧＥにより分離した。泳動条件は、２０ｍＡで２時間行った。泳動後のゲルは、セミドライ式トランスファー装置を用いて、１００ｍＡ、１時間反応させ、ｐｏｌｙｖｉｎｙｌｉｄｅｎｅ　ｄｉｆｌｕｏｒｉｄｅメンブレンに転写した。 <SDS-PAGE>
Proteins contained in the collected supernatant were separated by SDS-PAGE. The electrophoresis conditions were 20 mA for 2 hours. The gel after electrophoresis was reacted at 100 mA for 1 hour using a semi-dry transfer device and transferred to a polyvinylidene fluoride membrane.
　＜Ｗｅｓｔｅｒｎ　Ｂｌｏｔ＞
　５％スキムミルク／ＴＢＳ－０．１％Ｔでブロッキングを行った後、ＢＡＸ、ＢＣＬ２、ＢＣＬｘＬ、ＡＣＴの各遺伝子のコードするタンパク質に対する抗体を用いて４℃でオーバーナイト、１次抗体反応を行った。反応終了後、メンブレンをＴＢＳ－０．１％Ｔで３回ｗａｓｈした。次に、各１次抗体に対するＨＲＰ標識２次抗体（１：３，０００）を使用して室温で１時間、２次抗体反応を行った。反応終了後、ＴＢＳ－０．１％Ｔで３回ｗａｓｈした。その後、ＥＣＬ　Ｗｅｓｔｅｒｎ　Ｂｌｏｔｔｉｎｇ　Ｄｅｔｅｃｔｉｏｎ　Ｒｅａｇｅｎｔｓ、ＥＣＬ　Ｐｌｕｓ　Ｗｅｓｔｅｒｎ　Ｂｌｏｔｔｉｎｇ　Ｄｅｔｅｃｔｉｏｎ　Ｒｅｇｅｎｔｓ及びＬＡＳ－４０００　ｓｙｓｔｅｍを用いて、ＢＡＸ、ＢＣＬ２、ＢＣＬｘＬ、ＡＣＴのバンドを検出した。測定結果を電気泳動写真として図１１ｃに示す。 <Western Blot>
After blocking with 5% skim milk / TBS-0.1% T, overnight and primary antibody reaction was performed at 4 ° C. using antibodies against proteins encoded by BAX, BCL2, BCLxL, and ACT genes. . After completion of the reaction, the membrane was washed 3 times with TBS-0.1% T. Next, a secondary antibody reaction was performed at room temperature for 1 hour using an HRP-labeled secondary antibody (1: 3,000) against each primary antibody. After completion of the reaction, washing was performed 3 times with TBS-0.1% T. Thereafter, bands of BAX, BCL2, BCLxL, and ACT were detected using ECL Western Blotting Detection Reagents, ECL Plus Western Blotting Detection Reagents, and LAS-4000 system. The measurement results are shown in FIG.
　この実験では、ＢＡＸの発現量が増大しており、ＢＣＬ２、ＢＣＬｘＬの発現量が減少していれば、アポトーシスが亢進していると解釈している。ここで、Ｂａｘは細胞質に存在するが、ｄｅａｔｈシグナルに従ってミトコンドリアへと移動し、そこでシトクロムｃの放出を促進し、アポトーシスを誘導する。また、ＢＣＬ２、ＢＣＬｘＬは、ミトコンドリア外壁に存在し、シトクロムｃの放出を抑制して、アポトーシスを抑制する。そのため、ＢＡＸの発現量が増大しており、ＢＣＬ２、ＢＣＬｘＬの発現量が減少していれば、アポトーシスが亢進していると解釈できる。実験結果を見ると、明らかに、５－Ｆｕ単独又はＩＦＮ－ａ／５－ＦＵを添加した上で、ＴＧＦＢＲ２を過剰発現させた場合には、ＢＡＸの発現量が増大しており、ＢＣＬ２、ＢＣＬｘＬの発現量が減少しているため、アポトーシスが亢進していることが明らかである。なお、コントロールであるＡＣＴ（アクチン）の発現量は特に変化が見られない。 In this experiment, if the expression level of BAX is increased and the expression levels of BCL2 and BCLxL are decreased, it is interpreted that apoptosis is enhanced. Here, Bax exists in the cytoplasm, but moves to mitochondria according to the death signal, where it promotes the release of cytochrome c and induces apoptosis. BCL2 and BCLxL are present on the outer wall of mitochondria and suppress cytochrome c release to suppress apoptosis. Therefore, if the expression level of BAX is increased and the expression levels of BCL2 and BCLxL are decreased, it can be interpreted that apoptosis is enhanced. From the experimental results, it is clear that when TGFBR2 is overexpressed after adding 5-Fu alone or IFN-a / 5-FU, the expression level of BAX increases, and BCL2, BCLxL It is clear that apoptosis is enhanced because the expression level of is decreased. In addition, the expression level of ACT (actin) as a control is not particularly changed.
　＜実施例６：ＥＸＴ１過剰発現によるアポトーシス亢進に対するＥＲストレスの関与の確認＞
　（６－１）Ｒｅａｌ－Ｔｉｍｅ ＲＴ－ＰＣＲ
　ＥＸＴ１過剰発現によるアポトーシス亢進に対するＥＲストレスの関与について検討するために、実施例５の場合と同様の手法でＲｅａｌ－Ｔｉｍｅ ＲＴ－ＰＣＲを行った。測定結果をグラフにまとめて図１２ａおよび図１２ｂに示す。 <Example 6: Confirmation of involvement of ER stress in enhancement of apoptosis due to EXT1 overexpression>
(6-1) Real-Time RT-PCR
In order to examine the involvement of ER stress in the enhancement of apoptosis due to EXT1 overexpression, Real-Time RT-PCR was performed in the same manner as in Example 5. The measurement results are summarized in a graph and shown in FIGS. 12a and 12b.
　この実験では、アポトーシスを引き起こすＥＲストレスに応答して発現量が増大することが知られているＤＤＩＴ３（ＣＨＯＰ）のｍＲＮＡ発現量およびＨＳＰＡ５（ＢＩＰ）のｍＲＮＡ発現量を測定し、ＤＤＩＴ３（ＣＨＯＰ）およびＨＳＰＡ５（ＢＩＰ）のｍＲＮＡ発現量が上昇すればアポトーシスを引き起こすＥＲストレスが増大していると解釈している。実験結果を見ると、５－Ｆｕ単独又はＩＦＮ－ａ／５－ＦＵを添加した上で、ＥＸＴ１を過剰発現させた場合、ＤＤＩＴ３（ＣＨＯＰ）およびＨＳＰＡ５（ＢＩＰ）のｍＲＮＡ発現量が有意に上昇している。そのため、５－Ｆｕ単独又はＩＦＮ－ａ／５－ＦＵを添加した上で、ＥＸＴ１を過剰発現させた場合にアポトーシスを引き起こすＥＲストレスが増大していることが明らかである。 In this experiment, DDIT3 (CHOP) mRNA expression level and HSPA5 (BIP) mRNA expression level, which are known to increase in response to ER stress causing apoptosis, were measured, and DDIT3 (CHOP) and It is interpreted that the ER stress that causes apoptosis is increased when the mRNA expression level of HSPA5 (BIP) is increased. From the experimental results, when EXT1 was overexpressed after adding 5-Fu alone or IFN-a / 5-FU, the mRNA expression levels of DDIT3 (CHOP) and HSPA5 (BIP) increased significantly. ing. Therefore, it is apparent that ER stress causing apoptosis is increased when EXT1 is overexpressed after adding 5-Fu alone or IFN-a / 5-FU.
　（６－２）Ｗｅｓｔｅｒｎ　Ｂｌｏｔ
　ＥＸＴ１過剰発現によるアポトーシス関連タンパク質の発現の変化について検討するために、実施例５の場合と同様の手法でＷｅｓｔｅｒｎ　Ｂｌｏｔを行った。測定結果を電気泳動写真として図１２ｃに示す。 (6-2) Western Blot
In order to examine the change in the expression of apoptosis-related protein due to EXT1 overexpression, Western Blot was performed in the same manner as in Example 5. The measurement results are shown in FIG.
　この実験では、アポトーシスを引き起こすＥＲストレスに応答して発現量が増えるＡＴＦ４の発現量を測定し、ＡＴＦ４の発現量が上昇すればアポトーシスを引き起こすＥＲストレスが増大していると解釈する。実験結果を見ると、明らかに、５－Ｆｕ単独又はＩＦＮ－ａ／５－ＦＵを添加した上で、ＥＸＴ１を過剰発現させた場合には、ＡＴＦ４の発現量が増大しているため、アポトーシスを引き起こすＥＲストレスが増大していることが明らかである。なお、コントロールであるＡＣＴ（アクチン）の発現量は特に変化が見られない。 In this experiment, the expression level of ATF4 whose expression level increases in response to ER stress causing apoptosis is measured, and if the expression level of ATF4 increases, it is interpreted that ER stress causing apoptosis increases. From the experimental results, it is clear that when EXT1 is overexpressed after adding 5-Fu alone or IFN-a / 5-FU, the expression level of ATF4 increases, and thus apoptosis is suppressed. It is clear that the ER stress that causes is increasing. In addition, the expression level of ACT (actin) as a control is not particularly changed.
　＜実施例７：ＴＧＦＢＲ２、ＥＸＴ１過剰発現によるヒト肝癌モデルマウスへの効果＞
　ＴＧＦＢＲ２、ＥＸＴ１過剰発現によるヒト肝癌モデルマウスへの効果を確認するために、以下の手順でＩｎ　Ｖｉｖｏ実験を行った。まず、ＮＯＤ／ＳＣＩＤマウス皮下にヒト肝癌細胞株（ＨｅｐＧ２；６．８×１０６個／マウス）を移植した。移植約３週後に、腫瘍部へＬａｃＺ、ＴＧＦＢＲ２、ＥＸＴ１遺伝子をアデノウイルスを用いて直接導入した。アデノウイルス感染後１日後から、２０，０００Ｕ／ｂｏｄｙ／ｄａｙ　ＩＦＮ－αを皮下注射、３０ｍｇ／ｋｇ／ｄａｙ　５－ＦＵを腹腔内注射によって投与した。また、アデノウイルス、薬剤処理は３日ごとに行い、腫瘍径は、７日ごとにデジタルノギスを用いて長径と単径を測定し、以下の計算式によって求めた。腫瘍径（ＴＶ）＝長径×（短径）２／２。実験結果をまとめてグラフとして図１３に示す。 <Example 7: Effect on human liver cancer model mouse by overexpression of TGFBR2 and EXT1>
In order to confirm the effect of TGFBR2 and EXT1 overexpression on human liver cancer model mice, In Vivo experiments were performed according to the following procedure. First, a human liver cancer cell line (HepG2; 6.8 × 10 6 cells / mouse) was transplanted subcutaneously into NOD / SCID mice. About 3 weeks after transplantation, LacZ, TGFBR2, and EXT1 genes were directly introduced into the tumor site using adenovirus. One day after the adenovirus infection, 20,000 U / body / day IFN-α was subcutaneously injected and 30 mg / kg / day 5-FU was administered by intraperitoneal injection. Adenovirus and drug treatment were carried out every 3 days, and the tumor diameter was determined every 7 days using a digital caliper by measuring the long diameter and single diameter, and the following formula was used. Tumor diameter (TV) = major axis × (minor diameter) 2/2. The experimental results are summarized and shown as a graph in FIG.
　この実験では、ｉｎ　ｖｉｖｏでヒト肝癌モデルマウスに投与した場合に、腫瘍サイズの増加を抑制できれば、肝癌に対する抗腫瘍効果が得られていることを示す薬理データであると解釈する。実験結果を見ると、明らかに、５－Ｆｕ単独又はＩＦＮ－ａ／５－ＦＵを添加した上で、ＴＧＦＢＲ２およびＥＸＴ１を過剰発現させた場合には、腫瘍サイズの増加が有意に抑制されており、抗腫瘍効果が実証されている。 In this experiment, if administered to a human liver cancer model mouse in vivo, if the increase in tumor size can be suppressed, it is interpreted as pharmacological data indicating that an antitumor effect against liver cancer is obtained. From the experimental results, it is clear that when 5-Fu alone or IFN-a / 5-FU is added and TGFBR2 and EXT1 are overexpressed, the increase in tumor size is significantly suppressed. Antitumor effects have been demonstrated.
　＜実施例８：肝癌患者におけるＰＲＫＡＧ２、ＴＧＦＢＲ２、ＥＸＴ１の発現＞
　ＨＣＣ患者におけるＰＲＫＡＧ２、ＴＧＦＢＲ２、ＥＸＴ１のｍＲＮＡ発現を検討するために、以下の手順でｍＲＮＡ発現の解析を行った。まず、非Ｂ非Ｃ、Ｂ型、Ｃ型のＨＣＣ患者の癌部、非癌部それぞれ７、７、２７症例より採取した組織よりＲＮＡを回収した。次いで、これらのＲＮＡのサンプルを用いて、上記の実施例５および実施例６と同様の手法によって、Ｒｅａｌ－Ｔｉｍｅ　ＲＴ－ＰＣＲ法によりＰＲＫＡＧ２、ＴＧＦＢＲ２、ＥＸＴ１のｍＲＮＡ発現を解析した。 <Example 8: Expression of PRKAG2, TGFBR2, EXT1 in a liver cancer patient>
In order to examine mRNA expression of PRKAG2, TGFBR2, and EXT1 in HCC patients, mRNA expression was analyzed by the following procedure. First, RNA was collected from tissues collected from cancer cases and non-cancerous cases of non-B non-C, B-type, and C-type HCC patients, respectively. Next, using these RNA samples, mRNA expression of PRKAG2, TGFBR2, and EXT1 was analyzed by the Real-Time RT-PCR method in the same manner as in Examples 5 and 6 above.
　この実験では、非Ｂ非Ｃ、Ｂ型、Ｃ型の肝癌患者の癌部、非癌部でのＰＲＫＡＧ２、ＴＧＦＢＲ２およびＥＸＴ１のｍＲＮＡ発現量を測定した結果、Ｂ型の肝癌患者の癌部では非癌部に比べて有意にＴＧＦＢＲ２のｍＲＮＡ発現量が減少していることが判明した。また、非Ｂ非Ｃ型の肝癌患者の癌部では非癌部に比べて有意にＥＸＴ１のｍＲＮＡ発現量が減少していることが判明した。なお、図１４の＊印は、対応のあるｔ検定（Ｐａｉｒｅｄ－ｔ－ｔｅｓｔ）においてｐ＜０．０５（両側検定）であることを意味している。また、図１４の＊＊印は、対応のあるｔ検定（Ｐａｉｒｅｄ－ｔ－ｔｅｓｔ）においてｐ＜０．０１（両側検定）であることを意味している。 In this experiment, the mRNA expression levels of PRKAG2, TGFBR2, and EXT1 in non-B non-C, B-type and C-type liver cancer patients and non-cancerous areas were measured. It was found that the amount of TGFBR2 mRNA expression was significantly reduced compared to the cancerous part. It was also found that the EXT1 mRNA expression level was significantly decreased in the cancerous part of non-B non-C type liver cancer patients compared to the non-cancerous part. Note that the mark * in FIG. 14 means that p <0.05 (two-sided test) in a paired t-test. In addition, ** marks in FIG. 14 mean that p <0.01 (two-sided test) in the paired t-test (Paired-t-test).
　そのため、Ｂ型の肝癌患者の癌部において、５－Ｆｕ単独又はＩＦＮ－ａ／５－ＦＵを添加した上で、ＴＧＦＢＲ２を過剰発現させた場合には、特に抗腫瘍効果が増大する可能性が示唆される。また、非Ｂ非Ｃ型の肝癌患者の癌部において、５－Ｆｕ単独又はＩＦＮ－ａ／５－ＦＵを添加した上で、ＥＸＴ１を過剰発現させた場合には、特に抗腫瘍効果が増大する可能性が示唆される。 Therefore, when TGFBR2 is overexpressed after adding 5-Fu alone or IFN-a / 5-FU in the cancerous part of a type B liver cancer patient, the antitumor effect may be particularly increased. It is suggested. In addition, in the cancerous part of a non-B non-C type liver cancer patient, when 5-FU alone or IFN-a / 5-FU is added and EXT1 is overexpressed, the antitumor effect is particularly increased. The possibility is suggested.
　＜結果の考察＞
　以上の結果より、ランダムリボザイムライブラリーを用いたファンクショナルスクリーニングによって同定した５－ＦＵ感受性寄与遺伝子（５ＦＵＳＧｓ：ＰＲＫＡＧ２、ＴＧＦＢＲ２、ＥＸＴ１）の強制発現によって、５－ＦＵ感受性を増強されたことが分かる。すなわち、本研究で同定した３遺伝子（ＰＲＫＡＧ２、ＴＧＦＢＲ２、ＥＸＴ１）は、５－ＦＵ単独及びＩＦＮ－α／５－ＦＵ併用時の感受性を増強することが示された。 <Consideration of results>
From the above results, it can be seen that 5-FU sensitivity was enhanced by forced expression of 5-FU sensitivity contributing genes (5FUSGs: PRKAG2, TGFBR2, EXT1) identified by functional screening using a random ribozyme library. That is, it was shown that the three genes identified in this study (PRKAG2, TGFBR2, EXT1) enhance the sensitivity when 5-FU alone and IFN-α / 5-FU are used together.
　また、ＴＧＦＢＲ２、ＥＸＴ１のアデノウイルスベクターによる過剰発現は、ＨｅｐＧ２細胞のＩＦＮ－α／５－ＦＵ併用療法の感受性をｉｎ　ｖｉｔｒｏにおいて増強した。さらに、ＴＧＦＢＲ２のアデノウイルスベクターによる過剰発現は、マウスの腫瘍組織のＩＦＮ－α／５－ＦＵ併用療法への感受性をｉｎ　ｖｉｖｏにおいて増強した。そのため、これらの遺伝子を使用することによって、５－ＦＵ単独、ＩＦＮ－α／５－ＦＵ併用療法が適応されている種々の悪性腫瘍患者の長期予後改善が期待される。 Moreover, overexpression of TGFBR2 and EXT1 with an adenoviral vector enhanced the sensitivity of HepG2 cells to IFN-α / 5-FU combination therapy in vitro. Furthermore, overexpression of TGFBR2 by adenoviral vectors enhanced the sensitivity of mouse tumor tissues to IFN-α / 5-FU combination therapy in vivo. Therefore, the use of these genes is expected to improve the long-term prognosis of various malignant tumor patients to which 5-FU alone or IFN-α / 5-FU combination therapy is indicated.
　また、５－ＦＵおよびＩＦＮ－α／５－ＦＵは、種々の悪性腫瘍に使用されている抗がん剤であるため、ＰＲＫＡＧ２、ＴＧＦＢＲ２、ＥＸＴ１の３種類の遺伝子は、進行型肝細胞癌のみでなく、他の悪性腫瘍に対しても抗癌剤の増感剤として有効である可能性が期待される。 Further, since 5-FU and IFN-α / 5-FU are anticancer agents used in various malignant tumors, the three types of genes PRKAG2, TGFBR2, and EXT1 are only for advanced hepatocellular carcinoma. In addition, it is expected to be effective as a sensitizer for anticancer agents against other malignant tumors.
　以上、本発明を実施例に基づいて説明した。この実施例はあくまで例示であり、種々の変形例が可能なこと、またそうした変形例も本発明の範囲にあることは当業者に理解されるところである。 The present invention has been described based on the embodiments. It is to be understood by those skilled in the art that this embodiment is merely an example, and that various modifications are possible and that such modifications are within the scope of the present invention.

Claims (29)

  1. 　５－フルオロウラシル感受性増感剤であって、
    　ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子からなる群から選ばれる１以上の遺伝子をコードするヌクレオチド断片を含む、増感剤。     A 5-fluorouracil sensitive sensitizer,
    A sensitizer comprising a nucleotide fragment encoding one or more genes selected from the group consisting of a PRAK2 gene, a TGFBR2 gene and an EXT1 gene.
  2. 　請求項１記載の５－フルオロウラシル感受性増感剤において、
    　前記ＰＲＡＫＧ２遺伝子が、
    （ａ）ヒト野生型ＰＲＡＫＧ２遺伝子、および
    （ｂ）（ａ）の遺伝子の変異型遺伝子であって、哺乳動物細胞における５－フルオロウラシル感受性を増強させる遺伝子、
    　を含む、増感剤。     The 5-fluorouracil sensitive sensitizer according to claim 1,
    The PRAK2 gene is
    (A) a human wild-type PRAK2 gene, and (b) a mutant gene of the gene of (a), which enhances 5-fluorouracil sensitivity in mammalian cells,
    A sensitizer including
  3. 　請求項２記載の５－フルオロウラシル感受性増感剤において、
    前記変異型遺伝子が、
    　（ｂ－１）（ａ）の遺伝子がコードする蛋白質のアミノ酸配列をコードする塩基配列からなる変異型遺伝子、
    　（ｂ－２）（ａ）の遺伝子がコードする蛋白質のアミノ酸配列のうち１若しくは数個のアミノ酸残基を欠失・置換・付加してなるアミノ酸配列をコードする塩基配列からなる変異型遺伝子、
    　（ｂ－３）（ａ）の遺伝子がコードする蛋白質のアミノ酸配列に対して８０％以上の相同性を有するアミノ酸配列をコードする基配列からなる変異型遺伝子、
    　（ｂ－４）（ａ）の遺伝子の塩基配列に相補的な塩基配列に対してストリンジェントな条件でハイブリダイズする塩基配列からなる変異型遺伝子、
    を含む、増感剤。     The 5-fluorouracil sensitive sensitizer according to claim 2,
    The mutant gene is
    (B-1) a mutant gene comprising a base sequence encoding the amino acid sequence of the protein encoded by the gene of (a),
    (B-2) a mutant gene comprising a base sequence encoding an amino acid sequence obtained by deleting, substituting, or adding one or several amino acid residues in the amino acid sequence of the protein encoded by the gene of (a),
    (B-3) a mutant gene comprising a base sequence encoding an amino acid sequence having 80% or more homology to the amino acid sequence of the protein encoded by the gene of (a),
    (B-4) a mutant gene comprising a base sequence that hybridizes under stringent conditions to a base sequence complementary to the base sequence of the gene of (a),
    A sensitizer including
  4. 　請求項１記載の５－フルオロウラシル感受性増感剤において、
    　前記ＴＧＦＢＲ２遺伝子が、
    （ｃ）ヒト野生型ＴＧＦＢＲ２遺伝子、および
    （ｄ）（ｃ）の遺伝子の変異型遺伝子であって、哺乳動物細胞における５－フルオロウラシル感受性を増強させる遺伝子、
    　を含む、増感剤。     The 5-fluorouracil sensitive sensitizer according to claim 1,
    The TGFBR2 gene is
    (C) a human wild-type TGFBR2 gene, and (d) a mutant gene of the gene of (c), which enhances 5-fluorouracil sensitivity in mammalian cells,
    A sensitizer including
  5. 　請求項４記載の５－フルオロウラシル感受性増感剤において、
    前記変異型遺伝子が、
    　（ｄ－１）（ｃ）の遺伝子がコードする蛋白質のアミノ酸配列をコードする塩基配列からなる変異型遺伝子、
    　（ｄ－２）（ｃ）の遺伝子がコードする蛋白質のアミノ酸配列のうち１若しくは数個のアミノ酸残基を欠失・置換・付加してなるアミノ酸配列をコードする塩基配列からなる変異型遺伝子、
    　（ｄ－３）（ｃ）の遺伝子がコードする蛋白質のアミノ酸配列に対して８０％以上の相同性を有するアミノ酸配列をコードする基配列からなる変異型遺伝子、
    　（ｄ－４）（ｃ）の遺伝子の塩基配列に相補的な塩基配列に対してストリンジェントな条件でハイブリダイズする塩基配列からなる変異型遺伝子、
    を含む、増感剤。     The 5-fluorouracil sensitive sensitizer according to claim 4,
    The mutant gene is
    (D-1) a mutant gene comprising a base sequence encoding the amino acid sequence of the protein encoded by the gene of (c),
    (D-2) a mutant gene comprising a base sequence encoding an amino acid sequence obtained by deleting, substituting, or adding one or several amino acid residues in the amino acid sequence of the protein encoded by the gene of (c),
    (D-3) a mutant gene comprising a base sequence encoding an amino acid sequence having 80% or more homology to the amino acid sequence of the protein encoded by the gene of (c),
    (D-4) a mutant gene comprising a base sequence that hybridizes under stringent conditions to a base sequence complementary to the base sequence of the gene of (c),
    A sensitizer including
  6. 　請求項１記載の５－フルオロウラシル感受性増感剤において、
    　前記ＥＸＴ１遺伝子が、
    （ｅ）ヒト野生型ＥＸＴ１遺伝子、および
    （ｆ）（ｅ－１）の遺伝子の変異型遺伝子であって、哺乳動物細胞における５－フルオロウラシル感受性を増強させる遺伝子、
    　を含む、増感剤。     The 5-fluorouracil sensitive sensitizer according to claim 1,
    The EXT1 gene is
    (E) a human wild type EXT1 gene, and (f) a mutant gene of the gene of (e-1), which enhances 5-fluorouracil sensitivity in mammalian cells,
    A sensitizer including
  7. 　請求項６記載の５－フルオロウラシル感受性増感剤において、
    前記変異型遺伝子が、
    　（ｆ－１）（ｅ）の遺伝子がコードする蛋白質のアミノ酸配列をコードする塩基配列からなる変異型遺伝子、
    　（ｆ－２）（ｅ）の遺伝子がコードする蛋白質のアミノ酸配列のうち１若しくは数個のアミノ酸残基を欠失・置換・付加してなるアミノ酸配列をコードする塩基配列からなる変異型遺伝子、
    　（ｆ－３）（ｅ）の遺伝子がコードする蛋白質のアミノ酸配列に対して８０％以上の相同性を有するアミノ酸配列をコードする基配列からなる変異型遺伝子、
    　（ｆ－４）（ｅ）の遺伝子の塩基配列に相補的な塩基配列に対してストリンジェントな条件でハイブリダイズする塩基配列からなる変異型遺伝子、
    を含む、増感剤。     The 5-fluorouracil-sensitive sensitizer according to claim 6,
    The mutant gene is
    (F-1) a mutant gene comprising a base sequence encoding the amino acid sequence of the protein encoded by the gene of (e),
    (F-2) a mutant gene comprising a base sequence encoding an amino acid sequence obtained by deleting, substituting, or adding one or several amino acid residues in the amino acid sequence of the protein encoded by the gene of (e),
    (F-3) a mutant gene comprising a base sequence encoding an amino acid sequence having 80% or more homology to the amino acid sequence of the protein encoded by the gene of (e),
    (F-4) a mutant gene comprising a base sequence that hybridizes under stringent conditions to a base sequence complementary to the base sequence of the gene of (e),
    A sensitizer including
  8. 　５－フルオロウラシル感受性増感剤であって、
    　ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子からなる群から選ばれる１以上の遺伝子を発現するベクターを含む、増感剤。     A 5-fluorouracil sensitive sensitizer,
    A sensitizer comprising a vector that expresses one or more genes selected from the group consisting of a PRAK2 gene, a TGFBR2 gene, and an EXT1 gene.
  9. 　請求項８記載の５－フルオロウラシル感受性増感剤において、
    　前記ベクターが、アデノウイルスベクターである、
    　増感剤。     The 5-fluorouracil sensitive sensitizer according to claim 8,
    The vector is an adenovirus vector;
    Sensitizer.
  10. 　５－フルオロウラシル感受性増感剤であって、
    　ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子からなる群から選ばれる１以上の遺伝子のコードする蛋白質を含む、増感剤。     A 5-fluorouracil sensitive sensitizer,
    A sensitizer comprising a protein encoded by one or more genes selected from the group consisting of PRAKG2, TGFBR2 and EXT1 genes.
  11. 　請求項１０記載の５－フルオロウラシル感受性増感剤において、
    　前記ＰＲＡＫＧ２遺伝子のコードするＡＭＰ活性化蛋白質キナーゼが、
    （ｇ）ヒト野生型ＡＭＰ活性化蛋白質キナーゼ、および
    （ｈ）（ｇ）の変異型キナーゼであって、哺乳動物細胞における５－フルオロウラシル感受性を増強させるキナーゼ、
    　を含む、増感剤。     The 5-fluorouracil sensitive sensitizer according to claim 10,
    AMP-activated protein kinase encoded by the PRAK2 gene is
    (G) a human wild-type AMP-activated protein kinase, and (h) a mutant kinase of (g), which enhances 5-fluorouracil sensitivity in mammalian cells,
    A sensitizer including
  12. 　請求項１１記載の５－フルオロウラシル感受性増感剤において、
    前記変異型キナーゼが、
    　（ｈ－１）（ｇ）のヒト野生型ＡＭＰ活性化蛋白質キナーゼのアミノ酸配列のうち１若しくは数個のアミノ酸残基を欠失・置換・付加してなるアミノ酸配列からなる変異型キナーゼ、
    　（ｈ－２）（ｇ）のヒト野生型ＡＭＰ活性化蛋白質キナーゼのアミノ酸配列に対して８０％以上の相同性を有するアミノ酸配列からなる変異型キナーゼ、
    　（ｈ－３）（ａ）の遺伝子の塩基配列に相補的な塩基配列に対してストリンジェントな条件でハイブリダイズする塩基配列がコードするアミノ酸配列からなる変異型キナーゼ、
    を含む、増感剤。     The 5-fluorouracil sensitive sensitizer according to claim 11,
    The mutant kinase is
    (H-1) a mutant kinase comprising an amino acid sequence obtained by deleting, substituting, or adding one or several amino acid residues in the amino acid sequence of the human wild-type AMP-activated protein kinase of (g),
    (H-2) a mutant kinase comprising an amino acid sequence having 80% or more homology to the amino acid sequence of the human wild-type AMP-activated protein kinase of (g),
    (H-3) a mutant kinase comprising an amino acid sequence encoded by a base sequence that hybridizes under stringent conditions to a base sequence complementary to the base sequence of the gene of (a),
    A sensitizer including
  13. 　請求項１０記載の５－フルオロウラシル感受性増感剤において、
    　前記ＴＧＦＢＲ２遺伝子のコードするＴＧＦ－β２型受容体が、
    （ｉ）ヒト野生型ＴＧＦ－β２型受容体、および
    （ｊ）（ｉ）の変異型受容体であって、哺乳動物細胞における５－フルオロウラシル感受性を増強させる受容体、
    　を含む、増感剤。     The 5-fluorouracil sensitive sensitizer according to claim 10,
    The TGF-β2 type receptor encoded by the TGFBR2 gene is
    (I) a human wild type TGF-β2 type receptor, and (j) a mutant receptor of (i), which enhances 5-fluorouracil sensitivity in mammalian cells,
    A sensitizer including
  14. 　請求項１３記載の５－フルオロウラシル感受性増感剤において、
    前記変異型受容体が、
    　（ｊ－１）（ｉ）のヒト野生型ＴＧＦ－β２型受容体のアミノ酸配列のうち１若しくは数個のアミノ酸残基を欠失・置換・付加してなるアミノ酸配列からなる変異型受容体、
    　（ｊ－２）（ｉ）のヒト野生型ＴＧＦ－β２型受容体のアミノ酸配列に対して８０％以上の相同性を有するアミノ酸配列からなる変異型受容体、
    　（ｊ－３）（ｃ）の遺伝子の塩基配列に相補的な塩基配列に対してストリンジェントな条件でハイブリダイズする塩基配列がコードするアミノ酸配列からなる変異型受容体、
    を含む、増感剤。     The 5-fluorouracil sensitive sensitizer according to claim 13,
    The mutant receptor is
    (J-1) A mutant receptor comprising an amino acid sequence obtained by deleting, substituting, or adding one or several amino acid residues in the amino acid sequence of the human wild type TGF-β2 type receptor of (i),
    (J-2) a mutant receptor comprising an amino acid sequence having 80% or more homology to the amino acid sequence of the human wild-type TGF-β2 type receptor of (i),
    (J-3) a mutant receptor comprising an amino acid sequence encoded by a base sequence that hybridizes under stringent conditions to a base sequence complementary to the base sequence of the gene of (c),
    A sensitizer including
  15. 　請求項１０記載の５－フルオロウラシル感受性増感剤において、
    　前記ＥＸＴ１遺伝子のコードする小胞体内在性ＩＩ型膜貫通糖転移酵素が、
    （ｋ）ヒト野生型小胞体内在性ＩＩ型膜貫通糖転移酵素、および
    （ｌ）（ｋ）の変異型糖転移酵素であって、哺乳動物細胞における５－フルオロウラシル感受性を増強させる糖転移酵素、
    　を含む、増感剤。     The 5-fluorouracil sensitive sensitizer according to claim 10,
    Endoplasmic reticulum type II transmembrane glycosyltransferase encoded by the EXT1 gene is
    (K) a human wild-type endoplasmic reticulum type II transmembrane glycosyltransferase, and (l) a mutant glycosyltransferase of (k), which enhances 5-fluorouracil sensitivity in mammalian cells,
    A sensitizer including
  16. 　請求項１５記載の５－フルオロウラシル感受性増感剤において、
    前記変異型糖転移酵素が、
    　（ｌ－１）（ｋ）のヒト野生型小胞体内在性ＩＩ型膜貫通糖転移酵素のアミノ酸配列のうち１若しくは数個のアミノ酸残基を欠失・置換・付加してなるアミノ酸配列からなる変異型糖転移酵素、
    　（ｌ－２）（ｋ）のヒト野生型小胞体内在性ＩＩ型膜貫通糖転移酵素のアミノ酸配列に対して８０％以上の相同性を有するアミノ酸配列からなる変異型糖転移酵素、
    　（ｌ－３）（ｅ）の遺伝子の塩基配列に相補的な塩基配列に対してストリンジェントな条件でハイブリダイズする塩基配列がコードするアミノ酸配列からなる変異型糖転移酵素、
    を含む、増感剤。     The 5-fluorouracil sensitive sensitizer according to claim 15,
    The mutant glycosyltransferase is
    (L-1) It consists of an amino acid sequence obtained by deleting, substituting, or adding one or several amino acid residues in the amino acid sequence of human wild-type endoplasmic reticulum type II transmembrane glycosyltransferase in (k). Mutant glycosyltransferases,
    (1-2) a mutant glycosyltransferase comprising an amino acid sequence having 80% or more homology to the amino acid sequence of the human wild-type endoplasmic reticulum type II transmembrane glycosyltransferase of (k),
    (1-3) a mutant glycosyltransferase comprising an amino acid sequence encoded by a base sequence that hybridizes under stringent conditions to a base sequence complementary to the base sequence of the gene of (e),
    A sensitizer including
  17. 　請求項１に記載の増感剤において、
    　５－フルオロウラシルおよびインターフェロンαの併用時における５－フルオロウラシルおよびインターフェロンαへの感受性を増強する、増感剤。     The sensitizer according to claim 1,
    A sensitizer that enhances sensitivity to 5-fluorouracil and interferon α when 5-fluorouracil and interferon α are used in combination.
  18. 　請求項１に記載の増感剤において、
    　注射剤である、増感剤。     The sensitizer according to claim 1,
    A sensitizer that is an injection.
  19. 　抗癌剤キットであって、
    　　請求項１に記載の増感剤と、
    　５－フルオロウラシルと、
    　を含む、キット。     An anti-cancer agent kit,
    A sensitizer according to claim 1;
    5-fluorouracil,
    Including a kit.
  20. 　請求項１９記載の抗癌剤キットであって、
    　さらにインターフェロンαを含む、キット。     The anticancer agent kit according to claim 19,
    Furthermore, the kit containing interferon alpha.
  21. 　５－フルオロウラシル感受性抑制剤であって、
    　ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子からなる群から選ばれる１以上の遺伝子の発現を抑制するｓｉＲＮＡ、ｓｈＲＮＡまたはアンチセンスＲＮＡを含む、抑制剤。     A 5-fluorouracil sensitivity inhibitor,
    An inhibitor comprising siRNA, shRNA, or antisense RNA that suppresses the expression of one or more genes selected from the group consisting of a PRAK2 gene, a TGFBR2 gene, and an EXT1 gene.
  22. 　５－フルオロウラシル感受性を判定するための診断薬であって、
    　ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子からなる群から選ばれる１以上の遺伝子の発現量を測定するための試薬を含む、診断薬。     A diagnostic agent for determining 5-fluorouracil sensitivity,
    A diagnostic agent comprising a reagent for measuring the expression level of one or more genes selected from the group consisting of a PRAK2 gene, a TGFBR2 gene, and an EXT1 gene.
  23. 　請求項２２記載の診断薬において、
    　前記試薬が、ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子の塩基配列に相補的な塩基配列を含むプローブである、診断薬。     The diagnostic agent according to claim 22,
    A diagnostic agent, wherein the reagent is a probe comprising a base sequence complementary to the base sequences of the PRAKG2 gene, the TGFBR2 gene, and the EXT1 gene.
  24. 　請求項２３記載の診断薬において、
    　前記プローブが蛍光色素または放射性同位元素で標識されている、診断薬。     The diagnostic agent according to claim 23,
    A diagnostic agent, wherein the probe is labeled with a fluorescent dye or a radioisotope.
  25. 　５－フルオロウラシル感受性を判定するための診断薬であって、
    　ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子からなる群から選ばれる１以上の遺伝子がコードする蛋白質の発現量を測定するための試薬を含む、診断薬。     A diagnostic agent for determining 5-fluorouracil sensitivity,
    A diagnostic agent comprising a reagent for measuring an expression level of a protein encoded by one or more genes selected from the group consisting of a PRAK2 gene, a TGFBR2 gene, and an EXT1 gene.
  26. 　請求項２５記載の診断薬において、
    　前記試薬が、ＰＲＡＫＧ２遺伝子、ＴＧＦＢＲ２遺伝子およびＥＸＴ１遺伝子からなる群から選ばれる１以上の遺伝子のコードする蛋白質に特異的に結合する抗体である、診断薬。     The diagnostic agent according to claim 25,
    A diagnostic agent, wherein the reagent is an antibody that specifically binds to a protein encoded by one or more genes selected from the group consisting of PRAKG2 gene, TGFBR2 gene, and EXT1 gene.
  27. 　請求項２６記載の診断薬において、
    　前記抗体が蛍光色素または放射性同位元素で標識されている、診断薬。     The diagnostic agent according to claim 26,
    A diagnostic agent, wherein the antibody is labeled with a fluorescent dye or a radioisotope.
  28. 　抗癌剤の感受性増強作用を有する遺伝子のスクリーニング方法であって、
    　（ｍ）哺乳動物の所定の癌細胞株にリボザイムライブラリーを導入する工程と、
    　（ｎ）前記リボザイムライブラリーが導入された癌細胞を所定の抗癌剤で処理する工程と、
    　（ｏ）前記抗癌剤で処理された癌細胞のうち生存細胞からリボザイムを回収して新規なリボザイムライブラリーを得る工程と、
    　を含み、
    前記リボザイムライブラリーが、標的認識配列特異的にｍＲＮＡを切断し、遺伝子発現を抑制する複数のリボザイムを含み、各リボザイムが認識する標的認識配列がランダム化された複数の配列である、ランダム化リボザイムライブラリーであり、
    　（ｐ）前記（ｍ）、（ｎ）および（ｏ）の工程を複数回繰り返して得られるリボザイムライブラリーが認識する標的認識配列を検出する工程と、
    　（ｑ）前記検出された標的認識配列に対応する遺伝子情報をゲノムデータベースから抽出する工程と、
    　をさらに含む、スクリーニング方法。     A method for screening a gene having an effect of enhancing the sensitivity of an anticancer drug,
    (M) introducing a ribozyme library into a predetermined cancer cell line of a mammal;
    (N) treating the cancer cells into which the ribozyme library has been introduced with a predetermined anticancer agent;
    (O) recovering ribozymes from viable cells among the cancer cells treated with the anticancer agent to obtain a novel ribozyme library;
    Including
    Randomized ribozyme, wherein the ribozyme library includes a plurality of ribozymes that cleave mRNA specifically for a target recognition sequence and suppress gene expression, and the target recognition sequences recognized by each ribozyme are a plurality of randomized sequences. Library,
    (P) detecting a target recognition sequence recognized by a ribozyme library obtained by repeating the steps (m), (n) and (o) multiple times;
    (Q) extracting gene information corresponding to the detected target recognition sequence from a genome database;
    A screening method further comprising:
  29. 　請求項２８記載のスクリーニング方法において、
    　前記（ｐ）の工程が、各リボザイムに標識された各標的認識配列に特異的な波長を有する蛍光色素を検出する工程を含む、
    　スクリーニング方法。     The screening method according to claim 28, wherein
    The step (p) includes a step of detecting a fluorescent dye having a wavelength specific to each target recognition sequence labeled on each ribozyme.
    Screening method.
PCT/JP2011/059192 2010-04-14 2011-04-13 GENE GROUP CAPABLE OF ENHANCING ANTI-TUMOR EFFECT OF 5-FU ALONE OR IFN-α/5-FU COMBINATION WO2011129371A1 (en)

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WO2018230712A1 (en) * 2017-06-16 2018-12-20 学校法人同志社 NOVEL SCREENING METHOD FOR TGF-β INHIBITOR
JPWO2018230712A1 (en) * 2017-06-16 2020-04-16 学校法人同志社 Novel screening method for TGF-β inhibitor
US11433090B2 (en) 2017-06-16 2022-09-06 The Doshisha mTOR-inhibitor-containing medicine for treating or preventing ophthalmic symptoms, disorders, or diseases, and application thereof

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