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WO2002020770A1 - Method of screening antitumor agent by using interaction between arf protein and hk33 protein - Google Patents

Method of screening antitumor agent by using interaction between arf protein and hk33 protein Download PDF

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Publication number
WO2002020770A1
WO2002020770A1 PCT/JP2001/007732 JP0107732W WO0220770A1 WO 2002020770 A1 WO2002020770 A1 WO 2002020770A1 JP 0107732 W JP0107732 W JP 0107732W WO 0220770 A1 WO0220770 A1 WO 0220770A1
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Prior art keywords
protein
arf
gene
expression
interaction
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Ceased
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PCT/JP2001/007732
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French (fr)
Japanese (ja)
Inventor
Takashi Sugihara
Renu Wadhwa
Sunil C. Kaul
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Chugai Pharmaceutical Co Ltd
Chugai Research Institute for Molecular Medicine Inc
National Institute of Advanced Industrial Science and Technology AIST
Original Assignee
Chugai Pharmaceutical Co Ltd
Chugai Research Institute for Molecular Medicine Inc
National Institute of Advanced Industrial Science and Technology AIST
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Application filed by Chugai Pharmaceutical Co Ltd, Chugai Research Institute for Molecular Medicine Inc, National Institute of Advanced Industrial Science and Technology AIST filed Critical Chugai Pharmaceutical Co Ltd
Priority to JP2002525777A priority Critical patent/JPWO2002020770A1/en
Priority to AU2001284454A priority patent/AU2001284454A1/en
Publication of WO2002020770A1 publication Critical patent/WO2002020770A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • 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
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • G01N33/575—
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to a method for screening a drug having an antitumor effect utilizing an interaction between an ARF protein and a HK33 protein.
  • the present invention also relates to a method for regulating ARF protein-mediated signal transduction, p53 protein-mediated signal transduction, and cell proliferation by regulating the interaction or the expression of the HK33 gene.
  • Ryoaki Motomoto also relates to the use of the HK33 protein and the gene encoding the 33 protein for testing in diseases such as tumors.
  • pRb tumor suppressor retinoblas toma protein
  • p53 Tumor suppressor retinoblas toma protein
  • cdks cyclin dependent kinases
  • INK4A and ARF present in this INK4a gene locus are located on the same gene, and are translated as different splicing forms, and both factors arrest the cell cycle in the G1 phase and have cytostatic activity. It was revealed to have. The difference between the functions of the two factors is that INK4a inactivates cyclin-dependent kinase 4 (CDK4) and inhibits the above-mentioned phosphorylation of Rb, among other substrates. Arrested.
  • CDK4 cyclin-dependent kinase 4
  • ARF is thought to bind to MDM2 in the nucleus to suppress the degradation of p53 by MDM2, enhance the stability of p53 protein, and arrest the cell cycle (Serrano, M. et al. (1993) Nature, 366, 704-707; Jo, DE et al. (1993) Genes Dev., 7, 1559-1571).
  • HK33 was discovered by Andreeas Braun et al. In 1994, and is present in the Pero xisomal membrane.
  • HK33 is known as a human homologous gene to yeast Pexl9p, and its product has been suggested to be a protein that is phnesylated and involved in biosynthesis in peroxisomes (Ka Thigherer, S. et al. (1997) Genomics , 45, 200-21; Braun, A. et al. (1994) Gene, 146, 291-295). Recently, it was reported as a causal gene of the Zellweger syndrome group (Matsuzono, Y. et al., Proc. Natl. Acad. Sci. USA 96: 2116-21, 1999). However, no relationship with ARF or p53 is known. Disclosure of the invention
  • An object of the present invention is to provide a method for screening a drug having an antitumor effect utilizing an interaction between an ARF protein and a HK33 protein.
  • the present invention also provides a method for regulating the interaction or ARF protein-mediated signal transduction by regulating the expression of the HK33 gene, particularly regulating cell cycle and cell division, and a molecule used for the same.
  • the task is to The method of the present invention is useful for suppressing the growth of tumor cells. It is for.
  • Another object of the present invention is to provide the use of HK33 protein and a gene encoding the HK33 protein for testing in diseases such as tumors.
  • the present inventors have attempted to isolate a gene encoding a protein interacting with pl9ARF by the yeast Two-Hybrid method in order to solve the above problems.
  • a pharmacoprotein called the HK33 gene (Kammerer, S. et al. (1997) Genomics, 45, 200-21; Braun, A. et al. (1994) Gene, 146, 291-295) It was found to bind to 9ARF protein.
  • an experiment was performed by a mammalian two-hybrid method using C0S7 cells.
  • the interaction between these two proteins was also confirmed by the mammalian Two-Hybrid method.
  • a mutant that encodes P19ARF8-Stop, pl9ARF-80 did not bind to HK33 protein, and interaction was confirmed only at P19ARF81-Stop. did it. Therefore, it was shown that the HK33 protein interacts with a pl9ARF-specific site without binding to a site shared with pl6IM4a. Furthermore, the interaction was confirmed again by immunoprecipitation. As a result, a significant interaction between the HK33 protein and the ARF protein was found.
  • pl9ARF the localization of pl9ARF in cells was determined by co-transfection of GFP-HK33 and pl9ARf3 ⁇ 4yc, or transfected with pl9ARFmyc alone or GFP-HK33 alone, and the localization of each protein was observed. It was shown that the protein significantly inhibited pl9ARFmyc translocation to the nucleus. In addition, as a result of in vitro experiments using a reporter gene responsive to p53, it was found that HK33 significantly inhibited the p53-dependent transcriptional activity by pl9ARF.
  • HK33 may itself be a factor that causes immortalization. That is, the interaction between HK33 protein and ARF protein was suppressed. Screening for a drug that suppresses this will enable the development of a new drug that can suppress cell proliferation due to inactivation of ARF or suppress the proliferation of immortalized premalignant cells.
  • the HK33 protein and the gene encoding it are useful for controlling signal transduction via the ARF protein, as an index for diagnosis and testing of diseases such as cancer, and It can be used as a tool for elucidating pathological mechanisms and as a target for drug development for these diseases.
  • the present invention relates to a method for screening a drug having an antitumor effect utilizing an interaction between an ARF protein and a HK33 protein.
  • the present invention relates to a method for regulating ARF protein-mediated signal transduction and cell proliferation by regulating the interaction or expression of the HK33 gene.
  • the present invention relates to the use of the HK33 protein and the gene encoding the HK33 protein for testing for diseases such as tumors. The present invention more specifically relates to
  • a pharmaceutical composition comprising, as an active ingredient, a compound that regulates the expression of the HK33 gene, or a compound that regulates the interaction between the ARF protein and the HK33 protein.
  • the cell proliferative disease is a tumor, the test reagent of (d),
  • a method for examining abnormal ARF-mediated signal transduction, abnormal p53-mediated signal transduction, abnormal cell proliferation, cell proliferative disease, or tumor including the following steps (a) or (b):
  • the HK33 gene includes the housekeeping gene HK33 (Braun, A. et al., Gene 146: 291-295, 1994), its homologous gene, isoform, and derivatives thereof. HK33 is also called PXF or PEX19p.
  • the HK33 gene may be a human gene, or may be a gene derived from a non-human eukaryote. Examples of mammalian HK33 homologous genes include human PXF (ACCESSION X75535, Locus HSPXF), mouse PxF (ACCESSION Y09046, Locus drawing PXFBALB), rat PxF (ACCESSIO N Y09049, Locus RRPXFPRT) and the like.
  • the ARF gene includes ARF (alternative reading frame) (Quelle, DE et al., Cell 83: 993-1000, 1995), homologous genes, isoforms, and derivatives thereof.
  • ARF is also referred to as pl9 ARF or pl4 ARF .
  • ARF is known to be encoded at the chromosome INK4a site.
  • human pl4-CDK inhibitor pl4ARF
  • pl4ARF ACCESSION U17075, Locus HSU17 075
  • mouse pl9 ARF ACCESSION L76092, Locus MUSARF
  • protein refers to an action between proteins, including binding, modification, change in activity, induction of structural changes, and induction of changes in stability.
  • signal transduction via ARF protein refers to an action mediated by ARF protein.
  • signal transduction via p53 protein refers to an action mediated by p53 protein.
  • the present invention is based on the new finding that HK33 protein interacts with ARF protein and regulates the function of p53 protein. As shown in the Examples, HK33 protein exhibits an activity of binding to ARF protein, and nuclear localization of ARF protein is inhibited by expression of HK33 protein.
  • ARF is one of the factors regulating cell cycle-related signal transduction, and is known to bind to MDM2 and suppress the degradation of p53 by MDM2. It has also been suggested that the ARF protein modulates p53 function by interacting directly with p53.
  • HK33 protein binds to ARF protein by expression of HK33 gene, preventing nuclear localization of ARF By being harmed, the activity of ARF in cells is suppressed, and signal transmission via ARF is suppressed. Indeed, HK33 expression significantly inhibited p53-dependent transcriptional activity.From these facts, it was suggested that the interaction between ARF protein and HK33 protein or that the signal transduction through ARF protein could be increased by increasing the expression of HK33 gene. It is possible to inhibit the signal transduction through the ARF protein by reducing the interaction between the ARF protein and the HK33 protein or reducing the expression of the HK33 gene.
  • the interaction between the ARF protein and the HK33 protein includes the binding and modification of the ARF protein with the HK33 protein.
  • the interaction between the ARF protein and the HK33 protein can be detected by detecting the binding of the two proteins or by the ARF function or signal transduction (ARF nuclear translocation, MDM2 and ARF interaction) caused by the interaction between the ARF protein and the HK33 protein. Interaction, including stabilization of p53, promotion of p53-mediated signal transduction, etc.). Expression of the HK33 gene can be measured by detecting its transcript or protein.
  • a vector expressing the HK33 gene may be introduced into cells to increase the intracellular expression of the HK33 protein.
  • the expression level of the HK33 protein in the cell increases, and the activity of the HK33 protein in the cell can be increased.
  • the expression of the HK33 gene can be reduced by, for example, expression of antisense RNA against HK33 mRNA or expression of a liposome that cleaves HK33 raRNA.
  • it can be carried out by administering an antisense oligonucleotide or a decoy nucleic acid containing a part of the transcription regulatory region of HK33.
  • a mutation may be introduced into the HK33 gene.
  • the interaction between the ARF protein and the HK33 protein can be reduced by acting an antagonist of the HK33 protein.
  • the antagonist of the HK33 protein may be an antibody against HK33 or a fragment thereof. It may also be a partial peptide of the ARF protein. You.
  • the partial peptide of mouse ARF protein from the 81st amino acid onward has the activity of binding to HK33 protein.
  • Such a partial peptide of the ARF protein having an activity of binding to the HK33 protein can suppress the interaction of the HK33 protein with the intact ARF protein through binding to the HK33 protein.
  • ARF-mediated signal transduction regulators p53-mediated signal transduction regulators
  • cell growth regulator p53-mediated signal transduction regulators
  • Regulation of ARF-mediated signal transduction includes regulation of ARF protein activity.
  • Examples of the ARF activity include an activity of the ARF protein such as binding to a protein.
  • Regulation of signal transduction via the ARF protein includes regulation of changes in the activity of signaling molecules downstream from the ARF protein and concomitant changes in traits. That is, promotion of signal transduction via ARF protein includes suppression of MDM2 activity such as interaction between MDM2 protein and p53 protein, stabilization of p53 protein by suppression of MDM2 activity, and signal transduction through p53 protein by stabilization. Activation (including promotion of p53-dependent transcription, suppression of cell division and cell proliferation, induction of apoptosis, etc.).
  • the p53 protein is a protein that plays an important role in regulating cell functions and is also known as a tumor suppressor gene.
  • the p53 protein By increasing or decreasing the expression of the HK33 gene or the interaction between the ARF protein and the HK33 protein, it is possible to inhibit or promote this signal transmission via p53, respectively. If the expression of the HK33 gene or the interaction between the ARF protein and the HK33 protein is increased, the activity of p53 can be suppressed, and cell division and cell proliferation can be promoted, apoptosis can be suppressed, and the like.
  • the expression of the HK33 gene or the interaction between the ARF protein and the HK33 protein is reduced, the activity of p53 can be promoted, suppressing cell proliferation and inducing apoptosis. Inhibition of intracellular activity of HK33 protein is particularly useful for treating B-severe ulcer.
  • an increase in the expression level of the HK33 gene was observed. This fact makes the cells immortalize cancer This suggests that the HK33 protein is involved in metabolism, indicating that reducing the activity of the HK33 protein is important for suppressing tumor growth.
  • compounds that regulate (increase or decrease) the expression of the HK33 gene or the interaction between the ARF protein and the HK33 protein are useful for regulating signal transduction via the ARF protein.
  • the present invention provides a regulator of ARF protein-mediated signal transduction, including a compound that regulates (increases or decreases) the expression of the HK33 gene or the interaction between the ARF protein and the HK33 protein.
  • these compounds are useful as modulators of p53-mediated signal transmission and as cell growth regulators.
  • These compounds are also used as carcinogens (or cell immortalizing agents, carcinogens, etc.) or tumor suppressors (anticancer agents).
  • the above compounds include compounds that regulate the biochemical activity (eg, binding activity to ARF) of the HK33 protein, as well as compounds that regulate the expression level of the HK33 gene in cells or tissues. included.
  • Examples of the compound that increases the expression of the HK33 gene or the interaction between the ARF protein and the HK33 protein include the HK33 protein and a DNA encoding the protein.
  • the expression level of HK33 can be increased by introducing a vector expressing the HK33 protein into cells.
  • the present invention relates to the use of an HK33 protein, a DNA encoding the protein, and a vector containing the DNA, for controlling signal transduction via ARF protein or p53 or cell growth.
  • DNA encoding the HK33 protein can be prepared by a known method.
  • the amino acid sequence of human HK33 protein and the nucleotide sequence of cDNA encoding HK33 protein are already known (Kammerer, S. et al., Genomics 45: 200-210, 1997; Braun, A. Gene). 146: 291-295, 1994).
  • DNA encoding HK33 can be obtained, for example, by hybridization using a probe prepared based on this base sequence, or by polymerase chain reaction (PCR) using a primer prepared based on this base sequence. (Sambrook, J. et al , Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. Press, 1989).
  • DNA encoding the HK33 protein can be isolated, for example, from humans or non-human mammals (eg, proteins encoded by monkeys, mice, rats, guinea pigs, egrets, mice, pigs, etc.). HK33 is expressed in various tissues, and cDNA prepared from these tissues can be applied to screening using hybridization or PCR. Hybridization conditions can be appropriately selected by those skilled in the art. For example, the reaction can be performed under low stringent conditions, normal conditions, or stringent conditions.
  • Stringent hybridization conditions include, for example, washing after hybridization at 50 ° C., 2 ⁇ SSC, 0.1% SDS, preferably 50 ° C., 0.1 ⁇ SSC, 0.1 ⁇ SSC. Wash with 1% SDS. More preferably, for example, washing with 65 ° C., 0.1 ⁇ SSC and 0.1% SDS can be mentioned. Under these conditions, it can be expected that DNA with higher homology can be obtained more efficiently as the temperature is increased. However, a plurality of factors such as temperature and salt concentration can be considered as factors affecting the stringency of the hybridization, and those skilled in the art can realize the same stringency by appropriately selecting these factors. Is possible. As described above, proteins that inhibit the nuclear translocation of the ARF protein, which are proteins encoded by DNA that hybridizes with the cDNA encoding the known HK33 protein, are included in the HK33 protein in the present invention.
  • the protein encoded by the DNA isolated by these hybridization techniques or gene amplification techniques is usually the human HK33 protein (Braun, A. et al., Gene 146: 29 1-295, 1994) or other organisms.
  • Has high homology to homologous proteins and amino acid sequences Among the proteins having high homology to these proteins, those having the activity of inhibiting the nuclear translocation of the ARF protein are included in the HK33 protein in the present invention.
  • High homology generally means at least 40% or more identity, preferably 60% or more identity, more preferably 80% or more identity, at the amino acid level. Preferably, it indicates 90% or more identity.
  • the algorithm described in the literature Wang, WJ and Lipman, DJ, Pro Natl. Acad. Sci. USA (1983) 80, 726-730
  • the algorithm described in the literature Wang, WJ and Lipman, DJ, Pro Natl. Acad. Sci. USA (1983) 80, 726-730
  • a mutant of the natural HK33 protein can also be used. Such mutations may be artificial or may occur in nature.
  • a method well known to those skilled in the art for preparing a mutant of a certain protein a method of introducing a mutation into a protein is known. For example, those skilled in the art can use site-directed mutagenesis (Hashimot o-Gotoh, T. et al. (1995) Gene 152, 271-275, Zoller, MJ, and Smith, M. (1983) Methods Enzymol. 100, 468-500, Kramer, W. et al. (1984) Nucleic Acids Res. 12, 9441-9456, Kramer W., and Fritz HJ (1987) Methods.
  • a mutant protein can be prepared.
  • a protein having an amino acid sequence in which one or more amino acids are mutated in the amino acid sequence of the natural HK33 protein and having an activity of inhibiting nuclear translocation of the ARF protein is also referred to as the HK33 protein in the present invention. included.
  • the number of amino acids to be mutated in such a mutant is usually within 20 amino acids, preferably within 10 amino acids, more preferably within 5 amino acids, and still more preferably within 3 amino acids (eg, 1 amino acid). ). It is expected that the effect on the activity of the protein can be reduced if the amino acid residue to be mutated is mutated to another amino acid whose amino acid side chain properties are conserved.
  • amino acid side chains include hydrophobic amino acids (A, I, M, F, P, W, Y, V) and hydrophilic amino acids (R, D, N, E, Q, G, H, K, S, T), amino acids with aliphatic side chains (G, A, V, I, P), amino acids with hydroxyl-containing side chains (S, T, ⁇ ), sulfur atom-containing side chains (C, M) having amino acid, amino acid having carboxylic acid and amide-containing side chain (D, N, E, Q), amino acid having base side chain (R, K, ⁇ ), containing aromatic Amino acids having side chains (H, F, Y, W) can be mentioned (all brackets indicate one-letter designation of amino acid). Substitutions within amino acids that are similar in these properties are called conservative substitutions.
  • the HK33 protein includes a protein having a conservative amino acid substitution.
  • the HK33 protein may be, for example, a protein in which a plurality of amino acid residues are added to the amino acid sequence of a natural HK33 protein.
  • a fusion protein is a fusion of the natural H K33 protein and another peptide or protein, and is included in the H K33 protein of the present invention.
  • DNA encoding the HK33 protein and DNA encoding another peptide or protein are ligated in frame so that they are introduced into an expression vector and expressed in a host.
  • Well, techniques known to those skilled in the art can be used.
  • Other peptides or proteins to be subjected to the fusion are not particularly limited.
  • peptides to be fused include, for example, FLAG (Hopp, TP et al., BioTechnology (1988) 6, 1204-1210), 6 XHis consisting of 6 His (histidine) residues, 10 XHis, Influenza agglutinin (HA), human c-myc fragment, VSV-GP fragment, pl8HIV fragment, T7-tag, HSV_tag, E-tag, SV40T antigen fragment, lck tag, hy-tu bulin fragment, B
  • FLAG Hopp, TP et al., BioTechnology (1988) 6, 1204-1210
  • 6 XHis consisting of 6 His (histidine) residues
  • 10 XHis Influenza agglutinin (HA), human c-myc fragment, VSV-GP fragment, pl8HIV fragment, T7-tag, HSV_tag, E-tag, SV40T antigen fragment, lck tag, hy-tu bulin
  • proteins to be fused include, for example, GST (glutathione S-transferase), HA (influenza agglutinin), Region, ⁇ -galactosidase, ⁇ (maltose binding protein) and the like.
  • a fusion protein can be prepared by fusing commercially available DNA encoding these peptides or proteins with DNA encoding # 33 protein and expressing the fusion DNA thus prepared.
  • the amino acid sequence, molecular weight, isoelectric point, presence / absence and form of sugar chains, etc. of the produced protein may differ depending on the cell, host, or purification method that produces the protein described below. However, as long as the protein has the activity of inhibiting nuclear translocation of the ARF protein, the protein is included in the 33 protein in the present invention. For example, when the ⁇ 33 protein is expressed in a prokaryotic cell, for example, Escherichia coli, a methionine residue is added to the ⁇ terminus of the amino acid sequence of the original protein, and such a protein is also included.
  • # 33 protein can be prepared as a recombinant protein or a natural protein by methods known to those skilled in the art. If it is a recombinant protein, DNA encoding the ⁇ 33 protein (for example, see Braun, A. et al., Gene 146: 291-295, 1994 for human ⁇ 33 cDNA) is inserted into an appropriate expression vector, and this is inserted into an appropriate expression vector. After transformants obtained by introducing the cells into the host cell are collected and an extract is obtained, chromatography such as ion exchange, reverse phase, gel filtration, etc., or an antibody immobilized on a column with antibodies against HK33 protein It can be purified and prepared by subjecting it to tea chromatography or by further combining a plurality of these columns.
  • DNA encoding the ⁇ 33 protein for example, see Braun, A. et al., Gene 146: 291-295, 1994 for human ⁇ 33 cDNA
  • transformants obtained by introducing the cells into the host cell are collected and an extract is obtained
  • the HK33 protein When the HK33 protein is expressed in a host cell (for example, an animal cell or Escherichia coli) as a fusion protein with a glutathione S-transferase protein or as a recombinant protein to which multiple histidines are added, the expressed recombinant is used.
  • the protein can be purified using a glutathione column or a nickel column. After purification of the fusion protein, if necessary, regions other than the target protein in the fusion protein can be cleaved with thrombin or Factor Xa and removed.
  • the protein is a natural protein, a method known to those skilled in the art, for example, an affinity column to which an antibody that binds to the HK33 protein described below is allowed to act on a tissue or cell extract expressing the HK33 protein, as described below. And can be isolated by purification.
  • Antibodies may be polyclonal or monoclonal.
  • DNA encoding the HK33 protein is used for in vivo and in vitro production of the HK33 protein. Further, application to gene therapy for increasing the activity of HK33 protein is also considered.
  • the DNA encoding the HK33 protein may be in any form as long as it can encode the HK33 protein. That is, it does not matter whether it is cDNA synthesized from raRNA, genomic DNA, or chemically synthesized DNA. In addition, DNAs having any nucleotide sequence based on the degeneracy of the genetic code are included as long as they can encode the HK33 protein.
  • DNA encoding the HK33 protein can be prepared by a method known to those skilled in the art.
  • a cDNA library is prepared from cells expressing the HK33 protein, and a library of HK33 cDNA (for example, see Braun, A. et al., Gene 146: 291-295, 199 for human HK33 cDNA) is prepared. It can be prepared by performing hybridization using the part as a probe.
  • the cDNA library may be prepared, for example, by the method described in the literature (Sambrook, J. et al., Molecular Cloning ⁇ Cold Spring Harbor Laboratory Press (1989)), or a commercially available DNA library may be prepared. May be used.
  • RNA from cells expressing the HK33 protein synthesizing cDNA with reverse transcriptase, synthesizing oligo DNA based on the HK33 cDNA sequence, and performing PCR using this as a primer, It can also be prepared by amplifying a cDNA encoding a protein.
  • the translation region encoded by the cDNA can be determined, and the amino acid sequence of the HK33 protein can be obtained.
  • Genomic DNA can be isolated by screening the genomic DNA library using the obtained cDNA as a probe.
  • the gene encoding the HK33 protein Can also be obtained.
  • the transcriptional regulatory region of the human HK33 gene has been reported so far (Karamerer, S. et al., Genomics 45: 200-210, 1997) ⁇
  • mRNA is isolated from cells, tissues and organs that express the ⁇ 33 protein.
  • mRNA can be isolated by known methods, for example, guanidine ultracentrifugation (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299), and AGPC method (Chomczynski, P. and Sacchi, N., Anal.
  • Total RNA is prepared using Biochem. (1987) 162, 156-159) and the like, and the total RNA is purified using mRNA Purification Kit (Pharmacia) and the like.
  • mRNA can be directly prepared by using QuickPrep mRNA Purification Kit (Pharmacia).
  • CDNA is synthesized from the obtained mRNA using reverse transcriptase.
  • the cDNA can also be synthesized using AMV Reverse Transcriptase Fis- terstrand cDNA Synthesis Kit (Seikagaku Corporation) or the like.
  • the 5'-Afflpli FINDER RACE Kit manufactured by Clontech
  • the 5, -RACE and 3, -RACE methods using PCR Frohman, MA et al., Proc. Natl. Acad. Scad. USA (1988) 85, 8 998-9002
  • cDNA synthesis and amplification can be performed according to Belyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932).
  • a target DNA fragment is prepared from the obtained PCR product and ligated to a vector DNA. Further, a recombinant vector is prepared from this, introduced into E. coli, etc., and colonies are selected to prepare a desired recombinant vector.
  • the base sequence of the target DNA can be confirmed by a known method, for example, a dideoxynucleotide chain termination method.
  • a nucleotide sequence with higher expression efficiency can be designed in consideration of the codon usage of the host used for expression (Grantham, R. et al., Nucleic Acids Research (1981) 9 , r43-74).
  • the DNA sequence can be modified by a commercially available kit known method. Modifications include, for example, digestion with restriction enzymes, insertion of synthetic oligonucleotides--appropriate DNA fragments, Addition, insertion of an initiation codon (ATG) and Z or a termination codon (TAA, TGA, or TAG).
  • an expression vector is constructed and introduced into a host cell.
  • a vector for example, when E. coli is used as a host, the vector is amplified in E. coli (e.g., JM109, DH5, HB101, XLlBlue), etc. of
  • vectors include M13-based vectors, pUC-based vectors, pBR322, pBluescript, pCR-Script, and the like.
  • pGEM-T pDIRECT, pT7 and the like can be mentioned in addition to the above vectors.
  • Expression vectors are particularly useful when vectors are used to produce the 33 protein.
  • the expression vector when the expression vector is intended for expression in Escherichia coli, the expression vector may have the above-mentioned characteristics such that the vector is amplified in Escherichia coli, and may be used in a host such as E. coli such as JM109, DH5a, HB101, or XL1-Blue.
  • E. coli such as JM109, DH5a, HB101, or XL1-Blue.
  • a promoter that can be efficiently expressed in Escherichia coli such as the lacZ promoter (Ward et al., Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422 2427), the araB promoter
  • Such vectors include pGEX-5 ⁇ ⁇ -1 (Pharmacia), ⁇ QIAexpress systemj (Qiagen), pEGFP, or pET (in this case, the host is BL21 that expresses T7 RNA polymerase). Is preferred, etc.).
  • the vector may also include a signal sequence for polypeptide secretion.
  • a signal sequence for protein secretion the pelB signal sequence (Lei, SP et al J. Bacteriol. (1987) 16 9, 4379).
  • Introduction of a vector into a host cell can be performed using, for example, a calcium chloride method or an electroporation method.
  • a mammal-derived expression vector for example, pcDNA3 (manufactured by Invitrogen) or pEGF-BOS (Nucleic Acids. Res.
  • insect cell-derived expression vectors eg, “Bac-to-BAC baculovairus expression systemj (manufactured by Gibco BRL), pBacPAK8), plant-derived expression vectors (eg, ⁇ 1, pMH2), animal virus-derived expression vectors (eg, pHSV, pMV, pAdexLc), retrovirus-derived expression vectors (eg, pZIPneo), yeast-derived expression vectors (eg, “Pichia Expression KitJ (Invitrogen) ), PNVll, SP-Q01), and expression vectors derived from Bacillus subtilis (eg, pPL608, pKTH50).
  • Bacillus subtilis eg, pPL608, pKTH50.
  • a promoter necessary for expression in cells for example, SV40 promoter (Mulligan et al., Nature (1979) 277, 108) , The MMLV-LTR promoter, the EF1 promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), the CMV promoter, etc., are essential, and the genes for selecting for cell transformation ( For example, it is more preferable to have a drug resistance gene that can be identified by a drug (neomycin, G418, etc.).
  • vectors having such properties include pMAM, pDR2, pBK-RSV, pBK-CMV, p0PRSV, p0P13, and the like.
  • a vector having a DHFR gene that complements a nucleic acid synthesis pathway-deficient CH0 cell is used.
  • pCHOI a vector having a DHFR gene that complements a nucleic acid synthesis pathway-deficient CH0 cell
  • MTX methotrexate
  • the gene expressing the SV40 T There is a method of transforming a vector having a replication origin of SV40 (such as pcD) using the COS cell possessed above.
  • the replication origins include poliovirus, adenovirus, Viruses (BPV) and the like can also be used.
  • the expression vector is used as a selectable marker such as aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, Escherichia coli xanthinguanine phosphoribosyltransferase (Ecogpt) gene, It can contain a dihydrofolate reductase (dhfr) gene and the like.
  • APH aminoglycoside transferase
  • TK thymidine kinase
  • dhfr dihydrofolate reductase
  • the DNA is incorporated into an appropriate vector, and the DNA is expressed in vivo by the retrovirus method, ribosome method, catonic ribosome method, adenovirus method, or the like. And other methods.
  • the vector used include, but are not limited to, adenovirus vectors (eg, pAdexlcw) and retrovirus vectors (eg, pZIPneo).
  • HK33 protein can be produced in vitro by transforming a host cell with DNA encoding the HK33 protein and culturing the transformed cell.
  • the culture can be performed according to a known method.
  • DMEM, MEM, RPMI1640, IMDM and the like can be used as a culture solution of animal cells.
  • a serum replacement solution such as fetal calf serum (FCS) may be used in combination, or serum-free culture may be performed.
  • FCS fetal calf serum
  • the pH during culturing is preferably about 6-8. Culture is usually performed at about 30 to 40 ° C for about 15 to 200 hours, and the medium is replaced, aerated, and agitated as necessary.
  • the host cell into which the vector is introduced is not particularly limited, and for example, Escherichia coli and various eukaryotic cells can be used.
  • eukaryotic cells for example, Animal cells, plant cells, and fungal cells can be used as hosts.
  • Animal cells include mammalian cells, for example, CH0, COS, 3T3, myeloma, BHK (baby hamster kid ney), HeLa, Vero, amphibian cells, for example, African Megafrog oocytes (Valle, et al., Nature ( 1981) 291, 358-340), or insect cells such as Sf9, Sf21 and Tn5.
  • CH0 cells include DHfr-CHO (Proc. Natl.Acad.Sci.
  • the vector can be introduced into a host cell by, for example, the calcium phosphate method, the DEAE dextran method, the method using Cationic Ribosome D0TAP (Boehringer Mannheim), the electoporation method, or the ribofusion method. It is.
  • a plant cell for example, a cell derived from Nicotiana tapacum (Nicotiana ta bacum) is known as a protein production system, and it may be callus cultured.
  • Fungal cells include yeast, for example, the genus Saccharomyces, for example, Saccharomyces cerevisiae, filamentous fungi, for example, the genus Aspergillus, for example, Aspergillus niger (A spergill ni). Have been.
  • prokaryotic cells there is a production system using bacterial cells.
  • the bacterial cell include Escherichia coli (E. coli), for example, JM109, DH5o; and HB101.
  • Bacillus subtilis is also known.
  • examples of a system for producing a protein in vivo include a production system using animals and a production system using plants.
  • the desired DNA is introduced into these animals or plants, and proteins are produced and recovered in the animals or plants.
  • a transgenic animal When using animals, there are production systems using mammals and insects. Goats, pigs, sheep, mice, and mice can be used as mammals (Vicki Glasser, SPECTRUM Biotechnology Applications, 1993).
  • a transgenic animal can be used.
  • a target DNA is prepared as a fusion gene with a gene encoding a protein that is specifically produced in milk, such as goat jS casein.
  • the DNA fragment containing the fusion gene is injected into a goat embryo, and the embryo is transplanted into a female goat.
  • the target protein can be obtained from milk produced by the transgenic goat born from the goat that has received the embryo or its progeny.
  • Hormones may be used in transgenic goats as appropriate to increase the amount of milk containing proteins produced by transgenic goats (Ebert, KM et al., Bio / Technology (1994) 12 , 699-702
  • silkworms can be used as insects, for example.
  • the target protein can be obtained from the body fluid of the silkworm by infecting the silkworm with a paculovirus into which DNA encoding the protein of interest has been inserted (Susu rau, M. et al. , Nature (1985) 315, 592-59.4).
  • tobacco when using a plant, for example, tobacco can be used.
  • DNA encoding the protein of interest is inserted into a plant expression vector, for example, pM0N530, and this vector is introduced into a bacterium such as Agrobacterium tumefaciens.
  • This terrier is infected with tapaco, for example, Nicotiana tabacum, and the desired polypeptide can be obtained from the leaves of this tapaco (Julian K. -C. Ma et al., Eur. J. Immunol. (1994) 24, 131-138).
  • the HK33 protein thus obtained can be isolated from the inside or outside of the host cell (such as a medium) and purified as a substantially pure and homogeneous protein.
  • the separation and purification of the protein may be performed by any of the separation and purification methods used in ordinary protein purification, and is not particularly limited. For example, chromatographic columns, filters, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. When combined, proteins can be separated and purified.
  • chromatography examples include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory). Course Manual. Ed D anie ⁇ R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These chromatographys can be performed using liquid phase chromatography, for example, liquid phase chromatography such as HPLC and FPLC. Using these purification methods, the HK33 protein can be highly purified.
  • the protein can be arbitrarily modified or partially removed by reacting the protein with an appropriate protein modifying enzyme before or after purification.
  • an appropriate protein modifying enzyme for example, trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, dalcosidase and the like are used.
  • the present invention also relates to a compound that reduces the expression of the HK33 gene or the interaction between the ARF protein and the HK33 protein.
  • the present invention also relates to the use of the compound for regulating signal transduction via ARF protein, regulating signal transduction via p53 protein, regulating cell proliferation, and preventing or treating tumors.
  • Such compounds include, for example, antisense nucleotides against the HK33 gene.
  • Antisense nucleotides are nucleotides that include a region complementary to the nucleotide encoding the HK33 protein.
  • the antisense nucleotide is a polynucleotide comprising at least 15 nucleotides complementary to the nucleotides encoding the HK33 protein.
  • complementary strand refers to one strand of a double-stranded nucleic acid consisting of a base pair of A: T (U for RNA) and G: C, and the other strand.
  • the term “complementary” is not limited to the case where the complementary nucleotide region is a completely complementary sequence, and is at least 70%, preferably at least 80%, more preferably 90%, and still more preferably 95% or more. It is only necessary to have the homology on the base sequence. The algorithm for determining homology is described in the literature (Wilbur, WJ and Lipman, DJ, Proc. Natl. Acad. Sci. USA (1 983) 80, 726-730).
  • Antisense nucleotides are useful as modulators of ARF-mediated signaling, p53-mediated signaling, and cell growth regulators. That is, the antisense nucleotide for HK33 promotes signal transduction via ARF, promotes signal transduction via p53, and suppresses cell proliferation. In addition, antisense nucleotides against HK33 can be used for the detection of the present invention described below.
  • nucleotides include nucleotides or nucleotide derivatives for controlling the expression of the HK33 gene (for example, antisense oligonucleotide ⁇ ribozyme or DNA encoding the same).
  • Antisense oligonucleotides include, for example, antisense oligonucleotides that hybridize to any part of the DNA sequence on the genome of the HK33 gene or the mRNA sequence transcribed from the gene.
  • the antisense oligonucleotide is preferably an antisense oligonucleotide to at least 15 or more consecutive nucleotides in the HK33 gene sequence or mRNA sequence. More preferably, it is an antisense oligonucleotide in which at least 15 or more consecutive nucleotides contain a translation initiation codon.
  • the antisense oligonucleotide derivatives and modifications thereof can be used.
  • the modified product include a modified lower alkyl phosphonate such as a methylphosphonate type or an ethylphosphonate type, a phosphorothioate modified product, a phosphoroamidate modified product, and the like.
  • Antisense oligonucleotides include not only those in which all nucleotides corresponding to nucleotides constituting a predetermined region of DNA or mRNA are complementary sequences, and those in which DNA or mRNA and an oligonucleotide are the HK33 gene sequence or mRNA sequence. As long as it can be hybridized to, it includes one or more nucleotide mismatches.
  • Oligonucleotide derivatives act on HK33 protein producing cells By binding to DNA or mRNA encoding the protein, transcription or translation thereof is inhibited, or mRNA degradation is promoted, and the expression of the HK33 gene is suppressed, resulting in the activity of the HK33 protein. It has the effect of suppressing.
  • the antisense oligonucleotide derivative can be mixed with a suitable base material which is inactive against the derivative to prepare an external preparation such as a liniment or a poultice. If necessary, excipients, isotonic agents, solubilizing agents, stabilizing agents, preservatives, soothing agents, etc. are added to tablets, splinters, granules, capsules, ribosome capsules, and injections. , Lyophilized agent, such as drug, solution, nasal drop, etc. These can be prepared according to a conventional method.
  • the antisense oligonucleotide derivative is applied directly to the affected area of the patient, or applied to the patient so that it can reach the affected area as a result of intravenous administration.
  • an antisense encapsulating material that enhances durability and membrane permeability can be used.
  • ribosome, poly-L-lysine, lipid, cholesterol, lipofectin or derivatives thereof can be mentioned.
  • the dose of the antisense oligonucleotide derivative can be appropriately adjusted according to the condition of the patient, and a preferred amount can be used. For example, it can be administered in the range of 0.1 to 100 mg / kg, preferably 0.1 to 50 mg / kg.
  • Antisense oligonucleotides inhibit the expression of the HK33 gene and are therefore useful in suppressing the biological activity of the HK33 protein. Further, an expression inhibitor containing an antisense oligonucleotide is useful because it can suppress the biological activity of HK33 protein.
  • the antisense nucleic acid of the HK33 gene serves as a regulator of signal transfer via the ARF protein, a regulator of signal transmission via the p53 protein, and a regulator of cell proliferation.
  • the present invention also relates to the use of antisense nucleotides against the HK33 gene to regulate signal transmission via the ARF protein or p53, or cell growth.
  • Compounds that reduce the interaction between the ARF protein and the HK33 protein include, for example, antibodies against the HK33 protein.
  • Antibody to HK33 protein is ARF protein , Signaling regulators via p53 protein, and cell growth regulators. That is, if the activity of the HK33 protein is inhibited by an antibody against the HK33 protein, signal transduction via the ARF protein can be promoted, signal transduction via the p53 protein can be promoted, and cell proliferation can be further suppressed.
  • the invention also relates to the use of antibodies to the HK33 protein to regulate signaling through the ARF protein, signaling through the p53 protein, and cell growth. Further, an antibody against the HK33 protein is also used in the detection of the present invention described below.
  • An antibody against the HK33 protein can be prepared by a known method. There is no particular limitation on the form of the antibody. In addition to polyclonal antibodies, monoclonal antibodies are included. Also included are antisera obtained by immunizing immunized animals such as rabbits with antigenic proteins, polyclonal antibodies and monoclonal antibodies of all classes, as well as human antibodies and humanized antibodies obtained by genetic recombination.
  • the protein used as a sensitizing antigen for obtaining an antibody is not limited to the animal species from which it is derived, but is preferably a protein derived from a mammal, for example, a human, a mouse or a rat, and particularly preferably a protein derived from a human.
  • Human-derived HK33 protein can be prepared as described above.
  • the protein used as the sensitizing antigen may be a complete protein or a partial peptide of the protein.
  • partial peptides of proteins include amino (N) terminal fragments and carboxy (C) terminal fragments of proteins.
  • a binding region to the ARF protein may be mentioned.
  • antibody refers to an antibody that reacts with the full length or fragment of a protein.
  • a gene encoding the HK33 protein or a fragment thereof is inserted into a known expression vector system, and a host cell is transformed with the vector.
  • the target protein or a fragment thereof is obtained from inside or outside the host cell by a known method. These may be used as sensitizing antigens.
  • a cell expressing the protein, a lysate thereof, or a chemically synthesized HK33 protein may be used as the sensitizing antigen.
  • Short peptides can be transferred to keyhole limpets
  • An antigen can be obtained by appropriately binding to a carrier protein such as serum, albumin, or ovalbumin.
  • the mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion in the production of a monoclonal antibody. Typically, rodent, heron, and primate animals are used.
  • mice for example, mice, rats, hamsters and the like are used.
  • a heronoid animal for example, a heron is used.
  • monkeys are used as primates.
  • monkeys of the lower nose are used (old world monkeys), for example, cynomolgus monkeys, macaques, baboons, and chimpanzees.
  • Immunization of an animal with a sensitizing antigen is performed according to a known method.
  • a sensitizing antigen is injected intraperitoneally or subcutaneously into a mammal.
  • the sensitizing antigen is diluted and suspended in an appropriate amount with PBS (Phosphate-Buffered Saline), physiological saline, or the like, and then mixed with an appropriate amount of a normal adjuvant, for example, Freund's complete adjuvant, if desired. After emulsification, it is administered to mammals. Thereafter, it is preferable to administer the sensitizing antigen mixed with an appropriate amount of Freund's incomplete adjuvant several times every 4 to 21 days.
  • a suitable carrier can be used at the time of immunization with the sensitizing antigen. Immunization is performed in this manner, and an increase in the desired antibody level in the serum is confirmed by a conventional method.
  • the blood of a mammal sensitized with the antigen is taken after confirming that the level of the desired antibody in the serum has increased.
  • the serum is separated from the blood by a known method.
  • a serum containing the polyclonal antibody may be used.
  • a fraction containing the polyclonal antibody may be further isolated from the serum and used. For example, using an affinity column in which the protein used for the antigen is coupled
  • immunoglobulin G or M can be prepared by obtaining a fraction that recognizes only the antigen protein and further purifying this fraction using a protein A or protein G column.
  • the immune cells may be removed from the mammal and subjected to cell fusion.
  • preferred immune cells used for cell fusion include splenocytes in particular.
  • the other parent cell to be fused with the immunocyte is preferably a mammalian myeloma cell, more preferably a myeloma cell that has acquired the properties for fusion cell selection by a drug.
  • the cell fusion of the immune cells and myeoma cells is basically a known method, for example, the method of Milstein et al. (Galfre, G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46). ) And so on.
  • the hybridoma obtained by cell fusion is selected by culturing it in a normal selective culture medium, for example, a HAT culture medium (a culture medium containing hypoxanthine, aminopterin and thymidine). Culturing in the HAT culture solution is continued for a time sufficient to kill cells other than the target hybridoma (non-fused cells), usually for several days to several weeks. Next, screening and cloning of hybridomas producing the desired antibody are performed by the usual limiting dilution method.
  • a HAT culture medium a culture medium containing hypoxanthine, aminopterin and thymidine
  • human lymphocytes for example, human lymphocytes infected with EB virus, are sensitized in vitro with proteins, protein-expressing cells or lysates thereof. And sensitized lymphocytes are fused with human-derived myeloma cells capable of permanent division, such as U266, to obtain a hybridoma that produces a desired human antibody having protein binding activity (see Published in Sho 63-17688.
  • the obtained hybridoma is transplanted into the peritoneal cavity of a mouse, ascites is collected from the mouse, and the obtained monoclonal antibody is used, for example, for ammonium sulfate precipitation, protein A, It can be prepared by purifying it with a mouth tin G column, DEAE ion exchange chromatography, or an affinity column to which the protein used as the antigen has been coupled.
  • the obtained antibody is used for detecting the HK33 protein and is also a candidate for the HK33 protein antagonist.
  • Antibodies to the HK33 protein are candidates for drugs that suppress the interaction between the HK33 protein and the ARF protein.
  • the antibodies may be human or human antibodies.
  • a transgenic animal having a repertoire of human antibody genes is immunized with a protein serving as an antigen, a protein-expressing cell or a lysate thereof to obtain antibody-producing cells, which are then fused with myeloma cells.
  • a human antibody against the protein see International Publication Nos. W092-03918, W093-2227, W094-02602, W094-25585, W096-33735 and W096-34096).
  • cells in which immune cells such as sensitized lymphocytes that produce antibodies are immortalized with oncogenes may be used.
  • the monoclonal antibody thus obtained can also be obtained as a recombinant antibody produced using a genetic recombination technique (for example, Borrebaeck, CAK and Larrick, JW, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United States). Kingdom by MCMILLAN PUBLISHERS LTD, 1990).
  • Recombinant antibodies are produced by cloning DNA encoding them from immunized cells such as hybridomas or sensitized lymphocytes that produce the antibodies, incorporating the DNA into an appropriate vector, and introducing it into a host.
  • antibody fragments include Fab, F (ab,) 2, Fv, or a single-chain Fv (scFv) (Fuston, JS et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883).
  • an antibody is treated with an enzyme, for example, papain or pepsin, to generate an antibody fragment, or a gene encoding these antibody fragments is constructed and introduced into an expression vector. Expression in a suitable host cell (eg, Co, MS et al., J. Immunol. (1994) 152, 2968-2976; Better, M.
  • Antibodies bound to various molecules such as polyethylene glycol (PEG) can also be used as modified antibodies.
  • the “antibody” referred to in the present invention also includes these modified antibodies.
  • Such an antibody modification can be obtained by chemically modifying the obtained antibody.
  • Antibodies can be derived from chimeric or non-human antibody-derived CDRs (negative-capacity determining regions) and non-human antibody-derived CDRs using known techniques. It can be obtained as a humanized antibody consisting of FR (framework region) and constant region.
  • the antibody obtained as described above can be purified to homogeneity.
  • the separation and purification of the antibody may be performed by the separation and purification methods used for ordinary proteins. For example, if appropriate selection and combination of chromatography columns such as affinity chromatography, filter, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, and isoelectric focusing, antibodies can be separated. It can be purified (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988). Power is not limited to these.
  • the concentration of the antibody obtained as described above can be measured by measuring absorbance or by enzyme-linked immunosorbent assay (ELISA).
  • Columns used for affinity chromatography include a protein A column and a protein G column.
  • Columns include Hyper D, POROS, Sepharose FF (Pharmacia), etc.
  • Examples of chromatography other than water chromatography include, for example, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, Adsorption chromatography and the like (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These chromatography methods can be carried out using liquid phase chromatography such as HP1 and FPLC.
  • a method for measuring the antigen-binding activity of the antibody for example, absorbance measurement, ELISA, EIA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay) or a fluorescent antibody method can be used.
  • ELISA Enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • HK33 protein is added to a plate on which antibodies are immobilized, and then a sample containing the target antibody, for example, a culture supernatant of antibody-producing cells or a purified antibody is added.
  • a secondary antibody that recognizes an enzyme for example, an antibody labeled with alkaline phosphatase
  • an enzyme substrate such as p-ditophenyl phenylphosphate
  • the antigen binding activity can be evaluated.
  • a fragment of the HK33 protein for example, a fragment comprising the C-terminal thereof may be used.
  • BIAcore Pharmacia
  • the HK33 protein or the partial peptide of the ARF protein can regulate the interaction between the ARF protein and the HK33 protein by inhibiting the binding between the original ARF protein and the HK33 protein. Therefore, the HK33 protein or the partial peptide of the ARF protein is a compound that regulates the interaction between the ARF protein and the HK33 protein, and is a regulator of ARF protein-mediated signal transduction, a p53 protein-mediated signal transduction regulator, It can be used as the above-mentioned various regulators such as growth regulators and antitumor agents.
  • Such a partial peptide has at least 7 amino acids or more, preferably 8 amino acids or more, and more preferably 9 amino acids. It consists of an amino acid sequence of no acid or higher.
  • the partial peptide can be produced by a genetic engineering technique, a known peptide synthesis method, or by cutting the HK33 protein or ARF protein with an appropriate peptide.
  • the peptide may be synthesized by, for example, either a solid phase synthesis method or a liquid phase synthesis method.
  • the present invention also relates to a method for screening compounds that regulates the interaction between ARF protein and HK33 protein.
  • One of the screening methods of the present invention is to a method for screening compounds that regulates the interaction between ARF protein and HK33 protein.
  • the interaction between the ARF protein and the HK33 protein can be detected by directly detecting the binding of the two proteins or indirectly by detecting the change caused by the interaction between the two proteins. Detection of interaction with the protein can be performed.
  • a screening system can be constructed in a test tube or in a cell. When performed intracellularly, by incubating cells expressing ARF and HK33 proteins in the presence of a test sample, the ARF protein expressed in cells and HK33 protein come into contact, and screening is performed through detection of the interaction between the two. It can be carried out.
  • the screening method using the cell line is included in the screening method described above.
  • a step of bringing a test sample into contact with cells expressing the ARF protein and the HK33 protein (B) selecting a compound that regulates the interaction between the ARF protein and the HK33 protein in comparison with (c) detecting the interaction in the absence of the test sample (control) Process.
  • This screening method using cells includes a screening method using ARF protein or p53 protein-mediated signal transmission described later as an index, in addition to screening using the Two Hybrid method described below, and the like.
  • One embodiment of the screening method of the compound for regulating the interaction between the ARF protein and the HK33 protein of the present invention comprises detecting the interaction between the ARF protein and the HK33 protein using the binding between the ARF protein and the HK33 protein as an indicator.
  • this screening method comprises: (a) a step of bringing the ARF protein into contact with the HK33 protein in the presence of the test sample; (b) a step of detecting the binding between the ARF protein and the HK33 protein; ) A step of selecting a compound that modulates the interaction, as compared to a case where detection is performed in the absence of a test sample (control).
  • the HK33 protein and ARF protein used for screening may be a recombinant protein or a naturally-occurring protein.
  • the origin of the protein is not limited, and proteins derived from eukaryotes including humans and other animals can be used.
  • a human-derived protein is used.
  • the protein may be a mutant, a partial peptide, or a fusion protein with another peptide.
  • Proteins may be, for example, purified proteins, soluble proteins, forms bound to carriers, fusion proteins with other proteins, forms expressed on cell membranes, or screen fractions as membrane fractions. Can be used for ling.
  • mouse ARF protein from which 1-80 amino acids have been deleted retains the ability to bind to HK33 protein.
  • the screening of the present invention can be carried out using the partial peptide of the ARF protein of No. 81 or later.
  • the test sample is not particularly limited.
  • cell culture supernatant fermented microorganism product, marine organism extract , Plant extracts, prokaryotic cell extracts, eukaryotic single cell extracts or animal cell extracts or their libraries, purified or crude proteins, peptides, non-peptidic compounds, synthetic low-molecular compounds, and natural compounds.
  • a vector expressing a fusion protein in which either the HK33 protein or the ARF protein or a partial peptide thereof is fused to an SRF DNA binding region or a GAL4 DNA binding region, and the other protein construct a vector in which the partial peptide is fused to a transcriptional activation region such as VP16 or GAL4, and introduce these into a yeast cell together with a reporter gene-encoding vector to obtain a sample containing a test compound. The compounds are assayed below using the reporter activity as an indicator. The binding of the HK33 protein and the ARF protein induces the expression of the reporter gene.
  • reporter gene examples include, but are not limited to, HIS3 gene, Ade2 gene, LacZ gene, CAT gene, luciferase gene, PAI-1 (Plasminogen activator inhibitor typel) gene and the like.
  • a cytotoxic gene can be expressed as a reporter gene.
  • Screening can also be performed using, for example, immunoprecipitation.
  • Cells expressing the HK33 protein and the ARF protein are cultured in the presence of the sample containing the test compound, and after the cells are collected, the complex is recovered using an antibody against one of the proteins, and then the other protein is recovered. By detecting with an antibody against the protein, the binding of both proteins can be evaluated.
  • Both proteins may be proteins that are expressed endogenously by cells, but either or both proteins can be expressed in cells exogenously.
  • the gene encoding the HK33 protein and / or ARF protein can be inserted into an exogenous gene expression vector such as pSV2neo, pcDNA I, or pCD8.
  • the gene is expressed in cells or the like. Promoters used for expression include the SV40 early promoter (Rigby, In Willia rason (ed.), Genetic Engineering, Vol. 3. Academic Press, London, p. 83-141 (1982)) and the EF-1a promoter (Kim et al. , Gene, p. 217-223 (1990)), CAG proraoter (Niwa et al., Gene 108, p. 193-200 (1991)), RSV LTR promoter (Cullen, Methods in Enzyraology 152, p. 684-) 704 (1987)), SR promoter (Takebe et al., Mol. Cell. Biol. 8, p.
  • a method for preparing a fusion protein by introducing only a small epitope portion consisting of several to several tens of amino acids in order to minimize the properties of the original protein when making it into a fusion protein has been reported.
  • His-tag polyhistidine (His-tag) (for example, 6XHis or lOXHis), influenza agglutinin HA fragment, human c-myc fragment, FLAG (Hopp, TP et al., BioTechnology (1988) 6, 1204) -1210), fragment of Vesicular stomatitis virus glycoprotein (VSV-GP), fragment of T7 genelO protein (T7-tag), fragment of human simple herpesvirus glycoprotein (HSV-tag ⁇ , E-tag (monoclonal Epitopes on phage), known epitopes such as SV40T antigen fragment, lck tag, a-tubulin fragment, B-tag, Protein C fragment, etc. and monoclonal antibodies recognizing it, were
  • an immune complex is formed by adding these antibodies to a cell lysate prepared using an appropriate surfactant.
  • This immune complex contains HK33 protein, ARF protein, and antibodies.
  • immunoprecipitation can also be performed using an antibody against the HK33 protein or the ARF protein.
  • these antibodies are prepared by introducing a gene encoding a target protein into an appropriate E. coli expression vector, expressing the gene in E. coli, purifying the expressed protein, and then purifying the expressed protein. It can be prepared by immunizing goats and chickens. It can also be prepared by immunizing the above animal with the synthesized partial peptide.
  • the immune complex can be precipitated using Protein A Sepharose or Protein G Sepharose.
  • G When a fusion protein with an epitope such as ST is prepared, an immune complex can be formed using a substance that specifically binds to these epitopes, such as glutathione-Sepharose 4B.
  • the general method of sedimentation is described, for example, in the literature (Harlow, E. and Lane, D .: Antibodies, pp. 511-552, Cold Spring Harbor Labora tory publications, New York (1988)). Or in accordance with.
  • the screening of the present invention can be carried out using a pull-down assay without using a cell line.
  • HK33 protein and ARF protein are incubated in vitro in the presence of a sample containing a test compound, and a complex is formed with an antibody against one of the proteins or an antibody against a tag fused to these proteins. After recovery, the binding of the two proteins can be evaluated by detecting the other protein using an antibody against the protein or an antibody against a tag added to the protein. Also, one protein is bound to a support, the other protein is bound, and the test sample is applied thereto. By detecting whether the bound protein dissociates, the effect of the test sample can be examined. Screening can also be performed using ELISA.
  • a biosensor utilizing surface plasmon resonance can be used as a means for detecting or measuring the bound protein.
  • a biosensor utilizing the surface plasmon resonance phenomenon can observe the interaction between proteins in real time as a surface plasmon resonance signal using a small amount of protein sample and without labeling (for example, BIAcore, manufactured by Pharmacia). ). Therefore, it is possible to evaluate the binding of HK33 protein and ARF protein by using a biosensor such as BIAcore.
  • a compound that promotes or suppresses the binding between the HK33 protein and the ARF protein which is identified by the above method, is determined to be a compound that promotes or suppresses the interaction between the ARF protein and the HK33 protein, respectively.
  • These compounds can be used to regulate signaling through the ARF protein, signaling through the p53 protein, and cell growth. It can also be used as a drug such as an antitumor agent as described below.
  • the interaction between the ARF protein and the HK33 protein can be detected by using the nuclear translocation of the ARF protein as an index.
  • HK33 protein inhibits nuclear translocation of ARF protein. Therefore, by screening for a compound that regulates the inhibition of nuclear translocation of the ARF protein by the HK33 protein, a compound that regulates the interaction between the ARF protein and the HK33 protein can be obtained.
  • This screening method comprises the steps of (a) contacting a test sample with a cell expressing the ARF protein and the HK33 protein, (b) detecting the nuclear localization of the ARF protein, and (c) detecting the nuclear localization of the ARF protein. Selecting a compound that modulates (promotes or reduces) the inhibition of nuclear localization of the ARF protein by the HK33 protein as compared to the case where the detection is performed in the absence of the sample (control).
  • a compound that promotes the inhibition of nuclear localization of ARF protein by HK33 protein is determined to be a compound that promotes the interaction between ARF protein and HK33 protein, and also reduces the inhibition of nuclear localization of ARF protein by HK33 protein.
  • the compound is considered to be a compound that suppresses the interaction between the ARF protein and the HK33 protein.
  • the ARF protein and HK33 protein may be endogenous proteins or may be exogenously expressed.
  • the cells used are not limited as long as the nuclear translocation of the ARF protein is inhibited by the HK33 protein.
  • the NIH3T3 or COS cells used in Examples can be used.
  • nuclear translocation of ARF protein for example, an antibody against ARF protein was used. It can be detected by an immunocytochemical technique. The antibody is, for example, fluorescently labeled.
  • an ARF protein fused with a GFP protein or the like is exogenously expressed in a cell, the intracellular localization of the ARF protein can be easily detected.
  • Another embodiment of the above-described screening method for a compound that regulates the interaction between the ARF protein and the HK33 protein is to detect the interaction between the ARF protein and the HK33 protein using ARF-mediated signal transduction as an index.
  • the ARF protein is a signaling factor involved in cell cycle regulation, and signals mediated by the ARF protein are transmitted to other molecules such as MDM2, p53, and its downstream p21. Therefore, a compound that regulates the interaction between the ARF protein and the HK33 protein can be obtained by performing screening using these changes in signal transduction as an index.
  • Such screening can be performed using cell lines.
  • This screening includes, for example, (a) a step of bringing a test sample into contact with cells expressing the ARF protein and the HK33 protein, (b) a step of detecting suppression of ARF-mediated signal transduction by the HK33 protein, and (C) a step of selecting a compound that regulates (enhance or reduce) the inhibition as compared to the case where detection is performed in the absence of a test sample (control).
  • Such screening is also included in the screening of the present invention.
  • the test sample used for screening is not particularly limited.
  • ARF protein and HK33 protein may be endogenous proteins or may be expressed exogenously.
  • the cells to be used many cells having a signal transduction pathway through the ARF protein can be used. Examples include CH0 cells, COS cells, HeLa cells, NIH3T3 cells, BHK, Vero and the like. Cells with significantly increased HK33 gene expression compared to parent cells can be used favorably
  • Signaling through the ARF protein can be detected by the following indicators.
  • Binding of MDM2 protein to p53 protein • Modification of p53 protein (such as ubiquitination)
  • the flow of signal transmission through the ARF protein is as follows.
  • the nuclear translocation of the ARF protein is inhibited by the interaction between the HK33 protein and the ARF protein, the binding between the ARF protein and the MDM2 protein is suppressed.
  • Suppression of binding between ARF protein and MDM2 protein promotes the activity of MDM2 protein, which promotes binding of MDM2 protein to p53 protein and induces degradation of p53 protein, including ubiquitination of p53 protein Promote modification.
  • degradation of the p53 protein is promoted, and the stability of the p53 protein is reduced, so that the activity of the p53 protein in the cell is reduced, and the functions of the p53 protein, such as induction of G1 arrest, are inhibited.
  • HK33 can be screened for compounds that promote or suppress the signal transduction using each step in signal transduction via the ARF protein as an index. It is possible to obtain a compound that suppresses or promotes the interaction between the protein and the ARF protein, respectively.
  • screening is performed by a Western plotting method using an antibody to confirm the expression of a p53 target gene, such as P21, which is induced by activation of p53 protein through activation of ARF-mediated signal transduction.
  • Screening can also be performed by detecting mRNA of a target gene such as p21 by Northern blotting or RT-PCR.
  • screening can be performed using reporter activity as an indicator, by incorporating DNA into which a reporter gene is bound downstream of the expression control sequence of the target gene such as p21.
  • Compounds that promote the expression of p53 target genes are: A The compound is considered to be a compound that reduces the suppression of signal transduction via RF protein by HK33 protein.
  • a compound that suppresses the expression of a target gene is considered to be a compound that promotes suppression of signal transmission through the ARF protein by the HK33 protein.
  • screening can be performed using the inhibition of cell proliferation as an index. This screening can be carried out by measuring proliferation by measuring the number of cells, or by measuring cell proliferation using WST reagent (Roche Diagnostics). Using these methods, HK33 activity, which suppresses ARF activity, is measured by the above-mentioned method to measure cell growth inhibition due to cancellation of the test sample, and screening is performed using this as an index. I can.
  • a compound that promotes cell proliferation is considered to be a compound that promotes the suppression of signal transduction through the ARF protein by the HK33 protein.
  • a compound that suppresses cell proliferation is considered to be a compound that reduces suppression of ARF protein-mediated signal transduction by the HK33 protein.
  • screenings can be similarly carried out in the screening of compounds that regulate the suppression of p53 protein-mediated signal transduction by the HK33 protein as described below.
  • the expression of the HK33 protein suppresses p53-dependent transcription. This indicates that the HK33 protein suppresses p53 activity. It is important that the HK33 protein controls the activity of the p53 protein, which plays a central role as a cell cycle regulator.
  • Another embodiment of the screening method of the present invention is a method for screening by selecting a compound that regulates the suppression of signal transduction through the p53 protein by the HK33 protein.
  • This screening method comprises: (a) a step of bringing a test sample into contact with cells expressing the A RF protein and the HK33 protein; (b) a step of detecting suppression of p53-mediated signal transduction by the HK33 protein; ) Selecting a compound that modulates (enhance or reduce) the inhibition as compared to the case of detection in the absence of a test sample (control). Preferably, these screens are performed in cell lines.
  • the test sample used for screening is not particularly limited. As for cells, the same screening as above using ARF protein signal transduction as an index Similar cells can be used. Cells in which the expression of the HK33 gene is significantly increased as compared to the parent cells can be preferably used.
  • Signal transduction via the p53 protein can be evaluated by detecting the p53 protein or its action as described above. Specifically, for example, p53 protein binding to MDM2 protein, p53 protein modification, p53 protein stability, p53 protein target gene expression, apoptosis, DNA synthesis, cell division, cell proliferation, etc. Can be.
  • Compounds obtained by this screening can be used as cell growth regulators and antitumor agents, similarly to the compounds that regulate the interaction between the ARF protein and the HK33 protein. Therefore, this screening method can be used to obtain a compound that regulates cell proliferation, as well as an antitumor agent, in the same manner as the above-described method for screening a compound that regulates the interaction between ARF protein and HK33 protein. It is also suitably used for obtaining.
  • screening can be performed by the expression of the target gene of p53 such as p21 described above with an antibody (estrant blotting method or Northern plotting RT-PCR).
  • a compound that promotes the expression of a p53 target gene is considered to be a compound that reduces suppression of p53 protein-mediated signal transduction by the HK33 protein.
  • a compound that suppresses the expression of a target gene is considered to be a compound that promotes suppression of signal transduction through the p53 protein by the HK33 protein.
  • screening can be performed using the inhibition of cell proliferation as an index.
  • a compound that promotes cell proliferation is considered to be a compound that promotes suppression of signal transduction via the p53 protein by the HK33 protein.
  • a compound that suppresses cell proliferation is considered to be a compound that reduces suppression of signal transmission through the p53 protein by the HK33 protein.
  • the sample containing the test compound is added to, for example, a cell culture medium.
  • a gene is used as a test sample, it is introduced into cells.
  • the introduction can be carried out, for example, by a known gene introduction method using an expression vector or the like.
  • Screening in vivo system using excision In ning a sample containing the test compound is administered by an appropriate route.
  • the administration of the sample can be performed, for example, by transdermal, intraperitoneal, intramuscular, enteral, intravenous injection and the like.
  • the gene can be administered using an expression vector or the like.
  • the HK33 protein or ARF protein expressed in a cell, or various signaling molecules downstream from these proteins, may be endogenous or exogenously expressed.
  • the test compound can be assayed to determine the specificity of the compound for the ARF protein. It is possible to verify.
  • NIH3T3 cells lacking the INK4a site where ARF is present.
  • the present invention also provides a method for screening for a regulator of ARF protein-mediated signal transduction by selecting a compound that regulates the expression of the HK33 gene.
  • This screening can regulate the expression (transcription or translation) of the HK33 gene in a cell, in a living body, or in an in vitro cloning system, using a gene encoding the HK33 protein or its expression control region.
  • This is a method including a step of selecting a compound.
  • This screening can also be used, for example, to screen for modulators of signal transduction via the ARF protein or p53 protein, cell growth regulators, apoptosis regulators, or carcinogens, cell immortalization reagents, or antitumor agents.
  • the screening method described above includes, for example, (a) a step of bringing a test sample into contact with cells endogenously expressing the HK33 gene, (b) a step of detecting expression of the HK33 gene, and (c) a step of: Selecting a compound having an activity of regulating (enhancing or inhibiting) the expression as compared to the case where the cells are not brought into contact with the cells (control).
  • gene expression includes transcription and translation.
  • cells expressing the HK33 gene are cultured together with a test sample, and the expression of the gene is detected by mRNA analysis such as Northern analysis or RT-PCR, or by protein expression such as Western blotting, immunoprecipitation, and ELISA.
  • mRNA analysis such as Northern analysis or RT-PCR
  • protein expression such as Western blotting, immunoprecipitation, and ELISA.
  • the target compound can be screened by selecting a compound that promotes or inhibits the expression of the gene as compared with the case where the test sample is not added and detection is performed by an improved method.
  • This screening comprises: (a) contacting a test sample with cells into which a vector having a reporter gene operably linked downstream of the endogenous transcription control sequence of the HK33 gene has been introduced; (b) the reporter A step of detecting the expression of the gene, and (c) selecting a compound having an activity of regulating (promoting or inhibiting) the expression of the reporter gene, as compared with the case where the test sample is not brought into contact with the cells (control). And a method comprising the steps of:
  • the “endogenous transcription control sequence” refers to a sequence that controls transcription of the HK33 gene in a cell naturally retaining the gene. Such sequences include promoters, enhancers, and / or repressors. As these sequences, for example, DNA in the upstream region of the gene encoding the HK33 protein can be used. For example, a DNA fragment from the transcription initiation point (or translation initiation codon) of the gene encoding the HK33 protein to several kb upstream thereof is considered to contain an endogenous transcription control sequence of the gene. By linking this fragment with the reporter gene, the expression of the reporter gene can be placed under the transcriptional control of the gene encoding the HK33 protein.
  • transcription control activity is It is also possible to determine the sequence involved in transcription control by measuring the sex and use the fragment thereof.
  • many transcription control sequences to which transcription factors involved in transcription control are bound are known. From upstream region of the gene encoding the HK3 3 protein, by identifying these known transcription control sequences, it is conceivable to identify the endogenous transcriptional control sequences. Usually, a plurality of sequences controlling the transcription of a gene are present in one gene, but any one of them or a combination thereof can be used in the screening of the present invention.
  • the endogenous transcription control sequence may be a chimera with another promoter. Chimeric promoters are often used in transcriptional regulation studies.
  • promoters used for producing the chimeric promoter include, for example, a minimal promoter derived from the SV40 early promoter.
  • “functionally linked” means that the transcription control sequence and the reporter gene are linked so that the reporter gene linked downstream thereof can be expressed in response to activation of the transcription control sequence. Point.
  • a suitable reporter gene eg, chloramphenicol acetyltransferase gene, luciferase gene, etc.
  • a test sample is brought into contact with the cells, the reporter activity is detected, and a compound that increases or decreases the reporter activity compared to the reporter activity in the cells not contacted with the test sample is selected.
  • compounds that can regulate the expression of the gene of the present invention can be screened. This screening has a feature that it is simpler than the direct detection such as the above-described Northern analysis, because the expression of the HK33 gene is detected using the reporter activity as an index.
  • the transcription control sequence of human HK33 gene has already been cloned, Transcription analysis has been performed by linking genes (Ka Thigherer, S. et al., Genomics 45: 200-210, 1997) ⁇
  • the transcription control region of the human ⁇ 33 gene contains known transcription such as Spl sequence. A factor binding sequence was found.
  • the screening of the present invention can be performed using a region containing these sequences in the transcription control sequence of the HK33 gene.
  • Compounds that can be isolated by the above-described screening method of the present invention include compounds that regulate the interaction between HK33 protein and ARF protein, compounds that regulate HK33 gene expression, and that regulate signaling through ARF protein. And compounds that regulate signal transduction through the p53 protein.
  • Compounds that suppress the interaction between HK33 protein and ARF protein and compounds that suppress the expression of 33 gene increase the intracellular activity of ARF protein, stabilize p53 and cause cell arrest. Inhibits cell division and cell proliferation. It can also induce apoptosis.
  • compounds that promote the interaction between the HK33 protein and the ARF protein and compounds that promote the expression of the ⁇ ⁇ ⁇ 33 gene, etc. decrease the activity of the ARF protein in cells, promote the degradation of p53, and promote cell division. Releases arrest and promotes cell division and cell proliferation. It can also suppress apoptosis. Therefore, the above-mentioned screening of the present invention is useful for evaluating a compound that regulates cell proliferation or isolating the compound. In particular, compounds that suppress cell proliferation are expected to be used as antitumor agents. Therefore, the screening method of the present invention is suitably used for evaluating or isolating an antitumor agent.
  • a compound that regulates the activity of the HK33 protein, a compound that regulates the interaction between the HK33 protein and the ARF protein, a compound that regulates the signal transduction via the ARF protein, a compound that regulates the signal transduction via the p53 protein, and Compounds that regulate cell proliferation can be used as reagents and / or medicaments.
  • compounds that can be isolated by the above screening can also be used as reagents and / or medicaments.
  • Drugs include reagents and drugs. These compounds can also be used as agents for regulating cell proliferation.
  • the compound is considered to be applicable to, for example, treatment of cell proliferative diseases and diseases which can be treated by controlling cell proliferation.
  • compounds that suppress the action of HK33 protein such as compounds that suppress the interaction between HK33 protein and ARF protein and compounds that suppress the expression of HK33 gene, are expected to be used as tumor suppressors.
  • a compound that promotes the interaction between the HK33 protein and the ARF protein may be used, for example, as an apoptosis inhibitor. It is also useful as a cell growth promoter. Compounds that promote the interaction between the HK33 protein and the ARF protein can inhibit the cell growth suppression mechanism that is negatively controlled by the ARF protein, and thus may be used in organ regeneration and the like. In addition, these compounds are useful for preparing a tumor cell model and for analyzing the pathology of a tumor.
  • the above compounds can be used in humans, such as mammals, such as mice, rats, guinea pigs, egrets, chicks, cats, dogs, When used as a medicament for sheep, pigs, magpies, monkeys, baboons, and chimpanzees, the compound itself is administered directly to the patient, as well as a pharmaceutical composition formulated by a known pharmaceutical method. It is also possible.
  • tablets, capsules, elixirs, and microcapsules which are sugar-coated as necessary, orally, or aseptic solution or suspension in water or other pharmaceutically acceptable liquids It can be used parenterally in the form of injections.
  • pharmacologically acceptable carriers or vehicles specifically, sterile water or saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives Agent, binder, etc., as appropriate, generally accepted It may be formulated by mixing in the unit dosage form required for pharmaceutical practice. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.
  • excipients examples include binders such as gelatin, corn starch, tragacanth gum, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid.
  • binders such as gelatin, corn starch, tragacanth gum, gum arabic
  • excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid.
  • a suitable bulking agent, a lubricant such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharin, and a flavoring agent such as peppermint, cocoa oil or cherry are used.
  • the unit dosage form is a capsule, the above-mentioned materials may further contain a liquid carrier such as an oil or fat.
  • a sterile composition for injection can be formulated using a potable vehicle such as distilled water for injection according to normal pharmaceutical practice.
  • Aqueous injection solutions include, for example, saline, isotonic solutions containing dextrose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride. It may be used in combination with an agent, for example, an alcohol, specifically, ethanol, a polyalcohol, for example, propylene glycol, polyethylene glycol, a nonionic surfactant, for example, polysorbate 80 (TM), or HCO-50.
  • the oily liquid includes sesame oil and soybean oil, and may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizer.
  • a buffering agent such as a phosphate buffer, a sodium acetate buffer, a soothing agent such as proforce hydrochloride, a stabilizer, for example, benzyl alcohol, phenol, or an antioxidant may be used. Good.
  • the prepared injection solution is usually filled into an appropriate ampoule.
  • Administration to patients can be performed, for example, by intraarterial injection, intravenous injection, subcutaneous injection, etc., or intranasally, transbronchially, intramuscularly, transdermally, or orally by a method known to those skilled in the art. It can do better.
  • the dose varies depending on the weight and age of the patient, the administration method, and the like, but those skilled in the art can appropriately select an appropriate dose.
  • the compound can be encoded by DNA, the DNA is incorporated into a vector for gene therapy, It is also conceivable to perform gene therapy.
  • the dose and administration method vary depending on the patient's weight, age, symptoms, etc., and can be appropriately selected by those skilled in the art.
  • the dose of the compound varies depending on the administration subject, target organ, symptoms, and administration method.
  • oral administration in general, for an adult (assuming a body weight of 60 kg), about 0.1 to 100 mg / day is preferable. Is about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.
  • parenteral administration the single dose varies depending on the administration target, target organ, symptoms, and administration method.
  • parenteral injections usually for adults (with a body weight of 60 kg) per day, It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection.
  • the dose can be administered in terms of the amount converted per 60 kg body weight or the amount converted per body surface area.
  • the present invention provides a test reagent comprising an antibody that binds to the HK33 protein, or a polynucleotide containing at least 15 nucleotides complementary to DNA encoding the HK33 protein or its complementary strand.
  • Increased expression of the HK33 gene suppresses intracellular activity of the ARF protein, reduces p53 protein function, and enhances cell proliferation.
  • decreased expression of the HK33 gene may increase ARF protein activity, increase p53 function, suppress cell proliferation and induce apoptosis. Therefore, cell proliferation and apoptosis can be detected by examining abnormal expression of the HK33 gene in cells.
  • the test reagent of the present invention is useful for detecting abnormal signal transduction via the ARF protein, detecting abnormal signal transduction via the p53 protein, detecting abnormal cell proliferation, detecting apoptosis, etc. . It is also useful for examining diseases.
  • decreased expression of the HK33 gene or interaction with the ARF protein suggests that a decrease in cell proliferation or a disease associated with apoptosis may be caused. It suggests that it may be causing a disease accompanied by an increase in blood pressure.
  • the expression of the HK33 gene was significantly enhanced in the tumor-ridden cells. This means that the HK33 gene or HK33 protein is used for tumor testing Indicates that you can do it. For example, by detecting HK33 protein or mRNA encoding the protein in a test sample, it is possible to examine the presence of tumor cells, tumor progression, tumor malignancy, tumor type, etc. is there.
  • the term “tumor test” refers to not only the detection of a patient who has formed a tumor due to a mutation (mutation in the structure or expression level) of the HK33 gene, but also that the subject has expressed the HK33 gene. It also includes testing for the expression level of the HK33 gene and testing for mutations in the gene to determine whether it is susceptible to cancer due to abnormal amounts or mutations in the gene. In other words, increased expression of the HK33 gene, or the occurrence of an active mutation in one of the HK33 alleles, increases the risk of developing cancer even when symptoms have not yet appeared on the surface. It is thought that there is.
  • the test of the present invention can be performed using, for example, an antibody that binds to the HK33 protein, or a polynucleotide containing at least 15 nucleotides complementary to the DNA encoding the HK33 protein or its complementary strand.
  • the polynucleotide is useful for, for example, detection and amplification of DNA encoding the HK33 protein, and detection of expression of the DNA.
  • the detection of DNA includes the detection of DNA mutation.
  • phase capture strand refers to one strand of a double-stranded nucleic acid composed of A: T (U for RNA) and G: C base pairs, as described above.
  • complementary is not limited to the case where the complementary nucleotide region is a completely complementary sequence, and is at least 70%, preferably at least 80%, more preferably 90%, and still more preferably 95% or more. It is only necessary to have homology on the base sequence.
  • the algorithm for determining homology is according to the algorithm described in the literature (Wilbur, WJ and Lipman, DJ Pro Natl. Acad. Sci. USA (1983) 80, 726-730). By using this polynucleotide as a probe or primer and detecting the expression or mutation of the HK33 gene, a cell proliferation abnormality or a cell proliferative disease such as a tumor can be examined.
  • the present invention detects abnormalities in the expression level or structure of the HK33 gene using DNA encoding the HK33 protein or a polynucleotide containing at least 15 nucleotides that are complementary to its complementary chain.
  • A abnormal signal transduction via ARF
  • B abnormal signal transduction via p53
  • c abnormal cell proliferation
  • cell proliferative disease or
  • e examination of tumor Provide a way.
  • Gene expression may be detected by detecting mRNA or protein.
  • mutation of the gene structure may be examined using either chromosomal DNA or mRNA.
  • the test using an antibody that binds to the HK33 protein involves contacting the antibody with a sample expected to contain the HK33 protein, and detecting or measuring an immune complex between the antibody and the protein. Alternatively, it can be performed by a measuring method. Increased expression of the HK33 protein indicates enhanced cell proliferation or cancerous cells. Therefore, for example, by examining the expression level of the HK33 gene, it is possible to examine a cell proliferation abnormality or a cell proliferation disease such as a tumor. That is, the present invention comprises a step of detecting an abnormality in the expression level or structure of the HK33 protein using an antibody that binds to the HK33 protein, (a) an abnormality in signal transduction via ARF, and (b) a signal transmission through p53. ( C ) cell proliferation abnormality, (d) cell proliferative disease, or (e) tumor. .
  • solutes such as stabilizers, preservatives, salts, and buffering agents can be appropriately combined with water, physiological saline, and the like to dissolve them.
  • Polynucleotides can also be used to make DNA chips and microarrays. These DNA chips and microarrays can be used for the test of the present invention.
  • One of the detection methods of the present invention is a method including a step of detecting the expression level of DNA encoding HK33 protein in a test sample.
  • a test method comprises the steps of (a) contacting a DNA encoding the HK33 protein or a complementary strand thereof with a polynucleotide containing at least 15 nucleotides in a complementary manner, to an RNA sample derived from a patient; Detecting the binding of the polynucleotide to the RNA sample.
  • Such tests should be performed by, for example, Northern hybridization ⁇ ⁇ RT-PCR. Can be.
  • Inspection using RT-PCR includes the following steps: (a) the step of synthesizing cDNA from a patient-derived RNA sample; (b) the synthesized cDNA as a type I, and the above polynucleotide as a primer. A step of performing PCR, and (c) a step of detecting DNA amplified by PCR.
  • Northern hybridization and RT-PCR can be performed by known genetic engineering techniques. Detection by DNA chip or DNA microarray is also possible.
  • the test method of the present invention includes a method including a step of detecting HK33 protein content in a test sample derived from a patient.
  • a detection can be performed using, for example, an antibody against the HK33 protein.
  • the detection using an antibody against HK33 includes, specifically, (a) a step of contacting an antibody that binds to the HK33 protein with a patient-derived protein sample, and (b) detecting the binding of the antibody to the protein sample.
  • the protein can be detected by immunoprecipitation using an antibody against the HK33 protein, Western blot, immunohistochemistry, ELISA, or the like.
  • abnormal expression of the HK33 gene was caused, for example, by examining the expression of a tissue collected by biopsy, for example, by immunohistochemical staining or in situ hybridization. It is conceivable to identify the lesion. Increased expression of the HK33 gene indicates, for example, the potential for onset and / or progression of cancer. Diseases such as cancer are thought to be caused by various causes. For example, if the expression of the HK33 gene is increased in cancer, it is predicted that the p53 pathway will be suppressed. It is conceivable to use it for diagnosis when performing.
  • the test of the present invention can also be performed by detecting a mutation in the HK33 protein or a mutation in DNA encoding the protein. Since the HK33 gene is considered to be involved in the onset and / or progression of cancer, mutations in the protein and the DNA suggest a risk of onset and progression of cancer.
  • Mutations in the HK33 protein include structural and functional mutations.
  • HK By using an antibody against the 33 protein and comparing the molecular weight of the protein with a protein derived from a healthy subject by Western blot or the like of a protein sample derived from a patient, structural mutations of the protein can be examined.
  • mutations in the HK33 protein can also be detected using changes in protein modification, changes in the binding of a protein or antibody that binds to the HK33 protein, and the like as indices. For these tests, for example, ELISA using an antibody against the HK33 protein, immunoprecipitation, pull-down, and the like can be used.
  • the test of the present invention can also be performed by detecting the binding between the HK33 protein and the ARF protein.
  • Enhanced binding of HK33 protein and ARF protein is thought to contribute to the development and progression of cancer.
  • Protein binding can be evaluated, for example, by ELISA using antibodies to HK33 protein or ARF protein, immunoprecipitation, pull-down, and the like.
  • a polynucleotide containing at least 15 nucleotides complementary to the DNA encoding the HK33 protein or its complementary strand can be used.
  • the nucleotides are the nucleotide sequence of the cDNA encoding the HK33 protein, the nucleotide sequence of the genomic DNA sequence (including exons, introns, and endogenous transcription control sequences), or a polynucleotide (probe or probe) complementary to its complementary strand. Hoop primer).
  • the mutation test includes a test for identifying a patient (carrier) having a mutation in one of the HK33 alleles. It also includes testing to determine the type of single nucleotide polymorphism (SNP). Polymorphisms in the HK33 gene may be linked to cancer susceptibility.
  • the polynucleotide When used as a primer, the polynucleotide is usually 15 bp to 100 bp, preferably 17 bp to 30 bp.
  • the primer may be any primer as long as it can amplify at least a part of the HK33 gene or a region that regulates its expression. Such regions include, for example, the exon region, intron region, promoter region, and enhancer region of the HK33 gene.
  • a polynucleotide as a probe is a synthetic polynucleotide, Usually has a chain length of at least 15 bp or more. Double-stranded DNA obtained from a clone integrated into a vector such as a plasmid DNA can also be used as a probe.
  • the probe may be any probe as long as it is complementary to the base sequence of at least a part of the HK33 gene or a region that regulates its expression, or a complementary strand thereof. Examples of the region to which the probe hybridizes include an exon region, an intron region, a promoter region, and an enhancer region of the HK33 gene.
  • the polynucleotide or double-stranded DNA is appropriately labeled and used.
  • Labeling methods include, for example, labeling by phosphorylating the 5 'end of the polynucleotide with a radioisotope using T4 polynucleotide kinase, or random hexamer oligonucleotide using a DNA polymerase such as Klenow enzyme.
  • a method of incorporating a substrate base labeled with a radioisotope III such as 32 P using a nucleotide as a primer, a fluorescent dye or a biotin (random prime method, etc.) may be mentioned.
  • One embodiment of a method for detecting a mutation in the HK33 gene is a method for directly determining the nucleotide sequence of the HK33 gene in a patient. For example, using the above nucleotides as primers, DNA isolated from a patient suspected of having a disease caused by a mutation in the HK33 gene as type III, and a part or all of the HK33 gene (eg, exon, The region containing the intron, promoter and enhancer) is amplified and its nucleotide sequence is determined. By comparing this with the HK33 gene sequence of a healthy subject, a disease caused by a mutation in the HK33 gene can be detected.
  • a part or all of the HK33 gene eg, exon, The region containing the intron, promoter and enhancer
  • One embodiment is (a) a step of preparing a DNA sample from a patient, and (b) a patient using a DNA encoding HK33 protein or a polynucleotide containing at least 15 nucleotides that is complementary to its complementary strand as a primer. Amplifying the derived DNA, (c) dissociating the amplified DNA into single-stranded DNA, (d) separating the dissociated single-stranded DNA on a nondenaturing gel, and (e) separating Single-stranded DNA gel Comparing the mobility above with a healthy control.
  • This method has advantages such as relatively simple operation and small sample volume, and is particularly suitable for screening a large number of DNA samples.
  • the principle is as follows. When a double-stranded DNA fragment is dissociated into single strands, each strand forms a unique higher-order structure depending on its base sequence. When this dissociated DNA strand is electrophoresed in a polyacrylamide gel containing no denaturing agent, the single-stranded DNA of the same complementary length moves to a different position according to the difference in each higher-order structure. . The higher-order structure of this single-stranded DNA is also changed by single-base substitution, and shows different mobility in polyacrylamide gel electrophoresis. Therefore, by detecting this change in mobility, it is possible to detect the presence of a mutation due to a point mutation, deletion, insertion, or the like in the DNA fragment.
  • a part or all of the HK33 gene is amplified by PCR or the like.
  • the range to be amplified usually, a length of about 200 to 400 bp is preferable.
  • the region to be amplified include all exons and all introns of the HK33 gene, as well as the promoter and enhancer of the HK33 gene.
  • radioisotopes such as 32 P or fluorescent dyes Ya Biochin force used by connexion labeled primers etc., or radioisotopes such as 32 P in a PCR reaction solution, or by such as a fluorescent dye or Piochin, Add labeled substrate base Then, the DNA fragment synthesized by performing PCR is labeled.
  • labeling can also be carried out by adding a radioisotope such as 32 P or a substrate base labeled with a fluorescent dye or biotin to the synthesized DNA fragment using a Klenow enzyme after PCR.
  • the labeled DNA fragment thus obtained is denatured by applying heat, and electrophoresis is carried out on a polyacrylamide gel containing no denaturant such as urea.
  • the conditions for separating DNA fragments can be improved by adding an appropriate amount (about 5 to 10%) of glycerol to the polyacrylamide gel.
  • electrophoresis conditions vary depending on the properties of each DNA fragment.However, usually, the reaction should be performed at room temperature (20 to 25 ° C). Consider the temperature to be given.
  • the mobility of the DNA fragment is detected and analyzed by autoradiography using an X-ray film or a scanner that detects fluorescence. If a band having a difference in mobility is detected, this band can be directly excised from the gel, re-amplified by PCR, and directly sequenced to confirm the presence of the mutation. Even when labeled DNA is not used, the band can be detected by staining the gel after electrophoresis with ethidium-membranide silver silver staining.
  • test method of the present invention include: (a) a step of preparing a DNA sample from a patient; (b) a step of amplifying a patient-derived DNA using the above-mentioned polynucleotide as a primer; (c) an amplified DNA (D) separating a DNA fragment according to its size, (e) hybridizing the separated DNA fragment with a detectable labeled probe, and (f) detecting the detected DNA fragment Comparing the size of ⁇ with a control of a healthy individual.
  • Examples of such a method include a method using restriction fragment length polymorphism (ZRFLP) and a PCR-RFLP method.
  • Restriction enzymes are usually used as enzymes that cut DNA. Specifically, when there is a mutation at the recognition site of the restriction enzyme, or when a salt is included in the DNA fragment generated by the restriction enzyme treatment. When there is a base insertion or deletion, the size of the fragment generated after treatment with the restriction enzyme is changed as compared to a healthy person. By amplifying a portion containing this mutation by PCR and treating it with each restriction enzyme, these mutations can be detected as a difference in band mobility after electrophoresis.
  • the presence or absence of a mutation can be detected by treating chromosomal DNA with these restriction enzymes, electrophoresing, and performing Southern blotting using a probe.
  • the restriction enzyme to be used can be appropriately selected according to each mutation.
  • RNA prepared from a patient can be converted into cDNA using reverse transcriptase, which can be directly cut with restriction enzymes and then subjected to Southern blotting.
  • a part or all of the HK33 gene can be amplified by PCR, cut with restriction enzymes, and then examined for differences in mobility.
  • RNA instead of DNA prepared from patients.
  • Such methods include (a) the step of preparing an RNA sample from a patient, (b) the step of separating RNA prepared according to size, and (c) the detectable labeling of the isolated RNA. Hybridizing the polynucleotide as a probe, and (d) comparing the size of the detected RNA with a healthy control.
  • RNA prepared from a patient is electrophoresed, and Northern blotting is performed using a probe to detect a difference in mobility.
  • test method of the present invention includes: (a) a step of preparing a DNA sample from a patient, (b) a step of amplifying DNA derived from a patient using the polynucleotide as a primer, and (c) amplifying the amplified DNA. And (d) comparing the mobility of the separated DNA on the gel with a control of a healthy subject.
  • Such a method includes denaturant gradient gel electrophoresis (DGGE).
  • DGGE denaturant gradient gel electrophoresis
  • a part or all of the HK33 gene is amplified by PCR using the above polynucleotide as a primer. This is electrophoresed in a polyacrylamide gel, which gradually increases as the concentration of a denaturant such as urea moves, and is compared with a healthy person.
  • DGGE denaturant gradient gel electrophoresis
  • the Allele Specific 01igonucleotide / AS0 hybridization method can be used for the purpose of detecting only a mutation at a specific position.
  • an oligonucleotide containing a nucleotide sequence that is considered to have a mutation is prepared and hybridized with this and the sample DNA, the efficiency of hybridization is reduced in the presence of the mutation. This can be detected by the Southern plot method or a method utilizing the property of quenching by intercalating a special fluorescent reagent into the gap of the hybrid. Detection by the ribonuclease A mismatch cleavage method is also possible.
  • part or all of the HK33 gene is amplified by PCR or the like, and this is hybridized with labeled RNA prepared from HK33 cDNA or the like in which this is integrated into a plasmid vector or the like. Since the hybrid has a single-stranded structure in the portion where the mutation exists, the presence of the mutation can be detected by cleaving this portion with ribonuclease A and detecting this by autoradiography or the like.
  • FIG. 1 shows the results of confirming the interaction between the ARF protein and the HK33 protein using a mammalian Two-Hybrid system using C0S7 cells. It was confirmed that the two proteins interacted significantly.
  • FIG. 2 is a photograph showing the results of confirming the interaction between pl9ARFmyc protein and GFP-HK33 protein by immunoprecipitation using a cell extract of C0S7 cells into which pl9ARFmyc and GFP-HK33 expression vectors have been introduced. Interaction between GFP-HK33 protein and pl9ARF protein is immune It was also confirmed by the sedimentation method.
  • FIG. 3 is a diagram showing the results of confirming the interaction between the HK33 protein and a pl9ARF-deleted gene encoding P19ARF1-80 and pl9ARF8-Stop by the yeast Two-Hybrid method.
  • the HK33 protein was shown to interact with P19ARF81-Stop.
  • FIG. 4 is a photograph showing the results of expressing pl9ARFmyc protein and GFP-HK33 protein in NIH3T3 cells and examining the intracellular localization.
  • GFP-HK33 alone localized in the cytoplasm
  • pi9ARF protein alone accumulated in nucleoli.
  • pl9ARF + GFP-HK33 pl9ARF was not detected in the nucleus, indicating that it was localized in the cytoplasm.
  • Figure 5 shows HK33 in cells undergoing the immortalization process (BFT, BET, RKF-T * Lifroumani Syndrome-p53-deficient cells, before and during immortalization, respectively) by introducing the SV40 gene into normal human cells.
  • 4 is a photograph showing the result of analysis of gene expression by RT-PCR. The upper row shows the expression of the HK33 gene, and the lower row shows the expression of the GAPDH gene detected as a control.
  • the expression level of HK33 gene was compared between the senescent Precrisis cell and the postcrisis cell which escaped cell crisis from the senescent state by introducing the gene of SV40 Large T Antigen.
  • fibroblasts, vascular endothelial cells, and RKF-T * Lifroumani Syndrome-p53-deficient cells expression was significantly increased in Postcrisis cells, respectively.
  • FIG. 6 is a photograph and a diagram showing suppression of p53-dependent transcriptional activity by forced expression of the HK33 gene.
  • HA-pl9ARF HA-tagged pl9ARF
  • mouse HK33 gene sense His-max / mHK33-S
  • antisense His-fflax / mHK33-AS
  • An MDM2 expression vector was used as a positive control for p53 protein degradation activity.
  • the pG13Luc vector was used as an indicator of p53-dependent transcription activity and the p53-dependent transcription activity was measured by luciferase.
  • the top panel shows the cells transfected for 24 hours Later, l. OmM Zn 2+ was added :! : After exchanging the medium with the medium with or without the medium, the expression of mouse HK33 (mHK33) and ⁇ -pl9ARF (HA-pl9) protein in the cells after 24 hours was confirmed by Xpress antibody and HA polyclonal antibody, respectively. The result of detection by Western blotting is shown. The lower panel shows the results of analysis of p53 reporter activity. BEST MODE FOR CARRYING OUT THE INVENTION
  • the gene fragment was excised from the isolated yeast, and the DNA sequence was analyzed by the dideoxy chain termination method using an ABI377 automatic nucleotide sequencer. As a result, the sequence of the isolated gene fragment was obtained from the DNA sequence data bank. It had 100% identity with a gene known as Human Housekeeping gene 33 (HK33), which was registered in the US.
  • HK33 Human Housekeeping gene 33
  • pl9ARFmyc was detected by reacting with a myc antibody (Invirogen), and the antibody was reacted with a Texas Red-conjugated secondary antibody and detected.
  • HK33 protein was detected by luminescence of GFP itself. The results of the experiment showed that GFP-HK33 was localized in the cytoplasm, and that the P19ARF protein alone accumulated in the nucleolus. On the other hand, in co-transfection of p19ARF + GFP-HK33, pl9ARF was not detected in the nucleus, indicating that it was localized in the cytoplasm.
  • oligo primers of SEQ ID NOs: 3 and 4 were used and analyzed at 94 ° C for 1 minute, 58 ° C for 2 minutes, and 72 ° C for 3 minutes under the conditions of 30 cycles.
  • the HK33 gene expression level was compared between the aging cell Precrisis cells and the cells that escaped cell crisis from the aging state, and into which the SV40 Large T Antigen gene was introduced.
  • fibroblasts, vascular endothelial cells, and RKF-T * Lifroumani Syndrome-p53-deficient cells expression was significantly increased in Postcrisis cells, respectively.
  • HK33 may have inactivated pl9ARF in immortalized cells that escaped cell senescence (Fig. 5). This finding is consistent with reports that inactivation of pl9ARF causes prolonged cell life (Carner, A. et al. (2000) Nat. Cell Biol. (3): 148-55).
  • HA-pl9ARF HA-tagged pl9ARF
  • N IH / ARF cells HA-pl9ARF cells capable of controlling the expression of HA-tagged pl9ARF (HA-pl9ARF) by the action of zinc ion on the meta-mouth thionein promoter N IH / ARF cells
  • An HK33 gene sense or antisense was ligated to an expression vector (pcDNA4, Invitrogen) to which an Xpress tag was added, and used as an HK33 expression vector.
  • MDM2 expression vector was used as a positive control for p53 protein degradation activity.
  • pG13Luc beta one as an indicator of p53-dependent transcription activity
  • pRL-TK vector Promega
  • p53-dependent transcription activity was measured by luciferase. did. After introducing the gene into the cells, 24 hours later, replace the medium with a medium supplemented with L OmM Zn 2+ or a medium containing only the medium.After 24 hours, lyse the cells and analyze the expression of the protein and measure the reporter activity. went. ⁇ It was confirmed by estamplotting that HK33 protein was expressed in sense and HK33 protein was not expressed in antisense.
  • the present inventors have clarified that the HK33 protein interacts with the ARF protein, and that the interaction inhibits the nuclear translocation of the ARF protein, thereby inhibiting the function of ARF. Furthermore, the expression of the HK33 gene was higher in the immortalized cells (Postcrisis cells) that reached the stage in which cell senescence was escaped by the gene transfer of SV40 Large T Antigen than in the cells before the immortalized cells (Precrisis cells). It has shown that a rise will occur. According to the present invention, a drug having an antitumor effect utilizing the interaction between the ARF protein and the HK33 protein has been developed. Screening becomes possible.
  • a method for regulating ARF protein-mediated signal transduction, particularly cell cycle and cell proliferation, by regulating the interaction or the expression of the HK33 gene, and a molecule used for the same are disclosed. sponsored. Further, according to the present invention, it has become possible to use the HK33 protein or a gene encoding the HK33 protein to examine a disease such as a tumor. Screening for a drug that suppresses the interaction between HK33 protein and ARF protein will enable the development of a new drug that can suppress cell growth due to inactivation of ARF protein or growth of immortalized precancerous cells Can be

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Abstract

It is found out that HK33 protein binds to ARF protein. Nuclear transport of ARF protein is inhibited by the expression of HK33 gene and thus p53-dependent transcription is suppressed. In immortalized cells, moreover, the expression of HK33 gene is significantly elevated. A method of screening an antitumor agent by using the interaction between ARF protein and HK33 protein; a method of regulating HK33 protein-targeting signal transduction mediated by ARF protein and molecules employed therefor; and utilization of HK33 protein and a gene encoding HK33 protein in examining diseases such as tumor. Namely, it becomes possible to screen a novel antitumor agent with the use of the interaction between ARF protein and HK33 protein as an indication.

Description

ARFタンパクと HK33タンパクの相互作用を利用した  Utilizing the interaction between ARF protein and HK33 protein

抗腫瘍作用を持つ薬剤のスクリーニング方法 技術分野  Screening method for drugs with antitumor activity

本発明は ARF蛋白質と HK33蛋白質の相互作用を利用した抗腫瘍作用を持つ薬剤 のスクリーニング方法に関する。 本発明はまた、 該相互作用、 または HK33遺伝子 の発現を調節することにより ARF蛋白質を介するシグナル伝達、 p53蛋白質を介す るシグナル伝達、 および細胞の増殖を調節する方法に関する。 本亮明はさらに、 腫瘍等の疾患における検査のための HK33蛋白質およひ ΉΚ33蛋白質をコードする遺 伝子の利用にも関する。 景技術  The present invention relates to a method for screening a drug having an antitumor effect utilizing an interaction between an ARF protein and a HK33 protein. The present invention also relates to a method for regulating ARF protein-mediated signal transduction, p53 protein-mediated signal transduction, and cell proliferation by regulating the interaction or the expression of the HK33 gene. Ryoaki Motomoto also relates to the use of the HK33 protein and the gene encoding the 33 protein for testing in diseases such as tumors. Landscape technology

細胞周期の調節に深く関わっている因子として、 癌抑制因子である retinoblas toma protein (pRb) と p53があげられる （Weinberg, R. A. (1995) Cell, 81, 3 23-330 ; Levine, A. J. (1997) Cell, 88， 323-331 ; Sherr, C. J. (1996) Scie nce, 274, 1672-1677)。 pRbおよび p53はそれぞれ、 別の経路で細胞周期を調節し ていると考えられている。 二つの作用の違いは、 pRbは cyclin dependent kinase s (cdks) の作用によって Gl期から S期へと移行させ細胞増殖抑制へと導くのに対 して、 p53は化学物質や DNA障害によって発現することにより、 p21の転写を促進し 細胞周期を抑制すると言われている。また、 p53は MDM2との結合によって分解され るため、 MDM2の発現により p53の増殖制御の解除が起こると考えられている （Got tlieb. M. F. and Oren, M. (1996) Biochim. Biophys. Acta 1287， 77—102 ; Mo raand, J. et al. (1992) Cell, 69， 1237-1245 ; ubbutat, M. H. et al. (1997 ) Nature, 387, 299 - 303)。 一方、 INK4a gene locusの不活性化は多くの腫瘍において見いだされており、 その染色体上に存在する遺伝子の腫瘍との関係が深く研究されてきた。 近年、 こ の INK4a gene locusに存在する INK4Aと ARFはそれぞれ同一の遺伝子上にあり、 異 なったスプライシングフオームとして翻訳され、 どちらの因子も G1期に細胞周期 をァレス トし、 細胞増殖抑制活性を持つことが明らかにされた。 二つの因子の機 能の違いとしては、 INK4aはサイクリン依存性キナーゼ 4 (CDK4) を不活性ィ匕し、 基質の中でも特に前述した Rbのリン酸化を阻害し、 このリン酸化阻害によって細 胞周期をァレストする。 一方、 ARFは核内において MDM2と結合することによって M DM2による p53の分解を抑え、 p53蛋白質の安定性を高め、細胞周期をアレストして いると考えられている （Serrano, M. et al. (1993) Nature, 366， 704-707； Qu elle, D. E. et al. (1993) Genes Dev. , 7, 1559—1571)。 Factors deeply involved in cell cycle regulation include the tumor suppressor retinoblas toma protein (pRb) and p53 (Weinberg, RA (1995) Cell, 81, 323-330; Levine, AJ (1997) Cell, 88, 323-331; Sherr, CJ (1996) Science, 274, 1672-1677). Each of pRb and p53 is thought to regulate the cell cycle in other pathways. The difference between the two actions is that pRb shifts from Gl phase to S phase by the action of cyclin dependent kinases (cdks) and leads to suppression of cell growth, whereas p53 is expressed by chemical substances or DNA damage It is said that this promotes the transcription of p21 and suppresses the cell cycle. Since p53 is degraded by binding to MDM2, expression of MDM2 is thought to cause the release of p53 proliferation control (Got tlieb. MF and Oren, M. (1996) Biochim. Biophys. Acta 1287). Morand, J. et al. (1992) Cell, 69, 1237-1245; ubbutat, MH et al. (1997) Nature, 387, 299-303). On the other hand, inactivation of the INK4a gene locus has been found in many tumors, and the relationship between genes on its chromosomes and tumors has been studied in depth. In recent years, INK4A and ARF present in this INK4a gene locus are located on the same gene, and are translated as different splicing forms, and both factors arrest the cell cycle in the G1 phase and have cytostatic activity. It was revealed to have. The difference between the functions of the two factors is that INK4a inactivates cyclin-dependent kinase 4 (CDK4) and inhibits the above-mentioned phosphorylation of Rb, among other substrates. Arrested. ARF, on the other hand, is thought to bind to MDM2 in the nucleus to suppress the degradation of p53 by MDM2, enhance the stability of p53 protein, and arrest the cell cycle (Serrano, M. et al. (1993) Nature, 366, 704-707; Quelle, DE et al. (1993) Genes Dev., 7, 1559-1571).

HK33遺伝子は、 1994年に Andreeas Braunらによって発見された遺伝子で、 Pero xisomal膜に存在する。 HK33は酵母 Pexl9pのヒト相同遺伝子として知られ、その産 物はフアルネシル化される蛋白質でありペルォキシソームにおける生合成に関与 することが示唆されている （Ka腿 erer, S. et al. (1997) Genomics, 45, 200-2 1 ; Braun, A. et al. (1994) Gene, 146, 291-295)。 また近年、 Zellweger症候 群の原因遺伝子として報告された （Matsuzono, Y. et al.， Proc. Natl. Acad. Sci. USA 96 : 2116-21, 1999)。しかし ARFまたは p53との関係は全く知られていな い。 発明の開示  The HK33 gene was discovered by Andreeas Braun et al. In 1994, and is present in the Pero xisomal membrane. HK33 is known as a human homologous gene to yeast Pexl9p, and its product has been suggested to be a protein that is phnesylated and involved in biosynthesis in peroxisomes (Ka Thigherer, S. et al. (1997) Genomics , 45, 200-21; Braun, A. et al. (1994) Gene, 146, 291-295). Recently, it was reported as a causal gene of the Zellweger syndrome group (Matsuzono, Y. et al., Proc. Natl. Acad. Sci. USA 96: 2116-21, 1999). However, no relationship with ARF or p53 is known. Disclosure of the invention

本発明は ARF蛋白質と HK33蛋白質の相互作用を利用した抗腫瘍作用を持つ薬剤 のスクリーニング方法を提供することを課題とする。 本発明はまた、 該相互作用 、または HK33遺伝子の発現を調節することにより ARF蛋白質を介するシグナル伝達 、 特に細胞周期おょぴ細胞分裂を調節するための方法、 およびそのために用いら れる分子を提供することを課題とする。 本発明の方法は腫瘍細胞の増殖抑制に有 用である。 本発明はさらに、 腫瘍等の疾患における検査のための HK33蛋白質およ び HK33蛋白質をコードする遺伝子の利用を提供することを課題とする。 An object of the present invention is to provide a method for screening a drug having an antitumor effect utilizing an interaction between an ARF protein and a HK33 protein. The present invention also provides a method for regulating the interaction or ARF protein-mediated signal transduction by regulating the expression of the HK33 gene, particularly regulating cell cycle and cell division, and a molecule used for the same. The task is to The method of the present invention is useful for suppressing the growth of tumor cells. It is for. Another object of the present invention is to provide the use of HK33 protein and a gene encoding the HK33 protein for testing in diseases such as tumors.

本発明らは、 上記課題を解決するために、 酵母 Two - Hybrid法により pl9ARFと相 互作用する蛋白質をコードする遺伝子の単離を試みた。 その結果、 HK33遺伝子と 呼ばれるフアルネシル化蛋白質 （Kammerer, S. et al. (1997) Genomics, 45, 2 00-21； Braun, A. et al. (1994) Gene, 146, 291 - 295)力 pi 9ARF蛋白質と結合 することを見出した。 HK33蛋白質と pl9ARF蛋白質との相互作用を哺乳動物細胞に おいても確認するために、 C0S7細胞を用いた mammalian Two- Hybrid法により実験 を行った。 その結果、 mammalian Two-Hybrid法においても、 これら二つの蛋白質 の相互作用が確認された。 また、 P19ARF1- 80、及ぴ pl9ARF8卜 Stopをコードしたミ ユータント P19ARFを用いた酵母 Two- Hybrid法実験においては、 pl9ARF卜 80は HK33 蛋白質とは結合せず、 P19ARF81- Stopにおいてのみ相互作用が確認できた。そのた め、 HK33蛋白質は、 pl6IM4aと共有した部位とは結合せずに、 pl9ARF特異的な部 位と相互作用することが示された。 さらに、 免疫沈降法によっても、 互作用を 再度確認した結果、 HK33蛋白質と ARF蛋白質との有意な相互作用が見られた。一方 、細胞内における pl9ARFの局在性を GFP-HK33と pl9ARf¾ycの共遺伝子導入、あるい は pl9ARFmyc単独、もしくは GFP- HK33単独で遺伝導入し、それぞれの蛋白質の局在 性を観察したところ、 HK33蛋白質によって pl9ARFmycの核への移行が有意に阻害さ れることが示された。また、 p53反応性のレポーター遺伝子を用いた in vitro実験 を行った結果、 HK33は pl9ARFによる p53依存性の転写活性を有意に阻害することを 見いだした。 さらに、 不死化する過程のヒト細胞を用いた HK33遺伝子の発現解析 を行つた結果、 老化によるクライシスを乗り越えた不死化細胞において HK33遣伝 子の発現が高まることを見いだした。 この結果は、 pl9ARFアンチセンス遺伝子導 入によつて pl9ARFを不活性ィヒした細胞が細胞寿命の延長を引き起こすという報告 と一致する結果である。 これらの結果から、 HK33自身が不死化を引き起こす因子 である可能性が示唆された。すなわち、 HK33蛋白質と ARF蛋白質との相互作用を抑 制する薬剤のスクリーニングを行うことは、 ARFの不活性ィヒによる細胞増殖を抑制 したり、 あるいは不死ィ匕した前癌状態の細胞の増殖を抑制できる新規の薬剤開発 を可能とする。 The present inventors have attempted to isolate a gene encoding a protein interacting with pl9ARF by the yeast Two-Hybrid method in order to solve the above problems. As a result, a pharmacoprotein called the HK33 gene (Kammerer, S. et al. (1997) Genomics, 45, 200-21; Braun, A. et al. (1994) Gene, 146, 291-295) It was found to bind to 9ARF protein. In order to confirm the interaction between HK33 protein and pl9ARF protein also in mammalian cells, an experiment was performed by a mammalian two-hybrid method using C0S7 cells. As a result, the interaction between these two proteins was also confirmed by the mammalian Two-Hybrid method. In the yeast two-hybrid method using P19ARF1-80 and pl9ARF8-Stop, a mutant that encodes P19ARF8-Stop, pl9ARF-80 did not bind to HK33 protein, and interaction was confirmed only at P19ARF81-Stop. did it. Therefore, it was shown that the HK33 protein interacts with a pl9ARF-specific site without binding to a site shared with pl6IM4a. Furthermore, the interaction was confirmed again by immunoprecipitation. As a result, a significant interaction between the HK33 protein and the ARF protein was found. On the other hand, the localization of pl9ARF in cells was determined by co-transfection of GFP-HK33 and pl9ARf¾yc, or transfected with pl9ARFmyc alone or GFP-HK33 alone, and the localization of each protein was observed. It was shown that the protein significantly inhibited pl9ARFmyc translocation to the nucleus. In addition, as a result of in vitro experiments using a reporter gene responsive to p53, it was found that HK33 significantly inhibited the p53-dependent transcriptional activity by pl9ARF. Furthermore, as a result of analyzing the expression of the HK33 gene using human cells in the process of immortalization, they found that the expression of the HK33 gene was increased in immortalized cells that survived the aging-related crisis. This result is consistent with the report that cells inactivated pl9ARF by introduction of the pl9ARF antisense gene cause prolonged cell life. These results suggested that HK33 may itself be a factor that causes immortalization. That is, the interaction between HK33 protein and ARF protein was suppressed. Screening for a drug that suppresses this will enable the development of a new drug that can suppress cell proliferation due to inactivation of ARF or suppress the proliferation of immortalized premalignant cells.

このように、 HK33蛋白質およびこれをコードする遺伝子は、 ARF蛋白質を介する シグナル伝達を制御するために有用であり、 また、 癌などの疾患の診断 ·検査の ための指標として、 また、 これら疾患の病理機構の解明のためのツールとして、 さらには、 これら疾患に対する医薬品開発のための標的として利用することがで きる。  Thus, the HK33 protein and the gene encoding it are useful for controlling signal transduction via the ARF protein, as an index for diagnosis and testing of diseases such as cancer, and It can be used as a tool for elucidating pathological mechanisms and as a target for drug development for these diseases.

すなわち本発明は、 ARF蛋白質と HK33蛋白質の相互作用を利用した抗腫瘍作用を 持つ薬剤のスクリーニング方法に関する。 また本発明は、 該相互作用または HK33 遺伝子の発現を調節することにより ARF蛋白質を介するシグナル伝達おょぴ細胞 の増殖を調節する方法に関する。 さらに本発明は、 腫瘍等の疾患における検査の ための HK33蛋白質おょぴ HK33蛋白質をコードする遺伝子の利用に関する。 本発明 は、 より具体的には、  That is, the present invention relates to a method for screening a drug having an antitumor effect utilizing an interaction between an ARF protein and a HK33 protein. In addition, the present invention relates to a method for regulating ARF protein-mediated signal transduction and cell proliferation by regulating the interaction or expression of the HK33 gene. Furthermore, the present invention relates to the use of the HK33 protein and the gene encoding the HK33 protein for testing for diseases such as tumors. The present invention more specifically relates to

( 1 ) HK33遺伝子の発現を上昇または下降させることにより、 ARFを介するシグナ ル伝達を阻害または促進する方法、  (1) a method of inhibiting or promoting ARF-mediated signal transmission by increasing or decreasing the expression of HK33 gene,

( 2 ) HK33遺伝子の発現を上昇または下降させることにより、 p53を介するシグナ ル伝達を阻害または促進する方法、  (2) a method of inhibiting or promoting p53-mediated signal transmission by increasing or decreasing the expression of HK33 gene,

( 3 ) HK33遺伝子の発現を上昇または下降させることにより、 細胞増殖を促進ま たは阻害する方法、  (3) a method of promoting or inhibiting cell proliferation by increasing or decreasing the expression of HK33 gene,

( 4 ) ARF蛋白質と HK33蛋白質との相互作用を上昇または下降させることにより、 ARFを介するシグナル伝達を阻害または促進する方法、  (4) a method of inhibiting or promoting ARF-mediated signal transmission by increasing or decreasing the interaction between ARF protein and HK33 protein,

( 5 ) ARF蛋白質と HK33蛋白質との相互作用を上昇または下降させることにより、 p53を介するシグナル伝達を阻害または促進する方法、  (5) A method of inhibiting or promoting p53-mediated signal transduction by increasing or decreasing the interaction between ARF protein and HK33 protein,

( 6 ) ARF蛋白質と HK33蛋白質との相互作用を上昇または下降させることにより、 細胞増殖を促進または阻害する方法、 (7) 細胞が腫瘍細胞である、 （3) または （6) に記載の方法、(6) a method of promoting or inhibiting cell proliferation by increasing or decreasing the interaction between ARF protein and HK33 protein, (7) The method according to (3) or (6), wherein the cell is a tumor cell,

( 8 ) ARF蛋白質と HK33蛋白質との相互作用を調節する化合物のスクリーニング方 法であって、 (8) A method for screening for a compound that modulates the interaction between ARF protein and HK33 protein,

( a ) 被検試料の存在下で ARF蛋白質と HK33蛋白質とを接触させる工程、 ( b ) ARF蛋白質と HK33蛋白質との相互作用を検出する工程、  (a) contacting the ARF protein with the HK33 protein in the presence of the test sample, (b) detecting the interaction between the ARF protein and the HK33 protein,

(c) 被検試料非存在下で検出した場合と比較して、 該相互作用を調節する化 合物を選択する工程、 を含む方法、  (c) selecting a compound that modulates the interaction, as compared to the case where the compound is detected in the absence of the test sample,

(9) ARF蛋白質と HK33蛋白質との相互作用を、 ARF蛋白質と HK33蛋白質との結合 を指標に検出する、 （8) に記載の方法、  (9) The method according to (8), wherein the interaction between the ARF protein and the HK33 protein is detected using the binding between the ARF protein and the HK33 protein as an index.

(10) 細胞系を用いて行う、 （8) または （9) に記載の方法、  (10) The method according to (8) or (9), wherein the method is performed using a cell line.

(1 1) ARF蛋白質を介するシグナル伝達を調節する化合物のスクリーニング方法 であって、  (11) A method for screening a compound that regulates signal transduction through an ARF protein,

(a) HK33遺伝子を内因的に保持する細胞に被検試料を接触させる工程、 (a) contacting a test sample with cells endogenously retaining the HK33 gene,

(b) HK33遺伝子の発現を検出する工程、 (b) detecting the expression of the HK33 gene,

(c) 被検試料を該細胞に接触させない場合と比較して、 該発現を調節する活 性を有する化合物を選択する工程、 を含む方法、  (c) selecting a compound having an activity of regulating the expression as compared to a case where the test sample is not contacted with the cells,

(1 2) ARF蛋白質を介するシグナル伝達を調節する化合物のスクリーニング方法 であって、  (12) A method for screening a compound that regulates signal transduction via an ARF protein,

( a ) HK33遺伝子の内因的転写制御配列の下流に機能的に結合されたレポータ 一遺伝子を有するベクターが導入された細胞に、 被検試料を接触させる工程、 (a) contacting a test sample with cells into which a vector having a reporter gene operably linked downstream of the endogenous transcription control sequence of the HK33 gene has been introduced,

(b) 該レポーター遺伝子の発現を検出する工程、 (b) detecting the expression of the reporter gene,

(c) 被検試料を該細胞に接触させない場合と比較して、 該レポーター遺伝子 の発現を調節する活性を有する化合物を選択する工程、 を含む方法、  (c) selecting a compound having an activity of regulating the expression of the reporter gene as compared to a case where the test sample is not contacted with the cells,

(13) (8) から （1 2) のいずれかに記載の方法により単離されうる化合物、 (13) a compound which can be isolated by the method according to any one of (8) to (12),

(14) HK33遺伝子の発現を調節する化合物、または ARF蛋白質と HK33蛋白質との 相互作用を調節する化合物を含む、 下記 （a) から (c) のいずれかに記載の薬 剤： (14) The drug according to any one of the following (a) to (c), comprising a compound that regulates the expression of the HK33 gene or a compound that regulates the interaction between the ARF protein and the HK33 protein. Agent:

( a ) . ARFを介するシグナル伝達の調節剤、  (a) a modulator of ARF-mediated signaling;

(b) p53を介するシグナル伝達の調節剤、  (b) a modulator of p53-mediated signaling,

(c) 細胞増殖調節剤、  (c) a cell growth regulator,

(1 5) HK33遺伝子の発現を調節する化合物、または ARF蛋白質と HK33蛋白質との 相互作用を調節する化合物を有効成分とする医薬組成物、  (15) a pharmaceutical composition comprising, as an active ingredient, a compound that regulates the expression of the HK33 gene, or a compound that regulates the interaction between the ARF protein and the HK33 protein.

(1 6) 抗腫瘍剤である、 （1 5) に記載の医薬組成物、  (16) The pharmaceutical composition according to (15), which is an antitumor agent,

(1 7) HK33蛋白質をコードする DNAまたはその相補鎖に相捕的な少なくとも 15 ヌクレオチドを含むポリヌクレオチド、 あるいは HK33蛋白質に結合する抗体を含 む、 下記 （a) か (e) のいずれかに記載の検査試薬 ·'  (17) Any of the following (a) or (e), including a polynucleotide containing at least 15 nucleotides complementary to the DNA encoding the HK33 protein or its complementary strand, or an antibody that binds to the HK33 protein: Test reagents described

(a) ARFを介するシグナル伝達の異常の検查試薬、  (a) a reagent for detecting an abnormality in signal transmission via ARF,

(b) p53を介するシグナル伝達の異常の検查試薬、  (b) a reagent for detecting abnormality in signal transduction via p53,

(c) 細胞増殖異常の検査試薬、  (c) a test reagent for abnormal cell proliferation,

(d) 細胞増殖性疾患の検査試薬、  (d) a cell proliferative disease test reagent,

(e) 細胞増殖性疾患が腫瘍である、 （d) の検査試薬、  (e) the cell proliferative disease is a tumor, the test reagent of (d),

(1 8) 下記 （a) または （b) の工程を含む、 ARFを介するシグナル伝達の異常 、 p53を介するシグナル伝達の異常、細胞増殖異常、細胞増殖性疾患、 または腫瘍 の検査方法：  (18) A method for examining abnormal ARF-mediated signal transduction, abnormal p53-mediated signal transduction, abnormal cell proliferation, cell proliferative disease, or tumor, including the following steps (a) or (b):

(a) 患者由来の被検試料中における HK33遺伝子の発現量を検出し、 健常者と 比較する工程、  (a) detecting the expression level of the HK33 gene in a test sample derived from a patient and comparing the expression level with a healthy subject;

(b) HK33蛋白質または該蛋白質をコードする核酸における変異を検出するェ 程、 に関する。  (b) detecting a mutation in the HK33 protein or a nucleic acid encoding the protein.

本発明において HK33遺伝子とは、 housekeeping gene HK33 (Braun, A. et al. ， Gene 146: 291-295, 1994)、 その相同遺伝子、 ァイソフォーム、 およびそれら の誘導体が含まれる。 HK33は PXFまたは PEX19pとも称される。 HK33遺伝子はヒト遺 伝子であってもよく、 また、 ヒト以外の真核生物由来の遺伝子であってもよい。 哺乳動物の HK33相同遺伝子としては、 ヒト PXF (ACCESSION X75535, Locus HSPX F)、 マウス PxF (ACCESSION Y09046, Locus画 PXFBALB)、 ラット PxF (ACCESSIO N Y09049, Locus RRPXFPRT) などが挙げられる。 また、 他の真核生物の HK33相同 遺伝子としては、 Pichia pastoris の Pexl9 (ACCESSION AF133271)、 Saccharom yces cerevisiae の Pexl9 (ACCESSION Q07418) などが知られている。 In the present invention, the HK33 gene includes the housekeeping gene HK33 (Braun, A. et al., Gene 146: 291-295, 1994), its homologous gene, isoform, and derivatives thereof. HK33 is also called PXF or PEX19p. The HK33 gene may be a human gene, or may be a gene derived from a non-human eukaryote. Examples of mammalian HK33 homologous genes include human PXF (ACCESSION X75535, Locus HSPXF), mouse PxF (ACCESSION Y09046, Locus drawing PXFBALB), rat PxF (ACCESSIO N Y09049, Locus RRPXFPRT) and the like. As other eukaryotic HK33 homologous genes, Pexl9 of Pichia pastoris (ACCESSION AF133271), Pexl9 of Saccharomyces cerevisiae (ACCESSION Q07418) and the like are known.

本発明において ARF遺伝子とは、 ARF (alternative reading frame) (Quelle, D. E. et al.， Cell 83 : 993-1000, 1995)、 その相同遺伝子、 ァイソフォーム、 およびそれらの誘導体が含まれる。 ARFは pl9ARFまたは pl4ARFとも称される。 ARFは 染色体 INK4a部位にコードされていることが知られている。 哺乳動物の ARF遺伝子 としては、 ヒト pl4 - CDK inhibitor (pl4ARF) (ACCESSION U17075, Locus HSU17 075)、 マウス pl9 ARF (ACCESSION L76092, Locus MUSARF) などが知られている 本発明において蛋白質間の 「相互作用」 とは、 蛋白質の結合、 修飾、 活性の変 化、 構造変化の誘導、 安定性の変化の誘導などを含む、 蛋白質間の作用を言う。 本発明において 「ARF蛋白質を介するシグナル伝達」 とは、 ARF蛋白質が媒介す る作用を言う。 また、 本発明において 「p53蛋白質を介するシグナル伝達」 とは、 p53蛋白質が媒介する作用を言う。 これらの作用には、 ARF蛋白質または p53蛋白質 が関与する様々な蛋白質の局在変化、 修飾、 構造変化 （活性化、 不活性化を含む )、 安定性変化、 遺伝子発現、 および細胞の表現型変化などが含まれる。 In the present invention, the ARF gene includes ARF (alternative reading frame) (Quelle, DE et al., Cell 83: 993-1000, 1995), homologous genes, isoforms, and derivatives thereof. ARF is also referred to as pl9 ARF or pl4 ARF . ARF is known to be encoded at the chromosome INK4a site. As mammalian ARF genes, human pl4-CDK inhibitor (pl4ARF) (ACCESSION U17075, Locus HSU17 075), mouse pl9 ARF (ACCESSION L76092, Locus MUSARF) and the like are known. The term "protein" refers to an action between proteins, including binding, modification, change in activity, induction of structural changes, and induction of changes in stability. In the present invention, “signal transduction via ARF protein” refers to an action mediated by ARF protein. In the present invention, “signal transduction via p53 protein” refers to an action mediated by p53 protein. These effects include changes in localization, modification, structural changes (including activation and inactivation), stability changes, gene expression, and cell phenotype of various proteins involving the ARF or p53 proteins. And so on.

本発明は、 HK33蛋白質が ARF蛋白質と相互作用し、 p53蛋白質の機能を調節する という新たな知見に基づく。実施例に示したように、 HK33蛋白質は ARF蛋白質に結 合する活性を示し、 HK33蛋白質の発現により ARF蛋白質の核局在が阻害される。 A RFは細胞周期に関連するシグナル伝達を調節している因子の一つであり、 MDM2に 結合し、 MDM2による p53の分解を抑制することが知られている。 また、 ARF蛋白質 は p53と直接相互作用することにより p53の機能を修飾することも示唆されている 。 HK33遺伝子の発現により HK33蛋白質が ARF蛋白質と結合し、 ARFの核局在化が阻 害されることにより、 ARFの細胞内における活性が抑制され、 ARFを介するシグナ ル伝達が抑制される。実際、 HK33の発現は p53依存性の転写活性を有意に阻害した これらのこと力 ら、 ARF蛋白質と HK33蛋白質との相互作用、または HK33遺伝子の 発現を上昇させることにより ARF蛋白質を介するシグナル伝達を阻害することが 可能であり、逆に ARF蛋白質と HK33蛋白質との相互作用、または HK33遺伝子の発現 を低下させることにより、ARF蛋白質を介するシグナル伝達を促進することが可能 である。 The present invention is based on the new finding that HK33 protein interacts with ARF protein and regulates the function of p53 protein. As shown in the Examples, HK33 protein exhibits an activity of binding to ARF protein, and nuclear localization of ARF protein is inhibited by expression of HK33 protein. ARF is one of the factors regulating cell cycle-related signal transduction, and is known to bind to MDM2 and suppress the degradation of p53 by MDM2. It has also been suggested that the ARF protein modulates p53 function by interacting directly with p53. HK33 protein binds to ARF protein by expression of HK33 gene, preventing nuclear localization of ARF By being harmed, the activity of ARF in cells is suppressed, and signal transmission via ARF is suppressed. Indeed, HK33 expression significantly inhibited p53-dependent transcriptional activity.From these facts, it was suggested that the interaction between ARF protein and HK33 protein or that the signal transduction through ARF protein could be increased by increasing the expression of HK33 gene. It is possible to inhibit the signal transduction through the ARF protein by reducing the interaction between the ARF protein and the HK33 protein or reducing the expression of the HK33 gene.

ARF蛋白質と HK33蛋白質との相互作用には、 ARF蛋白質と HK33蛋白質との結合お ょぴ修飾などが含まれる。 ARF蛋白質と HK33蛋白質との相互作用は、両者の蛋白質 の結合を検出したり、あるいは、 ARF蛋白質と HK33蛋白質との相互作用により生じ る ARFの機能またはシグナル伝達 （ARF核移行、 MDM2と ARFとの相互作用、 p53の安 定化、 p53を介するシグナル伝達の促進などを含む）などの阻害を指標に検出する ことが可能である。 HK33遺伝子の発現は、 その転写産物または蛋白質を検出する ことにより測定することができる。  The interaction between the ARF protein and the HK33 protein includes the binding and modification of the ARF protein with the HK33 protein. The interaction between the ARF protein and the HK33 protein can be detected by detecting the binding of the two proteins or by the ARF function or signal transduction (ARF nuclear translocation, MDM2 and ARF interaction) caused by the interaction between the ARF protein and the HK33 protein. Interaction, including stabilization of p53, promotion of p53-mediated signal transduction, etc.). Expression of the HK33 gene can be measured by detecting its transcript or protein.

例えば HK33遺伝子の発現を上昇させるには、 例えば HK33遺伝子を発現するべク ターを細胞に導入して HK33蛋白質の細胞内発現を上昇させればよい。 これにより 、 細胞内における HK33蛋白質の発現レベルが上昇し、 HK33蛋白質の細胞内におけ る活性を上昇させることができる。 逆に HK33遺伝子の発現の低下は、 例えば HK33 mRNAに対するァンチセンス RNAの発現、または HK33 raRNAを切断するようなリポザ ィムの発現などにより行うことができる。 また、 アンチセンスオリゴヌクレオチ ドまたは HK33の転写調節領域の一部を含むデコィ型核酸の投与などによっても実 施できる。 また、 HK33遺伝子に変異を導入してもよい。  For example, to increase the expression of the HK33 gene, for example, a vector expressing the HK33 gene may be introduced into cells to increase the intracellular expression of the HK33 protein. As a result, the expression level of the HK33 protein in the cell increases, and the activity of the HK33 protein in the cell can be increased. Conversely, the expression of the HK33 gene can be reduced by, for example, expression of antisense RNA against HK33 mRNA or expression of a liposome that cleaves HK33 raRNA. Alternatively, it can be carried out by administering an antisense oligonucleotide or a decoy nucleic acid containing a part of the transcription regulatory region of HK33. Further, a mutation may be introduced into the HK33 gene.

また、 HK33蛋白質のアンタゴニストを作用させて ARF蛋白質と HK33蛋白質との相 互作用を低下させることができる。 HK33蛋白質のアンタゴニストは、 HK33に対す る抗体またはその断片などであり得る。また、 ARF蛋白質の部分べプチドであり得 る。マウス ARF蛋白質のアミノ酸 81番目以降の部分ペプチドは、 HK33蛋白質と結合 する活性を有している。 このような、 HK33蛋白質に結合する活性を有する ARF蛋白 質の部分べプチドは、 HK33蛋白質への結合を通して、インタクトな ARF蛋白質への HK33蛋白質の相互作用を抑制し得る。 ARF蛋白質と HK33蛋白質との相互作用を調節 する化合物、 および HK33遺伝子の発現を調節する化合物は、 以下に述べるように 、 ARFを介するシグナル伝達の調節剤、 p53を介するシグナル伝達の調節剤、 およ ぴ細胞増殖調節剤として用いることが可能である。 Further, the interaction between the ARF protein and the HK33 protein can be reduced by acting an antagonist of the HK33 protein. The antagonist of the HK33 protein may be an antibody against HK33 or a fragment thereof. It may also be a partial peptide of the ARF protein. You. The partial peptide of mouse ARF protein from the 81st amino acid onward has the activity of binding to HK33 protein. Such a partial peptide of the ARF protein having an activity of binding to the HK33 protein can suppress the interaction of the HK33 protein with the intact ARF protein through binding to the HK33 protein. Compounds that regulate the interaction between the ARF protein and the HK33 protein, and compounds that regulate the expression of the HK33 gene, include ARF-mediated signal transduction regulators, p53-mediated signal transduction regulators, and Furthermore, it can be used as a cell growth regulator.

ARFを介するシグナル伝達の調節には、 ARF蛋白質の活性の調節が含まれる。 AR F活性としては、 例えば 蛋白質との結合などの、 ARF蛋白質が持つ活性が挙げ られる。 また、 ARF蛋白質を介するシグナル伝達の調節には、 ARF蛋白質から下流 のシグナル伝達分子の活性の変化およびそれに伴う形質の変化などの調節が含ま れる。すなわち、 ARF蛋白質を介するシグナル伝達の促進としては、 MDM2蛋白質と p53蛋白質との相互作用等の MDM2活性の抑制、 MDM2活性の抑制による p53蛋白質の 安定化、その安定化による p53蛋白質を介するシグナル伝達の活性化 (p53依存性転 写の促進、 細胞分裂および細胞増殖の抑制、 アポトーシス誘導などを含む） 等が 挙げられる。特に p53蛋白質は細胞機能の調節に重要な機能を果たす蛋白質であり 、 癌抑制遺伝子としても知られる。 HK33遺伝子の発現、 または ARF蛋白質と HK33 蛋白質との相互作用を上昇または下降させることにより、この p53を介するシグナ ル伝達をそれぞれ阻害または促進することが可能である。 HK33遺伝子の発現、 ま たは ARF蛋白質と HK33蛋白質との相互作用を上昇させれば、 p53の活性は抑制され 、 細胞分裂および細胞増殖の促進、 アポトーシスの抑制などがもたらされ得る。 逆に HK33遺伝子の発現、または ARF蛋白質と HK33蛋白質との相互作用を低下させれ ば、 p53の活性が促進され、 細胞増殖の抑制、 アポトーシスの誘導を行い得る。 H K33蛋白質の細胞内における活性の抑制は、 特に B重瘍に対する治療に有用である。 実際、実施例に示すように、セルクライシスを乗り越えた不死化細胞においては H K33遺伝子の発現レベルの上昇が観察された。 この事実は、細胞の不死化おょぴ癌 化に HK33蛋白質が関与していることを示唆するものであり、 HK33蛋白質の活性を 低下させることが腫瘍の増殖を抑制するために重要であることを示している。 以上のことから、 HK33遺伝子の発現、または ARF蛋白質と HK33蛋白質との相互作 用を調節（上昇または下降）する化合物は、 ARF蛋白質を介するシグナル伝達を調 節するために有用である。 本発明は、 HK33遺伝子の発現、 または ARF蛋白質と HK3 3蛋白質との相互作用を調節 （上昇または下降） する化合物を含む、 ARF蛋白質を 介するシグナル伝達の調節剤を提供する。またこれらの化合物は、 p53を介するシ グナル伝達の調節剤として、 さらに、 細胞増殖調節剤として有用である。 また、 これらの化合物は癌化剤 （または細胞の不死化剤、 発癌剤など） あるいは腫瘍抑 制剤 （抗癌剤） としても使用される。 Regulation of ARF-mediated signal transduction includes regulation of ARF protein activity. Examples of the ARF activity include an activity of the ARF protein such as binding to a protein. Regulation of signal transduction via the ARF protein includes regulation of changes in the activity of signaling molecules downstream from the ARF protein and concomitant changes in traits. That is, promotion of signal transduction via ARF protein includes suppression of MDM2 activity such as interaction between MDM2 protein and p53 protein, stabilization of p53 protein by suppression of MDM2 activity, and signal transduction through p53 protein by stabilization. Activation (including promotion of p53-dependent transcription, suppression of cell division and cell proliferation, induction of apoptosis, etc.). In particular, the p53 protein is a protein that plays an important role in regulating cell functions and is also known as a tumor suppressor gene. By increasing or decreasing the expression of the HK33 gene or the interaction between the ARF protein and the HK33 protein, it is possible to inhibit or promote this signal transmission via p53, respectively. If the expression of the HK33 gene or the interaction between the ARF protein and the HK33 protein is increased, the activity of p53 can be suppressed, and cell division and cell proliferation can be promoted, apoptosis can be suppressed, and the like. Conversely, if the expression of the HK33 gene or the interaction between the ARF protein and the HK33 protein is reduced, the activity of p53 can be promoted, suppressing cell proliferation and inducing apoptosis. Inhibition of intracellular activity of HK33 protein is particularly useful for treating B-severe ulcer. In fact, as shown in the examples, in the immortalized cells that survived the cell crisis, an increase in the expression level of the HK33 gene was observed. This fact makes the cells immortalize cancer This suggests that the HK33 protein is involved in metabolism, indicating that reducing the activity of the HK33 protein is important for suppressing tumor growth. Based on the above, compounds that regulate (increase or decrease) the expression of the HK33 gene or the interaction between the ARF protein and the HK33 protein are useful for regulating signal transduction via the ARF protein. The present invention provides a regulator of ARF protein-mediated signal transduction, including a compound that regulates (increases or decreases) the expression of the HK33 gene or the interaction between the ARF protein and the HK33 protein. In addition, these compounds are useful as modulators of p53-mediated signal transmission and as cell growth regulators. These compounds are also used as carcinogens (or cell immortalizing agents, carcinogens, etc.) or tumor suppressors (anticancer agents).

上記の化合物には、 HK33蛋白質が持つ生化学的活性（例えば ARFとの結合活性な ど） を調節する化合物の他、 細胞内または組織内における HK33遺伝子の発現レべ ルを調節する化合物などが含まれる。 HK33遺伝子の発現、 または ARF蛋白質と HK3 3蛋白質との相互作用を上昇させる化合物としては、例えば HK33蛋白質、およぴ該 蛋白質をコードする DNAが挙げられる。例えば、 HK33蛋白質を発現するベクターを 細胞に導入することにより HK33の発現レベルを上昇させることができる。 本発明 は、 ARF蛋白質または p53を介するシグナル伝達、 あるいは細胞増殖を調節するた めの、 HK33蛋白質、 該蛋白質をコードする DNA、 およぴ該 DNAを含むベクターの使 用に関する。  The above compounds include compounds that regulate the biochemical activity (eg, binding activity to ARF) of the HK33 protein, as well as compounds that regulate the expression level of the HK33 gene in cells or tissues. included. Examples of the compound that increases the expression of the HK33 gene or the interaction between the ARF protein and the HK33 protein include the HK33 protein and a DNA encoding the protein. For example, the expression level of HK33 can be increased by introducing a vector expressing the HK33 protein into cells. The present invention relates to the use of an HK33 protein, a DNA encoding the protein, and a vector containing the DNA, for controlling signal transduction via ARF protein or p53 or cell growth.

HK33蛋白質をコードする DNAは、 公知の方法により調製することが可能である。 例えば、 ヒ ト HK33蛋白質のアミノ酸配列、 および HK33蛋白質をコードする cDNAの 塩基配列は既に知られている （Kammerer, S. et al.， Genomics 45 : 200-210， 1 997 ; Braun, A. Gene 146 : 291-295, 1994)。 HK33をコードする DNAは、 例えばこ の塩基配列を基に作製したプローブを用いたハイプリダイゼーション、 あるいは この塩基配列を基に作製したプライマーを用いたポリメラーゼ連鎖反応 （polyme rase chain reaction; PCR) により調製することができる （Sambrook, J. et al . , Molecular Cloning 2nd ed.， 9. 47 - 9. 58， Cold Spring Harbor Lab. press, 1989)。 HK33蛋白質をコードする DNAは、 例えば、 ヒトまたはヒト以外の哺乳動物 (例えば、 サル、 マウス、 ラット、 モルモット、 ゥサギ、 ゥシ、 ブタなどがコー ドする蛋白質） 等から単離し得る。 HK33は様々な組織に発現しており、 これらの 組織から調製した cDNAをハイプリダイゼーシヨンまたは PCRを用いたスクリー二 ングに適用することができる。 ハイブリダィゼーシヨンの条件としては、 当業者 であれば適宜選択することができる。 例えば低ストリンジェントな条件、 通常の 条件、 またはストリンジェントな条件で行うことができる。 DNA encoding the HK33 protein can be prepared by a known method. For example, the amino acid sequence of human HK33 protein and the nucleotide sequence of cDNA encoding HK33 protein are already known (Kammerer, S. et al., Genomics 45: 200-210, 1997; Braun, A. Gene). 146: 291-295, 1994). DNA encoding HK33 can be obtained, for example, by hybridization using a probe prepared based on this base sequence, or by polymerase chain reaction (PCR) using a primer prepared based on this base sequence. (Sambrook, J. et al , Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. Press, 1989). DNA encoding the HK33 protein can be isolated, for example, from humans or non-human mammals (eg, proteins encoded by monkeys, mice, rats, guinea pigs, egrets, mice, pigs, etc.). HK33 is expressed in various tissues, and cDNA prepared from these tissues can be applied to screening using hybridization or PCR. Hybridization conditions can be appropriately selected by those skilled in the art. For example, the reaction can be performed under low stringent conditions, normal conditions, or stringent conditions.

ストリンジヱントなハイブリダイゼーションの条件は、 例えばハイブリダイゼ ーシヨン後の洗浄において、 50°C、 2 XSSC、 0. 1%SDSでの洗浄が挙げられ、 好ま しくは 50°C、 0. 1 X SSC、 0. 1%SDSで洗浄する。 またより好ましくは、 例えば 65°C 、 0. 1 X SSC及ぴ 0. 1%SDSでの洗浄が挙げられる。 これらの条件において、 温度を 上げる程に高い相同性を有する DNAが効率的に得られることが期待できる。 但し、 ハイブリダイゼーシヨンのストリンジエンシーに影響する要素としては温度や塩 濃度など複数の要素が考えられ、 当業者であればこれら要素を適宜選択すること で同様のストリンジエンシーを実現することが可能である。 このように、既知の H K33蛋白質をコードする cDNAとハイブリダィズする DNAがコードする蛋白質であつ て、 ARF蛋白質の核移行を阻害する蛋白質は、本発明において HK33蛋白質に含まれ る。  Stringent hybridization conditions include, for example, washing after hybridization at 50 ° C., 2 × SSC, 0.1% SDS, preferably 50 ° C., 0.1 × SSC, 0.1 × SSC. Wash with 1% SDS. More preferably, for example, washing with 65 ° C., 0.1 × SSC and 0.1% SDS can be mentioned. Under these conditions, it can be expected that DNA with higher homology can be obtained more efficiently as the temperature is increased. However, a plurality of factors such as temperature and salt concentration can be considered as factors affecting the stringency of the hybridization, and those skilled in the art can realize the same stringency by appropriately selecting these factors. Is possible. As described above, proteins that inhibit the nuclear translocation of the ARF protein, which are proteins encoded by DNA that hybridizes with the cDNA encoding the known HK33 protein, are included in the HK33 protein in the present invention.

これらハイブリダィゼーション技術や遺伝子増幅技術により単離される DNAが コードする蛋白質は、 通常、 ヒト HK33蛋白質 （Braun, A. et al. , Gene 146 : 29 1-295, 1994)または他の生物における相同蛋白質とアミノ酸配列に対し高い相同 性を有する。 これらの蛋白質と高い相同性を有する蛋白質において、 ARF蛋白質の 核移行を阻害する活性を有する蛋白質は、 本発明において HK33蛋白質に含まれる 。 高い相同性とは、 アミノ酸レベルにおいて、 通常、 少なくとも 40%以上の同一 性、 好ましくは 60%以上の同一性、 さらに好ましくは 80%以上の同一性、 さらに 好ましくは 90%以上の同一性を指す。 蛋白質の相同性を決定するには、 文献 （Wi lbur, W. J. and Lipman, D. J.， Pro Natl. Acad. Sci. USA (1983) 80， 726 -730) に記載のアルゴリズムにしたがえばよい。 The protein encoded by the DNA isolated by these hybridization techniques or gene amplification techniques is usually the human HK33 protein (Braun, A. et al., Gene 146: 29 1-295, 1994) or other organisms. Has high homology to homologous proteins and amino acid sequences. Among the proteins having high homology to these proteins, those having the activity of inhibiting the nuclear translocation of the ARF protein are included in the HK33 protein in the present invention. High homology generally means at least 40% or more identity, preferably 60% or more identity, more preferably 80% or more identity, at the amino acid level. Preferably, it indicates 90% or more identity. In order to determine the homology of proteins, the algorithm described in the literature (Wilbur, WJ and Lipman, DJ, Pro Natl. Acad. Sci. USA (1983) 80, 726-730) may be used.

また、 天然の HK33蛋白質の変異体を用いることもできる。 このような変異は人 為的であってもよく、 また自然界においても生じうる。 ある蛋白質の変異体を調 製するための、 当業者によく知られた方法としては、 蛋白質に変異を導入する方 法が知られている。 例えば、 当業者であれば、 部位特異的変異誘発法 （Hashimot o-Gotoh, T. et al. (1995) Gene 152, 271 - 275、 Zoller, M. J. , and Smith, M . (1983) Methods Enzymol. 100， 468—500、 Kramer, W. et al. (1984) Nucleic Acids Res. 12， 9441-9456、 Kramer W. , and Fritz H. J. (1987) Methods. Enz ymol. 154, 350-367、 Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA. 82, 488-492、 Kunkel (1988) Methods Enzymol. 85， 2763-2766) などを用いて、 天 然の HK33蛋白質のァミノ酸に適宜変異を導入することにより、 変異蛋白質を調製 することができる。 このように、天然の HK33蛋白質のアミノ酸配列において 1もし くは複数のアミノ酸が変異したアミノ酸配列を有し、 ARF蛋白質の核移行を阻害す る活性を有する蛋白質もまた本発明でいう HK33蛋白質に含まれる。  In addition, a mutant of the natural HK33 protein can also be used. Such mutations may be artificial or may occur in nature. As a method well known to those skilled in the art for preparing a mutant of a certain protein, a method of introducing a mutation into a protein is known. For example, those skilled in the art can use site-directed mutagenesis (Hashimot o-Gotoh, T. et al. (1995) Gene 152, 271-275, Zoller, MJ, and Smith, M. (1983) Methods Enzymol. 100, 468-500, Kramer, W. et al. (1984) Nucleic Acids Res. 12, 9441-9456, Kramer W., and Fritz HJ (1987) Methods. Enz ymol. 154, 350-367, Kunkel, TA (1985) Proc. Natl. Acad. Sci. USA. 82, 488-492, Kunkel (1988) Methods Enzymol. 85, 2763-2766), etc., and appropriately introduce mutation into the amino acid of natural HK33 protein By doing so, a mutant protein can be prepared. As described above, a protein having an amino acid sequence in which one or more amino acids are mutated in the amino acid sequence of the natural HK33 protein and having an activity of inhibiting nuclear translocation of the ARF protein is also referred to as the HK33 protein in the present invention. included.

このような変異体における、 変異するアミノ酸数は、 通常、 20アミノ酸以内で あり、好ましくは 10ァミノ酸以内であり、 さらに好ましくは 5ァミノ酸以内であり 、 さらに好ましくは 3アミノ酸以内 （例えば 1アミノ酸） であると考えられる。 変異するアミノ酸残基においては、 アミノ酸側鎖の性質が保存されている別の アミノ酸に変異すれば、 蛋白質の活性への影響を少なくできると期待される。 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、 レ M、 F、 P、 W、 Y、 V )、 親水性アミノ酸 （R、 D、 N、 E、 Q、 G、 H、 K、 S、 T)、 脂肪族側鎖を有するァ ミノ酸 （G、 A、 V、 し、 I、 P)、 水酸基含有側鎖を有するアミノ酸 （S、 T、 Υ)、 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、 Η)、 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 ァミノ酸の一文字標記を表す)。これらの性質が似たァミノ酸内の置換を保存的置 換と言う。 本発明において HK33蛋白質には、 アミノ酸の保存的置換を受けた蛋白 質が含まれる。 The number of amino acids to be mutated in such a mutant is usually within 20 amino acids, preferably within 10 amino acids, more preferably within 5 amino acids, and still more preferably within 3 amino acids (eg, 1 amino acid). ). It is expected that the effect on the activity of the protein can be reduced if the amino acid residue to be mutated is mutated to another amino acid whose amino acid side chain properties are conserved. For example, the properties of amino acid side chains include hydrophobic amino acids (A, I, M, F, P, W, Y, V) and hydrophilic amino acids (R, D, N, E, Q, G, H, K, S, T), amino acids with aliphatic side chains (G, A, V, I, P), amino acids with hydroxyl-containing side chains (S, T, Υ), sulfur atom-containing side chains (C, M) having amino acid, amino acid having carboxylic acid and amide-containing side chain (D, N, E, Q), amino acid having base side chain (R, K, Η), containing aromatic Amino acids having side chains (H, F, Y, W) can be mentioned (all brackets indicate one-letter designation of amino acid). Substitutions within amino acids that are similar in these properties are called conservative substitutions. In the present invention, the HK33 protein includes a protein having a conservative amino acid substitution.

あるアミノ酸配列に対する 1又は複数個のアミノ酸残基の欠失、 付加及ぴ Z又 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蛋白質がその 生物学的活性を維持することはすでに知られている （Mark, D. F. et al.， Proc . Natl. Acad. Sci. USA (1984) 81, 5662 - 5666、 Zoller, M. J. & Smith, M. N ucleic Acids Research (1982) 10, 6487—6500、 Wang, A. et al.， Science 224 ， 143卜 1433、 Dalbadie— McFarland, G. et al. , Proc. Natl. Acad. Sci. USA ( 1982) 79, 6409-6413) 0 It is already known that a protein having an amino acid sequence modified by deletion or addition of one or more amino acid residues to a certain amino acid sequence and substitution by Z or substitution with another amino acid maintains its biological activity. Natl. Acad. Sci. USA (1984) 81, 5662-5666, Zoller, MJ & Smith, M. Nucleic Acids Research (1982) 10, 6487-6500, Wang , A. et al., Science 224, 143 1433, Dalbadie—McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409-6413) 0

HK33蛋白質としては、 例えば天然の HK33蛋白質のアミノ酸配列に複数個のアミ ノ酸残基が付加された蛋白質であってもよい。 このような融合蛋白質は、天然の H K33蛋白質と他のペプチド又は蛋白質とが融合したものであり、 本発明における H K33蛋白質に含まれる。 融合蛋白質を作製する方法は、 HK33蛋白質をコードする D NAと他のぺプチド又は蛋白質をコードする DNAをフレームが一致するように連結 してこれを発現ベクターに導入し、 宿主で発現させればよく、 当業者に公知の手 法を用いることができる。 融合に付される他のペプチド又は蛋白質としては、 特 に限定されない。  The HK33 protein may be, for example, a protein in which a plurality of amino acid residues are added to the amino acid sequence of a natural HK33 protein. Such a fusion protein is a fusion of the natural H K33 protein and another peptide or protein, and is included in the H K33 protein of the present invention. To prepare a fusion protein, DNA encoding the HK33 protein and DNA encoding another peptide or protein are ligated in frame so that they are introduced into an expression vector and expressed in a host. Well, techniques known to those skilled in the art can be used. Other peptides or proteins to be subjected to the fusion are not particularly limited.

融合に付される他のペプチドとしては、 例えば、 FLAG (Hopp, T. P. et al.， BioTechnology (1988) 6， 1204 - 1210)、 6個の His (ヒスチジン） 残基からなる 6 XHis、 10 XHis、 インフルエンザ凝集素 （HA)、 ヒト c- myc の断片、 VSV- GPの断片 、 pl8HIVの断片、 T7- tag、 HSV_tag、 E- tag、 SV40T抗原の断片、 lck tag、 ひ- tu bulinの断片、 B-tag、 Protein C の断片等の公知のペプチドを使用することがで きる。 また、 融合に付される他の蛋白質としては、 例えば、 GST (グルタチオン S -トランスフェラーゼ）、 HA (ィンフルェンザ凝集素）、 ィムノグロプリン定常領 域、 β -ガラク トシダーゼ、 ΜΒΡ (マルトース結合蛋白質） 等が挙げられる。 市販 されているこれらべプチドまたは蛋白質をコードする DNAを ΗΚ33蛋白質をコード する DNAと融合させ、 これにより調製された融合 DNAを発現させることにより、 融 合蛋白質を調製することができる。 Other peptides to be fused include, for example, FLAG (Hopp, TP et al., BioTechnology (1988) 6, 1204-1210), 6 XHis consisting of 6 His (histidine) residues, 10 XHis, Influenza agglutinin (HA), human c-myc fragment, VSV-GP fragment, pl8HIV fragment, T7-tag, HSV_tag, E-tag, SV40T antigen fragment, lck tag, hy-tu bulin fragment, B Known peptides such as -tag and a fragment of Protein C can be used. Other proteins to be fused include, for example, GST (glutathione S-transferase), HA (influenza agglutinin), Region, β-galactosidase, ΜΒΡ (maltose binding protein) and the like. A fusion protein can be prepared by fusing commercially available DNA encoding these peptides or proteins with DNA encoding # 33 protein and expressing the fusion DNA thus prepared.

後述する蛋白質を産生する細胞や宿主あるいは精製方法により、 生産させた蛋 白質のアミノ酸配列、 分子量、 等電点又は糖鎖の有無や形態などが異なり得る。 しかしながら、得られた蛋白質力 ARF蛋白質の核移行を阻害する活性を有してい る限り、 本発明において ΗΚ33蛋白質に含まれる。 例えば、 ΗΚ33蛋白質を原核細胞 、例えば大腸菌で発現させた場合、本来の蛋白質のアミノ酸配列の Ν末端にメチォ ニン残基が付加されるが、 このような蛋白質も包含される。  The amino acid sequence, molecular weight, isoelectric point, presence / absence and form of sugar chains, etc. of the produced protein may differ depending on the cell, host, or purification method that produces the protein described below. However, as long as the protein has the activity of inhibiting nuclear translocation of the ARF protein, the protein is included in the 33 protein in the present invention. For example, when the ΗΚ33 protein is expressed in a prokaryotic cell, for example, Escherichia coli, a methionine residue is added to the Ν terminus of the amino acid sequence of the original protein, and such a protein is also included.

ΗΚ33蛋白質は、 当業者に公知の方法により、 組み換え蛋白質として、 また天然 の蛋白質として調製することが可能である。 組み換え蛋白質であれば、 ΗΚ33蛋白 質をコードする DNA (例えばヒト ΗΚ33 cDNAについては Braun, A. et al.， Gene 146 : 291 - 295， 1994 を参照）を適当な発現ベクターに組み込み、これを適当な宿 主細胞に導入して得た形質転換体を回収し、 抽出物を得た後、 イオン交換、 逆相 、 ゲル濾過などのクロマトグラフィー、 あるいは HK33蛋白質に対する抗体をカラ ムに固定したァフィ二ティークロマトグラフィーにかけることにより、 または、 さらにこれらのカラムを複数組み合わせることにより精製し、 調製することが可 能である。  # 33 protein can be prepared as a recombinant protein or a natural protein by methods known to those skilled in the art. If it is a recombinant protein, DNA encoding the ΗΚ33 protein (for example, see Braun, A. et al., Gene 146: 291-295, 1994 for human ΗΚ33 cDNA) is inserted into an appropriate expression vector, and this is inserted into an appropriate expression vector. After transformants obtained by introducing the cells into the host cell are collected and an extract is obtained, chromatography such as ion exchange, reverse phase, gel filtration, etc., or an antibody immobilized on a column with antibodies against HK33 protein It can be purified and prepared by subjecting it to tea chromatography or by further combining a plurality of these columns.

また、 HK33蛋白質をグルタチオン S-トランスフェラーゼ蛋白質との融合蛋白質 として、 あるいはヒスチジンを複数付加させた組み換え蛋白質として宿主細胞 （ 例えば、 動物細胞や大腸菌など） 内で発現させた場合には、 発現させた組み換え 蛋白質はグルタチオンカラムあるいはニッケルカラムを用いて精製することがで きる。 融合蛋白質の精製後、 必要に応じて融合蛋白質のうち、 目的の蛋白質以外 の領域を、 トロンビンまたはファクター Xaなどにより切断し、 除去することも可 能である。 天然の蛋白質であれば、 当業者に周知の方法、 例えば、 HK33蛋白質を発現して いる組織や細胞の抽出物に対し、 後述する HK33蛋白質に結合する抗体が結合した ァフィ二ティーカラムを作用させて精製することにより単離することができる。 抗体はポリクローナル抗体であってもモノクローナル抗体であってもよい。 When the HK33 protein is expressed in a host cell (for example, an animal cell or Escherichia coli) as a fusion protein with a glutathione S-transferase protein or as a recombinant protein to which multiple histidines are added, the expressed recombinant is used. The protein can be purified using a glutathione column or a nickel column. After purification of the fusion protein, if necessary, regions other than the target protein in the fusion protein can be cleaved with thrombin or Factor Xa and removed. If the protein is a natural protein, a method known to those skilled in the art, for example, an affinity column to which an antibody that binds to the HK33 protein described below is allowed to act on a tissue or cell extract expressing the HK33 protein, as described below. And can be isolated by purification. Antibodies may be polyclonal or monoclonal.

HK33蛋白質をコードする DNAは、 HK33蛋白質の in vivoや in vitroにおける生 産に利用される。 また、 HK33蛋白質の活性を上昇させるための遺伝子治療などへ の応用も考えられる。 HK33蛋白質をコードする DNAは、 HK33蛋白質をコードしうる ものであればいかなる形態でもよい。 即ち、 raRNAから合成された cDNAであるか、 ゲノム DNAである力、 化学合成 DNAであるかなどを問わない。 また、 HK33蛋白質を コードしうる限り、遺伝暗号の縮重に基づく任意の塩基配列を有する DNAが含まれ る。  DNA encoding the HK33 protein is used for in vivo and in vitro production of the HK33 protein. Further, application to gene therapy for increasing the activity of HK33 protein is also considered. The DNA encoding the HK33 protein may be in any form as long as it can encode the HK33 protein. That is, it does not matter whether it is cDNA synthesized from raRNA, genomic DNA, or chemically synthesized DNA. In addition, DNAs having any nucleotide sequence based on the degeneracy of the genetic code are included as long as they can encode the HK33 protein.

HK33蛋白質をコードする DNAは、当業者に公知の方法により調製することができ る。 例えば、 HK33蛋白質を発現している細胞より cDNAライブラリーを作製し、 HK 33 cDNA (例えばヒト HK33 cDNAについては Braun, A. et al. , Gene 146 : 291 - 2 95, 199 を参照）の一部をプローブにしてハイブリダィゼーシヨンを行うことに より調製できる。 cDNAライブラリ一は、 例えば、 文献 （Sambrook, J. et al. , M olecular Cloning^ Cold Spring Harbor Laboratory Press (1989) ) ίこ fi載の方 法により調製してもよいし、 市販の DNAライプラリーを用いてもよい。 また、 HK 33蛋白質を発現している細胞より RNAを調製し、逆転写酵素により cDNAを合成した 後、 HK33 cDNA配列に基づいてオリゴ DNAを合成し、 これをプライマーとして用い て PCRを行い、 HK33蛋白質をコードする cDNAを増幅させることにより調製すること も可能である。  DNA encoding the HK33 protein can be prepared by a method known to those skilled in the art. For example, a cDNA library is prepared from cells expressing the HK33 protein, and a library of HK33 cDNA (for example, see Braun, A. et al., Gene 146: 291-295, 199 for human HK33 cDNA) is prepared. It can be prepared by performing hybridization using the part as a probe. The cDNA library may be prepared, for example, by the method described in the literature (Sambrook, J. et al., Molecular Cloning ^ Cold Spring Harbor Laboratory Press (1989)), or a commercially available DNA library may be prepared. May be used. In addition, after preparing RNA from cells expressing the HK33 protein, synthesizing cDNA with reverse transcriptase, synthesizing oligo DNA based on the HK33 cDNA sequence, and performing PCR using this as a primer, It can also be prepared by amplifying a cDNA encoding a protein.

また、 得られた cDNAの塩基配列を決定することにより、 それがコードする翻訳 領域を決定でき、 HK33蛋白質のアミノ酸配列を得ることができる。 また、 得られ た cDNAをプローブとしてゲノム DNAライプラリーをスクリーニングすることによ り、 ゲノム DNAを単離することができる。 同様に、 HK33蛋白質をコードする遺伝子 の内因的転写制御配列を得ることもできる。 例えば、 これまでにヒト HK33遺伝子 の転写制御領域が報告されている （Karamerer, S. et al. , Genomics 45 : 200-21 0, 1997) ο Also, by determining the nucleotide sequence of the obtained cDNA, the translation region encoded by the cDNA can be determined, and the amino acid sequence of the HK33 protein can be obtained. Genomic DNA can be isolated by screening the genomic DNA library using the obtained cDNA as a probe. Similarly, the gene encoding the HK33 protein Can also be obtained. For example, the transcriptional regulatory region of the human HK33 gene has been reported so far (Karamerer, S. et al., Genomics 45: 200-210, 1997) ο

具体的には、 次のようにすればよい。 まず、 ΗΚ33蛋白質を発現する細胞、 組織 、 臓器から mRNAを単離する。 mRNAの単離は、 公知の方法、 例えば、 グァニジン超 遠心法（Chirgwin, J. M. et al. , Biochemistry (1979) 18， 5294-5299)、 AGPC 法 （Chomczynski, P. and Sacchi, N.， Anal. Biochem. (1987) 162, 156-159) 等により全 RNAを調製し、 mRNA Purification Kit (Pharmacia) 等を使用して全 R NA力、ら mRNAを精製する。 また、 QuickPrep mRNA Purification Kit (Pharmacia) を用いることにより mRNAを直接調製することもできる。 得られた mRNAから逆転写 酵素を用いて cDNAを合成する。 cDNAの合成は、 AMV Reverse Transcriptase Firs t- strand cDNA Synthesis Kit (生化学工業）等を用いて行うこともできる。また 、 5' -Afflpli FINDER RACE Kit (Clontech製）および PCRを用いた 5， -RACE法や 3， - R ACE法 （Frohman, M. A. et al. , Proc. Natl. Acad. Sci. U. S. A. (1988) 85, 8 998-9002 ； Belyavsky, A. et al.， Nucleic Acids Res. (1989) 17, 2919—2932 ) にしたがい、 cDNAの合成おょぴ増幅を行うことができる。  Specifically, the following may be performed. First, mRNA is isolated from cells, tissues and organs that express the ΗΚ33 protein. mRNA can be isolated by known methods, for example, guanidine ultracentrifugation (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299), and AGPC method (Chomczynski, P. and Sacchi, N., Anal. Total RNA is prepared using Biochem. (1987) 162, 156-159) and the like, and the total RNA is purified using mRNA Purification Kit (Pharmacia) and the like. Alternatively, mRNA can be directly prepared by using QuickPrep mRNA Purification Kit (Pharmacia). CDNA is synthesized from the obtained mRNA using reverse transcriptase. The cDNA can also be synthesized using AMV Reverse Transcriptase Fis- terstrand cDNA Synthesis Kit (Seikagaku Corporation) or the like. The 5'-Afflpli FINDER RACE Kit (manufactured by Clontech) and the 5, -RACE and 3, -RACE methods using PCR (Frohman, MA et al., Proc. Natl. Acad. Scad. USA (1988) 85, 8 998-9002; cDNA synthesis and amplification can be performed according to Belyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932).

得られた PCR産物から目的とする DNA断片を調製し、ベクター DNAと連結する。 さ らに、 これより組換えベクターを作製し、 大腸菌等に導入してコロニーを選択し て所望の組換えベクターを調製する。 目的とする DNAの塩基配列は、 公知の方法、 例えば、 ジデォキシヌクレオチドチェインターミネーション法により確認するこ とができる。  A target DNA fragment is prepared from the obtained PCR product and ligated to a vector DNA. Further, a recombinant vector is prepared from this, introduced into E. coli, etc., and colonies are selected to prepare a desired recombinant vector. The base sequence of the target DNA can be confirmed by a known method, for example, a dideoxynucleotide chain termination method.

HK33蛋白質をコードする DNAは、発現に使用する宿主のコドン使用頻度を考慮し て、 より発現効率の高い塩基配列を設計することができる （Grantham, R. et al つ Nucleic Acids Research (1981) 9, r43- 74)。また、市販のキットゃ公知の方 法によって DNA配列を改変することができる。改変としては、例えば、制限酵素に よる消化、合成ォリゴヌクレオチドゃ適当な DNAフラグメントの挿入、 リンカーの 付加、 開始コドン （ATG) 及ぴ Z又は終止コドン （TAA、 TGA、 又は TAG) の揷入等 が挙げられる。 For the DNA encoding the HK33 protein, a nucleotide sequence with higher expression efficiency can be designed in consideration of the codon usage of the host used for expression (Grantham, R. et al., Nucleic Acids Research (1981) 9 , r43-74). The DNA sequence can be modified by a commercially available kit known method. Modifications include, for example, digestion with restriction enzymes, insertion of synthetic oligonucleotides--appropriate DNA fragments, Addition, insertion of an initiation codon (ATG) and Z or a termination codon (TAA, TGA, or TAG).

HK33蛋白質をコードする DNAを用いて HK33蛋白質を発現させるためには、例えば 発現ベクターを構築し、 宿主細胞に導入する。 ベクターとしては、 例えば、 大腸 菌を宿主とする場合には、 ベクターを大腸菌 （例えば、 JM109、 DH5ひ、 HB101、 X LlBlue) などで大量に増幅させ大量調製するために、 大腸菌で増幅されるための In order to express the HK33 protein using the DNA encoding the HK33 protein, for example, an expression vector is constructed and introduced into a host cell. As a vector, for example, when E. coli is used as a host, the vector is amplified in E. coli (e.g., JM109, DH5, HB101, XLlBlue), etc. of

「ori」 をもち、 さらに形質転換された大腸菌の選抜遺伝子 （例えば、 なんらかの 薬剤 （アンピシリンやテトラサイクリン、 カナマイシン、 クロラムフエ二コール ) により判別できるような薬剤耐性遺伝子） を有すれば特に制限はない。 ベクタ 一の例としては、 M13系ベクター、 pUC系ベクター、 pBR322、 pBluescript, pCR- S criptなどが挙げられる。 また、 cDNAのサブクローニング、切り出しを目的とした 場合、 上記ベクターの他に、例えば、 pGEM-T、 pDIRECT、 pT7などが挙げられる。 Η Κ33蛋白質を生産する目的においてベクターを使用する場合には、特に、発現べク ターが有用である。 発現ベクターとしては、 例えば、 大腸菌での発現を目的とし た場合は、ベクターが大腸菌で増幅されるような上記特徴を持つほかに、宿主を J M109、 DH5 a、 HB101、 XLl- Blueなどの大腸菌とした場合においては、 大腸菌で効 率よく発現できるようなプロモーター、 例えば、 lacZプロモーター （Wardら， Na ture (1989) 341, 544 - 546； FASEB J. (1992) 6, 2422 2427)、 araBプロモーターThere is no particular limitation as long as it has "ori" and further has a transformed gene for selection of Escherichia coli (eg, a drug resistance gene that can be identified by any drug (ampicillin, tetracycline, kanamycin, chloramphenicol)). Examples of vectors include M13-based vectors, pUC-based vectors, pBR322, pBluescript, pCR-Script, and the like. Further, in the case of subcloning and excision of cDNA, for example, pGEM-T, pDIRECT, pT7 and the like can be mentioned in addition to the above vectors. Expression vectors are particularly useful when vectors are used to produce the 33 protein. For example, when the expression vector is intended for expression in Escherichia coli, the expression vector may have the above-mentioned characteristics such that the vector is amplified in Escherichia coli, and may be used in a host such as E. coli such as JM109, DH5a, HB101, or XL1-Blue. In this case, a promoter that can be efficiently expressed in Escherichia coli, such as the lacZ promoter (Ward et al., Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422 2427), the araB promoter

(Betterら， Science (1988) 240， 1041 - 1043)、 または T7プロモーターなどを持 つていることが不可欠である。 このようなベクターとしては、 上記ベクターの他 に pGEX- 5Χ- 1 (フアルマシア社製）、 Γ QIAexpress systemj (キアゲン社製）、 pEGF P、 または pET (この場合、 宿主は T7 RNAポリメラーゼを発現する BL21が好ましレ、） などが挙げられる。 (Better et al., Science (1988) 240, 1041-1043), or have a T7 promoter. Such vectors include pGEX-5 上 記 -1 (Pharmacia), Γ QIAexpress systemj (Qiagen), pEGFP, or pET (in this case, the host is BL21 that expresses T7 RNA polymerase). Is preferred, etc.).

また、 ベクターには、 ポリペプチド分泌のためのシグナル配列が含まれていて もよい。 蛋白質分泌のためのシグナル配列としては、 大腸菌のペリブラズムに産 生させる場合、 pelBシグナル配列 （Lei， S. P. et al J. Bacteriol. (1987) 16 9， 4379) を使用すればよレ、。宿主細胞へのベクターの導入は、例えば塩化カルシ ゥム法、 エレクトロポレーション法を用いて行うことができる。 The vector may also include a signal sequence for polypeptide secretion. As a signal sequence for protein secretion, the pelB signal sequence (Lei, SP et al J. Bacteriol. (1987) 16 9, 4379). Introduction of a vector into a host cell can be performed using, for example, a calcium chloride method or an electroporation method.

大腸菌以外にも、 例えば、 HK33蛋白質を製造するためのベクターとしては、 哺 乳動物由来の発現ベクター （例えば、 pcDNA3 (インビトロゲン社製） や、 pEGF- B OS (Nucleic Acids. Res. 1990, 18 (17)， p5322)、 pEF、 pCDM8)、昆虫細胞由来の発 現べクタ一 (例えば 「Bac- to - BAC baculovairus expression systemj (ギブコ BR L社製）、 pBacPAK8)、 植物由来の発現ベクター （例えば ρΜΗ1、 pMH2)、 動物ウィル ス由来の発現ベクター （例えば、 pHSV、 pMV、 pAdexLc ) , レトロウイルス由来の 発現ベクター （例えば、 pZIPneo)、 酵母由来の発現ベクター （例えば、 「Pichia Expression KitJ (インビトロゲン社製）、 pNVll、 SP- Q01)、 枯草菌由来の発現べ クタ一 （例えば、 pPL608、 pKTH50) が挙げられる。  In addition to Escherichia coli, for example, as a vector for producing the HK33 protein, a mammal-derived expression vector (for example, pcDNA3 (manufactured by Invitrogen) or pEGF-BOS (Nucleic Acids. Res. 1990, 18) (17), p5322), pEF, pCDM8), insect cell-derived expression vectors (eg, “Bac-to-BAC baculovairus expression systemj (manufactured by Gibco BRL), pBacPAK8), plant-derived expression vectors (eg, ρΜΗ1, pMH2), animal virus-derived expression vectors (eg, pHSV, pMV, pAdexLc), retrovirus-derived expression vectors (eg, pZIPneo), yeast-derived expression vectors (eg, “Pichia Expression KitJ (Invitrogen) ), PNVll, SP-Q01), and expression vectors derived from Bacillus subtilis (eg, pPL608, pKTH50).

CH0細胞、 COS細胞、 NIH3T3細胞等の動物細胞での発現を目的とした場合には、 細胞内で発現させるために必要なプロモーター、 例えば SV40プロモーター （Mull iganら， Nature (1979) 277, 108)、 MMLV-LTRプロモーター、 EF1ひプロモーター (Mizushimaら， Nucleic Acids Res. (1990) 18， 5322)、CMVプロモーターなどを 持っていることが不可欠であり、 細胞への形質転換を選抜するための遺伝子 （例 えば、 薬剤 （ネオマイシン、 G418など） により判別できるような薬剤耐性遺伝子 ) を有すればさらに好ましい。 このような特性を有するベクターとしては、 例え ば、 pMAM、 pDR2、 pBK - RSV、 pBK- CMV、 p0PRSV、 p0P13などが挙げられる。  When the expression is intended for expression in animal cells such as CH0 cells, COS cells, and NIH3T3 cells, a promoter necessary for expression in cells, for example, SV40 promoter (Mulligan et al., Nature (1979) 277, 108) , The MMLV-LTR promoter, the EF1 promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), the CMV promoter, etc., are essential, and the genes for selecting for cell transformation ( For example, it is more preferable to have a drug resistance gene that can be identified by a drug (neomycin, G418, etc.). Examples of vectors having such properties include pMAM, pDR2, pBK-RSV, pBK-CMV, p0PRSV, p0P13, and the like.

さらに、 遺伝子を安定的に発現させ、 かつ、 細胞内での遺伝子のコピー数の増 幅を目的とする場合には、 核酸合成経路を欠損した CH0細胞にそれを相補する DHF R遺伝子を有するベクター （例えば、 pCHOIなど） を導入し、 メトトレキセート （M TX) により増幅させる方法が挙げられ、 また、 遺伝子の一過性の発現を目的とす る場合には、 SV40 T抗原を発現する遺伝子を染色体上に持つ COS細胞を用いて SV4 0の複製起点を持つベクター （pcDなど） で形質転換する方法が挙げられる。 複製 開始点としては、 また、 ポリオ一マウィルス、 アデノウイルス、 ゥシパピローマ ウィルス （BPV) 等の由来のものを用いることもできる。 さらに、宿主細胞系で遺 伝子コピー数増幅のため、 発現ベクターは選択マーカーとして、 アミノグリコシ ドトランスフェラーゼ （APH) 遺伝子、 チミジンキナーゼ （TK) 遺伝子、 大腸菌キ サンチングァニンホスホリボシルトランスフェラーゼ （Ecogpt) 遺伝子、 ジヒ ド 口葉酸還元酵素 （dhfr) 遺伝子等を含むことができる。 Furthermore, when the gene is to be stably expressed and the copy number of the gene is to be increased in a cell, a vector having a DHFR gene that complements a nucleic acid synthesis pathway-deficient CH0 cell is used. (E.g., pCHOI) and then amplify it with methotrexate (MTX). For transient expression of the gene, the gene expressing the SV40 T There is a method of transforming a vector having a replication origin of SV40 (such as pcD) using the COS cell possessed above. The replication origins include poliovirus, adenovirus, Viruses (BPV) and the like can also be used. In addition, for amplification of gene copy number in the host cell system, the expression vector is used as a selectable marker such as aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, Escherichia coli xanthinguanine phosphoribosyltransferase (Ecogpt) gene, It can contain a dihydrofolate reductase (dhfr) gene and the like.

一方、 動物の生体内で HK33蛋白質をコードする DNAを発現させる方法としては、 該 DNAを適当なベクターに組み込み、 例えば、 レトロウイルス法、 リボソーム法、 カチォニックリボソーム法、 アデノウィルス法などにより生体内に導入する方法 などが挙げられる。 これにより、 HK33遺伝子の変異に起因する疾患、 または HK33 遺伝子の発現により治療可能な疾患等に対する遺伝子治療を行うことも可能であ る。 用いられるベクターとしては、 例えば、 アデノウイルスベクター （例えば pA dexlcw) やレトロウイルスベクター（例えば pZIPneo) などが挙げられる力 これ らに制限されない。 ベクターへの HK33 cDNAの揷入などの一般的な遺伝子操作は、 常法に従って行うことが可能である （Molecular Cloning , 5. 61-5. 63) 0生体内へ の投与は、 ex vivo法または in vivo法であり得る。 HK33蛋白質をコードするこれ らのベクターは、 本発明において HK33遺伝子の発現を上昇させる薬剤として用い ることができる。 On the other hand, as a method for expressing DNA encoding the HK33 protein in an animal body, the DNA is incorporated into an appropriate vector, and the DNA is expressed in vivo by the retrovirus method, ribosome method, catonic ribosome method, adenovirus method, or the like. And other methods. This makes it possible to perform gene therapy for diseases caused by mutations in the HK33 gene, or diseases that can be treated by expression of the HK33 gene. Examples of the vector used include, but are not limited to, adenovirus vectors (eg, pAdexlcw) and retrovirus vectors (eg, pZIPneo). General genetic manipulations such as揷入of HK33 cDNA into a vector, it can be performed according to conventional methods (Molecular Cloning, 5. 61-5. 63 ) 0 administration in vivo, ex vivo method or It may be an in vivo method. These vectors encoding the HK33 protein can be used in the present invention as an agent for increasing the expression of the HK33 gene.

HK33蛋白質をコードする DNAにより宿主細胞を形質転換し、形質転換された細胞 を培養することにより、 HK33蛋白質を in vitroで生産することができる。 培養は 、 公知の方法に従い行うことができる。 例えば、 動物細胞の培養液としては、 DM EM、 MEM、 RPMI1640, IMDMなどを使用することができる。 その際、 牛胎児血清 （F CS)等の血清補液を併用することもできるし、無血清培養してもよい。培養時の p Hは、 約 6〜8であるのが好ましい。 培養は、 通常、 約 30〜40°Cで約 15〜200時間行 い、 必要に応じて培地の交換、 通気、 攪拌を加える。  HK33 protein can be produced in vitro by transforming a host cell with DNA encoding the HK33 protein and culturing the transformed cell. The culture can be performed according to a known method. For example, DMEM, MEM, RPMI1640, IMDM and the like can be used as a culture solution of animal cells. At that time, a serum replacement solution such as fetal calf serum (FCS) may be used in combination, or serum-free culture may be performed. The pH during culturing is preferably about 6-8. Culture is usually performed at about 30 to 40 ° C for about 15 to 200 hours, and the medium is replaced, aerated, and agitated as necessary.

ベクターが導入される宿主細胞としては特に制限はなく、 例えば、 大腸菌や種 々の真核細胞などを用いることが可能である。 真核細胞を使用する場合、 例えば 、 動物細胞、 植物細胞、 真菌細胞を宿主に用いることができる。 動物細胞として は、 哺乳類細胞、 例えば、 CH0、 COS, 3T3、 ミエローマ、 BHK (baby hamster kid ney)、 HeLa、 Vero、 両生類細胞、 例えばアフリカッメガエル卵母細胞 （Valle, e t al. , Nature (1981) 291, 358 - 340)、 あるいは昆虫細胞、 例えば、 Sf9、 Sf21 、 Tn5が知られている。 CH0細胞としては、 特に、 DHFR遺伝子を欠損した CH0細胞 である dhfr- CHO (Proc. Natl. Acad. Sci. USA (1980) 77, 4216-4220) や CHO K - 1 (Proc. Natl. Acad. Sci. USA (1968) 60， 1275) を好適に使用することがで きる。動物細胞において、大量発現を目的とする場合には特に CH0細胞が好ましい 。 宿主細胞へのベクターの導入は、 例えば、 リン酸カルシウム法、 DEAEデキスト ラン法、 カチォニックリボソーム D0TAP (ベーリンガーマンハイム社製） を用いた 方法、 エレクト口ポーレーション法、 リボフヱクシヨンなどの方法で行うことが 可能である。 植物細胞としては、 例えば、 ニコチアナ 'タパカム （Nicotiana ta bacum) 由来の細胞が蛋白質生産系として知られており、これをカルス培養すれば よい。 真菌細胞としては、 酵母、 例えば、 サッカロミセス （Saccharomyces) 属、 例えば、 サッカロミセス -セレビシェ (Saccharomyces cerevisiae) , 糸状菌、 例 えば、 ァスペルギルス （Aspergillus) 属、 例えば、 ァスペルギノレス ·二ガー （A spergillus niger) が知られている。 原核細胞を使用する場合、 細菌細胞を用い る産生系がある。 細菌細胞としては、 大腸菌 （E. coli)、 例えば、 JM109、 DH5 o; 、 HB101 等が挙げられ、 その他、 枯草菌が知られている。 The host cell into which the vector is introduced is not particularly limited, and for example, Escherichia coli and various eukaryotic cells can be used. When using eukaryotic cells, for example, Animal cells, plant cells, and fungal cells can be used as hosts. Animal cells include mammalian cells, for example, CH0, COS, 3T3, myeloma, BHK (baby hamster kid ney), HeLa, Vero, amphibian cells, for example, African Megafrog oocytes (Valle, et al., Nature ( 1981) 291, 358-340), or insect cells such as Sf9, Sf21 and Tn5. Examples of the CH0 cells include DHfr-CHO (Proc. Natl.Acad.Sci. USA (1980) 77, 4216-4220) and CHO K-1 (Proc. Natl. Acad. Sci. USA (1968) 60, 1275) can be suitably used. For the purpose of expressing a large amount in animal cells, CH0 cells are particularly preferable. The vector can be introduced into a host cell by, for example, the calcium phosphate method, the DEAE dextran method, the method using Cationic Ribosome D0TAP (Boehringer Mannheim), the electoporation method, or the ribofusion method. It is. As a plant cell, for example, a cell derived from Nicotiana tapacum (Nicotiana ta bacum) is known as a protein production system, and it may be callus cultured. Fungal cells include yeast, for example, the genus Saccharomyces, for example, Saccharomyces cerevisiae, filamentous fungi, for example, the genus Aspergillus, for example, Aspergillus niger (A spergill ni). Have been. When prokaryotic cells are used, there is a production system using bacterial cells. Examples of the bacterial cell include Escherichia coli (E. coli), for example, JM109, DH5o; and HB101. Bacillus subtilis is also known.

一方、 in vivo で蛋白質を産生させる系としては、 例えば、 動物を使用する産 生系や植物を使用する産生系が挙げられる。これらの動物又は植物に目的とする D NAを導入し、 動物又は植物の体内で蛋白質を産生させ、 回収する。  On the other hand, examples of a system for producing a protein in vivo include a production system using animals and a production system using plants. The desired DNA is introduced into these animals or plants, and proteins are produced and recovered in the animals or plants.

動物を使用する場合、 哺乳類動物、 昆虫を用いる産生系がある。 哺乳類動物と しては、 ャギ、 ブタ、 ヒッジ、 マウス、 ゥシを用いることができる （Vicki Glas er, SPECTRUM Biotechnology Applications, 1993)。 また、 哺乳類動物を用いる 場合、 トランスジエニック動物を用いることができる。 例えば、 目的とする DNAを、ャギ jSカゼインのような乳汁中に固有に産生される 蛋白質をコードする遺伝子との融合遺伝子として調製する。 次いで、 この融合遺 伝子を含む DNA断片をャギの胚へ注入し、この胚を雌のャギへ移植する。胚を受容 したャギから生まれるトランスジエニックャギ又はその子孫が産生する乳汁から 、 目的の蛋白質を得ることができる。 トランスジエニックャギから産生される蛋 白質を含む乳汁量を増加させるために、 適宜ホルモンをトランスジエニックャギ に使用してもよい （Ebert, K. M. et al.， Bio/Technology (1994) 12, 699-702 When using animals, there are production systems using mammals and insects. Goats, pigs, sheep, mice, and mice can be used as mammals (Vicki Glasser, SPECTRUM Biotechnology Applications, 1993). When a mammal is used, a transgenic animal can be used. For example, a target DNA is prepared as a fusion gene with a gene encoding a protein that is specifically produced in milk, such as goat jS casein. Next, the DNA fragment containing the fusion gene is injected into a goat embryo, and the embryo is transplanted into a female goat. The target protein can be obtained from milk produced by the transgenic goat born from the goat that has received the embryo or its progeny. Hormones may be used in transgenic goats as appropriate to increase the amount of milk containing proteins produced by transgenic goats (Ebert, KM et al., Bio / Technology (1994) 12 , 699-702

)。 ).

また、 昆虫としては、 例えばカイコを用いることができる。 カイコを用いる場 合、目的の蛋白質をコードする DNAを挿入したパキュロウィルスをカイコに感染さ せることにより、 このカイコの体液から目的の蛋白質を得ることができる （Susu rau, M. et al. , Nature (1985) 315, 592-59.4)。  In addition, silkworms can be used as insects, for example. When a silkworm is used, the target protein can be obtained from the body fluid of the silkworm by infecting the silkworm with a paculovirus into which DNA encoding the protein of interest has been inserted (Susu rau, M. et al. , Nature (1985) 315, 592-59.4).

さらに、 植物を使用する場合、 例えばタバコを用いることができる。 タバコを 用いる場合、 目的とする蛋白質をコードする DNAを植物発現用ベクター、 例えば p M0N 530に挿入し、 このベクターをァグロバクテリゥム 'ッメファシエンス （Agr obacterium tumefaciens) のようなバクテリアに導入する。 このパクテリアをタ パコ、 例えば、 ニコチアナ 'タパカム （Nicotiana tabacum) に感染させ、 本タパ コの葉より所望のポリペプチドを得ることができる （Julian K. -C. Ma et al. , Eur. J. Immunol. (1994) 24, 131-138)。  Furthermore, when using a plant, for example, tobacco can be used. When tobacco is used, DNA encoding the protein of interest is inserted into a plant expression vector, for example, pM0N530, and this vector is introduced into a bacterium such as Agrobacterium tumefaciens. This terrier is infected with tapaco, for example, Nicotiana tabacum, and the desired polypeptide can be obtained from the leaves of this tapaco (Julian K. -C. Ma et al., Eur. J. Immunol. (1994) 24, 131-138).

これにより得られた HK33蛋白質は、 宿主細胞内または細胞外 （培地など） から 単離し、 実質的に純粋で均一な蛋白質として精製することができる。 蛋白質の分 離、 精製は、 通常の蛋白質の精製で使用されている分離、 精製方法を使用すれば よく、 何ら限定されるものではない。 例えば、 クロマトグラフィーカラム、 フィ ルター、 限外濾過、 塩析、 溶媒沈殿、 溶媒抽出、 蒸留、 免疫沈降、 SDS-ポリアク リルアミドゲル電気泳動、 等電点電気泳動法、 透析、 再結晶等を適宜選択、 組み 合わせれば蛋白質を分離、 精製することができる。 クロマトグラフィーとしては、 例えばァフィ二ティークロマトグラフィー、 ィ オン交換クロマトグラフィー、 疎水性クロマトグラフィー、 ゲル濾過、 逆相クロ マトグラフィー、 吸着クロマトグラフィー等が挙げられる （Strategies for Pro tein Purification and Characterization: A Laboratory Course Manual. Ed D anie丄 R. Marshak et al. , Cold Spring Harbor Laboratory Press, 1996)。これ らのクロマトグラフィーは、 液相クロマトグラフィー、 例えば HPLC、 FPLC等の液 相クロマトグラフィーを用いて行うことができる。 これらの精製方法を用い、 HK 33蛋白質を高度に精製することもできる。 The HK33 protein thus obtained can be isolated from the inside or outside of the host cell (such as a medium) and purified as a substantially pure and homogeneous protein. The separation and purification of the protein may be performed by any of the separation and purification methods used in ordinary protein purification, and is not particularly limited. For example, chromatographic columns, filters, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. When combined, proteins can be separated and purified. Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory). Course Manual. Ed D anie 丄 R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These chromatographys can be performed using liquid phase chromatography, for example, liquid phase chromatography such as HPLC and FPLC. Using these purification methods, the HK33 protein can be highly purified.

なお、 蛋白質を精製前又は精製後に適当な蛋白質修飾酵素を作用させることに より、 任意に修飾を加えたり部分的にペプチドを除去することもできる。 蛋白質 修飾酵素としては、 例えば、 トリプシン、 キモトリブシン、 リシルエンドべプチ ダーゼ、 プロテインキナーゼ、 ダルコシダーゼなどが用いられる。  The protein can be arbitrarily modified or partially removed by reacting the protein with an appropriate protein modifying enzyme before or after purification. As the protein modifying enzyme, for example, trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, dalcosidase and the like are used.

また、本発明は、 HK33遺伝子の発現、または ARF蛋白質と HK33蛋白質との相互作 用を低下させる化合物に関する。 また本発明は、該化合物の、 ARF蛋白質を介する シグナル伝達の調節、 p53蛋白質を介するシグナル伝達の調節、細胞増殖調節、並 ぴに腫瘍の予防または治療のための使用にも関する。 このような化合物としては 、 例えば、 HK33遺伝子に対するアンチセンスヌクレオチド等が挙げられる。 アン チセンスヌクレオチドは、 HK33蛋白質をコードするヌクレオチドに相捕的な領域 を含むヌクレオチドである。 好ましくは、 アンチセンスヌクレオチドは HK33蛋白 質をコードするヌクレオチドに相捕的な少なくとも 15ヌクレオチドを含むポリヌ クレオチドである。 ここで 「相補鎖」 とは、 A:T (ただし RNAの場合は U)、 G:Cの塩 基対からなる 2本鎖核酸の一方の鎖に対する他方の鎖を指す。 また、 「相捕的」 と は、 相補的ヌクレオチド領域が完全に相補配列である場合に限られず、 少なくと も 70%、 好ましくは少なくとも 80%、 より好ましくは 90%、 さらに好ましくは 95% 以上の塩基配列上の相同性を有すればよい。 相同性を決定するためのアルゴリズ ムは文献 (Wilbur, W. J. and Lipman, D. J. , Proc. Natl. Acad. Sci. USA (1 983) 80， 726-730) に記載のアルゴリズムにしたがえばよい。 アンチセンスヌク レオチドは、 ARFを介するシグナル伝達の調節剤、 p53を介するシグナル伝達の調 節剤、 および細胞増殖の調節剤として有用である。 すなわち、 HK33に対するアン チセンスヌクレオチドは、 ARFを介するシグナル伝達を促進し、 p53を介するシグ ナル伝達を促進し、 さらに細胞増殖を抑制する。 また、 HK33に対するアンチセン スヌクレオチドは、 後述の本発明の検查にも利用され得る。 The present invention also relates to a compound that reduces the expression of the HK33 gene or the interaction between the ARF protein and the HK33 protein. The present invention also relates to the use of the compound for regulating signal transduction via ARF protein, regulating signal transduction via p53 protein, regulating cell proliferation, and preventing or treating tumors. Such compounds include, for example, antisense nucleotides against the HK33 gene. Antisense nucleotides are nucleotides that include a region complementary to the nucleotide encoding the HK33 protein. Preferably, the antisense nucleotide is a polynucleotide comprising at least 15 nucleotides complementary to the nucleotides encoding the HK33 protein. As used herein, the term “complementary strand” refers to one strand of a double-stranded nucleic acid consisting of a base pair of A: T (U for RNA) and G: C, and the other strand. The term “complementary” is not limited to the case where the complementary nucleotide region is a completely complementary sequence, and is at least 70%, preferably at least 80%, more preferably 90%, and still more preferably 95% or more. It is only necessary to have the homology on the base sequence. The algorithm for determining homology is described in the literature (Wilbur, WJ and Lipman, DJ, Proc. Natl. Acad. Sci. USA (1 983) 80, 726-730). Antisense nucleotides are useful as modulators of ARF-mediated signaling, p53-mediated signaling, and cell growth regulators. That is, the antisense nucleotide for HK33 promotes signal transduction via ARF, promotes signal transduction via p53, and suppresses cell proliferation. In addition, antisense nucleotides against HK33 can be used for the detection of the present invention described below.

このようなヌクレオチドには、 HK33遺伝子の発現を制御するためのヌクレオチ ド又はヌクレオチド誘導体 （例えば、 アンチセンスオリゴヌクレオチドゃリボザ ィム、 またはこれらをコードする DNA等） が含まれる。  Such nucleotides include nucleotides or nucleotide derivatives for controlling the expression of the HK33 gene (for example, antisense oligonucleotide ゃ ribozyme or DNA encoding the same).

アンチセンスオリゴヌクレオチドとしては、例えば、 HK33遺伝子のゲノム上の D NA配列、 または該遺伝子から転写される mRNAの配列のいずれかの箇所にハイプリ ダイズするァンチセンスォリゴヌクレオチドが含まれる。 このアンチセンスオリ ゴヌクレオチドは、 好ましくは該 HK33遺伝子配列または mRNA配列中の連続する少 なくとも 15個以上のヌクレオチドに対するアンチセンスオリゴヌクレオチドであ る。 さらに好ましくは、 連続する少なくとも 15個以上のヌクレオチドが翻訳開始 コドンを含むアンチセンスオリゴヌクレオチドである。  Antisense oligonucleotides include, for example, antisense oligonucleotides that hybridize to any part of the DNA sequence on the genome of the HK33 gene or the mRNA sequence transcribed from the gene. The antisense oligonucleotide is preferably an antisense oligonucleotide to at least 15 or more consecutive nucleotides in the HK33 gene sequence or mRNA sequence. More preferably, it is an antisense oligonucleotide in which at least 15 or more consecutive nucleotides contain a translation initiation codon.

アンチセンスオリゴヌクレオチドとしては、 それらの誘導体や修飾体を使用す ることができる。 修飾体として、 例えばメチルホスホネート型又はェチルホスホ ネート型のような低級アルキルホスホネート修飾体、 ホスホロチォエート修飾体 又はホスホロアミデート修飾体等が挙げられる。  As the antisense oligonucleotide, derivatives and modifications thereof can be used. Examples of the modified product include a modified lower alkyl phosphonate such as a methylphosphonate type or an ethylphosphonate type, a phosphorothioate modified product, a phosphoroamidate modified product, and the like.

アンチセンスオリゴヌクレオチドは、 DNA又は mRNAの所定の領域を構成するヌク レオチドに対応するヌクレオチドが全て相補配列であるもののみならず、 DNA ま たは mRNAとオリゴヌクレオチドとが該 HK33遺伝子配列または mRNA配列にハイプリ ダイズできる限り、 1 又は複数個のヌクレオチドのミスマッチが存在しているも のも含まれる。  Antisense oligonucleotides include not only those in which all nucleotides corresponding to nucleotides constituting a predetermined region of DNA or mRNA are complementary sequences, and those in which DNA or mRNA and an oligonucleotide are the HK33 gene sequence or mRNA sequence. As long as it can be hybridized to, it includes one or more nucleotide mismatches.

.オリゴヌクレオチド誘導体は、 HK33蛋白質の産生細胞に作用して 、 該蛋白質をコードする DNA又は mRNAに結合することにより、 その転写又は翻訳 を阻害したり、 mRNA の分解を促進したりして、 HK33遺伝子の発現を抑制すること により結果的に HK33蛋白質の活性を抑制する効果を有する。 Oligonucleotide derivatives act on HK33 protein producing cells By binding to DNA or mRNA encoding the protein, transcription or translation thereof is inhibited, or mRNA degradation is promoted, and the expression of the HK33 gene is suppressed, resulting in the activity of the HK33 protein. It has the effect of suppressing.

アンチセンスオリゴヌクレオチド誘導体は、 それらに対して不活性な適当な基 剤と混和して塗布剤、 パップ剤等の外用剤とすることができる。 また、 必要に応 じて、 賦形剤、 等張化剤、 溶解補助剤、 安定化剤、 防腐剤、 無痛化剤等を加えて 錠剤、 散財、 顆粒剤、 カプセル剤、 リボソームカプセル剤、 注射剤、 液剤、 点鼻 剤など、 さらに凍結乾燥剤とすることができる。 これらは常法にしたがって調製 することができる。 アンチセンスオリゴヌクレオチド誘導体は患者の患部に直接 適用するか、 又は血管内に投与するなどして結果的に患部に到達し得るように患 者に適用する。 さらには、 持続性、 膜透過性を高めるアンチセンス封入素材を用 いることもできる。 例えば、 リボソーム、 ポリ - L -リジン、 リピッド、 コレステロ ール、 リポフエクチン又はこれらの誘導体が挙げられる。 アンチセンスオリゴヌ クレオチド誘導体の投与量は、 患者の状態に応じて適宜調整し、 好ましい量を用 いることができる。 例えば、 0. 1 〜100mg/kg、 好ましくは 0. 1 〜50mg/kg の範囲 で投与することができる。  The antisense oligonucleotide derivative can be mixed with a suitable base material which is inactive against the derivative to prepare an external preparation such as a liniment or a poultice. If necessary, excipients, isotonic agents, solubilizing agents, stabilizing agents, preservatives, soothing agents, etc. are added to tablets, splinters, granules, capsules, ribosome capsules, and injections. , Lyophilized agent, such as drug, solution, nasal drop, etc. These can be prepared according to a conventional method. The antisense oligonucleotide derivative is applied directly to the affected area of the patient, or applied to the patient so that it can reach the affected area as a result of intravenous administration. Furthermore, an antisense encapsulating material that enhances durability and membrane permeability can be used. For example, ribosome, poly-L-lysine, lipid, cholesterol, lipofectin or derivatives thereof can be mentioned. The dose of the antisense oligonucleotide derivative can be appropriately adjusted according to the condition of the patient, and a preferred amount can be used. For example, it can be administered in the range of 0.1 to 100 mg / kg, preferably 0.1 to 50 mg / kg.

アンチセンスオリゴヌクレオチドは HK33遺伝子の発現を阻害し、 従って HK33蛋 白質の生物学的活性を抑制することにおいて有用である。 また、 アンチセンス才 リゴヌクレオチドを含有する発現阻害剤は、 HK33蛋白質の生物学的活性を抑制す ることが可能である点で有用である。 HK33遺伝子のアンチセンス核酸は、 ARF蛋白 質を介するシグナノレ伝達の調節剤、 p53蛋白質を介するシグナル伝達の調節剤、お よび細胞増殖の調節剤となる。 また本発明は、 ARF蛋白質または p53を介するシグ ナル伝達、 あるいは細胞増殖を調節するための、 HK33遺伝子に対するアンチセン スヌクレオチドの使用にも関する。  Antisense oligonucleotides inhibit the expression of the HK33 gene and are therefore useful in suppressing the biological activity of the HK33 protein. Further, an expression inhibitor containing an antisense oligonucleotide is useful because it can suppress the biological activity of HK33 protein. The antisense nucleic acid of the HK33 gene serves as a regulator of signal transfer via the ARF protein, a regulator of signal transmission via the p53 protein, and a regulator of cell proliferation. The present invention also relates to the use of antisense nucleotides against the HK33 gene to regulate signal transmission via the ARF protein or p53, or cell growth.

また、 ARF蛋白質と HK33蛋白質との相互作用を低下させる化合物としては、例え ば HK33蛋白質に対する抗体が挙げられる。 HK33蛋白質に対する抗体は、 ARF蛋白質 を介するシグナル伝達の調節剤、 p53蛋白質を介するシグナル伝達の調節剤、およ ぴ細胞増殖の調節剤となり得る。 すなわち、 HK33蛋白質に対する抗体により HK33 蛋白質の活性を阻害すれば、 ARF蛋白質を介するシグナル伝達を促進し、 p53蛋白 質を介するシグナル伝達を促進し、 さらに細胞増殖を抑制することができる。 本 発明は、 ARF蛋白質を介するシグナル伝達、 p53蛋白質を介するシグナル伝達、 お よび細胞増殖を調節するための、 HK33蛋白質に対する抗体の使用にも関する。 ま た、 HK33蛋白質に対する抗体は、 後述の本発明の検查にも使用される。 Compounds that reduce the interaction between the ARF protein and the HK33 protein include, for example, antibodies against the HK33 protein. Antibody to HK33 protein is ARF protein , Signaling regulators via p53 protein, and cell growth regulators. That is, if the activity of the HK33 protein is inhibited by an antibody against the HK33 protein, signal transduction via the ARF protein can be promoted, signal transduction via the p53 protein can be promoted, and cell proliferation can be further suppressed. The invention also relates to the use of antibodies to the HK33 protein to regulate signaling through the ARF protein, signaling through the p53 protein, and cell growth. Further, an antibody against the HK33 protein is also used in the detection of the present invention described below.

HK33蛋白質に対する抗体は、 公知の方法により作製することができる。 抗体の 形態には特に制限はない。 ポリクローナル抗体の他、 モノクローナル抗体も含ま れる。 また、 ゥサギなどの免疫動物に抗原蛋白質を免疫して得た抗血清、 すべて のクラスのポリクローナル抗体おょぴモノクローナル抗体、 さらにヒト抗体や遺 伝子組み換えによるヒト型化抗体も含まれる。  An antibody against the HK33 protein can be prepared by a known method. There is no particular limitation on the form of the antibody. In addition to polyclonal antibodies, monoclonal antibodies are included. Also included are antisera obtained by immunizing immunized animals such as rabbits with antigenic proteins, polyclonal antibodies and monoclonal antibodies of all classes, as well as human antibodies and humanized antibodies obtained by genetic recombination.

抗体取得の感作抗原として使用される蛋白質は、 その由来となる動物種に制限 されないが哺乳動物、 例えばヒト、 マウス又はラット由来の蛋白質が好ましく、 特にヒ ト由来の蛋白質が好ましい。 ヒ ト由来の HK33蛋白質は、 上述のようにして 調製することができる。  The protein used as a sensitizing antigen for obtaining an antibody is not limited to the animal species from which it is derived, but is preferably a protein derived from a mammal, for example, a human, a mouse or a rat, and particularly preferably a protein derived from a human. Human-derived HK33 protein can be prepared as described above.

本発明において、 感作抗原として使用される蛋白質は、 完全な蛋白質であって もよいし、 また、 蛋白質の部分ペプチドであってもよい。 蛋白質の部分ペプチド としては、 例えば、 蛋白質のアミノ基 （N) 末端断片やカルボキシ (C) 末端断片 が挙げられる。 また、 ARF蛋白質との結合領域が挙げられる。本明細書で述べる 「 抗体」 とは蛋白質の全長又は断片に反応する抗体を意味する。  In the present invention, the protein used as the sensitizing antigen may be a complete protein or a partial peptide of the protein. Examples of partial peptides of proteins include amino (N) terminal fragments and carboxy (C) terminal fragments of proteins. Further, a binding region to the ARF protein may be mentioned. As used herein, “antibody” refers to an antibody that reacts with the full length or fragment of a protein.

HK33蛋白質又はその断片をコードする遺伝子を公知の発現べクタ一系に揷入し 、 該ベクターで宿主細胞を形質転換させ、 該宿主細胞内外から目的の蛋白質又は その断片を公知の方法で得て、 これらを感作抗原として用いればよい。 また、 蛋 白質を発現する細胞又はその溶解物あるいは化学的に合成した HK33蛋白質を感作 抗原として使用してもよい。 短いペプチドは、 キーホールリンペットへモシァニ ン、 ゥシ血清アルブミン、 卵白アルブミンなどのキャリア蛋白質と適宜結合させ て抗原とすることができる。 A gene encoding the HK33 protein or a fragment thereof is inserted into a known expression vector system, and a host cell is transformed with the vector.The target protein or a fragment thereof is obtained from inside or outside the host cell by a known method. These may be used as sensitizing antigens. Alternatively, a cell expressing the protein, a lysate thereof, or a chemically synthesized HK33 protein may be used as the sensitizing antigen. Short peptides can be transferred to keyhole limpets An antigen can be obtained by appropriately binding to a carrier protein such as serum, albumin, or ovalbumin.

感作抗原で免疫される哺乳動物としては、 特に限定されるものではないが、 モ ノクローナル抗体の作製においては細胞融合に使用する親細胞との適合性を考慮 して選択するのが好ましく、 一般的には、 げっ歯目、 ゥサギ目、 霊長目の動物が 使用される。  The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion in the production of a monoclonal antibody. Typically, rodent, heron, and primate animals are used.

げっ歯目の動物としては、 例えば、 マウス、 ラット、 ハムスター等が使用され る。 ゥサギ目の動物としては、 例えば、 ゥサギが使用される。 霊長目の動物とし ては、 例えば、 サルが使用される。 サルとしては、 狭鼻下目のサル （旧世界ザル )、例えば、 力二クイザル、 ァカゲザル、 マントヒヒ、 チンパンジー等が使用され る。  As rodent animals, for example, mice, rats, hamsters and the like are used.動物 As an heronoid animal, for example, a heron is used. For example, monkeys are used as primates. As monkeys, monkeys of the lower nose are used (old world monkeys), for example, cynomolgus monkeys, macaques, baboons, and chimpanzees.

感作抗原を動物に免疫するには、 公知の方法にしたがって行われる。 一般的方 法としては、 感作抗原を哺乳動物の腹腔内又は皮下に注射する。 具体的には、 感 作抗原を PBS (Phosphate-Buffered Saline) や生理食塩水等で適当量に希釈、 懸 濁したものに対し、 所望により通常のアジュバント、 例えば、 フロイント完全ァ ジュバントを適量混合し、 乳化後、 哺乳動物に投与する。 さらに、 その後、 フロ イント不完全アジュパントに適量混合した感作抗原を、 4〜21日毎に数回投与する ことが好ましい。 また、 感作抗原免疫時に適当な担体を使用することができる。 このように免疫し、 血清中に所望の抗体レベルが上昇するのを常法により確認す る。  Immunization of an animal with a sensitizing antigen is performed according to a known method. As a general method, a sensitizing antigen is injected intraperitoneally or subcutaneously into a mammal. Specifically, the sensitizing antigen is diluted and suspended in an appropriate amount with PBS (Phosphate-Buffered Saline), physiological saline, or the like, and then mixed with an appropriate amount of a normal adjuvant, for example, Freund's complete adjuvant, if desired. After emulsification, it is administered to mammals. Thereafter, it is preferable to administer the sensitizing antigen mixed with an appropriate amount of Freund's incomplete adjuvant several times every 4 to 21 days. In addition, a suitable carrier can be used at the time of immunization with the sensitizing antigen. Immunization is performed in this manner, and an increase in the desired antibody level in the serum is confirmed by a conventional method.

ここで、 ある蛋白質に対するポリクローナル抗体を得るには、 血清中の所望の 抗体レベルが上昇したことを確認した後、 抗原を感作した哺乳動物の血液を取り 出す。 この血液から公知の方法により血清を分離する。 ポリクローナル抗体とし ては、 ポリクローナル抗体を含む血清を使用してもよいし、 必要に応じこの血清 からポリクローナル抗体を含む画分をさらに単離して、 これを使用してもよい。 例えば、 抗原に用いた蛋白質をカツプリングさせたァフィ二ティーカラムを用い て、抗原蛋白質のみを認識する画分を得て、 さらにこの画分をプロテイン Aあるい はプロテイン Gカラムを利用して精製することにより、免疫グロプリン Gあるいは M を調製することができる。 Here, in order to obtain a polyclonal antibody against a certain protein, the blood of a mammal sensitized with the antigen is taken after confirming that the level of the desired antibody in the serum has increased. The serum is separated from the blood by a known method. As the polyclonal antibody, a serum containing the polyclonal antibody may be used. If necessary, a fraction containing the polyclonal antibody may be further isolated from the serum and used. For example, using an affinity column in which the protein used for the antigen is coupled Thus, immunoglobulin G or M can be prepared by obtaining a fraction that recognizes only the antigen protein and further purifying this fraction using a protein A or protein G column.

モノクローナル抗体を得るには、 上記抗原を感作した哺乳動物の血清中に所望 の抗体レベルが上昇するのを確認した後に、 哺乳動物から免疫細胞を取り出し、 細胞融合に付せばよい。 この際、 細胞融合に使用される好ましい免疫細胞として 、 特に脾細胞が挙げられる。 前記免疫細胞と融合される他方の親細胞としては、 好ましくは哺乳動物のミエローマ細胞、 より好ましくは、 薬剤による融合細胞選 別のための特性を獲得したミエローマ細胞が挙げられる。 前記免疫細胞とミエ口 一マ細胞の細胞融合は基本的には公知の方法、 例えば、 ミルスティンらの方法 (G alfre, G. and Milstein, C.， Methods Enzymol. (1981) 73, 3- 46)等に準じて 行うことができる。  To obtain a monoclonal antibody, after confirming that the desired antibody level is increased in the serum of the mammal sensitized with the antigen, the immune cells may be removed from the mammal and subjected to cell fusion. At this time, preferred immune cells used for cell fusion include splenocytes in particular. The other parent cell to be fused with the immunocyte is preferably a mammalian myeloma cell, more preferably a myeloma cell that has acquired the properties for fusion cell selection by a drug. The cell fusion of the immune cells and myeoma cells is basically a known method, for example, the method of Milstein et al. (Galfre, G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46). ) And so on.

細胞融合により得られたハイプリ ドーマは、 通常の選択培養液、 例えば、 HAT 培養液 （ヒポキサンチン、 アミノプテリンおょぴチミジンを含む培養液） で培養 することにより選択される。当該 HAT培養液での培養は、 目的とするハイプリ ドー マ以外の細胞 （非融合細胞） が死滅するのに十分な時間、 通常、 数日〜数週間継 続して行う。 次いで、 通常の限界希釈法を実施し、 目的とする抗体を産生するハ イブリ ドーマのスクリ一二ングおよびクローニングを行う。  The hybridoma obtained by cell fusion is selected by culturing it in a normal selective culture medium, for example, a HAT culture medium (a culture medium containing hypoxanthine, aminopterin and thymidine). Culturing in the HAT culture solution is continued for a time sufficient to kill cells other than the target hybridoma (non-fused cells), usually for several days to several weeks. Next, screening and cloning of hybridomas producing the desired antibody are performed by the usual limiting dilution method.

また、 ヒ ト以外の動物に抗原を免疫して上記ハイプリドーマを得る他に、 ヒ ト リンパ球、 例えば EBウィルスに感染したヒトリンパ球を in vitroで蛋白質、 蛋白 質発現細胞又はその溶解物で感作し、 感作リンパ球をヒト由来の永久分裂能を有 するミエローマ細胞、 例えば U266と融合させ、 蛋白質への結合活性を有する所望 のヒト抗体を産生するハイプリ ドーマを得ることもできる （特開昭 63- 17688号公 報)。  In addition, in addition to obtaining the above-mentioned hybridoma by immunizing animals other than humans with the antigen, human lymphocytes, for example, human lymphocytes infected with EB virus, are sensitized in vitro with proteins, protein-expressing cells or lysates thereof. And sensitized lymphocytes are fused with human-derived myeloma cells capable of permanent division, such as U266, to obtain a hybridoma that produces a desired human antibody having protein binding activity (see Published in Sho 63-17688.

次いで、 得られたハイプリドーマをマウス腹腔内に移植し、 同マウスより腹水 を回収し、 得られたモノクローナル抗体を、 例えば、硫安沈殿、 プロテイン A、 プ 口ティン Gカラム、 DEAEイオン交換クロマトグラフィー、抗原とした蛋白質をカツ プリングしたァフィ二ティーカラムなどにより精製することで調製することが可 能である。 得られる抗体は、 HK33蛋白質の検出に用いられる他、 HK33蛋白質のァ ンタゴニストの候補になる。 HK33蛋白質に対する抗体は、 HK33蛋白質と ARF蛋白質 との相互作用を抑制する薬剤の候補となる。 Next, the obtained hybridoma is transplanted into the peritoneal cavity of a mouse, ascites is collected from the mouse, and the obtained monoclonal antibody is used, for example, for ammonium sulfate precipitation, protein A, It can be prepared by purifying it with a mouth tin G column, DEAE ion exchange chromatography, or an affinity column to which the protein used as the antigen has been coupled. The obtained antibody is used for detecting the HK33 protein and is also a candidate for the HK33 protein antagonist. Antibodies to the HK33 protein are candidates for drugs that suppress the interaction between the HK33 protein and the ARF protein.

抗体は、 ヒト抗体またはヒト型抗体であってもよい。 例えば、 ヒト抗体遺伝子 のレパートリーを有するトランスジヱニック動物に抗原となる蛋白質、 蛋白質発 '現細胞又はその溶解物を免疫して抗体産生細胞を取得し、 これをミエローマ細胞 と融合させたハイプリドーマを用いて蛋白質に対するヒト抗体を取得することが できる （国際公開番号 W092-03918、 W093 - 2227、 W094- 02602、 W094- 25585、 W096- 33735およぴ W096- 34096参照）。  The antibodies may be human or human antibodies. For example, a transgenic animal having a repertoire of human antibody genes is immunized with a protein serving as an antigen, a protein-expressing cell or a lysate thereof to obtain antibody-producing cells, which are then fused with myeloma cells. Can be used to obtain a human antibody against the protein (see International Publication Nos. W092-03918, W093-2227, W094-02602, W094-25585, W096-33735 and W096-34096).

ハイプリ ドーマを用いて抗体を産生する以外に、 抗体を産生する感作リンパ球 等の免疫細胞を癌遺伝子 （oncogene) により不死化させた細胞を用いてもよい。 このように得られたモノクローナル抗体はまた、 遺伝子組換え技術を用いて産 生させた組換え型抗体として得ることができる （例えば、 Borrebaeck, C. A. K. and Larrick, J. W.， THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MCMILLAN PUBLISHERS LTD, 1990参照）。 組換え型抗体は、 それをコードする DNAをハイプリ ドーマ又は抗体を産生する感作リンパ球等の免 疫細胞からクローニングし、 適当なベクターに組み込んで、 これを宿主に導入し 産生させる。  In addition to producing antibodies using hybridomas, cells in which immune cells such as sensitized lymphocytes that produce antibodies are immortalized with oncogenes may be used. The monoclonal antibody thus obtained can also be obtained as a recombinant antibody produced using a genetic recombination technique (for example, Borrebaeck, CAK and Larrick, JW, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United States). Kingdom by MCMILLAN PUBLISHERS LTD, 1990). Recombinant antibodies are produced by cloning DNA encoding them from immunized cells such as hybridomas or sensitized lymphocytes that produce the antibodies, incorporating the DNA into an appropriate vector, and introducing it into a host.

さらに、 HK33蛋白質に結合する限り、 抗体断片や抗体修飾物であってよい。 例 えば、 抗体断片としては、 Fab、 F (ab，）2、 Fv又は H鎖と L鎖の Fvを適当なリンカ一 で連結させたシングルチェイン Fv (scFv) (Huston, J. S. et al.， Proc. Natl. Acad. Sci. U. S. A. (1988) 85, 5879-5883) が挙げられる。 具体的には、 抗体を 酵素、 例えば、 パパイン、 ペプシンで処理し抗体断片を生成させるか、 又は、 こ れら抗体断片をコードする遺伝子を構築し、 これを発現ベクターに導入した後、 適当な宿主細胞で発現させる （例えば、 Co, M. S. et al.， J. Immunol. (1994) 152, 2968-2976 ； Better, M. and Horwitz, A. H.， Methods Enzymol. (1989) 178, 476-496 ； Pluckthun, A. and Skerra, A. , Methods Enzymol. (1989) 17 8， 497-515 ； Lamoyi, E.， Methods Enzymol. (1986) 121, 652 - 663 ； Rousseau x, J. et al. , Methods Enzymol. (1986) 121， 663-669 ； Bird, R. E. and Wal ker, B. W. , Trends Biotechnol. (1991) 9, 132- 137参照）。 Furthermore, as long as it binds to the HK33 protein, it may be an antibody fragment or a modified antibody. For example, antibody fragments include Fab, F (ab,) 2, Fv, or a single-chain Fv (scFv) (Fuston, JS et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883). Specifically, an antibody is treated with an enzyme, for example, papain or pepsin, to generate an antibody fragment, or a gene encoding these antibody fragments is constructed and introduced into an expression vector. Expression in a suitable host cell (eg, Co, MS et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, AH, Methods Enzymol. (1989) 178, 476-496. Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663; Rousseau x, J. et al., Methods Enzymol. (1986) 121, 663-669; Bird, RE and Walker, BW, Trends Biotechnol. (1991) 9, 132-137).

抗体修飾物として、ポリエチレングリコール（PEG)等の各種分子と結合した抗 体を使用することもできる。 本発明で言う 「抗体」 にはこれらの抗体修飾物も包 含される。 このような抗体修飾物を得るには、 得られた抗体に化学的な修飾を施 すことによって得ることができる。 これらの方法はこの分野において既に確立さ れている。  Antibodies bound to various molecules such as polyethylene glycol (PEG) can also be used as modified antibodies. The “antibody” referred to in the present invention also includes these modified antibodies. Such an antibody modification can be obtained by chemically modifying the obtained antibody. These methods are already established in this field.

また、 抗体は公知の技術を使用して非ヒト抗体由来の可変領域とヒト抗体由来 の定常領域からなるキメラ抗体又は非ヒト抗体由来の CDR (ネ目捕性決定領域） とヒ ト抗体由来の FR (フレームワーク領域） 及び定常領域からなるヒト型化抗体とし て得ることができる。  Antibodies can be derived from chimeric or non-human antibody-derived CDRs (negative-capacity determining regions) and non-human antibody-derived CDRs using known techniques. It can be obtained as a humanized antibody consisting of FR (framework region) and constant region.

前記のように得られた抗体は、 均一にまで精製することができる。 抗体の分離 、 精製は通常の蛋白質で使用されている分離、 精製方法を使用すればよい。 例え ば、 ァフィ二ティークロマトグラフィー等のクロマトグラフィーカラム、 フイスレ ター、 限外濾過、塩析、 透析、 SDSポリアクリルアミドゲル電気泳動、 等電点電気 泳動等を適宜選択、 組み合わせれば、 抗体を分離、 精製することができる（Antib odies ： A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbo r Laboratory, 1988) 力 これらに限定されるものではない。 上記で得られた抗 体の濃度測定は吸光度の測定又は酵素結合免疫吸着検定法 (Enzyme- linked immun osorbent assay； ELISA) 等により行うことができる。  The antibody obtained as described above can be purified to homogeneity. The separation and purification of the antibody may be performed by the separation and purification methods used for ordinary proteins. For example, if appropriate selection and combination of chromatography columns such as affinity chromatography, filter, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, and isoelectric focusing, antibodies can be separated. It can be purified (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988). Power is not limited to these. The concentration of the antibody obtained as described above can be measured by measuring absorbance or by enzyme-linked immunosorbent assay (ELISA).

ァフィ二ティークロマトグラフィ一に用いるカラムとしては、 プロテイン A力 ラム、 プロテイン Gカラムが挙げられる。 例えば、 プロテイン Aカラムを用いた カラムとして、 Hyper D, POROS, Sepharose F. F. (Pharmacia) 等が挙げられる 了フイエティークロマトグラフィー以外のクロマトグラフィーとしては、 例え ば、 イオン交換クロマトグラフィー、 疎水性クロマトグラフィー、 ゲル濾過、 逆 相クロマトグラフィー、 吸着クロマトグラフィー等が挙げられる（Strategies fo r Protein Purification and Characterization ： A Laboratory Course Manual . Ed Daniel R. Marshak et al. , Cold Spring Harbor Laboratory Press, 1996 )。 これらのクロマトグラフィ一は HP1 、 FPLC等の液相クロマトグラフィーを用い て行うことができる。 Columns used for affinity chromatography include a protein A column and a protein G column. For example, using a protein A column Columns include Hyper D, POROS, Sepharose FF (Pharmacia), etc. Examples of chromatography other than water chromatography include, for example, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, Adsorption chromatography and the like (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These chromatography methods can be carried out using liquid phase chromatography such as HP1 and FPLC.

抗体の抗原結合活性を測定する方法としては、 例えば、 吸光度の測定、 ELISA 、 EIA (酵素免疫測定法）、 RIA (放射免疫測定法） あるいは蛍光抗体法を用いるこ とができる。 ELISAを用いる場合、抗体を固相化したプレートに HK33蛋白質を添加 し、 次いで目的の抗体を含む試料、 例えば、 抗体産生細胞の培養上清や精製抗体 を加える。 酵素、 例えば、 アルカリフォスファターゼ等で標識した抗体を認識す る二次抗体を添加し、 プレートをインキュベーションし、 次いで洗浄した後、 p - 二ト口フエニル燐酸などの酵素基質を加えて吸光度を測定することで抗原結合活 性を評価することができる。 蛋白質として HK33蛋白質の断片、 例えばその C末端 からなる断片等を使用してもよい。 抗体の活性評価には、 BIAcore (Pharmacia製） を使用することもできる。  As a method for measuring the antigen-binding activity of the antibody, for example, absorbance measurement, ELISA, EIA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay) or a fluorescent antibody method can be used. When using ELISA, HK33 protein is added to a plate on which antibodies are immobilized, and then a sample containing the target antibody, for example, a culture supernatant of antibody-producing cells or a purified antibody is added. After adding a secondary antibody that recognizes an enzyme, for example, an antibody labeled with alkaline phosphatase, incubate the plate, wash the plate, and then add an enzyme substrate such as p-ditophenyl phenylphosphate to measure the absorbance. In this way, the antigen binding activity can be evaluated. As the protein, a fragment of the HK33 protein, for example, a fragment comprising the C-terminal thereof may be used. BIAcore (Pharmacia) can also be used to evaluate the activity of the antibody.

HK33蛋白質または ARF蛋白質の部分べプチドは、 本来の ARF蛋白質と HK33蛋白質 との結合を阻害することにより、ARF蛋白質と HK33蛋白質との相互作用を調節し得 る。 従って、 HK33蛋白質または ARF蛋白質の部分ペプチドは、 ARF蛋白質と HK33蛋 白質との相互作用を調節する化合物となり、ARF蛋白質を介するシグナル伝達の調 節剤、 p53蛋白質を介するシグナル伝達の調節剤、細胞増殖調節剤、抗腫瘍剤等の 上記の各種調節剤として使用することができる。 このような部分ペプチドは、 少 なくとも 7アミノ酸以上、 好ましくは 8アミノ酸以上、 さらに好ましくは 9アミ ノ酸以上のアミノ酸配列からなる。 部分ペプチドは、 遺伝子工学的手法、 公知の ぺプチド合成法、あるいは HK33蛋白質または ARF蛋白質を適切なぺプチダーゼで切 断することによって製造することができる。 ペプチドの合成は、 例えば、 固相合 成法、 液相合成法のいずれによってもよい。 The HK33 protein or the partial peptide of the ARF protein can regulate the interaction between the ARF protein and the HK33 protein by inhibiting the binding between the original ARF protein and the HK33 protein. Therefore, the HK33 protein or the partial peptide of the ARF protein is a compound that regulates the interaction between the ARF protein and the HK33 protein, and is a regulator of ARF protein-mediated signal transduction, a p53 protein-mediated signal transduction regulator, It can be used as the above-mentioned various regulators such as growth regulators and antitumor agents. Such a partial peptide has at least 7 amino acids or more, preferably 8 amino acids or more, and more preferably 9 amino acids. It consists of an amino acid sequence of no acid or higher. The partial peptide can be produced by a genetic engineering technique, a known peptide synthesis method, or by cutting the HK33 protein or ARF protein with an appropriate peptide. The peptide may be synthesized by, for example, either a solid phase synthesis method or a liquid phase synthesis method.

また本発明は、 ARF蛋白質と HK33蛋白質との相互作用を調節する化合物のスクリ 一ユング方法に関する。 本発明のスクリーニング方法の一つは、  The present invention also relates to a method for screening compounds that regulates the interaction between ARF protein and HK33 protein. One of the screening methods of the present invention,

( a ) 被検試料の存在下で ARF蛋白質と HK33蛋白質とを接触させる工程、  (a) contacting the ARF protein with the HK33 protein in the presence of the test sample,

( b ) ARF蛋白質と HK33蛋白質との相互作用を検出する工程、  (b) detecting an interaction between the ARF protein and the HK33 protein,

( c ) 被検試科非存在下で検出した場合 （対照） と比較して、 該相互作用を調節 する化合物を選択する工程、 を含む方法である。 このスクリーニングにより、 AR F蛋白寳または HK33蛋白質の活性を調節する化合物を単離することもできる。  (c) a step of selecting a compound that modulates the interaction, as compared to a case where the compound is detected in the absence of the test sample (control). By this screening, compounds that regulate the activity of ARF protein or HK33 protein can also be isolated.

ARF蛋白質と HK33蛋白質との相互作用の検出は、両者の蛋白質の結合を直接的に 検出することにより、 または両者の蛋白質の相互作用により生じる変化を検出す ることにより間接的に ARF蛋白質と HK33蛋白質との相互作用の検出を行い得る。こ のようなスクリーニング系は、 試験管内または細胞内等で構築することが可能で ある。細胞内で行う場合、 ARF蛋白質と HK33蛋白質を発現する細胞を被検試料存在 下でインキュベートすることにより、細胞内で発現した ARF蛋白質と HK33蛋白質が 接触し、 両者の相互作用の検出を通してスクリーニングを行うことができる。 細 胞系を用いたスクリーニング方法は上記に記載したスクリーニング方法に含まれ るが、 より具体的には、 （a ) ARF蛋白質と HK33蛋白質とを発現する細胞に、 被検 試料を接触させる工程、 （b ) ARF蛋白質と HK33蛋白質との相互作用を検出するェ 程、 および （c ) 被検試料非存在下で検出した場合 （対照） と比較して、 該相互 作用を調節する化合物を選択する工程、 を含む。 細胞を用いたこのスクリーニン グ方法は、以下に述べる Twoハイプリッド法によるスクリーユング等の他、後述す る ARF蛋白質または p53蛋白質を介するシグナル伝達を指標とするスクリーニング 方法を含む。 本発明の ARF蛋白質と HK33蛋白質との相互作用を調節する化合物のスクリー二 ング方法の一態様は、 ARF蛋白質と HK33蛋白質との相互作用を、 ARF蛋白質と HK33 蛋白質との結合を指標に検出する方法である。 具体的には、 このスクリーニング 方法は、 （a )被検試料の存在下で ARF蛋白質と HK33蛋白質とを接触させる工程、 （ b ) ARF蛋白質と HK33蛋白質との結合を検出する工程、 および（c )被検試料非存 在下で検出した場合 （対照） と比較して、 該相互作用を調節する化合物を選択す る工程、 を含む方法である。 The interaction between the ARF protein and the HK33 protein can be detected by directly detecting the binding of the two proteins or indirectly by detecting the change caused by the interaction between the two proteins. Detection of interaction with the protein can be performed. Such a screening system can be constructed in a test tube or in a cell. When performed intracellularly, by incubating cells expressing ARF and HK33 proteins in the presence of a test sample, the ARF protein expressed in cells and HK33 protein come into contact, and screening is performed through detection of the interaction between the two. It can be carried out. The screening method using the cell line is included in the screening method described above. More specifically, (a) a step of bringing a test sample into contact with cells expressing the ARF protein and the HK33 protein, (B) selecting a compound that regulates the interaction between the ARF protein and the HK33 protein in comparison with (c) detecting the interaction in the absence of the test sample (control) Process. This screening method using cells includes a screening method using ARF protein or p53 protein-mediated signal transmission described later as an index, in addition to screening using the Two Hybrid method described below, and the like. One embodiment of the screening method of the compound for regulating the interaction between the ARF protein and the HK33 protein of the present invention comprises detecting the interaction between the ARF protein and the HK33 protein using the binding between the ARF protein and the HK33 protein as an indicator. Is the way. Specifically, this screening method comprises: (a) a step of bringing the ARF protein into contact with the HK33 protein in the presence of the test sample; (b) a step of detecting the binding between the ARF protein and the HK33 protein; ) A step of selecting a compound that modulates the interaction, as compared to a case where detection is performed in the absence of a test sample (control).

スクリーニングに用いられる HK33蛋白質およぴ ARF蛋白質は組換え蛋白質であ つても、 天然由来の蛋白質であってもよい。 蛋白質の由来に制限はなく、 ヒトぉ ょぴ他の動物を含む真核生物由来の蛋白質を用いることができる。 好ましくは、 ヒト由来の蛋白質が用いられる。 また蛋白質は変異体、 部分ペプチド、 または他 のペプチドとの融合蛋白質であってもよい。 蛋白質は、 例えば、 精製した蛋白質 として、 可溶型蛋白質として、 担体に結合させた形態として、 他の蛋白質との融 合蛋白質として、 細胞膜上に発現させた形態として、 または膜画分としてスクリ 一二ングに用いることができる。 実施例に示すように、 1-80アミノ酸を欠失させ たマウス ARF蛋白質は HK33蛋白質との結合能を保持している。 従って、 ARF蛋白質 の 81番以降の部分ぺプチドを用いて本発明のスクリーニングを行うことができる 被検試料としては特に制限はなく、 例えば、 細胞培養上清、 発酵微生物産生物 、 海洋生物抽出物、 植物抽出物、 原核細胞紬出物、 真核単細胞抽出物又は動物細 胞抽出物あるいはそれらのライブラリー、 精製若しくは粗精製蛋白質、 ペプチド 、 非ペプチド性化合物、 合成低分子化合物、 天然化合物が挙げられる。  The HK33 protein and ARF protein used for screening may be a recombinant protein or a naturally-occurring protein. The origin of the protein is not limited, and proteins derived from eukaryotes including humans and other animals can be used. Preferably, a human-derived protein is used. The protein may be a mutant, a partial peptide, or a fusion protein with another peptide. Proteins may be, for example, purified proteins, soluble proteins, forms bound to carriers, fusion proteins with other proteins, forms expressed on cell membranes, or screen fractions as membrane fractions. Can be used for ling. As shown in the Examples, mouse ARF protein from which 1-80 amino acids have been deleted retains the ability to bind to HK33 protein. Therefore, the screening of the present invention can be carried out using the partial peptide of the ARF protein of No. 81 or later. The test sample is not particularly limited. For example, cell culture supernatant, fermented microorganism product, marine organism extract , Plant extracts, prokaryotic cell extracts, eukaryotic single cell extracts or animal cell extracts or their libraries, purified or crude proteins, peptides, non-peptidic compounds, synthetic low-molecular compounds, and natural compounds. Can be

蛋白質同士の結合を調節する化合物をスクリーニングする方法としては、 当業 者に公知の多くの方法を用いることが可能である。 このような方法は、 例えば酵 母または動物細胞などを用いた 2ハイプリッド法を用いて行うことができる （Fi elds, S.， and Sternglanz, R.， Trends. Genet. (1994) 10， 286—292、 Dal ton S, and Treisman R (1992) Characterization of SAP - 1， a protein recruited by serum response factor to the c-fos serum response element. Cell 68， 597 - 612、 rMATCHMARKER Two-Hybrid SystemJ ， 「Mammalian MATCHMAKER Two-Hyb rid Assay Kit；」， 「MATCHMAKER One-Hybrid SystemJ (いずれもクロンテック社製 ) , 「HybriZAP Two-Hybrid Vector SystemJ (ストラタジーン社製））。 Many methods known to those skilled in the art can be used as a method of screening for a compound that regulates the binding between proteins. Such a method can be performed using, for example, a two-hybrid method using yeast or animal cells (Fields, S., and Sternglanz, R., Trends. Genet. (1994) 10, 286- 292, Dal ton S, and Treisman R (1992) Characterization of SAP-1, a protein recruited by serum response factor to the c-fos serum response element.Cell 68, 597-612, rMATCHMARKER Two-Hybrid SystemJ, `` Mammalian MATCHMAKER Two-Hyb rid Assay Kit; ”,“ MATCHMAKER One-Hybrid SystemJ (all manufactured by Clontech), ”“ HybriZAP Two-Hybrid Vector SystemJ (manufactured by Stratagene) ”.

酵母 2-ハイプリッドシステムにおいては、 HK33蛋白質または ARF蛋白質のどちら か一方の蛋白質またはその部分ペプチドを SRF DNA結合領域または GAL4 DNA結合 領域等と融合させた融合蛋白質を発現するベクター、 そして他方の蛋白質または その部分べプチドを VP16または GAL4等の転写活性化領域と融合させたベクターを 構築し、 これらを、 レポーター遺伝子をコードするベクターと共に酵母細胞に導 入して、 被検化合物を含む試料の存在下でレポーター活性を指標に化合物のアツ セィを行う。 HK33蛋白質と ARF蛋白質との結合によりレポーター遺伝子の発現が誘 導されるが、 被検化合物により両者の蛋白質の結合が阻害されると、 レポーター 遺伝子の発現が抑制される。 レポーター遺伝子としては、 例えば、 HIS3遺伝子の 他、 Ade2遺伝子、 LacZ遺伝子、 CAT遺伝子、 ルシフェラーゼ遺伝子、 PAI- 1 (Plas minogen activator inhibitor typel) 遺伝子等が挙げられるが、 これらに制限 されない。 レポーター遺伝子として、 細胞毒性のある遺伝子を発現させることも できる。 2ハイプリッド法によるスクリーニングは、 酵母の他、 哺乳動物細胞な どを使って行うこともできる。 '  In the yeast 2-hybrid system, a vector expressing a fusion protein in which either the HK33 protein or the ARF protein or a partial peptide thereof is fused to an SRF DNA binding region or a GAL4 DNA binding region, and the other protein Alternatively, construct a vector in which the partial peptide is fused to a transcriptional activation region such as VP16 or GAL4, and introduce these into a yeast cell together with a reporter gene-encoding vector to obtain a sample containing a test compound. The compounds are assayed below using the reporter activity as an indicator. The binding of the HK33 protein and the ARF protein induces the expression of the reporter gene. However, if the binding of both proteins is inhibited by the test compound, the expression of the reporter gene is suppressed. Examples of the reporter gene include, but are not limited to, HIS3 gene, Ade2 gene, LacZ gene, CAT gene, luciferase gene, PAI-1 (Plasminogen activator inhibitor typel) gene and the like. A cytotoxic gene can be expressed as a reporter gene. (2) Screening by the hybrid method can be performed using mammalian cells in addition to yeast cells. '

また、 例えば免疫沈降を用いてスクリーニングを行うことも可能である。 被検 化合物を含む試料の存在下で、 HK33蛋白質と ARF蛋白質とを発現する細胞を培養し 、 細胞を回収後、 一方の蛋白質に対する抗体等で複合体を回収したのち、 他方の 蛋白質を、 その蛋白質に対する抗体等を用いて検出することにより、 両者の蛋白 質の結合を評価することができる。 両者の蛋白質は細胞が内因的に発現する蛋白 質であってもよいが、 どちらかまたは両者の蛋白質を外来的に細胞で発現させる こともできる。 動物細胞内で蛋白質を外来的に発現させる場合、 例えば、 HK33蛋白質および/ または ARF蛋白質をコードする遺伝子を、 pSV2neo， pcDNA I， pCD8 などの外来遺 伝子発現用のベクターに挿入することで動物細胞などで当該遺伝子を発現させる 。 発現に用いるプロモーターとしては SV40 early promoter (Rigby, In Willia rason (ed. ) , Genetic Engineering, Vol. 3. Academic Press, London, p. 83-141 (1982) )， EF-1 a promoter (Kimら， Gene ， p. 217-223 (1990) ) , CAG proraot er (Niwa et al.， Gene 108, p. 193-200 (1991) ) , RSV LTR promoter (Cullen, Methods in Enzyraology 152, p. 684-704 (1987) ) ， SR promoter (Takebe et al.， Mol. Cell. Biol. 8, p. 466 (1988) )， CMV immediate early promoter (S eed and Aruffo, Proc. Natl. Acad. Sci. USA 84, p. 3365- 3369 (1987) ) , SV40 late promoter (Gheysen and Fiers, J. Mol. Appl. Genet. 1, p. 385- 394 (19 82) )， Adenovirus late promoter (Kaufman et al. , Mol. Cell. Biol. 9， p. 9 46 (1989) ) , HSV TK promoter等の一般的に使用できるプロモーターであれば何 を用いてもよい。 動物細胞に遺伝子を導入することで外来遺伝子を発現させるた めには、 エレクトロポレーシヨン法 (Chu, G. et al. , Nucl. Acid Res. 15， 13 11-1326 (1987) )、 リン酸カルシウム法 （Chen, C and Okayama, H. , Mol. Cell.Screening can also be performed using, for example, immunoprecipitation. Cells expressing the HK33 protein and the ARF protein are cultured in the presence of the sample containing the test compound, and after the cells are collected, the complex is recovered using an antibody against one of the proteins, and then the other protein is recovered. By detecting with an antibody against the protein, the binding of both proteins can be evaluated. Both proteins may be proteins that are expressed endogenously by cells, but either or both proteins can be expressed in cells exogenously. When a protein is exogenously expressed in animal cells, for example, the gene encoding the HK33 protein and / or ARF protein can be inserted into an exogenous gene expression vector such as pSV2neo, pcDNA I, or pCD8. The gene is expressed in cells or the like. Promoters used for expression include the SV40 early promoter (Rigby, In Willia rason (ed.), Genetic Engineering, Vol. 3. Academic Press, London, p. 83-141 (1982)) and the EF-1a promoter (Kim et al. , Gene, p. 217-223 (1990)), CAG proraoter (Niwa et al., Gene 108, p. 193-200 (1991)), RSV LTR promoter (Cullen, Methods in Enzyraology 152, p. 684-) 704 (1987)), SR promoter (Takebe et al., Mol. Cell. Biol. 8, p. 466 (1988)), CMV immediate early promoter (Sed and Aruffo, Proc. Natl. Acad. Sci. USA 84 , p. 3365-3369 (1987)), SV40 late promoter (Gheysen and Fiers, J. Mol. Appl. Genet. 1, p. 385-394 (1982)), Adenovirus late promoter (Kaufman et al., Mol Cell. Biol. 9, p. 946 (1989)) and any commonly used promoter such as the HSV TK promoter may be used. In order to express a foreign gene by introducing the gene into animal cells, the electroporation method (Chu, G. et al., Nucl. Acid Res. 15, 13 11-1326 (1987)), calcium phosphate Method (Chen, C and Okayama, H., Mol. Cell.

Biol. 7, 2745-2752 (1987) )、 DEAEデキストラン法 （Lopata， M. A. et al. , N ucl. Acids Res. 12, 5707-5717 (1984)； Sussman, D. J. and Milraan, G. , Mol . Cell. Biol. 4, 1642-1643 (1985) )、カチォニックリボソーム D0TAP (ベーリン ガーマンハイム社製） を用いた方法、 リポフエクチン法 （Derijard, B. , Cell 7 , 1025-1037 (1994)； Lamb, B. T. et al. , Nature Genetics 5, 22-30 (1993)；Biol. 7, 2745-2752 (1987)), DEAE dextran method (Lopata, MA et al., Nucl. Acids Res. 12, 5707-5717 (1984); Sussman, DJ and Milraan, G., Mol. Cell). Biol. 4, 1642-1643 (1985)), a method using catonic ribosome D0TAP (manufactured by Boehringer-Germanheim), a lipofectin method (Derijard, B., Cell 7, 1025-1037 (1994); Lamb, BT et al., Nature Genetics 5, 22-30 (1993);

Rabindran, S. K. et al. , Science 259, 230-234 (1993) )等の方法があるが、 レ、ずれの方法によってもよレ、。 特異性の明らかとなっているモノクローナル抗体 の認識部位 （ェピトープ） を HK33および/または ARF蛋白質の N末または C末に導 入することにより、 モノクローナル抗体の認識部位を有する融合蛋白質とするこ とができる。 用いるェピトープー抗体系としては市販されているものを利用する ことができる （実験医学 ， 85-90 (1995) )。 マルチクローユングサイトを介し て、 j8 -ガラクトシダーゼ、 マルトース結合蛋白質、 ィムノグロプリン定常領域、 ダルタチオン S-トランスフェラーゼ、 緑色蛍光蛋白質 （GFP) などとの融合蛋白 質を発現することができるベクターが市販されている。 Rabindran, SK et al., Science 259, 230-234 (1993)) and the like. By introducing a monoclonal antibody recognition site (epitope) whose specificity is known into the N-terminal or C-terminal of HK33 and / or ARF protein, it is possible to obtain a fusion protein having a monoclonal antibody recognition site. it can. Use commercially available epitope antibody system (Experimental Medicine, 85-90 (1995)). Vectors capable of expressing a fusion protein with j8-galactosidase, maltose binding protein, imnoglobulin constant region, daltathione S-transferase, green fluorescent protein (GFP), etc. through a multicloning site are commercially available.

融合蛋白質にする場合に、 元の蛋白質の性質をできるだけ変化させないように するために数個から十数個のアミノ酸からなる小さなェピトープ部分のみを導入 して、融合蛋白質を調製する方法も報告されている。例えば、 ポリヒスチジン （H is- tag) (例えば 6 XHisまたは lO XHisなど）、 インフルエンザ凝集素 HAの断片、 ヒト c- mycの断片、 FLAG (Hopp, T. P. et al. , BioTechnology (1988) 6, 1204- 1210)、 Vesicular stomatitis ウィルス糖蛋白質 （VSV- GP) の断片、 T7 genelO 蛋白質の断片 （T7- tag)、 ヒト単純へルぺスウィルス糖蛋白質の断片 （HSV- tag^ 、 E-tag (モノクローナルファージ上のェピトープ）、 SV40T抗原の断片、 lck ta g、 a - tubulinの断片、 B- tag、 Protein C の断片等の公知のェピトープとそれを 認識するモノクローナル抗体を、 上記のスクリーニングのためのェピトープー抗 体系として利用できる （実験医学 ， 85-90 (1995) ) 0 A method for preparing a fusion protein by introducing only a small epitope portion consisting of several to several tens of amino acids in order to minimize the properties of the original protein when making it into a fusion protein has been reported. I have. For example, polyhistidine (His-tag) (for example, 6XHis or lOXHis), influenza agglutinin HA fragment, human c-myc fragment, FLAG (Hopp, TP et al., BioTechnology (1988) 6, 1204) -1210), fragment of Vesicular stomatitis virus glycoprotein (VSV-GP), fragment of T7 genelO protein (T7-tag), fragment of human simple herpesvirus glycoprotein (HSV-tag ^, E-tag (monoclonal Epitopes on phage), known epitopes such as SV40T antigen fragment, lck tag, a-tubulin fragment, B-tag, Protein C fragment, etc. and monoclonal antibodies recognizing it, were used for the above-mentioned screening. Can be used as an anti-system (Experimental Medicine, 85-90 (1995)) 0

免疫沈降においては、 これらの抗体を適当な界面活性剤を利用して調製した細 胞溶解液に添加することにより免疫複合体を形成させる。 この免疫複合体は HK33 蛋白質、 ARF蛋白質、および抗体を含む。上記ェピトープに対する抗体を用いる以 外に、 HK33蛋白質または ARF蛋白質に対する抗体を利用して免疫沈降を行うことも 可能である。 これらの抗体は、 上記のように、 例えば目的の蛋白質をコードする 遺伝子を適当な大腸菌発現ベクターに導入して大腸菌内で発現させ、 発現させた 蛋白質を精製し、 これをゥサギやマウス、 ラット、 ャギ、 ニヮトリなどに免疫す ることで調製することができる。 また、 合成した部分ペプチドを上記の動物に免 疫することによって調製することもできる。  In immunoprecipitation, an immune complex is formed by adding these antibodies to a cell lysate prepared using an appropriate surfactant. This immune complex contains HK33 protein, ARF protein, and antibodies. In addition to using an antibody against the above-mentioned epitope, immunoprecipitation can also be performed using an antibody against the HK33 protein or the ARF protein. As described above, for example, these antibodies are prepared by introducing a gene encoding a target protein into an appropriate E. coli expression vector, expressing the gene in E. coli, purifying the expressed protein, and then purifying the expressed protein. It can be prepared by immunizing goats and chickens. It can also be prepared by immunizing the above animal with the synthesized partial peptide.

免疫複合体は、 例えば、 抗体がマウス IgG抗体であれば、 Protein A Sepharo seや Protein G Sepharose を用いて沈降させることができる。 また、例えば、 G STなどのェピトープとの融合蛋白質を調製した場合には、 グルタチオン- Sepharo se 4Bなどのこれらェピトープに特異的に結合する物質を利用して免疫複合体を 形成させることができる。 免 ¾沈降の一般的な方法については、例えば、文献 （H arlow, E. and Lane, D.： Antibodies, pp. 511-552, Cold Spring Harbor Labora tory publications, New York (1988) ) 記載の方法に従って、 または準じて行え ばよい。 If the antibody is a mouse IgG antibody, for example, the immune complex can be precipitated using Protein A Sepharose or Protein G Sepharose. Also, for example, G When a fusion protein with an epitope such as ST is prepared, an immune complex can be formed using a substance that specifically binds to these epitopes, such as glutathione-Sepharose 4B. The general method of sedimentation is described, for example, in the literature (Harlow, E. and Lane, D .: Antibodies, pp. 511-552, Cold Spring Harbor Labora tory publications, New York (1988)). Or in accordance with.

また、 細胞系を使わずにプルダウンアツセィを利用して本発明のスクリーニン グを行うこともできる。 例えば、 被検化合物を含む試料の存在下で、 HK33蛋白質 と ARF蛋白質とをインビトロでインキュべしトし、一方の蛋白質に対する抗体、ま たはこれらの蛋白質に融合させたタグに対する抗体等で複合体を回収したのち、 他方の蛋白質を、 その蛋白質に対する抗体または該蛋白質に付加したタグに対す る抗体等を用いて検出することにより、 両者の蛋白質の結合を評価することがで きる。 また、 一方の蛋白質を支持体に結合させておき、 他方の蛋白質を結合させ 、 そこに被検試料を適用する。 結合していた蛋白質が解離するかを検出すること により被検試科の効果を調べることができる。また、 ELISAを用いてスクリーニン グを行うことも可能である。  In addition, the screening of the present invention can be carried out using a pull-down assay without using a cell line. For example, HK33 protein and ARF protein are incubated in vitro in the presence of a sample containing a test compound, and a complex is formed with an antibody against one of the proteins or an antibody against a tag fused to these proteins. After recovery, the binding of the two proteins can be evaluated by detecting the other protein using an antibody against the protein or an antibody against a tag added to the protein. Also, one protein is bound to a support, the other protein is bound, and the test sample is applied thereto. By detecting whether the bound protein dissociates, the effect of the test sample can be examined. Screening can also be performed using ELISA.

さらに、 結合した蛋白質を検出又は測定する手段として表面プラズモン共鳴現 象を利用したバイオセンサーを使用することもできる。 表面プラズモン共鳴現象 を利用したバイオセンサーは、 蛋白質間の相互作用を微量の蛋白質試料を用いて かつ標識することなく、 表面プラズモン共鳴シグナルとしてリアルタイムに観察 することが可能である （例えば BIAcore、 Pharmacia製）。 したがって、 BIAcore等 のパイォセンサーを用いることにより HK33蛋白質およぴ ARF蛋白質の結合を評価 することが可能である。 さらに、 コンビナトリアルケミストリーを利用したハイ スループットスクリーニング （Wrighton NC ; Farrell FX; Chang R; Kashyap AK ； Barbone FP; Mulcahy LS ; Johnson DL; Barrett RW; Jolliffe LK; Dower WJ.， Small peptides as potent mimetics of the protein hormone erythropoietin ， Science (UNITED STATES) Jul 26 1996， 273 p458- 64、 Verdine GL. , The com binatorial chemistry of nature. Nature (ENGLAND) Nov 7 1996, 384 piト 13 、 Hogan JC Jr. , Directed combinatorial chemistry. Nature (ENGLAND) Nov 7 1996， 384 pl7-9) などにより本発明のスクリーニングを行うことも可能である。 以上の方法により同定される、 HK33蛋白質と ARF蛋白質の結合を促進または抑制 する化合物は、 ARF蛋白質と HK33蛋白質との相互作用をそれぞれ促進または抑制す る化合物と判定される。 これらの化合物は、 ARF蛋白質を介するシグナル伝達、 p 53蛋白質を介するシグナル伝達、 およぴ細胞増殖を調節するために使用され得る 。 また、 後述のように抗腫瘍剤などの医薬としても使用され得る。 Further, as a means for detecting or measuring the bound protein, a biosensor utilizing surface plasmon resonance can be used. A biosensor utilizing the surface plasmon resonance phenomenon can observe the interaction between proteins in real time as a surface plasmon resonance signal using a small amount of protein sample and without labeling (for example, BIAcore, manufactured by Pharmacia). ). Therefore, it is possible to evaluate the binding of HK33 protein and ARF protein by using a biosensor such as BIAcore. Furthermore, high-throughput screening using combinatorial chemistry (Wrighton NC; Farrell FX; Chang R; Kashyap AK; Barbone FP; Mulcahy LS; Johnson DL; Barrett RW; Jolliffe LK; Dower WJ .; Small peptides as potent mimetics of the protein hormone erythropoietin , Science (UNITED STATES) Jul 26 1996, 273 p458-64, Verdine GL., The com binatorial chemistry of nature.Nature (ENGLAND) Nov 7 1996, 384 pi 13, Hogan JC Jr., Directed combinatorial chemistry. ENGLAND) Nov 7 1996, 384 pl7-9) and the like, and the screening of the present invention can be performed. A compound that promotes or suppresses the binding between the HK33 protein and the ARF protein, which is identified by the above method, is determined to be a compound that promotes or suppresses the interaction between the ARF protein and the HK33 protein, respectively. These compounds can be used to regulate signaling through the ARF protein, signaling through the p53 protein, and cell growth. It can also be used as a drug such as an antitumor agent as described below.

上記の ARF蛋白質と HK33蛋白質との相互作用を調節する化合物のスクリーニン グにおいて、 ARF蛋白質と HK33蛋白質との相互作用の検出を、 ARF蛋白質の核移行 を指標として行うことも可能である。実施例に示すように、 HK33蛋白質は ARF蛋白 質の核移行を阻害する。従って、 HK33蛋白質による ARF蛋白質の核移行の阻害を調 節する化合物をスクリーニングすることにより、 ARF蛋白質と HK33蛋白質との相互 作用を調節する化合物を得ることができる。 このスクリーニング方法は、 ( a ) A RF蛋白質と HK33蛋白質とを発現する細胞に、 被検試料を接触させる工程、 （b ) A RF蛋白質の核局在を検出する工程、 および （c ) 被検試料非存在下で検出した場 合 （対照） と比較して、 HK33蛋白質による ARF蛋白質の核局在の阻害を調節 （促進 または低減）する化合物を選択する工程、 を含む。 HK33蛋白質による ARF蛋白質の 核局在の阻害を促進させる化合物は、 ARF蛋白質と HK33蛋白質との相互作用を促進 する化合物と判断され、また、 HK33蛋白質による ARF蛋白質の核局在の阻害を低減 させる化合物は、 ARF蛋白質と HK33蛋白質との相互作用を抑制する化合物と判断さ れる。 ARF蛋白質や HK33蛋白質は、 内因的蛋白質であってもよく、 また外来的に発 現させてもよレ、。用いられる細胞としては、 HK33蛋白質により ARF蛋白質の核移行 が阻害される限り制限はな 1/、。例えば実施例で用いられた NIH3T3または COS細胞等 が用いられ得る。 ARF蛋白質の核移行は、 例えば ARF蛋白質に対する抗体を用いた 免疫細胞化学的手法により検出することが可能である。 抗体は、 例えば蛍光標識 される。 また、 GFP蛋白質などを融合させた ARF蛋白質を外来的に細胞で発現させ れば、 ARF蛋白質の細胞内局在を容易に検出することができる。 In the above-mentioned screening of a compound that regulates the interaction between the ARF protein and the HK33 protein, the interaction between the ARF protein and the HK33 protein can be detected by using the nuclear translocation of the ARF protein as an index. As shown in the Examples, HK33 protein inhibits nuclear translocation of ARF protein. Therefore, by screening for a compound that regulates the inhibition of nuclear translocation of the ARF protein by the HK33 protein, a compound that regulates the interaction between the ARF protein and the HK33 protein can be obtained. This screening method comprises the steps of (a) contacting a test sample with a cell expressing the ARF protein and the HK33 protein, (b) detecting the nuclear localization of the ARF protein, and (c) detecting the nuclear localization of the ARF protein. Selecting a compound that modulates (promotes or reduces) the inhibition of nuclear localization of the ARF protein by the HK33 protein as compared to the case where the detection is performed in the absence of the sample (control). A compound that promotes the inhibition of nuclear localization of ARF protein by HK33 protein is determined to be a compound that promotes the interaction between ARF protein and HK33 protein, and also reduces the inhibition of nuclear localization of ARF protein by HK33 protein. The compound is considered to be a compound that suppresses the interaction between the ARF protein and the HK33 protein. The ARF protein and HK33 protein may be endogenous proteins or may be exogenously expressed. The cells used are not limited as long as the nuclear translocation of the ARF protein is inhibited by the HK33 protein. For example, the NIH3T3 or COS cells used in Examples can be used. For nuclear translocation of ARF protein, for example, an antibody against ARF protein was used. It can be detected by an immunocytochemical technique. The antibody is, for example, fluorescently labeled. In addition, if an ARF protein fused with a GFP protein or the like is exogenously expressed in a cell, the intracellular localization of the ARF protein can be easily detected.

上記の ARF蛋白質と HK33蛋白質との相互作用を調節する化合物のスクリーニン グ方法の他の態様は、 ARF蛋白質と HK33蛋白質との相互作用を、 ARF蛋白質を介す るシグナル伝達を指標に検出する方法である。 ARF蛋白質は細胞周期調節に関連す るシグナル因子であり、 ARF蛋白質を介するシグナルは MDM2、 p53、 およびその下 流の p21などの他の分子に伝達される。従って、これらのシグナル伝達の変化を指 標にしてスクリーニングを行うことにより、ARF蛋白質と HK33蛋白質との相互作用 を調節する化合物を得ることができる。  Another embodiment of the above-described screening method for a compound that regulates the interaction between the ARF protein and the HK33 protein is to detect the interaction between the ARF protein and the HK33 protein using ARF-mediated signal transduction as an index. Is the way. The ARF protein is a signaling factor involved in cell cycle regulation, and signals mediated by the ARF protein are transmitted to other molecules such as MDM2, p53, and its downstream p21. Therefore, a compound that regulates the interaction between the ARF protein and the HK33 protein can be obtained by performing screening using these changes in signal transduction as an index.

このようなスクリーニングは細胞系を用いて行うことができる。 このスクリー ニングは、 例えば、 （a ) ARF蛋白質と HK33蛋白質とを発現する細胞に、 被検試料 を接触させる工程、 （b ) ARFを介するシグナル伝達の HK33蛋白質による抑制を検 出する工程、 および （c ) 被検試料非存在下で検出した場合 （対照） と比較して 、 該抑制を調節 （増強または低減） する化合物を選択する工程、 を含む方法であ る。 このようなスクリーニングも、 本発明のスクリーニングに含まれる。 スクリ 一二ングに用いる被検試料は特に制限はない。 ARF蛋白質や HK33蛋白質は、内因的 蛋白質であってもよく、 また外来的に発現させてもよい。 用いられる細胞として は、 ARF蛋白質を介するシグナル伝達経路を持つ多くの細胞が用いられ得る。例え ば、 CH0細胞、 COS細胞、 HeLa細胞、 NIH3T3細胞、 BHK、 Veroなどが挙げられる。 親 細胞に比べ HK33遺伝子の発現が有意に上昇した細胞を好適に用いることができる  Such screening can be performed using cell lines. This screening includes, for example, (a) a step of bringing a test sample into contact with cells expressing the ARF protein and the HK33 protein, (b) a step of detecting suppression of ARF-mediated signal transduction by the HK33 protein, and (C) a step of selecting a compound that regulates (enhance or reduce) the inhibition as compared to the case where detection is performed in the absence of a test sample (control). Such screening is also included in the screening of the present invention. The test sample used for screening is not particularly limited. ARF protein and HK33 protein may be endogenous proteins or may be expressed exogenously. As the cells to be used, many cells having a signal transduction pathway through the ARF protein can be used. Examples include CH0 cells, COS cells, HeLa cells, NIH3T3 cells, BHK, Vero and the like. Cells with significantly increased HK33 gene expression compared to parent cells can be used favorably

ARF蛋白質を介するシグナル伝達は、以下の指標により検出することが可能であ る。 Signaling through the ARF protein can be detected by the following indicators.

• ARF蛋白質と MDM2蛋白質との結合  • Binding of ARF protein to MDM2 protein

• MDM2蛋白質と p53蛋白質との結合 • p53蛋白質の修飾 （ュビキチン化など） • Binding of MDM2 protein to p53 protein • Modification of p53 protein (such as ubiquitination)

• p53蛋白質の分解  • Degradation of p53 protein

• p53蛋白質活性  • p53 protein activity

• p53蛋白質が標的とする遺伝子の発現 （例えば p53依存的転写など） • DNA合成、 細胞周期、 細胞分裂、 細胞増殖、 アポトーシス  • Expression of genes targeted by p53 protein (eg, p53-dependent transcription) • DNA synthesis, cell cycle, cell division, cell proliferation, apoptosis

ARF蛋白質を介するシグナル伝達の流れは以下のようになっている。 HK33蛋白質 と ARF蛋白質との相互作用により ARF蛋白質の核移行が阻害されると、ARF蛋白質と MDM2蛋白質との結合が抑制される。 ARF蛋白質と MDM2蛋白質との結合の抑制は、 M DM2蛋白質の活性を促進し、 これにより MDM2蛋白質と p53蛋白質との結合が促進さ れ、 p53蛋白質のュビキチン化を含む p53蛋白質の分解を誘導する修飾を促進する 。 結果として p53蛋白質の分解が促進され、 p53蛋白質の安定性が低下することに より細胞内における p53蛋白質の活性は低下し、 p53蛋白質が持つ G1停止おょぴァ ポトーシスの誘導などの機能が抑制される。 その結果、 DNA合成の上昇、 アポトー シスの抑制、および/または細胞分裂の促進がもたらされ、細胞増殖が促進される 。 これらの指標は、公知の方法により検出することが可能である。以上のような A RF蛋白質を介するシグナル伝達の流れに基づき、 ARF蛋白質を介するシグナル伝達 における各段階を指標に、 該シグナル伝達を促進または抑制する化合物をスクリ 一二ングすることによつて、 HK33蛋白質と ARF蛋白質との相互作用をそれぞれ抑制 または促進する化合物を得ることが可能である。  The flow of signal transmission through the ARF protein is as follows. When the nuclear translocation of the ARF protein is inhibited by the interaction between the HK33 protein and the ARF protein, the binding between the ARF protein and the MDM2 protein is suppressed. Suppression of binding between ARF protein and MDM2 protein promotes the activity of MDM2 protein, which promotes binding of MDM2 protein to p53 protein and induces degradation of p53 protein, including ubiquitination of p53 protein Promote modification. As a result, degradation of the p53 protein is promoted, and the stability of the p53 protein is reduced, so that the activity of the p53 protein in the cell is reduced, and the functions of the p53 protein, such as induction of G1 arrest, are inhibited. Is done. The result is increased DNA synthesis, reduced apoptosis, and / or enhanced cell division, and promoted cell proliferation. These indices can be detected by a known method. Based on the above-described flow of signal transduction via the ARF protein, HK33 can be screened for compounds that promote or suppress the signal transduction using each step in signal transduction via the ARF protein as an index. It is possible to obtain a compound that suppresses or promotes the interaction between the protein and the ARF protein, respectively.

具体的には、 例えば ARF蛋白質を介するシグナル伝達の活性化により p53蛋白質 が活性ィ匕して誘導される P21等の p53の標的遺伝子の発現を抗体で確認するウェス タンプロッティング法によりスクリーニングすることができる。また、 p21等の標 的遺伝子の mRNAをノーザンブロッテイングまたは RT- PCRにより検出してスクリー ニングすることもできる。あるいは、 p21等の標的遺伝子の発現制御配列の下流に レポータ一遺伝子を結合した DNAを細胞に組み込み、レポータ一活性を指標にスク リーユングを行うこともできる。 p53の標的遺伝子の発現を促進する化合物は、 A RF蛋白質を介するシグナル伝達の HK33蛋白質による抑制を低減する化合物と判断 される。逆に、標的遺伝子の発現を抑制する化合物は、 ARF蛋白質を介するシグナ ル伝達の HK33蛋白質による抑制を促進する化合物と判断される。 あるいは、 細胞 増殖の抑制を指標にスクリーニングすることができる。 このスクリーニングは、 細胞数の計測による増殖測定、 または WST試薬 （ロシュダイァゴニスティック社） 等を用いた細胞増殖測定により実施することができる。 これらの方法を用いて、 A RFの活性を抑制している HK33の活性を、 加えた被検試料がキャンセルすることに よる細胞増殖抑制を上記の方法により計測し、 これを指標にスクリーニングを行 うことができる。細胞増殖を促進する化合物は、 ARF蛋白質を介するシグナル伝達 の HK33蛋白質による抑制を促進する化合物と判断される。 逆に、 細胞増殖を抑制 する化合物は、 ARF蛋白質を介するシグナル伝達の HK33蛋白質による抑制を低減す る化合物と判断される。 これらのスクリーニングは、以下に述べる p53蛋白質を介 するシグナル伝達の HK33蛋白質による抑制を調節する化合物のスクリーニングに おいても同様に実施することができる。 Specifically, for example, screening is performed by a Western plotting method using an antibody to confirm the expression of a p53 target gene, such as P21, which is induced by activation of p53 protein through activation of ARF-mediated signal transduction. Can be. Screening can also be performed by detecting mRNA of a target gene such as p21 by Northern blotting or RT-PCR. Alternatively, screening can be performed using reporter activity as an indicator, by incorporating DNA into which a reporter gene is bound downstream of the expression control sequence of the target gene such as p21. Compounds that promote the expression of p53 target genes are: A The compound is considered to be a compound that reduces the suppression of signal transduction via RF protein by HK33 protein. Conversely, a compound that suppresses the expression of a target gene is considered to be a compound that promotes suppression of signal transmission through the ARF protein by the HK33 protein. Alternatively, screening can be performed using the inhibition of cell proliferation as an index. This screening can be carried out by measuring proliferation by measuring the number of cells, or by measuring cell proliferation using WST reagent (Roche Diagnostics). Using these methods, HK33 activity, which suppresses ARF activity, is measured by the above-mentioned method to measure cell growth inhibition due to cancellation of the test sample, and screening is performed using this as an index. I can. A compound that promotes cell proliferation is considered to be a compound that promotes the suppression of signal transduction through the ARF protein by the HK33 protein. Conversely, a compound that suppresses cell proliferation is considered to be a compound that reduces suppression of ARF protein-mediated signal transduction by the HK33 protein. These screenings can be similarly carried out in the screening of compounds that regulate the suppression of p53 protein-mediated signal transduction by the HK33 protein as described below.

本発明において、 HK33蛋白質の発現が、 p53依存性の転写を抑制することが示さ れた。 このことは、 HK33蛋白質が p53の活性を抑制することを示している。細胞周 期の調節因子として中心的な役割を果たす p53蛋白質の活性を HK33蛋白質が制御 していることは重要である。本発明のスクリーニング方法の他の態様は、 p53蛋白 質を介するシグナル伝達の HK33蛋白質による抑制を調節する化合物を選択するこ とによりスクリーニングを行う方法である。 このスクリーニング方法は、 （a ) A RF蛋白質と HK33蛋白質とを発現する細胞に、 被検試料を接触させる工程、 （b ) p 53を介するシグナル伝達の HK33蛋白質による抑制を検出する工程、 （c )被検試料 非存在下で検出した場合 （対照） と比較して、 該抑制を調節 （増強または低減） する化合物を選択する工程、 を含む方法である。 これらのスクリーニングは細胞 系で行うことが好ましい。 スクリーニングに用いる被検試料は特に制限はない。 細胞としては、上記の ARF蛋白質のシグナル伝達を指標としたスクリーニングと同 様の細胞が用いられ得る。 親細胞に比べ HK33遺伝子の発現が有意に上昇した細胞 を好適に用いることができる。 In the present invention, it has been shown that the expression of the HK33 protein suppresses p53-dependent transcription. This indicates that the HK33 protein suppresses p53 activity. It is important that the HK33 protein controls the activity of the p53 protein, which plays a central role as a cell cycle regulator. Another embodiment of the screening method of the present invention is a method for screening by selecting a compound that regulates the suppression of signal transduction through the p53 protein by the HK33 protein. This screening method comprises: (a) a step of bringing a test sample into contact with cells expressing the A RF protein and the HK33 protein; (b) a step of detecting suppression of p53-mediated signal transduction by the HK33 protein; ) Selecting a compound that modulates (enhance or reduce) the inhibition as compared to the case of detection in the absence of a test sample (control). Preferably, these screens are performed in cell lines. The test sample used for screening is not particularly limited. As for cells, the same screening as above using ARF protein signal transduction as an index Similar cells can be used. Cells in which the expression of the HK33 gene is significantly increased as compared to the parent cells can be preferably used.

p53蛋白質を介するシグナル伝達は、 上記に示したように p53蛋白質またはその 作用を検出することにより評価することができる。具体的には、例えば p53蛋白質 と MDM2蛋白質との結合、 p53蛋白質の修飾、 p53蛋白質の安定性、 p53蛋白質の標的 遺伝子の発現、 アポトーシス、 DNA合成、細胞分裂、細胞増殖などを指標とするこ とができる。 このスクリーニングにより得られる化合物は、 上記の ARF蛋白質と H K33蛋白質との相互作用を調節する化合物と同様、細胞増殖の調節剤として、また 抗腫瘍剤として利用され得る。 従ってこのスクリーニング方法は、 上記に記載し た ARF蛋白質と HK33蛋白質のと相互作用を調節する化合物のスクリーニング方法 と同様、 細胞増殖を調節する化合物を得るために用いることができる他、 抗腫瘍 剤を得るためにも好適に用いられる。  Signal transduction via the p53 protein can be evaluated by detecting the p53 protein or its action as described above. Specifically, for example, p53 protein binding to MDM2 protein, p53 protein modification, p53 protein stability, p53 protein target gene expression, apoptosis, DNA synthesis, cell division, cell proliferation, etc. Can be. Compounds obtained by this screening can be used as cell growth regulators and antitumor agents, similarly to the compounds that regulate the interaction between the ARF protein and the HK33 protein. Therefore, this screening method can be used to obtain a compound that regulates cell proliferation, as well as an antitumor agent, in the same manner as the above-described method for screening a compound that regulates the interaction between ARF protein and HK33 protein. It is also suitably used for obtaining.

具体的には、 例えば上記の p21等の p53の標的遺伝子の発現を抗体で確認するゥ エスタンプロッティング法またはノーザンプロッティングゃ RT- PCR等によりスク リーユングすることができる。 p53の標的遺伝子の発現を促進する化合物は、 p53 蛋白質を介するシグナル伝達の HK33蛋白質による抑制を低減する化合物と判断さ れる。逆に、標的遺伝子の発現を抑制する化合物は、 p53蛋白質を介するシグナル 伝達の HK33蛋白質による抑制を促進する化合物と判断される。 また上記と同様に 、 細胞増殖の抑制を指標にスクリーニングすることができる。 細胞増殖を促進す る化合物は、 p53蛋白質を介するシグナル伝達の HK33蛋白質による抑制を促進する 化合物と判断される。逆に、細胞増殖を抑制する化合物は、 p53蛋白質を介するシ グナル伝達の HK33蛋白質による抑制を低減する化合物と判断される。  Specifically, for example, screening can be performed by the expression of the target gene of p53 such as p21 described above with an antibody (estrant blotting method or Northern plotting RT-PCR). A compound that promotes the expression of a p53 target gene is considered to be a compound that reduces suppression of p53 protein-mediated signal transduction by the HK33 protein. Conversely, a compound that suppresses the expression of a target gene is considered to be a compound that promotes suppression of signal transduction through the p53 protein by the HK33 protein. Similarly to the above, screening can be performed using the inhibition of cell proliferation as an index. A compound that promotes cell proliferation is considered to be a compound that promotes suppression of signal transduction via the p53 protein by the HK33 protein. Conversely, a compound that suppresses cell proliferation is considered to be a compound that reduces suppression of signal transmission through the p53 protein by the HK33 protein.

以上に記載したスクリーニングにおいて細胞系を用いる場合は、 被検化合物を 含む試料は、 例えば細胞の培地に添カ卩される。 また、 遺伝子を被検試料として用 いる場合は、 これを細胞へ導入する。 導入には、 例えは発現ベクター等を用いた 公知の遺伝子導入法により行い得る。 摘体を用いたインビボ系におけるスクリー ニングにおいては、 被検化合物を含む試料を適当な経路で投与する。 試料の投与 は、 例えば経皮、 腹腔内、 筋肉内、 経腸、 静脈注射等により行い得る。 また、 発 現ベクター等により遺伝子を投与することもできる。 When a cell line is used in the screening described above, the sample containing the test compound is added to, for example, a cell culture medium. When a gene is used as a test sample, it is introduced into cells. The introduction can be carried out, for example, by a known gene introduction method using an expression vector or the like. Screening in vivo system using excision In ning, a sample containing the test compound is administered by an appropriate route. The administration of the sample can be performed, for example, by transdermal, intraperitoneal, intramuscular, enteral, intravenous injection and the like. In addition, the gene can be administered using an expression vector or the like.

細胞内において発現する、 HK33蛋白質または ARF蛋白質、あるいはこれらの蛋白 質から下流の各種のシグナル伝達分子は、 内因性であっても、 外来的に発現させ たものであったもよい。例えば、 ARF遺伝子を欠損する細胞を用いて、 この細胞と 、これに外来的に ARF遺伝子を導入した細胞とを用レ、て被検化合物のアツセィを行 えば、化合物の ARF蛋白質に対する特異性を検証することが可能である。 このため の最も簡便な細胞として、 ARFの存在する INK4a部位を欠損した NIH3T3細胞を用い ることが適当である。  The HK33 protein or ARF protein expressed in a cell, or various signaling molecules downstream from these proteins, may be endogenous or exogenously expressed. For example, by using an ARF gene-deficient cell and using the cell and the cell into which the ARF gene has been introduced exogenously, the test compound can be assayed to determine the specificity of the compound for the ARF protein. It is possible to verify. As the simplest cell for this purpose, it is appropriate to use NIH3T3 cells lacking the INK4a site where ARF is present.

また、本発明は、 HK33遺伝子の発現を調節する化合物の選択により、 ARF蛋白質 を介するシグナル伝達の調節剤をスクリーニングする方法を提供する。 このスク リーニングは、 HK33蛋白質をコードする遺伝子またはその発現制御領域を利用し て、 細胞内、 生体内、 または in vitro癸現系等において HK33遺伝子の発現 （転 写または翻訳など） を調節しうる化合物を選択する工程を含む方法である。 この スクリーニングは、 例えば、 ARF蛋白質または p53蛋白質を介するシグナル伝達の 調節剤、 細胞増殖調節剤、 アポトーシス調節剤、 あるいは発癌剤、 細胞の不死化 試薬、 または抗腫瘍剤のスクリーニングにも利用し得る。  The present invention also provides a method for screening for a regulator of ARF protein-mediated signal transduction by selecting a compound that regulates the expression of the HK33 gene. This screening can regulate the expression (transcription or translation) of the HK33 gene in a cell, in a living body, or in an in vitro cloning system, using a gene encoding the HK33 protein or its expression control region. This is a method including a step of selecting a compound. This screening can also be used, for example, to screen for modulators of signal transduction via the ARF protein or p53 protein, cell growth regulators, apoptosis regulators, or carcinogens, cell immortalization reagents, or antitumor agents.

上記のスクリーニング方法は、 例えば （a ) HK33遺伝子を内因的に発現する細 胞に被検試料を接触させる工程、 （b ) HK33遺伝子の発現を検出する工程、および ( c ) 被検試料を該細胞に接触させない場合 （対照） と比較して、 該発現を調節 (促進または阻害） する活性を有する化合物を選択する工程、 を含む方法により 実施しうる。 ここで遺伝子の発現には、 転写おょぴ翻訳が含まれる。  The screening method described above includes, for example, (a) a step of bringing a test sample into contact with cells endogenously expressing the HK33 gene, (b) a step of detecting expression of the HK33 gene, and (c) a step of: Selecting a compound having an activity of regulating (enhancing or inhibiting) the expression as compared to the case where the cells are not brought into contact with the cells (control). Here, gene expression includes transcription and translation.

例えば、 HK33遺伝子を発現している細胞を被検試料と共に培養し、 該遺伝子の 発現をノーザン解析や RT- PCR法などの mRNAを検出する方法、 あるいはウエスタン ブロッテイング、免疫沈降、 ELISAなどの蛋白質を検出する方法、 またはこれらを 改良した方法により検出し、 被検試科を添加しない場合と比較して、 該遺伝子の 発現を促進あるいは阻害する化合物を選択することにより、 目的の化合物をスク リ一二ングすることができる。 For example, cells expressing the HK33 gene are cultured together with a test sample, and the expression of the gene is detected by mRNA analysis such as Northern analysis or RT-PCR, or by protein expression such as Western blotting, immunoprecipitation, and ELISA. How to detect or these The target compound can be screened by selecting a compound that promotes or inhibits the expression of the gene as compared with the case where the test sample is not added and detection is performed by an improved method.

スクリーニングに用いる細胞に特に制限はないが、 癌治療の観点からは HK33遺 伝子の発現を抑制することが治療につながると予想されるため、 HK33遺伝子の発 現宂進が見られる種々の癌細胞株または不死化細胞等を用いて、 HK33遺伝子の発 現を抑制する化合物をスクリ一二ングすることが考えられる。 逆に発現を上昇さ せる化合物のスクリーニングを目的とした場合は、 HK33遺伝子の発現レベルが低 い正常細胞等を用いることができる。  There are no particular restrictions on the cells used for screening, but from the perspective of cancer treatment, suppression of the expression of the HK33 gene is expected to lead to treatment. It is conceivable to screen a compound that suppresses the expression of the HK33 gene using a cell line or an immortalized cell. Conversely, when the purpose is to screen for a compound that increases expression, normal cells or the like having a low expression level of the HK33 gene can be used.

また、 HK33遺伝子の発現制御領域の活性化または不活性化を指標とする方法に よって、 生体内または細胞内において HK33遺伝子の発現を調節しうる化合物をス クリーニングすることも考えられる。 このスクリーニングは、 （a ) HK33遺伝子の 内因的転写制御配列の下流に機能的に結合されたレポーター遺伝子を有するベタ ターが導入された細胞に、被検試料を接触させる工程、 （b )該レポーター遺伝子 の発現を検出する工程、 および （c ) 被検試料を該細胞に接触させない場合 （対 照） と比較して、 該レポーター遺伝子の発現を調節 （促進または阻害） する活性 を有する化合物を選択する工程、 を含む方法により実施しうる。  It is also conceivable to screen a compound that can regulate the expression of the HK33 gene in vivo or in a cell by a method using the activation or inactivation of the expression control region of the HK33 gene as an index. This screening comprises: (a) contacting a test sample with cells into which a vector having a reporter gene operably linked downstream of the endogenous transcription control sequence of the HK33 gene has been introduced; (b) the reporter A step of detecting the expression of the gene, and (c) selecting a compound having an activity of regulating (promoting or inhibiting) the expression of the reporter gene, as compared with the case where the test sample is not brought into contact with the cells (control). And a method comprising the steps of:

ここで 「内因的転写制御配列」 とは、 HK33遺伝子を天然に保持している細胞に おいて、 該遺伝子の転写を制御している配列のことを言う。 このような配列は、 プロモーター、 ェンハンサー、および/またはリプレッサーなどが含まれる。 これ ら配列としては、例えば、 HK33蛋白質をコードする遺伝子の上流領域の DNAを用い ることができる。 例えば、 HK33蛋白質をコードする遺伝子の転写開始点 （または 翻訳開始コドン）からその上流数 kbまでの DNA断片には、該遺伝子の内因的転写制 御配列が含まれていると考えられる。 この断片をレポータ一遺伝子と連結するこ とで、 レポーター遺伝子の発現を、 HK33蛋白質をコードする遺伝子の転写制御下 に置くことが可能である。 上流領域は適宜欠失または変異を導入して転写制御活 性を測定し、 転写制御に関与している配列を特定してその断片を用いることも可 能である。 現在、 転写制御に関与する転写因子が結合する多くの転写制御配列が 知られている。 HK33蛋白質をコードする遺伝子の上流域から、 これら既知の転写 制御配列を同定することで、 内因的転写制御配列を特定することも考えられる。 通常、 遺伝子の転写を制御している配列は、 1つの遺伝子に複数存在しているが 、 本発明のスクリーニングにおいては、 それらのいずれか、 またはその組み合わ せを用いることができる。 内因的転写制御配列は、 他のプロモーターとのキメラ にしてもよい。 キメラプロモーターは、 転写制御の試験においてはよく用いられ ている。キメラプロモーターの作製に用いる他のプロモーターとしては、例えば S V40初期プロモーター由来の最小プロモーターなどが挙げられる。 なお、 「機能的 に結合された」 とは、 該転写制御配列の活性化に応答して、 その下流に結合され たレポーター遺伝子が発現しうるように、 該転写制御配列とレポーター遺伝子が 結合していることを指す。 Here, the “endogenous transcription control sequence” refers to a sequence that controls transcription of the HK33 gene in a cell naturally retaining the gene. Such sequences include promoters, enhancers, and / or repressors. As these sequences, for example, DNA in the upstream region of the gene encoding the HK33 protein can be used. For example, a DNA fragment from the transcription initiation point (or translation initiation codon) of the gene encoding the HK33 protein to several kb upstream thereof is considered to contain an endogenous transcription control sequence of the gene. By linking this fragment with the reporter gene, the expression of the reporter gene can be placed under the transcriptional control of the gene encoding the HK33 protein. In the upstream region, transcription control activity is It is also possible to determine the sequence involved in transcription control by measuring the sex and use the fragment thereof. At present, many transcription control sequences to which transcription factors involved in transcription control are bound are known. From upstream region of the gene encoding the HK3 3 protein, by identifying these known transcription control sequences, it is conceivable to identify the endogenous transcriptional control sequences. Usually, a plurality of sequences controlling the transcription of a gene are present in one gene, but any one of them or a combination thereof can be used in the screening of the present invention. The endogenous transcription control sequence may be a chimera with another promoter. Chimeric promoters are often used in transcriptional regulation studies. Other promoters used for producing the chimeric promoter include, for example, a minimal promoter derived from the SV40 early promoter. Here, “functionally linked” means that the transcription control sequence and the reporter gene are linked so that the reporter gene linked downstream thereof can be expressed in response to activation of the transcription control sequence. Point.

具体的には、 例えば、 HK33蛋白質をコードする配列若しくはその一部をプロ一 ブとしたゲノム DNAライブラリーのスクリーニングにより、 HK33遺伝子の転写制御 領域 （プロモーター、 ェンハンサーなど） をクローニングし、 これを適当なレポ 一ター遺伝子 （'クロラムフエニコールァセチルトランスフェラーゼ遺伝子、 ルシ フェラーゼ遺伝子など） の上流に揷入した発現ベクターを作製し、 これを哺¾動 物細胞に導入する。 次いで、 被検試料を該細胞に接触させ、 レポーター活性を検 出し、 被検試料を接触させない細胞におけるレポーター活性と比較して、 レポ一 ター活性を増加または減少させる化合物を選択することにより、 細胞内において 本発明の遺伝子の発現を調節しうる化合物をスクリーニングすることができる。 このスクリーニングは、 HK33遺伝子の発現を、 レポーター活性を指標として検出 するため、 上記したノーザン解析などの直接的な検出と比較して、 簡便であると いう特徴を有する。  Specifically, for example, by cloning the transcription control region (promoter, enhancer, etc.) of the HK33 gene by screening a genomic DNA library using the sequence encoding the HK33 protein or a part thereof as a probe, An expression vector is inserted upstream of a suitable reporter gene (eg, chloramphenicol acetyltransferase gene, luciferase gene, etc.), and is introduced into a mammalian cell. Then, a test sample is brought into contact with the cells, the reporter activity is detected, and a compound that increases or decreases the reporter activity compared to the reporter activity in the cells not contacted with the test sample is selected. Within, compounds that can regulate the expression of the gene of the present invention can be screened. This screening has a feature that it is simpler than the direct detection such as the above-described Northern analysis, because the expression of the HK33 gene is detected using the reporter activity as an index.

ヒト HK33遺伝子の転写制御配列は既にクローニングされており、 レポーター遺 伝子を連結させ転写解析が行われている （Ka腿 erer， S. et al. , Genomics 45 : 200-210, 1997) ο ヒト ΗΚ33遺伝子の転写制御領域には、 Spl配列などの既知の転写 因子結合配列が見出された。 HK33遺伝子の転写制御配列中のこれらの配列を含む 領域を用いて本発明のスクリーニングを行うことができる。 The transcription control sequence of human HK33 gene has already been cloned, Transcription analysis has been performed by linking genes (Ka Thigherer, S. et al., Genomics 45: 200-210, 1997) ο The transcription control region of the human ΗΚ33 gene contains known transcription such as Spl sequence. A factor binding sequence was found. The screening of the present invention can be performed using a region containing these sequences in the transcription control sequence of the HK33 gene.

以上に記載した本発明のスクリーニング方法により単離され得る化合物は、 HK 33蛋白質と ARF蛋白質との相互作用を調節する化合物、 HK33遺伝子の発現を調節す る化合物、 ARF蛋白質を介するシグナル伝達を調節する化合物、 および p53蛋白質 を介するシグナル '伝達を調節する化合物などを含む。 HK33蛋白質と ARF蛋白質との 相互作用を抑制する化合物およひ ΉΚ33遺伝子の発現を抑制する化合物等は、 ARF 蛋白質の細胞内における活性を上昇させ、 p53を安定化し細胞分裂の停止を引き起 こし細胞分裂および細胞増殖を抑制する。またアポトーシスを誘導し得る。逆に H K33蛋白質と ARF蛋白質との相互作用を促進する化合物およひ ΉΚ33遺伝子の発現を 促進する化合物等は、 ARF蛋白質の細胞内における活性を低下させ、 p53の分解を 促進し細胞分裂の停止を解除し細胞分裂および細胞増殖を促進する。 またアポト 一シスを抑制し得る。 従って、 本発明の上記のスクリーニングは、 細胞增殖を調 節する化合物を評価したり、 または該化合物を単離するために有用である。 特に 、 細胞増殖を抑制する化合物は抗腫瘍剤としての利用が期待される。 従って、 本 発明のスクリーニング方法は、 抗腫瘍剤の評価または単離のために好適に用いら れる。  Compounds that can be isolated by the above-described screening method of the present invention include compounds that regulate the interaction between HK33 protein and ARF protein, compounds that regulate HK33 gene expression, and that regulate signaling through ARF protein. And compounds that regulate signal transduction through the p53 protein. Compounds that suppress the interaction between HK33 protein and ARF protein and compounds that suppress the expression of 33 gene increase the intracellular activity of ARF protein, stabilize p53 and cause cell arrest. Inhibits cell division and cell proliferation. It can also induce apoptosis. Conversely, compounds that promote the interaction between the HK33 protein and the ARF protein and compounds that promote the expression of the 遺 伝 子 33 gene, etc., decrease the activity of the ARF protein in cells, promote the degradation of p53, and promote cell division. Releases arrest and promotes cell division and cell proliferation. It can also suppress apoptosis. Therefore, the above-mentioned screening of the present invention is useful for evaluating a compound that regulates cell proliferation or isolating the compound. In particular, compounds that suppress cell proliferation are expected to be used as antitumor agents. Therefore, the screening method of the present invention is suitably used for evaluating or isolating an antitumor agent.

上記の HK33蛋白質の活性を調節する化合物、 HK33蛋白質と ARF蛋白質との相互作 用を調節する化合物、 ARF蛋白質を介するシグナル伝達を調節する化合物、 p53蛋 白質を介するシグナル伝達を調節する化合物、 および細胞増殖を調節する化合物 等は試薬および/または医薬として利用することができる。また、上記スクリー二 ングにより単離しうる化合物も、同様に試薬および/または医薬として利用するこ とができる。 本発明のスクリーニング方法を用いて単離しうる化合物の構造の一 部を、 付加、 欠失及ぴ Z又は置換により変換される化合物も、 本発明のスクリー ニングにより単離され得る化合物に含まれる。 このような化合物も同様に試薬お よび/または医薬として利用することができる。 これらの化合物は、 例えば ARF活 性調節剤または P53活性調節剤などの薬剤として有用である。薬剤には、試薬およ ぴ医薬が含まれる。 またこれらの化合物は、 細胞増殖を調節するための薬剤とし て用いられ得る。 該化合物は、 例えば細胞増殖性疾患や細胞増殖を制御すること により治療可能な疾患の治療への応用が考えられる。特に、 HK33蛋白質と ARF蛋白 質との相互作用を抑制する化合物および HK33遺伝子の発現を抑制する化合物など の HK33蛋白質の作用を抑制する化合物は、 腫瘍の抑制剤としての利用が期待され る.。 A compound that regulates the activity of the HK33 protein, a compound that regulates the interaction between the HK33 protein and the ARF protein, a compound that regulates the signal transduction via the ARF protein, a compound that regulates the signal transduction via the p53 protein, and Compounds that regulate cell proliferation can be used as reagents and / or medicaments. In addition, compounds that can be isolated by the above screening can also be used as reagents and / or medicaments. Compounds in which a part of the structure of a compound that can be isolated using the screening method of the present invention is converted by addition, deletion, Included in compounds that can be isolated by tanning. Such compounds can also be used as reagents and / or medicaments. These compounds are useful as agents, for example, ARF activity modulators or P53 activity modulators. Drugs include reagents and drugs. These compounds can also be used as agents for regulating cell proliferation. The compound is considered to be applicable to, for example, treatment of cell proliferative diseases and diseases which can be treated by controlling cell proliferation. In particular, compounds that suppress the action of HK33 protein, such as compounds that suppress the interaction between HK33 protein and ARF protein and compounds that suppress the expression of HK33 gene, are expected to be used as tumor suppressors.

また、 HK33蛋白質と ARF蛋白質との相互作用を促進する化合物は例えばアポトー シスの抑制剤としての利用が考えられる。 また、 細胞増殖促進剤としても有用で ある。 HK33蛋白質と ARF蛋白質との相互作用を促進する化合物は、 ARF蛋白質によ り負に制御されている細胞増殖の抑制機構を阻害する事ができるため、 臓器再生 等での利用なども考えられる。 またこれらの化合物は、 腫瘍細胞モデルの作製や 腫瘍の病態 §析に有用である。  A compound that promotes the interaction between the HK33 protein and the ARF protein may be used, for example, as an apoptosis inhibitor. It is also useful as a cell growth promoter. Compounds that promote the interaction between the HK33 protein and the ARF protein can inhibit the cell growth suppression mechanism that is negatively controlled by the ARF protein, and thus may be used in organ regeneration and the like. In addition, these compounds are useful for preparing a tumor cell model and for analyzing the pathology of a tumor.

ΗΚ33遺伝子の発現を調節する化合物、または ARF蛋白質と ΗΚ33蛋白質との相互作 用を調節する化合物等の上記化合物をヒトゃ哺乳動物、 例えばマウス、 ラット、 モルモット、 ゥサギ、 ニヮトリ、 ネコ、 ィヌ、 ヒッジ、 ブタ、 ゥシ、 サル、 マン トヒヒ、 チンパンジーの医薬として使用する場合には、 化合物自体を直接患者に 投与する以外に、 公知の製剤学的方法により製剤化した医薬組成物として投与を 行うことも可能である。 例えば、 必要に応じて糖衣を施した錠剤、 カプセル剤、 エリキシル剤、 マイクロカプセル剤として経口的に、 あるいは水もしくはそれ以 外の薬学的に許容し得る液との無菌性溶液、 又は懸濁液剤の注射剤の形で非経口 的に使用できる。 例えば、 薬理学上許容される担体もしくは媒体、 具体的には、 滅菌水や生理食塩水、 植物油、 乳化剤、 懸濁剤、 界面活性剤、 安定剤、 香味剤、 賦形剤、 べヒクル、 防腐剤、 結合剤などと適宜組み合わせて、 一般に認められた 製薬実施に要求される単位用量形態で混和することによつて製剤化することが考 えられる。 これら製剤における有効成分量は指示された範囲の適当な容量が得ら れるようにするものである。 The above compounds, such as compounds that regulate the expression of the ΗΚ33 gene, or compounds that regulate the interaction between the ARF protein and the ΗΚ33 protein, can be used in humans, such as mammals, such as mice, rats, guinea pigs, egrets, chicks, cats, dogs, When used as a medicament for sheep, pigs, magpies, monkeys, baboons, and chimpanzees, the compound itself is administered directly to the patient, as well as a pharmaceutical composition formulated by a known pharmaceutical method. It is also possible. For example, tablets, capsules, elixirs, and microcapsules, which are sugar-coated as necessary, orally, or aseptic solution or suspension in water or other pharmaceutically acceptable liquids It can be used parenterally in the form of injections. For example, pharmacologically acceptable carriers or vehicles, specifically, sterile water or saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives Agent, binder, etc., as appropriate, generally accepted It may be formulated by mixing in the unit dosage form required for pharmaceutical practice. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.

錠剤、 カプセノレ剤に混和することができる添加剤としては、 例えばゼラチン、 コーンスターチ、 トラガントガム、 アラビアゴムのような結合剤、 結晶性セル口 ースのような賦形剤、 コーンスターチ、 ゼラチン、 アルギン酸のような膨化剤、 ステアリン酸マグネシゥムのような潤滑剤、 ショ糖、 乳糖又はサッカリンのよう な甘味剤、 ペパーミント、 ァカモノ油又はチェリーのような香味剤が用いられる 。 調剤単位形態がカプセルである場合には、 上記の材料にさらに油脂のような液 状担体を含有することができる。 注射のための無菌組成物は注射用蒸留水のよう なべヒクルを用いて通常の製剤実施に従って処方することができる。  Examples of excipients that can be mixed with tablets and capsules include binders such as gelatin, corn starch, tragacanth gum, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid. A suitable bulking agent, a lubricant such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharin, and a flavoring agent such as peppermint, cocoa oil or cherry are used. When the unit dosage form is a capsule, the above-mentioned materials may further contain a liquid carrier such as an oil or fat. A sterile composition for injection can be formulated using a potable vehicle such as distilled water for injection according to normal pharmaceutical practice.

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 ゥムが挙げられ、 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 ィォン性界面活性剤、 例えばポリソルベート 80 (TM)、 HCO-50と併用してもよい。 油性液としてはゴマ油、 大豆油があげられ、 溶解補助剤として安息香酸ベンジ ル、 ベンジルアルコールと併用してもよい。 また、 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、 塩酸プロ力イン、 安定剤、 例え ばべンジルアルコール、 フエノール、 酸ィ匕防止剤と配合してもよい。 調製された 注射液は通常、 適当なアンプルに充填させる。  Aqueous injection solutions include, for example, saline, isotonic solutions containing dextrose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride. It may be used in combination with an agent, for example, an alcohol, specifically, ethanol, a polyalcohol, for example, propylene glycol, polyethylene glycol, a nonionic surfactant, for example, polysorbate 80 (TM), or HCO-50. The oily liquid includes sesame oil and soybean oil, and may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizer. Also, a buffering agent such as a phosphate buffer, a sodium acetate buffer, a soothing agent such as proforce hydrochloride, a stabilizer, for example, benzyl alcohol, phenol, or an antioxidant may be used. Good. The prepared injection solution is usually filled into an appropriate ampoule.

患者への投与は、 例えば、 動脈内注射、 静脈内注射、 皮下注射などのほか、 鼻 腔内的、 経気管支的、 筋内的、 経皮的、 または経口的に当業者に公知の方法によ り行いうる。 投与量は、 患者の体重や年齢、 投与方法などにより変動するが、 当 業者であれば適当な投与量を適宜選択することが可能である。また、該化合物が D NAによりコードされうるものであれば、 該 DNAを遺伝子治療用ベクターに組込み、 遺伝子治療を行うことも考えられる。 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。 化合物の投与量は、 投与対象、 対象臓器、 症状、 投与方法により差異はあるが 、 経口投与の場合、 一般的に成人 （体重 60kgとして） においては、 1日あたり約 0 . 1から 100mg、 好ましくは約 1. 0から 50mg、 より好ましくは約 1. 0から 20mgである。 非経口的に投与する場合は、 その 1回投与量は投与対象、 対象臓器、 症状、 投 与方法によっても異なるが、 例えば注射剤の形では通常成人 （体重 60kgとして） においては、 1日あたり約 0. 01から 30mg、 好ましくは約 0. 1から 20mg、 より好まし くは約 0. 1から 10mg程度を静脈注射により投与するのが好都合である。他の動物の 場合も、 体重 60kg当たりに換算した量、 あるいは体表面積あたりに換算した量を 投与することができる。 Administration to patients can be performed, for example, by intraarterial injection, intravenous injection, subcutaneous injection, etc., or intranasally, transbronchially, intramuscularly, transdermally, or orally by a method known to those skilled in the art. It can do better. The dose varies depending on the weight and age of the patient, the administration method, and the like, but those skilled in the art can appropriately select an appropriate dose. If the compound can be encoded by DNA, the DNA is incorporated into a vector for gene therapy, It is also conceivable to perform gene therapy. The dose and administration method vary depending on the patient's weight, age, symptoms, etc., and can be appropriately selected by those skilled in the art. The dose of the compound varies depending on the administration subject, target organ, symptoms, and administration method. However, in the case of oral administration, in general, for an adult (assuming a body weight of 60 kg), about 0.1 to 100 mg / day is preferable. Is about 1.0 to 50 mg, more preferably about 1.0 to 20 mg. For parenteral administration, the single dose varies depending on the administration target, target organ, symptoms, and administration method. For example, in the case of parenteral injections, usually for adults (with a body weight of 60 kg) per day, It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection. In the case of other animals, the dose can be administered in terms of the amount converted per 60 kg body weight or the amount converted per body surface area.

本発明は、 HK33蛋白質に結合する抗体、 あるいは HK33蛋白質をコードする DNA またはその相補鎖に相補的な少なくとも 15ヌクレオチドを含むポリヌクレオチド を含む検査試薬を提供する。 HK33遺伝子の発現の亢進は、 ARF蛋白質の細胞内にお ける活性を抑制し、 p53蛋白質の機能を低下させ細胞増殖を亢進させる。 逆に HK3 3遺伝子の発現の低下は、 ARF蛋白質の活性を上昇させ、 p53の機能を上昇させ細胞 増殖を抑制しアポトーシスを誘導し得る。 従って、 細胞における HK33遺伝子の変 異ゃ発現異常を検査することにより、 細胞増殖おょぴアポトーシス等の検查を行 うことができる。 具体的には、本発明の検査試薬は、 ARF蛋白質を介するシグナル 伝達の異常の検查、 p53蛋白質を介するシグナル伝達の異常の検査、細胞増殖異常 の検查、 アポトーシスの検査等に有用である。 また疾患の検査にも有用である。 すなわち、 HK33遺伝子の発現または ARF蛋白質に対する相互作用の低下は、細胞増 殖の低下またはアポトーシスを伴う疾患を引き起こしている可能性を示唆し、 HK 33遺伝子の発現または該相互作用の亢進は細胞増殖の亢進を伴う疾患を引き起こ している可能性を示唆する。 実際、 腫瘍ィ匕した細胞において HK33遺伝子の発現は 有意に亢進していた。 これは、 HK33遺伝子または HK33蛋白質が腫瘍の検査に利用 できることを示している。 例えば、 被検試料において、 HK33蛋白質または該蛋白 質をコードする mRNAを検出することにより、 腫瘍細胞の存在、 腫瘍の進行度、 腫 瘍の悪性度、 腫瘍の種類等を検査することが可能である。 The present invention provides a test reagent comprising an antibody that binds to the HK33 protein, or a polynucleotide containing at least 15 nucleotides complementary to DNA encoding the HK33 protein or its complementary strand. Increased expression of the HK33 gene suppresses intracellular activity of the ARF protein, reduces p53 protein function, and enhances cell proliferation. Conversely, decreased expression of the HK33 gene may increase ARF protein activity, increase p53 function, suppress cell proliferation and induce apoptosis. Therefore, cell proliferation and apoptosis can be detected by examining abnormal expression of the HK33 gene in cells. Specifically, the test reagent of the present invention is useful for detecting abnormal signal transduction via the ARF protein, detecting abnormal signal transduction via the p53 protein, detecting abnormal cell proliferation, detecting apoptosis, etc. . It is also useful for examining diseases. In other words, decreased expression of the HK33 gene or interaction with the ARF protein suggests that a decrease in cell proliferation or a disease associated with apoptosis may be caused. It suggests that it may be causing a disease accompanied by an increase in blood pressure. In fact, the expression of the HK33 gene was significantly enhanced in the tumor-ridden cells. This means that the HK33 gene or HK33 protein is used for tumor testing Indicates that you can do it. For example, by detecting HK33 protein or mRNA encoding the protein in a test sample, it is possible to examine the presence of tumor cells, tumor progression, tumor malignancy, tumor type, etc. is there.

本発明において 「腫瘍の検査」 とは、 HK33遺伝子の変異 （構造的または発現量 の変異） に起因して腫瘍を形成している患者の検查のみならず、 被験者が HK33遺 伝子の発現量の異常または遺伝子の変異に起因して癌にかかりやすいか否かを判 断するために行う、 HK33遺伝子の発現量の検査、 および遺伝子の変異の検査も含 まれる。 すなわち、 HK33遺伝子の発現の亢進や、 HK33対立遺伝子の片方に活性型 の変異が生じることなどにより、 表面上は未だ症状を発現していない場合におい ても、 癌にかかる危険性が増大しているものと考えられる。  In the present invention, the term “tumor test” refers to not only the detection of a patient who has formed a tumor due to a mutation (mutation in the structure or expression level) of the HK33 gene, but also that the subject has expressed the HK33 gene. It also includes testing for the expression level of the HK33 gene and testing for mutations in the gene to determine whether it is susceptible to cancer due to abnormal amounts or mutations in the gene. In other words, increased expression of the HK33 gene, or the occurrence of an active mutation in one of the HK33 alleles, increases the risk of developing cancer even when symptoms have not yet appeared on the surface. It is thought that there is.

本発明の検査は、 例えば HK33蛋白質に結合する抗体、 あるいは HK33蛋白質をコ ードする DNAまたはその相補鎖に相捕的な少なくとも 15ヌクレオチドを含むポリ ヌクレオチドを利用して行うことができる。 該ポリヌクレオチドは、 例えば HK33 蛋白質をコードする DNAの検出、 増幅、 該 DNAの発現の検出に有用である。 ここで 、 DNAの検出には、 DNAの変異の検出も含まれる。 ここで 「相捕鎖」 とは、 上記と 同様、 A:T (ただし RNAの場合は U)、 G:Cの塩基対からなる 2本鎖核酸の一方の鎖に 対する他方の鎖を指す。 また、 「相補的」 とは、相補的ヌクレオチド領域が完全に 相捕配列である場合に限られず、 少なくとも 70%、 好ましくは少なくとも 80%、 よ り好ましくは 90%、さらに好ましくは 95%以上の塩基配列上の相同性を有すればよ い。 相同性を決定するためのアルゴリズムは文献 （Wilbur, W. J. and Lipman, D. J. Pro Natl. Acad. Sci. USA (1983) 80， 726- 730)に記載のアルゴリズム にしたがえばよレ、。 このポリヌクレオチドをプローブまたはプライマー等として 用い、 HK33遺伝子の発現または変異を検出することにより、 細胞増殖の異常また は腫瘍等の細胞増殖性疾患の検査を行うことができる。 すなわち本発明は、 HK33 蛋白質をコードする DNAまたはその相捕鎖に相捕的な少なくとも 15ヌクレオチド を含むポリヌクレオチドを用いて、 HK33遺伝子の発現量または構造の異常を検出 する工程を含む、 （a ) ARFを介するシグナル伝達の異常、 （b ) p53を介するシグ ナル伝達の異常、 （c ) 細胞増殖異常、 （d ) 細胞増殖性疾患、 または （e ) 腫瘍 の検査方法を提供する。 遺伝子の発現は mRNAを検出しても蛋白質を検出してもよ い。 また、遺伝子の構造の変異は、染色体 DNAおよび mRNAのどちらを用いて調べて あよい。 The test of the present invention can be performed using, for example, an antibody that binds to the HK33 protein, or a polynucleotide containing at least 15 nucleotides complementary to the DNA encoding the HK33 protein or its complementary strand. The polynucleotide is useful for, for example, detection and amplification of DNA encoding the HK33 protein, and detection of expression of the DNA. Here, the detection of DNA includes the detection of DNA mutation. As used herein, the term “phase capture strand” refers to one strand of a double-stranded nucleic acid composed of A: T (U for RNA) and G: C base pairs, as described above. Further, the term "complementary" is not limited to the case where the complementary nucleotide region is a completely complementary sequence, and is at least 70%, preferably at least 80%, more preferably 90%, and still more preferably 95% or more. It is only necessary to have homology on the base sequence. The algorithm for determining homology is according to the algorithm described in the literature (Wilbur, WJ and Lipman, DJ Pro Natl. Acad. Sci. USA (1983) 80, 726-730). By using this polynucleotide as a probe or primer and detecting the expression or mutation of the HK33 gene, a cell proliferation abnormality or a cell proliferative disease such as a tumor can be examined. That is, the present invention detects abnormalities in the expression level or structure of the HK33 gene using DNA encoding the HK33 protein or a polynucleotide containing at least 15 nucleotides that are complementary to its complementary chain. (A) abnormal signal transduction via ARF, (b) abnormal signal transduction via p53, (c) abnormal cell proliferation, (d) cell proliferative disease, or (e) examination of tumor Provide a way. Gene expression may be detected by detecting mRNA or protein. In addition, mutation of the gene structure may be examined using either chromosomal DNA or mRNA.

HK33蛋白質に結合する抗体による検査は、 この抗体を HK33蛋白質が含まれると 予想される試料と接触せしめ、 該抗体と該蛋白質との免疫複合体を検出又は測定 することからなる、 HK33蛋白質の検出又は測定方法により実施することができる 。 HK33蛋白質の発現の亢進は、 細胞増殖の亢進、 または細胞の癌化を示唆する。 従って、 例えば HK33遺伝子の発現量を検查することにより、 細胞増殖の異常また は腫瘍等の細胞増殖性疾患の検査を行うことができる。 すなわち本発明は、 HK33 蛋白質に結合する抗体を用いて、 HK33蛋白質の発現量または構造の異常を検出す る工程を含む、 （a ) ARFを介するシグナル伝達の異常、 （b ) p53を介するシグナ ル伝達の異常、 （c ) 細胞増殖異常、 （d ) 細胞増殖性疾患、 または （e ) 腫瘍の 検査方法を提供する。 . The test using an antibody that binds to the HK33 protein involves contacting the antibody with a sample expected to contain the HK33 protein, and detecting or measuring an immune complex between the antibody and the protein. Alternatively, it can be performed by a measuring method. Increased expression of the HK33 protein indicates enhanced cell proliferation or cancerous cells. Therefore, for example, by examining the expression level of the HK33 gene, it is possible to examine a cell proliferation abnormality or a cell proliferation disease such as a tumor. That is, the present invention comprises a step of detecting an abnormality in the expression level or structure of the HK33 protein using an antibody that binds to the HK33 protein, (a) an abnormality in signal transduction via ARF, and (b) a signal transmission through p53. ( C ) cell proliferation abnormality, (d) cell proliferative disease, or (e) tumor. .

これらの抗体やポリヌクレオチドを検査試薬として用いる場合は、 適宜安定剤 、 保存剤、 塩、 緩衝剤などの溶質と、 それを溶かすものとしての水や生理食塩水 などを組み合わせることができる。ポリヌクレオチドは、 DNAチップやマイクロア レイの作製に利用することもできる。 これらの DNAチップやマイクロアレイは、本 発明の検査に用いられ得る。  When these antibodies and polynucleotides are used as test reagents, solutes such as stabilizers, preservatives, salts, and buffering agents can be appropriately combined with water, physiological saline, and the like to dissolve them. Polynucleotides can also be used to make DNA chips and microarrays. These DNA chips and microarrays can be used for the test of the present invention.

本発明の検查方法の一つは、 被検試料中における HK33蛋白質をコードする DNA の発現量を検出する工程を含む方法である。 このような検査方法は、 （a )患者由 来の RNA試料に HK33蛋白質をコードする DNAまたはその相補鎖に相捕的な少なくと も 15ヌクレオチドを含むポリヌクレオチドを接触させる工程、 （b ) 該 RNA試料へ の該ポリヌクレオチドの結合を検出する工程、 を含む方法が含まれる。 このよう な検査は、 例えばノーザンハイブリダイゼーションゃ RT-PCRなどにより行うこと ができる。 RT - PCRを利用した検査は、 具体的には （a ) 患者由来の RNA試料から c DNAを合成する工程、 （b ) 合成した cDNAを铸型に、 上記ポリヌクレオチドをプラ イマ一として用いて PCRを行う工程、 （c ) PCRにより増幅された DNAを検出するェ 程、 を含む。 ノーザンハイプリダイゼーシヨンや RT- PCRは、 公知の遺伝子工学技 術により行うことができる。 また、 DNAチップまたは DNAマイクロアレイによる検 出も可能である。 One of the detection methods of the present invention is a method including a step of detecting the expression level of DNA encoding HK33 protein in a test sample. Such a test method comprises the steps of (a) contacting a DNA encoding the HK33 protein or a complementary strand thereof with a polynucleotide containing at least 15 nucleotides in a complementary manner, to an RNA sample derived from a patient; Detecting the binding of the polynucleotide to the RNA sample. Such tests should be performed by, for example, Northern hybridization ハ イ RT-PCR. Can be. Inspection using RT-PCR includes the following steps: (a) the step of synthesizing cDNA from a patient-derived RNA sample; (b) the synthesized cDNA as a type I, and the above polynucleotide as a primer. A step of performing PCR, and (c) a step of detecting DNA amplified by PCR. Northern hybridization and RT-PCR can be performed by known genetic engineering techniques. Detection by DNA chip or DNA microarray is also possible.

また、 本発明の検査方法には、 患者由来の被検試料中における HK33蛋白質量を 検出する工程を含む方法が含まれる。 このような検查は、 例えば HK33蛋白質に対 する抗体を用いて行うことができる。 HK33に対する抗体を用いた検查は、 具体的 には、 （a )患者由来の蛋白質試料に HK33蛋白質に結合する抗体を接触させる工程 、 ( b )該蛋白質試料への該抗体の結合を検出する工程、 を含む。 蛋白質の検出は 、 HK33蛋白質に対する抗体を用いた免疫沈降、 ウェスタンプロット、 免疫組織化 学、 ELISAなどにより行うことができる。  Further, the test method of the present invention includes a method including a step of detecting HK33 protein content in a test sample derived from a patient. Such a detection can be performed using, for example, an antibody against the HK33 protein. The detection using an antibody against HK33 includes, specifically, (a) a step of contacting an antibody that binds to the HK33 protein with a patient-derived protein sample, and (b) detecting the binding of the antibody to the protein sample. Process. The protein can be detected by immunoprecipitation using an antibody against the HK33 protein, Western blot, immunohistochemistry, ELISA, or the like.

これらの検查は、 具体的には、 例えばバイオプシーにより採取した組織に対し て、 免疫組織染色あるいは in situハイプリダイゼーシヨン等の方法で発現を調 ベる事により HK33遺伝子の発現異常を起した病巣を特定することが考えられる。 H K33遺伝子の発現の亢進は、 例えば癌の発症および/または進展の可能性を示唆す る。 また、 癌などの疾患は、 種々の原因で起こっていると考えられる。 例えば癌 において HK33遺伝子の発現の亢進が認められた場合、 p53経路が抑制されているこ とが予測される事から、 この経路を標的とした治療法を施行する、 と云ったテー ラーメード療法を行う際の診断に利用することが考えられる。  Specifically, in these tests, abnormal expression of the HK33 gene was caused, for example, by examining the expression of a tissue collected by biopsy, for example, by immunohistochemical staining or in situ hybridization. It is conceivable to identify the lesion. Increased expression of the HK33 gene indicates, for example, the potential for onset and / or progression of cancer. Diseases such as cancer are thought to be caused by various causes. For example, if the expression of the HK33 gene is increased in cancer, it is predicted that the p53 pathway will be suppressed. It is conceivable to use it for diagnosis when performing.

本発明の検査は、また、 HK33蛋白質における変異または該蛋白質をコードする D NAにおける変異を検出することによって行うことも考えられる。 HK33遺伝子は癌 の発症および/または進展に関与していると考えられることから該蛋白質および 該 DNAの変異は、 癌の発症や進展の危険を示唆する。  The test of the present invention can also be performed by detecting a mutation in the HK33 protein or a mutation in DNA encoding the protein. Since the HK33 gene is considered to be involved in the onset and / or progression of cancer, mutations in the protein and the DNA suggest a risk of onset and progression of cancer.

HK33蛋白質の変異には、 構造的変異および機能的変異が含まれる。 例えば、 HK 33蛋白質に対する抗体を用いて、 患者由来の蛋白質試料のウェスタンブロット等 により蛋白質の分子量を健常者由来の蛋白質と比較することにより、 蛋白質の構 造的変異を検査することができる。 また、 蛋白質の修飾の変化、 HK33蛋白質と結 合する蛋白質または抗体の結合性の変化などを指標に、 HK33蛋白質の変異を検出 することもできる。 これらの検査には、例えば HK33蛋白質に対する抗体を用いた ELISA、 免疫沈降法、 pull-down法などを利用することができる。 Mutations in the HK33 protein include structural and functional mutations. For example, HK By using an antibody against the 33 protein and comparing the molecular weight of the protein with a protein derived from a healthy subject by Western blot or the like of a protein sample derived from a patient, structural mutations of the protein can be examined. In addition, mutations in the HK33 protein can also be detected using changes in protein modification, changes in the binding of a protein or antibody that binds to the HK33 protein, and the like as indices. For these tests, for example, ELISA using an antibody against the HK33 protein, immunoprecipitation, pull-down, and the like can be used.

本発明の検査は、 HK33蛋白質と ARF蛋白質との結合を検出することにより行うこ ともできる。 HK33蛋白質と ARF蛋白質との結合の亢進は、癌の発症や進展に寄与し ていると考えられる。蛋白質の結合は、例えば HK33蛋白質または ARF蛋白質に対す る抗体を用いた ELISA、免疫沈降法、 pull-down法などにより評価することができ る。  The test of the present invention can also be performed by detecting the binding between the HK33 protein and the ARF protein. Enhanced binding of HK33 protein and ARF protein is thought to contribute to the development and progression of cancer. Protein binding can be evaluated, for example, by ELISA using antibodies to HK33 protein or ARF protein, immunoprecipitation, pull-down, and the like.

HK33蛋白質をコードする DNAにおける変異の検出には、 HK33蛋白質をコードする DNAまたはその相補鎖に相補的な少なくとも 15ヌクレオチドを含むポリヌクレオ チドが利用され得る。 該ヌクレオチドは、 HK33蛋白質をコードする cDNAの塩基配 列、 およびゲノム DNA配列 （ェキソン、 イントロン、 および内因性転写制御配列を 含む） の塩基配列、 またはその相補鎖と相補的なポリヌクレオチド （プローブお ょぴプライマー） である。 なお、 変異の検査には、 HK33対立遺伝子の片方のァレ ルに変異を持つ患者 （キャリアー） を特定するための検查も含まれる。 また、 一 塩基多型（SNP) の型を決定するための検査も含まれる。 HK33遺伝子の多型は、癌 の罹りやすさに関連する可能性がある。  For detecting a mutation in the DNA encoding the HK33 protein, a polynucleotide containing at least 15 nucleotides complementary to the DNA encoding the HK33 protein or its complementary strand can be used. The nucleotides are the nucleotide sequence of the cDNA encoding the HK33 protein, the nucleotide sequence of the genomic DNA sequence (including exons, introns, and endogenous transcription control sequences), or a polynucleotide (probe or probe) complementary to its complementary strand. Hoop primer). The mutation test includes a test for identifying a patient (carrier) having a mutation in one of the HK33 alleles. It also includes testing to determine the type of single nucleotide polymorphism (SNP). Polymorphisms in the HK33 gene may be linked to cancer susceptibility.

プライマーとして用いられる場合、 ポリヌクレオチドは、 通常、 15bp〜100bp であり、 好ましくは 17bp〜30bpである。 プライマーは、 HK33遺伝子またはその発 現を調節する領域の少なくとも一部を増幅しうるものであればいかなるものでも よい。 このような領域としては、 例えば、 HK33遺伝子のェキソン領域、 イントロ ン領域、 プロモーター領域、 ェンハンサー領域が含まれる。  When used as a primer, the polynucleotide is usually 15 bp to 100 bp, preferably 17 bp to 30 bp. The primer may be any primer as long as it can amplify at least a part of the HK33 gene or a region that regulates its expression. Such regions include, for example, the exon region, intron region, promoter region, and enhancer region of the HK33 gene.

一方、 プローブとしてのポリヌクレオチドは、 合成ポリヌクレオチドであれば 、通常、少なくとも 15bp以上の鎖長を有する。 プラスミド DNAなどのベクターに組 み込んだクローンから得た二本鎖 DNAをプローブとして用いることも可能である。 プローブとしては、 HK33遺伝子またはその発現を調節する領域の少なくとも一部 の塩基配列またはそれらの相補鎖に相補的であればいかなるものでもよい。 プロ ーブがハイプリダイズする領域としては、 例えば、 HK33遺伝子のェキソン領域、 イントロン領域、 プロモーター領域、 ェンハンサー領域が含まれる。 プローブと して用いる場合、ポリヌクレオチドあるいは二本鎖 DNAは適宜標識して用いられる 。 標識する方法としては、 例えば、 T4ポリヌクレオチドキナーゼを用いてポリヌ クレオチドの 5'端をラジォアイソトープの でリン酸化することにより標識す る方法や、クレノウ酵素などの DNAポリメラーゼを用い、ランダムへキサマーオリ ゴヌクレオチドなどをプライマーとして32 Pなどのラジオァイソトープゃ、 蛍光色 素あるいはピオチンなどによつて標識された基質塩基を取り込ませる方法 （ラン ダムプライム法など） が挙げられる。 On the other hand, if a polynucleotide as a probe is a synthetic polynucleotide, Usually has a chain length of at least 15 bp or more. Double-stranded DNA obtained from a clone integrated into a vector such as a plasmid DNA can also be used as a probe. The probe may be any probe as long as it is complementary to the base sequence of at least a part of the HK33 gene or a region that regulates its expression, or a complementary strand thereof. Examples of the region to which the probe hybridizes include an exon region, an intron region, a promoter region, and an enhancer region of the HK33 gene. When used as a probe, the polynucleotide or double-stranded DNA is appropriately labeled and used. Labeling methods include, for example, labeling by phosphorylating the 5 'end of the polynucleotide with a radioisotope using T4 polynucleotide kinase, or random hexamer oligonucleotide using a DNA polymerase such as Klenow enzyme. A method of incorporating a substrate base labeled with a radioisotope III such as 32 P using a nucleotide as a primer, a fluorescent dye or a biotin (random prime method, etc.) may be mentioned.

HK33遺伝子の変異を検出する方法の一つの態様は、 患者の HK33遺伝子の塩基配 列を直接決定する方法である。例えば、上記ヌクレオチドをプライマーとして、 H K33遺伝子の変異に起因する疾患の疑いのある患者から単離した DNAを铸型として 、 PCR法などにより、患者の HK33遺伝子の一部もしくは全部 （例えばェキソン、ィ ントロン、 プロモーター、 ェンハンサーを含む領域） を増幅し、 その塩基配列の 決定を行う。 これを健常者の HK33遺伝子の配列と比較することにより、 HK33遺伝 子の変異に起因する疾患を検查することができる。  One embodiment of a method for detecting a mutation in the HK33 gene is a method for directly determining the nucleotide sequence of the HK33 gene in a patient. For example, using the above nucleotides as primers, DNA isolated from a patient suspected of having a disease caused by a mutation in the HK33 gene as type III, and a part or all of the HK33 gene (eg, exon, The region containing the intron, promoter and enhancer) is amplified and its nucleotide sequence is determined. By comparing this with the HK33 gene sequence of a healthy subject, a disease caused by a mutation in the HK33 gene can be detected.

本発明の検査方法としては、このように直接患者由来の DNAの塩基配列を決定す る方法以外に種々の方法が用いられる。 その一つの態様は、 （a)患者から DNA試料 を調製する工程、 （b) HK33蛋白質をコードする DNAまたはその相捕鎖に相捕的な少 なくとも 15ヌクレオチドを含むポリヌクレオチドをプライマーとして患者由来の DNAを増幅する工程、 （c)増幅した DNAを一本鎖 DNAに解離させる工程、（d)解離させ た一本鎖 DNAを非変性ゲル上で分離する工程、および (e)分離した一本鎖 DNAのゲル 上での移動度を健常者の対照と比較する工程、 を含む。 As the test method of the present invention, various methods are used other than the method of directly determining the nucleotide sequence of DNA derived from a patient. One embodiment is (a) a step of preparing a DNA sample from a patient, and (b) a patient using a DNA encoding HK33 protein or a polynucleotide containing at least 15 nucleotides that is complementary to its complementary strand as a primer. Amplifying the derived DNA, (c) dissociating the amplified DNA into single-stranded DNA, (d) separating the dissociated single-stranded DNA on a nondenaturing gel, and (e) separating Single-stranded DNA gel Comparing the mobility above with a healthy control.

このよつな方法として、 PCR-SSCP ^single-strand conformation polymorphism 、 一本 Ik it)次 1¾造多型)法 (Cloning and polymerase chain reaction - single- str and conformation polymorphism analysis of anonymous Alu repeats on chrom osome 11. Genomics. 1992 Jan 1 ; 12 (1) : 139-146.、 Detection of p53 gene m utations in human brain tumors by single-strand conformation polymorphis m analysis of polymerase chain reaction products. Oncogene. 1991 Aug 1； 6 (8) : 1313 - 1318.、 Multiple fluorescence-based PCR - SSCP analysis with pos tlabeling.、 PCR Methods Appl. 1995 Apr 1 ; 4 (5)： 275- 282. )が挙げられる。 こ の方法は操作が比較的簡便であり、 また試料の量も少なくてすむなどの利点を有 するため、特に多数の DNAサンプルをスクリーユングするのに好適である。その原 理は以下の如くである。二本鎖 DNA断片を一本鎖に解離すると、各鎖はその塩基配 列に依存した独自の高次構造を形成する。この解離した DNA鎖を変性剤を含まなレ、 ポリアクリルアミドゲル中で電気泳動すると、 それぞれの高次構造の差に応じて 、相補的な同じ鎖長の一本鎖 DNAが異なる位置に移動する。一塩基の置換によって もこの一本鎖 DNAの高次構造は変化し、ポリアクリルアミドゲル電気泳動において 異なる移動度を示す。従って、この移動度の変化を検出することにより DNA断片に 点突然変異や欠失、 あるいは揷入などによる変異が存在することを検出すること ができる。  One such method is the PCR-SSCP ^ single-strand conformation polymorphism, Ik it) next 1¾ ¾ ¾ polymorphism) method (Cloning and polymerase chain reaction-single- str and conformation polymorphism analysis of anonymous Alu repeats on chrom osome 11. Genomics. 1992 Jan 1; 12 (1): 139-146., Detection of p53 gene mutations in human brain tumors by single-strand conformation polymorphis m analysis of polymerase chain reaction products.Oncogene.1991 Aug 1; 6 ( 8): 1313-1318., Multiple fluorescence-based PCR-SSCP analysis with poster labeling., PCR Methods Appl. 1995 Apr 1; 4 (5): 275-282.). This method has advantages such as relatively simple operation and small sample volume, and is particularly suitable for screening a large number of DNA samples. The principle is as follows. When a double-stranded DNA fragment is dissociated into single strands, each strand forms a unique higher-order structure depending on its base sequence. When this dissociated DNA strand is electrophoresed in a polyacrylamide gel containing no denaturing agent, the single-stranded DNA of the same complementary length moves to a different position according to the difference in each higher-order structure. . The higher-order structure of this single-stranded DNA is also changed by single-base substitution, and shows different mobility in polyacrylamide gel electrophoresis. Therefore, by detecting this change in mobility, it is possible to detect the presence of a mutation due to a point mutation, deletion, insertion, or the like in the DNA fragment.

具体的には、 まず、 HK33遺伝子の一部、 あるいは全部を PCR法などによって増巾; する。 増幅される範囲としては、 通常 200〜400bp程度の長さが好ましい。 また、 増幅される領域としては、 HK33遺伝子の全てのェキソン、 全てのイントロンの他 、 HK33遺伝子のプロモーター、ェンハンサーなどが挙げられる。 PCRによる遺伝子 断片増幅の際、 32Pなどのラジオアイソトープ、 あるいは蛍光色素ゃビォチンなど によつて標識したプライマーを用いる力、 あるいは PCR反応液に32 Pなどのラジオ アイソトープ、 あるいは蛍光色素やピオチンなどによって標識した基質塩基を加 えて PCRを行うことによって合成される DNA断片を標識する。あるいは PCR後にクレ ノゥ酵素などを用いて32 Pなどのラジオアイソトープ、 あるいは蛍光色素やビォチ ンなどによつて標識した基質塩基を合成された DNA断片に付加することによって も標識を行うことができる。 こうして得られた標識された DNA断片を、熱を加える ことなどにより変性し、 尿素などの変性剤を含まないポリアクリルアミドゲルに よって電気泳動を行う。 この際、 ポリアクリルアミドゲルに適量（5から 10%程度 )のグリセ口ールを添加することにより、 DNA断片の分離の条件を改善することが できる。 また、 泳動条件は各 DNA断片の性質により変動するが、 通常、 室温 （20 から 25°C)で行い、好ましい分離が得られないときには 4から 30°Cまでの温度で最 適の移動度を与える温度の検討を行う。電気泳動後、 DNA断片の移動度を、 X線フ イルムを用いたオートラジオグラフィーや、 蛍光を検出するスキャナ一等で検出 し、 解析する。 移動度に差があるバンドが検出された場合、 このバンドを直接ゲ ルから切り出し、 PCRによって再度増幅し、それを直接シークェンシングすること により、変異の存在を確認することができる。 また、標識した DNAを使わない場合 においても、 電気泳動後のゲルをェチジゥムブ口マイドゃ銀染色法などによって 染色することにより、 バンドを検出することができる。 Specifically, first, a part or all of the HK33 gene is amplified by PCR or the like. As the range to be amplified, usually, a length of about 200 to 400 bp is preferable. Examples of the region to be amplified include all exons and all introns of the HK33 gene, as well as the promoter and enhancer of the HK33 gene. During PCR by gene fragments amplified, radioisotopes such as 32 P or fluorescent dyes Ya Biochin force used by connexion labeled primers etc., or radioisotopes such as 32 P in a PCR reaction solution, or by such as a fluorescent dye or Piochin, Add labeled substrate base Then, the DNA fragment synthesized by performing PCR is labeled. Alternatively, labeling can also be carried out by adding a radioisotope such as 32 P or a substrate base labeled with a fluorescent dye or biotin to the synthesized DNA fragment using a Klenow enzyme after PCR. The labeled DNA fragment thus obtained is denatured by applying heat, and electrophoresis is carried out on a polyacrylamide gel containing no denaturant such as urea. At this time, the conditions for separating DNA fragments can be improved by adding an appropriate amount (about 5 to 10%) of glycerol to the polyacrylamide gel. In addition, electrophoresis conditions vary depending on the properties of each DNA fragment.However, usually, the reaction should be performed at room temperature (20 to 25 ° C). Consider the temperature to be given. After electrophoresis, the mobility of the DNA fragment is detected and analyzed by autoradiography using an X-ray film or a scanner that detects fluorescence. If a band having a difference in mobility is detected, this band can be directly excised from the gel, re-amplified by PCR, and directly sequenced to confirm the presence of the mutation. Even when labeled DNA is not used, the band can be detected by staining the gel after electrophoresis with ethidium-membranide silver silver staining.

本発明の検査方法の他の態様は、 （a)患者から DNA試料を調製する工程、 （b)上 記ポリヌクレオチドをプライマーとして用いて患者由来の DNAを増幅する工程、 （ c)増幅した DNAを切断する工程、 （d) DNA断片をその大きさに応じて分離する工程 、 (e)分離した DNA断片に対し、検出可能な標識プローブでハイブリダィズさせる 工程、 および （f) 検出された DNA断片の大きさを、 健常者の対照と比較する工程 、 を含む。  Other aspects of the test method of the present invention include: (a) a step of preparing a DNA sample from a patient; (b) a step of amplifying a patient-derived DNA using the above-mentioned polynucleotide as a primer; (c) an amplified DNA (D) separating a DNA fragment according to its size, (e) hybridizing the separated DNA fragment with a detectable labeled probe, and (f) detecting the detected DNA fragment Comparing the size of と with a control of a healthy individual.

このような方法としては、 制限酵素断片長多型 （Restriction Fragment Lengt h PolymorphismZRFLP) を利用した方法、 PCR— RFLP法などが挙げられる。 DNAを 切断する酵素としては、 通常、 制限酵素を用いる。 具体的には、 制限酵素の認識 部位に変異が存在する場合、あるいは制限酵素処理によつて生じる DNA断片内に塩 基挿入、 または欠失がある場合、 制限酵素処理後に生じる断片の大きさが健常者 と比較して変化する。この変異を含む部分を PCR法によって増幅し、それぞれの制 限酵素で処理することによって、 これらの変異を電気泳動後のパンドの移動度の 差として検出することができる。あるいは、染色体 DNAをこれらの制限酵素によつ て処理し、 電気泳動した後、 プローブを用いてサザンブロッテイングを行うこと により、 変異の有無を検出することができる。 用いられる制限酵素は、 それぞれ の変異に応じて適宜選択することができる。 この方法では、ゲノム DNA以外にも患 者から調製した RNAを逆転写酵素で cDNAにし、これをそのまま制限酵素で切断した 後サザンブロッテイングを行うこともできる。 また、 この cDNAを錶型として PCR で HK33遺伝子の一部、 あるいは全部を増幅し、 それを制限酵素で切断した後、 移 動度の差を調べることもできる。 Examples of such a method include a method using restriction fragment length polymorphism (ZRFLP) and a PCR-RFLP method. Restriction enzymes are usually used as enzymes that cut DNA. Specifically, when there is a mutation at the recognition site of the restriction enzyme, or when a salt is included in the DNA fragment generated by the restriction enzyme treatment. When there is a base insertion or deletion, the size of the fragment generated after treatment with the restriction enzyme is changed as compared to a healthy person. By amplifying a portion containing this mutation by PCR and treating it with each restriction enzyme, these mutations can be detected as a difference in band mobility after electrophoresis. Alternatively, the presence or absence of a mutation can be detected by treating chromosomal DNA with these restriction enzymes, electrophoresing, and performing Southern blotting using a probe. The restriction enzyme to be used can be appropriately selected according to each mutation. In this method, in addition to genomic DNA, RNA prepared from a patient can be converted into cDNA using reverse transcriptase, which can be directly cut with restriction enzymes and then subjected to Southern blotting. Alternatively, using this cDNA as type II, a part or all of the HK33 gene can be amplified by PCR, cut with restriction enzymes, and then examined for differences in mobility.

また、 患者から調製した DNAの代わりに RNAを用いても同様に検出を行うことが 可能である。 このような方法は、 （a) 患者から RNA試料を調製する工程、 （b)大き さに応じて調製した RNAを分離する工程、 （c) 分離した RNAに対し、 検出可能な標 識をした上記ポリヌクレオチドをプローブとしてハイブリダィズさせる工程、 お よび （d) 検出された RNAの大きさを、 健常者の対照と比較する工程、 を含む。 具 体的な方法の一例としては、患者から調製した RNAを電気泳動し、プローブを用い てノーザンブロッテイングを行い、 移動度の差を検出する。  Similarly, detection can be performed by using RNA instead of DNA prepared from patients. Such methods include (a) the step of preparing an RNA sample from a patient, (b) the step of separating RNA prepared according to size, and (c) the detectable labeling of the isolated RNA. Hybridizing the polynucleotide as a probe, and (d) comparing the size of the detected RNA with a healthy control. As an example of a specific method, RNA prepared from a patient is electrophoresed, and Northern blotting is performed using a probe to detect a difference in mobility.

本発明の検査方法の他の態様は、 （a)患者から DNA試料を調製する工程、 （b)上 記ポリヌクレオチドをプライマーとして患者由来の DNAを増幅する工程、 （c)増幅 した DNAを、 DNA変性剤の濃度が次第に高まるゲル上で分離する工程、 および （d )分離した DNAのゲル上での移動度を健常者の対照と比較する工程、を含む方法で ある。  Another embodiment of the test method of the present invention includes: (a) a step of preparing a DNA sample from a patient, (b) a step of amplifying DNA derived from a patient using the polynucleotide as a primer, and (c) amplifying the amplified DNA. And (d) comparing the mobility of the separated DNA on the gel with a control of a healthy subject.

このような方法としては、 変性剤濃度勾配ゲル電気泳動法 （denaturant gradi ent gel electrophoresis： DGGE) が挙げられる。 HK33遺伝子の一部あるいは全 部を、上記ポリヌクレオチドをプライマーとして用いた PCR法などによって増幅し 、 これを尿素などの変性剤の濃度が移動するに従って徐々に高くなっているポリ アクリルアミドゲル中で電気泳動し、健常者と比較する。変異が存在する DNA断片 の場合、 より低い変性剤濃度位置で DNA断片が一本鎖になり、極端に移動速度が遅 くなるため、 この移動度の差を検出することにより変異の有無を検出することが できる。 Such a method includes denaturant gradient gel electrophoresis (DGGE). A part or all of the HK33 gene is amplified by PCR using the above polynucleotide as a primer. This is electrophoresed in a polyacrylamide gel, which gradually increases as the concentration of a denaturant such as urea moves, and is compared with a healthy person. In the case of a DNA fragment containing a mutation, the DNA fragment becomes single-stranded at a lower denaturant concentration position, and the movement speed is extremely slow.Therefore, the presence or absence of the mutation is detected by detecting the difference in the mobility. can do.

これら方法以外にも、 特定位置の変異のみを検出する目的にはアレル特異的ォ リゴヌクレオチド （Allele Specific 01igonucleotide/AS0) ハイプリダイゼー シヨン法が利用できる。 変異が存在すると考えられる塩基配列を含むオリゴヌク レオチドを作製し、 これと試料 DNAでハイブリダィゼーシヨンを行わせると、変異 が存在する場合、 ハイブリッド形成の効率が低下する。 それをサザンプロット法 や、 特殊な蛍光試薬がハイブリッドのギャップにインターカレーシヨンすること により消光する性質を利用した方法などにより検出できる。 また、 リボヌクレア ーゼ Aミスマッチ切断法による検出も可能である。具体的には、 HK33遺伝子の一部 あるいは全部を PCR法などによって増幅し、これをプラスミドベクタ一等に組み込 んだ HK33 cDNA等から調製した標識 RNAとハイブリダイゼーシヨンを行う。 変異が 存在する部分においてはハイプリッドが一本鎖構造となるので、 この部分をリボ ヌクレアーゼ Aによって切断し、これをオートラジオグラフィーなどで検出するこ とによって変異の存在を検出することができる。 図面の簡単な説明  In addition to these methods, the Allele Specific 01igonucleotide / AS0 hybridization method can be used for the purpose of detecting only a mutation at a specific position. When an oligonucleotide containing a nucleotide sequence that is considered to have a mutation is prepared and hybridized with this and the sample DNA, the efficiency of hybridization is reduced in the presence of the mutation. This can be detected by the Southern plot method or a method utilizing the property of quenching by intercalating a special fluorescent reagent into the gap of the hybrid. Detection by the ribonuclease A mismatch cleavage method is also possible. Specifically, part or all of the HK33 gene is amplified by PCR or the like, and this is hybridized with labeled RNA prepared from HK33 cDNA or the like in which this is integrated into a plasmid vector or the like. Since the hybrid has a single-stranded structure in the portion where the mutation exists, the presence of the mutation can be detected by cleaving this portion with ribonuclease A and detecting this by autoradiography or the like. BRIEF DESCRIPTION OF THE FIGURES

図 1は、 C0S7細胞を用いた哺乳動物 Two-Hybrid系により、 ARF蛋白質と HK33蛋白 質との相互作用を確認した結果を示す図である。 二つの蛋白質は有意に相互作用 していることが確認された。  FIG. 1 shows the results of confirming the interaction between the ARF protein and the HK33 protein using a mammalian Two-Hybrid system using C0S7 cells. It was confirmed that the two proteins interacted significantly.

図 2は、 pl9ARFmycおよび GFP-HK33発現ベクターを導入した C0S7細胞の細胞抽出 液を用いた免疫沈降により、pl9ARFmyc蛋白質と GFP-HK33蛋白質との相互作用を確 認した結果を示す写真である。 GFP - HK33蛋白質と pl9ARF蛋白質の相互作用が免疫 沈降法によっても確認された。 FIG. 2 is a photograph showing the results of confirming the interaction between pl9ARFmyc protein and GFP-HK33 protein by immunoprecipitation using a cell extract of C0S7 cells into which pl9ARFmyc and GFP-HK33 expression vectors have been introduced. Interaction between GFP-HK33 protein and pl9ARF protein is immune It was also confirmed by the sedimentation method.

図 3は、 P19ARF1- 80、及ぴ pl9ARF8卜 Stopをコードした pl9ARF欠失体と HK33蛋白 質との相互作用を酵母 Two-Hybrid法によって確認した結果を示す図である。 HK33 蛋白質は、 P19ARF81 - Stopと相互作用することが示された。  FIG. 3 is a diagram showing the results of confirming the interaction between the HK33 protein and a pl9ARF-deleted gene encoding P19ARF1-80 and pl9ARF8-Stop by the yeast Two-Hybrid method. The HK33 protein was shown to interact with P19ARF81-Stop.

図 4は、 pl9ARFmyc蛋白質と GFP-HK33蛋白質を NIH3T3細胞で発現させ、細胞内局 在を調べた結果を示す写真である。 GFP-HK33単独では細胞質に局在しており、 pi 9ARF蛋白質単独では核小体に蓄積していた。一方、 pl9ARF + GFP-HK33の共発現に おいては、 pl9ARFが核では検出されず、 細胞質に局在していることが示された。 この結果より、 GFP-HK33と pl9ARFの結合によって、 pl9ARFの核移行が阻害される ことが明らかになった。  FIG. 4 is a photograph showing the results of expressing pl9ARFmyc protein and GFP-HK33 protein in NIH3T3 cells and examining the intracellular localization. GFP-HK33 alone localized in the cytoplasm, and pi9ARF protein alone accumulated in nucleoli. On the other hand, in co-expression of pl9ARF + GFP-HK33, pl9ARF was not detected in the nucleus, indicating that it was localized in the cytoplasm. These results revealed that binding of pl9ARF to GFP-HK33 inhibits nuclear translocation of pl9ARF.

図 5は、 正常ヒト細胞に SV40遺伝子を導入し、 不死化させる過程 （BFT、 BET, R KF-T*Lifroumani Syndrome- p53欠損細胞、 それぞれの不死化前と不死化中） の細 胞における HK33遺伝子の発現を RT- PCRにより解析した結果を示す写真である。 上 段が HK33遺伝子、 下段がコントロールとして検出した GAPDH遺伝子の発現を表す。 SV40 Large T Antigenの遺伝子を導入し、 老化状態である細胞 Precrisis細胞と 、老化状態からセルクライシスを逃れた細胞 Postcrisis細胞との HK33遺伝子発現 量を比較した。繊維芽細胞、血管内皮細胞、 RKF- T*Lifroumani Syndrome- p53欠損 細胞において、 それぞれ有意に Postcrisis細胞において発現が亢進していること が判明した。  Figure 5 shows HK33 in cells undergoing the immortalization process (BFT, BET, RKF-T * Lifroumani Syndrome-p53-deficient cells, before and during immortalization, respectively) by introducing the SV40 gene into normal human cells. 4 is a photograph showing the result of analysis of gene expression by RT-PCR. The upper row shows the expression of the HK33 gene, and the lower row shows the expression of the GAPDH gene detected as a control. The expression level of HK33 gene was compared between the senescent Precrisis cell and the postcrisis cell which escaped cell crisis from the senescent state by introducing the gene of SV40 Large T Antigen. In fibroblasts, vascular endothelial cells, and RKF-T * Lifroumani Syndrome-p53-deficient cells, expression was significantly increased in Postcrisis cells, respectively.

図 6は、 HK33遺伝子の強制発現による p53依存性転写活性の抑制を示す写真およ ぴ図である。 HAタグが付加された pl9ARF (HA- pl9ARF) の発現を亜鉛イオンの添カロ により制御できるようにした NIH/ARF細胞に、 マウス HK33遺伝子のセンス （His- m ax/mHK33-S) またはアンチセンス （His-fflax/mHK33- AS) を発現させた。 p53蛋白質 の分解活性のポジティブコントロールとして、 MDM2発現ベクターを用いた。 また 、 p53依存性転写活~生の指標として pG13Lucベクターを用いて、 ルシフェラーゼに よつて p53依存性転写活性を測定した。上パネルは細胞に遺伝子を導入し、 24時間 後に、 l. OmMの Zn2+を加えた:！:咅地あるいは加えない培地で培地交換し、 24時間後の 細胞のマウス HK33 (mHK33) およひ ΉΑ- pl9ARF (HA-pl9) 蛋白質の発現を、 それぞ れ Xpress抗体および HAポリク口ーナル抗体によるウェスタンブロッテイングで検 出した結果を示す。 下パネルは、 p53レポーター活性の解析結果を示す。 発明を実施するための最良の形態 FIG. 6 is a photograph and a diagram showing suppression of p53-dependent transcriptional activity by forced expression of the HK33 gene. In the NIH / ARF cells where expression of HA-tagged pl9ARF (HA-pl9ARF) can be controlled by the addition of zinc ions, mouse HK33 gene sense (His-max / mHK33-S) or antisense (His-fflax / mHK33-AS) was expressed. An MDM2 expression vector was used as a positive control for p53 protein degradation activity. Further, the pG13Luc vector was used as an indicator of p53-dependent transcription activity and the p53-dependent transcription activity was measured by luciferase. The top panel shows the cells transfected for 24 hours Later, l. OmM Zn 2+ was added :! : After exchanging the medium with the medium with or without the medium, the expression of mouse HK33 (mHK33) and ΉΑ-pl9ARF (HA-pl9) protein in the cells after 24 hours was confirmed by Xpress antibody and HA polyclonal antibody, respectively. The result of detection by Western blotting is shown. The lower panel shows the results of analysis of p53 reporter activity. BEST MODE FOR CARRYING OUT THE INVENTION

以下、 本発明を実施例により具体的に説明するが、 本発明はこれら実施例に制 限されるものではない。 なお、 本明細書中に引用された文献は、 すべて本明細書 の一部として組み込まれる。  Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples. All documents cited in this specification are incorporated as a part of this specification.

[実施例 1 ] cDNAのクローニングとシークェンス  [Example 1] Cloning and sequence of cDNA

酵母 Two - Hybr i d法によりマウス ARF蛋白質と相互作用する蛋白質をコードする 遺伝子の単離を試みた。 具体的には、 マウス pl9ARFをコードする遺伝子 （Access ion No. L76092) を酵母 Two-Hybrid用ベクター p0DB8に組み込み、 酵母株 PJ69/2A に遺伝子導入した。 遺伝子導入した酵母は、 トリプトフアンを除いた基礎培地で 選択し、 クローニングした。 この pl9ARFを導入した酵母とプレトランスフォーム ドライブラリー酵母 （クロンテック社） とを接合させ、 トリブトファン、 口イシ ンを除いた基礎培地で生育する酵母を単離した。 単離した酵母より、 遺伝子断片 を摘出し、 DNA配列をジデォキシチェーンターミネーシヨン法により、 ABI377自動 塩基配列決定機を用い分析した結果、単離された遺伝子断片の配列は、 DNA配列デ ータバンク内に登録されているヒト Housekeeping gene 33 (HK33) として知られ ている遺伝子と 100%の同一性を有していた。  An attempt was made to isolate a gene encoding a protein that interacts with the mouse ARF protein by the yeast Two-Hybrid method. Specifically, the gene encoding mouse pl9ARF (Accession No. L76092) was incorporated into the yeast Two-Hybrid vector p0DB8, and the gene was introduced into the yeast strain PJ69 / 2A. The transfected yeast was selected and cloned on a basal medium without tryptophan. The yeast into which pl9ARF had been introduced was conjugated with a pre-transformed library yeast (Clontech), and yeasts that grew on a basal medium excluding tributofan and porcine were isolated. The gene fragment was excised from the isolated yeast, and the DNA sequence was analyzed by the dideoxy chain termination method using an ABI377 automatic nucleotide sequencer. As a result, the sequence of the isolated gene fragment was obtained from the DNA sequence data bank. It had 100% identity with a gene known as Human Housekeeping gene 33 (HK33), which was registered in the US.

[実施例 2 ] 遺伝子導入による C0S7細胞での相互作用の確認  [Example 2] Confirmation of interaction in C0S7 cells by gene transfer

P19ARFおよびマウス HK33遺伝子 （Accession No. Y09046) をコードした配列を 哺乳動物用 Two - Hybridベクターに組み換え、 C0S7細胞に GAL4-レポーター遺伝子と 共にそれぞれの Two-Hybridベタターと遺伝子導入することによって、 相互作用を 確認した。 相互作用の確認にはレポーター蛋白であるルシフエラーゼの活性によ つて測定した。 測定には、 Ludferaseレポーターアツセィシステム （プロメガ社 ) を用いた。 実験の結果、 これら二つの蛋白質は有意に相互作用していることが 確認された （図 1 )。 Interaction by recombination of the sequences encoding P19ARF and mouse HK33 gene (Accession No. Y09046) into a mammalian two-hybrid vector, and transfection of C0S7 cells together with the GAL4-reporter gene with each of the two-hybrid vectors. It was confirmed. The interaction was confirmed by the activity of the reporter protein, luciferase. Was measured. For the measurement, a Ludferase Reporter Atsushi System (Promega) was used. As a result of the experiment, it was confirmed that these two proteins interacted significantly (Fig. 1).

[実施例 3 ] 免疫沈降法による相互作用の確認  [Example 3] Confirmation of interaction by immunoprecipitation method

実際に二つの蛋白質が相互作用をしているかどうかを確認するために、 pl9ARF 町 c、 GFP - HK33発現ベクターを C0S7細胞に遺伝子導入、蛋白質発現させ、モノクロ ーナル myc抗体によって蛋白質複合体を沈降させた。 さらに、 この複合体を SDS- P AGEにて分離後、 モノクローナル GFP抗体で相互作用を検出した（図 2 )。 対照とし て、 GST Tesmin、 LKB Myc、 MPD Mycを組み合わせて用いた。 この実験の結果、 pi 9ARF蛋白質により GFPは免疫沈降しなかつたが、 GFP- HK33蛋白質においては免疫沈 降された。 この結果から、 GFP-HK33蛋白質と pl9ARF蛋白質の相互作用が免疫沈降 法によっても確認された。  To confirm whether the two proteins are actually interacting, pl9ARF town c, GFP-HK33 expression vector was transfected into C0S7 cells, the proteins were expressed, and the protein complex was precipitated with a monoclonal myc antibody. Was. After separating this complex by SDS-PAGE, the interaction was detected with a monoclonal GFP antibody (Fig. 2). As a control, a combination of GST Tesmin, LKB Myc and MPD Myc was used. As a result of this experiment, GFP was not immunoprecipitated by pi9ARF protein, but was immunoprecipitated by GFP-HK33 protein. From these results, the interaction between the GFP-HK33 protein and the pl9ARF protein was also confirmed by immunoprecipitation.

[実施例 4 ] pl9ARF結合部位の同定  [Example 4] Identification of pl9ARF binding site

P19ARF1- 80、 及ぴ pl9ARF81- Stopをコードしたミュータント pl9ARFを pODB8ベタ ターに組み換えクローニングし、 PACT- 2ベクターに組み込んだ HK33遺伝子を酵母 T wo - Hybrid法によって相互作用を確認した。その結果、 pACT- 2/HK33遺伝子産物と p 19ARF1- 80クローンではレポーターの j8 - GAL活性はネガティブであつたが、 pl9AR F81- Stop遺伝子産物においては β -GAL活性がポジティプであった（図 3 )。 この結 果より、 HK33蛋白質は、 pl9ARF中の pl6INK4aと共有した部位とは結合せずに、 pi 9ARF特異的な部位と相互作用することが示された。  Mutants pl9ARF encoding P19ARF1-80 and pl9ARF81-Stop were recombinantly cloned into pODB8 vector, and the interaction of the HK33 gene incorporated into the PACT-2 vector was confirmed by the yeast Two-Hybrid method. As a result, the reporter j8-GAL activity was negative in the pACT-2 / HK33 gene product and the p19ARF1-80 clone, but the β-GAL activity was positive in the pl9AR F81-Stop gene product (Fig. 3). ). This result indicates that the HK33 protein interacts with the pi9ARF-specific site without binding to the site shared with pl6INK4a in pl9ARF.

[実施例 5 ] 細胞内での局在  [Example 5] Localization in cells

哺乳動物細胞発現ベクター（pcDNA3， Invitrogen社）に pi 9ARFに mycタグを付加 した遺伝子を、 また、 GFP蛋白と融合させて発現させるベクター （pEGFPCl, Clon tech社） に HK33遺伝子をそれぞれ組み込み、 蛋白質発現ベクター GFP-HK33、 およ ぴ pl9ARFmycを構築した。 pl9ARFmyc、 GFP - HK33、 pl9ARFmyc + GFP- HK33を NIH3T3 細胞に遺伝子導入し、 蛍光レーザー顕微鏡 （ォリンパス社） を用いて細胞内での 局在を調べた。 pl9ARFmycの検出には myc抗体（Invirogen社） で反応させ、 その抗 体をテキサスレッド結合二次抗体で反応させ検出した。 一方、 HK33蛋白質は GFP 自身の発光により検出した。 実験の結果、 GFP- HK33は細胞質に局在していること が示され、 P19ARF蛋白質単独では核小体に蓄積する事が明らかとなった。一方、 p 19ARF + GFP-HK33の共遺伝子導入においては、 pl9ARFが核では検出されず、 細胞 質に局在していることが示された。 この結果より、 GFP - HK33と pl9ARFの結合によ つて、 pl9ARFの核移行を阻害することを明らかにした （図 4 )。 Integrate the pi9ARF-tagged myc-tagged gene into a mammalian cell expression vector (pcDNA3, Invitrogen) and the HK33 gene into a vector (pEGFPCl, Clontech) that is fused and expressed with the GFP protein to express the protein. The vectors GFP-HK33 and pl9ARFmyc were constructed. pl9ARFmyc, GFP-HK33, and pl9ARFmyc + GFP-HK33 were transfected into NIH3T3 cells and transfected using a fluorescence laser microscope (Olympus). The localization was checked. pl9ARFmyc was detected by reacting with a myc antibody (Invirogen), and the antibody was reacted with a Texas Red-conjugated secondary antibody and detected. On the other hand, HK33 protein was detected by luminescence of GFP itself. The results of the experiment showed that GFP-HK33 was localized in the cytoplasm, and that the P19ARF protein alone accumulated in the nucleolus. On the other hand, in co-transfection of p19ARF + GFP-HK33, pl9ARF was not detected in the nucleus, indicating that it was localized in the cytoplasm. These results revealed that binding of GFP-HK33 to pl9ARF inhibits the nuclear translocation of pl9ARF (Fig. 4).

[実施 ί列 6 ] Postcrisis、 Precrisis細胞における HK33遺伝子の発現  [Experiment column 6] Expression of HK33 gene in Postcrisis and Precrisis cells

正常ヒト細胞に SV40遺伝子を導入し、 不死化させる過程 （BFT、 BET, RKF - T*Li froumani Syndrome_p53欠損細胞、それぞれの不死化前と不死化中）の細胞からト 一タル RNAを抽出した。 RNAは 65DCで変性させた後、 リバーストランスクリプター ゼ：スーパースクリプト 2 (GIBC0/BRL社） を用い cDNAを作成した。 これらの cDNA をテンプレートに用い、 HK33の反応には、 配列番号： 1および 2のオリゴプライ マーを用い、 94°C 1分， 55°C 2分， 72°C 3分を 32サイクルの条件で、 GAPDHの反応 には配列番号： 3および 4のオリゴプライマーを用い、 94°C 1分， 58°C 2分， 72 °C 3分を 30サイクルの条件で解析した。 SV40 Large T Antigenの遺伝子を導入し 、 老化状態である細胞 Precrisis細胞と、 老化状態からセルクライシスを逃れた 細胞 Postcrisis細胞との HK33遺伝子発現量を比較した。 その結果、 繊維芽細胞、 血管内皮細胞、 RKF-T*Lifroumani Syndrome- p53欠損細胞において、それぞれ有意 に Postcrisis細胞において発現が亢進した。 この結果より、 細胞老化から逃れた 不死化細胞において HK33による pl9ARFの不活性化が起きている可能性が考えられ た（図 5 )。 この知見は pl9ARFの不活性化が細胞寿命延長を引き起こすという報告 と一致する (Carner , A. et al. (2000) Nat. Cell Biol. (3)： 148 - 55)。 Total RNA was extracted from cells undergoing the immortalization process by introducing the SV40 gene into normal human cells (BFT, BET, RKF-T * Li froumani Syndrome_p53 deficient cells, before and during immortalization, respectively). After RNA is denatured with 65 D C, reverse transcriptase: creating the cDNA using Superscript 2 (GIBC0 / BRL, Inc.). Using these cDNAs as templates, the HK33 reaction was performed using the oligo primers of SEQ ID NOs: 1 and 2 under the conditions of 32 cycles of 94 ° C for 1 minute, 55 ° C for 2 minutes, and 72 ° C for 3 minutes. In the reaction of GAPDH, oligo primers of SEQ ID NOs: 3 and 4 were used and analyzed at 94 ° C for 1 minute, 58 ° C for 2 minutes, and 72 ° C for 3 minutes under the conditions of 30 cycles. The HK33 gene expression level was compared between the aging cell Precrisis cells and the cells that escaped cell crisis from the aging state, and into which the SV40 Large T Antigen gene was introduced. As a result, in fibroblasts, vascular endothelial cells, and RKF-T * Lifroumani Syndrome-p53-deficient cells, expression was significantly increased in Postcrisis cells, respectively. These results suggest that HK33 may have inactivated pl9ARF in immortalized cells that escaped cell senescence (Fig. 5). This finding is consistent with reports that inactivation of pl9ARF causes prolonged cell life (Carner, A. et al. (2000) Nat. Cell Biol. (3): 148-55).

[実施例 7 ] HK33遺伝子の強制発現による p53依存性転写活性の抑制  [Example 7] Suppression of p53-dependent transcriptional activity by forced expression of HK33 gene

本実施例にはメタ口チォネインプロモーターに亜鉛ィオンが働くことによって 、 HAタグが付加された pl9ARF (HA-pl9ARF) の発現を制御できるようにした細胞 N IH/ARF細胞を用いた （Kurokawa, K. et al. , Oncogene 18 : 2718-27， 1999参照） 。 Xpressタグを付加した発現ベクター （pcDNA4， Invitrogen社） に HK33遺伝子の センスまたはアンチセンスを結合させ HK33発現ベクターとして用いた。 p53蛋白質 の分解活性のポジティブコント口ールとして、 MDM2発現べクタ一を用いた。 また 、 p53依存性転写活性の指標として pG13Lucベタタ一、 遺伝子導入効率のコント口 ールとして pRL - TKベクター （プロメガ社） を共に遺伝子導入し、 細胞溶解後ルシ フェラーゼによって p53依存性転写活性を測定した。 細胞に遺伝子を導入し、 24 時間後に、 L OmMの Zn2+を加えた培地あるいは、 培地のみのもので培地交換し、 24 時間後に細胞を溶解し、 蛋白質の発現解析とレポーター活性の測定を行った。 ゥ エスタンプロッティング法により、 センスにおいて HK33蛋白質が発現し、 アンチ センスによっては HK33蛋白質が発現していないことが確認された。 また、 Zn2+に よつて HAタグが付加された pl9ARFの発現が誘導されたことがウェスタンプロッテ イングによって示された （図 6上パネル）。 さらに、 p53のレポーター活性の解析 の結果、 MDM2による p53分解活性をわずかに HA_pl9ARFが抑制することが判明した. 。 また HK33遺伝子のセンスを導入したとき、 アンチセンスに比べて有意に pl9ARF による p53の発現上昇を抑制することが示された。 この実験結果より、 HK33蛋白質 の発現によつて有意に pl9ARFによる p53依存性転写活性を抑制できることを示し た。 産業上の利用の可能性 In the present example, cells capable of controlling the expression of HA-tagged pl9ARF (HA-pl9ARF) by the action of zinc ion on the meta-mouth thionein promoter N IH / ARF cells were used (see Kurokawa, K. et al., Oncogene 18: 2718-27, 1999). An HK33 gene sense or antisense was ligated to an expression vector (pcDNA4, Invitrogen) to which an Xpress tag was added, and used as an HK33 expression vector. MDM2 expression vector was used as a positive control for p53 protein degradation activity. In addition, pG13Luc beta one as an indicator of p53-dependent transcription activity and pRL-TK vector (Promega) as a control of gene transfer efficiency were introduced together, and after cell lysis, p53-dependent transcription activity was measured by luciferase. did. After introducing the gene into the cells, 24 hours later, replace the medium with a medium supplemented with L OmM Zn 2+ or a medium containing only the medium.After 24 hours, lyse the cells and analyze the expression of the protein and measure the reporter activity. went.ゥ It was confirmed by estamplotting that HK33 protein was expressed in sense and HK33 protein was not expressed in antisense. In addition, it was shown by western blotting that the expression of pl9ARF with HA tag added was induced by Zn 2+ (Fig. 6, upper panel). Furthermore, analysis of the reporter activity of p53 revealed that HA_pl9ARF slightly suppressed the p53-degrading activity of MDM2. In addition, it was shown that when sense of the HK33 gene was introduced, pl9ARF-induced increase in p53 expression was significantly suppressed as compared with antisense. These experimental results showed that the expression of HK33 protein can significantly suppress the p53-dependent transcriptional activity by pl9ARF. Industrial applicability

本発明者らは、 HK33蛋白質と ARF蛋白質とが相互作用すること、またその相互作 用によって ARF蛋白質の核移行が阻害され、 ARFの機能を阻害することを明らかに した。 さらに、 SV40 Large T Antigenの遺伝子導入により細胞老化を逃れるステ ージに達した不死ィ匕細胞 （Postcrisis細胞） において、 不死ィ匕前のステージの細 胞（Precrisis細胞） に比べて HK33遺伝子の発現上昇が起こることを示した。本発 明により、ARF蛋白質と HK33蛋白質の相互作用を利用した抗腫瘍作用を持つ薬剤の スクリーニングが可能となる。 また、 本発明により、 該相互作用または HK33遺伝 子の発現を調節することにより、 ARF蛋白質を介するシグナル伝達、特に細胞周期 や細胞増殖を調節するための方法、 およぴそのために用いられる分子が提供され た。 また本発明により、 HK33蛋白質または HK33蛋白質をコードする遺伝子を利用 して、腫瘍等の疾患の検査を行うことが可能となった。 HK33蛋白質と ARF蛋白質と の相互作用を抑制する薬剤のスクリーニングは、 ARF蛋白質の不活性化による細胞 増殖、 あるいは不死化した前癌状態の細胞の増殖を抑制できる新規の薬剤開発を 可能とすると考えられる。 The present inventors have clarified that the HK33 protein interacts with the ARF protein, and that the interaction inhibits the nuclear translocation of the ARF protein, thereby inhibiting the function of ARF. Furthermore, the expression of the HK33 gene was higher in the immortalized cells (Postcrisis cells) that reached the stage in which cell senescence was escaped by the gene transfer of SV40 Large T Antigen than in the cells before the immortalized cells (Precrisis cells). It has shown that a rise will occur. According to the present invention, a drug having an antitumor effect utilizing the interaction between the ARF protein and the HK33 protein has been developed. Screening becomes possible. Further, according to the present invention, a method for regulating ARF protein-mediated signal transduction, particularly cell cycle and cell proliferation, by regulating the interaction or the expression of the HK33 gene, and a molecule used for the same, are disclosed. sponsored. Further, according to the present invention, it has become possible to use the HK33 protein or a gene encoding the HK33 protein to examine a disease such as a tumor. Screening for a drug that suppresses the interaction between HK33 protein and ARF protein will enable the development of a new drug that can suppress cell growth due to inactivation of ARF protein or growth of immortalized precancerous cells Can be

Claims

請求の範囲 The scope of the claims I . HK33遺伝子の発現を上昇または下降させることにより、 ARFを介するシグナル 伝達を阻害または促進する方法。 -I. A method of inhibiting or promoting ARF-mediated signal transduction by increasing or decreasing the expression of the HK33 gene. - 2 . HK33遺伝子の発現を上昇または下降させることにより、 p53を介するシグナル 伝達を阻害または促進する方法。 2. A method of inhibiting or promoting p53-mediated signal transduction by increasing or decreasing the expression of the HK33 gene. 3 . HK33遺伝子の発現を上昇または下降させることにより、 細胞増殖を促進また は阻害する方法。  3. A method of promoting or inhibiting cell proliferation by increasing or decreasing the expression of the HK33 gene. 4 . ARF蛋白質と HK33蛋白質との相互作用を上昇または下降させることにより、 A RFを介するシグナル伝達を阻害または促進する方法。  4. A method for inhibiting or promoting ARF-mediated signal transduction by increasing or decreasing the interaction between ARF protein and HK33 protein. 5 . ARF蛋白質と HK33蛋白質との相互作用を上昇または下降させることにより、 p 53を介するシグナル伝達を阻害または促進する方法。  5. A method for inhibiting or promoting p53-mediated signal transduction by increasing or decreasing the interaction between ARF protein and HK33 protein. 6 . ARF蛋白質と HK33蛋白質との相互作用を上昇または下降させることにより、細 胞増殖を促進または阻害する方法。  6. A method of promoting or inhibiting cell proliferation by increasing or decreasing the interaction between ARF protein and HK33 protein. 7 . 細胞が腫瘍細胞である、 請求項 3または 6に記載の方法。  7. The method according to claim 3 or 6, wherein the cells are tumor cells. 8 . ARF蛋白質と HK33蛋白質との相互作用を調節する化合物のスクリーニング方法 であって、  8. A method for screening for a compound that regulates the interaction between ARF protein and HK33 protein, ( a ) 被検試料の存在下で ARF蛋白質と HK33蛋白質とを接触させる工程、  (a) contacting the ARF protein with the HK33 protein in the presence of the test sample, ( b ) ARF蛋白質と HK33蛋白質との相互作用を検出する工程、  (b) detecting an interaction between the ARF protein and the HK33 protein, ( c ) 被検試料非存在下で検出した場合と比較して、 該相互作用を調節する化合 物を選択する工程、 を含む方法。  (c) selecting a compound that modulates the interaction as compared to a case where the compound is detected in the absence of a test sample. 9 . ARF蛋白質と HK33蛋白質との相互作用を、 ARF蛋白質と HK33蛋白質との結合を 指標に検出する、 請求項 8に記載の方法。  9. The method according to claim 8, wherein the interaction between the ARF protein and the HK33 protein is detected using the binding between the ARF protein and the HK33 protein as an indicator. 1 0 . 細胞系を用いて行う、 請求項 8または 9に記載の方法。  10. The method of claim 8 or 9, which is performed using a cell line. I I . ARF蛋白質を介するシグナル伝達を調節する化合物のスクリーニング方法で あって、 (a) HK33遺伝子を内因的に保持する細胞に被検試料を接触させる工程、II. A method for screening a compound that modulates signal transduction via an ARF protein, (a) contacting a test sample with cells endogenously retaining the HK33 gene, (b) HK33遺伝子の発現を検出する工程、 (b) detecting the expression of the HK33 gene, (c) 被検試料を該細胞に接触させない場合と比較して、 該発現を調節する活性 を有する化合物を選択する工程、 を含む方法。  (c) selecting a compound having an activity of regulating the expression as compared to a case where the test sample is not contacted with the cells. 12. ARF蛋白質を介するシグナル伝達を調節する化合物のスクリーニング方法で あって、 1 2. A method for screening a compound that modulates signaling through ARF protein, (a) HK33遺伝子の内因的転写制御配列の下流に機能的に結合されたレポーター 遺伝子を有するベクターが導入された細胞に、 被検試料を接触させる工程、 (a) contacting a test sample with cells into which a vector having a reporter gene operably linked downstream of an endogenous transcription control sequence of the HK33 gene has been introduced; (b) 該レポーター遺伝子の発現を検出する工程、 (b) detecting the expression of the reporter gene, (c) 被検試料を該細胞に接触させない場合と比較して、 該レポーター遺伝子の 発現を調節する活性を有する化合物を選択する工程、 を含む方法。  (c) selecting a compound having an activity of regulating the expression of the reporter gene as compared to a case where the test sample is not contacted with the cells. 1 3. 請求項 8から 1 2のいずれかに記載の方法により単離されうる化合物。 1 3. A compound that can be isolated by the method of any of claims 8 to 12. 14. HK33遺伝子の発現を調節する化合物、または ARF蛋白質と HK33蛋白質との相 互作用を調節する化合物を含む、 下記 （a) 力 ら (c) のいずれかに記載の薬剤 14. A drug according to any one of the following (a) Riki et al. (C), comprising a compound that regulates the expression of the HK33 gene or a compound that regulates the interaction between the ARF protein and the HK33 protein (a) ARFを介するシグナル伝達の調節剤。 (a) A regulator of ARF-mediated signaling. (b) p53を介するシグナル伝達の調節剤。  (b) Modulators of p53-mediated signal transduction. (c) 細胞増殖調節剤。  (c) Cell growth regulator. 1 5. HK33遺伝子の発現を調節する化合物、または ARF蛋白質と HK33蛋白質との相 互作用を調節する化合物を有効成分とする医薬組成物。  1 5. A pharmaceutical composition comprising, as an active ingredient, a compound that regulates HK33 gene expression or a compound that regulates the interaction between ARF protein and HK33 protein. 16. 抗腫瘍剤である、 請求項 1 5に記載の医薬組成物。  16. The pharmaceutical composition according to claim 15, which is an antitumor agent. 17. HK33蛋白質をコードする DNAまたはその相補鎖に相補的な少なくとも 15ヌク レオチドを含むポリヌクレオチド、 あるいは HK33蛋白質に結合する抗体を含む、 下記 （a) から （e) のいずれかに記載の検査試薬：  17. The test according to any one of the following (a) to (e), including a polynucleotide containing at least 15 nucleotides complementary to the DNA encoding the HK33 protein or its complementary strand, or an antibody that binds to the HK33 protein. Reagents: ( a ) ARFを介するシグナル伝達の異常の検査試薬。  (a) A test reagent for abnormalities in signal transduction via ARF. ( b ) p53を介するシグナル伝達の異常の検査試薬。 (c) 細胞増殖異常の検查試薬。 (b) A test reagent for abnormality in signal transduction via p53. (c) A reagent for detecting abnormal cell proliferation. (d) 細胞増殖性疾患の検査試薬。  (d) Test reagent for cell proliferative disease. (e) 細胞増殖性疾患が腫瘍である、 （d) の検査試薬。  (e) The test reagent of (d), wherein the cell proliferative disease is a tumor. 18. 下記 （a) または （b) の工程を含む、 ARFを介するシグナル伝達の異常、 P53を介するシグナル伝達の異常、細胞増殖異常、細胞增殖性疾患、または腫瘍の 検査方法：  18. Methods for testing ARF-mediated signaling abnormalities, P53-mediated signaling abnormalities, cell proliferation abnormalities, cell proliferative disorders, or tumors, including the following steps (a) or (b): (a) 々者由来の被検試料中における HK33遺伝子の発現量を検出し、 健常者と比 較する工程。  (a) a step of detecting the expression level of the HK33 gene in a test sample derived from various individuals and comparing the expression level with that of a healthy individual. (b) HK33蛋白質または該蛋白質をコードする核酸における変異を検出する工程  (b) detecting a mutation in the HK33 protein or a nucleic acid encoding the protein
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