WO2002020770A1 - Methode de criblage d'un agent antitumoral a l'aide d'une interaction entre une proteine arf et une proteine hk33 - Google Patents

Methode de criblage d'un agent antitumoral a l'aide d'une interaction entre une proteine arf et une proteine hk33 Download PDF

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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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protein
arf
gene
expression
interaction
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PCT/JP2001/007732
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Japanese (ja)
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Takashi Sugihara
Renu Wadhwa
Sunil C. Kaul
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Chugai Seiyaku Kabushiki Kaisha
National Institute Of Advanced Industrial Science And Technology
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Priority to AU2001284454A priority Critical patent/AU2001284454A1/en
Priority to JP2002525777A priority patent/JPWO2002020770A1/ja
Publication of WO2002020770A1 publication Critical patent/WO2002020770A1/fr

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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
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—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

Selon l'invention, on a découvert que la protéine HK33 se lie à la protéine ARF. Le transport nucléaire de la protéine ARF est inhibé par l'expression du gène HK33, d'où la suppression de la transcription dépendant de p53. De plus, dans les cellules immortalisées, l'expression du gène HK33 s'intensifie sensiblement. L'invention concerne une méthode destinée à cribler un agent antitumoral à l'aide d'une interaction entre la protéine ARF et la protéine HK33, une méthode de régulation de la transduction de signal de ciblage de la protéine HK33 induite par la protéine ARF et des molécules utilisées à cet effet, ainsi que l'utilisation de la protéine HK33 et d'un gène codant pour la protéine HK33 dans l'analyse de maladies telles qu'une tumeur. En d'autres termes, il est possible de cribler un nouvel agent antitumoral en utilisant, comme indication, une interaction entre la protéine ARF et la protéine HK33.
PCT/JP2001/007732 2000-09-08 2001-09-06 Methode de criblage d'un agent antitumoral a l'aide d'une interaction entre une proteine arf et une proteine hk33 WO2002020770A1 (fr)

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WO2003087395A2 (fr) * 2002-04-15 2003-10-23 Inserm Procedes d'identification d'agents antitumoraux inhibiteurs des interactions de rasgap avec la kinase aurora
WO2005054870A2 (fr) * 2003-11-26 2005-06-16 Vanderbilt University Methodes d'estimation des interactions entre p19-arf et cmyc
US7045913B2 (en) 2002-06-18 2006-05-16 Ingersoll Rand Energy Systems Microturbine engine system
JP2008513805A (ja) * 2004-09-22 2008-05-01 オデュッセイ セラ インコーポレイテッド 新しい薬物リード及び、既知の薬物の新しい治療的用途を同定する方法
JP2009052942A (ja) * 2007-08-24 2009-03-12 Konica Minolta Holdings Inc シグナル伝達調節剤のスクリーニング方法
JP2013526829A (ja) * 2009-06-11 2013-06-27 サイノファーム タイワン リミテッド 細胞クローンのための阻害に基づくハイスループットスクリーニング方法
CN105238833A (zh) * 2014-06-20 2016-01-13 浙江海洋学院 一种泥螺寡肽在抗前列腺癌中的应用

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003087395A2 (fr) * 2002-04-15 2003-10-23 Inserm Procedes d'identification d'agents antitumoraux inhibiteurs des interactions de rasgap avec la kinase aurora
WO2003087395A3 (fr) * 2002-04-15 2004-03-04 Inst Nat Sante Rech Med Procedes d'identification d'agents antitumoraux inhibiteurs des interactions de rasgap avec la kinase aurora
US7045913B2 (en) 2002-06-18 2006-05-16 Ingersoll Rand Energy Systems Microturbine engine system
WO2005054870A2 (fr) * 2003-11-26 2005-06-16 Vanderbilt University Methodes d'estimation des interactions entre p19-arf et cmyc
WO2005054870A3 (fr) * 2003-11-26 2006-01-05 Univ Vanderbilt Methodes d'estimation des interactions entre p19-arf et cmyc
US7625716B2 (en) 2003-11-26 2009-12-01 Vanderbilt University Methods for assessing p19-Arf interactions in cMyc
JP2008513805A (ja) * 2004-09-22 2008-05-01 オデュッセイ セラ インコーポレイテッド 新しい薬物リード及び、既知の薬物の新しい治療的用途を同定する方法
JP2012154938A (ja) * 2004-09-22 2012-08-16 Odyssey Thera Inc 新しい薬物リード及び、既知の薬物の新しい治療的用途を同定する方法
JP2009052942A (ja) * 2007-08-24 2009-03-12 Konica Minolta Holdings Inc シグナル伝達調節剤のスクリーニング方法
JP2013526829A (ja) * 2009-06-11 2013-06-27 サイノファーム タイワン リミテッド 細胞クローンのための阻害に基づくハイスループットスクリーニング方法
CN105238833A (zh) * 2014-06-20 2016-01-13 浙江海洋学院 一种泥螺寡肽在抗前列腺癌中的应用
CN105238833B (zh) * 2014-06-20 2021-01-15 浙江海洋学院 一种泥螺寡肽在抗前列腺癌中的应用

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