+

WO2002065116A1 - Methode de criblage d'un medicament regulant l'interaction entre une proteine arf et une proteine a10 - Google Patents

Methode de criblage d'un medicament regulant l'interaction entre une proteine arf et une proteine a10 Download PDF

Info

Publication number
WO2002065116A1
WO2002065116A1 PCT/JP2002/001194 JP0201194W WO02065116A1 WO 2002065116 A1 WO2002065116 A1 WO 2002065116A1 JP 0201194 W JP0201194 W JP 0201194W WO 02065116 A1 WO02065116 A1 WO 02065116A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
arf
gene
interaction
expression
Prior art date
Application number
PCT/JP2002/001194
Other languages
English (en)
Japanese (ja)
Inventor
Takashi Sugihara
Renu Wadhwa
Sunil C. Kaul
Original Assignee
Chugai Seiyaku Kabushiki Kaisha
National Institute Of Advanced Industrial Science And Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Chugai Seiyaku Kabushiki Kaisha, National Institute Of Advanced Industrial Science And Technology filed Critical Chugai Seiyaku Kabushiki Kaisha
Priority to JP2002564583A priority Critical patent/JP4189456B2/ja
Publication of WO2002065116A1 publication Critical patent/WO2002065116A1/fr

Links

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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

Definitions

  • the present invention relates to a method for screening a compound that regulates the interaction between an ARF protein and an A10 protein. This screening is particularly useful for screening antitumor drugs.
  • the present invention further relates to the use of the A10 protein and the gene encoding the A10 protein for examining the interaction between the ARF protein and the A10 protein.
  • pRb tumor suppressor retinoblastoma protein
  • p53 Tumor suppressor retinoblastoma protein
  • pRb and p53 are thought to regulate the cell cycle in different ways. The difference between the two actions is that pRb binds to E2F, which controls the expression of proteins essential for DNA replication and cell proliferation, and suppresses its apoptosis, leading to suppression of cell growth. It is regulated in the transition from Gl phase to S phase by the action of dependent kinases (cdks).
  • p53 is said to promote transcription of p21 and suppress the cell cycle by being expressed by chemical substances or DNA damage.
  • p53 is degraded by binding to MDM2, and activation of p53 by this mechanism is thought to contribute to tumor growth in the amplification of MDM2 found in certain cancers (Gott Kunststoff.MF and Oren, M. (1996) Biochim. Biophys. Acta 1287, 77-102; Momand, J. et al. (1992) Cell, 69, 1237-1245; Kubbutat, MH et al. (1997) Nature, 387, 299-303).
  • INK4a inactivates cyclin-dependent kinase 4 (CDK4) and inhibits the phosphorylation of Rb, among other substrates, and arrests the cell cycle by inhibiting phosphorylation. I do.
  • CDK4 cyclin-dependent kinase 4
  • ARF is thought to inhibit the degradation of p53 by MDM2 by binding to MDM2 in the nucleus, 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). Disclosure of the invention
  • An object of the present invention is to provide a method for screening a compound that regulates the interaction between an ARF protein and an MO protein.
  • Compounds that modulate the interaction of ARF and A10 proteins are particularly useful for inhibiting tumor cell growth.
  • the present invention further provides the use of the A10 protein and the gene encoding the A10 protein for examining the interaction between the ARF protein and the A10 protein. This test is useful for testing diseases such as tumors that involve signal transduction via the ARF protein.
  • the present inventors have attempted to specifically isolate a gene encoding a protein that interacts with pi9ARF by the yeast Two-Hybrid method in order to solve the above problems. As a result, they found that the protein encoded by the A10 gene binds to the P19ARF protein.
  • FIG. 1 The A10 gene was the same as the gene registered as GenBank Accession Number NM_017632 [Homo sapiens hypothetical protein FLJ20036 (FLJ20036), mRNA (GenBank Accession Number NMJ17632)].
  • GenBank Accession Number NM_017632 Homo sapiens hypothetical protein FLJ20036 (FLJ20036), mRNA (GenBank Accession Number NMJ17632)].
  • the interaction between A10 protein and pl9ARF protein was confirmed again by immunoprecipitation.
  • Figure 2 A10 Significant interaction between white matter and P19ARF protein was observed (Figure 2).
  • pl9ARFmyc significantly promoted the translocation of the M0 protein to the nucleus (FIG. 3). Therefore, screening for a drug that suppresses the interaction between the A10 protein and the ARF protein has the potential to screen for a drug that is important for controlling the signaling of the ARF-P53-p21 pathway.
  • the ARF-p53-p21 pathway is known to be involved in cell carcinogenesis. Can be used.
  • the A10 gene can be used as an index for diagnosis and examination of diseases such as cancer, and as a tool for elucidating the pathological mechanism of these diseases.
  • the present invention relates to a method for screening a compound that regulates the interaction between an ARF protein and an A10 protein.
  • the present invention also relates to the use of the A10 protein and a gene encoding the A10 protein for examining the interaction between the ARF protein and the A10 protein.
  • the present invention more specifically relates to
  • composition comprising as an active ingredient a compound that regulates the interaction between the ARF protein and the M0 protein,
  • a method for testing the interaction between ARF protein and A10 protein including the following steps (a) or (b):
  • a test reagent for the interaction between ARF protein and A10 protein comprising a polynucleotide containing at least 15 nucleotides complementary to DNA encoding A10 protein or its complementary strand, or an antibody binding to A10 protein.
  • the A10 gene includes a gene encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2, its homologous gene, isoform, and derivatives thereof.
  • the A10 gene is ⁇ Homo sapiens hypothetical protein FLJ20036 (FLJ20036), mRNAj (GenBank Accession Number NM_017632), ⁇ Homo sapiens cDNA FLJ20036 fis, clone C0L00219J (AK000043), and ⁇ sapiens putative serine-rich protein mRNA and partial cdsj (AF246705).
  • the ARF gene includes ARF (alternative reading frame) (Quelle, DE et al., Cell 83: 993-1000, 1995), its homologous gene, isoform, 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 P14-CDK inhibitor pi-hibiki
  • mouse 19 ARF ACCESSION L76092, Locus MUSARF
  • the "interaction" between proteins refers to an action between proteins, including binding, modification, transport, change in activity, induction of structural change, induction of stability change, and the like.
  • 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 ARF protein modulates p53 function by interacting directly with P53.
  • the A10 protein binds to the ARF protein and the function of the ARF is modified, whereby the signal transduction via the ARF can be controlled. Therefore, by controlling the interaction between the ARF protein and the A10 protein or regulating the expression of the A10 gene, It is thought that it is possible to control signal transduction through white matter.
  • the interaction between the ARF protein and the A10 protein includes binding and modification of the ARF protein with the A10 protein.
  • the interaction between the ARF protein and the A10 protein can be detected by detecting the binding of the two proteins, or by altering the intracellular distribution or activity of the A10 protein caused by the interaction between the ARF protein and the A10 protein, or by altering the function or function of ARF. Changes in signal transduction (including ARF nuclear translocation, interaction between MDM2 and ARF, stabilization of p53, promotion of p53-mediated signal transduction, etc.) can be detected as indicators.
  • A10 gene expression can be measured by detecting its transcript or protein.
  • the present invention provides a method for screening a compound that regulates the interaction between ARF protein and A10 protein.
  • One of the screening methods of the present invention comprises: (a) a step of bringing an ARF protein into contact with an A10 protein in the presence of a test sample; (b) a step of detecting an interaction between the ARF protein and the A10 protein; and (C) 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 compounds obtained by this screening can also be used to modulate the activity of ARF or A10 protein.
  • the interaction between the ARF protein and the A10 protein can be detected either directly by detecting the binding of the two proteins, or indirectly through the detection of changes caused by the interaction of the two proteins.
  • Such a screening system can be constructed in a test tube, in a cell, or the like. When performed intracellularly, by incubating cells expressing the ARF protein and the A10 protein in the presence of the test compound, the interaction between the ARF protein expressed in the cell and the A10 protein can be detected by contacting the two. Screening can be performed. The screening method using a 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 A10 protein b) a step of detecting an interaction between the ARF protein and the A10 protein; and (c) a step of detecting the interaction between the ARF protein and the A10 protein in the absence of the test sample.
  • This screening method using cells includes a screening method using signal transduction via ARF protein or P53 protein as an index, which will be described later, in addition to screening using the Two eight-bridging method described below.
  • One embodiment of the above-described screening method for a compound that regulates the interaction between the ARF protein and the A10 protein includes detecting the interaction between the ARF protein and the A10 protein by using the binding between the ARF protein and the A10 protein as an index. Is the way. Specifically, this screening method comprises: (a) a step of bringing the ARF protein into contact with the A10 protein in the presence of the test sample; (b) a step of detecting the binding between the ARF protein and the A10 protein; and (c) ) A step of selecting a compound that modulates the binding as compared to a case where detection is performed in the absence of a test sample (control).
  • the A10 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.
  • the protein can be used for screening, for example, as a purified protein, as a soluble protein, as a form bound to a carrier, as a form expressed on a cell membrane, or as a membrane fraction.
  • the test sample is not particularly limited, and a sample containing a desired test compound can be used.
  • test samples include, for example, cell culture supernatants, fermented microorganism products, marine organism extracts, plant extracts, prokaryotic cell extracts, eukaryotic single cell extracts, animal cell extracts, or live forms thereof. Rallies, purified or partially purified proteins, peptides, non-peptidic compounds, synthetic low-molecular compounds, and natural compounds.
  • a method of screening for a compound that regulates the binding between proteins can be performed using, for example, a two-hybrid method using yeast or animal cells ( Fields, S., and Sternglanz, R. 'Trends.Genet. (1994) 10, 286-292, Dalton S, and Treisman R (1992) Characierization of SAP-1, a protein recruited by serum response factor to the c -fos serum response element.
  • a vector for expressing a fusion protein in which the other protein or a partial peptide thereof is fused to a transcription activation region such as VP16 or GAL4 is constructed, and these are used to construct a reporter gene.
  • the compound is introduced into yeast cells together with the encoding vector, and the compound is assayed in the presence of a sample containing the test compound using the reporter activity as an index.
  • the binding of the A10 protein and the ARF protein induces the expression of the reporter gene, but if the test compound inhibits the binding of both proteins, the expression of the reporter gene is suppressed.
  • the repo overnight gene include, but are not limited to, the HIS3 gene, the Ade2 gene, the LacZ gene, the CAT gene, the Luciferase gene, the PA1 (Plasminogen activator inhibitor typel) gene, and the like. Not done.
  • a repo overnight gene a cytotoxic gene can be expressed.
  • Screening can also be performed using, for example, immunoprecipitation.
  • Cells expressing the A10 protein and the ARF protein are cultured in the presence of the sample containing the test compound, and after the cells are recovered, the complex is recovered using an antibody or the like against one protein, and then the other protein is recovered.
  • the binding of both proteins can be evaluated.
  • Both proteins may be proteins that are endogenously expressed by the cell, but either or both proteins are exogenously expressed in the cell You can also.
  • a tag peptide or other protein can be appropriately fused to the protein used for screening to facilitate detection.
  • any commonly used promoter such as the HSV TK promoter may be used.
  • electroporation Cho, G. et al. Nucl. Acid Res. 15, 1311-1326 (1987)
  • phosphate Calcium method Choen, C and Okayama, H. Mol. Cell. Biol. 7, 2745-2752 (1987)
  • DEAE dextran method Lipata, MA et al. Nucl. Acids Res. U, 5707-5717 ( 1984); Sussman, DJ and Milman, G. Mol. Cell. Biol.
  • a method for preparing a fusion protein by introducing only a small epitope portion consisting of several to a dozen or so amino acids in order to minimize the properties of the original protein when making it into a fusion protein has been reported.
  • polyhistidine for example, 6XHis or lOXHis
  • influenza agglutinin HA fragment human c-myc fragment
  • FLAG FLAG
  • VSV-GP Vesicular stomatitis virus glycoprotein
  • T7 genelO protein fragment T7 genelO protein fragment
  • HSV-tag human simple herpesvirus glycoprotein fragment
  • E-tag on monoclonal phage
  • Well-known epitopes such as SV40T antigen fragment, lck tag, ⁇ -tubulin fragment, B-tag, and Protein C fragment, and monoclonal antibodies recognizing it are analyzed by the above screening method.
  • an immune complex is formed by adding an antibody against these to a cell lysate prepared using an appropriate surfactant.
  • This immune complex contains A10 protein, ARF protein, and antibodies.
  • immunoprecipitation can also be performed using antibodies against the A10 protein or ARF protein.
  • these antibodies are prepared by introducing a gene encoding a target protein into an appropriate Escherichia coli expression vector and expressing it in Escherichia coli, purifying the expressed protein, It can be prepared by immunizing rats, goats, and chickens. It can also be prepared by immunizing the above animal with a synthesized partial peptide.
  • the immune complex can be precipitated using Protein A Sepharose or Protein G Sepharose.
  • an immune complex can be formed using a substance that specifically binds to these epitopes, such as daltathione-Sepharose 4B.
  • the screening of the present invention can be performed using a pull-down assay without using a cell line.
  • A10 protein and ARF protein were incubated in vitro in the presence of a sample containing a test compound, and a complex was recovered using an antibody against one of the proteins or an antibody against a tag fused to these proteins. That is, by detecting the other protein using an antibody against the protein or an antibody against a tag added to the protein, the binding between the two proteins can be evaluated.
  • one protein is bound to a support, the other protein is bound, and the test sample is applied thereto. The effect of the test sample can be examined by detecting whether the bound protein is dissociated. 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 using 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, Pharmacia Made). Therefore, it is possible to evaluate the binding of A10 protein and ARF protein by using a biosensor such as BIAcore.
  • a compound that promotes or suppresses the binding between the A10 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 A10 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 A10 protein can be detected by using the nuclear translocation of the A10 protein as an index.
  • the A10 protein is present alone in the cytoplasm, but is localized in the nucleus (nucleolus) by co-expression with the ARF protein. Therefore, a compound that regulates the interaction between the ARF protein and the A10 protein can be obtained by screening a compound that regulates the nuclear translocation of the A10 protein by the ARF protein.
  • This screening method comprises: (a) a step of bringing a test sample into contact with cells expressing the ARF protein and the M0 protein; (b) a step of detecting the nuclear localization of the A10 protein; Selecting a compound that modulates (promotes or reduces) the nuclear localization of the A10 protein as compared to when detected in the presence (control).
  • a compound that promotes the nuclear localization of the A10 protein under the expression of the A10 protein is judged to be a compound that promotes the interaction between the ARF protein and the M0 protein, while a compound that inhibits the nuclear localization of the A10 protein is the ARF protein. It is considered a compound that suppresses the interaction between A10 and A10.
  • the ARF protein or A10 protein may be an endogenous protein or may be expressed exogenously.
  • the cells used are those that express A10 protein. There is no restriction as long as nuclear translocation of the A10 protein occurs.
  • the NIH3T3 cells and COS cells used in the examples can be used.
  • the nuclear translocation of the A10 protein can be detected by, for example, an immunocytochemical technique using an antibody against the A10 protein.
  • the antibody is, for example, fluorescently labeled.
  • the A10 protein fused with the GFP protein or the like is exogenously expressed in a cell, the intracellular localization of the A10 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 A10 protein includes detecting the interaction between the ARF protein and the A10 protein by using the ARF protein-mediated signal transmission as an index. Is the way.
  • the ARF protein is a signaling factor involved in cell cycle regulation, and signals mediated by the ARF protein are transmitted to other molecules such as MDM2, p53, and P21 downstream thereof. Therefore, a compound that regulates the interaction between the ARF protein and the A10 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 A10 protein, (b) a step of detecting ARF-mediated signal transmission, and (c) a test sample. Selecting a compound that modulates (enhances or suppresses) the signal transduction as compared to the case of detection in the absence (control). Such a screening is also included in the screening of the present invention.
  • the test sample used for screening is not particularly limited.
  • the ARF protein or A10 protein may be an endogenous protein or may be expressed exogenously. Many cells having a signal transduction pathway through the ARF protein can be used as the cells to be used.
  • Examples include CH0 cells, COS cells, HeLa cells, NIH3T3 cells, BHK (baby hamster kidney), Vero and the like. Cells in which the expression of the A10 gene is significantly increased as compared to the parent cells can be suitably used.
  • Signaling via the ARF protein can be detected, for example, by the following indicators: It is possible.
  • the flow of signal transmission via the ARF protein is as follows.
  • the binding between the ARF protein and the MDM2 protein is promoted.
  • Promotion of binding between the ARF protein and the MDM2 protein suppresses the activity of the MDM2 protein, thereby suppressing the binding of the MDM2 protein to the P53 protein, and inducing degradation of the p53 protein, including ubiquitination of the p53 protein Is suppressed.
  • the degradation of the p53 protein is suppressed, and the stability of the P53 protein is increased, whereby the activity of the P53 protein in the cell is increased and the functions of the ⁇ 53 protein, such as G1 arrest and induction of apoptosis, are promoted.
  • the result is reduced DNA synthesis, enhanced apoptosis, and / or reduced cell division, and reduced cell proliferation.
  • the opposite effect occurs when the function of the ARF protein is inhibited.
  • These indices can be detected by a known method. Based on the flow of signal transduction via the ARF protein as described above, it is possible to screen for compounds that regulate the effect of A10 protein on these signal transductions.
  • ⁇ 53 protein is activated by the activation of signal transduction via the ARF protein, and the ⁇ 53 protein is induced. it can.
  • screening can be performed by detecting mRNRN of a target gene such as ⁇ 21 by Northern blotting or RT-PCR.
  • WST reagent Roche Diagnostics
  • This screening can be performed by measuring proliferation by measuring the number of cells, or by measuring cell proliferation using WST reagent (Roche Diagnostics) or the like. Using these methods, changes in cell proliferation caused by the added test compound promoting or canceling the effect of A10 on ARF activity can be measured by the above method, and screening can be performed using this as an index. .
  • These screenings can be similarly carried out in the screening of compounds that regulate the effect of the A10 protein on P53-mediated signal transduction described below.
  • a key signaling molecule for ARF-mediated signaling is p53. It is considered that the expression of the A10 protein regulates the p53 signal by regulating the function of the ARF protein, thereby controlling p53-dependent transcription. It is important that the A10 protein regulates 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 of performing screening by selecting a compound that controls the regulation of P53-mediated signal transduction by the A10 protein.
  • This screening method comprises: (a) a step of bringing a test sample into contact with cells expressing the ARF protein and M0 protein; (b) a step of detecting signal transduction via p53; Selecting a compound that modulates (enhances or suppresses) the signal transmission as compared to the case of detection in the absence (control). Preferably, these screens are performed in cell lines.
  • the test sample used for screening is not particularly limited.
  • the same cells as those used in the screening using the above-described signal transmission of the ARF protein as an index can be used. Cells in which the expression of the A10 gene is significantly increased as compared to the parent cells can be suitably used.
  • Signaling via the P53 protein can be evaluated by detecting the P53 protein or its action as described above. Specifically, for example, binding between P53 protein and MDM2 protein, modification of P53 protein, stability of p53 protein, expression of target gene of P53 protein, apoptosis, DNA synthesis, cell division, cell proliferation, etc. When can do.
  • the 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 A10 protein. Therefore, this screening method can be used to obtain a compound that regulates cell proliferation, as in the above-described screening method for a compound that regulates the interaction between the ARF protein and the A10 protein. It is also suitably used to obtain an agent.
  • screening can be performed by an estrangement method or a Northern plotting RT-PCR in which the expression of a target gene of p53 such as P21 or the like described above is confirmed with an antibody.
  • a compound that promotes p53 target gene expression is a compound that activates signal transduction via the P53 protein.
  • a compound that suppresses target gene expression is a compound that suppresses signal transduction via the P53 protein. is there.
  • screening can be performed using cell growth suppression as an index. The promotion of cell proliferation reflects the suppression of signal transduction through the P53 protein, and the suppression of cell proliferation is thought to reflect the promotion of signal transduction through the P53 protein.
  • a sample containing a 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 gene can be introduced by a known gene introduction method using an expression vector or the like.
  • a sample containing a 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 as a test sample using an expression vector or the like.
  • the A10 protein or ARF protein expressed in cells, or various signaling molecules downstream from these proteins, may be endogenous or exogenously expressed.
  • the assay of the test compound is performed. By doing so, it is possible to verify the specificity of the compound for the ARF protein.
  • the simplest cell for this purpose it is appropriate to use NIH3T3 cells deficient in the INK4a site where ARF is present.
  • the present invention also provides a method for screening for a modulator of the interaction between the ARF protein and the A10 protein by selecting a compound that regulates the expression of the A10 gene.
  • This screening can regulate the expression (such as transcription or translation) of the A10 gene in a cell, in a living body, or in an in vitro expression system using a gene encoding the A10 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 ARF or p53-mediated signal transduction, cell growth regulators, apoptosis regulators, or carcinogens, cell immortalization reagents, or antitumor agents. obtain.
  • the screening method described above includes, for example, (a) a step of bringing a test sample into contact with cells endogenously retaining the A10 gene, (b) a step of detecting expression of the A10 gene, and (c) a step of detecting the expression of the A10 gene. Selecting a compound having an activity of regulating (enhancing or inhibiting) the expression as compared to the case where the sample is not contacted with the cells (control).
  • expression of a gene includes transcription and translation.
  • cells expressing the A10 gene are cultured together with a test sample, and the expression of the gene is detected by niRNA such as Northern analysis or RT-PCR, or by Western blotting, immunoprecipitation, ELISA, etc.
  • the method of detecting the target compound can be carried out by selecting a compound which promotes or inhibits the expression of the gene by detecting the protein by the method of detecting the above-mentioned protein or by improving the method of the present invention, and comparing with the case where the test sample is not added. Can be screened.
  • Screening is expected to lead to the treatment of cancer. It is also conceivable to screen a compound that can regulate the expression of the A10 gene in vivo or in a cell by a method using the activation or inactivation of the expression control region of the A10 gene as an index.
  • This screening comprises: (a) contacting a test sample with cells holding a reporter gene operably linked downstream of the endogenous transcription control sequence of the A10 gene; (b) 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 contacted with the cells (control). And a method comprising the steps of:
  • the term “endogenous transcription control sequence” refers to a sequence that controls the cultivation of the A10 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 a gene encoding A10 protein can be used. For example, a DNA fragment from the transcription start point (or translation initiation codon) of a gene encoding the A10 protein to several kb upstream (eg, 2 kb, 3 kb, 5 kb, or 10 kb) of the gene encodes the endogenous transcriptional control of the gene. It is thought that the sequence is included.
  • the expression of the reporter gene can be placed under the transcriptional control of the gene encoding A10 protein.
  • the upstream region may be appropriately deleted or mutated to measure its transcription control activity, to identify a sequence involved in transcription control, and to use a fragment thereof.
  • many transcription control sequences to which transcription factors involved in transcription control are bound are known. By identifying these known transcription control sequences from the upstream region of the gene encoding the A10 protein, it is possible to identify the endogenous transcription control sequences. Usually, a plurality of sequences controlling gene transcription are present in one gene, but in the screening of the present invention, any of them or a combination thereof can be used.
  • the endogenous transcription control sequence may be chimeric with other promoters.
  • Chimeric promoters are often used in transcriptional regulation studies. Construction of chimeric promoter Examples of other promoters to be used include a minimal promoter derived from the SV40 early promoter and the like.
  • “functionally linked” means that the transcription control sequence is linked to the reporter gene so that the reporter gene linked downstream can be expressed in response to activation of the transcription control sequence. Point.
  • the transcription control region (promoter, enhancer, etc.) of the A10 gene is cloned, and this is cloned.
  • 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 is selected by comparing with the reporter activity in the cells not contacted with the test sample.
  • Compounds that can regulate the expression of the A10 gene in cells can be screened. This screening has the characteristic that the expression of the A10 gene is detected using the reporter activity as an index, and is therefore simpler than direct detection such as the Northern analysis described above.
  • a compound that increases A10 gene expression becomes a compound that increases the interaction between A10 protein and ARF protein, while a compound that decreases A10 gene expression decreases the interaction between A10 protein and ARF protein It becomes a compound.
  • a knock-in animal in which the coding region of the endogenous A10 gene has been replaced with a reporter gene may be used.
  • the screening described above can be performed using such an animal or cells derived from the animal.
  • Knock-in animals can be prepared according to a known method.
  • the compounds that can be isolated by the above-described screening method of the present invention are used as compounds that regulate the interaction between A10 protein and ARF protein. These compounds may be compounds that regulate signal transduction through the ARF protein, and compounds that regulate signal transduction through the p53 protein.
  • the present invention relates to the present invention.
  • the present invention also relates to a modulator of the interaction between the A10 protein and the ARF protein, including a compound that can be isolated by the cleaning method, and a modulator of the signal transduction via the ARF protein or the P53 protein.
  • a compound or the like that suppresses the interaction between the A10 protein and the ARF protein can regulate cell division and cell growth by controlling the activity of the ARF protein in cells and modifying downstream signals such as P53.
  • the above-described screens of the present invention are useful for evaluating or isolating compounds that modulate cell proliferation.
  • 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.
  • Compounds that regulate the interaction between A10 protein and ARF protein, and compounds that regulate signal transmission via ARF protein can be used as reagents and / or pharmaceuticals .
  • Compounds that can be isolated by the above screening can also be used as reagents and / or medicaments.
  • a compound in which a part of the structure of a compound that can be isolated using the screening method of the present invention is converted by addition, deletion, or substitution is also included in the compound that can be isolated by the screening of the present invention.
  • Such compounds can also be used as reagents and / or medicaments. These compounds are useful as agents such as modulators of signal transduction via ARF protein or P53 protein.
  • Drugs include reagents and medicaments.
  • these compounds can be used as agents for regulating cell proliferation.
  • the compound is considered to be applicable, for example, to the treatment of cell proliferative diseases and diseases that can be treated by controlling cell proliferation.
  • these compounds are expected to be used as tumor suppressors (anticancer agents).
  • Compounds that regulate the interaction between the ARF protein and the M0 protein include humans and mammals, such as mice, rats, guinea pigs, egrets, chicks, cats, dogs, higgins, bushes, sea lions, monkeys, baboons, and chimpanzees.
  • a drug When used as a medicament, a drug may be prepared by a known pharmaceutical method in addition to directly administering the compound to a patient. It can also be administered as a pharmaceutical composition. For example, sterile solutions or suspensions in tablets, capsules, elixirs, and microcapsules, orally coated with sugar or water or other pharmaceutically acceptable liquids, as appropriate. It can be used parenterally in the form of liquid injections.
  • pharmacologically acceptable carriers or vehicles specifically, sterile water or physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles It can be formulated by combining with a preservative, a binder and the like as appropriate and mixing in a unit dosage form required for generally accepted pharmaceutical practice.
  • the amount of active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.
  • Additives that can be incorporated into tablets and capsules include, for example, binders such as gelatin, corn starch, tragacanth gum, acacia, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid.
  • binders such as gelatin, corn starch, tragacanth gum, acacia
  • excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid.
  • Useful bulking agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents 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 solutions for injection include, for example, saline, isotonic solutions containing dextrose and other auxiliaries, e.g.!)-Sorbitol, D-mannose, D-mannitol, sodium chloride, and suitable dissolution Auxiliaries, such as alcohols, specifically ethanol, polyalcohols, such as propylene glycol, polyethylene glycol, nonionic surfactants, such as polysorbate 80 (TM), HC0-50 Examples include sesame oil and soybean oil, which may be used in combination with solubilizers such as benzoic acid and benzyl alcohol.
  • buffers such as phosphate buffers, sodium acetate buffers, soothing agents such as proforce hydrochloride, stabilizers, such as It may be combined with benzyl alcohol, phenol, and antioxidants.
  • 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 may be incorporated into a vector for gene therapy to perform gene therapy.
  • the dose and the administration method vary depending on the patient's body weight, age, symptoms, and the like, but can be appropriately selected by those skilled in the art.
  • the dose of the compound varies depending on the administration subject, target organ, symptoms, and administration method. However, in the case of oral administration, in general, for an adult (assuming a body weight of 60 kg), about 0.1 to 100 mg per day is preferable. Is about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.
  • the single dose will vary depending on the subject, target organ, symptoms, and administration method.
  • the dose can be administered in terms of the amount converted per 60 kg body weight or the amount converted per body surface area.
  • ARF-mediated signaling involves regulation of ARF protein activity.
  • ARF activity include an activity of the ARF protein such as binding to the MDM2 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.
  • the promotion of signal transmission through the ARF protein includes the promotion of MDM2 activity such as the interaction between the MDM2 protein and the P53 protein.
  • the P53 protein plays an important role in regulating cell functions, and is also known as a tumor suppressor gene.
  • P53-mediated signal transduction can be regulated.
  • Compounds that promote the interaction between ARF protein and A10 protein include, for example, compounds that increase the expression level of A10 protein.
  • Such compounds include A10 protein or a nucleic acid (DNA) encoding the protein. Or RNA) itself.
  • the expression level of the A10 protein can be increased by introducing the vector expressing the A10 gene into the cell to increase the intracellular expression of the A10 protein. As a result, the expression level of the A10 protein in the cell increases, and the interaction between the A10 protein and the ARF protein can be increased.
  • the present invention provides an A10 protein, an ARF protein, a DNA encoding the protein, and a DNA encoding the protein for regulating the interaction between the ARF protein and the M0 protein, or the signal transduction via the ARF protein or p53, or the cell growth.
  • the expression of the A10 gene can be reduced by, for example, expression of antisense RNA against A10 mRNA, or expression of a liposome that cleaves AlO mRNA.
  • it can be carried out by administration of an antisense oligonucleotide or a decoy nucleic acid containing a part of the transcriptional regulatory region of A10.
  • a mutation may be introduced into the A10 gene.
  • the expression level of the A10 protein is reduced, the interaction between the A10 protein and the ARF protein can be reduced.
  • the interaction between the ARF protein and the M0 protein can be reduced by the action of an agonist of the A10 protein.
  • An agonist of the A10 protein may be an antibody against A10 or a fragment thereof. It can also be a partial peptide of the ARF protein.
  • the partial peptide of the ARF protein having the activity of binding to the A10 protein is the A10 protein Through binding to A10 can inhibit the interaction of the A10 protein with the intact ARF protein.
  • Compounds that regulate the interaction between the ARF protein and the A10 protein, and compounds that regulate the expression of the A10 gene include, as described below, regulators of ARF-mediated signal transduction, P53-mediated signal transduction regulators, And it can be used as a cell growth regulator.
  • DNA encoding the A10 protein can be prepared by a known method.
  • the amino acid sequence of human A10 protein and the base sequence of cDNA encoding A10 protein are already known (GenBank Accession Number NM_017632, AK000043, and AF246705).
  • the DNA encoding the A10 protein can be prepared, for example, by hybridization using a probe prepared based on this base sequence or by polymerase chain reaction (PCR) using a primer (Sambrook, J. Am. et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. press, 1989).
  • DNA encoding the A10 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.).
  • the cDNA prepared from the tissue expressing the A10 protein can be applied to screening using hybridization or PCR.
  • Hybridization conditions can be appropriately selected by those skilled in the art. For example, it can be performed under low stringent conditions, normal conditions, or stringent conditions.
  • Stringent hybridization conditions are, for example, using ExpressHyb Hybridization Solution (manufactured by Clontech) as a hybridization buffer at 42 ° C, preferably 60 ° C, and particularly preferably 68 ° C. After the hybridization for 1 hour, washing with 50 ° C, 2XSS 0.1% SDS is preferable, and washing with 50 ° C, 0.1XSS 0.1% SDS is preferable. More preferably, in washing after hybridization, for example, washing with 65 ° (:, washing with 0.1X SSC and 0.1% SDS can be mentioned. Under these conditions, DNA having higher homology as the temperature is increased. Is efficient Can be expected.
  • the protein encoded by the DNA that hybridizes with the cDNA encoding the human M0 protein (SEQ ID NO: 2) and that interacts with the ARF protein can be used in the present invention in the same manner as the M0 protein. it can. Such a protein can also be used in the above-described screening of the present invention.
  • the protein encoded by the DNA isolated by these hybridization techniques or gene amplification techniques usually has high homology to human A10 protein (SEQ ID NO: 2) or a homologous protein in other organisms with the amino acid sequence.
  • proteins having high homology to these proteins those having the activity of interacting with the ARF protein can be used in the present invention in the same manner as the A10 protein.
  • High homology usually means at least 40% identity, preferably 60% identity, more preferably 80% identity, and even more preferably 90% identity at the amino acid level. .
  • the algorithm described in the literature Wang, WJ and Lip DJ, Proc. Natl. Acad. Sci. USA (1983) 80, 726-730
  • the algorithm described in the literature Wang, WJ and Lip DJ, Proc. Natl. Acad. Sci. USA (1983) 80, 726-730
  • a mutant of the natural A10 protein can be used. Such mutations may be artificial or may have occurred 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 a site-directed mutagenesis method (Hashimoto-Gotoh T. et al. (1995) Gene 152, 27 275 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 Enzymol.
  • a mutant protein can be prepared by appropriately introducing a mutation into the amino acid of the A10 protein.
  • a protein having one or more amino acids mutated by substitution, deletion and / or insertion in the amino acid sequence of the natural A10 protein and interacting with the ARF protein is In the present invention, it can be used similarly to the A10 protein.
  • 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 conceivable that. 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, L, M, F, P, W, Y, V) and hydrophilic amino acids (R, D, N, E, Q, G, H , K, S, T), an amino acid having an aliphatic side chain (G, A, V, L, I, P), an amino acid having a hydroxyl-containing side chain (S, T, ⁇ ), a sulfur atom-containing side Amino acids with chain (C, M), Amino acids with carboxylic acid and amide containing side chains (D, N, E, Q), Amino acids with base containing side chains (R, K, ⁇ ), Aromatic containing side Amino acids having a chain (H, F, Y, W) can be mentioned (all brackets indicate one letter of amino acids).
  • the protein may be a protein in which amino acids have been conservatively substituted.
  • the A10 protein includes, for example, a plurality of amino acids in the amino acid sequence of a natural A10 protein. It may be a protein to which an acid residue has been added.
  • a fusion protein is a fusion of the natural A10 protein and another peptide or protein, and is included in the A10 protein of the present invention.
  • the fusion protein is prepared by ligating the DNA encoding the A10 protein and the DNA encoding the other peptide or protein so that their frames match, introducing the DNA into an expression vector, and expressing it in a host. Any method known to those skilled in the art can be used. Other peptides or proteins to be fused 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 six His (histidine) residues, and lO XHis, influenza agglutinin (HA), human c-myc fragment, VSV-GP fragment, P18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, CK-tubul
  • HA influenza agglutinin
  • human c-myc fragment VSV-GP fragment
  • P18HIV fragment T7-tag
  • HSV-tag HSV-tag
  • E-tag E-tag
  • SV40T antigen fragment lck tag
  • CK-tubul CK-tubul
  • peptides such as an in fragment, a B-tag, and a protein C fragment can be used.
  • proteins to be fused include, for example, GST (daltathione S-transferase), HA (influenza agglutinin), immunoglobulin constant region, -galactosidase, MBP (maltosis binding protein) and the like.
  • Can be A fusion protein can be prepared by fusing a commercially available DNA encoding the peptide or protein with a DNA encoding the A10 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 vary depending on the cell, host, or purification method that produces it as described below. However, as long as the obtained protein has the activity of interacting with the ARF protein, it can be used in the present invention in the same manner as the A10 protein. For example, when the A10 protein is expressed in a prokaryotic cell, for example, Escherichia coli, a methionine residue is added to the N-terminal of the amino acid sequence of the original protein, but such a protein may be used.
  • the A10 protein can be prepared as a recombinant protein or a natural protein by methods known to those skilled in the art.
  • A10 protein if it is a recombinant protein DNA (see, for example, SEQ ID NO: 1 for human A10 cDNA) is inserted into an appropriate expression vector, which is then introduced into an appropriate host cell, and the resulting transformant is recovered. Once obtained, they are subjected to chromatography such as ion exchange, reverse phase, gel filtration, etc., or affinity chromatography in which an antibody against the A10 protein is immobilized on a column, or a combination of these columns. It is possible to purify and prepare by combining.
  • the A10 protein When the A10 protein is expressed in a host cell (for example, an animal cell or Escherichia coli) as a fusion protein with the daltathione S-transferase protein or as a recombinant protein to which a plurality of histidines are added, the expressed recombinant is used.
  • the protein can be purified using a daltathione column or a nickel column. After purifying the fusion protein, if necessary, a region 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 A10 protein described below is applied to an extract of a tissue or a cell expressing the A10 protein. It can be isolated by purification.
  • Antibodies may be polyclonal or monoclonal.
  • DNA encoding the A10 protein is used for in vivo and in vitro production of the A10 protein. Further, application to gene therapy for regulating the interaction between the A10 protein and the ARF protein is also considered.
  • the DNA encoding the A10 protein may be in any form as long as it can encode the A10 protein. That is, it does not matter whether it is cDNA synthesized from mRNA, genomic DNA, chemically synthesized DNA, or the like. Further, as long as it can encode the A10 protein, a DNA having an arbitrary base sequence based on the degeneracy of the genetic code is included.
  • DNA encoding the A10 protein can be prepared by a method known to those skilled in the art.
  • a cDNA library is prepared from cells expressing A10 protein, and A10 It can be prepared by performing hybridization using a part of cDNA (for example, human A10 cDNA) 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 by using a commercially available DNA library. Good.
  • RNA is prepared from cells expressing the A10 protein, cDNA is synthesized using reverse transcriptase, oligo DNi is synthesized based on the A10 cDNA sequence, and a PCR reaction is performed 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 A10 protein can be obtained.
  • Genomic DNA can be isolated by screening a genomic DNA library using the obtained cDNA as a probe.
  • an endogenous transcription control sequence of the gene encoding the A10 protein can be obtained.
  • mRNA is isolated from cells, tissues and organs that express the A10 protein.
  • mRNA can be isolated by known methods, for example, guanidine ultracentrifugation (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299), AGPC method (Chomczynski, P. and Sacci, N., Anal. Prepare total RNA according to Biochem. (1987) 162, 156-159) and purify mRNA from total RNA using mRNA Purification Kit (Pharmacia).
  • mRNA can be directly prepared by using QuickPrep mRNA Purification Kit (Pharmacia). CMA is synthesized from the obtained mRNA using reverse transcriptase.
  • cDNA can also be synthesized using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation) or the like.
  • AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation) or the like.
  • 5'-RACE method and 3'-RACE method (Frohman, MA et al., Proc.) Using 5 'Ampli FINDER RACE Kit (Clontech) and polymerase chain reaction (PCR). Natl. Acad. Sci. USA (1988) 85, 8998-9002; Beiyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932). And amplification can be performed.
  • a target DNA fragment is prepared from the obtained PCR product, and ligated to the 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, the 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 or a known method. Modifications include, for example, digestion with restriction enzymes, insertion of synthetic oligonucleotides or appropriate DNA fragments, addition of a linker, insertion of an initiation codon (ATG) and Z or a stop codon (TAA, TGA, or TAG). No.
  • an expression vector is constructed and introduced into a host cell.
  • Escherichia coli when Escherichia coli is used as a host, the vector is amplified in Escherichia coli for large-scale amplification and large-scale preparation in Escherichia coli (eg, JM109, DH5o; HB10K XLlBlue).
  • Escherichia coli for example, a drug resistance gene that can be distinguished by any drug (ampicillin-tetracycline, kanamycin, chloramphenicol)). No restrictions.
  • vectors examples include M13-based vectors, pUC-based vectors, pBR322, pBluescript, pCR-Script, and the like.
  • pGEM-T for the purpose of subcloning and excision of cDNA, pGEM-T, pDIRECT, pT7 and the like can be mentioned in addition to the above vectors.
  • an expression vector is particularly useful.
  • the expression vector when the expression vector is intended for expression in Escherichia coli, the expression vector has the above-mentioned characteristics such that the vector is amplified in Escherichia coli
  • the host is Escherichia coli such as JM109, DH5Q !, HBIOK XU-Blue, etc.
  • promoters that can be efficiently expressed in E. coli such as lacZ promoter Ichiichi (Ward et al., Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427), araB promoter (Better et al., Science (1988) 240, 1041-1043), or having the T7 promoter overnight is essential .
  • Such vectors include PGEX-5X-1 (Pharmacia), “QIAexpress system” (Qiagen), pEGFP, or pET (in this case, the host is T7 RNA polymerase) in addition to the vector described above.
  • BL21 which expresses lyase, is preferred).
  • 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) 169, 4379) may be used for production in E. coli periplasm.
  • the introduction of the vector into a host cell can be carried out, for example, using a calcium chloride method or an electroporation method.
  • one vector for producing the A10 protein includes a mammalian expression vector (eg, PCDNA3 (manufactured by Invitrogen), pEGF-B0S (Nucleic Acids.
  • a mammalian expression vector eg, PCDNA3 (manufactured by Invitrogen), pEGF-B0S (Nucleic Acids.
  • insect cell-derived expression vectors for example, ⁇ Bac-to-BAC baculovairus expression systemj (manufactured by Gibco BRL), pBacPAK8), plant-derived expression vectors (for example pMH2), animal virus-derived expression vectors (eg, pHSV, pMV, pAdexLcw), retrovirus-derived expression vectors (eg, pZIPneo), yeast-derived expression vectors (eg, HPichia Expression Kit (Invitrogen) And pNVll, SP-Q01), and Bacillus subtilis-derived expression vectors (eg, pPL608, pKTH50).
  • insect cell-derived expression vectors for example, ⁇ Bac-to-BAC baculovairus expression systemj (manufactured by Gibco BRL), pBacPAK8), plant-derived expression vectors (for example pMH2), animal virus-derived expression vectors (eg, pHSV, pMV, pAdexLc
  • promoters required for expression in cells such as SV40 promoter (Muligan et al., Nature (1979) 277, 108), Awake LV-LTR Promo Yuichi, EFla Promo Yuichi (Mizushima et al., Ucleic Acids Res. (1990) 18, 5322), CMV Promoter It is essential to have a gene for selection of transformation into cells (for example, a drug resistance gene that can be identified by a drug (neomycin, G418, etc.)). preferable.
  • Vectors having such properties include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, pOP13, etc.Furthermore, the gene is stably expressed and the gene is copied in a cell.
  • a vector eg, pCHOI
  • a vector containing the DHFR gene that complements the nucleic acid synthesis pathway is introduced into CH0 cells lacking the nucleic acid synthesis pathway, and amplification is performed using methotrexate ( ⁇ ).
  • a vector having an SV40 origin of replication using COS cells having a gene expressing the SV40 T antigen on the chromosome (eg, pcD) Transformation method When transient expression of a gene is intended, a vector having an SV40 origin of replication using COS cells having a gene expressing the SV40 T antigen on the chromosome (eg, pcD) Transformation method.
  • origin of replication those derived from poliovirus, adenovirus, ⁇ papilloma virus (BPV) and the like can also be used.
  • the expression vector is used as a selectable marker for amplification of gene copy number in the host cell system, such as the aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, and Escherichia coli xanthinguanine phosphoribosyl transferase (Ecogp )
  • APH aminoglycoside transferase
  • TK thymidine kinase
  • Ecogp The gene and the dihydrofolate reductase (dh fr) gene can be included.
  • the DM is incorporated into an appropriate vector, for example, by a retrovirus method, a ribosome method, a catonic ribosome method, an adenovirus method, or the like.
  • a retrovirus method for example, a retrovirus method, a ribosome method, a catonic ribosome method, an adenovirus method, or the like.
  • a method for introduction into a living body This makes it possible to carry out gene therapy for diseases and the like that can be treated by regulating the interaction between the A10 protein and the ARF protein.
  • the vector to be used include, but are not limited to, an adenovirus vector (eg, pAdexl cw) and a retrovirus vector (eg, pZIPneo).
  • A10 protein can be produced in vitro by transforming a host cell with DNA encoding the A10 protein and culturing the transformed cell.
  • the culturing can be performed according to a known method.
  • DMEM, MEM, RPMI 1640, IMDM, and the like can be used as a culture solution for 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.
  • 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, such as CH0, COS, 3T3, Mie Kazuma, BHK (baby hams ter ki dney), HeLa, Vero, and amphibian cells, such as African omega oocytes (Val le, et. al., Nature (1981) 291, 358-340), or insect cells such as Sf9 and Sf2K Tn5.
  • Examples of CH0 cells include dhfr-CHO (Proc. Natl. Acad.Sci. USA (1980) 77, 4216-4220) and CH0K-1 (Proc. Natl. Acad. Sc i.
  • the vector can be introduced into a host cell by, for example, a calcium phosphate method, a DEAE dextran method, a method using Cationic ribosome D0TAP (manufactured by Boehringer Mannheim), an electoral poration method, or a lipofection method. is there.
  • a plant cell for example, a cell derived from Nicotiana tabacum is known as a protein production system, and this may be callus cultured.
  • fungal cells include yeast, for example, the genus Saccharomyces, eg, For example, Saccharomyces cerevisiae and filamentous fungi, for example, the genus Aspergillus, for example, Aspergillus niger are known.
  • yeast for example, the genus Saccharomyces, eg, Saccharomyces cerevisiae
  • filamentous fungi for example, the genus Aspergillus, for example, Aspergillus niger are known.
  • prokaryotic cells some production systems use bacterial cells.
  • bacterial cells include Escherichia coli (E. coli), for example, ⁇ 9, DH5, HB101, and the like, and Bacillus subtilis.
  • examples of a system for producing a protein in vivo include a production system using animals and a production system using plants.
  • the target DNA is introduced into these animals or plants, and proteins are produced and recovered in the animals or plants.
  • mice When using animals, there are production systems using mammals and insects. Goats, pigs, sheep, mice and mice can be used as mammals (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). When a mammal is used, a transgenic animal can be used.
  • the DNA of interest is prepared as a fusion gene with a gene encoding a protein that is specifically produced in milk, such as goat j3 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 baculovirus into which DNA encoding the protein of interest has been inserted (Susurau, M. et al., Ature ( 1985) 315, 592-594).
  • tobacco when using a plant, for example, tobacco can be used.
  • a DNA encoding the protein of interest is converted into a plant expression vector, for example, Insert it into pMON530 and introduce this vector into bacteria such as Agrobacterium tumefaciens.
  • the bacterium is infected with tobacco, for example, Nicotiana tabacum, and the desired polypeptide can be obtained from the leaves of the tobacco (Ma, J.. El al., Eur. J. Immunol. (1994). 24, 131-138).
  • the A10 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 Daniel 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 A10 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 A10 protein, a nucleic acid encoding the A10 protein, and a vector containing the nucleic acid can be formulated according to a known method.
  • Examples of the compound that regulates the expression of the A10 gene or the interaction between the ARF protein and the A10 protein include antisense nucleotides against the A10 gene.
  • Antisense nucleotides are nucleotides that include a region that is complementary to the nucleotide encoding A10 protein.
  • the antisense nucleotide is a polynucleotide comprising at least 15 nucleotides complementary to the nucleotides encoding the A10 protein.
  • the “complementary strand” refers to one strand of a double-stranded nucleic acid consisting of A: T ( ⁇ ⁇ ⁇ in the case of RNA) and G: C base pairs with respect to the other strand.
  • the term “complementary” is not limited to a case where the complementary nucleotide region is a completely complementary sequence, and has a nucleotide sequence of at least 70%, preferably at least 80%, more preferably 90 °, and still more preferably 95% or more. It is only necessary to have the above homology.
  • the algorithm for determining homology may be in accordance with the algorithm described in the literature (Wibur, WJ and Lipman, DJ Proc. Natl. Acad. Sci.
  • Antisense nucleotides are useful as modulators of ARF-mediated signal transduction, p53-mediated signal transduction, and cell growth regulators. That is, antisense nucleotides against the nucleic acid encoding the A10 protein reduce the interaction between the ARF protein and the M0 protein by suppressing the expression of the A10 protein, and regulate ARF-mediated signal transduction and p53-mediated signal transduction. It also controls cell proliferation. In addition, antisense nucleotides against the nucleic acid encoding the A10 protein can be used for the test of the present invention described later.
  • nucleotides include nucleotides or nucleotide derivatives that control the expression of the A10 gene (for example, antisense oligonucleotides, liposome, 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 A10 gene or the sequence of mRNA transcribed from the gene.
  • This antisense oligonucleotide is preferably a continuous one in the A10 gene sequence or mRNA sequence. It is an antisense oligonucleotide for at least 15 nucleotides or more. 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 modified products 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 whose nucleotides corresponding to the nucleotides constituting the predetermined region of DNA or mRNA are all complementary sequences, but also those in which DNA or mRNA and the oligonucleotide hybridize to the A10 gene sequence or mRNA sequence. As far as the resizing can be carried out, a case where one or more nucleotide mismatches are present is also included. ⁇
  • the antisense oligonucleotide derivative acts on A10 protein-producing cells to bind to DNA or mRNA encoding the protein, thereby inhibiting its transcription or translation or accelerating the degradation of mRNA, By suppressing the expression of the A10 gene, it has the effect of suppressing the interaction between the A10 protein and the ARF protein.
  • 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.
  • 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, ribofectin or a derivative thereof can be mentioned.
  • Antisense oligo The dosage of the nucleotide derivative can be appropriately adjusted depending on the condition of the patient, and a preferable 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 A10 gene expression and are therefore useful in suppressing the interaction between A10 and ARF proteins.
  • An expression inhibitor containing an antisense oligonucleotide is useful in that it can suppress the interaction between the A10 protein and the ARF protein.
  • the antisense nucleic acid of the A10 gene serves as an inhibitor of the interaction between the ARF protein and the A10 protein. In addition, they act as regulators of ARF protein-mediated signal transduction, p53 protein-mediated signal transduction regulators, and cell growth regulators.
  • the present invention also relates to the use of antisense nucleotides against the A10 gene to regulate the interaction between the ARF protein and the A10 protein, signal transduction via the ARF protein or p53, or cell growth.
  • Examples of the compound that reduces the interaction between the ARF protein and the A10 protein include, for example, an antibody against the A10 protein or the ARF protein.
  • An antibody against the A10 protein or the ARF protein serves as an inhibitor of the interaction between the ARF protein and the A10 protein.
  • an antibody against the A10 protein or the ARF protein can be a regulator of ARF protein-mediated signal transduction, a P53 protein-mediated signal transduction regulator, and a cell growth regulator. That is, if the interaction between the A10 protein and the ARF protein is inhibited by the antibody, signal transmission via the ARF protein and signal transmission via the p53 protein can be regulated, and further, cell proliferation can be controlled.
  • the present invention also relates to the use of antibodies to the A10 or ARF protein to regulate the interaction between the ARF protein and the A10 protein, signal transduction via the ARF protein, signal transduction via the p53 protein, and cell growth. .
  • the antibody is also used in the test of the present invention described later.
  • Antibodies to the A10 protein or ARF protein can be prepared by known methods.
  • the form of the antibody is not particularly limited.
  • monoclonal antibodies are also included. Also obtained by immunizing an immunized animal such as a heron with an antigenic protein. It includes antisera, all classes of polyclonal and monoclonal antibodies, as well as human and recombinant humanized antibodies.
  • 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.
  • A10 protein of human origin can be prepared as described above.
  • the protein used as the sensitizing antigen may be a full-length protein or a partial peptide of the protein.
  • the partial peptide of the protein include an amino (N) terminal fragment and a carboxy (C) terminal fragment of the protein.
  • a binding region between the A10 protein and 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 A10 protein or a fragment thereof is inserted into a known expression vector system, a host cell is transformed with the vector, and the protein of interest is transferred from inside or outside the host cell.
  • fragments thereof may be obtained by a known method, and these may be used as a sensitizing antigen.
  • a cell expressing the protein, a lysate thereof, or a chemically synthesized A10 protein may be used as the sensitizing antigen.
  • the short peptide can be appropriately combined with a carrier protein such as keyhole limpet hemosinin, human serum albumin, or ovalbumin to obtain an antigen.
  • 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, In general, rodents, ephedra and primates are used.
  • mice for example, mice, rats, and hamsters are used.
  • a heronoid animal for example, a heron is used.
  • monkeys are used as primate animals. Monkeys include monkeys in the lower nose (old world monkeys), such as the cynomolgus monkey, the macaque monkey, the baboon, and the chimpanzee — Etc. are used.
  • 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) or physiological saline, and then added to a normal adjuvant, if desired, for example, Freund's complete adjuvant. Is mixed in an appropriate amount, emulsified, and administered to a mammal. 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.
  • an appropriate 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 collected 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, a fraction that recognizes only the antigen protein is obtained, and this fraction is further purified using a protein A or protein G column.
  • immunoglobulin G or M can be prepared.
  • 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. (Gal fre, G. and Milsiein, C., Methods Enzymol. (1981) 73, 3-46).
  • the hybridomas obtained by cell fusion are selected by culturing them in a conventional selective culture medium, for example, a HAT culture medium (a culture medium containing hypoxanthine, aminopterin and thymidine). Culturing in the HAT medium 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, a conventional limiting dilution method is performed to screen and clone a hybridoma producing the desired antibody.
  • a conventional selective culture medium for example, a HAT culture medium (a culture medium containing hypoxanthine, aminopterin and thymidine). Culturing in the HAT medium is continued for a time sufficient to kill cells other than the target hybridoma (non-fused cells), usually for several days to several weeks.
  • a conventional limiting dilution method is performed to screen and clone a hybridoma producing the desired antibody.
  • human lymphocytes for example, human lymphocytes infected with the EB virus
  • human lymphocytes can be obtained by in vitro protein, protein-expressing cells or lysates thereof. After sensitization, the sensitized lymphocytes are fused with human-derived myeloma cells capable of permanent division, for example, U266, to obtain a hybridoma that produces a desired human antibody having a protein binding activity.
  • the obtained hybridoma is implanted intraperitoneally into a mouse, ascites is collected from the mouse, and the obtained monoclonal antibody is subjected to, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, antigen It can be prepared by purifying the purified protein using a coupled affinity column.
  • the obtained antibody is used for detection of A10 protein and is also a candidate for an antagonist of A10 protein.
  • An antibody against the A10 protein is a candidate for a drug that suppresses the interaction between the A10 protein and the ARF protein.
  • the antibodies may be human or human antibodies.
  • a transgenic animal having a human antibody gene repertoire was immunized with a protein serving as an antigen, a protein-expressing cell or a lysate thereof to obtain an antibody-producing cell, which was fused with a myeloma cell.
  • a human antibody against a protein can be obtained using a hybridoma (International Publication Nos.WO92_03918, W093-2227, W094-02602, W094-25585, W096-33735 and W096-34096).
  • cells in which immune cells such as sensitized lymphocytes producing 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 MACMILLAN PUBLISHERS LTD, 1990).
  • Recombinant antibodies are produced by cloned DNA encoding the antibodies from immunized cells such as hybridomas or sensitized lymphocytes that produce the antibodies, incorporated into an appropriate vector, and introduced into a host to produce them.
  • the A10 protein may be an antibody fragment or a modified antibody.
  • Fab, F (ab ') 2, Fv, or a single chain Fv (scFv) in which an Fv of an H chain and an L chain are linked by an appropriate linker Huston, JS et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883.
  • the antibody was treated with an enzyme, for example, papain or pepsin, to generate an antibody fragment, or a gene encoding these antibody fragments was constructed and introduced into an expression vector.
  • a suitable host cell for example, Co, MS et al., J. I. thigh unol. (1994) 152, 2968-2976; Better, M. and Horwitz, AH, Methods Enzymol. (1989) 178) Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663; Rousseaux, J. et al. , Methods Enzyiol. (1986) 121, 663-669; Bird, RE and Walker, BW, Trends Biotechnol. (1991) 9, 132-137).
  • a suitable host cell for example, Co, MS et al., J. I. thigh unol. (1994) 152, 2968-2976; Better, M. and Horwitz, AH, Methods Enzymol. (1989) 178) Pluckthun, A. and Skerra, A., Method
  • modified antibody an antibody bound to various molecules such as polyethylene glycol (PEG) can be used.
  • PEG polyethylene glycol
  • the “antibody” referred to in the present invention also includes these modified antibodies.
  • Such a modified antibody can be obtained by subjecting the obtained antibody to chemical modification. These methods are already established in this field Have been.
  • a chimeric antibody composed of a variable region derived from a non-human antibody and a constant region derived from a human antibody, or a CDR (complementarity determining region) derived from a non-human antibody and a FR (frame) derived from a human antibody, using known techniques. (Work region) and a 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, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, etc. Can be separated and purified (Antibodies: A Laboratory Manual, Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988), but are not limited thereto.
  • the concentration of the antibody obtained above can be measured by measuring absorbance or by enzyme-linked immunosorbent assay (ELISA). Columns used for affinity chromatography include protein A column and protein G column.
  • columns using Protein A column include Hyper D, POROS, Sepharose FF (Pharmacia), etc.
  • chromatography other than affinity chromatography include ion exchange chromatography, hydrophobic chromatography, and gel. Filtration, reverse phase chromatography, adsorption chromatography, etc. (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These chromatographys can be performed using liquid phase chromatography such as HPLC and FPLC.
  • Methods for measuring the antigen-binding activity of an antibody include, for example, measurement of absorbance, Enzyme-linked immunosorbent assay (ELISA), EIA (enzyme immunoassay), RIA (radioimmunoassay) or fluorescent antibody method can be used.
  • ELISA Enzyme-linked immunosorbent assay
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • fluorescent antibody method can be used.
  • ELISA Enzyme-linked immunosorbent assay
  • a protein of interest is added to a plate on which the antibody is immobilized, and then a sample containing the antibody of interest, for example, a culture supernatant of antibody-producing cells or a purified antibody is added.
  • a secondary antibody that recognizes an enzyme such as an antibody labeled with alkaline phosphatase
  • an enzyme substrate such as p-nitrophenyl phosphate
  • Activity can be assessed.
  • a fragment of the A10 protein for example, a fragment comprising the C-terminus thereof may be used.
  • BIAcore Pieracia
  • the A10 protein or a partial peptide of the ARF protein can suppress the interaction between the ARF protein and the A10 protein by inhibiting the binding between the original ARF protein and the A10 protein. Therefore, the A10 protein or a partial peptide of the ARF protein becomes a compound that regulates the interaction between the ARF protein and the A10 protein, and is a regulator of ARF protein-mediated signal transduction, a P53 protein-mediated signal transduction regulator, and a cell growth regulator. Agents, antitumor agents and the like as the above-mentioned various regulators.
  • Such a partial peptide has an amino acid sequence of at least 7 amino acids or more, preferably 8 amino acids or more, and more preferably 9 amino acids or more.
  • the partial peptide can be produced by a genetic engineering technique, a known peptide synthesis method, or by cleaving the A10 protein or ARF protein with an appropriate peptide.
  • the peptide may be synthesized by, for example, any of a solid phase synthesis method and a liquid phase synthesis method.
  • the present invention relates to a reagent for testing the interaction between an ARF protein and an A10 protein, comprising an antibody that binds to the A10 protein, or a DNA encoding the A10 protein or a polynucleotide containing at least 15 nucleotides complementary to a complementary strand thereof.
  • a reagent for testing the interaction between an ARF protein and an A10 protein comprising an antibody that binds to the A10 protein, or a DNA encoding the A10 protein or a polynucleotide containing at least 15 nucleotides complementary to a complementary strand thereof.
  • Abnormal A10 gene expression It induces a change in the interaction between the protein and the Alt) protein, modifies the intracellular function of the ARF protein, and can alter the regulation of cell proliferation and apoptosis.
  • the test reagent for the interaction between the ARF protein and the A10 protein of the present invention includes a test for abnormal signal transduction via the ARF protein, a test for abnormal signal transduction via the P53 protein, a test for abnormal cell proliferation, It is useful for testing for apoptosis. It is also useful for examining diseases. That is, the expression of the A10 gene or the interaction of the A10 protein with the ARF protein may be involved in diseases involving cell proliferation or apoptosis.
  • the A10 gene or A10 protein can be used for tumor testing. For example, by detecting or analyzing the A10 protein or mRNA or DNA encoding the protein in a test sample, the risk of tumor development, presence of tumor cells, tumor progression, tumor malignancy, tumor type Etc. can be inspected.
  • tumor test means not only a test for a patient who has formed a tumor due to a mutation (structural or expression level mutation) of the A10 gene, but also a subject whose A10 gene expression level is high. Includes testing for A10 gene expression levels and testing for gene mutations to determine whether cancer is more likely to result from abnormalities or mutations in the gene. That is, even if the symptom has not yet been expressed on the surface, cancer is caused by increased expression of the A10 gene or mutation (including polymorphism) in one or both of the A10 alleles The danger is considered to be affected.
  • the test of the present invention can be performed using, for example, an antibody that binds to the A10 protein, or a polynucleotide that contains at least 15 nucleotides complementary to the DNA encoding the A10 protein or its complementary strand.
  • the polynucleotide may be DNA or RNA, or a derivative thereof.
  • the polynucleotide is useful for, for example, detection and amplification of DNA encoding the M0 protein, and detection of expression of the DNA.
  • detection of DNA includes detection of DNA mutation.
  • complementary strand is the same as above A: T (however, in the case of RNA, U) and G: C refers to one strand of a double-stranded nucleic acid composed of base pairs and the other strand.
  • the term “complementary” is not limited to the case where the complementary nucleotide region is a completely complementary sequence, and has at least 70, preferably at least 80%, more preferably still more preferably 9 or more homology on the base sequence. It is only necessary to have The algorithm for determining homology is based on the algorithm described in the literature (Wibur, WJ and Lipman, DJ, Proc. Natl. Acad. Sci. USA (1983) 80, 726-730). Good.
  • this polynucleotide as a probe or primer and detecting the expression or mutation of the A10 gene, the interaction between the ARF protein and the A10 protein can be examined. As a result, abnormalities in cell proliferation or cell proliferative diseases such as tumors can be examined and diagnosed.
  • the test using an antibody that binds to the A10 protein comprises contacting the antibody with a sample expected to contain the M0 protein, and detecting or measuring an immune complex between the antibody and the protein. Can be carried out by a detection or measurement method.
  • Significant changes compared to A10 protein expression in normal tissues are indicative of abnormal cell proliferation or cancerous cells. Therefore, for example, by examining the expression level of the A10 gene, the interaction between the ARF protein and the A10 protein can be examined. This makes it possible to test for abnormal cell growth or cell proliferative diseases such as tumors.
  • these antibodies and polynucleotides are used as test reagents, they may be used as appropriate, such as stabilizers, preservatives, salts, and buffers.
  • test reagent of the present invention can be such a composition.
  • 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 test methods of the present invention is a method including a step of detecting the expression level of a nucleic acid encoding an M0 protein in a test sample. Expression can be detected using transcription or translation as an indicator.
  • a test method can be performed by (a) adding A10 protein to a patient-derived RNA sample. Contacting a polynucleotide containing at least 15 nucleotides complementary to DNA encoding white matter or its complementary strand; and (b) detecting the binding of the polynucleotide to the RNA sample.
  • Such a test can be performed by, for example, Northern hybridization—RT-PCR.
  • Inspections using RT-PCR include (a) the process of synthesizing cDNA from a patient-derived RNA sample, (b) using the synthesized cDNA as type III, and the above polynucleotide as a primer, Performing a polymerase chain reaction; and (c) detecting DNA amplified by the polymerase chain reaction.
  • Northern hybridization and RT-PCR can be performed by known genetic engineering techniques. Detection by DNA chip or DNA microarray is also possible. Abnormalities can be detected by comparing the results with those of healthy subjects as controls. Increased expression of the nucleic acid encoding the A10 protein suggests enhanced interaction between the A10 protein and the ARF protein, and decreased expression of the nucleic acid encoding the A10 protein indicates decreased interaction between the A10 protein and the ARF protein Suggest.
  • the test method of the present invention includes a method including a step of detecting the amount of A10 protein in a test sample derived from a patient.
  • a test can be performed using, for example, an antibody against the A10 protein.
  • the test using an antibody against A10 includes (a) contacting an antibody that binds to the A1G 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 A10 protein, Western blot, immunohistochemistry, ELISA, or the like.
  • abnormal expression of the A10 gene was caused, for example, by examining the expression of a tissue collected by biopsy by immunohistochemical staining or in situ hybridization. It is conceivable to identify the lesion.
  • An abnormal expression of the A10 gene indicates, for example, the possibility of onset and / or progression of cancer. Diseases such as cancer are thought to be caused by various causes. For example, cancer If the expression of the A10 gene is abnormal in, the p53 pathway is predicted to be suppressed, and therapies that target this pathway will be administered. It can be used for diagnosis when performing
  • the test of the present invention can also be performed by detecting a mutation in the A10 protein or a mutation in a nucleic acid encoding the protein. Since the A10 gene may be involved in the onset and Z or progression of cancer, mutations in the protein and the nucleic acid suggest a risk of onset and progression of cancer.
  • Mutations in the A10 protein include structural and functional mutations.
  • a structural variation of a protein can be examined by comparing the molecular weight of the protein with a protein derived from a healthy subject by Western blotting or the like of a protein sample derived from a patient using an antibody against the A10 protein.
  • mutations in the A10 protein can be detected using as an index a change in the modification of the protein, a change in the binding of the protein or antibody that binds to the A10 protein, and the like.
  • ELISA using an antibody against the A10 protein, immunoprecipitation, and pull-down can be used.
  • the test of the present invention can also be performed by detecting the binding between the A10 protein and the ARF protein.
  • Changes in the binding ability between the A10 protein and the ARF protein may be involved in the development and progression of cancer. Protein binding can be evaluated, for example, by ELISA using an antibody against the A10 protein or ARF protein, immunoprecipitation, and pull-down.
  • a polynucleotide containing at least 15 nucleotides complementary to the DNA encoding the A10 protein or its complementary strand can be used.
  • the nucleotide is the nucleotide sequence of the cDNA encoding A10 protein (eg, SEQ ID NO: 1) and the nucleotide sequence of the genomic DNA sequence (including exons, introns, and endogenous transcription control sequences), or the complement thereof.
  • Complementary polynucleotides probes and primers. Mutations in nucleic acids can be determined using mRNA, cDNA, or genomic MA.
  • the mutation test was performed to identify patients (carrier 1) who had a mutation in one of the A10 alleles. Inspections are also included. It also includes testing to determine the type of single nucleotide polymorphism (SNP). Polymorphisms in the A10 gene may be related to susceptibility to cancer.
  • the polynucleotide When used as a primer, the polynucleotide is usually between 15 bp and 100 bp, preferably between 17 bp and 30 bp.
  • the primer may be any primer that can amplify at least a part of the A10 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 A10 gene.
  • a polynucleotide as a probe generally has a chain length of at least 15 bp if it is a synthetic polynucleotide. It is also possible to denature double-stranded DNA obtained from a clone integrated into a vector such as a plasmid DNA and use it as a probe.
  • the probe may be any probe as long as it is complementary to at least a part of the base sequence of the A10 gene or a region that regulates its expression, or its complementary strand. 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 A10 gene.
  • Labeling methods include, for example, a method of labeling by phosphorylating the end of the polynucleotide with 3 using T4 polynucleotide kinase, or a random hexamer oligonucleotide using a DNA polymerase such as Klenow enzyme.
  • the primer include a method of incorporating a substrate base labeled with an isotope II, a fluorescent dye, or a biotin (eg, a random prime method).
  • One embodiment of a method for detecting a mutation in the A10 gene is a method for directly determining the nucleotide sequence of the A10 gene in a patient. For example, using the above nucleotides as primers and MA isolated from a patient suspected of having a disease caused by a mutation in the A10 gene as type ⁇ ⁇ , a PCR (Polymerase Chain Reaction) method, etc. Part or all (for example, regions containing exons, introns, promoters, enhancers) ) Is amplified and its base sequence is determined. By comparing this with the sequence of the A10 gene of a healthy subject, a disease caused by mutation of the A10 gene can be examined.
  • PCR Polymerase Chain Reaction
  • test method of the present invention various methods are used in addition to the method of directly determining the nucleotide sequence of DNA derived from a patient.
  • One embodiment is (a) a step of preparing a DNA sample from a patient, and (b) a patient-derived method using, as a primer, a polynucleotide containing at least 15 nucleotides complementary to DNA encoding A10 protein or its complementary strand.
  • C dissociating the amplified MA into single-stranded DNA, (d) separating the dissociated single-stranded MA on a nondenaturing gel, and (e) separating Comparing the mobility of the single-stranded DNA on the gel with a control of a healthy subject.
  • Such methods include the PCR-SSCP ⁇ single-strand conformation polymorp ism, the cloning and polymerase chain reaction-single-strand conformation polymorphism analysis of anonymous Alu repeats on chromosome 11, Genomics , 1992 Jan 1,12 (1): 139-146; Detection of p53 gene mutations in human brain tumors by single-strand conf ormat ion polymorphism analysis of polymerase chain react ion products, Oncogene, 1991 Aug 1, 6 (8): 1313-1318; Multiple fluorescence-based PCR-SSCP analysis with post labeling, PCR Methods Appl., 1995 Ar 1, (5): 275-282).
  • This method has advantages such as relatively simple operation and small sample size, 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 the dissociated DNA strands are electrophoresed in a polyacrylamide gel containing no denaturing agent, the single-stranded DNAs with the same complementary length move to different positions according to the difference in their higher-order structures. The single-stranded substitution also changes the higher-order structure of the single-stranded DNA, indicating different mobilities in polyacrylamide gel electrophoresis. Therefore, by detecting this change in mobility, the presence of mutation due to point mutation, deletion, And can be detected.
  • first, part or all of the A10 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 / or all introns of the A10 gene, as well as the promoter or enhancer of the A10 gene.
  • amplifying gene fragments by PCR use an isotope such as 32 P or a primer labeled with a fluorescent dye or biotin, etc., or use a substrate salt labeled with an isotope such as 32 P or a fluorescent dye or biotin in the PCR reaction solution.
  • the DNA fragment to be synthesized is labeled by adding groups and performing PCR.
  • labeling can also be performed by adding an isotope such as 32 P or the like with a Klenow enzyme or a substrate base labeled with a fluorescent dye or biotin to the synthesized DNA fragment after the PCR reaction.
  • the labeled DNA fragment thus obtained is denatured by applying heat or the like, and electrophoresis is carried out using a polyacrylamide gel containing no denaturing agent 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, but are usually performed at room temperature (20 to 25 ° C), and when the desired separation cannot be obtained, optimal mobility is provided at a temperature of 4 to 30 ° C. Investigate the temperature. After electrophoresis, the mobility of the DNA fragments is detected and analyzed by autoradiography using an X-ray film or a scanner that detects fluorescence. If a band with 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.
  • the band can be detected by staining the gel after electrophoresis with ethidium gel, silver staining, or the like.
  • A preparing a DNA sample from a patient, (b) ) Amplifying the patient-derived DNA using the above polynucleotide as a primer, (c) cleaving the amplified DNA, (d) separating the DNA fragment according to its size, (e) separating The DNA fragment thus obtained is hybridized with a labeled probe capable of detecting the amplified fragment, and (0 a step of comparing the size of the detected DNA fragment with a control of a healthy subject).
  • Examples of such a method include a method using a restriction enzyme fragment length polymorphism (Restriction Fragment Length Polymorphism / RFLP) and a PCR-RFLP method.
  • Restriction enzymes are usually used to cut off employment. Specifically, when there is a mutation at the recognition site of the restriction enzyme, or when there is a base insertion or deletion in the DNA fragment generated by the restriction enzyme treatment, the size of the fragment generated after the restriction enzyme treatment is healthy Change compared to the person. By amplifying the portion containing this mutation by PCR and treating it with each restriction enzyme, these mutations can be detected as a difference in the mobility of the band after electrophoresis.
  • the presence or absence of a mutation can be detected by treating chromosome DN / V 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.
  • this cDNA can be used as type III to amplify part or all of the A10 gene by PCR, cut it with restriction enzymes, and then examine the difference in mobility.
  • RNA instead of DNA prepared from a patient.
  • methods include (a) preparing an RNA sample from a patient, (b) separating RNA prepared according to size, and (c) labeling the isolated RNA with a detectable label. And (d) comparing the size of the detected RNA with a control of a healthy individual.
  • RNA prepared from a patient is electrophoresed, and Perform Northern Plotting using a probe to detect differences in mobility.
  • 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 polynucleotide as a primer, and (c) amplifying the MA A step of separating on a gel in which the concentration of a denaturing agent is gradually increased, and (d) a step of comparing the mobility of the separated DNA on the gel with a control of a healthy person.
  • DGGE denaturant gradient gel electrophoresis
  • an allele-specific oligonucleotide (Allele Specific 01 igonuclide / ASO) 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 a 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 liponuclease A mismatch cleavage method is also possible.
  • part or all of the A10 gene is amplified by PCR or the like, and this is hybridized with a labeled RNA prepared from A10 cDNA or the like in which this is inserted into a plasmid vector or the like. Since the hybrid has a single-stranded structure in the portion where the mutation is present, this portion is cleaved with liponuclease A, and this is analyzed by autoradiography or the like. The presence of the mutation can be detected.
  • FIG. 1 is a photograph showing the results of screening for proteins that interact with the ARF protein using a two-hybrid system using yeast cells. Filters of yeast cells transfected with the plasmid shown in the figure. 1) S-gal assembly. In the cells transfected with clones A-10 and P19ARF, activation of the 3-gal repo overnight gene, which indicates the interaction between the two, was observed.
  • FIG. 2 is a photograph showing the in vivo binding of pl9AR protein and A10 protein.
  • An expression vector encoding pl9ARF-myc and A10-V5 was transfected into C0S7 cells. The interaction between pl9ARFmyc protein and A10-V5 protein was confirmed by immunoprecipitation of the cell extract using an anti-Myc antibody (anti-myc) or an anti-V5 antibody (anti-V5).
  • lanes 3 and 8 represent the test samples in which the transfected cells were immunoprecipitated
  • lanes 1, 2, 6, and 7 are the pre-immunoprecipitation controls (Input)
  • lanes 4, 5, 9, and 10 are the Untransfected control (Unt.) Is shown.
  • FIG. 3 is a photograph showing the intracellular localization of pl9ARFmyc protein and GFP-A10 protein.
  • pl 9 A single expression vector encoding the ARFmyc protein or the GFP-tagged A10 protein (GFP-A10), or both, were transfected into NIH3T3 cells. The cells were fixed 24 hours after the transfusion and observed under a microscope.
  • a gene encoding mouse pl9ARF (Accession No. L76092) was incorporated into pODB8, a vector for yeast Two-Hybrid, and transfected into yeast strain 69 / 2A.
  • the transfected yeast was selected and cloned on a basic medium excluding Tributofan.
  • the yeast into which the P19ARF was introduced was conjugated with a pre-transformed library yeast (Clontech), and a yeast growing on a basal medium excluding tributofan and leucine was isolated.
  • a gene fragment was extracted from the isolated yeast, and the DNA sequence was analyzed using the ABI 377 automatic sequence base determinator by the dideoxy chain yuichi minion method.As a result, the A10 gene was successfully isolated. did. When the sequence of the isolated gene fragment was searched, it was found that the gene was registered in the DNA sequence data bank (GenBank Accession Number Thigh—017632). The nucleotide sequence of Al 2 O cDNA and the amino acid sequence of the protein encoded thereby are shown in SEQ ID NOs: 1 and 2, respectively.
  • the expression vector for mammalian cells contains the gene with myc tag added to P19ARF, and the vector (pcDNA3-V5, Invitrogen) expresses it by fusing it with V5 protein.
  • the protein expression vectors AlO-V5 and pl9ARFmyc were respectively integrated and constructed. To confirm whether the two proteins are actually interacting, pl9ARFmyc, AlO-V5 expression vector was transfected into C0S7 cells, the protein was expressed, and the protein complex was precipitated with a monoclonal inyc antibody. I let it. After separating this complex by SDS-PAGE, the interaction was detected by Western blotting using the V5 antibody (Fig. 2, left).
  • pl9ARFmyc Integrate the myc-tagged gene with pl9ARF into an expression vector for mammalian cells (PCDNA3 Invitrogen) and the A10 gene into a vector (pEGFPCl, Clontech) that is fused to GFP protein for expression.
  • the protein expression vectors GFP-A10 and pl9ARFmyc were constructed.
  • pl9ARFmyc, GFP-A10, or l9ARFmyc + GFP-A10 was transfected into NIH3T3 cells, and their intracellular localization was examined using a fluorescence laser microscope (Olympus).
  • pl9ARFmyc was detected by reacting with an anti-myc antibody (Invitrogen), and the antibody was reacted with a Texas Red-conjugated secondary antibody and detected.
  • the present inventors have clarified that the A10 protein and the ARF protein interact with each other, and that the interaction causes the A10 protein to translocate to the nucleolus and affect the function of ARF. Based on these findings, screening for a drug that suppresses the interaction between the A10 protein and the ARF protein is a novel method that can induce cell growth suppression or growth promotion by activating or inactivating ARF. It will be possible to develop new drugs.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne une méthode de criblage d'un médicament effectuée au moyen de l'interaction entre une protéine ARF et une protéine A10. On a remarqué que la protéine A10 se lie à la protéine ARF, on a également détecté que la protéine A10 interagit avec la protéine ARF et migre dans le nucléole. Au moyen du ciblage du gène A10 ou de son produit d'expression, on peut cribler un composé qui régule l'interaction entre la protéine A10 et la protéine ARF. Un médicament régulant l'interaction entre la protéine A10 et la protéine ARF constitue un nouveau médicament qui exerce un effet antitumoral par le biais de la régulation de la fonction ARF.
PCT/JP2002/001194 2001-02-14 2002-02-13 Methode de criblage d'un medicament regulant l'interaction entre une proteine arf et une proteine a10 WO2002065116A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2002564583A JP4189456B2 (ja) 2001-02-14 2002-02-13 Ａｒｆタンパクとａ１０タンパクの相互作用を調節する薬剤のスクリーニング方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2001-36580 2001-02-14
JP2001036580 2001-02-14

Publications (1)

Publication Number Publication Date
WO2002065116A1 true WO2002065116A1 (fr) 2002-08-22

Family

ID=18899841

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2002/001194 WO2002065116A1 (fr) 2001-02-14 2002-02-13 Methode de criblage d'un medicament regulant l'interaction entre une proteine arf et une proteine a10

Country Status (2)

Country Link
JP (1) JP4189456B2 (fr)
WO (1) WO2002065116A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005054870A3 (fr) * 2003-11-26 2006-01-05 Univ Vanderbilt Methodes d'estimation des interactions entre p19-arf et cmyc

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000008153A2 (fr) * 1998-08-06 2000-02-17 St. Jude Children's Research Hospital L'arf-p19, regulateur du cycle cellulaire mammifere
WO2001042302A1 (fr) * 1999-12-06 2001-06-14 Japan Science And Technology Corporation PROTEINE ET ADNc HUMAINS

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000008153A2 (fr) * 1998-08-06 2000-02-17 St. Jude Children's Research Hospital L'arf-p19, regulateur du cycle cellulaire mammifere
WO2001042302A1 (fr) * 1999-12-06 2001-06-14 Japan Science And Technology Corporation PROTEINE ET ADNc HUMAINS

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Also Published As

Publication number Publication date
JPWO2002065116A1 (ja) 2004-06-17
JP4189456B2 (ja) 2008-12-03

Similar Documents

Publication Publication Date Title
JP2020036596A (ja) 新規ヘモポエチン受容体蛋白質、ｎｒ１０
US20080207549A1 (en) Genes and polypeptides relating to hepatocellular or colorectal carcinoma
WO2001023556A1 (fr) Nouvelle proteine receptrice d'hemopoietine (nr12)
JP5395767B2 (ja) トランスポーター遺伝子ｏａｔｐ−ｂ、ｃ、ｄ、およびｅ
WO2001009316A1 (fr) Nouveaux genes codant la proteine kinase / proteine phosphatase
WO2001009345A1 (fr) Nouveaux genes codant des proteines kinase et des proteines phosphatase
WO2002020770A1 (fr) Methode de criblage d'un agent antitumoral a l'aide d'une interaction entre une proteine arf et une proteine hk33
US8067540B2 (en) Human ALEX1 proteins
JP4189456B2 (ja) Ａｒｆタンパクとａ１０タンパクの相互作用を調節する薬剤のスクリーニング方法
JP4590107B2 (ja) 新規胎児性遺伝子
EP1215284A1 (fr) Gene de type tsg
US20030157501A1 (en) Novel human rna helicase, helicain
JP2008099690A (ja) 新規ヘモポエチン受容体蛋白質、ｎｒ１２
JP4499926B2 (ja) 腫瘍抑制遺伝子
EP1215283A1 (fr) NOUVEAU GENE DEC2 DE FACTEUR DE TRANSCRIPTION DE TYPE bHLH
EP1559786A1 (fr) Proteine membranaire produite dans les mastocytes
JP4942904B2 (ja) ｚｉｎｃフィンガードメインを有する新規転写因子
CN100497624C (zh) Wt1相互作用蛋白质wtip
JP2005046024A (ja) 新規なプロテインキナーゼおよびプロテインキナーゼスプライス変異体をコードする遺伝子
JP2004267003A (ja) ヒト白血球型抗原領域に存在する新規遺伝子
EP1260584A1 (fr) Nouvelle proteine comportant une sequence signal
JP2005521421A (ja) 結腸直腸癌を診断および治療する方法
WO2001027270A1 (fr) Gene ys68 associe a l'hematopoiese precoce
JP2002000279A (ja) 初期造血に関わるｙｓ６８遺伝子
WO2001083738A1 (fr) Facteur de regulation de differenciation et de determination du sexe

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2002564583

Country of ref document: JP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载