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WO2013035896A1 - Utilisations inédites de kiaa1764 en tant que régulateur de l'apoptose et du vieillissement - Google Patents

Utilisations inédites de kiaa1764 en tant que régulateur de l'apoptose et du vieillissement Download PDF

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WO2013035896A1
WO2013035896A1 PCT/KR2011/006559 KR2011006559W WO2013035896A1 WO 2013035896 A1 WO2013035896 A1 WO 2013035896A1 KR 2011006559 W KR2011006559 W KR 2011006559W WO 2013035896 A1 WO2013035896 A1 WO 2013035896A1
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apoptosis
gene
kiaa1764
protein
cells
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Korean (ko)
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신득용
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Industry Academic Cooperation Foundation of Dankook University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the present invention relates to the use of KIAA1764 as a novel apoptosis and aging regulator, and more particularly, a disease associated with apoptosis under activity including KIAA1764, a gene for regulating novel apoptosis or cell aging, the gene or a protein thereof. And a method for screening apoptosis promoters or inhibitors using transformed cells or in vitro expression systems comprising a transcriptional control region of said gene and said gene or reporter gene. .
  • Apoptosis refers to the destruction or suicide of cells in eukaryotic cells and is a basic intracellular process for maintaining homeostasis in individuals, including controlling the normal development of animals, or eliminating unnecessary or abnormal cells. to be.
  • Apoptosis occurs in response to various external and internal stimuli, with the progression of apoptosis, such as cytoplasmic breakdown, blebbing of cell membranes, changes in the cytoskeleton, cell contraction, chromosome condensation and DNA fragmentation. Characteristic changes occur 8 .
  • Such apoptosis is a physiologically important phenomenon and is precisely regulated by in vivo complex mechanisms, and induction of inappropriate cell death, ie, inhibition or promotion of apoptosis due to abnormal regulation, is associated with many diseases.
  • apoptosis can lead to the destruction of certain cells and thereby the loss of function in vivo, which can lead to central nervous system diseases such as a number of acute and chronic degenerative diseases (eg, Alzheimer's disease, Parkinson's disease, cerebral ischemia). / Stroke) 13, 20, 21, 18 , cardiovascular disease 27 , and autoimmune diseases 16,12 such as Graves' disease and type 2 diabetes.
  • central nervous system diseases such as a number of acute and chronic degenerative diseases (eg, Alzheimer's disease, Parkinson's disease, cerebral ischemia). / Stroke) 13, 20, 21, 18 , cardiovascular disease 27 , and autoimmune diseases 16,12 such as Graves' disease and type 2 diabetes.
  • a representative disease associated with abnormal inhibition of cell death is cancer. That is, the accumulation of tumor cells that could not be properly removed due to the underactivation of apoptosis is known to be closely related to the development of cancer 22 .
  • Pathways leading to apoptosis can be classified into pathways through and / or not through apoptosis receptors.
  • the former is apoptosis induced by the binding of a specific ligand to its receptor, and the receptors involved therein include Fas, tumor necrosis factor receptor 1 (TNFR), and TRAIL (TNF-related apoptosis-inducing ligand).
  • the latter is apoptosis induced by stress, and stresses that can cause apoptosis include ultraviolet rays, heat shock, gamma irradiation, and hypoxia.
  • Apoptosis caused by this stimulus is completed via factors acting in the downstream stages, a representative caspase protease. They are a group of proteins that act sequentially in the signaling pathway leading to cell death, and are involved in cell death by cleaving specific amino acid (aspartate) sites to activate a series of proteins.
  • TRAIL protein as a cancer therapeutic agent as apoptosis promoting factor induced via cellular receptors.
  • Fulda et al. 4 also proposed the use of Fas-L as a target of cell receptor-induced apoptosis regulation.
  • p53 a representative protein associated with induction of apoptosis
  • p53 is involved in the removal of these cells as mediators of apoptosis in cells showing abnormal cell growth by oncogenic genes such as Myc . 23 .
  • p53 is a representative tumor suppressor protein involved in repairing damaged DNA and cell cycle regulation. It acts as a transcriptional regulator with high affinity for a target gene in a target gene for regulation of expression of the target gene at the transcription level. It plays an important role in inhibiting the division of cells or selectively destroying abnormal cells with damaged DNA or abnormal division, thereby preventing their progression to cancer 15 .
  • WO 03/076647 discloses JADE genes, proteins that function as cell death and cell cycle regulators downstream of cell death, and methods for screening apoptosis control substances using the same.
  • Korean Patent Application Publication No. 2001-113088 discloses CIA proteins, genes and their use to interact with specific DNases activated by caspases to regulate apoptosis.
  • Korean Patent Application Publication No. 2002-40521 discloses an anticancer agent comprising microlactone using apoptosis.
  • Patent Document 1 International Patent Publication WO 03/076647
  • Patent Document 2 Korean Patent Application Publication 2001-113088
  • Patent Document 3 Korean Patent Application Publication 2002-40521
  • Non-Patent Document 1 Arendt, C. S., and Hochstrasser, M. (1997). Identification of the yeast 20S proteasome catalytic centers and subunit interactions required for active-site formation. Proc Natl Acad Sci U S A 94: 7156-7161.
  • Non-Patent Document 2 Altschul, S and Gish, W (1990). Basic Local Alignment Search Tool J. Mol. Biol. 215: 403-410.
  • Non-Patent Document 3 Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001). Duplexes of 21 nucleotide RNA mediate RNA interference in cultured mammalian cells. Nature 411: 494-8
  • Non-Patent Document 4 Fulda, S., Strauss, G., Meyer, E., and Debatin, K.M. (2000). Functional CD95 ligand and CD95 death-inducing signaling complex in activation-induced cell death and doxorubicin-induced apoptosis in leukemic T cells. Blood 95 (1), 301-308
  • Non-Patent Document 5 Fire, A.S. (1998). RNA-triggered gene silencing Nature 391: 806-811.
  • Non-Patent Document 6 Harvey, K. F., and Kumar, S. (1999). Nedd4-like proteins: an emerging family of ubiquitin-protein ligases implicated in diverse cellular functions. Trends Cell Biol 9: 166-169.
  • Non-Patent Document 7 Huibregtse, J., Scheffner, M., Beaudenon, S., and Howley, P. (1995). A Family of Proteins Structurally and Functionally Related to the E6-AP Ubiquitin-Protein Ligase. PNAS 92: 2563-2567.
  • Non-Patent Document 8 Kerr JF, Wyllie AH and Currie AR (1972). Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26: 239-257
  • Non-Patent Document 9 Karlin, S. and Altschul, SF (1993). Applications and statistics for multiple high-scoring segments in molecular sequences. Proc. Natl. Acad. Sci. USA 90: 5873-5877.
  • Non-Patent Document 10 (Non-Patent Document 10) Jung MS, Jin DH, Chae HD, Kang S, Kim SC, Bang YJ, Choi TS, Choi KS, and Shin DY. (2004). Bcl-xL and E1B-19K Proteins Inhibit p53-induced Irreversible Growth Arrest and Senescence by Preventing Reactive Oxygen Species-dependent p38 Activation. J. Biol. Chem. 2004 279: 17765-17771.
  • Non-Patent Document 11 Lin, X., Liang, M., and Feng, X. H. (2000).
  • Smurf2 is a ubiquitin E3 ligase mediating proteasome-dependent degradation of Smad2 in transforming growth factor-beta signaling. J Biol Chem 275 ,: 36818-36822.
  • Non-Patent Document 12 Magge. C.A. (1998). Therapeutic opportunities from the pharmacological manipulation of the Fas system. Pharmacol. Res. 38: 1-34
  • Non-Patent Document 13 Mochizuki, H., Goto, K., Mori, H., and Mixuno, Y. (1996). Histochemical detection of apoptosis in Parkinsons disease. J. Neurol. Sci. 131: 120-23
  • Non-Patent Document 14 Montgomery, Mark K. and Fire, Andrew (1998). Double-stranded RNA as a mediator in sequence-specific genetic silencing and co-suppression. Trends in Genetics 14: 255-258
  • Non-Patent Document 15 Oda K, Arakawa H, Tanaka T, Matsuda K, Tanikawa C, Mori T, Nishimori H, Tamai K, Tokino T, Nakamura Y, Taya Y (2000).
  • p53AIP1 a potential mediator of p53-dependent apoptosis, and its regulation by Ser-46-phosphorylated p53. Cell 15; 102 (6): 849-62.
  • Non-Patent Document 16 O'Reilly, L.A., and Strasser, A. (1999). Apoptosis and autoimmune disease. Inflam. Res. 48: 5-21
  • Non-Patent Document 17 Michieli, P., Li, W., Lorenzi, M. V., Miki, T., Zakut, R., Givol, D. & Pierce, J. H. (1996). Inhibition of oncogene-mediated transformation by ectopic expression of p21 waf in NIH 3T3 Oncogene 12: 775-784.
  • Non-Patent Document 18 Robertson, G.S., Crocker, S.J., Nicholson, D.W., and Schulz, J.B. (2000). Neuroprotection by the inhibition of Apoptosis. Brain Pathology 10, 283-292
  • Non-Patent Document 19 Sharp P A. (1999). RNAi and double stranded RNA. Genes Dev; 13 (2): 139-41.
  • Non-Patent Document 20 Male, G., Nichols, N.R., Brady, D. R., Finch, C. E., and Horton, W. E. (1995). Evidence for apoptotic cell death in Alzheimers disease. Exp. Neurol. 133: 225-230
  • Non-Patent Document 21 Thomas, L.B., Gates, D. J., Richfield. E. K., O'Brien, T. F., Schweitzer, J. B., and Steindler, D. A. (1995). DNA end labeling (TUNEL) in Huntingtons disease and other neuropathological conditions. Exp. Neurol. 133, 265-27
  • Non-Patent Document 22 Vogelstein, B., Lane, D., and Levine, AJ Surfing (2000). The p53 network, Nature 408: 307-310.
  • Non-Patent Document 23 Vousden KH and Lu X (2002). Live or let die: the cell's response to p53. Nat Rev Cancer 8: 594-604.
  • Non-Patent Document 24 Walzakzak, H., Miller, RE, Ariail, K., Gliniak, B., Griffith, TS, Kubin, M., CHin, W., Jones, J., Woodward, A., Le , T., Smith, C., Smolak, P., Goodwin, RG, Rauch, CT, Schuh, JC, and Lynch, DH (1999). Tumoricidal activity of tumor necorsis factor-related apoptosis-inducing ligand in vivo. Nat. Med. 5: 157-163
  • Non-Patent Document 25 Yamanaka. Y., Nlumenthal. R., Lorenzi M.V., Tatsumoto, T and Miki T. (2001). Ostip2, a Novel Oncoprotein that Associates with the Rho Exchange Factor Ost. DNA and Cell Biology 20: 383-390
  • Non-Patent Document 26 You, J., and Pickart, C. M. (2001). A HECT domain E3 enzyme assembles novel polyubiquitin chains. J Biol Chem 276: 19871-19878.
  • Non-Patent Document 27 Yue, T. L., Ohlsein, E. H., and Ruffolo, R. R. (1999). Apoptosis: A potential target for discovering novel therapies for cardiovascular diseases. Curr. Opin. Chem. Biol. 3: 474-480
  • Non-Patent Document 28 Zou, H., Li, Y., Liu, X., and Wang, X. (1999).
  • An APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J Biol Chem 274: 11549-11556.
  • the present invention provides a protein for controlling cell growth, apoptosis or cell aging composed of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
  • the present invention also provides a gene for regulating apoptosis or cell aging that codes for a protein composed of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4.
  • the present invention provides a recombinant vector comprising the gene for regulating apoptosis.
  • the present invention also provides a pharmaceutical composition comprising the apoptosis or apoptosis control protein, the apoptosis or apoptosis control gene, or the recombinant vector.
  • the present invention comprises the steps of contacting the candidate substance to a transduction control region or in vitro expression system comprising the transcriptional control region of the gene and the gene or reporter (report) gene; Detecting the expression level of the gene or reporter gene; And when the expression level is increased, the candidate substance is selected as a substance for the treatment or prevention of a disease associated with apoptosis under activity, and when the expression level is decreased, the candidate substance is treated as a treatment or disease associated with apoptosis hyperactivity or It provides a method for screening apoptosis promoter or inhibitor, characterized in that selected as a prophylactic material.
  • the KIAA1764 gene of the present invention promotes cell growth, death and / or cell aging, and is useful for the treatment and prevention of diseases related thereto. In addition, by screening for substances involved in the expression of KIAA1764, it is possible to develop therapeutic agents for diseases related to apoptosis and / or cell aging.
  • Figure 1 shows three cDNAs, including KIAA1764-1 (EAW87120), KIAA1764-2 (NP_208325.3), KIAA1764 homologue (AK004646), and truncated cDNA of mice, 11-61, which are genes derived from human KIAA1764. It is a schematic representation of the primary structure of.
  • Figure 2 compares the amino acid sequences of proteins encoded by the two types of cDNA derived from human KIAA1764 and mouse KIAA1764 gene.
  • Figure 3 shows the protein sequence differences encoded by two types of cDNA from KIAA1764 based on the exon and intron structure on the chromosome. Both cDNAs appear to originate from ATG of exon 1 for KIAA1764-1 and ATG of 2 exons for KIAA1764-1.
  • Fig. 4 shows mRNAs of KIAA1764-1 and -2 mRNAs of human fibroblasts, WI38 and human cancer-derived cells, human meat cancer cancer cell line (SaOs-2) and colon cancer cell line (HCT116) by RT-PCR. The existence of the will be confirmed. These results suggest that both mRNAs are present in normal and cancer cells.
  • 5 is 6 days after 0, 50, 150 MOI of p53 adenovirus infected with SaOs-2 cells, a human sarcoma cancer cell line expressing truncated cDNA, designated 11-61, and SaOs-2 cells of the control group.
  • the activity of aging-related beta-galactosidase (SA-beta-Gal), which measures the aging of cells, is measured.
  • SA-beta-Gal beta-galactosidase
  • Expression of the p53 gene in cells expressing 11-61 cDNA shows an increase in senescence marker SA-galactosidase activity.
  • FIG. 6 shows that after a p53 adenovirus is infected with SaOs-2 cells, which are human sarcoma cancer cell lines expressing two types of KIAA1674, KIAA1764-1 and KIAA1764-2, and SaOs-2 cells of the control group, respectively, Afterwards, SA-beta-Gal activity was measured to measure the aging of cells. Expression of the p53 gene in cells expressing KIAA1764-1, -2 shows an increase in senescence marker SA-galactosidase activity.
  • Figure 7 shows that after infection of p53 adenovirus with SaOs-2 cells, which are human sarcoma cancer cell lines expressing two types of cDNA derived from KIAA1674, KIAA1764-1 and KIAA1764-2, and control SaOs-2 cells, respectively, After this, apoptosis was measured. In a cell expressing cDNA derived from KIAA1764, apoptosis by expression of the p53 gene is increased.
  • FIG. 8 shows cell growth after treatment of HCT116 colon cancer cells expressing KIAA1764-1 gene and control HCT116 cells for 24 hours with anticancer drug doxorubicin, which causes DNA damage. Cancer cell lines expressing KIAA1764-1 significantly reduced cell growth compared to the control.
  • FIG. 9 shows SA-galactosidase measuring aging after 6 days of treatment with anticancer agent doxorubicin, which causes DNA damage, in osteosarcoma cells Saos2 expressing human-derived KIAA1764-1, colon cancer cell line HCT116, and control cell lines. The activity was measured. DNA damage in cells expressing KIAA1764 increases SA-galactosidase activity, an aging marker.
  • FIG. 10 compares tumor formation by injecting colon cancer cell line HCT116 expressing KIAA1764 gene and control HCT116 cells into nude mice, and shows that the tumor formation ability of KIAA1764 expressing cancer cells is significantly reduced compared to the control group. .
  • FIG. 11 shows that the HCT116 cell line expressing KIAA1764 gene and control HCT116 cells were transplanted to nude mice for 100 days, and then treated with doxorubicin. This is a marked increase.
  • the present invention provides an apoptosis and / or cell aging modulator comprising a KIAA1764 protein, a gene encoding the same, or a variant functionally equivalent thereto.
  • the apoptosis or apoptosis regulator is apoptosis or apoptosis promoting regulator.
  • the KIAA1764 protein can be used from a variety of mammals, but is preferably human derived, and they are purified from natural sequence sources that are present in all cell types of human and non-human mammal species. It includes all, chemically synthesized, produced by DNA recombination techniques, or prepared by a combination of these and / or other methods.
  • the KIAA1764 protein is composed of an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4
  • the KIAA1764 gene is a protein consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4 It consists of a nucleic acid sequence encoding a, more preferably consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3.
  • the KIAA1764 protein of the present invention and the gene encoding the same, include its functionally equivalent variants.
  • Functionally equivalent variants for the purposes of the present invention refer to compounds that exhibit biological activity comparable to the naturally occurring sequences found.
  • a functionally equivalent variant means that the cell death can be inhibited.
  • functionally equivalent variants are those derived from SEQ ID NOs: 1 or 3 in the case of genes.
  • such functionally equivalent variants include nucleotides constituting the gene sequence or sequence variants in which the amino acid sequence constituting the protein sequence is substituted, deleted, added, and / or inserted as long as it has the activity of inhibiting apoptosis.
  • Such variations in sequence may or may not involve amino acid sequence variations that make up the protein.
  • degeneracy mutations in which protein sequence variations do not involve amino acid variations in polypeptides, and such degeneracy mutants are also included in the genes of the invention.
  • Genes encoding variants that are functionally equivalent to the KIAA1764 protein are known in the art, such as hybridization techniques (Southern, EM, Journal of Molecular Biology, 98, 503, 1975) or PCR methods (Saiki et al., Science). 230: 1350-1354, 1985; Saiki et al., Science, 239: 487-491, 1988).
  • those skilled in the art will be able to isolate a gene having high homology with the KIAA1764 gene by designing a primer that can hybridize specifically to the KIAA1764 gene from the above-mentioned KIAA1764 protein sequence.
  • the protein encoded by this isolated gene has high homology with the native KIAA1764 protein at the amino acid level.
  • High homology refers to the identity of at least 50%, in particular at least 70%, more particularly at least 90% (eg, at least 95%) of sequences throughout an amino acid sequence.
  • the homology of amino acid sequences or nucleotide sequences can be analyzed using program 2 , called BLASTN or BLASTX, developed based on BLAST (Proc. Natl. Acad. Sci. USA, 90, 5873-5877, 1993). It is known at the following website (http://www.ncbi.nlm.nih.gov.).
  • the KIAA1764 protein, and the gene encoding it also include fragments having biological activity equivalent to its native sequence.
  • fragment refers to a sequence corresponding to a part of a gene or protein, and, in the case of a gene, includes a physical, endonuclease, or chemical cleavage, and in the case of a protein, a cleavage by a protease; Includes all chemical cuts.
  • variants for altering the safety, shelf life, solubility, etc. of the KIAA1764 protein, or variants for altering interaction with other proteins that interact with are also included in the functionally equivalent variants.
  • the present invention also provides a recombinant vector comprising the KIAA1764 gene.
  • the KIAA1764 gene of the present invention may be used by itself or in a vector for the purpose of the present invention, and methods for introducing DNA into the vector are known in the art ( Molecular Cloning: A Laboratory Manual, 3rd Ed., Sambrook and Russel, Cold Spring Harbor Laboratory Press, 2001; Current Protocols in Molecular Biology Ausubel, Brent, guitarist, More, Feidman, Smith and Guatemala eds, Greene Publ. Assoc., Wiley -Interscience, 1992). In one embodiment of the invention it was introduced and used as pCEV29 (Michieli et al., 1996; Yamanaka et al., 2001) vector for expression in eukaryotic cells.
  • vectors which can be expressed and / or amplified in cells, and otherwise introduced into the chromosome or present in the cell independently of the chromosome, and the vector is suitable for linear DNA, plasmid vector, or other purposes.
  • such vectors include chemical conjugate vectors, viruses, including ligands or nucleic acid binding moieties (eg, polylysine binding sites) to receptors on the cell surface (eg, those described in WO 93/04701).
  • Vector e.g., DNA or RNA virus
  • fusion protein moiety e.g., antibody moiety that recognizes the antigen of the cell, Glutathione S-transferase for ease of isolation and detection
  • a fusion protein expression vector comprising a fluorescent protein moiety selection of a vector appropriate for the purpose will be apparent to those skilled in the art.
  • the KIAA1764 gene of the present invention may be introduced into an expression vector used in a gene therapy system or the like, such as an adenovirus vector, to be included in a virus particle as a carrier according to a known method.
  • KIAA1764 polynucleotides according to the present invention and all variants described above can be prepared using known organic chemical methods for polypeptide synthesis, and can also be combined with each other in sequence synthesized into fragments to obtain the desired full length sequence. (The Peptides, Analysis, Synthesis, Biology, Vol. 1-9, Gross, Udenfriend and Meienhofer Ed. 1979-1987, Academic Press Inc.).
  • the KIAA1764 polynucleotide, fragment thereof, or functionally equivalent variant thereof according to the present invention preferably uses genetic recombination techniques.
  • the native KIAA1764 polynucleotides can be expressed in a suitable host cell to make cell lysates, or the KIAA1764 mRNA can be translated in vitro and purified by protein isolation methods known in the art, and can be purified by general genetic recombination techniques. And protein purification methods are described, for example, in Sambrook et al., Molecular Clonning: A Laboratory Mannual, Second Edition, Cold Spring Harbor Laboratory Press, 1989; Current Protocols in Molecular Biology, Ausubel et al Ed., Greene Publishing Associates and Wiley Interscience 1991).
  • Such KIAA1764 polypeptides of the invention include, for example, those in the form of fused proteins or amino acid residues bound to a carrier for delivery or administration to purified proteins, water soluble proteins, or target cells.
  • Intracellular expression of KIAA1764 of the present invention significantly promoted cell aging and cell death in p53 and the like.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a recombinant vector comprising a protein for regulating KIAA1764 apoptosis or cell aging, a gene for regulating KIAA1764 apoptosis or cell aging, or KIAA1764.
  • the pharmaceutical composition may be for the prophylaxis or treatment of a disease associated with apoptotic underactivity.
  • Diseases associated with such underactivation of apoptosis include, but are not limited to, cancers in particular by inactivation of p53, such as cancer 21, 22 .
  • the pharmaceutical composition of the present invention may further comprise an anticancer agent or may be used in combination with an anticancer agent.
  • the anticancer agent may be used in the art as long as it is used in combination with the KIAA1764 gene or protein expression inhibitor of the present invention to achieve the above effects, including but not limited to cisplatin, doxorubicin and etoposide. .
  • KIAA1764 gene is closely related to apoptosis
  • specific mutations such as substitution of a specific nucleic acid sequence, deletion addition, etc. exist due to the KIAA1764 coding sequence or a regulatory region such as a promoter and an enhancer.
  • the function of KIAA1764 may be reduced, or the expression of genes or proteins may be reduced. Therefore, by examining the mutation of a gene including the KIAA1764 coding sequence and its regulatory region, and comparing the difference with a normal gene, the risk of apoptosis-related diseases can be determined in advance.
  • Mutations in gene sequences and differences from normal sequences can be performed using methods known in the art, including but not limited to, for example, cDNA sequencing, single strand confimration polymorphism (PCR-SSCP), Methods such as PCR-SSO (sequence-specific oligonucleotide) can be used.
  • PCR-SSCP single strand confimration polymorphism
  • Methods such as PCR-SSO sequence-specific oligonucleotide
  • the present invention also provides a method for screening a substance which can be usefully used as an apoptosis and / or apoptosis promoter or inhibitor, in particular a therapeutic agent for apoptosis-related diseases.
  • the present invention comprises the steps of contacting a candidate substance with a transcriptional control region of a KIAA1764 gene and a transformed cell or in vitro expression system comprising said gene or reporter gene; Detecting the expression level of the gene or reporter gene; And when the expression level is increased, the candidate substance is selected as a substance for the treatment or prevention of a disease associated with apoptosis under activity, and when the expression level is decreased, the candidate substance is treated as a treatment or disease associated with apoptosis hyperactivity or It provides a method for screening apoptosis promoter or inhibitor, characterized in that selected as a prophylactic material.
  • the substance is preferably a substance which is a natural, synthetic, semisynthetic compound, a natural, recombinant, synthetic biological material, or a derivative thereof.
  • the reporter gene in this step for example, chloramphenicol acetyl transferase (CAT), beta-galactosidase ( ⁇ -Gal), luciferase (luciferase) and the like can be used.
  • an expression regulator of the KIAA1764 gene for example, an expression vector connecting the transcriptional regulatory region (promoter, enhancer) of the KIAA1764 gene to an upstream or downstream of the translation region of the reporter gene is constructed, introduced into an appropriate culture cell, and the like.
  • the cells can be screened by adding a compound obtained by a synthetic or genetic recombination technique or a compound derived from nature and derivatives thereof and measuring the expression amount of the reporter gene or the amount of the reporter protein after a certain time.
  • the regulatory region of the KIAA1764 gene can be obtained from a commercial genomic library by carrying out plaque hybridization using a fragment of KIAA1764 cDNA as a probe by a known method.
  • the amount of reporter protein may be measured as enzyme activity, or may be measured using an antibody or the like as the expression level of the protein.
  • Diseases associated with apoptotic underactivity may be cancer, and examples thereof include gastric cancer, colorectal cancer, liver cancer, breast cancer, and lung cancer, but are not limited thereto.
  • apoptosis hyperactivity diseases associated with such apoptosis hyperactivity include central nervous system diseases such as a number of acute and chronic degenerative diseases (eg, Alzheimer's disease, Parkinson's disease, cerebral ischemia / stroke), cardiovascular disease, and Graves' disease and type 2 Autoimmune diseases such as diabetes, but are not limited thereto.
  • central nervous system diseases such as a number of acute and chronic degenerative diseases (eg, Alzheimer's disease, Parkinson's disease, cerebral ischemia / stroke), cardiovascular disease, and Graves' disease and type 2 Autoimmune diseases such as diabetes, but are not limited thereto.
  • Apoptosis inducing agents according to the present invention, and pharmaceutical compositions for treating apoptosis-related diseases may be administered in a suitable formulation together with a carrier, diluent or excipient known in the art.
  • the pharmaceutical composition of the present invention may be administered via a general route as long as it can reach the desired tissue, and may be administered parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) according to the desired method.
  • parenterally eg, intravenously, subcutaneously, intraperitoneally or topically
  • Oral administration, in particular parenteral administration is preferred, and more particularly intravenous injection, the dosage form depending on the method of administration chosen.
  • Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and one or more of these components, as necessary. And other conventional additives such as buffers and bacteriostatic agents can be added.
  • diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions, and the like, and may act specifically on target organs.
  • Target organ specific antibodies or other ligands may be used in combination with the carriers so as to be used. Furthermore, it may be suitably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). have.
  • compositions such as liposomes and emulsions well known in the art may be employed.
  • Certain organic solvents such as dimethylsulfoxide may also be employed.
  • the dosage will vary depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and severity of the disease.
  • therapeutically effective dosages can be initially determined using in vitro assays through cell culture, and performed in animal models to determine IC 50 (in vitro) in which concentration ranges of blood KIAA1764 were determined in cell culture.
  • IC 50 in vitro
  • drug treatments the concentration of the test compound at a lethal dose relative to 50% of the cultured cells
  • Those skilled in the art will be able to determine the amount effective for treatment without undue experimentation, and this information can be used to more accurately determine the dosage useful in humans.
  • the pharmaceutical composition of the present invention may be provided alone or in combination with drugs such as surgery, hormonal therapy, and anticancer agents for the treatment of cancer.
  • Cell lines SaOs2, HCT116, HEK 293, H460, and A549 are DMEM medium (Dulbecco's) containing 10% fetal bovine serum (Fetal Bovine Serum, Hyclone), glucose, penicillin (100 units / ml) and streptomycin (100 ⁇ g / ml) Modified Eagle's Medium, Hycolon), incubated in 37 °C, 5% CO 2 environment. Cells were washed twice with Dulbecco's modified phosphate buffer saline (D-PBS, GIBCO BRL), treated with 1 x trypsin, and incubated by replacing the medium every 3-4 days in plates.
  • D-PBS Dulbecco's modified phosphate buffer saline
  • Transfectants were selected in culture medium containing 500 ⁇ g / ⁇ l of G418 (DMEM + 10% FBS) and cultured with changing medium every 3-4 days. Each clone of the stably introduced transfectant was selected for further analysis. Cell transformation was performed in serum and lipofectamine 2000 (Invitrogene) was used.
  • Cultured cells were treated with lipa lysis buffer (50 mM Tris-Cl / pH 7.5, 50 mM NaCl, 1% Triton X-100, 1 mM EGTA, 50 mM NaF, 10 mM Na 4 P 2 O 7 , 5 mM Na 3 VO 4 , 1 mM DTT, After treatment with aprotinin, a protease inhibitor cocktail consisting of lupeptin, phenylmethylsulfonyl fluoride (PMSF) and stored on ice for 30 minutes, it was centrifuged at 13,000 rpm for 20 minutes at 4 ° C.
  • lipa lysis buffer 50 mM Tris-Cl / pH 7.5, 50 mM NaCl, 1% Triton X-100, 1 mM EGTA, 50 mM NaF, 10 mM Na 4 P 2 O 7 , 5 mM Na 3 VO 4 , 1 mM DTT
  • Protein quantification was performed using a protein assay kit (Bio-Rad).
  • PVDF membrane Perkin Elmer life science. PVDF was blocked with 5% skim milk / 0.1% Tween 20 / TBS and each antibody was reacted for 4 days. After washing three times with TBS-T for 20 minutes, and then incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 hour.
  • Total cell RNA was isolated using Trizol solution (Invitrogen, co.). 20 ⁇ g of total RNA per sample was denatured, developed on a 1.2% agarose-formaldehyde gel, then transferred to a nylon membrane and hybridized according to the manufacturer's instructions (Expresshyb, clonethec Inc,). Probes were 32 P-labeled using a random primer DNA label kit (Roche). The blot was exposed to X-lane film, washed and then left overnight at -70 ° C.
  • SA- ⁇ -Gal staining was performed as described in Dimiri et al (27). Cells incubated for the indicated periods were washed with DPBS and fixed in 0.25% glutaraldehyde in PBS for 15 minutes at room temperature. SA- ⁇ -gal activity at pH 6.0 was detected by incubation with SA- ⁇ -gal staining solution washed for 6-12 hours at 37 °C. The solution is 1 mg of 5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside (X-gal) per 1 ml, 20 mg of dimethyl-formamide, 40 mM citric acid, 5 mM potassium ferri Cyanide, 150 mM NaCl, 2 mM MgCl 2 . Cells were rinsed with D-PBS and SA-Gal staining positive rate of each sample was determined using brightfield microscopy.
  • the nucleotide sequence of the gene was sequenced using the sequencing kit (Applied Biosystmes, USA) as recommended by the manufacturer, and the BLASTN program (Altschul et al, 1990; http: //www,ncbi.nlm.nih.gov) homology analysis in coming, as the sequence that forms part of the GenBank EAW87120 (man), NP_208325.3 (man), AK004646 (mouse) in mice, specific functions are not known.
  • the EAW87120 was named KIAA1764-1 (gene sequence: SEQ ID NO: 1, protein sequence: SEQ ID NO: 2), and NP_208325.3 was named KIAA1764-2 (gene sequence: SEQ ID NO: 3, protein sequence 4).
  • KIAA1764-1 gene sequence: SEQ ID NO: 1, protein sequence: SEQ ID NO: 2
  • NP_208325.3 was named KIAA1764-2 (gene sequence: SEQ ID NO: 3, protein sequence 4).
  • the primary structures of KIAA1764-1, KIAA1764-2, mouse KIAA1764 homologue (AK004646), and truncated 11-61 cDNA of mouse are shown in FIG.
  • KIAA1764-1 Sequence differences between KIAA1764-1 and KIAA1764-2 are shown in FIG. 3 based on the exon and intron structures on the chromosome of the protein sequence encoded by the cDNA. KIAA1764-1 was found to start at ATG of exon 1 and KIAA1764-2 was found to start at ATG of 2 axons.
  • MRNAs of human fibroblasts WI38, IMR90, human sarcoma cancer cell line (SaOs-2) and colorectal cancer cell line (HCT116) were extracted and RT-PCR was performed.
  • RT-PCR RT-PCR was performed.
  • the presence of KIAA1764-1 and -2 mRNA was confirmed.
  • the results are shown in FIG. In both normal and cancer cells, the presence of KIAA1764-1 and -2 mRNAs was confirmed.
  • Cleaved cDNAs designated 11-61 were constructed and cloned into expression vectors, which were then transfected into SaOs-2 cells. Aging-related beta-galactosidase measuring 11-61 SaOs-2 cells and control SaOs-2 cells, 0, 50, 150 MOI of p53 adenovirus 6 days after infection The activity of the agent (SA-beta-Gal) was measured. As a result, as shown in Figure 5, the expression of the p53 gene in cells expressing 11-61 cDNA was shown to increase the SA-galactosidase activity, an aging marker.
  • KIAA1764-1 and KIAA1764-2 cDNAs were prepared and cloned into expression vectors, which were then transfected into SaOs-2 cells. Aging to measure senescence of cells after a certain period of time after infection with 0, 50, 150 MOI of p53 adenovirus to SaOs-2 cells and control SaOs-2 cells expressing KIAA1764-1 and KIAA1764-2, respectively The activity of related beta-galactosidase (SA-beta-Gal) was measured. As a result, as shown in Figure 6, the expression of the p53 gene in cells expressing KIAA1764-1, -2, SA-galactosidase activity, which is an aging marker, was increased.
  • SA-beta-Gal beta-galactosidase
  • Cells were placed in 24-well plates at a total of 4 ⁇ 10 4 cells / well, 48 hours later the cells were rinsed with 1 ⁇ D-PBS and then infected with adenovirus 50 to 150 multiplicity of infection (MOI) expressing p53. All cells in each well were collected in their respective containers and stained with 0.4% trypan blue (Sigma) to quantify cell death by measuring the number of cells killed under a microscope.
  • MOI multiplicity of infection
  • FIG. 7 shows apoptosis after a certain time after infection with p53 adenovirus in SaOs-2 cells expressing two types of cDNA, KIAA1764-1 and KIAA1764-2, and control SaOs-2 cells derived from KIAA1674, respectively.
  • cDNA derived from KIAA1764 was shown to promote apoptosis by expression of the p53 gene.
  • Expression vectors expressing KIAA1764-1 and KIAA1764-2, respectively, were expressed in colon cancer cell line HCT116, respectively, and injected into nude mice to measure tumor formation.
  • the cells were resuspended in 100 ⁇ l sterile water and then injected subcutaneously into the 4 week old Balb / c female nude mouse flanks. Tumor size was measured by measuring the length (A) and width (B) using a caliper, and the tumor volume was calculated as (A x B 2 ) / 2.
  • FIG. 10 shows tumor cell size of KIAA1764-expressing cancer cells expressing KIAA1764-1 and KIAA1764-2 colonic cancer cell lines HCT116 and control HCT116 cells, respectively.
  • the size was found to be significantly smaller than the control (HCT116). Therefore, it was found that KIAA1764 effectively inhibited tumor formation.
  • Doxorubicin is an anticancer agent that causes DNA damage and inhibits the growth of cells. Therefore, it was confirmed that growth inhibition of cancer cells by treatment with doxorubicin to control HCT116 cells and HCT116 cells expressing KIAA1764.
  • FIG. 8 shows cell growth after treatment of HCT116 colon cancer cells expressing KIAA1764-1 gene and control HCT116 cells for 24 hours with anticancer drug doxorubicin, which causes DNA damage. Cancer cell lines expressing KIAA1764-1 significantly reduced cell growth compared to the control.
  • FIG. 9 shows SA-galactosidase measuring aging after 6 days of treatment with anticancer agent doxorubicin, which causes DNA damage, in osteosarcoma cells Saos2 expressing human-derived KIAA1764-1, colon cancer cell line HCT116, and control cell lines. The activity was measured. DNA damage in cells expressing KIAA1764 increases SA-galactosidase activity, an aging marker.
  • the cells were resuspended in 100 ⁇ l sterile water and then injected subcutaneously into the 4 week old Balb / c female nude mouse flanks.
  • Doxorubicin was dissolved in PBS and then injected intraperitoneally. Tumor size was measured by measuring the length (A) and width (B) using a caliper, and the tumor volume was calculated as (A x B 2 ) / 2.
  • FIG. 11 shows the tumor size when colon cancer cell lines HCT116 expressing KIAA1764 gene and control HCT116 cells were transplanted into nude mice, grown for 100 days, and treated with doxorubicin.

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Abstract

La présente invention concerne le gène KIAA1764 qui constitue un gène inédit de régulation de l'apoptose et du vieillissement cellulaire ; une composition pharmaceutique contenant ledit gène ou la protéine associée et utilisable en vue de la prévention ou du traitement de maladies liées à un niveau d'apoptose trop faible ; et un procédé de criblage permettant de rechercher un promoteur ou un inhibiteur de l'apoptose en faisant appel à KIAA1764.
PCT/KR2011/006559 2011-09-05 2011-09-05 Utilisations inédites de kiaa1764 en tant que régulateur de l'apoptose et du vieillissement Ceased WO2013035896A1 (fr)

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KR20110126851A (ko) * 2010-05-18 2011-11-24 단국대학교 산학협력단 신규한 세포사멸 및 노화 조절제로서의 ｋｉａａ1764 용도

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20110126851A (ko) * 2010-05-18 2011-11-24 단국대학교 산학협력단 신규한 세포사멸 및 노화 조절제로서의 ｋｉａａ1764 용도

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK 18 December 2006 (2006-12-18), accession no. AW 87120.1 *
DATABASE GENBANK 30 March 2010 (2010-03-30), accession no. M 033402.4 *
DATABASE GENBANK 30 March 2010 (2010-03-30), accession no. P_208325.3 *

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