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WO2013048211A2 - Composition induisant l'apoptose, comprenant une protéine kaiso - Google Patents

Composition induisant l'apoptose, comprenant une protéine kaiso Download PDF

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Publication number
WO2013048211A2
WO2013048211A2 PCT/KR2012/007960 KR2012007960W WO2013048211A2 WO 2013048211 A2 WO2013048211 A2 WO 2013048211A2 KR 2012007960 W KR2012007960 W KR 2012007960W WO 2013048211 A2 WO2013048211 A2 WO 2013048211A2
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Prior art keywords
kais0
protein
kaiso
amino acid
expression
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WO2013048211A3 (fr
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허만욱
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Industry Academic Cooperation Foundation of Yonsei University
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Industry Academic Cooperation Foundation of Yonsei 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
    • A61K38/02—Peptides of undefined number of amino acids; Derivatives thereof
    • 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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • composition for inducing aptosis including KAIS0 protein
  • the present invention relates to a composition for inducing apatosis comprising an KAIS0 protein as an active ingredient and an agent for inhibiting or treating damage caused by genotoxicity.
  • the P0K family of proteins consisting of BTB / P0Z-domain and KrUppel-like zinc-finger fingers, has a variety of regulatory functions including cell proliferation, differentiation, development, tumor suppression and tumor formation. 1 ⁇ 3 Recently, we and other researchers have discovered the function of P0K family proteins in tumor suppression or tumor formation. 3 We have found that some P0K family transcription factors are induced by DNA damage and are involved in tumor suppression or tumor formation. Immortalized normal human HEK293A cells induce expression of P0K transcription factors when exposed to DNA damaging agents such as etoposide (ET0).
  • E0 etoposide
  • P 53 and p300 are key regulators of cell cycle arrest and aloptosis.
  • p53 is acetylated by proteins consisting of HAT moieties such as CBP / p300, PCAF and TIP60, and later acetylated p53 regulates various cell functions, including cell fate.
  • KAIS0 acts as an upstream regulator of early cellular defense systems against genotoxicity and acts on p53 to induce cell cycle arrest and aptosis programs.
  • Post-translation of p53 protein such as inhibition of K381 acetylation by KAIS0 and acetylation of K320 and K382 Post-translational modification modulates cell cycle arrest and aptosis programs by p53.
  • KAIS0 is a master regulator of apoptosis and can function as a tumor suppressor.
  • the present invention suggests that a small molecule anticancer agent capable of strongly inducing KAIS0 and an inhibitor of the interaction of KAIS0-pl20ctn, which inhibits the action of KAIS0 by trapping KAIS0 in the cytoplasm by pl20ctn, can be used as an anticancer agent.
  • a number of papers and patent documents are referenced and their citations are indicated.
  • the disclosures of cited papers and patent documents are incorporated herein by reference in their entirety and the level of the technical field to which the present invention belongs. The content of the present invention is clearly explained.
  • KAIS0 was expressed prior to expression of P 53, P 21, preapoptotic MBcl-2-associated X protein and / M ( ⁇ 53 upregulated modulator of apoptosis) genes.
  • KAIS0 is the master regulator of the p53 pathway that regulates the expression of genes of the p53 pathway (eg, activates expression of CDKN1A, BAX and PUMA genes; and inhibits the expression of the Be 1-2 gene, an anti-apoptotic gene).
  • the present invention has been completed by discovering that complexes with p53 and p300 activate p53 to induce cell cycle arrest and apoptosis.
  • Another object of the present invention is to provide a composition for inhibiting or treating damage caused by genotoxicity.
  • Another object of the present invention is damage caused by genotoxicity. To provide a method for screening an inhibitor or a therapeutic agent.
  • Another object of the present invention is to provide a p53 variant protein. Another object of the present invention is to provide a nucleic acid molecule encoding a p53 variant protein.
  • the present invention provides an apoptosis comprising a polypeptide having an amino acid sequence of SEQ ID NO: 2 or a nucleotide sequence encoding an amino acid sequence of SEQ ID NO: 2 as an active ingredient. It provides a composition for induction.
  • the present invention provides a genotoxicity comprising a polypeptide having the amino acid sequence of SEQ ID NO: 2 (KAIS0 protein) or a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 as an active ingredient ( It provides a composition for inhibiting or treating damage caused by genotoxicity.
  • the present invention provides a composition comprising a polypeptide having an amino acid sequence of SEQ ID NO: 2 (KAIS0 protein) or a pharmaceutically effective amount of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 It provides a method for inducing atoposis of cells in the subject comprising administering to the subject (subject).
  • the present invention provides a composition comprising a polypeptide (KAIS0 protein) having an amino acid sequence of SEQ ID NO: 2 or a pharmaceutically effective amount of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2
  • a composition comprising a polypeptide (KAIS0 protein) having an amino acid sequence of SEQ ID NO: 2 or a pharmaceutically effective amount of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2
  • Administering to the subject Provided are methods for inhibiting or treating damage by genotoxicity.
  • the present inventors have made extensive efforts to discover novel regulators of the p53 pathway that are important in cell cycle regulation.
  • p53, p21, preapoptotic i Bcl-2-associated X protein and ⁇ Prior to expression of the ⁇ 53 upregulated modulator of apoptosis gene, we found that KAIS0 expressed and KAIS0 regulated the expression of genes of the P 53 pathway (eg, activated expression of CDKN1A, and PUMA genes; and anti-apoptotic genes). Inhibitors of the expression of the Bcl-2 gene) were found to be master regulators of the p53 pathway, acetylating P53 and complexing with p53 and p300 to induce cell cycle arrest and aptosis.
  • p53 and p300 are major regulators of cell cycle arrest and aptosis.
  • 6 ⁇ 53 is acetylated by proteins consisting of HAT domains such as CBP / p300, PCAF and TIP60, and later acetylated p53 regulates various cell functions, including cell fate.
  • 5 ' 8 — 11 p21 gene expression is regulated by p53, a tumor suppressor protein, which mediates p53-dependent cell cycle G1 arrest against a variety of stresses.
  • the term 'apoptosis' refers to programmed cell death (PCD) that occurs in multicells (About Apoptosis.
  • the atopic body produced by athetosis is encircled and removed by phagocytic eel Is before it flows out of the cell and damages the cell.
  • the term 'genetic toxicity' refers to genotoxicity having a deleterious effect on the genetic material of the cell. Genotoxic agents are usually known to induce mutations or cause cancer, and in particular can cause genetic mutations and form tumors. Genotoxicity affects sperm and eggs, which can lead to genetic variation.
  • Subsequent gene toxicity factors include internal factors such as attack of free radicals produced by byproducts of normal metabolism and errors in DNA replication, and ultraviolet ( 200-300 nm), radiation such as ⁇ -rays and gamma rays, plant toxins, aromatic compounds intercalating DNA, cancer chemotherapy and radiotherapy and viruses.
  • internal factors such as attack of free radicals produced by byproducts of normal metabolism and errors in DNA replication, and ultraviolet ( 200-300 nm), radiation such as ⁇ -rays and gamma rays, plant toxins, aromatic compounds intercalating DNA, cancer chemotherapy and radiotherapy and viruses.
  • KAIS0 of the present invention functions as a master modulator that regulates the genes of the ⁇ 53 pathway (eg, ⁇ 53, ⁇ 21, priacetic and ⁇ 2 expression, which is a small molecule anticancer agent that can strongly induce KAIS0).
  • ⁇ 53 pathway eg, ⁇ 53, ⁇ 21, priacetic and ⁇ 2 expression
  • composition for inducing apatosis and the composition for inhibiting or treating damage due to genotoxicity may be mounted and used in a gene delivery system.
  • the gene delivery system of the present invention can be produced in a variety of forms, including (i) naked recombinant DNA molecules, (ii) plasmids (iii) viral vectors, and (iv) the Naked recombinant DNA molecules. Or in the form of liposomes or niosomes containing plasmids.
  • the nucleotide sequence of KAIS0 can be applied to all gene delivery systems used in conventional gene therapy, preferably plasmids, adenoviruses (Lockett LJ, et al., Clin. Cancer Res. 3: 2075-2080 (1997)). , Adeno—associated viruses: AAV, Lashford LS., Et al., Gene Therapy Technologies, Applications and Regulations Ed.A. Meager, 1999), retroviruses (Gunzburg WH, et al., Retroviral vectors. Gene Therapy Technologies, Appli c ions and Regulations Ed.A. Meager, 1999), lentiviruses (Wang G. et al., J, Clin. Invest.
  • Adenoviruses are widely used as gene transfer vectors due to their genome size, ease of manipulation, high titers, wide range of target cells and excellent infectivity. Both ends of the genome contain 100-200 bp inverted terminal repeats (ITRs), which are cis elements essential for DNA replication and packaging.
  • ITRs inverted terminal repeats
  • the genome El region (E1A and E1B) encodes proteins that regulate transcription and transcription of host cell genes.
  • the E2 regions (E2A and E2B) encode proteins that are involved in viral DNA replication.
  • the KAIS0 gene of the present invention is preferably inserted into the deleted E1 region (E1A region and / or E1B region, preferably E1B region) or E3 region, and more preferably inserted into the deleted E3 region.
  • the term "deletion" as used in connection with a viral genome sequence has the meaning including not only a complete deletion of the sequence, but also a partial deletion.
  • adenovirus can pack up to about 105% of the wild-type genome, about 2 kb can be additionally packaged (Ghosh-Choudhury et al., ⁇ J., 6: 1733-1739 (1987)).
  • the KAIS0 variant gene inserted into the adenovirus may additionally bind to the genome of the adenovirus.
  • Adenoviruses have 42 different serotypes and subgroups of AF. Of these, adenovirus type 5 belonging to subgroup C is the most preferred starting material for obtaining the adenovirus vector of the present invention. Biochemical and genetic information for adenovirus type 5 is well known.
  • KAIS0 variant genes carried by adenoviruses are replicated in the same way as episomes, and have very low genetic toxicity to host cells. Therefore, gene therapy using the adenovirus gene delivery system of the present invention is considered to be very safe. ⁇ . Retrovirus
  • Retroviruses are widely used as gene transfer vectors because they insert their genes into the host genome, carry large amounts of foreign genetic material, and have a broad spectrum of cells that can infect them.
  • the AIS0 gene is inserted into the retrovirus genome instead of the sequence of the retrovirus to produce a nonreplicating virus.
  • a packaging cell line containing the gag, pol and env genes but without the LTRGong terminal repeat) and sequence was constructed (Mann et al., Cell, 33: 153-159 (1983)).
  • the recombinant plasmid containing the ⁇ 53 variant gene, LTR, and ⁇ sequences is introduced into the cell line, the ⁇ sequences allow the production of RNA transcripts of the recombinant plasmids, which are packaged with a virus and the virus is discharged into the medium.
  • Nicolas and Rubinstein “Retroviral vectors,” In: Vectors: A survey of molecular cloning vectors and their uses, Rodriguez and Denhardt (eds.), Stoneham: Butter worth, 494-513 (1988)). The medium containing the recombinant retrovirus is collected, concentrated and used as a gene delivery system.
  • Adeno-associated virus can infect non-dividing cells, It is suitable as the gene delivery system of the present invention because it has the ability to infect various kinds of cells. Details of the manufacture and use of MV vectors are disclosed in detail in US Pat. Nos. 5,139,941 and 4,797,368.
  • the AAV virus is a plasmid (McLaughlin et al., J. Virol., 62: 1963-1973 (1988)) comprising a gene sequence of interest (KAIS0 gene) with two AAV terminal repeats located next to it; and Samulski et al., J. Virol., 63: 3822-3828 (1989)) and expression plasmids comprising wild type AAV coding sequences without terminal repeats (McCarty et aL, J. Virol., 65: 2936-2945 (1991) Prepared by cotransformation).
  • a plasmid (McLaughlin et al., J. Virol., 62: 1963-1973 (1988)) comprising a gene sequence of interest (KAIS0 gene) with two AAV terminal repeats located next to it; and Samulski et al., J. Virol., 63: 3822-3828 (1989)) and expression plasmids comprising wild type AAV
  • Liposomes are automatically formed by phospholipids dispersed in the aqueous phase.
  • Lipofectamine (Gibco BRL) is the most used reagent for the transformation of animal cells using liposomes. Liposomes containing the KAIS0 gene interact with cells through mechanisms such as endocytosis, adsorption to the cell surface, or fusion with plasma cell membranes to transport the KAIS0 gene into cells.
  • the method of introducing the gene delivery system of the present invention as described above may be carried out through various methods known in the art.
  • the gene delivery system when the gene delivery system is manufactured based on a viral vector, the gene delivery system is performed according to a virus infection method known in the art. Infection of host cells with viral vectors is described in the above cited references.
  • the gene delivery system is a naked recombinant DNA molecule or plasmid
  • microinjection method Capecchi, MR, Cell, 22: 479 (1980); and Harland and Weintraub, J. Cell Biol. 101: 1094 1099 (1985)
  • calcium phosphate precipitation Graham, FL et al., Virology, 52: 456 (1973); and Chen and Okayama, Mol. Cell. Biol. 7: 2745—2752 (1987)
  • electric Perforation Nemann, E. et al., EMBO J., 1: 841 (1982); and Tur-Kaspa et al., Mol.
  • composition for inducing apatosis and the composition for inhibiting or treating damage by genotoxicity may be delivered into cells through cell-penetrating peptides.
  • cell penetrating peptide refers to a peptide having the ability to carry a cargo of delivery into a cell in vitro and / or in vivo
  • CPP cell penetrating peptide
  • Cell membrane permeable peptides are themselves peptides having an amino acid sequence that can pass through the cell membrane of a phospholipid bilayer, such as Tat-derived peptides, signal sequences (eg, cell membrane permeable sequences), arginine-rich peptides, VP22, transpotanes. Or amphiphilic peptide carriers, and the like (Morris, MC et al., Nature Biotechnol. 19: 1173- 1176 (2001); Dupont, AJ and Prochiantz, A., CRC Handbook on Cell Penetrating Peptides, Lange 1, Editor, CRC Press, (2002); Cha loin, L.
  • the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 is a nucleotide of SEQ ID NO: 1.
  • the KAIS0 activates the expression of p21, Bcl-2-associated X protein (BAX), Dr5, FAS or p53 upregulated modulator of apoptosis (PUMA).
  • BAX Bcl-2-associated X protein
  • Dr5 FAS
  • PUMA p53 upregulated modulator of apoptosis
  • the KAIS0 forms a complex with p53 protein and p300 protein.
  • the KAIS0-P53—p300 complex of the present invention interacts with the C-terminal DNA-binding domain of p53 via the P0Z domain and zinc-finger DNA binding domain (ZFDBD) of KAIS0.
  • the P0Z domain of KAIS0 is equal to the DIF906 domain (# 3) of p300. Interact with it to surround the HATQiistone acetyltransferase.
  • the KAIS0 induces acetylation of the 320th and 382th Lys residues of p53 and inhibits the acetylation of the 381th Lys residues of p53 to increase the stability of p53.
  • KAIS0 is complexed with p300 and p53, and K320 of p53 is acetylated and K381 inhibits acetylation, which shows the result of increasing the stability and PIMA target specificity of p53.
  • the composition is used for the prevention or treatment of hyperproliferative disease by inducing apatosis
  • the hyperproliferative disease may be cancer, abnormal growth, keloids, Cushing syndrome, hyperaldosteronemia, erythematosis, Include, but are not limited to, erythrocytosis, vitiligo, hypertrophic scars, lichen planus, atherosclerosis, arteriosclerosis, atherosclerosis, restenosis or stenosis.
  • the present invention provides a method for screening an athetosis inducer comprising the following steps: (a) encoding a polypeptide (KAIS0 protein) having the amino acid sequence of SEQ ID NO: 2 Contacting a sample to be analyzed with a cell comprising a nucleotide sequence; And (b) measuring the expression level or activity of the KAIS0 protein; When the expression level or activity of the AIS0 protein is increased in comparison with the control that does not process the sample, the sample is determined to be an aptosis inducing agent.
  • KAIS0 protein polypeptide having the amino acid sequence of SEQ ID NO: 2
  • a method for screening a therapeutic agent for inhibiting damage or treating damage by genotoxicity comprising the following steps: (a) having an amino acid sequence of SEQ ID NO: 2 Contacting a sample to be analyzed with a cell comprising a nucleotide sequence encoding a polypeptide (KAIS0 protein); And (b) measuring the expression level or activity of the KAIS0 protein; Expression level of the KAIS0 protein Or if the activity is increased compared to a control that has not been treated with the sample, the sample is considered to be an agent for inhibiting or treating damage due to genotoxicity.
  • an expression vector comprising a KAIS0-encoding nucleotide sequence or a promoter that regulates expression of KAIS0 and DNA in which a GFP (Green fluorescence protein) or luciferase gene is fused to a reporter are stabilized in a cell.
  • the injected cells are contacted with the sample to be analyzed to control the expression of KAIS0.
  • a promoter of the KAIS0 gene is preferably located upstream of the KAIS0-coding nucleotide sequence.
  • sample used while referring to the screening method of the present invention
  • the sample includes, but is not limited to, chemicals, nucleotides, antisense-RNAs, small interference RNAs (SiRNAs), peptides and natural extracts.
  • the sample analyzed by the screening method of the present invention is a single compound or a combination of compounds (eg, a cell or tissue culture). Samples can be obtained from libraries of synthetic or natural compounds. 6 Methods of obtaining libraries of these compounds are known in the art. Synthetic compound libraries are described in Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA), and Sigina-Aldr ich (USA) commercially available.
  • the expression level of the KAIS0 nucleotide sequence and the expression level of the reporter gene are measured in the cells treated with the sample.
  • the expression level is measured through a conventional method as described below, and as a result of the measurement, increasing the expression of the above-described KAIS0 nucleotide sequence can be determined as an agent for inhibiting or treating damage caused by apoptosis and genotoxicity.
  • the expression analysis of KAIS0 in the above step (b) can be carried out through various methods known in the art.
  • RT-PCR Standardbrook, such as, Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)
  • northern blotting Peter B.
  • RNA is isolated from the cells treated with the sample, and then the first-chain cDNA is prepared using oligo dT primers and reverse transcriptase. Subsequently, the first chain cDNA is used as a template : PCR reaction is performed using a KAIS0 gene-specific primer. A sequence included in a KAIS0-encoding nucleotide sequence. Then, PCR amplification products are electrophoresed, and the formed bands are analyzed to measure the change in expression level of the KAIS0 gene.
  • the change in the amount of KAIS0 protein can be carried out through various immunoassay methods known in the art.
  • changes in the amount of KAIS0 protein can be attributed to immunostaining, radioimmunoassay, radioimmunoprecipitation, western blotting, immunoprecipitation, ELISA (linked immunosorbent assay), capture-ELISA, inhibition or competition assay, and sandwiches. Include, but are not limited to, analysis
  • the method of immunoassay or immunostaining is Enzyme Immunoassay, ET Maggio, ed. , CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-1 inked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, JM ed. , Humana Press, NJ, 1984; and Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.
  • an antibody labeled with a radioisotope detects a marker molecule of the present invention. It can be used to.
  • the present invention comprises the steps of: (i) coating the surface of the solid substrate of the unknown cell sample to be analyzed; (ii) reacting said cell lysate with an antibody against a target as a primary antibody; (iii) reacting the resultant of step (ii) with the secondary antibody to which the enzyme is bound; And ⁇ ) further comprising measuring the activity of the enzyme.
  • Suitable as the solid substrate are hydrocarbon polymers (such as pylene and polypropylene), glass, metals or gels, most preferably microtiter plates.
  • Enzymes bound to the secondary antibody include, but are not limited to, enzymes catalyzing color reaction, fluorescent reaction, luminescence or infrared reaction, for example, alkaline phosphatase, ⁇ -galactosidase, hose radish peroxidase, and includes to-Shipper la kinase and cytochrome ⁇ 450.
  • alkaline phosphatase is used as the enzyme that binds to the secondary antibody, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium ( ⁇ ), and naph are -AS ⁇ B1 ⁇ phosphate (naphthol-) as substrates.
  • Genine bis-N-methylacridinium nitrate
  • resorphin benzyl ether luminol
  • amplex red reagent (10—acetyl—3,7-dihydroxyphenoxazine)
  • HYR p-pheny 1 ened i am i ne-HC 1 and pyrocatechol
  • TMB tetramethyl benzidine
  • ABTS 2,2 ' ⁇ Azine ⁇ di [3-ethylbenzthiazol ine sulfonate]
  • OPD o-phenylenediamine
  • nap / pyronine glucose oxidase and t-NBT (nitroblue tetrazolium)
  • m-PMS phenzaine methosulfate
  • certain embodiments of the invention comprise (i) coating an antibody against a target of the invention as a capturing antibody on the surface of a solid substrate; (ii) reacting the capture antibody with the cell sample; (Hi) reacting the result of step (H) with a detecting antibody that has a label that generates a signal and specifically reacts with granulation factor proteins; And (iv) measuring the signal resulting from the label.
  • the detection antibody carries a label which generates a detectable signal.
  • the label may include chemicals (e.g. biotin), enzymes (alkaline phosphatase, ⁇ -galactosidase, horse radish peroxidase and cytochrome ⁇ 450 ), radioactive substances (e.g., C 14 , I 125 , ⁇ 32 and S 35 ), fluorescent materials (eg, fluorescein), luminescent materials, chemi luminescent and fluorescence resonance energy transfer (FRET), including but not limited to various labels and labeling methods Are described in Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999.
  • Measurement of the final enzyme activity or signal in the ELISA method and capture-ELISA method can be carried out according to various methods known in the art. Detection of such signals allows for qualitative or quantitative analysis of the targets of the present invention. If biotin is used as a label, the signal can be easily detected with streptavidin and luciferin if luciferase is used. According to another aspect of the present invention, there is provided a p53 variant protein having increased stability compared to the wild type p53 protein in which the 381th Lys of the wild type p53 protein is substituted with Ala or Arg. According to another aspect of the invention, a nucleic acid encoding a p53 variant protein Provide a molecule.
  • the p53 variant protein of the present invention has about three times increased stability than wild-type p53 and aptogenic activity of at least p53.
  • nucleic acids and proteins of the p53 variant can be used directly in gene therapy or other forms.
  • P53 variant p53K381A wherein 381 Lys of wild-type p53 protein is substituted with Ala has the nucleotide sequence of SEQ ID NO: 3 encoding the amino acid sequence of SEQ ID NO: 4, and 381 Lys of the wild-type p53 protein is substituted with Arg
  • the p53 variant p53K381R has a nucleotide of SEQ ID NO: 5 encoding the amino acid sequence of SEQ ID NO: 6.
  • the present invention provides the function of KAIS0 as a master regulator of the p53 pathway associated with cell cycle arrest and apatosis.
  • the present invention can provide a method for treating cancer by inhibiting trapping of KAIS0 by developing and using an inhibitor for the interaction of pl20ctn with KAIS in the cytoplasm and a low molecular weight anticancer agent that can strongly induce KAIS0.
  • the present invention can provide p53K381R and p53K381A protein and nucleic acid sequences that are about three times more stable than R 53WT and exhibit atoptotic activity of at least p53.
  • the present invention can provide a composition for inducing aptosis and a composition for inhibiting or treating damage caused by genotoxicity.
  • FIG. La to 11 show the results confirming that KAIS0 induces cell cycle arrest and apatosis.
  • Fig. La and lb show the results of confirming the expression profile of KAISO, ⁇ 53, ⁇ 21, ⁇ and PUMA mRNA by qRT-PCR by treating HEK293A cells with etoposide (5 ⁇ ).
  • Fig. Lc shows the results obtained by Western blot of the cell lysates of Figs. La and lb.
  • FIG. ID shows FACS analysis of aformosis induced by KAISO in HEK293A
  • FIG. Le is shown in Kaiso WT and Kaiso K0 MEF.
  • Figure If shows TUNEL analysis of MEF treated with etoposide. Cracked DNA strands were labeled in green and nuclei were stained with DAPI. Apoptotic cells appear in cyan color.
  • Figure lg is the result of confirming the expression of endogenous p21, PUMA and BAX by Western blot by transfection of HEIS293A cells with KAIS0 expression vector or KAISO siRNA.
  • Figure lh shows the results of co-immunoprecipitation and Western blot of endogenous KAIS0, p53 and HAT proteins.
  • FIG. Li shows the results of coimmunoprecipitation of KAIS0 and p53 tested with cell lysate after 48 hours transfecting KAK0 expression vector to HEK293A cells.
  • Fig lj is recombinant GST, GST-P0Z or GST-ZF proteins [3 3 ⁇ 4] - shows the results of analysis down-methionine labeled p53 or the reaction in vitro GST- p300 fusion protein pool.
  • Ik shows the structure of KAIS0 and the mutation KAIS0-K8. KAIS0 and mutant KAISO-K8 were identically transcribed and proteins were similarly expressed.
  • FIG. 11 shows the results of RT-qPCR analysis of mRNA expression of p53 target apoptotic genes in HEK293A cells transfected with KAIS0 or mutant KAIS0-K8 expression vectors.
  • 2A-2H show that acetylation of K320, 381 and 382 residues of p53 by p300 is regulated by KAIS0.
  • 2A is a flowchart of p53 acetylation and LC / MS / MS analysis on in vitro.
  • FIG. 2B graphically shows LC / MS / MS analysis of acetylation of p53 by p300 or p300 + KAIS0 on in vitro (red ⁇ , lysine acetylated by p300; blue Y with red dot) ⁇ p300 + KAISO acetylated
  • Acetylation of K381 was measured using XIC of acetylated peptides containing K381 and K382 acetylation sites of the other three groups (p53, p53 + P 300 and p53 + p300 + KAIS0).
  • XICs of precursor peptides were plotted in the mass window (calculated with acetylated peptide mass ⁇ 10 ppm) using the Xcalibur program.
  • 2F is the extracted ion chromatogram (XIC) of the acetylated peptide K (ac) LMFKTEGPDSD (mass to charge ratio (m / z): 713.33, charge 2+), and on the right is the acetylated peptide HKK (ac
  • LMFKTEGPDSD mass to charge ratio (m / z): 845.91, charge 2+).
  • Acetylation of K382 was measured using XIC of acetylated peptide HKK (ac) LMF TEGPDSD (mass to charge ratio (m / z): 845.91, charge 2+). Finally, relative differences in the amount of all acetylated peptides, including acetylation at K381 or K382, were plotted on the Y-axis.
  • 2G shows Western blot results confirming acetylated p53 (K320, 381 and ⁇ 382), total ⁇ 53 and other proteins of HEK293A cells transfected with pcDNA3.1-i (AIS0 and / or p300 expression vector).
  • FIG. 2H Shows Western blot results confirming the acetylated p53 (K320, K381), total p53, KAIS0 and ⁇ 300 proteins of HEK293A cells transfected with KAIS0 siRNA.
  • 3A-3I show that KAIS0 inhibits ubiquitination and degradation of p53, thereby increasing the stability of p53 and also increasing p53 binding to p53Res at 2 / bit and in vivo.
  • 3A shows the relative protein stability of p53 ′ and P53K381A.
  • H1299 p53 ⁇ / ⁇ cells transfected with p53WT or P53K381A expression vector were Western blotted cell lysates obtained by treatment of cyclonucleamide (CHX). Evaluation was based on GAPDH.
  • 3b shows ⁇ 53? ⁇ or P53 mRNA expression level of H1299 cells transfected with P53K381A expression vector was confirmed by RT-PCR.
  • FIG. 3C shows the results of polyubiquitination analysis of p53 by treatment with MG132 3 hours before the HEK293A cells transfected with His—ubiquitin, p53 and KAIS0 expression vectors.
  • FIG. 3D shows the results of poly-ubiquitination analysis of p53WT and p53K381A of His-ubiquitin, KAIS0, and H1299 cells transfected with p53WT or p53K381A expression vector.
  • 3E and 3F show oligonucleotide pulldown analysis of p53RE of p21MF / CDKN1A and PUMA promoters.
  • FIG. 3G shows the results of oligonucleotide pull-down analysis of p53 binding to the in vitro P 53 acetylation reaction complex of p53 + p300 and KAISO + p53 + p300.
  • Full-length protein (FL) means full-length protein.
  • 3H shows the results of ChIP / re-ChIP analysis of P53-KAIS0 that binds to the p53 target promoter.
  • H1299 cells were transfected with both p53WT and KAIS0 expression vectors and analyzed for binding of KAIS0-p53 complexes that bind to the aromatic gene promoter.
  • 3I shows the results of ChIP / re-ChIP analysis of the p53-KAIS0 complex that binds to the p53 target promoter.
  • H1299 cells were transfected with p53WT and p53K381A expression vectors, and the P53K381A modifications appear partially similar to p53 generated by the P53-KAIS0—p300 interaction.
  • FIGS. 4A and 4B show the mRNA expression levels were analyzed by RT—qPCR p53WT, P 53K381A, p53381R and P 53K320R / 382R expression vectors were transfected into H1299 cells and analyzed for mRNA expression, quantified based on 18S RNA.
  • FIG. 4D shows the results of ChIP analysis of RNA polymerase ⁇ enrichment downstream of the genes of the apoptosis pathway, Kaiso and Trp53 genes in Kato WT and Kaiso K0 MEF cells treated with etoposide (Eto). IgG was used as a ChIP control antibody.
  • ChIP enrichment was based on input DNA. RNA polymerase ⁇ binding enrichment revealed by ChIP reflects the transcriptional activity of the target gene. Increased RNA polymerase ⁇ binding of Trp53 by etoposide was not affected by the presence of Kaiso.
  • pl20ctn is p21 / CDKN1A, PUMA by KAIS0 and
  • FIG. 5a shows the results of analysis of the expression of KAIS0 by qRT—PCR and Western blot by treating HCT116 p53 ⁇ / ⁇ cells with various cytotoxic agents.
  • 5B shows the results of RT-qPCR confirming the KAIS0 target genes P 21, PUMA and BAX ⁇ mRNA expression levels in HEK293A cells in which KAIS0 and / or pl20ctn were ectopically expressed.
  • 5C shows immunohistochemical results of KAIS0 and / or pl20ctn expression in normal and cancerous tissues of lung and colorectal cancer patients, with black arrowheads representing KAIS0 and red arrowheads representing pl20ctn.
  • FIG. 5D shows the ratio of KAIS0 in the nucleus in normal and cancerous tissue observed with a microscope (Olympus BX51 Virtual microscope, 200 ' magnification).
  • Figures 6A-61 show that AIS0 is important for the transcriptional activity of p21 / CDI (NU and acetostatic genes) in Kaiso MEFs and mice.
  • Figure 6A is a theoretical model of the role of KAIS0 in aptosis.
  • Figure 6e confirms the mRNA expression of p53 and p53 target genes p21 and 3 ⁇ 4 4 in RT-PCR in Kaiso 0 mouse tissue, quantified based on 18S RNA RNA
  • Figure 6f is Kaiso Protein expression of p53 and p53 target genes p21, PUMA and p53 in K0 mouse tissues was confirmed by Western blot. 6G and 6H show immunohistochemistry results in Kaiso WT and K0 mouse skin, kidneys, colon, testis and spleen tissue. In Kaiso K0 mice, expression levels of target genes of Kaiso, such as P 21 and Bax ', were decreased, but expression of Pcna, which is an indicator of cell proliferation, was increased. .
  • FIG. 61 is a schematic diagram of KAIS0 activity in apoptosis. DNA damage strongly induces KAIS0 expression prior to induction of p53. AIS0 interacts with p53 and p300 to form the KAIS ()-p53-p300 complex and give the kill code to p53.
  • P53 with a death code (Ac-K320 / Ac-382 / nonacetylated K381) obtains protein stability, increased DNA binding activity and p53 target gene specificity.
  • the encoded p53, KAISO and p300 bind to the promoters of the cell cycle arrest genes p21WAF / CDKN1A and the genes of the endogenous acetopathic pathway.
  • the p21-Luc plasmid was provided by Dr. Yoshihiro Sowa of Kyoto Perpetual Medical University (Kyoto, Japan).
  • pcDNA3.1-p53 and pcDNA3.1—KAIS0 plasmids were incorporated into the cDNA fragment (KIAA0354) into pcDNA3.1 (Invitrogen, USA).
  • Prepared by cloning To prepare recombinant GST ⁇ P0ZKAIS0 and GST-ZFKAIS0 proteins, cDNA fragments encoding «) 2-domain (3.3. 1-107) and zinc finger (aa 492-672) were converted to pGEX4T3 (Amersham Biosciences, USA). Cloned into. Recombinant full length His-tagged KAISO.a. 1-672) and His-tagged P 53 were cloned into the pET21a vector. All plasmid constructs were confirmed by DNA sequencing. Antibodies and Recombinant Proteins
  • Antibodies to P 21 (sc-397), DR5 (sc-57084), FASL (sc-956), p53 (FL-393: sc-6243) and p73 (sc-7957) are described in Santa Cruz Biotech (Santa Cruz, From CA).
  • Polyclonal anti-p53 antibody (D ()-1) was purchased from BD Biosciences (San Jose, CA, USA).
  • Recombinant mouse Fas protein was purchased from Leinco Technologies, Inc., and recombinant murine TRAIL and human TRAIL were purchased from PEPR0TECH (PeproTech, NJ).
  • Recombinant His-KAIS0 (aa 1-672, full length) and His-p53 Proteins (aa 1-393, full length) were expressed in E. coli DH5 and purified using affinity chromatography.
  • Full length recombinant p300 was purchased from Active Moti f (Carlsbad, Calif.) And Upstate Biotechnology (Charlottesville, Va.).
  • Cell culture HEK293A, HCT116, H1299 and MEF cells were supplemented with 10% fetal bovine serum (FBS, Gibco-BRL, USA). cultured in modified Eagle's medium, Gibco-BRL, USA). Transcriptional analysis of the p21- and p53 ⁇ banung promoters
  • Lipofectamine plus in various cell lines (HEK293A, HCT116 and MB352) with various pGL2—p21—Lu promoter reporter fusion plasmids and pcDNA3.1-Kaiso, pcDNA3.1-p53 and / or pcDNA3—HADubiquitin expression plasmids in various combinations. Transfection was temporarily performed using a reagent (lipofectamine plus reagents, Invitrogen, USA). After incubation for 24 to 36 hours, cells were harvested and analyzed for luciferase activity. Reporter activity was corrected for transfection efficiency with co-transfected ⁇ -galactoxidase and showed three independent experimental results. Western blot analysis
  • HCT116 and HCT116 P 53 ⁇ / ⁇ cells were lysed with HKMG buffer (10 mM HEPES (pH 7.9), 100 mM KC1, 5 mM MgCl 2 , 10% glycerol, 1 mM DTT and 0.5% NP40).
  • Cell extracts were biotinylated double-stranded o 1 i gonuc 1 eo ti des (53RE-1, p53RE-2) for 1 hour with p21MF / CDKN1A and HMA biotinylated double-stranded oligonucleotides. Incubated.
  • DNA-binding protein To obtain a DNA-binding protein, the mixture was incubated for 2 hours with straptavidin-agarose beads, washed 5 times with HKMG buffer and precipitated using centrifugation. Precipitates were analyzed using Western blot, as described above using antibodies against KAIS0 and p53. GST-fusion protein purification, in vitro transcription and translation, and GST-fusion protein pull-down analysis of p53 and p300
  • Recombinant GST, GST-P0ZKAIS0 and GST-ZF AISO fusion proteins were prepared by incubating E. coli BL2KDE3) at 37 ° C for 4 hours in a culture medium with ImM IPTG. E. coli was dissolved and the protein was purified using glutathione-agarose 4-bead affinity chromatography (Peptron, South Korea). 1 lig P cDNA3.1-p53 or pcDNA3.1—p300 expression plasmids were constructed with 40 ⁇ TNT Quick Master Mix and 2, [ 3 3 ⁇ 4] -methionine (1175.0 Ci / mol) (Perkin Elmer Life Sciences, USA).
  • HEK293A and H1299 cells were transfected with KAIS0 expression vector or AISO siRNA. Cells were washed and then fixed with methane and stained for 30 minutes in 371: dark with a solution added propidium iodide (50 iig / mi) and ribonuclease A (100; «g / m«). DNA content, cell cycle profile and forward scatter were analyzed by FACS caliber (BD Bioscience, USA) (488 nm and 575 nm) The results were ModFit LT 2 dislike0 (Verity Software House, USA) and Wind MDI 2.8 (Joseph).
  • KAISO siRNA was transfected and transferred to 96-well culture dishes (4 ⁇ 10 3 ) and incubated for 0-6 days. Cells transfected with a KAIS0 expression vector reacted with 100 / well MTT (2 mg /) for 1 hour at 37 ° C. on days 0, 2, 4 and 6 of culture. Cells transfected with KAISO siRNA were subjected to ⁇ analysis as described above on culture days 0, 1, 2 and 3.
  • KAISO WT and K0 MEFs were seeded in 6-3 ⁇ 4 culture crate (6—well culture dishes overlaid with glass cover siides) at a density of about 1 ⁇ 10 4 cells / well and incubated overnight. MEF was treated with etoposide for 24 hours and TUNEL analysis was performed using a phosphorus killing detection kit (POE Roche, NJ).
  • SiRNA targeting Kaiso mRNA was prepared and purchased from Bioneer (Daejeon, Korea). siRNA (sense 5′-GUCUAGACCUUCAAACCAU-3 ′, antisense 5′-AUGGUUUGAAGGUCUAGAC-3 ′) was transfected into HEK293A and HCT116 cells using lipofectamine iMax (Invitrogen, USA). After tracking, cells were harvested to separate total RNA, and mRNA expression was analyzed by RT-qPCR as follows. Kaiso knockout mice and MEF cells
  • Kaiso WT alleles were identified by PCR using KLS and KES primer sets (Prokhortchouk et al., 2006).
  • Kaiso K0 alleles were detected by PCR amplification using KLS and KI2R primer sets (Prokhortchouk et al., 2006).
  • p53 stability analysis
  • PcDNA3—p53WT or pcDNA3- P 53K381A plasmid was transfected into H1299 p53 null cells grown in 10 cm culture dishes and incubated for 24 hours. Cells were treated with cyclonucleamide (100 ⁇ ) for 0, 1, 2 and 3 hours. Cells were harvested and lysed in 100 ⁇ SDS lysis buffer and cell lysates (50 proteins) were analyzed by Western blot. In vitro Protein Acetylation Assay
  • GST, GST-KAIS0P0Z, GST—KAISOZF and GST-p53 proteins were expressed in E. coli and purified by glutathione beads.
  • the HAT domain of purified p300 was purchased from Upstate Biotechnology (USA). In vitro p53 acetylation analysis was performed according to the manufacturer's protocol.
  • Recombinant His-KAIS0 (aa 1-672, full length) and His—p53 (aa 1-393, full length) proteins were expressed in E. coli in DH5a and purified by affinity chromatography.
  • Full length p300 is derived from Active motif (Carlsbad, CA) Purchased.
  • KAISO protein (5 yg) with p300 (2 U g) and His— p53 (6 yg) acetylation assay buffer (100 niM Tris-HCKpH 8), 20% glycerol, 2 mM DTT, 0.1 M NaCl, 20 mM butyl ic acid, 10 mM acetyl CoA) was incubated at 37 ° C.
  • the extracted gel pieces were destained and dried and hydrated again.
  • the dried gel pieces were reduced with 10 mM DTT and alkylated with 50 mM iodoacetamide (IAA).
  • Gel pieces were digested overnight with an sequencing grade grade trypsin (Promega, USA) at an enzyme: protein ratio of 1: 100 w / w (%). After digestion, peptides were extracted from the gel pieces and desalted with C18 stageTip 5 .
  • the parameters of mass spectrometry are as follows: injection voltage 1.8 kV; No sheath and auxiliary gas flow; Ion-transfer tube temperature, 200 ° C. All MS / MS spectra were obtained using the following parameters: normalized collision energy 35%; ion selection threshold, 1,000 counts; Activation Q, 0.25; Activation time, 20 ms. Dynamic exclusion was performed under conditions of repeat count 1, 30-s repeated persistence exclusion list size 500, exclusion persistence 45s, and ⁇ 1,5 m / z exclusion mass width. Data analysis of acetylation of full-length p53 obtained by high resolution mass spectrometry
  • Peptide identification was performed using the Andromeda search engine 6 from SEQUEST and MaxQuant software version 1.2.2.5 on the Sorcerer 2 platform. The same database and search parameters were used for all individual runs for the two search engines. . All MS / MS data were analyzed using a Targeted Decoy database search strategy against the International Protein Index (IPI) human v3.74 database. Searches included cysteine carbamidomethylation as a fixed variable and acetylation of Lys as a variable, oxidation of methionine and deamidation of asparagine and glutamine.
  • IPI International Protein Index
  • Database search parameters are as follows: semi-enzyme cleavage by trypsin up to 4 missing cleavage; Precursor ion mass tolerance 20 ppm; And acetylated peptides identified by fragment ion mass tolerance 0.5 Da ⁇ SEQUEST and Andromeda were filtered and verified using Scaffold 3 software (Proteome Software Inc., Portland, OR) and MaxQuant software, respectively.
  • LC-MS / MS analysis was performed twice for each sample group (p53, P 53 + p300, KAIS0 + p53 + p300) to quantify the acetylated peptide. Histology and immunohistochemistry
  • KAIS0 and pl20ctn (dilution 1) were placed on the slides.
  • the primary antibody against: 10 was incubated at 4 ° C. overnight, washed and then incubated with biotin-coupled universal secondary antibody for 1 hour at room temperature.
  • KAIS0 can arrest the cell cycle and induce aptosis. Since KAISO strongly arrests the cell cycle and induces acetosis, the present inventors investigated whether AIS0 can regulate the expression of CDKN1A, PUMA, BAX, and anti-athetostatic genes and what mechanisms are involved. KAIS0 increased W4 and murine gene expression (FIG. Lg). Interestingly, KAIS0 did not significantly affect p53 expression.
  • KAIS0 may affect the activity of p53, thus increasing the expression of p53 target genes involved in cell cycle arrest and apoptosis.
  • Post-translational modi f icat ion of p53 by p53 and acetyltransferase protein (HATs; p300, PCAF and TIP60) is known to play an important role in cell cycle arrest and aformosis 13 '17 -18 .
  • HATs acetyltransferase protein
  • KAISO, p53 and p300 are major regulators of cell cycle arrest and aloptosis
  • the inventors examined their interaction with KAIS0.
  • KAISO, p53 and p300 interacted with each other to form a complex was confirmed by GST-fusion pull-down analysis and co-ii munoprecipitation (Fig. Li and Fig. Lj).
  • the complex interacts with the C′-terminal DNA—binding domain of p53 via the P0Z domain and zinc-finger DNA binding domain (ZFDBD) of KAIS0.
  • ZFDBD zinc-finger DNA binding domain
  • the P0Z domain also interacts with the DUF906 domain (# 3), which includes the hi stone acetyltransferase (HAT) portion of p300.
  • HAT hi stone acetyltransferase
  • RT-QPCR confirmed the mRNA expression level of the representative aptotic gene in cells transfected with KAIS0 or KAISO—K8, but KAIS0 significantly increased p21 and acetotropic genes, but the control gene, ⁇ gene, did not.
  • AIS0-K8 lacks the P0Z domain and the zinc-finger domain, which are characteristic domains that interact with p53 and p300, and dominates KAIS0 It is in dominant negative form.
  • KAIS0-K8 failed to activate the transcription of apoptotic genes and P21 / CDKN1A, KAIS0 and KAISO-K8 expressed similarly (FIG. Lk and FIG. 11).
  • p53K381A substituted K at position 381 with A
  • p53K381R mutated K to R and retains positive charge, but can be methylated
  • KIAS0 inhibits p53 ubiquitation when KAIS0 and ubiquitin expression vectors are present It was confirmed through the ubiquitination analysis of p53 (Fig.
  • KAIS0 strongly restrains the cell cycle and induces apoptosis
  • KAIS0 increases the expression of PUMA and genes, while inhibiting the expression of the anti-apoptotic gene BcI-2.
  • KAIS0 activates the transcription of CDKN1A by acting on a distal expression regulatory site composed of p53 binding elements.
  • KAIS0 binds to p53RE, a p53 binding element near exon la, and AIS0 has shown the ability to bind to this element, requiring p53 to bind to this element, through nucleotide pull-down analysis (FIGS. 3E and FIG. 3f).
  • the ligation of nucleotide pull-down analysis of the in vitro p53 acetylation reaction complex comprising p53 + p300 or KAISO + p53 + p300 shows that p53 binding to the p53RE probe is significantly increased by KAIS0 (FIG. 3g).
  • the p53 acetylation code, or "death code” is used to regulate cell cycle arrest and aptosis.
  • p53K381A or p53K381R binds more strongly to the p53 target gene promoter than p53WT and increased the transcription of CDKN1A and aformotic genes more strongly.
  • KAIS0 affects the whole process of aptosis.
  • RNA polymerase ⁇ binding to the downstream axon of Trp53 was increased by etoposide in both Kaiso T and Kaiso K0 MEF (FIG. 4D).
  • ChIP analysis indicates that transcription of the p53 target gene of the atopic pathway is activated by etoposide and that the RNA polymerase ⁇ is actively involved in transcription in the presence of Kaiso. "on and off, may be one of the key regulators of regulation. Induction of expression of KAIS0
  • chemotherapeutic agents ionizing radiation or ultraviolet light, which are known to induce the expression of p53, can induce the expression of KAIS0 (FIG. 5A).
  • chemotherapeutic agents ionizing radiation or ultraviolet light
  • KAIS0 the expression of KAIS0
  • all p53 inducers induced the expression of KAIS0, which influenced the stability and activity of p53 in the regulation of cell cycle arrest and aptosis genes.
  • the ⁇ -catenin analogue pl20ctn of the noncanonical Wnt signaling pathway interacts with KAIS0 and changes the intracellular location of KAIS0 from the nucleus to the cytoplasm, which is important for inhibiting KAIS0 activity in transcription and tumor growth.
  • AIS0-activated genes such as p21, PUMA and BAX, which regulate cell cycle arrest and apoptosis, was significantly reduced by pl20ctn (FIG. 5B).
  • pl20ctn pl20ctn
  • P 120ctn was observed in the nucleus and inner cell membrane, respectively.
  • AIS0 was predominantly observed in the nucleus (40-50%); Only 10-16% of KAIS0 was observed in the nucleus in cancerous tissues (FIG. 5D).
  • 120ctn was expressed in the cytoplasm at low levels in normal tissues; 120ctn was significantly increased in cancer tissues and observed in the cytoplasm and nucleus.
  • lung and colorectal cancer In tissues pl20ctn expression was high, in 16 samples (60%) of 27 lung cancer samples, in 20 samples (62.5%) of 32 colorectal cancer samples, and KAIS0 and pl20ctn were co-located in the cytoplasm.
  • the apoptosis activity or tumor suppression activity of KAIS0 was inhibited by P 120ctn (FIG. 5C).
  • KAISO and / or p53-mediated endogenous and exogenous acetotropic phenomena initiated by DNA damaging, etoposide or cispoline killing receptor ligands in KAISO YJd MEF cells change. It remained similar in WT and K0 ME and similarly induced by etoposide in both KAISO MEFs. Even when cells were treated with etoposide, the expression levels of endogenous Puma, Bax and KAISO were not significantly down regulated or detected compared to the protein levels of WT MEF (FIGS. 6A and 6B).
  • the present inventors also studied whether apoptotic phenomena including activation of death receptors and caspase activation by ligands such as Trail or FasL are affected by deficiency of KAIS0.
  • apoptotic phenomena including activation of death receptors and caspase activation by ligands such as Trail or FasL are affected by deficiency of KAIS0.
  • the expression of some endogenous killing receptors was significantly lower or relatively less induced compared to Kaiso WT MEF cells.
  • activation of killing receptors activated the expression of KAIS0, killing receptor and caspase, and this change was not observed in KAISO K0 MEF cells (FIGS. 6C and 6D).
  • KAIS0 regulates overall atoptoses, including the exogenous pathways involved in killing receptors, as well as the endogenous apoptosis pathway in which p53 plays a major role.

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Abstract

La présente invention concerne une composition induisant l'apoptose, qui comprend une protéine KAISO en tant que principe actif. L'invention porte également sur un inhibiteur ou un agent thérapeutique permettant d'inhiber ou de traiter les lésions provoquées par la génotoxicité. Plus particulièrement, la présente invention concerne une composition induisant l'apoptose qui comprend, en tant que principe actif, un polypeptide (protéine KAISO) possédant une séquence d'acides aminés d'une seconde séquence sur une liste de séquences ou une séquence nucléotide codant pour une séquence d'acides aminés d'une seconde séquence sur une liste de séquences. L'invention a également trait à un inhibiteur ou une composition thérapeutique permettant d'inhiber ou de traiter des lésions provoquées par la génotoxicité. La présente invention permet de procurer à KAISO une fonctionnalité en tant que régulateur principal de la voie p53 associée à l'arrêt du cycle cellulaire et à l'apoptose, ainsi qu'une méthode de traitement du cancer ; pour ce faire, l'invention porte sur des médicaments anticancéreux à petites molécules aptes à induire fortement l'apoptose au moyen de KAISO, ainsi que sur un inhibiteur d'interaction KAISO-p120ctn, dans les cas où KAISO dans un cytoplasme est piégée par p120ctn pour inhiber l'activité de KAISO. Par ailleurs, l'invention a trait à une composition induisant l'apoptose comprenant la protéine KAISO spécifiée ici en tant que principe actif, et à une composition destinée à inhiber ou traiter les lésions provoquées par la génotoxicité.
PCT/KR2012/007960 2011-09-28 2012-09-28 Composition induisant l'apoptose, comprenant une protéine kaiso Ceased WO2013048211A2 (fr)

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Title
DATABASE NCBI 18 May 2010 'Homo sapiens zinc finger and BTB domain containing 33 (ZBTB33), transcript variant 1, mRNA.' Database accession no. NM_001184742.1 *
HUH, MAN UK: 'KAISO, a unique modifier of the p53 code, is a master regulator of apoptosis' ABSTRACT ON SEMINAR OF RESEARCH CENTER FOR CONTROLLING PROTEIN FUNCTION 23 September 2011, *
ZHAO, L. Y. ET AL.: 'Negative Regulation of p53 Functions by Daxx and the involvement of MDM2' THE JOURNAL OF BIOLOGICAL CHEMISTRY vol. 279, no. 48, 2004, pages 50566 - 50579 *

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