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WO2008108521A1 - Nouvelle utilisation de kiaa0317 en tant que régulateur de l'apoptose - Google Patents

Nouvelle utilisation de kiaa0317 en tant que régulateur de l'apoptose Download PDF

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Publication number
WO2008108521A1
WO2008108521A1 PCT/KR2007/004553 KR2007004553W WO2008108521A1 WO 2008108521 A1 WO2008108521 A1 WO 2008108521A1 KR 2007004553 W KR2007004553 W KR 2007004553W WO 2008108521 A1 WO2008108521 A1 WO 2008108521A1
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kiaa0317
apoptosis
gene
protein
treating
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Deug-Yong Shin
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GENCROSS CO
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GENCROSS CO
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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
    • 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
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1761—Apoptosis related proteins, e.g. Apoptotic protease-activating factor-1 (APAF-1), Bax, Bax-inhibitory protein(s)(BI; bax-I), Myeloid cell leukemia associated protein (MCL-1), Inhibitor of apoptosis [IAP] or Bcl-2
    • 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
    • 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
    • C07K14/4747—Apoptosis related proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2510/00—Detection of programmed cell death, i.e. apoptosis

Definitions

  • the present invention relates to a cell growth regulator using a particular protein, and more particularly an apoptosis regulator using KIAA0317. [Background of the invention]
  • Apoptosis means a cell disruption or a cell suicide found in a eukaryotic cell (Kerr et al., 1972), which is a fundamental intercellular process for maintaining homeostasis, for example, to regulate normal generation, to remove unnecessary cells or abnormal cells, and so on.
  • Apoptosis is caused by responding to various external and internal stimuli. While apoptosis is processing, the characterized changes are carried out in a cell as follows: the cytoplasm is disrupted; the cell membrane forms a bleb; the cytoskeleton is changed; the cell is shrunken; the chromosome is condensed; and DNA is fractionized (Kerr et al., 1972).
  • Such apoptosis is a physiologically important phenomenon, and it is elaborately regulated by a complicatedly intercellular mechanism.
  • apoptosis is inappropriately regulated, unsuitable apoptosis is induced. That is, the suppression or the promotion of apoptosis is related to many diseases. Specifically, excessive apoptosis may cause a certain cell to be disrupted; thereby, it may also cause the loss of in vivo functions that are mediated by the same.
  • central nervous system disease for example a plurality of acute and chronic degenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, cerebral ischemia/stroke) (Mochizuki et al., 1996; Smale et al.,1995; Thomas et al., 1995; Robertson, et al., 2000), cardiovascular disease (Yue et al., 1999), and autoimmune disease such as Graves' disease and type Il diabetes (O'Reilly et al., 1999; Magge, 1998).
  • the representative disease relating to abnormal suppression of apoptosis is cancer. If a tumor cell is not removed due to hypoactivity of apoptosis, tumor cells will be accumulated, which is related to occurrence of cancer (Vogelstein et al., 2000; Vousden et al., 2002).
  • the understanding of the pathogenesis of the various diseases may be increased, which is useful in the development of agents for treating/diagnosing the diseases relating to apoptosis.
  • the pathway to apoptosis can be divided into one passage through an apoptosis receptor and another path not through the receptor.
  • the former is apoptosis that is induced by binding a certain intercellular ligand with its receptor.
  • the related receptor may include Fas, TNFR (tumor necrosis factor receptor 1 ), TRAIL (TNF-related apoptosis-inducing ligand), and so on.
  • the latter is apoptosis that is induced by stress.
  • the stress causing apoptosis includes ultraviolet rays, heat shock, gamma-ray irradiation, and a hypo-oxygenated state.
  • the apoptosis caused by these stimuli is completed through factors functioning in the downstream.
  • the representative example of the factors is a caspase which is a protease.
  • a protease affects a group of proteins sequentially functioning in a signaling pathway toward the apoptosis, and it regulates the apoptosis by cleaving a certain amino acid (aspatate) region and activating a series of proteins.
  • Walczak et al. (1999) disclosed a use for treating a cancer of TRAIL protein, which is an apoptosis-promoting factor induced through a cell receptor.
  • Fas-L as a target for regulating the apoptosis induced by a cell receptor.
  • WO03/076647 disclosed a method of screening a JADE gene, protein, and a material regulating the apoptosis using the same that function as a regulator of the apoptosis and the cell period in the downstream of apoptosis.
  • Korean Patent Laid-open Publication No. 2001-113088 disclosed a CIA protein, a gene, and a use thereof that regulate the apoptosis by interacting with a certain DNase activated by a caspase.
  • Korean Patent Laid-open Publication No. 2002-0040521 disclosed an anticancer drug that includes a microlactone using apoptosis.
  • the representative protein relating to the apoptosis induction is p53.
  • p53 is known as a medium of the apoptosis in a cell showing abnormal cell growth due to a carcinogen such as Myc, and p53 regulates the removal of such cell (Vogelstein et al., 2000; Vousden et al., 2002).
  • p53 is a representative tumor suppression protein that repairs damaged DNA or regulates a cell period. It acts as a transcription regulator having high affinity for a target sequence in a promoter of a certain gene, so that it regulates the target gene expression at the transcription level.
  • p53 suppresses cell cleavage or selectively destroys abnormal cells having damaged DNA or abnormal cells that are abnormally cleaved, so that it plays an important role in prevention of cells from progressing to become cancer cells (Oda et al., 2000).
  • the present inventors accomplished the present invention by cloning a gene that applies resistance to apoptosis induced by p53 using a p53-deficient cell, in order to isolate a novel factor that is capable of regulating the apoptosis that is induced by a cell receptor or various exogenous stressors.
  • the present invention relates to a novel use of a KIAA0317 protein and a gene encoding the same as an apoptosis regulator, and particularly relates to: a method of regulating apoptosis by using a KIAA0317 gene, a protein, or a mutant that is functionally equivalent to the same; an apoptosis regulator including an active ingredient of a KIAA0317 gene, a protein, or a mutant that is functionally equivalent to the same; and an agent for treating a disease relating to apoptosis.
  • the present invention provides an apoptosis regulator including an effective ingredient of a KIAA0317 protein, a gene encoding the same, or a mutant that is functionally equivalent to the same.
  • the present invention further provides an agent for treating or supplementarily treating a cancer including an effective ingredient of a KIAA0317 gene or a protein expression or functional regulator.
  • the present invention still further provides a pharmaceutical composition for diagnosing, treating, or preventing a disease relating to the hypoactivity of apoptosis including an effective ingredient of a KIAA0317 gene or protein expression or functional regulator.
  • the present invention still further provides a method of screening a gene that regulates apoptosis.
  • the present invention still further provides a method of treating, diagnosing, or preventing a disease related to hyperactivity of apoptosis including the step of administrating an effective amount, for the treatment, the diagnosis, or prevention, of a KIAA0317 protein, a gene encoding the same, or a mutant that is functionally equivalent to the same to a subject.
  • the present invention still further provides a method of regulating apoptosis including the step of contacting a KIAA0317 protein, a gene encoding the same, or a mutant that is functionally equivalent to the same to the cell.
  • the present invention still further provides a method of treating or supplementarily treating a cancer including a step of administrating an effective amount, for treating or supplementarily treating a cancer, of an effective ingredient of a KIAA0317 gene or protein expression inhibitor to a subject requiring treatment or supplementary treatment of the disease.
  • the present invention provides a method of screening a gene for suppressing apoptosis, including steps of: (a) introducing a mammalian origin cDNA library to a eukaryotic cell;
  • step (c) collecting cells in which the apoptosis is not induced from the step (b);
  • step (d) isolating cDNA from cells of step (c), wherein the cDNA isolated from cells of the step (d) has a function of regulating apoptosis.
  • the term "regulation" used in the present invention means to include both downward regulation such as suppression and upward regulation such as facilitation.
  • the regulation means to suppress.
  • the mammal includes a human, a mouse, a monkey, and so on, but it is not limited thereto.
  • the mammal is particularly a human or a mouse.
  • the cDNA library includes various eukaryotic animal origins including a human origin and a mouse origin, but it is not limited thereto.
  • the present invention includes various commercially available libraries or various origin libraries prepared by using mRNA extracted from a tissue of interest in accordance with the conventional methods.
  • the eukaryotic cell is a malfunctioning cell in which the p53 gene is missing, or in which the p53 protein is inactivated.
  • the eukaryotic cell is of human origin, but it is not limited thereto.
  • the eukaryotic cell may be an osteosarcoma origin Saos-2 cell.
  • the p53 gene in step (b) can be obtained by various conventional methods of inducing DNA into a eukaryotic cell, for example, by virus transportation using a virus vector, by transfection using a calcium phosphate precipitation, and by transfection using a lipid such as liposome, but it is not limited thereto.
  • the method of separating cDNA from cells in which apoptosis is not performed in accordance with said method may include any conventional methods known in this art.
  • the references mentioned can be referred to (Sambrook, ibid; Miller & Calos, ibid; Current Protocols in Molecular Biology).
  • the apoptosis inhibitory gene cloned by the method can be reconfirmed regarding whether the apoptosis is performed or not by inducing the p53 into other cells having the same or similar characteristics as those used in the screening step.
  • Measurements for confirming whether the apoptosis has occurred are taken by a conventional method such as a DNA ladder phenomenon, TUNNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling) staining and so on due to a nuclear dye, and chromosome DNA fractioning.
  • 2.2 kb cDNA is separated from clones (9-51), which provides strong resistance against apoptosis that is induced by p53, and the base sequence was determined (SEQ ID No. 5).
  • the determined DNA sequence was applied to a homology analysis using a computer with a program called BLASTX (Altschul et al., 1990; http://www.ncbi.nlm.nih.gov.) or BLASTN which were developed from BLAST (Karlin et al., 1993). From the results (FIG. 13), 9-51 corresponds to KIAA0317 (SEQ ID NO.
  • KIAA0317 has very low similarity with a HECT (homology to E6-AP carboxy terminus) region (FIG. 2 and FIG. 3).
  • HECT homology to E6-AP carboxy terminus
  • the HECT (homology to E6-AP carboxy terminus) region is a conservational region composed of approximately 350 amino acid residues, and is mainly found in E3 ubiquitin ligase relating to protein ubiquitination, for example, E6-AP (Huibregtse et al., 1995), Nedd4 (Harvey and Kumar, 1999), SMURF2 (Lin et al., 2000), and PUP1 (Arendt and Hochstrasser, 1997) (Huibregtse et al., 1995).
  • E3 ubiquitin ligase including around 20 HECT regions has been found thus far, and the functions thereof are extremely varied depending upon the intercellular functions in which the ubiquitinization is involved (You and Pickart, 2001 ). Therefore, many tests are required to investigate the specific intercellular functions, which is not easily cognized by a person having ordinary skill in the art, even though it is a protein including the HECT region, and particularly, it is less easily cognized, as in KIAA0317 according to the present invention, in the case that it has low homology of less than 35% with the HECT region of the known E6-AP, hNedct ⁇ , hSMURF2, hPUP1 , and yRSP ⁇ (FIG. 2 and FIG. 3). However, as a result of continued studies, the present inventors found that KIAA0317 can effectively suppress apoptosis with the E3 ubiquitin ligase including the HECT region.
  • the KIAA0317 protein of the present invention has E3 ubiquitin ligase activity (FIG. 4).
  • E3 ubiquitin ligase activity FIG. 4
  • over-expression of the KIAA0317 gene remarkably suppresses the apoptosis that is caused by p53, TNF-alpha, staurosporine (STS), and so on (about 50% or more, FIG. 5 and FIG. 6).
  • KIAA0317 is over-expressed in various cancers such as a stomach cancer, colon cancer, a hepatoma, breast cancer, and lung cancer at 48%, 46%, 33%, 41%, and 42%, respectively (FIG. 9).
  • siRNA small interfering RNA
  • the expression suppression of endogenic KIAA0317 protein due to siRNA induces the apoptosis, which suppresses the cancer cell growth and induces sensitivity to anticancer drugs (FIG. 10 and FIG. 11 ).
  • siRNA small interfering RNA
  • a KIAA0317 gene according to present invention can be useful as an apoptosis regulator, an agent for treating or supplementarily treating a cancer, a target material for developing a new anticancer drug, and an agent for diagnosing cancer.
  • the present invention provides a novel apoptosis regulator including an effective ingredient of a KIAA0317 protein, a gene encoding the same, or a mutant that is functionally equivalent to the same.
  • the KIAA0317 protein and a gene sequence encoding the same can use various mammalian origins, and preferably, it is of human origin. It includes one provided by purifying the natural sequence source that is present in all kinds of cells of human and non-human mammalian species, chemically synthesizing it, or producing it through DNA recombination technology, or associating these methods and/or other methods.
  • the KIAA0317 protein and the gene encoding the same includes "a mutant that is functionally equivalent" to the same.
  • the mutant that is functionally equivalent indicates a compound having biological activity that is equivalent to the sequence that is commonly found in nature.
  • the mutant that is functionally equivalent indicates that it has effects of the present invention including the activity of apoptosis suppression.
  • the mutant that is functionally equivalent includes a gene of GenBank Accession No. AB002315 (human) (SEQ ID NO 1 ) and AK077015 (mouse) (SEQ ID NO 2), and a 29470 sequence disclosed in U.S. Patent Publication No.
  • the mutant that is functionally equivalent includes a sequence mutant in which a nucleotide including a gene sequence or amino acid sequence including a protein sequence is substituted, missed, added, and/or inserted.
  • the protein sequence mutation can be provided by any conventional method known by a person of ordinary skill in the art, for example by site-directed mutagenesis (Kramer, W. & Fritz, H-J. Oligonucleotide-directed Construction of Mutagenesis via Gapped Duplux DNA. Methods in Enzymology, 154, 350-367, 1987) and so on.
  • site-directed mutagenesis Karlin, W. & Fritz, H-J. Oligonucleotide-directed Construction of Mutagenesis via Gapped Duplux DNA. Methods in Enzymology, 154, 350-367, 1987
  • the polynucleotide sequence mutation can naturally occur.
  • sequence mutation is carried out either together with an amino acid sequence mutation or not.
  • the gene according to the present invention includes such degeneracy mutants.
  • the gene encoding the mutant that is functionally equivalent to a KIAA0317 protein may be obtained in accordance with the conventional method such as hybridization (Southern, E. M., Journal of Molecular Biology, 98, 503, 1975) or a PCR method (Saiki et al., Science, 230: 1350-1354, 1985; Saiki et al., Science, 239: 487-491 , 1988).
  • a person of ordinary skill in the art could separate a gene having high homology with the KIAA0317 gene from the KIAA0317 protein sequence by providing a primer that is capable of hybridizing specifically to KIAA0317 gene.
  • the protein coded by the separated gene has high homology to a natural KIAA0317 protein in an amino acid level.
  • High homology refers to homology of 50% or more with respect to the entire amino acid sequence, particularly 70% or more, and more particularly 90% or more (e.g., 95% or more).
  • the homology of the amino acid sequence or base sequence may be analyzed by BLASTN that was developed from BLAST (Proc. Natl. Acad. Sci. USA, 90, 5873-5877, 1993) or a program called BLASTX (Altschul et al, J. MoI. Biol., 215, 403-410, 1990), of which the specific method is open to the public in the following website (http://www.ncbi.nlm.nih.gov.).
  • the KIAA0317 protein and the gene encoding the same further include a fragment having biological activity that is equivalent to the natural sequence.
  • fragment indicates a sequence corresponding to a part of a gene or a protein.
  • a gene it includes a physically, endonuclease, or chemically-cleaved one
  • a protein in the case of a protein, it includes both a protease-cleaved one and a chemically-cleaved one.
  • the protein it includes a mutant for modifying the safety, preservation, and solubility characteristics of the KIAA0317 protein or a mutation for modifying interaction with other interactive proteins.
  • the KIAA0317 gene or the mutant that is functionally equivalent to the same may be used by itself or by being involved in a vector.
  • the method of introducing DNA into the vector is known in this 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 Sea Eds., Greene Publ. Assoc, Wiley-lnterscience, 1992).
  • a pCEV29 Movity et al.
  • Various vectors that are capable of being present independent from a chromosome in a cell or inducing into a chromosome, or expressing and/or amplifying in a cell are disclosed.
  • the vectors include a plasmid vector, linear DNA, or a certain vector that is suitable for accomplishing the object.
  • Such vectors include a chemical conjugate vector including a ligand to a receptor on the cell surface or a nucleic acid binding moiety (for example a polylysine binding portion) (for example disclosed in WO 93/04701); a virus vector (for example, a DNA or RNA virus); and a fused protein expression vector including a fusion protein moiety that is fused with an introduced protein and expressed (for example an antibody moiety recognizing the antibody of the cell glutathione S-transferase for compliance to separate and detect, or a fluorescent protein moiety).
  • a person of ordinary skill in the art can easily select a vector that is suitable for the purpose.
  • the KIAA0317 gene of the present invention may be involved in a virus moiety as a transformer by being introduced in an expression vector, for example an adenovirus vector, which is used in a gene therapy system.
  • the KIAA0317 polynucleotide according to the present invention and all mentioned mutants may be prepared by using a conventional organochemical method of polypeptide synthesis. In order to obtain a desired full-length sequence, the sequences synthesized from fragments may be bound to each other (The Peptides, Analysis, Synthesis, Biology, Vol. 1-9, Gross, Udenfriend, and Meienhofer Eds. 1979-1987, Academic Press Inc.). Particularly, the KIAA0317 polynucleotide according to the present invention, a fragment thereof, or a mutant that is functionally equivalent to the same is preferably prepared by using gene recombination technology.
  • KIAA0317 polypeptide After natural KIAA0317 polynucleotide is expressed in a suitable host cell, it can be provided as a cell fusion; or after KIAA0317 mRNA is translated in a test tube, it can be purified according to a conventional method of separating a protein.
  • the general gene recombination techniques and the protein purification methods may be found by referred to the following: Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989; and Current Protocols in Molecular Biology, Ausubel et al., Eds., Greene Publishing Associates and Wiley-lnterscience 1991.
  • the KIAA0317 polypeptide according to the present invention includes a purified protein, a water-soluble protein, or a fusion with a protein or amino acid residue that is bound with a carrier for carrying to the target cell or for administration.
  • the intercellular expression of KIAA0317 according to the present invention remarkably suppressed apoptosis induced by p53, TNF-a, and STS (by around 50% or more, FIG. 4).
  • the suppression of endogenous KIAA0317 expression by siRNA specific to KIAA0317 results in sensitization to apoptosis inducers.
  • the KIAA0317 gene according to the present invention has a novel function for suppressing apoptosis.
  • the KIAA0317 protein having a function for suppressing apoptosis can be used for suppressing abnormally-induced apoptosis. Therefore, the results indicate that it can be useful for an agent for treating a disease that is induced from the abnormal apoptosis.
  • the apoptosis regulator according to the present invention is characterized in a use for treating and preventing a disease relating to hyperactivity of apoptosis.
  • disease relating to the hyperactivity of apoptosis may include, but is not limited to, central nervous diseases such as a plurality of acute and chronic degenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, cerebral ischemia/stroke) (Mochizuki et al., 1996; Smale et al., 1995; Thomas et al., 1995; Robertson et al., 2000), cardiovascular diseases (Yue et al., 1999), and autoimmune diseases such as Graves' disease and type Il diabetes (O'Reilly et al.,1999; Magge et al., 1998).
  • central nervous diseases such as a plurality of acute and chronic degenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, cerebral ischemia/stroke) (Mochizuki et al., 1996
  • a KIAA0317 protein, a gene encoding the same, or a mutant that is functionally equivalent to the same is characterized in being particularly related to the HECT region of the KIAA0317 protein. According to one embodiment of the present invention, it is proven that the apoptosis can be sufficiently suppressed by the HECT region of only the C-terminal region of KIAA0317.
  • the present invention has been fundamentally designed from the findings that KIAA0317 is useful as the apoptosis inhibitor and KIAA0317 is over-expressed in diseases relating to the hypoactivity of apoptosis such as in cancer cells (referring to FIG. 6).
  • the present invention therefore provides a pharmaceutical composition for treating or preventing a disease relating to the hypoactivity of apoptosis including an active ingredient of the KIAA0317 gene or the protein expression inhibitor.
  • a disease relating to hypoactivity of apoptosis may include, but is not limited to, a cancer due to, particularly, the inactivation of p53 (Vogelstein et al., 2000; Vousden et al., 2002).
  • KIAA0317 is overly expressed in stomach cancer, a hepatoma, colon cancer, and lung cancer, which means the apoptosis is suppressed in a cancer cell including the cancer tissue. Therefore, it is suggested that inducing apoptosis is effective in treatment of a cancer due to the KIAA0317 function loss.
  • the term "inhibitor” indicates any material that suppresses the expression of the KIAA0317 gene, the expression of the KIAA0317 protein, and/or the activity of the expressed protein without limitation.
  • it may include, but is not limited to, a material obtained by a synthesis or a gene recombination technique, a natural origin material, or a derivative thereof such as an antibody, an antisense nucleotide, and so on.
  • an inhibitor of si RNA can effectively suppress the function of the KIAA0317 apoptosis inhibitor.
  • the siRNA indicates a double-stranded RNA molecule inhibiting its translation by binding with the target mRNA, which includes a sense strand and an antisense strand.
  • the sense strand includes a ribonucleotide sequence corresponding to the target sequence selected from KIAA0317 gene sequences, for example SEQ ID NO 1 or 2, and the antisense strand includes a ribonucleotide sequence complementary to the sense strand.
  • siRNA is expressed during a bio-developing process or a bio-preventing process against the virus, and so on.
  • siRNA When it is bound with mRNA, it induces binding with an intracellular enzyme composite recognizing this, and it decomposes the corresponding mRNA (Fire, 1998; Montgomery et al., 1998; Sharp PA, 1999).
  • siRNA is generally a double-stranded RNA of 20 or 21 mer, and it is composed of 19 complementary sequences and a dimer of 3'-terminal non-complementary thymidine or uridine.
  • Such double-stranded RNA specifically suppresses the corresponding gene when it is introduced in the cell (Elbashir et al., 2001 ).
  • siRNA according to the present invention may include two complementary nucleotide molecules or one molecule (transcript) of a sense and antisense sequence as in a hairpin structure.
  • the length of siRNA is around 10 or more nucleotides, and the length of natural KIAA0317 transcript can be longer.
  • the siRNA has a length of around 100 or less nucleotides, and, most particularly, it ranges from 15 to around 25 nucleotides.
  • the function of KIAA0317 is effectively suppressed by the double-stranded molecule having a sense strand and antisense strand of SEQ ID NO. 3 or 4, respectively.
  • the complementary polynucleotide sequence can form a double-stranded molecule or a hairpin-structured double-stranded molecule having several mismatches or no mismatch by hybridizing under suitable conditions. Considering the object of the present invention, two sequences having 5 or fewer mismatches may be considered as complementary.
  • the method of introducing siRNA into a cell is publically known.
  • siRNA is expressed by transfecting a vector including a DNA template that is capable of being transcribed to siRNA, into a cell.
  • siRNA is obtained from a region corresponding to a human KIAA0317 gene, and it is introduced into a 293 cell line in which KIAA0317 is highly expressed. As a result, it specifically suppresses the expression of KIAA0317.
  • the results indicate that the apoptosis increase is observed in the 293 cells, it increases the sensitivity to TNF-alpha which is one of apoptosis promoters, and it also increases sensitivity to the conventional anticancer drugs (Example 7, FIG. 7 and 8). Particularly, the phenomenon in which the sensitivity to conventional anticancer drugs is increased due to the KIAA0317 suppression is highly effective in a clinical aspect.
  • KIAA0317 inhibitor to KIAA0317 such as siRNA
  • an anticancer drug as a supplemental drug for increasing the sensitivity of the conventional anticancer drug
  • it can considerably reduce the administrating amount of the conventional anticancer drug that has many side effects. Therefore, it improves the patient's welfare and economic aspects.
  • the pharmaceutical composition that is useful for treating or preventing a disease relating to the hypoactivity of apoptosis including an effective ingredient of the KIAA0317 gene or protein expression inhibitor according to the present invention may further include an anticancer drug.
  • the present invention provides an agent for treating or supplementahly treating a cancer including an effective ingredient of the KIAA0317 gene or protein expression inhibitor according to the present invention.
  • the present invention provides an anticancer drug characterized in associating the KIAA0317 gene or protein expression inhibitor according to the present invention with an anticancer drug.
  • the inhibitor is characterized in increasing the sensitivity of a cancer cell to an anticancer drug.
  • the anticancer drug may include any conventional anticancer drugs.
  • it includes, but is not limited to, cisplatin, doxorubicin, and etoposide.
  • the present invention further provides a kit for diagnosing a disease related to the apoptosis including a means for detecting a KIAA0317 gene or KIAA0317 protein expression or KIAA0317 gene mutation.
  • the present inventors found that the KIAA0317 gene or protein not only could suppress the apoptosis in a cell in which the apoptosis is induced, but is also over-expressed in a cancer in which the apoptosis is hypoactivated. Accordingly, the KIAA0317 gene and/or protein are measured for expression amount, so the apoptosis-related diseases are diagnosed and/or evaluated for progression degree.
  • the diseases relating to apoptosis are as above.
  • the expression amount may be measured by the conventional detection means.
  • it includes, but is not limited to, in the case of gene expression, an RT-PCR method in which RNA is separated from biosyp, cDNA is synthesized through reverse transcription, then PCR (polymerase chain reaction) is carried out with a suitable primer that is capable of amplifying specific to KIAA0317; or real-time quantitative PCR 1 cDNA array, Northern blot, and so on.
  • PCR polymerase chain reaction
  • the function of KIAA0317 is deteriorated or the expression of a gene or protein is decreased by a certain mutation, for example the presence of a substitution, a subtraction, an addition, and so on, to the KIAA0317 coding sequence or regulation region such as a promoter and an enhancer.
  • the function of KIAA0317 is deteriorated, or the expression of the gene or protein is decreased.
  • a risk of having a disease relating to the apoptosis could be preliminarily predicted by analyzing the mutation of a gene including the KIAA coding sequence, the regulation region, and comparing a differentiation to the general gene.
  • the differentiation between the mutant gene sequence and the normal sequence is determined by any conventional method such as cDNA base sequence analysis, PCR-SSCP (single strand confirmation polymorphism), PCR-SSO (sequence-specific oligonucleotide), and so on, but it is not limited thereto.
  • the present invention further provides a method of screening a material that is useful as an agent for treating a disease relating to the apoptosis.
  • the present invention provides a method of screening at least one material selected from the group consisting of a material regulating KIAA0317 gene expression, a material regulating KIAA0317 protein expression, and a material regulating KIAA0317 protein function.
  • the material is preferably a material obtained from a synthesis or gene recombination technology, a natural origin material, or a derivative material thereof.
  • a method of screening the material includes the step of (1) contacting the material with a region controlling KIAA0317 gene transcription and a transformation cell including a KIAA0317 gene or a reporter gene, or an in vitro expression system, to detect the expression amount of the KIAA0317 gene or reporter gene; or (2) contacting the material with a target molecule in the presence of a KIAA0317 protein to detect the apoptosis suppression function of the KIAA0317 protein.
  • the report gene may include, for example, chloramphenicol acetyl transferase enzyme(CAT), ⁇ -galactosidase ( ⁇ -Gal), luciferase, and so on.
  • the material regulating the KIAA0317 gene expression may be screened by, for example, providing an expression vector in which a region regulating the KIAA0317 gene transcription (promoter, enhancer) is bound with an upstream or downstream side of a region translating the reporter gene; introducing the same to a suitable culture cell; adding the culture cell with a compound obtained from the synthesis or gene recombination technology or a natural origin compound, and a derivative thereof; and measuring the expression amount of the report gene or the protein amount of a reporter after a certain period of time.
  • CAT chloramphenicol acetyl transferase enzyme
  • ⁇ -Gal ⁇ -galactosidase
  • luciferase luciferase
  • the region regulating a KIAA0317 gene may be obtained by probing a KIAA0317 cDNA fragment from the commercially available genome library in accordance with a conventional method, and performing plaque hybridization.
  • the protein amount of reporter may be measured by enzyme activity or by an antibody such as an amount of protein expression.
  • the method of the second step can screen a material for regulating KIAA0317 expression by binding a cell extract (fraction) including KIAA0317 or KIAA0317 that is over-expressed in a cell according to a method such as recombination and purifying it with a ubiquitin in a test tube, and measuring the ubiquitinization activity in accordance with, for example, the same process as in Example 2.
  • An apoptosis inducer and a pharmaceutical composition for treating a disease relating to the apoptosis according to the present invention may be administrated as a suitable formulation together with a conventional carrier, diluent, or excipient.
  • the pharmaceutical composition according to the present invention may be administrated through a general pathway as long as it can reach the target tissue. It includes parenteral administration (e.g., intravenous, hypodermic, intraperitoneal, or topical administration) or oral administration depending upon the target method. Particularly, the parenteral administration is preferable, and more particularly, it may be administrated by intravenous injection.
  • the formulation is changed depending on the selected administration method.
  • a pharmaceutically acceptable carrier includes saline, sterilized water, a Ringer's solution, buffered saline, a dextrose solution, a maltodextrin solution, a glycerol, an ethanol, a liposome, and a mixture of at least one of the components.
  • It further includes a common additive such as an antioxidant, a buffer solution, a bacteriostatic agent, and so on. It may be formulated in an injection formulation such as an aqueous solution, a dispersion, an emulsion, and so on by further adding a diluent, a dispersing agent, a surfactant, a binder, and a lubricant, or it may be formed as a pill, a capsule, granules, or a tablet. In order to cause it operate specifically to the target organ, it can be used by binding an antibody or other ligands specific to the target organ with the carrier. In addition, it can be preferably formulated depending upon the disease or ingredient by using a suitable method for this art or a method disclosed in Remington's Pharmaceutical Science (new edition) (Mack Publishing Company, Easton PA).
  • a common additive such as an antioxidant, a buffer solution, a bacteriostatic agent, and so on. It may be formulated in an injection formulation such as an aqueous
  • it may employ another pharmaceutical delivery system, for example liposome and emulsion, which are known in this art. It may employ a certain organic solvent, for example dimethylsulfoxide.
  • the administration amount depends on a patient's weight, age, gender, health status, diet, administration time, administration way, excretion, and severity of the disease.
  • the pharmaceutically effective amount can be determined by, in the early stage, an in vitro analysis through a cell culture, or it can be determined through an animal model test and an IC50 (test compound concentration of 50% lethal dose of the culture cell when it is treated with a test material and in vitro analyzed) in which the blood-KIAA0317 concentration is determined in the cell culture.
  • the pharmaceutically effective amount can be determined by a person of ordinary skill in the art without performing excessive tests. From the information, the administration amount for a human is more precisely determined.
  • the pharmaceutical composition according to the present invention may be supplied singularly or in association with a surgical operation, a hormone therapy, and a drug such as an anticancer drug in order to treat a cancer.
  • the general molecular genetic and genetic engineering methods are described in the new version of Molecular Cloning: A Laboratory Manual, (Sambrook et al., Cold Spring Harbor); Gene Transfer Vectors for Mammalian Cells (Miller & Calos Eds.); and Current Protocols in Molecular Biology (F. M. Ausubel et al., Eds., Wiley & Sons).
  • the reagents, the cloning vectors, and a kit for modifying genes may be purchased in the market, for example Gibco/BRL, Bio-Rad, Stratagene, Invitrogen, ClonTech, and Sigma-Aldrich Co.
  • the general cell culture method is described in new versions of Culture of Animal Cells: A Manual of Basic Techniques (R.I.
  • the present invention provides a method of treating, diagnosing, or preventing a disease relating to hyperactivity of apoptosis, including administrating a KIAA0317 protein, a gene encoding the same, or a mutant that is functionally equivalent to the same in an effective amount for treating, diagnosing, or preventing the disease relating to apoptosis to a subject.
  • the KIAA0317 protein, the gene encoding the same, or the mutant that is functionally equivalent to the same is related to a HECT region of the KIAA0317 protein.
  • the KIAA0317 protein, the gene encoding the same, or the mutant that is functionally equivalent to the same is the same as in the above.
  • a method of administrating a KIAA0317 protein, a gene encoding the same, or a mutant that is functionally equivalent to the same according to the present invention may be carried out in accordance with the method of introducing the gene or the protein into a cell and the various conventional methods, which is clearly understood by a person of ordinary skill in the art.
  • the effective amount for treating, diagnosing, or preventing a disease relating to the hyperactivity of apoptosis is the same as described in the pharmaceutical composition.
  • the disease relating to the hyperactivity of apoptosis includes a degenerative cerebral nervous disease, a stroke, and an autoimmune disease, but it is not limited thereto.
  • the present invention provides a method of regulating the apoptosis that is characterized in comprising the step of: contacting a KIAA0317 protein, a gene encoding the same, or a mutant that is functionally equivalent to the same with a cell having an abnormal gene relating to apoptosis.
  • the abnormal gene relating to the apoptosis indicates that the gene is abnormal due to the substitution, subtraction, or addition of a nucleotide, and as a result, the corresponding expression protein is abnormal, for example, it exhibits inactivation.
  • the gene relating to the apoptosis is, for example, p53, but it is not limited thereto.
  • the cell having the abnormal gene relating to the apoptosis may include a cancer cell, but it is not limited thereto.
  • the contacting to the cell is carried out by the conventional method of introducing a protein or gene into a cell, for example by cloning a gene of interest to a vector and transfecting with the various medium materials such as liposome, calcium phosphate, and so on, which is clearly understood by a person of ordinary skill in the art.
  • the present invention provides a method of treating or supplementarily treating a cancer, including the step of administrating an effective ingredient of the KIAA0317 gene or protein expression inhibitor, in an effective amount for treating or supplementarily treating a cancer, to a subject requiring the treatment or the supplementary treatment for the disease.
  • the inhibitor increases the sensitivity of cancer cells to the anticancer drug.
  • the inhibitor is a double-stranded molecule including a sense strand and antisense strand.
  • the sense strand includes a ribonucleotide sequence corresponding to a target sequence selected from SEQ ID NO 1 or 2, and the antisense strand includes a ribonucleotide sequence corresponding to the sense strand.
  • the double-stranded molecule inhibits the KIAA0317 gene and protein from being expressed.
  • the double-stranded molecule may include SEQ ID NO 3 or 4, but it is not limited thereto.
  • FIG. 1 is a view showing a process of isolating a cell line having resistance against apoptosis which is induced by p53, and cloning an apoptosis-suppressing gene.
  • FIG. 2 and FIG. 3 show the protein homology between a c-terminal portion of a KIAA0317 protein and various protein origin HECT regions.
  • FIG. 2 is a view showing a comparison of the sequences of the protein including a HECT region and KIAA0317, and FIG. 3 shows the homology percentage in the HECT region. * indicates a cysteine residue which is important in forming a thioester bond in the conventionally-known HECT region.
  • FIG. 4 is a view showing an activity of the KIAA0317 protein as an E3 ubiquitin ligase.
  • cysteine residue in the HECT region is substituted with alanine residue, it loses its activity as an E3 ubiquitin ligase.
  • FIG. 5 is a schematic view showing a structure of the KIAA0317 protein for the apoptosis suppression analysis.
  • FIG. 6 is a view showing the apoptosis suppression of the KIAA0317 protein induced by p53, TNF ⁇ , and STS (strausporine).
  • FIG. 7 is a view showing that the KIAA0317 protein suppresses the activity of a protease, caspase-9, in an apoptosis cascade.
  • FIG. 8 is a view showing that the activity of a protease, caspase-3, activated by a protease, caspase-9, is suppressed.
  • FIG. 9 is a view showing the expression of KIAA0317 in various organ-origin cancers and normal tissues.
  • FIG. 10 is a view showing that the apoptosis of cancer cells is promoted by siRNA (small interfering RNA) suppressing KIAA0317 gene expression.
  • siRNA small interfering RNA
  • FIG. 11 is a view showing that the sensitivity of cancer cells to the anticancer drug is increased by siRNA (small interfering RNA) suppressing KIAA0317 gene expression.
  • FIG. 12 is a view showing that tumor growth is suppressed by siRNA suppressing KIAA0317 gene expression.
  • FIG. 13 is a view showing a result of the homology analysis of 9-51 , human KIAA0317, and mouse KIAA0317 protein sequences cloned in accordance with the method as shown in FIG. 1.
  • each amino acid corresponding to a single-lettered amino acid code is referenced as in Molecular Cloning: A Laboratory Manual, (Sambrook et al., Cold Spring Harbor). [Examples]
  • Example 1 Cloning Apoptosis Suppression Gene
  • Example 1-1 Cell Incubation and Transfection Cells used for this example were incubated in a culture media including
  • a murine testicle cDNA library was introduced into human osteosarcoma origin Saos-2 cells (American Type Culture Collection, ATCC; Cat. No. HTB-85), in which apoptosis is efficiently induced by p53.
  • the cDNA library was obtained by extracting mRNA from the murine testicle by using Trizol (Invitrogen) according to the recommendations of the manufacturer; purifying the same and providing it as a template; synthesizing cDNA with the same by using Superscipt Il (Invitrogen) according to the recommendations of the manufacturer; and cloning the same at the Sfi I restriction enzyme region of a pCEV29 expression vector (Michieli et al., 1996; Yamanaka et al., 2001).
  • the pCEV29 expression vector expressed cDNA was cloned through a MuMLV-LTR (Moloney murine leukemia virus long terminal repeat) promoter and expressed a Neomycin-resistant gene to show resistance against G
  • 1x10 6 Saos-2 cells were introduced into a plate having a diameter of 100 mm and incubated in 10 ml culture medium (DMEM+10% FBS) for two days. Then, the obtained 2 ⁇ g cDNA library was induced into cells through a CaPO 4 method, and 500 ⁇ g/ml G418 was added into a culture medium. It was then subjected to incubation. After a cDNA-non introduced cell line was removed around two weeks later, a G418 addition was disconnected. The cell was forming a G418 resistant colony including around 100-2000 cells. When each colony grew to have a diameter of 1mm, it was separated and put into two wells of two 24 well plates and subjected to incubation.
  • 10 ml culture medium DMEM+10% FBS
  • adenovirus expressing p53 (Jung et al., 2004) was infected to cells in one well of the two wells, and the culture medium was changed 3 hours later. 3 days later, the apoptosis was observed by p53 expression. A week later, after the cells were immobilized and dyed with Giemsa (Sigma USA) solution, the cell growth was compared between the p53 virus-infected well and the non-infected well.
  • the colony in the well in which apoptosis was not actively occurring due to the p53 expression was isolated and moved to a plate having a diameter of 60mm and allowed to grow. Such isolated cells were infected with the p53 virus and it was reconfirmed that the resistance to apoptosis was induced by p53. Then the introduced cDNA was isolated from the apoptosis-confirmed cells as follows.
  • chromosome DNA was extracted from p53-resistant cells in accordance with the conventional method.
  • the chromosome DNA 50 ⁇ g was cleaved by one of the restriction enzymes Xhol or Notl that existed in a vector, and DNA was purified by the conventional phenol extraction method.
  • 1.2 ⁇ g of DNA was bound with T4 ligase for 16 to 24 hours, and it was purified again in accordance with the phenol extraction method.
  • the bound DNA concentration was determined by agarose gel electrophoresis.
  • the DNA was introduced into a cell by electric shock therapy in accordance with the recommendations of the manufacturer of the E.
  • coli DH10B cell line (Gibco-BRL, USA).
  • the pCEV29 expression vector includes an ampicillin (Sigma, USA) resistant gene
  • the resistant colony was selected in the medium including ampicillin.
  • a total of 300 to 500 colonies were collected in an LB medium (Luria-Bertani medium, Difco) by washing the whole plate, then ampicillin was input thereto (100 ⁇ g/ml) and incubated at 37 0 C for a night. The next day, cells were collected and plasmid DNA was isolated in accordance with the conventional alkaline-lysis method.
  • a primer forward: 5'-CGACTGGAGCACGAGGACACTGA-3'; reverse: 5'-CATCAAAAATAGCCAAAAGG-S'
  • a primer composed of base sequences that are present adjacent to each 5' and 3' of the inserted genes of the vector was used.
  • the pCEV29 expression vector extracted from each isolated apoptosis resistant Saos-2 cell line induced by p53 was used as a template, and PCR reaction in which 94 1 min, 48 1 min, and 72 10 min were set for one cycle was repeated for 30 cycles.
  • a 2.2 kb gene was isolated from eight p53 induced apoptosis resistant Saos-2 clones. Then, the base sequence was analyzed by a base sequence analysis kit (Applied Biosystems, USA) in accordance with the recommendations of the manufacturer, and the homology analysis was processed by using a computer with a BLASTN program (Altschul et al., 1990; http://www.ncbi.nlm.nih.gov). From the results, it was found to be a sequence constructing a part of GenBank AK077015 (mouse) and AB002315 (human), which is confirmed to have an estimated HECT region. However, it has not been known to have the specific functions as an E3-ubiquitin ligase. The gene was named KIAA0317 (Resistance to apoptosis induction).
  • the KIAA0317 was measured for a composite formation of ubiquitin and thioester in the presence of ubiquitin-active enzymes, E1 and E2, by carrying out the following process.
  • the ubiquitin-bound KIAA0317 protein may be separated from unbound protein by the difference of moving distance under electrophoresis because the bound protein has a higher molecular weight than the unbound protein.
  • the KIAA0317 protein according to the present invention formed a composite with ubiquitin in the presence of E1 and E2. Such composite was lost by a reducing agent (DDT) that prevented thioester binding with ubiquitin and a mutation (KIAA0317-Hc/a) in which cysteine residue was substituted for alanine residue.
  • DDT reducing agent
  • the E3-ubiquitin ligase relating to the intercellular ubiquitinization of the target protein generally formed a thioester bond with ubiquitin in the presence of E1 and E2. Accordingly, the results proved that the KIAA0317 protein according to the present invention, particularly the HECT region, can function as an E3-ubiquitin ligase.
  • an expression vector pCDNA6-v5 including KIAA0317 cDNA 9-51 , human origin KIAA0317 cDNA, the KIAA0317-HECT region, and the KIAA0317-HECT region in which each 720th cysteine residue was substituted with alanine residue were induced into Saos-2 cells. Accordingly, a cell line that stably expressed the KIAA0317 protein or its HECT region in accordance with the process of Example 1 was produced. Each of the cells was input into 24 well plates at 4x10 4 , and had 1ml of cell culture medium added thereto.
  • an adenovirus expressing p53 (Jung et al., 2004), 250 MOI (multiplicity of infection); TNF-alpha (0.1 ng/ml) including 50//g/ml of cyclohexamide; and 25nM of storosporin were added to each well.
  • the culture medium was changed after 2 hours, and in the case of the others, all cells in each well were collected in individual containers after 24 hours and were dyed with 0.4% Trypan blue (Sigma). The number of dead cells was counted under a microscope and the apoptosis was quantified. Untreated Saos-2 was used as a negative control, while the conventional bclXL that is known for suppressing apoptosis was used for a positive control (Jung et al., 2004).
  • cells expressing KIAA0317 and KIAA0317-H (amino acid sequences 455 to 823), and 9-51 (amino acid sequences 551 to 823) shown in FIG. 5, suppressed apoptosis about as much as conventional apoptosis inhibitors such as bclXL as a positive control group, which was induced by various signals of p53, TNF-alpha, and storosporine, which are known as apoptosis inducers.
  • Example 4 Suppression of Protease, Capase-9, and Caspase-3, by KIAA0317 TNF alpha (0.1 ng/ml) was added to cells expressing the normal and mutant KIAA0317 genes or the Bcl-xL gene that is already known for suppressing apoptosis, and 24 hours later, it was measured for caspase activity.
  • the caspase activity was analyzed with a substrate of DEVD (D: aspartic acid, E: glutamic acid, V: valine) and LEHD (L: leucine, H: histidine)-AFC conjugated to 7-amino-4-trifluoromethyl coumarin (AFC) for caspase 3 and 9, respectively, by a fluorescent analysis kit (R&D Systems, USA) in accordance with the recommendations of the manufacturer.
  • the fluorescent signal was measured in an exciting wavelength of 400nm and an emission wavelength of 505nm by a microplate fluorescent detector.
  • KIAA0317 expression in cells of a cancer which is a representative disease for the apoptosis suppression, was measured.
  • Normal and cancer origin tissue samples from the stomach, large intestine, and liver were measured on a tissue microarray
  • Tyramide Signal Amplification Kit (NEN Life Science) in accordance with the recommendations of the manufacturer.
  • KIAA0317 included in the tissues.
  • the antibody for KIAA0317 was prepared by epitoping a peptide including a certain region of the KIAA0317 protein sequence. After binding the antibody to the tissue samples, it was colored with diaminobenzidine (Sigma, USA) and control-dyed with Haematoxylin A. Non-immunized rabbit serum was used as a negative control. The results were independently read by three different pathologies, and the reaction degrees to the KIAA0317 antibody were classified into the following four steps: (1 ) negative for 0-5%; (2) low for 5-30%; (3) intermediate for 30-50%; (4) high for 50% or more. Since KIAA0317 expression was read as negative or low in the normal tissue, those evaluated as intermediate and high reactivity were considered as over-expression.
  • Example 6 Effect of KIAA0317 siRNA on Cancer Cell Apoptosis
  • Example 6-1 siRNA Preparation A siRNA sequence was designed by using the Ambion website (www. ambion.com/techlib/misc/siRNA_finder.html). In brief, it was scanned in the 3' direction from the AUG sequence of KIAA0317 cDNA until the AA sequence appeared. The sequence and 19 nucleic acid sequences thereafter were selected as a potential siRNA target region, but sequences of 5' and 3' non-translated regions and translating initiation region (in 75 base) were omitted from the target. The selected sequence was compared with the conventional sequences recorded in the database, and sequences having high homology to coding sequences of other genes were omitted.
  • the KIAA0317 target sequence used in this example was ⁇ '-AATTGGTCCCTGAGAACCTTT-S', and the siRNA forming sequence was as follows: sense, ⁇ '-UUGGUCCCUGAGAACCUUUTT-S' (SEQ ID NO: 3); antisense, 5'-AAAGGUUCUCAGGGACCAATT-3 1 (SEQ ID NO: 4).
  • a control siRNA that has no effect on the KIAA0317 gene expression was purchased and used, i.e., Scrambled siRNA from PROLIGO Inc.
  • siRNA having a hairpin structure including the sense and antisense sequences In order to intracellular express siRNA having a hairpin structure including the sense and antisense sequences, the Knockout RNAi System (BD-Clontech, USA) BD was used in accordance with the recommendations of the manufacturer thereof. In brief, oligonucleotides including the sense and antisense sequences were synthesized in accordance with the recommendations of the manufacturer, and were cloned in pSIREN-Retro Q (BD-Clontech, USA) to provide a vector (pSIREN-KIAA0317si) expressing siRNA targeting KIAA0317 and to use it for the transfection of cells.
  • pSIREN-KIAA0317si a vector expressing siRNA targeting KIAA0317 and to use it for the transfection of cells.
  • Example 7 Inducing Apoptosis and Increasing Sensitivity of KIAA0317 siRNA to Anticancer siRNA-treated cells (colon cancer cell line H1299) were treated with a conventional anticancer drug such as cisplatin (Cis) (Bristol-Myers Squibb Company, USA), doxorubicin (Doxo) (ALZA Pharmaceuticals, USA), and eptoposide (Eto) (Bristol-Myers Squibb Company, USA) in a concentration of 73 ⁇ M, 2 ⁇ M, and 5 ⁇ M, respectively, in accordance with the same process as in Example 6-2, and the apoptosis effect was observed. As shown in FIG. 11 , when it was treated with a combination of siRNA and the anticancer drug, its effects were improved to about four times more than the case of treating with siRNA alone or the anticancer drug alone.
  • a conventional anticancer drug such as cisplatin (Cis) (Bristol-Myers Squibb Company,
  • Example 8 Tumor Suppression Effect of Cancer Cell Expressing KIAA0317 siRNA
  • a pSIREN-KIAA0317si vector expressing KIAA0317 siRNA obtained from Example 6-1 was transfected to a colon cancer cell line H 1299 in accordance with the conventional method (General Techniques of Cell Culture, M.A. Harrison & I. F. Rae, Cambridge Univ. Press).
  • H 1299 cells and pSIREN-KIA0317si-induced H1299 cells were injected to Balb/c nude mice (Jackson Laboratories, U.S.A.) at 1x10 6 each, and weight and tumor size were observed to obtain a tumor growth ratio. Tumor size was measured with a vernier caliper in accordance with the conventional method.
  • the tumors grew normally in control mice (A) that were only injected with the H 1299 cells, and on the other hand, the tumor size increased little in the presence of H1299 cells (B) expressing KIAA0317 siRNA. This proves that suppressing KIAA0317 expression can effectively suppress tumor formation.
  • Nedd4-like Proteins An Emerging Family of Ubiquitin-protein Ligases Implicated in Diverse Cellular Functions. Trends Cell Biol 9: 166-169.
  • Smurf2 is a Ubiquitin E3 Ligase Mediating Proteasome-dependent Degradation of Smad2 in Transforming
  • p53AIP1 a Potential Mediator of p53-dependent Apoptosis, and its Regulation by Ser-46-phosphorylated p53. Cell 15; 102(6): 849-62.
  • KIAA0317 protein according to the present invention effectively suppresses the activity of the protease caspase, and apoptosis.
  • KIAA0317 inhibitor effectively induces apoptosis in cancer cells in which apoptosis is suppressed
  • KIAA0317 can be used as an agent for treating abnormal apoptosis-related diseases such as degenerative cerebral nervous diseases, autoimmune diseases, and cancers.

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Abstract

La présente invention concerne une nouvelle utilisation de KIAA0317 en tant que régulateur de l'apoptose. Selon la présente invention, le gène KIAA0317 peut inhiber l'activité d'une protéase, d'une caspase, qui est nécessaire pour l'apoptose, de sorte qu'il est possible de supprimer l'apoptose. Un gène KIAA0317 ou une protéine codée par ce dernier, ou une matière régulant l'activité de ces derniers, par exemple un anticorps, un ARNsi, une matière chimique, etc., selon la présente invention peuvent être appliqués de manière utile pour traiter une maladie liée à l'apoptose.
PCT/KR2007/004553 2007-03-02 2007-09-19 Nouvelle utilisation de kiaa0317 en tant que régulateur de l'apoptose Ceased WO2008108521A1 (fr)

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