WO2005061007A1 - Method of suppressing cancer - Google Patents

Method of suppressing cancer Download PDF

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Publication number
WO2005061007A1
WO2005061007A1 PCT/JP2004/019797 JP2004019797W WO2005061007A1 WO 2005061007 A1 WO2005061007 A1 WO 2005061007A1 JP 2004019797 W JP2004019797 W JP 2004019797W WO 2005061007 A1 WO2005061007 A1 WO 2005061007A1
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seq
cancer
synoviolin
sequence
suppressor gene
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PCT/JP2004/019797
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French (fr)
Japanese (ja)
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Toshihiro Nakajima
Satoshi Yamasaki
Naoko Yagishita
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St. Marianna University School Of Medicine
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Priority to JP2005516543A priority Critical patent/JPWO2005061007A1/en
Publication of WO2005061007A1 publication Critical patent/WO2005061007A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

  • the present invention relates to a method for activating the p53 tumor suppressor gene and localizing it to the nucleus.
  • the present invention also relates to a pharmaceutical composition containing a substance that promotes the activity of a p53 tumor suppressor gene.
  • Synopiolin is a novel protein discovered as a membrane protein overexpressed in synovial cells from rheumatic patients (WO02 / 052007). In addition, studies using genetically modified products have revealed that Synoviolin is an essential molecule for the development of rheumatoid arthritis.
  • Synoviolin has a RING finger motif. This motif is often found in an enzyme called E3 ubiquitin ligase, which plays an important role in protein ubiquitination, but in fact, syobiolin is one of the features of E3 ubiquitin ligase, It has been shown to have activity (WO02 / 052007).
  • p53 is located on chromosome 17 pl3 and is a very important tumor suppressor gene in the development and proliferation of cancer cells.
  • p53 recognizes a specific base sequence [5 '-(A / T) GPyPyPy-3')] on DNA and promotes the transcriptional activation of specific genes such as wafl / cipl, GADD45, and BAX . It also (i) suppresses the transcription of many other genes; (ii) interacts with viral oncogenes such as SV40 large T antigen, adenovirus EIB protein, papillomavirus E6, or cellular oncogenes such as mdm2. Physiological functions such as binding and (iii) specific binding to DNA containing mismatches are known.
  • An object of the present invention is to provide a method for promoting the activation of a p53 tumor suppressor gene, and a pharmaceutical composition for promoting the activation of a p53 tumor suppressor gene.
  • the present inventor has conducted intensive studies in order to solve the above problems.
  • synoviolin homo-knockout animal was analyzed in detail, more apoptotic cells were observed than in the wild type, and p53, which is deeply involved in apoptosis induction, was localized in the nucleus. However, it was found to be strongly expressed. Then, they found that by suppressing the function of synoviolin, p53 was activated and the growth of cancer cells was inhibited, thereby completing the present invention.
  • the present invention is as follows.
  • a pharmaceutical composition comprising an siRNA designed using, as a target sequence, at least one sequence selected from the group consisting of the nucleotide sequences shown in SEQ ID NOs: 2 to 15 among the nucleotide sequences shown in SEQ ID NO: 1.
  • the pharmaceutical composition is used for treating cancer.
  • the p53 tumor suppressor gene localized in the nucleus can be further irradiated with radiation.
  • FIG. 1 is a photograph showing the result of immunohistochemical staining of Synoviolin homonockato mouse embryo fibroblasts (MEFs).
  • FIG. 2 is a photograph showing the results of immunohistochemical staining with anti-p53 antibody in syno ⁇ / ⁇ embryos.
  • FIG. 3 is a photograph showing the results of Western blotting on an apoptosis-related protein.
  • FIG. 4 is a photograph showing the results of identifying p53 phosphorylation sites in syno- MEF cultured cells.
  • the present invention is characterized by inhibiting cancer by inhibiting the function of synoviolin to localize and activate the p53 tumor suppressor gene (sometimes simply referred to as p53) in the nucleus.
  • p53 tumor suppressor gene sometimes simply referred to as p53
  • p53 in the cells When normal cells are exposed to ultraviolet light, etc., p53 in the cells is activated, and as a result, cell growth is stopped. Therefore, by increasing the concentration of p53, the growth of cancer cells can be stopped. In other words, when p53 does not function, the growth of cancer cells is not stopped and cancer progresses. In fact, p53 is rarely found in cells of normal individuals, but about half of cells from cancer patients have this p53 deficient mutation. Even when such mutations have not occurred, some mutations have occurred in the control mechanism of p53, and the tumor suppressor function has been lost. Therefore, it is necessary to make p53 function effectively to suppress the progression of cancer.
  • the function of synoviolin was focused on in order to make activation of p53 one of the effective methods for cancer treatment. Then, a synopiolin homo-knockout animal was prepared and analyzed in detail. As a result, a greater number of apoptotic cells were observed than in the wild type. In other words, they found that inhibiting the function of synoviolin promoted the activation of p53, which is deeply involved in apoptosis, and that inhibiting the function of synoviolin leads to cancer suppression.
  • apoptosis refers to programmed cell death caused by the cell itself, chromosome aggregation of the cell nucleus, fragmentation of the cell nucleus, loss of cell surface microvilli, aggregation of the cytoplasm, activation of caspase, activation of mitochondrial membrane potential It is characterized by disappearance. When the above characteristics occur in the cell, it is determined that apoptosis has been induced.
  • p53 when immunostaining of p53 in a fetal embryo is performed, p53 is strongly expressed in the whole body of a Synoviolin homo-knockout mouse embryo.
  • fetal fibroblasts (MEFs) isolated from the fetal embryo of Synoviolin homonockato mouse are more strongly expressed than those isolated from the wild type, and p53 is strongly localized in the nucleus. I do. This nuclear localization is not observed at all in the wild type. This means that p53 can be translocated into the nucleus by inhibiting the expression of synopiolin.
  • Synoviolin homo-knockout mouse embryo MEFs show high radiosensitivity. Therefore, in the present invention, when irradiation is performed after inhibiting the expression of synoviolin to transfer p53 to the nucleus, the growth of cancer cells can be effectively suppressed.
  • the present invention is characterized in that p53 is activated by phosphorylating a part of amino acids of p53 (p53 protein).
  • the target of p53 phosphorylation is preferably a serine residue in the amino acid sequence of p53, and more preferably the 15th serine residue.
  • p53 is phosphorylated by kinases such as ATR and ATM, and phosphorylation is thought to increase synthobiolin's activity by inhibiting synoviolin.
  • RNA interference can be used.
  • RNAi can be induced by designing and synthesizing siRNA (small interfering RNA) for the synoviolin gene and introducing it into cells.
  • RNAi means that dsRNA (double-strand RNA) is specific and selective for the target gene. It is a phenomenon that binds and cleaves the target gene to efficiently inhibit its expression. For example, when dsRNA is introduced into a cell, expression of a gene having a sequence homologous to the RNA is suppressed (knocked down).
  • siRNA The design of siRNA can be performed as follows.
  • the gene is not particularly limited as long as it encodes synoviolin, and any arbitrary region can be used as a candidate.
  • any region of Gen Bank Accession number AB024690 SEQ ID NO: 1 can be a candidate.
  • a sequence starting with AA is selected from the selected region, and the length of the sequence is 19 to 25 bases, preferably 19 to 21 bases.
  • the sequence should have a GC content of, for example, 40 to 60%.
  • a DNA containing at least one sequence selected from the following nucleotide sequences among the nucleotide sequences shown in SEQ ID NO: 1 can be used as a target sequence of siRNA. In particular, it targets (i) (SEQ ID NO: 2), (ii) (SEQ ID NO: 3), (vi) (SEQ ID NO: 7), (vii) (SEQ ID NO: 8), (viii) (SEQ ID NO: 9) Is preferred.
  • siRNA synthesized in vitro is ligated to plasmid DNA and introduced into cells
  • a method in which two RNAs are annealed, and the like can be employed.
  • shRNA is an RNA molecule having a stem-loop structure because a part of a single strand forms a complementary strand with another area, which is called a short hairpin RNA (short hairpin RNA).
  • shRNAs can be designed such that a portion forms a stem-loop structure. For example, if the sequence of a certain region is sequence A and the complementary strand to sequence A is sequence B, these sequences are present in a single RNA strand so that sequence A, spacer, and sequence B are arranged in this order. And design the total length to be 45-60 bases.
  • Sequence A is a sequence of a partial region of the target Synoviolin gene (SEQ ID NO: 1), and the target region is not particularly limited, and any region can be a candidate.
  • the length of sequence A is 19 to 25 bases, preferably 19 to 21 bases.
  • the shRNA and siRNA produced in the present invention are substances that suppress the expression of Synoviolin and can be used as a pharmaceutical composition for activating p53 (particularly a gene therapy agent for cancer).
  • the site of application is not particularly limited, and it may be brain tumor, tongue cancer, pharynx cancer, lung cancer, breast cancer, esophagus cancer, stomach cancer, knee cancer, biliary tract.
  • gallbladder cancer duodenal cancer, colon cancer, liver cancer, uterine cancer, ovarian cancer, prostate cancer, kidney cancer, bladder cancer, rhabdomyosarcoma, fibrosarcoma, osteosarcoma, chondrosarcoma, skin cancer, various leukemias (eg (Acute myeloid leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, adult T-cell leukemia, malignant lymphoma).
  • various leukemias eg (Acute myeloid leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, adult T-cell leukemia, malignant lymphoma).
  • the above-mentioned cancer may be a primary tumor, a metastasized tumor, or a disease associated with another disease.
  • a vector incorporating a nucleic acid is administered.
  • Method. examples include adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, vaccinia virus vectors, retrovirus vectors, lentivirus vectors, and the like. Efficient use of these virus vectors Can be administered well.
  • the pharmaceutical composition of the present invention into phospholipid vesicles such as ribosomes and administer the vesicles.
  • the endoplasmic reticulum retaining the siRNA or shRNA is introduced into predetermined cells by the lipofection method. Then, the obtained cells are systemically administered, for example, into a vein or an artery. It can also be administered locally to the brain and the like.
  • the dosage of the pharmaceutical composition of the present invention for example a dose in the case of adenovirus 10 6 ⁇ per day:! OIS pieces About 1 to 8 weeks.
  • a commercially available gene transfer kit for example, AdenoExpress: Clontech
  • AdenoExpress: Clontech a commercially available gene transfer kit
  • apoptosis-related proteins in synoviolin homo-knockout mouse (syno) fetal fibroblasts (MEFs) were detected by Western blotting, and the cells were confirmed by immunohistochemical staining.
  • the immunostaining method involves fixing MEFs on a slide glass according to a conventional method, and using two types of anti-p53 antibodies (mouse monoclonal antibody BD: Becton, Dickinson, Inc. ⁇ Sagi polyclonal antibody FL393: Santa cruz). Immunostaining was performed. Specimens that had been blocked with 3% bovine serum albumin (BSA) for 30 minutes were immunoreacted with an anti-p53 antibody (BD: 10 pg / ml, FL393: 5 g / ml) diluted with 0.3% BSA at room temperature for 60 minutes. .
  • BSA bovine serum albumin
  • the specimen was washed with PBS, and immunoreacted with fluorescein isothiocyanate-labeled anti-Peagle IgG antibody or TRITC-labeled anti-mouse IgG antibody (Dako) as a secondary antibody. Confirmation of antigen immunoreactive with anti-p53 antibody is performed with a fluorescence microscope It was.
  • This example is an examination of p53 activation in syno mice.
  • p53 activation in syno mice was examined by immunostaining using embryos.
  • immunostaining of syno-/-fetuses was performed using a Vectostin ABC kit (VECTOR) with the tissue fixed on a slide glass according to a conventional method.
  • the specimen blocked with the blocking reagent for 30 minutes was immunoreacted with the anti-p53 antibody FL393 diluted to 5 pg / ml at room temperature for 60 minutes.
  • the specimen after the reaction was washed with PBS, and immunoreacted with an HRP-labeled anti-Peacock IgG antibody as a secondary antibody.
  • the antigen immunoreactive with the anti-p53 antibody was confirmed by color development of 3,3'-diaminobenzidine tetrahydrochloride based on HRP activity. Methyl green staining was performed as counterstain.
  • Apoptosis-related proteins in syno-/-cultured MEF cells were detected by Western blotting.
  • cell lysate 15 mM Tris-HCl (pH 7.5), 200 mM NaCL 0.5% NP40, 1 mM PMSF, 0.1% sodium dodecyl sulfate (SDS), 2 pg / ml leptin, 2 pg /
  • SDS sodium dodecyl sulfate
  • the cell-fractionated fraction was prepared using (ml aprotinin, 2 pg / ml peptide). Then, the cell-fractionated fraction was separated by SDS-polyacrylamide electrophoresis (SDS-PAGE). After SDS-PAGE, cell-derived proteins were transferred to a nitrocellulose (NC) membrane by the electroblotting method.
  • NC nitrocellulose
  • the NC membrane was treated with Tris-HCl (TBS) containing 5% skim milk for 1 hour at room temperature. After locking, the anti-p53 antibody C-tarminal aa; 195_393 or FL393 was diluted with TBS supplemented with 5% skim milk and immunoreacted at room temperature for 1 hour. After the reaction, the NC membrane was washed with 0.1% Tween20 / TBS, and a horse radish peroxidase (HRP) -labeled anti-Egret Ig G antibody was used as a secondary antibody for immunoreaction at room temperature for 1 hour, and then washed with 0.1% Tween20 / TBS. The target antigen was detected by detecting the HRP activity. For detection of HRP activity, an ECL kit (Amersham) was used (Clinical Chemistry. 25, pl531, 1979).
  • HRP activity For detection of HRP activity, an ECL kit (Amersham) was used (Clinical Chemistry. 25, pl531, 1979).
  • the phosphorylation site of p53 was probed by Western plotting using an anti-p53 antibody.
  • a substance that promotes the activity of a p53 tumor suppressor gene is provided. Since this substance can activate p53 and transfer p53 to the nucleus, it is useful as a pharmaceutical composition for treating cancer. Further, the present invention suppresses the function of synoviolin. Will enable cancer treatment

Abstract

A method of activating a cancer suppressor gene p53 and localizing it in nucleus; a medicinal composition containing a substance promoting the activity of the cancer suppressor gene p53; a medicinal composition containing siRNA which is designed as referring at least one sequence selected from a group consisting of base sequences represented by SEQ ID NOS:2 to 15 in the base sequence represented by SEQ ID NO:1 to as the target sequence; and a method of activating the cancer suppressor gene p53 characterized by inhibiting the expression of synoviolin with the use of the siRNA as described above.

Description

明 細 書 癌の抑制方法 技術分野  Description Method of cancer control Technical field
本発明は、 p53癌抑制遺伝子を活性化させて核に局在化させる方法に関する。 また、 本発明は、 p53癌抑制遺伝子の活性を促進する物質を含む医薬組成物に関 する。 背景技術  The present invention relates to a method for activating the p53 tumor suppressor gene and localizing it to the nucleus. The present invention also relates to a pharmaceutical composition containing a substance that promotes the activity of a p53 tumor suppressor gene. Background art
シノピオリンは、 リウマチ患者由来滑膜細胞で過剰発現している膜タンパク質 として発見された新規タンパク質である （WO02/052007) 。 そして、 遺伝子改 変動物を用いた研究により、 シノビオリンは関節リウマチの発症に必須の分子で あることが判明した。  Synopiolin is a novel protein discovered as a membrane protein overexpressed in synovial cells from rheumatic patients (WO02 / 052007). In addition, studies using genetically modified products have revealed that Synoviolin is an essential molecule for the development of rheumatoid arthritis.
タンパク質構造予測システムにより、 シノビオリンは RING finger モチーフ を有することが示唆されている。 このモチーフはタンパク質のュピキチン化に重 要な役割を果たす E3ュビキチンライゲースという酵素に多く見出されているが、 実際、 シオビオリンが E3ュビキチンライゲースの特徴のひとつである自己ュビ キチン化活性を有することが証明されている （WO02/052007) 。  The protein structure prediction system suggests that Synoviolin has a RING finger motif. This motif is often found in an enzyme called E3 ubiquitin ligase, which plays an important role in protein ubiquitination, but in fact, syobiolin is one of the features of E3 ubiquitin ligase, It has been shown to have activity (WO02 / 052007).
ところで、 p53は、 第 17染色体 pl3に位置しており、 癌細胞の発生及び増殖 においてきわめて重要な癌抑制遺伝子である。 p53 は、 DNA 上の特異的塩基配 列 [ 5'-(A/T)GPyPyPy-3')]を認識し、 wafl/cipl、 GADD45, BAX等の特定の遺 伝子の転写活性化を促す。 また、 （i )その他の多くの遺伝子の転写を抑制するこ と、 （i i ) SV40ラージ T抗原、 アデノウイルス EIBタンパク質、 パピローマウ ィルス E6 などのウィルス性癌遺伝子、 あるいは mdm2 等の細胞性癌遺伝子と 結合すること、 （i i i )ミスマッチを含む DNAと特異的に結合すること等の生理 機能が知られている。  By the way, p53 is located on chromosome 17 pl3 and is a very important tumor suppressor gene in the development and proliferation of cancer cells. p53 recognizes a specific base sequence [5 '-(A / T) GPyPyPy-3')] on DNA and promotes the transcriptional activation of specific genes such as wafl / cipl, GADD45, and BAX . It also (i) suppresses the transcription of many other genes; (ii) interacts with viral oncogenes such as SV40 large T antigen, adenovirus EIB protein, papillomavirus E6, or cellular oncogenes such as mdm2. Physiological functions such as binding and (iii) specific binding to DNA containing mismatches are known.
従って、 癌の抑制物質を見出すには、 p53の機能を解析することが重要であ る。 発明の開示 Therefore, it is important to analyze the function of p53 in order to find cancer suppressors. Disclosure of the invention
本発明は、 p53癌抑制遺伝子の活性化を促進する方法、 及び p53癌抑制遺伝子 の活性化を促進する医薬組成物を提供することを目的とする。  An object of the present invention is to provide a method for promoting the activation of a p53 tumor suppressor gene, and a pharmaceutical composition for promoting the activation of a p53 tumor suppressor gene.
本発明者は、 上記課題を解決するために鋭意研究を行った。 そして、 シノビォ リンホモノックアウト動物を詳細に解析したところ、 野生型に比し、 アポトーシ スを起こしている細胞が多数観察され、 また、 アポトーシスの誘導に深く関与し ている p53が核内に局在し、 強く発現していることが判明した。 そして、 シノビ ォリンの機能を抑制することにより、 p53が活性化され、 癌細胞の増殖が阻止さ れることを見出し、 本発明を完成するに至った。  The present inventor has conducted intensive studies in order to solve the above problems. When the synoviolin homo-knockout animal was analyzed in detail, more apoptotic cells were observed than in the wild type, and p53, which is deeply involved in apoptosis induction, was localized in the nucleus. However, it was found to be strongly expressed. Then, they found that by suppressing the function of synoviolin, p53 was activated and the growth of cancer cells was inhibited, thereby completing the present invention.
すなわち、 本発明は以下の通りである。  That is, the present invention is as follows.
(1) 配列番号 1に示される塩基配列のうち配列番号 2〜15に示す塩基配列からな る群から選択される少なくとも 1つの配列を標的配列として設計された siRNA を含む医薬組成物。  (1) A pharmaceutical composition comprising an siRNA designed using, as a target sequence, at least one sequence selected from the group consisting of the nucleotide sequences shown in SEQ ID NOs: 2 to 15 among the nucleotide sequences shown in SEQ ID NO: 1.
上記医薬組成物は、 癌を治療するために使用される。  The pharmaceutical composition is used for treating cancer.
(2) 配列番号 1に示される塩基配列のうち配列番号 2〜： 15に示す塩基配列からな る群から選択される少なくとも 1つの配列を標的配列として設計された siRNA を用いて、 シノビオリンの発現を阻害することを特徴とする p53癌抑制遺伝子の 活性化方法。  (2) Synoviolin expression using an siRNA designed with at least one sequence selected from the group consisting of the nucleotide sequences shown in SEQ ID NOs: 2 to 15 of the nucleotide sequence shown in SEQ ID NO: 1 as a target sequence A method for activating a p53 tumor suppressor gene, which comprises inhibiting the p53 tumor suppressor gene.
(3) 配列番号 1に示される塩基配列のうち配列番号 2〜： 15に示す塩基配列からな る群から選択される少なくとも 1つの配列を標的配列として設計された siRNA を用いて、 シノビオリンの発現を阻害することを特徴とする p53癌抑制遺伝子の 核への局在化方法。  (3) Expression of Synoviolin using at least one sequence selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 2 to 15 among the nucleotide sequences of SEQ ID NO: 1 as a target sequence A method for localizing a p53 tumor suppressor gene to the nucleus, wherein the p53 tumor suppressor gene is inhibited.
上記局在化方法は、 核に局在化した p53癌抑制遺伝子に、 さらに放射線照射を 行うこともできる。  In the above localization method, the p53 tumor suppressor gene localized in the nucleus can be further irradiated with radiation.
(4) p53の第 15番目のセリン残基をリン酸化することを特徴とする p53の活性化 方法。 図面の簡単な説明 図 1は、 シノビオリンホモノックァゥトマウス胎児線維芽細胞（MEFs)におけ る免疫組織染色の結果を示す写真である。 (4) A method for activating p53, which comprises phosphorylating the 15th serine residue of p53. Brief Description of Drawings FIG. 1 is a photograph showing the result of immunohistochemical staining of Synoviolin homonockato mouse embryo fibroblasts (MEFs).
図 2は、 syno-/ -の胚における抗 p53抗体による免疫組織染色の結果を示す写 真である。  FIG. 2 is a photograph showing the results of immunohistochemical staining with anti-p53 antibody in syno − / − embryos.
図 3 は、 アポト一シス関連タンパク質に関するウェスタンブロッテイングの 結果を示す写真である。  FIG. 3 is a photograph showing the results of Western blotting on an apoptosis-related protein.
図 4は、 syno- の MEF培養細胞における p53 のリン酸化部位を同定した結 果を示す写真である。 発明を実施するための最良の形態  FIG. 4 is a photograph showing the results of identifying p53 phosphorylation sites in syno- MEF cultured cells. BEST MODE FOR CARRYING OUT THE INVENTION
以下、 本発明を詳細に説明する。 '  Hereinafter, the present invention will be described in detail. '
本発明は、 シノビォリンの機能を阻害して p53癌抑制遺伝子 （単に p53という こともある） を核に局在化及び活性化させることにより、 癌を抑制することを特 徵とする。  The present invention is characterized by inhibiting cancer by inhibiting the function of synoviolin to localize and activate the p53 tumor suppressor gene (sometimes simply referred to as p53) in the nucleus.
1 . p53の活性化  1. Activation of p53
正常細胞を紫外線等にさらすと、 細胞内の p53が活性化して、 その結果細胞増 殖が阻止されることから、 p53の濃度を上昇させることにより、 癌細胞の増殖を 止めることができる。 つまり、 p53が機能しない場合は、 癌細胞の増殖が止めら れず、 癌が進行することになる。 事実、 p53は正常な個体の細胞にはほとんど見 られないが、 癌患者由来の細胞の約半数においてはこの p53の欠損変異が起こつ ている。 また、 このような変異が起こっていない場合でも、 p53の制御機構に何 らかの変異が生じて癌抑制機能が失われている。 したがって、 癌の進行を抑える には p53を有効に機能させることが必要である。  When normal cells are exposed to ultraviolet light, etc., p53 in the cells is activated, and as a result, cell growth is stopped. Therefore, by increasing the concentration of p53, the growth of cancer cells can be stopped. In other words, when p53 does not function, the growth of cancer cells is not stopped and cancer progresses. In fact, p53 is rarely found in cells of normal individuals, but about half of cells from cancer patients have this p53 deficient mutation. Even when such mutations have not occurred, some mutations have occurred in the control mechanism of p53, and the tumor suppressor function has been lost. Therefore, it is necessary to make p53 function effectively to suppress the progression of cancer.
本発明においては、 p53の活性化を癌治療の有効な方法の一つとするため、 シ ノビォリンの機能に着目した。 そして、 シノピオリンホモノックアウト動物を作 製し、 詳細に解析したところ、 野生型に比してアポトーシスを起こしている細胞 が多数観察された。 すなわち、 シノビォリンの機能を阻害すると、 アポトーシス に深く関与している p53の活性化が促進され、 シノビオリンの機能阻害が癌抑制 につながることを見出した。 なお、 アポトーシスとは、 細胞が自ら引き起こすプログラムされた細胞死を意 味し、 細胞核の染色体凝集、 細胞核の断片化、 細胞表面微絨毛の消失、 細胞質の 凝集、 カスパーゼの活性化、 ミトコンドリア膜電位の消失等を特徴とする。 細胞 に上記特徴が生じたときに、 アポト一シスが引き起こされたと判断する。 In the present invention, the function of synoviolin was focused on in order to make activation of p53 one of the effective methods for cancer treatment. Then, a synopiolin homo-knockout animal was prepared and analyzed in detail. As a result, a greater number of apoptotic cells were observed than in the wild type. In other words, they found that inhibiting the function of synoviolin promoted the activation of p53, which is deeply involved in apoptosis, and that inhibiting the function of synoviolin leads to cancer suppression. In addition, apoptosis refers to programmed cell death caused by the cell itself, chromosome aggregation of the cell nucleus, fragmentation of the cell nucleus, loss of cell surface microvilli, aggregation of the cytoplasm, activation of caspase, activation of mitochondrial membrane potential It is characterized by disappearance. When the above characteristics occur in the cell, it is determined that apoptosis has been induced.
本発明において、 胎児胚における p53の免疫染色を行うと、 シノビオリンホモ ノックアウトマウス胎児胚では全身において p53が強く発現する。 また、 シノビ オリンホモノックァゥトマウス胎児胚から単離した胎仔線維芽細胞 （MEFs) も、 野生型から単離したものに比して強く発現しており、 しかも p53は核内に強 く局在する。 この核局在は野生型ではまったく観察されない。 このことは、 シノ ピオリンの発現を阻害することにより、 p53を核内へ移行させることができるこ とを意味する。 さらに、 シノビオリンホモノックアウトマウス胎仔 MEFsでは、 高い放射線感受性を示す。 従って、 本発明において、 シノビォリンの発現を阻害 して p53を核に移行させた後に放射線照射を行うと、 癌細胞の増殖を効果的に抑 制することができる。  In the present invention, when immunostaining of p53 in a fetal embryo is performed, p53 is strongly expressed in the whole body of a Synoviolin homo-knockout mouse embryo. In addition, fetal fibroblasts (MEFs) isolated from the fetal embryo of Synoviolin homonockato mouse are more strongly expressed than those isolated from the wild type, and p53 is strongly localized in the nucleus. I do. This nuclear localization is not observed at all in the wild type. This means that p53 can be translocated into the nucleus by inhibiting the expression of synopiolin. Furthermore, Synoviolin homo-knockout mouse embryo MEFs show high radiosensitivity. Therefore, in the present invention, when irradiation is performed after inhibiting the expression of synoviolin to transfer p53 to the nucleus, the growth of cancer cells can be effectively suppressed.
さらに、 本発明においては、 p53 (p53タンパク質） のアミノ酸の一部をリン 酸化することにより p53を活性化させることを特徴とする。 p53のリン酸化の対 象は、 p53のアミノ酸配列のうちセリン残基であることが好ましく、 第 15番目の セリン残基がさらに好ましい。  Furthermore, the present invention is characterized in that p53 is activated by phosphorylating a part of amino acids of p53 (p53 protein). The target of p53 phosphorylation is preferably a serine residue in the amino acid sequence of p53, and more preferably the 15th serine residue.
p53は ATRおよび ATM等のキナーゼによりリン酸化されることが知られてお り、 リン酸化は、 シノビオリンを阻害することによりこれらのキナーゼ活性を上 昇させることが考えられる。  It is known that p53 is phosphorylated by kinases such as ATR and ATM, and phosphorylation is thought to increase synthobiolin's activity by inhibiting synoviolin.
2 . シノピオリン発現阻害及び活性阻害 2. Inhibition of synopiolin expression and activity
p53を活性化するためには、 シノビオリンの発現を阻害する方法が採用され る。  In order to activate p53, a method of inhibiting the expression of Synoviolin is employed.
シノビォリンの発現阻害には、 特に限定されるものではないが、 例えば RNA 干渉 （RNAi) を利用することができる。 シノビオリン遺伝子に対する siRNA (s mall interfering RNA)を設計及び合成し、 これを細胞内に導入させることによ つて、 RNAiを引き起こすことができる。  Inhibition of synoviolin expression is not particularly limited, and for example, RNA interference (RNAi) can be used. RNAi can be induced by designing and synthesizing siRNA (small interfering RNA) for the synoviolin gene and introducing it into cells.
RNAiとは、 dsRNA (double-strand RNA)が標的遺伝子に特異的かつ選択的に 結合し、 当該標的遺伝子を切断することによりその発現を効率よく阻害する現象 である。 例えば、 dsRNAを細胞内に導入すると、 その RNAと相同配列の遺伝子 の発現が抑制 （ノックダウン） される。 RNAi means that dsRNA (double-strand RNA) is specific and selective for the target gene. It is a phenomenon that binds and cleaves the target gene to efficiently inhibit its expression. For example, when dsRNA is introduced into a cell, expression of a gene having a sequence homologous to the RNA is suppressed (knocked down).
siRNAの設計は、 以下の通り行なうことができる。  The design of siRNA can be performed as follows.
(a) シノビオリンをコードする遺伝子であれば特に限定されるものではなく、 任意の領域を全て候補にすることが可能である。 例えば、 ヒトの場合では、 Gen Bank Accession number AB024690 (配列番号 1 ) の任意の領域を候補にする ことができる。  (a) The gene is not particularly limited as long as it encodes synoviolin, and any arbitrary region can be used as a candidate. For example, in the case of human, any region of Gen Bank Accession number AB024690 (SEQ ID NO: 1) can be a candidate.
(b) 選択した領域から、 AAで始まる配列を選択し、 その配列の長さは 19〜25 塩基、 好ましくは 19〜21塩基である。 その配列の GC含量は、 例えば 40〜60%と なるものを選択するとよい。 具体的には、 配列番号 1に示される塩基配列のう ち、 以下の塩基配列から選ばれる少なくとも 1つの配列を含む DNAを siRNAの標 的配列として使用することができる。 特に、 （i) (配列番号 2) 、 (i i) (配列 番号 3) 、 (v i ) (配列番号 7) 、 (v i i ) (配列番号 8) 、 (v i i i) (配列番号 9) を標的とすることが好ましい。  (b) A sequence starting with AA is selected from the selected region, and the length of the sequence is 19 to 25 bases, preferably 19 to 21 bases. The sequence should have a GC content of, for example, 40 to 60%. Specifically, a DNA containing at least one sequence selected from the following nucleotide sequences among the nucleotide sequences shown in SEQ ID NO: 1 can be used as a target sequence of siRNA. In particular, it targets (i) (SEQ ID NO: 2), (ii) (SEQ ID NO: 3), (vi) (SEQ ID NO: 7), (vii) (SEQ ID NO: 8), (viii) (SEQ ID NO: 9) Is preferred.
(i) AA TGTCTGCATCATCTGCCGA GA (配列番号 2)  (i) AA TGTCTGCATCATCTGCCGA GA (SEQ ID NO: 2)
(ii) AA GCTGTGACAGATGCCATCA TG (配列番号 3)  (ii) AA GCTGTGACAGATGCCATCA TG (SEQ ID NO: 3)
(iii) AA AGCTGTGACAGATGCCATC AT (配列番号 4)  (iii) AA AGCTGTGACAGATGCCATC AT (SEQ ID NO: 4)
(iv) AA GAAAGCTGTGACAGATGCC AT (配列番号 5)  (iv) AA GAAAGCTGTGACAGATGCC AT (SEQ ID NO: 5)
(v) AA GGTTCTGCTGTACATGGCC TT (配列番号 6) (v) AA GGTTCTGCTGTACATGGCC TT (SEQ ID NO: 6)
(vi) AA CAAGGCTGTGTACATGCTC TA (配列番号 7)  (vi) AA CAAGGCTGTGTACATGCTC TA (SEQ ID NO: 7)
(vii) AA ATGTTTCCACTGGCTGGCT GA (配列番号 8)  (vii) AA ATGTTTCCACTGGCTGGCT GA (SEQ ID NO: 8)
(viii) AA GGTGTTCTTTGGGCAACTG AG (配列番号 9)  (viii) AA GGTGTTCTTTGGGCAACTG AG (SEQ ID NO: 9)
(ix) AA CATCCACACACTGCTGGAC GC (配列番号 10)  (ix) AA CATCCACACACTGCTGGAC GC (SEQ ID NO: 10)
(x) AA CACCCTGTATCCAGATGCC AC (配列番号 11) (x) AA CACCCTGTATCCAGATGCC AC (SEQ ID NO: 11)
(xi) AA GGTGCACACCTTCCCACTC TT (配列番号 12)  (xi) AA GGTGCACACCTTCCCACTC TT (SEQ ID NO: 12)
(xii) AA TGTTTCCACTGGCTGGCTG AG (配列番号 13)  (xii) AA TGTTTCCACTGGCTGGCTG AG (SEQ ID NO: 13)
(xiii) AA GAGACTGCCCTGCAACCAC AT (配列番号 14)  (xiii) AA GAGACTGCCCTGCAACCAC AT (SEQ ID NO: 14)
(xiv) AA CGTTCCTGGTACGCCGTCA CA (配列番号 15) siRNAを細胞に導入するには、 インビトロで合成した siRNAをプラスミド DN Aに連結してこれを細胞に導入する方法、 2本の RNAをァニールする方法などを 採用することができる。 (xiv) AA CGTTCCTGGTACGCCGTCA CA (SEQ ID NO: 15) In order to introduce siRNA into cells, a method in which siRNA synthesized in vitro is ligated to plasmid DNA and introduced into cells, a method in which two RNAs are annealed, and the like can be employed.
また、 本発明は、 RNAi効果をもたらすために shRNAを使用することもでき る。 shRNAとは、 ショートヘアピン RNA(short hairpin RNA)と呼ばれ、 一本 鎖の一部の領域が他の領域と相補鎖を形成するためにステムループ構造を有する RNA分子である。  The present invention can also use shRNA to produce an RNAi effect. shRNA is an RNA molecule having a stem-loop structure because a part of a single strand forms a complementary strand with another area, which is called a short hairpin RNA (short hairpin RNA).
shRNAは、 その一部がステムループを構造を形成するように設計することが できる。 例えば、 ある領域の配列を配列 Aとし、 配列 Aに対する相補鎖を配列 B とすると、 配列 A、 スぺーサ一、 配列 Bの順になるようにこれらの配列が一本の RNA鎖に存在するようにし、 全体で 45〜60塩基の長さとなるように設計する。 配列 Aは、 標的となるシノビオリン遺伝子 （配列番号 1) の一部の領域の配列で あり、 標的領域は特に限定されるものではなく、 任意の領域を候補にすることが 可能である。 そして配列 Aの長さは 19〜25塩基、 好ましくは 19〜21塩基であ る。  shRNAs can be designed such that a portion forms a stem-loop structure. For example, if the sequence of a certain region is sequence A and the complementary strand to sequence A is sequence B, these sequences are present in a single RNA strand so that sequence A, spacer, and sequence B are arranged in this order. And design the total length to be 45-60 bases. Sequence A is a sequence of a partial region of the target Synoviolin gene (SEQ ID NO: 1), and the target region is not particularly limited, and any region can be a candidate. The length of sequence A is 19 to 25 bases, preferably 19 to 21 bases.
3 . 医薬組成物  3. Pharmaceutical composition
本発明において作製された shRNA、 siRNAは、 シノビォリンの発現を抑制す る物質であり、 p53を活性化させる医薬組成物 （特に癌の遺伝子治療剤） として 使用することができる。  The shRNA and siRNA produced in the present invention are substances that suppress the expression of Synoviolin and can be used as a pharmaceutical composition for activating p53 (particularly a gene therapy agent for cancer).
本発明の医薬組成物を癌の遺伝子治療剤として使用する場合は、 適用部位は特 に限定されず、 脳腫瘍、 舌癌、 咽頭癌、 肺癌、 乳癌、 食道癌、 胃癌、 膝臓癌、 胆 道癌、 胆嚢癌、 十二指腸癌、 大腸癌、 肝癌、 子宮癌、 卵巣癌、 前立腺癌、 腎癌、 膀胱癌、 横紋筋肉腫、 線維肉腫、 骨肉腫、 軟骨肉腫、 皮膚癌、 各種白血病 （例え ば急性骨髄性白血病、 急性リンパ性白血病、 慢性骨髄性白血病、 慢性リンパ性白 血病、 成人型 T細胞白血病、 悪性リンパ腫） 、 等を対象として適用される。  When the pharmaceutical composition of the present invention is used as a gene therapy agent for cancer, the site of application is not particularly limited, and it may be brain tumor, tongue cancer, pharynx cancer, lung cancer, breast cancer, esophagus cancer, stomach cancer, knee cancer, biliary tract. Cancer, gallbladder cancer, duodenal cancer, colon cancer, liver cancer, uterine cancer, ovarian cancer, prostate cancer, kidney cancer, bladder cancer, rhabdomyosarcoma, fibrosarcoma, osteosarcoma, chondrosarcoma, skin cancer, various leukemias (eg (Acute myeloid leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, adult T-cell leukemia, malignant lymphoma).
上記癌は、 原発巣であっても、 転移したものであっても、 他の疾患と併発した ものであってもよい。  The above-mentioned cancer may be a primary tumor, a metastasized tumor, or a disease associated with another disease.
本発明の医薬組成物を遺伝子治療剤として使用する場合は、 本発明の医薬組成 物を注射により直接投与する方法のほか、 核酸が組込まれたベクターを投与する 方法が挙げられる。 上記ベクターとしては、 アデノウイルスベクター、 アデノ随 伴ウィルスベクタ一、 ヘルぺスウィルスベクター、 ワクシニアウィルスベクター 、 レトロウイルスベクタ一、 レンチウィルスベクタ一等が挙げられ、 これらのゥ ィルスベクターを用いることにより効率よく投与することができる。 When the pharmaceutical composition of the present invention is used as a gene therapy agent, in addition to a method of directly administering the pharmaceutical composition of the present invention by injection, a vector incorporating a nucleic acid is administered. Method. Examples of the above vectors include adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, vaccinia virus vectors, retrovirus vectors, lentivirus vectors, and the like. Efficient use of these virus vectors Can be administered well.
また、 本発明の医薬組成物をリボソームなどのリン脂質小胞体に導入し、 その 小胞体を投与することも可能である。 siRNAや shRNAを保持させた小胞体をリ ポフエクシヨン法により所定の細胞に導入する。 そして、 得られる細胞を例えば 静脈内、 動脈内等の全身投与する。 脳等に局所投与することもできる。  It is also possible to introduce the pharmaceutical composition of the present invention into phospholipid vesicles such as ribosomes and administer the vesicles. The endoplasmic reticulum retaining the siRNA or shRNA is introduced into predetermined cells by the lipofection method. Then, the obtained cells are systemically administered, for example, into a vein or an artery. It can also be administered locally to the brain and the like.
本発明の医薬組成物の投与量は、 年齢、 性別、 症状、 投与経路、 投与回数、 剤 型によって異なるが、 例えばアデノウイルスの場合の投与量は 1日 1回あたり 106 〜： !Ois個程度であり、 1週〜 8週間隔で投与される。 The dosage of the pharmaceutical composition of the present invention, age, sex, symptoms, administration route, frequency of administration, and the dosage form, for example a dose in the case of adenovirus 10 6 ~ per day:! OIS pieces About 1 to 8 weeks.
siRNA又は shRNAを目的の組織又は器官に導入するために、 市販の遺伝子導 入キット (例えばアデノエクスプレス ： クローンテック社) を用いることもでき る。  In order to introduce the siRNA or shRNA into a target tissue or organ, a commercially available gene transfer kit (for example, AdenoExpress: Clontech) can also be used.
以下、 実施例により本発明をさらに具体的に説明する。 但し、 本発明はこれら 実施例に限定されるものではない。  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
〔実施例 1〕 (Example 1)
本実施例は、 シノビオリンホモノックアウトマウス （syno ） 胎児線維芽細 胞 (MEFs)におけるアポトーシス関連タンパク質をウエスタンプロッティングに より検出し、 さらに細胞を免疫組織染色により確認した。  In this example, apoptosis-related proteins in synoviolin homo-knockout mouse (syno) fetal fibroblasts (MEFs) were detected by Western blotting, and the cells were confirmed by immunohistochemical staining.
すなわち、 免疫染色法は、 MEFsを常法に従いスライドガラス上に固定し、 2 種類の抗 p53抗体 （マウスモノクローナル抗体 BD： Becton， Dickinson 社、 ゥ サギポリクロ一ナル抗体 FL393： Santa cruz社） を用いて免疫染色を行った。 3 % 牛血清アルブミン （BSA) で 30分ブロッキングを行った標本に、 0.3%BSAで 希釈した抗 p53抗体 （BD： 10pg/ml、 FL393： 5 g/ml) を室温で 60分免疫反応 させた。 反応後の標本を PBSで洗浄後、 フルォレセインイソチオシァネート標識 抗ゥサギ IgG抗体または TRITC標識抗マウス IgG抗体 （Dako社） を 2次抗体とし て免疫反応させた。 抗 p53抗体に免疫反応する抗原の確認は、 蛍光顕微鏡で行つ た。 In other words, the immunostaining method involves fixing MEFs on a slide glass according to a conventional method, and using two types of anti-p53 antibodies (mouse monoclonal antibody BD: Becton, Dickinson, Inc. ゥ Sagi polyclonal antibody FL393: Santa cruz). Immunostaining was performed. Specimens that had been blocked with 3% bovine serum albumin (BSA) for 30 minutes were immunoreacted with an anti-p53 antibody (BD: 10 pg / ml, FL393: 5 g / ml) diluted with 0.3% BSA at room temperature for 60 minutes. . After the reaction, the specimen was washed with PBS, and immunoreacted with fluorescein isothiocyanate-labeled anti-Peagle IgG antibody or TRITC-labeled anti-mouse IgG antibody (Dako) as a secondary antibody. Confirmation of antigen immunoreactive with anti-p53 antibody is performed with a fluorescence microscope It was.
その結果、 野生型に比し、 syno- の MEF培養細胞では p53の活性化を起こし ている細胞が多数確認された （図 1 ) 。 〔実施例 2〕  As a result, compared with the wild-type, a greater number of p53-activated cells were found in the syno- MEF culture cells (Fig. 1). (Example 2)
本実施例は、 syno マウスにおける p53活性化の検討である。  This example is an examination of p53 activation in syno mice.
synoチマウスにおける p53活性化の検討を、 胚を用いて免疫染色により行つ た。  p53 activation in syno mice was examined by immunostaining using embryos.
すなわち、 syno-/-の胎仔における免疫染色は、 常法に従い組織をスライドガ ラス上に固定し、 ベクタスティン ABCキット（VECTOR社）を用いて行った。 ブ ロッキング試薬で 30分ブロッキングした標本に対して、 5pg/mlに希釈した抗 p 53 抗体 FL393を室温で 60分間免疫反応させた。 反応後の標本を PBSで洗浄し、 HR P標識抗ゥサギ IgG抗体を 2次抗体として免疫反応させた。 抗 p53抗体に免疫反 応する抗原は、 HRP活性に基づく 3，3'-ジァミノべンジジン四塩酸塩の発色によ り確認した。 対比染色としてメチルグリーン染色を行った。  That is, immunostaining of syno-/-fetuses was performed using a Vectostin ABC kit (VECTOR) with the tissue fixed on a slide glass according to a conventional method. The specimen blocked with the blocking reagent for 30 minutes was immunoreacted with the anti-p53 antibody FL393 diluted to 5 pg / ml at room temperature for 60 minutes. The specimen after the reaction was washed with PBS, and immunoreacted with an HRP-labeled anti-Peacock IgG antibody as a secondary antibody. The antigen immunoreactive with the anti-p53 antibody was confirmed by color development of 3,3'-diaminobenzidine tetrahydrochloride based on HRP activity. Methyl green staining was performed as counterstain.
この結果、 synoチの胚における p53が活性化していることが確認された （図 2 ) 。  As a result, it was confirmed that p53 was activated in the syno embryo (Fig. 2).
〔実施例 3〕 (Example 3)
本実施例では、 p53に対するシノビォリンの効果を検討した。  In this example, the effect of synoviolin on p53 was examined.
syno-/-の MEF培養細胞におけるアポトーシス関連タンパク質をウエスタンブ ロッティングにより検出した。  Apoptosis-related proteins in syno-/-cultured MEF cells were detected by Western blotting.
すなわち、 各種細胞を細胞破砕液 （15 mM Tris-HCl (pH7.5) 、 200 mM NaCL 0.5% NP40、 1 mM PMSF、 0.1%ドデシル硫酸ナトリウム （SDS) 、 2 pg/mlロイぺプチン、 2pg/mlァプロチニン、 2pg/mlぺプス夕チン） を用いて細胞 破碎画分を調製した。 その後、 SDS ポリアクリルアミド電気泳動 （SDS-PAG E) により細胞破砕画分を分離した。 SDS-PAGE後、 細胞由来タンパク質は、 ェ レクトロブロッテイング法によりニトロセルロース （NC) 膜に転写した。 この NC膜に対し、 5%スキムミルクを加えたトリス塩酸 （TBS) で室温、 1時間プロ ッキングした後、 抗 p53抗体 C-tarminal aa;195_393または FL393を 5%スキムミ ルクを加えた TBSで希釈して室温、 1時間免疫反応させた。 反応後の NC膜を 0. 1% Tween20/TBSで洗浄し、 horse radish peroxidase (HRP) 標識抗ゥサギ Ig G抗体を 2次抗体として室温、 1時間免疫反応させ、 0.1% Tween20/TBSで洗浄 し、 HRP活性を検出することにより目的抗原を検出した。 HRP活性の検出には ECLキット （Amersham社） を用いた （Clinical Chemistry. 25, pl531， 197 9) 。 That is, various cells were lysed with cell lysate (15 mM Tris-HCl (pH 7.5), 200 mM NaCL 0.5% NP40, 1 mM PMSF, 0.1% sodium dodecyl sulfate (SDS), 2 pg / ml leptin, 2 pg / The cell-fractionated fraction was prepared using (ml aprotinin, 2 pg / ml peptide). Then, the cell-fractionated fraction was separated by SDS-polyacrylamide electrophoresis (SDS-PAGE). After SDS-PAGE, cell-derived proteins were transferred to a nitrocellulose (NC) membrane by the electroblotting method. The NC membrane was treated with Tris-HCl (TBS) containing 5% skim milk for 1 hour at room temperature. After locking, the anti-p53 antibody C-tarminal aa; 195_393 or FL393 was diluted with TBS supplemented with 5% skim milk and immunoreacted at room temperature for 1 hour. After the reaction, the NC membrane was washed with 0.1% Tween20 / TBS, and a horse radish peroxidase (HRP) -labeled anti-Egret Ig G antibody was used as a secondary antibody for immunoreaction at room temperature for 1 hour, and then washed with 0.1% Tween20 / TBS. The target antigen was detected by detecting the HRP activity. For detection of HRP activity, an ECL kit (Amersham) was used (Clinical Chemistry. 25, pl531, 1979).
その結果、 ウェスタンブロッティングにより syno の MEF培養細胞における p 53発現量が増加していることが確認された （図 3 ) 。  As a result, it was confirmed by western blotting that p53 expression in syno MEF cultured cells was increased (Fig. 3).
〔実施例 4〕  (Example 4)
本実施例は、 syno- の MEF培養細胞における p53のリン酸化部位を同定し た。  In this example, the phosphorylation site of p53 in the MEF cultured cell of syno- was identified.
抗 p53抗体を用いたウエスタンプロッティングにより p53のリン酸化部位の同 疋を行った。  The phosphorylation site of p53 was probed by Western plotting using an anti-p53 antibody.
すなわち、 p53 (配列番号 16) の異なるセリン残基のリン酸化を認識する 4種 の抗リン酸化 p53モノクロ一ナル抗体 （P53serl5-P 、 P53ser20-P 、 P53ser37- Pおよび P53ser46-P； Becton, Dickinson 社） を用いて、 MEF細胞の蛋白質を S DS-PAGEで分離し、 ウエスタンブロッテイング法を行った。 ウエスタンブロッ ティング法の操作は、 一次抗体として抗リン酸化 p53モノクローナル抗体を、 そ して標識抗体として抗マウス IgGヒッジ -HRPを用いる他は実施例 3に記載のとお りである。 図 4において、'左上のパネルが第 15番目のセリン残基がリン酸化さ れたものである。 53kDa付近のバンドが顕著に濃く表れている。  That is, four anti-phosphorylated p53 monoclonal antibodies that recognize phosphorylation of different serine residues of p53 (SEQ ID NO: 16) (P53serl5-P, P53ser20-P, P53ser37-P and P53ser46-P; Becton, Dickinson The proteins of the MEF cells were separated by SDS-PAGE using Western blotting. The operation of the Western blotting method is as described in Example 3 except that an anti-phosphorylated p53 monoclonal antibody is used as a primary antibody and an anti-mouse IgG hidge-HRP is used as a labeled antibody. In FIG. 4, the upper left panel shows the 15th serine residue phosphorylated. The band around 53 kDa is remarkably dark.
その結果、 syno- の MEF培養細胞において、 p53のアミノ酸配列 （配列番号 1 6 ) 中、 第 15番目のセリン残基のリン酸化が顕著であった （図 4 ) 。 産業上の利用可能性  As a result, phosphorylation of the 15th serine residue in the amino acid sequence of p53 (SEQ ID NO: 16) was remarkable in the syno- MEF cultured cells (FIG. 4). Industrial applicability
本発明により、 p53 癌抑制遺伝子の活性を促進する物質が提供される。 この物 質は、 p53 を活性化して p53 を核に移行させることができるため、 癌の治療用 医薬組成物として有用である。 また、 本発明によりシノビォリンの機能を抑制す ることにより、 癌の治療が可能となる According to the present invention, a substance that promotes the activity of a p53 tumor suppressor gene is provided. Since this substance can activate p53 and transfer p53 to the nucleus, it is useful as a pharmaceutical composition for treating cancer. Further, the present invention suppresses the function of synoviolin. Will enable cancer treatment

Claims

1 . 配列番号 1に示される塩基配列のうち配列番号 2〜： 15に示す塩基配列から なる群から選択される少なくとも 1つの配列を標的配列として設計された siR NAを含む医薬組成物。 . 1 SEQ ID NO: 1 SEQ ID NO of the nucleotide sequence shown in 2: Pharmaceutical composition comprising a s iR NA designed at least one sequence selected from the group consisting of the nucleotide sequence shown in 15 as the target sequence.
2 . 癌を治療するための請求項 1記載の医薬組成物。  2. The pharmaceutical composition according to claim 1, for treating cancer.
3 . 配列番号 1に示される塩基配列のうち配列番号 2〜： 15に示す塩基配列からな る群から選択される少なくとも 1つの配列を標的配列として設計された siRN 3. siRN designed with at least one sequence selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 2 to 15 among the nucleotide sequences of SEQ ID NO: 1 as a target sequence
Aを用いて、 シノビオリンの発現を阻害することを特徴とする p53癌抑制遺 伝子の活性化方法。 の A method for activating a p53 tumor suppressor gene, which comprises using A to inhibit the expression of Synoviolin. of
4. 配列番号 1に示される塩基配列のうち配列番号 2〜： 15に示す塩基配列からな る群から選択される少なくとも 1つの配列囲を標的配列として設計された siRN 4. siRN designed with at least one sequence selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 2 to 15 among the nucleotide sequences of SEQ ID NO: 1 as the target sequence
Aを用いて、 シノビオリンの発現を阻害することを特徴とする p53癌抑制遺 伝子の核への局在化方法。 A method for localizing a p53 tumor suppressor gene to the nucleus using A to inhibit the expression of Synoviolin.
5 . 核に局在化した p53癌抑制遺伝子に、 さらに放射線照射を行うことを特徴と する請求項 4記載の方法。 5. The method according to claim 4, wherein the p53 tumor suppressor gene localized in the nucleus is further irradiated with radiation.
6 . p53の第 15番目のセリン残基をリン酸化することを特徴とする p53の活性化 方法。' 6. A method for activating p53, which comprises phosphorylating the 15th serine residue of p53. '
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* Cited by examiner, † Cited by third party
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