WO2007073005A1 - Regulator of methyl transfer reaction - Google Patents

Regulator of methyl transfer reaction Download PDF

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Publication number
WO2007073005A1
WO2007073005A1 PCT/JP2006/326306 JP2006326306W WO2007073005A1 WO 2007073005 A1 WO2007073005 A1 WO 2007073005A1 JP 2006326306 W JP2006326306 W JP 2006326306W WO 2007073005 A1 WO2007073005 A1 WO 2007073005A1
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carbon monoxide
methylation
biopolymer
present
cell
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PCT/JP2006/326306
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French (fr)
Japanese (ja)
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Makoto Suematsu
Takehiro Yamamoto
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Keio University
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Publication of WO2007073005A1 publication Critical patent/WO2007073005A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection

Definitions

  • the present invention relates to a substance that affects the methylation of biopolymers. Specifically, the present invention relates to a substance that regulates a methyl group transfer reaction in a biopolymer.
  • BACKGROUND ART Methylation of biopolymers plays an important role as a regulatory system for important biological control mechanisms involved in the protection of cells and organs. Such biopolymer modifications occur on proteins, enzymes, DNA, and the like. In particular, in the case of DNA, acetylation is a mechanism for inserting a switch for gene expression, whereas methylation is a mechanism for cutting an expression switch and is extremely important.
  • CO carbon monoxide
  • NO nitric oxide
  • This gas molecule is known to be involved in unknown cytoprotective effects, suppression of inflammatory responses, transplantation tolerance, induction of cell differentiation, etc., but the mechanism has not been elucidated (Reference:! ⁇ Four ) .
  • the problem to be solved by the present invention is to provide a substance capable of adjusting a methyl group transfer reaction in a biopolymer and a method for adjusting the methyl group transfer reaction. .
  • Another object of the present invention is to provide a substance that induces cell differentiation and a method for inducing cell differentiation.
  • CO carbon monoxide
  • the present invention is as follows.
  • a biopolymer methylation regulator comprising carbon monoxide.
  • a method for regulating methylation of a biopolymer which comprises contacting carbon monoxide with the biopolymer.
  • a cell differentiation inducer characterized by containing carbon monoxide.
  • a method for inducing cell differentiation comprising bringing carbon monoxide into contact with a cell and enhancing demethylation of a protein in the cell.
  • this invention relates to the following.
  • a biopolymer acetylation regulator containing carbon monoxide containing carbon monoxide.
  • a method for regulating acetylation of a biopolymer characterized by bringing carbon monoxide into contact with the biopolymer.
  • Fig. 1 is a flow diagram showing the outline of the verification method for CO gene modification by Chromatin immhoprecipitation.
  • FIG. 2 shows changes in chromatin dynamics due to CO administration of CDlla, which is the differentiation marker for U937.
  • Fig. 3 shows the analysis of global protein methylation by Western blotting using ADMA antibody and the effect of CO addition (Ru: Ru (DMSO) 4 Cl 2 , CO donor: CORM 2).
  • Figure 4 shows the same strategy for proteins that undergo methylation modification by carbon monoxide (CO) treatment.
  • FIG. 5 shows the detection of methylated protein by Western blotting using ADMA antibody. Spots 3, 5, 6, 34, 35, and 36 show spots with enhanced methylation by CO treatment.
  • FIG. 6 is a diagram showing a MALDI-TOF MS analysis result of a spot cut out from a two-dimensional electrophoresis gel.
  • FIG. 7 shows the detection of methylated protein by Western blotting using ADMA antibody.
  • Figure 8 summarizes the results of MALDI-TQF / MS analysis.
  • Gas mediators are a group of molecules that can easily pass through cell membranes and bind to biopolymers such as proteins and DNA to give specific functions. Carbon monoxide is one of these mediators, and heme is decomposed into biliary pigments such as biliverdin and pyryrubin, carbon monoxide, and reduced iron (Fe 2+ ) by heme oxygenase. Is known to occur. However, the specific function of carbon monoxide has not been fully clarified.
  • the present invention relates to a biopolymer methylation regulator containing carbon monoxide and a biopolymer methylation regulation method using carbon monoxide.
  • carbon monoxide has an action of controlling acetylation of a biopolymer, particularly an action of enhancing acetylation of a biopolymer. That is, in the present invention, it has been shown that carbon monoxide can be used to control acetylation of biopolymers. Therefore, the present invention relates to a biopolymer acetylation regulator for carbon monoxide and a method for regulating biopolymer acetylation with carbon monoxide. Since acetylation of biopolymers is known to be related to the control of gene expression, it can be said that carbon monoxide can control gene expression through regulation of acetylation of biopolymers.
  • the present invention relates to a cell differentiation-inducing agent containing carbon monoxide or a method for inducing cell differentiation by carbon monoxide.
  • the present invention also relates to a method for producing carbon monoxide in a biopolymer by carbon monoxide.
  • “regulation of methylation” or “regulation of methyl group transfer reaction” means control of methyl group modification state in biological polymer, enhancement of methyl group transfer or withdrawal, or methylation or desorption of biopolymer. Means increased methylation.
  • modulation of acetylation means enhancement of acetylation or withdrawal of a acetyl group in a biopolymer, or enhancement of acetylation or deacetylation of a biopolymer.
  • the degree of enhancement is not particularly limited, but is 5% or more of the degree of methylation or acetylation of the control biopolymer not treated with carbon monoxide, preferably 10% or more, more preferably 30% or more, More preferably, it is 40% or more.
  • the biopolymer is not particularly limited as long as it is a molecule derived from a living body having a side chain that can be methylated or acetylated.
  • protein protein, nucleic acid (DNA, RNA, etc.), lipid, carbohydrate Etc.
  • the biological high molecule is preferably a protein or DNA, more preferably a protein. 1.
  • biopolymers methylated by carbon oxide include transketolase or alpha-enolase, which are glycolytic enzymes.
  • the side chain methylated or acetylated by carbon monoxide A side chain of an amino acid, more preferably a side chain of arginine can be mentioned, but it is not particularly limited. Further, the number of methylation or acetylation per molecule is not particularly limited.
  • carbon monoxide may be used as it is, or in a ribosome containing carbon monoxide, heme and heme protein containing carbon monoxide, erythrocytes containing carbon monoxide or an aqueous solution.
  • Modified carbon monoxide such as a carbon monoxide-containing complex that can release carbon monoxide, or a compound that is metabolized in the body to release CO (such as aryl hydride carbon) may be used.
  • Preference is given to using gaseous carbon monoxide. From the viewpoint of operational safety, it is preferable to use modified carbon monoxide. You may use the pharmaceutical composition containing the carbon monoxide mentioned later.
  • Carbon monoxide-containing complexes include CORM-K CORM-2 ([Ru (CO) 3 Cl2] 2) (Ozawa N, Goda N, Makino N, Yamaguchi T, Yoshimura Y, Suematsu M (2002) Leydig cell-derived heme oxygenase-1 regulates apoptosis of premeiotic germ cells in response to stress. J Clin Invest 109: 457-467.)), and CO releasing molecules such as CORM-3, C0RM-A1, and CORM F3.
  • the central metals are manganese (CORM-1), ruthenium (C0RM-2, -3), boron (C0RM-A1) and iron (CORM-F3), respectively.
  • the carbon monoxide-containing complex is preferably C0RM-2 in that the action of the central metal on the biopolymer is relatively small.
  • the regulation of methylation or acetylation of a biopolymer by carbon monoxide can be performed by bringing carbon monoxide into contact with the biopolymer. More specifically, it can be carried out by bringing carbon monoxide into contact with a cell, tissue, body fluid or the like containing a biopolymer to be subjected to methylation regulation or acetylation regulation.
  • “contact” means exposing a biopolymer or a cell, tissue, or body fluid containing the biopolymer to an environment in which carbon monoxide exists.
  • a mode in which cells or tissues are cultured in the presence of carbon monoxide a mode in which cells or tissues are cultured in a medium containing carbon monoxide, or carbon monoxide gas is blown onto cells, tissues or body fluids
  • concentration of carbon monoxide when added to a cell or tissue culture is 0.1 to: ⁇ ⁇ mol / L, preferably l lOOO mol / L, more preferably 10 to: 100 / i mol L, more preferably 25 ⁇ mol L.
  • the concentration when carbon monoxide is exposed to cells, tissues or body fluids is 5-100 i mol / L, more preferably 10.30 ⁇ mol / L.
  • Contact is from 10 ° C to 60 ° C, preferably 20 ° C. C to 50 ° C, more preferably 30 ° C to 40 ° C, most preferably 37 ° C, 10 minutes to 72 hours, preferably 20 minutes to 60 hours, more preferably Can be performed from 30 minutes to 48 hours.
  • the ability to regulate methylation or acetylation of biopolymers in this way The degree of methylation or acetylation is determined by Western blot analysis using anti-methylated antibody or anti-acetylated antibody, ELISA (Enzyme It can be detected by immunochemical methods such as -linked immunosorbent assay) and immunoprecipitation. Further, methylation or acetylation of a target biopolymer can be detected by appropriately combining known biopolymer extraction, separation, and purification methods with an immunochemical method.
  • the anti-methylated antibody used for the detection of methylation may be an antibody that recognizes a methylated biopolymer or an antibody that recognizes a methyl group.
  • an anti-methylated cytosine antibody Frazier, ASB
  • Anti-methylated lysine antibody Anti-dimethylarginine antibody
  • ADMA anti-methylated histone H3 (K9) antibody
  • K9 antibody Upstate, Abeam, SIGMA
  • anti-dimethylhistone H3 (K9) antibody upstate # 07) -441
  • anti-methylated DNA antibody and anti-methyl dalixoxal antibody (Nippon Yushi, Japan Aging Control Research Laboratories).
  • the anti-acetylated antibody used for the detection of acetylation may be any antibody that recognizes a acetylated biological polymer or an antibody that recognizes a acetyl group, and is not limited to, for example, anti-acetylyl And histone H3 (Lys9) antibody (Upstate # 07-352).
  • histone methylation status can be detected by chromatin immunoprecipitation.
  • chromatin immunoprecipitation method cells that have been contacted with carbon monoxide are first fixed with formaldehyde, etc., and then enzymatically cleaved to fragment the DNA to an average of 250 to 500 bp.
  • the immunoprecipitation is performed using the anti-dimethyl histone H3 (Lys 9) antibody (upstate # 07-441). Next, reverse-crosslinking and elution are performed, and the target gene is amplified by PCR using the eluate. The amplified product can be electrophoresed to determine the methylation status of histone 113.
  • This chromatin immunoprecipitation method can also be used to detect cell differentiation induction by carbon monoxide, which will be described later. Proteins in which the modification state by methyl group or acetyl group is changed by carbon monoxide treatment can be identified by a known protein identification method.
  • proteins in spots with altered modification compared to controls can be identified using a mass spectrometer.
  • the protein in the spot may be digested with trypsin or the like and then analyzed with a mass spectrometer.
  • Mass spectrometers include, for example, matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF / MS), electrospray mass spectrometry (ESI / MS), liquid chromatography Matographic mass spectrometry (LC / MS), gas chromatography matrix spectrometry (GCMS), etc. can be used.
  • the present invention provides a cell differentiation-inducing agent containing carbon monoxide (hereinafter also referred to as “differentiation-inducing agent of the present invention”).
  • the present invention also provides a method for inducing cell differentiation, characterized in that carbon monoxide is brought into contact with a cell to enhance demethylation or acetylation of the protein in the cell.
  • the differentiation inducer of the present invention can be used for a cell differentiation induction method and a cell differentiation 'induction therapy.
  • carbon monoxide is related to induction of differentiation of lymphoid cells, and thus artificial control of inflammatory reaction and rejection in transplantation, and a pharmaceutical composition used for this control (hereinafter referred to as “the present invention”). Also referred to as “immunomodulator”). Moreover, in the present invention, when carbon monoxide is brought into contact with a biopolymer, the expression of HO-1 is induced, so that carbon monoxide can be used for the induction of HO-1. In addition, carbon monoxide enhances the production of carbon monoxide ⁇ in vivo via HO-1 with increased expression level, so carbon monoxide produces endogenous carbon monoxide in biopolymers. Can be used for That is, exogenous administration of CO increases endogenous CO and may additively regulate cell function control.
  • carbon monoxide is considered to be involved in various biological functions that biopolymers play, it is possible to artificially control all proteins or genes subject to methylation control or acetylation control according to the present invention. Became possible.
  • the present invention can also be applied to proteins or genes that will be elucidated in the future to undergo methylation control or acetylation control.
  • carbon monoxide, a carbon monoxide-containing complex, or the like can be used for the carbon monoxide that is the active ingredient of the differentiation-inducing agent of the present invention or the immunomodulator of the present invention. It is not something. CO binds to hemoglobin and is transported to peripheral tissues.
  • a gaseous carbon monoxide when using gaseous carbon monoxide, adults (60 kg), 0.1 to 300 ppm per day, preferably 10 to 200 ppm, more preferably lOOppm at a concentration of 1 minute to 24 hours, preferably 5 minutes Administer by inhalation for ⁇ 12 hours, more preferably from 10 minutes to 6 hours. Inhalation can be performed using a mask, nasal catheter, or nasal force neurone connected to a carbon monoxide source (eg, a carbon monoxide cylinder or concentrator). It is also possible to administer a saturated solution of CO (saline) through blood vessels.
  • a carbon monoxide source eg, a carbon monoxide cylinder or concentrator
  • modified hemoglobin compounds that release CO through metabolism, and carbon monoxide bound to phospho-encapsulated hemoglobin (modified carbon monoxide), adults (60 kg)
  • 0.1 nmol to 1000 ju mol preferably lnmol to 100 ⁇ mol, more preferably lOnmol to lO yu mol per day.
  • modified carbon monoxide When using modified carbon monoxide, it may be used as it is, or it may be formulated in combination with a pharmacologically acceptable carrier.
  • protoneme IX or fma protoporphyrin IX, or fma dopamine or dobutamine When used as it is, or it may be used as a preparation.
  • Examples of pharmacologically acceptable carriers include excipients, binders, disintegrants, lubricants, lubricants, emulsifiers, coloring agents, flavoring agents, surfactants, and the like, which are usually used in medicine.
  • examples include solubilizers, suspending agents, isotonic agents, stabilizers, buffers, and antioxidants.
  • preparations may include tablets, powders, granules, capsules, oral preparations such as syrups, suppositories, ointments, eye drops, external preparations such as eye drops, and injections.
  • the above injections can be used by methods such as infusion, intramuscular injection, subcutaneous injection, and intravenous injection.
  • An injection may be formulated as a ribosome preparation by appropriately combining the above pharmacologically acceptable carriers.
  • the present invention also provides a kit containing carbon monoxide (hereinafter also referred to as “kit of the present invention”).
  • kit of the present invention is used for regulation of methylation or acetylation of biopolymers or induction of cell differentiation.
  • the carbon monoxide contained in the kit of the present invention is not particularly limited, and is, for example, gaseous carbon monoxide, modified carbon monoxide, or formulated carbon monoxide.
  • the kit of the present invention can be used to regulate methylation or acetylation of biopolymers or to induce and detect differentiation of cells, such as methylation detection reagents (for example, antimethylation reagents).
  • methylation detection reagents for example, antimethylation reagents
  • Acetylated antibodies for example, acetylated detection reagents
  • biopolymers or cells enzyme substrates (chromogenic substrates, etc.), and enzyme reaction terminators.
  • the kit of the present invention may contain various buffers, sterilized water, culture vessels, reaction vessels, experimental operation instructions, and the like.
  • Example 1 Detection of DNA modification by Chromatin immnoprecipitation method
  • CDlla is a differentiation-inducing marker for liii sphere cells, and is expressed on the cell membrane when leukemia cell U937 differentiates into monocyte macrophage.
  • the cells were cultured for 30 minutes in the presence of Ru (DMSO) 4 Cl 2 ( ⁇ ) as a control or CORM-2 (100 ⁇ ) as a CO donor, and the cells were collected.
  • whole cell lysate (lGO g / gel) prepared from the collected cells was electrophoresed two-dimensionally (pH 6-ll), and the separated protein was treated with anti-dimethyl alcohol used in Example 2.
  • Western blot analysis was performed using ginin (ADMA) antibody.
  • the spot of the sample treated with Ru (DMSO) 4 Cl 2 was compared with the spot of the sample treated with CORM-2, and spots with different degrees of methylation were cut out.
  • the excised spot protein was digested in gel with trypsin and then subjected to mass spectrometry using MALDI-TOF / MS. From the results of mass spectrometry, proteins were identified by the PMF method. Result>
  • cytoplasmic fractions were separated from human monocyte-derived U937 cells treated with CORM-2 or control Ru (DMSO) 4 Cl 2 for 30 minutes, and two-dimensional electrophoresis was performed. Western blot analysis was performed using ADMA antibody. As a result, spots with increased methylation (arrows) and spots with demethylation (open arrows) were confirmed by CO treatment (Fig. 7).
  • carbon monoxide can be used for differentiation-inducing therapy in which undifferentiated cells such as cancer cells are differentiated into normal cells by carbon monoxide.
  • the present invention provides a biopolymer methylation regulator and a biopolymer methylation regulation method.
  • the present invention also provides a cell differentiation inducer and a cell differentiation induction method.
  • the present invention makes it possible to control the biological functions of carbon monoxide, the present invention can be applied to control of inflammatory reactions and rejection in transplantation, for example.

Abstract

Disclosed are: a regulator of the methylation of a biological polymer, wherein the regulator comprises carbon monooxide; a method for regulating the methylation of a biological polymer; an inducer of the differentiation of a cell; and a method for inducing the differentiation of a cell.

Description

明 細 書  Specification
メチル基転移反応調節物質 Methyl group transfer regulator
技術分野 Technical field
' 本発明は、 生体高分子のメチル化に影響を与える物質に関する。 詳しくは、 生 体高分子におけるメチル基転移反応を調節する物質に関する。  'The present invention relates to a substance that affects the methylation of biopolymers. Specifically, the present invention relates to a substance that regulates a methyl group transfer reaction in a biopolymer.
背景技術 . 生体高分子のメチル化は、 細胞や臓器の保護にかかわる重要な生体制御機構の 調節系として重要な役割を果たしている。 このような生体高分子の修飾は、 蛋白 . 質、 酵素及び DNAなどに対して起こる。 特に DNAの場合には、 ァセチル化が 遺伝子発現のスィツチを入れる機構であるのに対して、 メチル化は発現スィツチ を切る機構であり、 極めて重要である。 BACKGROUND ART Methylation of biopolymers plays an important role as a regulatory system for important biological control mechanisms involved in the protection of cells and organs. Such biopolymer modifications occur on proteins, enzymes, DNA, and the like. In particular, in the case of DNA, acetylation is a mechanism for inserting a switch for gene expression, whereas methylation is a mechanism for cutting an expression switch and is extremely important.
ところで、 一酸化炭素 （CO) は、 低酸素、 活性酸素、 過剰な一酸化窒素 (NO) 、 重金属、 熱ショック、 ホルモン産生などにより各種の細胞に誘導される heme oxygenase から生成されるガスメディエーターである。 このガス分子は未 知の細胞保護効果や炎症反応の抑制、 移植免疫寛容、 細胞の分化誘導などに関与 していることが知られているが、 そのメカニズムは明らかにされていない （文献 ：！〜 4 ) 。  By the way, carbon monoxide (CO) is a gas mediator produced from heme oxygenase induced in various cells by hypoxia, active oxygen, excess nitric oxide (NO), heavy metals, heat shock, hormone production, etc. is there. This gas molecule is known to be involved in unknown cytoprotective effects, suppression of inflammatory responses, transplantation tolerance, induction of cell differentiation, etc., but the mechanism has not been elucidated (Reference:! ~ Four ) .
(文献） (Reference)
1. Hayashi S, Takamiya R, ϊ amaguchi Τ， Matsumoto Κ， Tojo SJ， Tamatani T， Kitajima M, Makino N, Ishimura Y， Suematsu M (1999) Induction of heme oxygenase- 1 suppresses venular leukocyte adhesion elicited by oxidative stress: role of bilirubin generated by the enzyme. Circ Res 85: 663-671.  1. Hayashi S, Takamiya R, ϊ amaguchi Τ, Matsumoto Κ, Tojo SJ, Tamatani T, Kitajima M, Makino N, Ishimura Y, Suematsu M (1999) Induction of heme oxygenase-1 suppresses venular leukocyte adhesion elicited by oxidative stress: role of bilirubin generated by the enzyme.Circ Res 85: 663-671.
2. Goda N， Suzuki K， Naito M, Takeoka S, Tsuchida E, Ishimura Y, Tamatani T， Suematsu M (1998) Distribution of heme oxygenase isoforms in rat liver. Topographic basis for carbon monoxide-mediated microvascular relaxation. J Clin Invest 101: 604-612. 2. Goda N, Suzuki K, Naito M, Takeoka S, Tsuchida E, Ishimura Y, Tamatani T, Suematsu M (1998) Distribution of heme oxygenase isoforms in rat liver.Topographic basis for carbon monoxide-mediated microvascular relaxation.J Clin Invest 101: 604-612.
3. Kyokane T, Norimizu S， Taniai H, Yamaguchi T, Takeoka S, Tsuchida E， Naito M, Nimura Y， Ishimura Y， Suematsu M (2001) Carbon monoxide from heme catabolism protects against hepatobiliary dysfunction in endotoxin-treated rat liver. Gastroenterology 120: 1227- 1240. 3. Kyokane T, Norimizu S, Taniai H, Yamaguchi T, Takeoka S, Tsuchida E, Naito M, Nimura Y, Ishimura Y, Suematsu M (2001) Carbon monoxide from heme catabolism protects against hepatobiliary dysfunction in endotoxin-treated rat liver. Gastroenterology 120 : 1227-1240.
4. Wakaba ashi Y, Takamiya R， Mizuki A， Kyokane T， Goda N, Yamaguchi T, Takeoka S， Tsuchida E， Suematsu M, Ishimura Y (1999) Carbon monoxide overproduced by heme oxygenase- 1 causes a reduction of vascular resistance in perfused rat liver. Am J Physiol 277(5 Pt 4. Wakaba ashi Y, Takamiya R, Mizuki A, Kyokane T, Goda N, Yamaguchi T, Takeoka S, Tsuchida E, Suematsu M, Ishimura Y (1999) Carbon monoxide overproduced by heme oxygenase-1 causes a reduction of vascular resistance in perfused rat liver. Am J Physiol 277 (5 Pt
1):G1088- 1096. 発明の開示 1): G1088-1096. Disclosure of the Invention
本発明が解決しようとする課題は、 生体高分子におけるメチル基転移反応を調 節し得る物質及びメチル基転移反応の調節方法を提供することにある。.  The problem to be solved by the present invention is to provide a substance capable of adjusting a methyl group transfer reaction in a biopolymer and a method for adjusting the methyl group transfer reaction. .
また、 本発明の別の課題は、 細胞の分化を誘導する物質及び細胞の分化誘導方 法を提供することにある。  Another object of the present invention is to provide a substance that induces cell differentiation and a method for inducing cell differentiation.
本発明者は、 上記課題を解決するべく鋭意検討を行った。 その結果、 生体内で 一酸化炭素 （CO) が、 蛋白質や DNA等の生体高分子におけるメチル化に関与し、 メチル基転移反応を調節し得る物質であることを見出し、 本発明を完成した。  The present inventor has intensively studied to solve the above problems. As a result, the present inventors have found that carbon monoxide (CO) is a substance that can participate in methylation in biological macromolecules such as proteins and DNA and regulate the methyl group transfer reaction in vivo.
すなわち本発明は、 以下のとおりである。  That is, the present invention is as follows.
( 1 ) 一酸化炭素を含有することを特徴とする、 生体高分子のメチル化調節剤。 (1) A biopolymer methylation regulator comprising carbon monoxide.
( 2 ) 生体高分子が、 タンパク質又は DNAである、 （1 ) に記載の調節剤。 (2) The regulator according to (1), wherein the biopolymer is protein or DNA.
( 3 ) 生体高分子が、 transketorase 又は alpha-enolase である、 （1 ) に記載 の調節剤。  (3) The regulator according to (1), wherein the biopolymer is transketorase or alpha-enolase.
( 4 ) 生体高分子に一酸化炭素.を接触させることを特徴とする、 生体高分子のメ チル化調節方法。  (4) A method for regulating methylation of a biopolymer, which comprises contacting carbon monoxide with the biopolymer.
( 5 ) 一酸化炭素を含有することを特徴とする、 細胞の分化誘導剤。  (5) A cell differentiation inducer characterized by containing carbon monoxide.
( 6 ) 細胞に一酸化炭素を接触させ、 細胞中のタンパク質の脱メチル化を亢進す ることを特徴とする、 細胞の分化誘導方法。 - また、 本発明は以下に関する。 ( 7 ) 一酸化炭素を含有する生体高分子のァセチル化調節剤。 (6) A method for inducing cell differentiation, comprising bringing carbon monoxide into contact with a cell and enhancing demethylation of a protein in the cell. -Moreover, this invention relates to the following. (7) A biopolymer acetylation regulator containing carbon monoxide.
( 8 ) 生体高分子に一酸化炭素を接触させることを特徴とする、 生体高分子のァ セチル化調節方法。  (8) A method for regulating acetylation of a biopolymer, characterized by bringing carbon monoxide into contact with the biopolymer.
( 9 ) 一酸化炭素を含有する heme oxygenase- 1の発現誘導方法。  (9) A method for inducing expression of heme oxygenase-1 containing carbon monoxide.
( 1 0 ) 生体高分子に一酸化炭素を接触させ、 heme oxygenase- 1の発現を 誘導することを特徴とする、 生体高分子における一酸化炭素の産生方法。 図面の詳細な説明  (10) A method for producing carbon monoxide in a biopolymer, characterized in that carbon monoxide is brought into contact with the biopolymer to induce the expression of heme oxygenase-1. Detailed description of the drawings
図 1は、 Chromatin immhoprecipitationによる COの遺伝子修飾作用の検証方 法の概要を示すフロー図である。  Fig. 1 is a flow diagram showing the outline of the verification method for CO gene modification by Chromatin immhoprecipitation.
図 2は、 U937の分化マーカ一である CDl laの CO投与によるクロマチン動態 の変化を示す図である。  FIG. 2 shows changes in chromatin dynamics due to CO administration of CDlla, which is the differentiation marker for U937.
図 3は、 ADMA 抗体を用いた Western blotting による global protein met ylation の解析と CO 添加による効果を示す図である （Ru: Ru(DMSO)4Cl2, CO donor: CORM 2) 。 Fig. 3 shows the analysis of global protein methylation by Western blotting using ADMA antibody and the effect of CO addition (Ru: Ru (DMSO) 4 Cl 2 , CO donor: CORM 2).
図 4は、 一酸化炭素 （CO)処理により、 メチル化修飾を受けるタンパク質の同 定のストラテジーを示す図である。  Figure 4 shows the same strategy for proteins that undergo methylation modification by carbon monoxide (CO) treatment.
図 5は、 ADMA抗体を用いた Western blottingによるメチル化タンパク質の検 出を示す図である。 スポット 3，5，6,34,35，36 は CO処理でメチル化の増強したス ポットを示す。  FIG. 5 shows the detection of methylated protein by Western blotting using ADMA antibody. Spots 3, 5, 6, 34, 35, and 36 show spots with enhanced methylation by CO treatment.
図 6は、 2次元電^泳動ゲルから切り出したスポッ トの MALDI-TOF MS解析 結果を示す図である。  FIG. 6 is a diagram showing a MALDI-TOF MS analysis result of a spot cut out from a two-dimensional electrophoresis gel.
図 7は、 ADMA抗体を用レ、た Western blottingによるメチル化タンパク質の検 出を示す図である。  FIG. 7 shows the detection of methylated protein by Western blotting using ADMA antibody.
図 8は、 MALDI-TQF/MS解析結果をまとめた図である。 発明を実施するための最良の形態 以下、 本発明について詳しく説明するが、 本発明の範囲はこれらの説明に拘束 されることはなく、 以下の例示以外についても、 本発明の趣旨を損なわない範囲 で適宜変更し実施し得る。 Figure 8 summarizes the results of MALDI-TQF / MS analysis. BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. However, the scope of the present invention is not limited to these explanations, and the examples other than the following examples can be appropriately modified and implemented without departing from the spirit of the present invention.
なお、 本明細書において引用された全ての刊行物、 例えば先行技術文献、 及び 公開公報、 特許公報その他の特許文献は、 その全体が参照として本明細書に組み '入れられる。 また、 本明細書は、 本願優先権主張の基礎となる米国仮出願第 60/753,388号の開示内容を包含する。 ガスメディエータは、 細胞膜を容易に通過することが可能であり、 蛋白質や DNAなどの生体高分子と結合して特異的な機能を付与する分子群である。 一酸化 炭素は、 このガスメデイエ一タの一つであり、 ヘムがヘムォキシゲナーゼにより、 ビリベルジン、 ピリルビンなどの胆汁色素と一酸化炭素と還元鉄 （Fe2+) とに分 解されることにより生じることが知られている。 しカゝし、 一酸化炭素の特異的な 機能については十分に明らかにされていなかった。 It should be noted that all publications cited in the present specification, for example, prior art documents, and published publications, patent publications, and other patent documents, are incorporated herein by reference in their entirety. This specification also includes the disclosure of US Provisional Application No. 60 / 753,388, which is the basis for claiming priority. Gas mediators are a group of molecules that can easily pass through cell membranes and bind to biopolymers such as proteins and DNA to give specific functions. Carbon monoxide is one of these mediators, and heme is decomposed into biliary pigments such as biliverdin and pyryrubin, carbon monoxide, and reduced iron (Fe 2+ ) by heme oxygenase. Is known to occur. However, the specific function of carbon monoxide has not been fully clarified.
本発明において、 一酸化炭素は、 生体高分子のメチル化又は脱メチル化を亢進 する作用を有することが明らかになった。 すなわち、 本発明おいて、 一酸化炭素 は、 生体高分子のメチル化調節に使用できることが示された。 したがって、 本発 明は、 一酸化炭素を含有する生体高分子のメチル化調節剤及び一酸化炭素による 生体高分子のメチル化調節方法に関する。  In the present invention, it has been clarified that carbon monoxide has an action of enhancing methylation or demethylation of biopolymers. That is, in the present invention, it was shown that carbon monoxide can be used for regulating methylation of biopolymers. Accordingly, the present invention relates to a biopolymer methylation regulator containing carbon monoxide and a biopolymer methylation regulation method using carbon monoxide.
また、 本発明において、 一酸化炭素は、 生体高分子のァセチル化を制御する作 用、 特に生体高分子のァセチル化を亢進する作用を有することが明らかになった。 すなわち、 本発明において、 一酸化炭素は生体高分子のァセチル化調節に使用で きることが示された。 したがっ 、 本発明は、 一酸化炭素を含有する生体高分子 のァセチル化調節剤及び一酸化炭素による生体高分子のァセチル化調節方法に関 する。 生体高分子のァセチル化は、 遺伝子発現の制御に関係することが知られて. いるため、 一酸化炭素は生体高分子のァセチル化調節を介して遺伝子発現を制御 することが可能といえる。  In the present invention, it has also been clarified that carbon monoxide has an action of controlling acetylation of a biopolymer, particularly an action of enhancing acetylation of a biopolymer. That is, in the present invention, it has been shown that carbon monoxide can be used to control acetylation of biopolymers. Therefore, the present invention relates to a biopolymer acetylation regulator for carbon monoxide and a method for regulating biopolymer acetylation with carbon monoxide. Since acetylation of biopolymers is known to be related to the control of gene expression, it can be said that carbon monoxide can control gene expression through regulation of acetylation of biopolymers.
また、 生体高分子のメチル化若しくは脱メチル化またはァセチル化は、 遺伝子 発現の制御に関係することが知られているため、 一酸化炭素は、 生体高分子のメ チル化またはァセチル化の調節を介して、 遺伝子発現、 さらに細胞の分化誘導を 制御することが可能といえる。 実際、 実施例において、 白血病細胞株 U937 を一 酸化炭素で処理することにより、 細胞の分化誘導が引き起こされることが示され た。 したがって、 本発明は、 一酸化炭素を含有する細胞の分化誘導剤又は一酸化 '炭素による細胞の分化誘導方法に関する。 In addition, methylation or demethylation or acetylation of biopolymers Since it is known to be involved in the regulation of expression, carbon monoxide can control gene expression and further induction of cell differentiation through the regulation of methylation or acetylation of biopolymers. I can say that. In fact, in the examples, it was shown that treatment of leukemic cell line U937 with carbon monoxide induces cell differentiation. Therefore, the present invention relates to a cell differentiation-inducing agent containing carbon monoxide or a method for inducing cell differentiation by carbon monoxide.
さらにまた、 本発明において、 一酸化炭素は、 heme oxygenase- 1 (ΗΟ·1) の 発現を誘導することが示された。 HO- 1 はヘムを分解する酵素であり、 ヘムの分 解により」酸化炭素が生成される。 すなわち、 本発明において、 一酸化炭素は HO I の発現誘導を介する一酸化炭素の産生に使用できることが示された。 した がって、 本発明は、 一酸化炭素による生体高分子における一酸化炭素の産生方法 にも関する。 本発明において、 「メチル化の調節」 又は 「メチル基転移反応の調節」 は、 生 体高分子におけるメチル基修飾状態の制御、 メチル基の転移もしくは離脱の亢進、 又は生体高分子のメチル化若しくは脱メチル化の亢進を意味する。 また、 「ァセ チル化の調節」 は、 生体高分子におけるァセチル基の転移もしくは離脱の亢進、 又は生体高分子のァセチル化もしくは脱ァセチル化の亢進を意味する。 亢進の程 度は、 特に限定されないが、 一酸化炭素で処理しない対照生体高分子のメチル化 またはァセチル化の程度の 5 %以上、.好ましくは 1 0 %以上、 より好ましくは 3 0 %以上、 さらに好ましくは 4 0 %以上である。  Furthermore, in the present invention, it was shown that carbon monoxide induces the expression of heme oxygenase-1 (ΗΟ · 1). HO-1 is an enzyme that breaks down heme, and the decomposition of heme produces carbon oxides. That is, in the present invention, it was shown that carbon monoxide can be used for the production of carbon monoxide through induction of HO I expression. Therefore, the present invention also relates to a method for producing carbon monoxide in a biopolymer by carbon monoxide. In the present invention, “regulation of methylation” or “regulation of methyl group transfer reaction” means control of methyl group modification state in biological polymer, enhancement of methyl group transfer or withdrawal, or methylation or desorption of biopolymer. Means increased methylation. Further, “modulation of acetylation” means enhancement of acetylation or withdrawal of a acetyl group in a biopolymer, or enhancement of acetylation or deacetylation of a biopolymer. The degree of enhancement is not particularly limited, but is 5% or more of the degree of methylation or acetylation of the control biopolymer not treated with carbon monoxide, preferably 10% or more, more preferably 30% or more, More preferably, it is 40% or more.
本発明において、 生体高分子は、 メチル化またはァセチル化可能な側鎖を有す る生体由来の分子であれば特に限定されないが、 例えば、 タンパク質、 核酸 (DNA、 RNA など） 、 脂質、 糖質などが挙げられる。 本発明において、 生体高 分子は、 好ましくはタンパク質又は DNA、 より好ましくはタンパク質である。 一. 酸化炭素によりメチル化される生体高分子としては、 例えば、 解糖系の酵素であ transketolase又は alpha-enolase力挙けられる。  In the present invention, the biopolymer is not particularly limited as long as it is a molecule derived from a living body having a side chain that can be methylated or acetylated. For example, protein, nucleic acid (DNA, RNA, etc.), lipid, carbohydrate Etc. In the present invention, the biological high molecule is preferably a protein or DNA, more preferably a protein. 1. Examples of biopolymers methylated by carbon oxide include transketolase or alpha-enolase, which are glycolytic enzymes.
本発明において、 一酸化炭素によりメチル化またはァセチル化される側鎖には、 アミノ酸の側鎖、 より好ましくはアルギニンの側鎖を挙げることができるが、 特 に限定されるものではない。 また、 一分子あたりのメチル化またはァセチル化の 数は特に限定されない。 In the present invention, the side chain methylated or acetylated by carbon monoxide A side chain of an amino acid, more preferably a side chain of arginine can be mentioned, but it is not particularly limited. Further, the number of methylation or acetylation per molecule is not particularly limited.
本発明において、 一酸化炭素は、 そのまま使用してもよいし、 あるいは、 一酸 化炭素を含有するリボソーム、 一酸化炭素を含有するヘムおよびヘム蛋白質、 一 酸化炭素を含有する赤血球又は水溶液中で一酸化炭素を放出できる一酸化炭素含 有錯体等の修飾型一酸化炭素、 さらには体内で代謝されて CO を放出する化^^物 (ァリルハイ ド口カーボンなど） を使用してもよい。 気体の一酸化炭素を使用す ることが好ましい。 また、 操作の安全性の点から、 修飾型一酸化炭素を使用する ことが好ましい。 後述する一酸化炭素を含有する医薬組成物を使用しても良い。 一酸化炭素含有錯体には、 CORM-K CORM-2 ([Ru(CO)3Cl2】2 ) (Ozawa N， Goda N, Makino N, Yamaguchi T, Yoshimura Y, Suematsu M (2002) Leydig cell- derived heme oxygenase- 1 regulates apoptosis of premeiotic germ cells in response to stress. J Clin Invest 109: 457-467.)) 、 CORM-3、 C0RM-A1、 CORM F3などの CO releasing molecule を挙げることができる。 それぞれ中心 金属は、 マンガン （CORM- 1) 、 ルテニウム （C0RM-2、 -3) 、 ホウ素 （C0RM- A1) 及び鉄 （CORM-F3) である。 本発明において、 一酸化炭素含有錯体は、 生 体高分子に対する中心金属の作用が比較的小さい点で、 C0RM-2が好ましい。 本発明において、 一酸化炭素による生体高分子のメチル化またはァセチル化の 調節は、 生体高分子に一酸化炭素を接触させることにより行うことができる。 よ り具体的には、 メチル化調節またはァセチル化調節の対象となる生体高分子を含 む細胞、 組織、 体液などに一酸化炭素を接触させることにより行うことができる。 本発明において、 「接触」 は、 生体高分子又は生体高分子を含む細胞、 組織、 体液を一酸化炭素の存在する環境にさらすことを意味する。 接触には、 例えば、 一酸化炭素の存在下で細胞若しくは組織を培養する態様、 一酸化炭素を含む培地 で細胞若しくは組織を培養する態様、 又は細胞、 組織若しくは体液に一酸化炭素 ガスを吹きつける態様などがある。 細胞又は組織の培養液に添加する場合の一酸化炭素の濃度は、 0.1〜： ΙΟΟΟΟ μ mol/L, 好ましくは l lOOO mol/L より好ましくは 10〜： 100 /i mol L、 さらに 好ましくは 25 ^ mol L である。 また、 細胞、 組織又は体液に一酸化炭素を暴露す る場合の濃度は、 5-100 i mol/L、 より好ましくは 10·30 μ mol/Lである。 In the present invention, carbon monoxide may be used as it is, or in a ribosome containing carbon monoxide, heme and heme protein containing carbon monoxide, erythrocytes containing carbon monoxide or an aqueous solution. Modified carbon monoxide, such as a carbon monoxide-containing complex that can release carbon monoxide, or a compound that is metabolized in the body to release CO (such as aryl hydride carbon) may be used. Preference is given to using gaseous carbon monoxide. From the viewpoint of operational safety, it is preferable to use modified carbon monoxide. You may use the pharmaceutical composition containing the carbon monoxide mentioned later. Carbon monoxide-containing complexes include CORM-K CORM-2 ([Ru (CO) 3 Cl2] 2) (Ozawa N, Goda N, Makino N, Yamaguchi T, Yoshimura Y, Suematsu M (2002) Leydig cell-derived heme oxygenase-1 regulates apoptosis of premeiotic germ cells in response to stress. J Clin Invest 109: 457-467.)), and CO releasing molecules such as CORM-3, C0RM-A1, and CORM F3. The central metals are manganese (CORM-1), ruthenium (C0RM-2, -3), boron (C0RM-A1) and iron (CORM-F3), respectively. In the present invention, the carbon monoxide-containing complex is preferably C0RM-2 in that the action of the central metal on the biopolymer is relatively small. In the present invention, the regulation of methylation or acetylation of a biopolymer by carbon monoxide can be performed by bringing carbon monoxide into contact with the biopolymer. More specifically, it can be carried out by bringing carbon monoxide into contact with a cell, tissue, body fluid or the like containing a biopolymer to be subjected to methylation regulation or acetylation regulation. In the present invention, “contact” means exposing a biopolymer or a cell, tissue, or body fluid containing the biopolymer to an environment in which carbon monoxide exists. For the contact, for example, a mode in which cells or tissues are cultured in the presence of carbon monoxide, a mode in which cells or tissues are cultured in a medium containing carbon monoxide, or carbon monoxide gas is blown onto cells, tissues or body fluids There are aspects. The concentration of carbon monoxide when added to a cell or tissue culture is 0.1 to: ΙΟΟΟΟ μmol / L, preferably l lOOO mol / L, more preferably 10 to: 100 / i mol L, more preferably 25 ^ mol L. The concentration when carbon monoxide is exposed to cells, tissues or body fluids is 5-100 i mol / L, more preferably 10.30 μmol / L.
接触は、 1 0 °C〜6 0 °C、 好ましくは 2 0。C〜5 0 °C、 より好ましくば 3 0 °C 〜4 0 °C、 最も好ましくは 3 7 °Cで、 1 0分〜 7 2時間、 好ましくは 2 0分〜 6 0時間、 さらに好ましくは 3 0分〜 4 8時間行うことができる。 このように生体高分子のメチル化またはァセチル化を調節することができる力 メチル化またはァセチル化の程度は、 抗メチル化抗体または抗ァセチル化抗体を 用レヽるウェスタ ンブロ ッ ト解析、 ELISA ( Enzyme-linked immunosorbent assay) 法、 免役沈降法などの免疫化学的方法により検出することができる。 また、 免疫化学的方法に、 公知の生体高分子の抽出、 分離、 精製方法を適宜組み合わせ て、 目的の生体高分子のメチル化またはァセチル化を検出することもできる。  Contact is from 10 ° C to 60 ° C, preferably 20 ° C. C to 50 ° C, more preferably 30 ° C to 40 ° C, most preferably 37 ° C, 10 minutes to 72 hours, preferably 20 minutes to 60 hours, more preferably Can be performed from 30 minutes to 48 hours. The ability to regulate methylation or acetylation of biopolymers in this way The degree of methylation or acetylation is determined by Western blot analysis using anti-methylated antibody or anti-acetylated antibody, ELISA (Enzyme It can be detected by immunochemical methods such as -linked immunosorbent assay) and immunoprecipitation. Further, methylation or acetylation of a target biopolymer can be detected by appropriately combining known biopolymer extraction, separation, and purification methods with an immunochemical method.
メチル化の検出に使用する抗メチル化抗体は、 メチル化された生体高分子を認 識する抗体又はメチル基を認識する抗体であればよく、 例えば、 抗メチル化シト シン抗体 （Filgen社、 ASB社） 、 抗メチル化リジン抗体、 抗ジメチルアルギニン 抗体 (ADMA)、 抗メチル化ヒス トン H3 (K9) 抗体 （Upstate 社、 Abeam 社、 SIGMA社） 、 抗ジメチルヒス トン H3 (K9)抗体 (upstate # 07-441)、 抗メチル化 DNA抗体、 抗メチルダリオキサール抗体 （日本油脂、 日本老化制御研究所） を挙 げることができる。  The anti-methylated antibody used for the detection of methylation may be an antibody that recognizes a methylated biopolymer or an antibody that recognizes a methyl group. For example, an anti-methylated cytosine antibody (Filgen, ASB ), Anti-methylated lysine antibody, anti-dimethylarginine antibody (ADMA), anti-methylated histone H3 (K9) antibody (Upstate, Abeam, SIGMA), anti-dimethylhistone H3 (K9) antibody (upstate # 07) -441), anti-methylated DNA antibody, and anti-methyl dalixoxal antibody (Nippon Yushi, Japan Aging Control Research Laboratories).
ァセチル化の検出に使用する抗ァセチル化抗体は、 ァセチル化された生体高分 子を認識する抗体又はァセチル基を認識する抗体であればよく、 限定されるわけ ではないが、 例えば、 抗ァセチルヒストン H3 (Lys9) 抗体 （Upstate #07-352) を挙げることができる。 例えば、 ヒストンのメチル化状態 （修飾状態） 'は、 クロマチン免疫沈降法によ り検出することができる。 クロマチン免疫沈降法は、 まず、 一酸化炭素に接触させた細胞を formaldehyde などで固定し、 酵素的に切断して 250〜500 bp平均になるように DNAを断片化 する。 このサンプノレカ ら、 anti-dimethyl histone H3 (Lys 9) antibody (upstate # 07-441)を用いて免疫沈降する。 次に、 reverse-crosslinking及び溶出を行い、 溶 出液を用いて PCRにより目的遺伝子の増幅を行う。 増幅産物を電気泳動し、' ヒス ン113のメチル化状態を判断することができる。 The anti-acetylated antibody used for the detection of acetylation may be any antibody that recognizes a acetylated biological polymer or an antibody that recognizes a acetyl group, and is not limited to, for example, anti-acetylyl And histone H3 (Lys9) antibody (Upstate # 07-352). For example, histone methylation status (modification status) can be detected by chromatin immunoprecipitation. In the chromatin immunoprecipitation method, cells that have been contacted with carbon monoxide are first fixed with formaldehyde, etc., and then enzymatically cleaved to fragment the DNA to an average of 250 to 500 bp. The immunoprecipitation is performed using the anti-dimethyl histone H3 (Lys 9) antibody (upstate # 07-441). Next, reverse-crosslinking and elution are performed, and the target gene is amplified by PCR using the eluate. The amplified product can be electrophoresed to determine the methylation status of histone 113.
このクロマチン免疫沈降法は、 後述する一酸化炭素による細胞の分化誘導を検 出するために使用することもできる。 一酸化炭素処理により、 メチル基またはァセチル基による修飾状態が変動した タンパク質は、 公知のタンパク質.同定手法により同定することができる。  This chromatin immunoprecipitation method can also be used to detect cell differentiation induction by carbon monoxide, which will be described later. Proteins in which the modification state by methyl group or acetyl group is changed by carbon monoxide treatment can be identified by a known protein identification method.
例えば、 2次元電気泳動とウェスタンブロッ ト解析において、 対照に比べて修 飾状態が変動したスポット中のタンパク質は、 質量分析計を用いて同定すること ができる。 場合により、 スポッ ト中のタンパク質をトリプシンなどにより消化し てから質量分析計で解析してもよい。 質量分析計は、 例えば、 マトリ ックス支援 レーザー脱離イオン化一飛行時間型マススぺク ト ロメ ト リ一 （MALDI- TOF/MS) 、 エレク トロスプレーマススぺク トロメ トリー （ESI/MS) 、 液体クロ マトグラフィーマススぺク トロメ トリー （LC/MS) 、 ガスク口マ トグラフィーマ ススぺク トロメ トリー （GCMS) 等を使用することができる。 MALDI-TOF/MS で質量分析した場合は、 解析結果からぺプチドマスフィンガープリント法 （PMF 法） によりタンパク質を同定することができる。 また、 本発明者は、 ヒ ト白血病細胞株 U937 において、 一酸化炭素処理により U937 の分化が誘導されることを、 上記のクロマチン免疫沈降法を用いて示した c つまり、 分化誘導剤である PMAで U937を処理すると、 U937の分化マーカーで ある CDllaの転写調節領域中のヒストン H3の脱メチル化およびァセチル化が亢 進するが、 このヒストン H3 の脱メチル化およびァセチル化の亢進は、 一酸化炭 素処理によっても同様に確認された。 このことは、 一酸化炭素は白血病細胞など の未分化な細胞の分化を誘導できること示している。 したがって、 本発明は、 一 酸化炭素を含有する細胞の分化誘導剤 （以下、 「本発明の分化誘導剤」 とも称す る） を提供する。 また、 本発明は、 細胞に一酸化炭素を接触させ、 細胞中のタン パク質の脱メチル化又はァセチル化を亢進することを特徴とする、 細胞の分化誘 導方法も提供する。 本発明の分化誘導剤は、 細胞の分化誘導方法及び細胞の分化 '誘導療法に使用することができる。 For example, in two-dimensional electrophoresis and Western blot analysis, proteins in spots with altered modification compared to controls can be identified using a mass spectrometer. In some cases, the protein in the spot may be digested with trypsin or the like and then analyzed with a mass spectrometer. Mass spectrometers include, for example, matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF / MS), electrospray mass spectrometry (ESI / MS), liquid chromatography Matographic mass spectrometry (LC / MS), gas chromatography matrix spectrometry (GCMS), etc. can be used. When mass spectrometry is performed with MALDI-TOF / MS, proteins can be identified from the analysis results by the peptide mass fingerprint method (PMF method). In addition, the present inventor has shown that differentiation of U937 is induced by treatment with carbon monoxide in the human leukemia cell line U937 c, i.e., PMA which is a differentiation inducer. Treatment with U937 increases histone H3 demethylation and acetylation in the transcriptional regulatory region of CDlla, a marker of U937 differentiation, and this increased histone H3 demethylation and acetylation The same was confirmed by the carbon treatment. This means that carbon monoxide is leukemia cells It has been shown that differentiation of undifferentiated cells can be induced. Therefore, the present invention provides a cell differentiation-inducing agent containing carbon monoxide (hereinafter also referred to as “differentiation-inducing agent of the present invention”). The present invention also provides a method for inducing cell differentiation, characterized in that carbon monoxide is brought into contact with a cell to enhance demethylation or acetylation of the protein in the cell. The differentiation inducer of the present invention can be used for a cell differentiation induction method and a cell differentiation 'induction therapy.
また、 本発明において、 一酸化炭素は、 リンパ系細胞の分化誘導に関係するた め、 炎症反応や移植における拒絶反応の人為的制御、 及びこの制御に用いる医薬 組成物 （以下、 「本発明の免疫調節剤」 とも称する） に使用することができる。 また、 本発明において、 生体高分子に一酸化炭素を接触させると . HO- 1 の発現 が誘導されるため、 一酸化炭素は HO-1 の誘導に使用することができる。 さらに、 一酸化炭素により、 発現量の増加した HO-1 を介して、 生体内で一酸^ ^炭素の産 生が亢進するため、 一酸化炭素は生体高分子における内因性一酸化炭素の産生に 使用することができる。 すなわち、 CO の外因性投与は内因性の CO を増加させ、 細胞機能制御を相加的に制御する可能性がある。  In the present invention, carbon monoxide is related to induction of differentiation of lymphoid cells, and thus artificial control of inflammatory reaction and rejection in transplantation, and a pharmaceutical composition used for this control (hereinafter referred to as “the present invention”). Also referred to as “immunomodulator”). Moreover, in the present invention, when carbon monoxide is brought into contact with a biopolymer, the expression of HO-1 is induced, so that carbon monoxide can be used for the induction of HO-1. In addition, carbon monoxide enhances the production of carbon monoxide ^^ in vivo via HO-1 with increased expression level, so carbon monoxide produces endogenous carbon monoxide in biopolymers. Can be used for That is, exogenous administration of CO increases endogenous CO and may additively regulate cell function control.
さらに、 一酸化炭素は生体高分子が担う多様な生物機能に関与していると考え られるため、 本発明により、 メチル化制御またはァセチル化制御を受ける全ての タンパク質又は遺伝子を人為的に制御することが可能となった。 本発明は、 メチ ル化制御またはァセチル化制御を受けることが将来的に解明されるタンパク質又 は遺伝子についても、 適用することができる。 本発明の分化誘導剤又は本発明の免疫調節剤の有効成分である一酸化炭素には、 前述の通り、 一酸化炭素又は一酸化炭素含有錯体等を使用することができるが、 これに限定されるものではない。 なお、 CO は hemoglobin に結合して末梢組織に運ばれるものである。 そのた め、 CO を生体に投与する方法に関しては、 （1) 低濃度 （lOOppm 程度） の吸入 とすること、 （2) 赤血球、 修飾ヘモグロ ビン及び liposome-encapsulated hemoglobin等に結合させて血管内投与すること、 並びに (3) protoheme IX及び protoporphyrin IX等を体内に投与すること （経口投与、 又は腹腔内若しくは筋 肉内注射 ： 40〜： 100 μιηοΙ/L ) が好適であると考えられ、 さ らには、 （4) dopamine及び dobutamine等の、 細胞の cyclic AMPを増加させて HO- 1を誘 導する能力のあるすベての薬剤等により代替投与が可能であると考えられる。 本発明の分化誘導剤又は本発明の免疫調節剤の投与量は、 年齢、 性別、 症状、 投与経路、 投与回数、 剤形によって異なる。 Furthermore, since carbon monoxide is considered to be involved in various biological functions that biopolymers play, it is possible to artificially control all proteins or genes subject to methylation control or acetylation control according to the present invention. Became possible. The present invention can also be applied to proteins or genes that will be elucidated in the future to undergo methylation control or acetylation control. As described above, carbon monoxide, a carbon monoxide-containing complex, or the like can be used for the carbon monoxide that is the active ingredient of the differentiation-inducing agent of the present invention or the immunomodulator of the present invention. It is not something. CO binds to hemoglobin and is transported to peripheral tissues. Therefore, regarding the method of administering CO to the living body, (1) Inhalation at a low concentration (about 10 ppm), (2) Red blood cells, modified hemoglobin, and liposome-encapsulated (3) Protoheme IX and protoporphyrin IX etc. are administered into the body (oral administration or intraperitoneal or intramuscular injection: 40 ~: 100 μιηοΙ / L) In addition, (4) Alternative administration is possible with all drugs that have the ability to induce HO-1 by increasing cyclic AMP in cells, such as dopamine and dobutamine. It is thought that. The dose of the differentiation inducer of the present invention or the immunomodulator of the present invention varies depending on age, sex, symptom, administration route, administration frequency, and dosage form.
例えば、'気体の一酸化炭素を使用する場合は、 成人 （60 kg) 、 1 日当たり 0.1 〜300ppm、 好ましくは 10〜200ppm、 より好ましくは lOOppmの濃度で 1分〜 2 4時間、 好ましくは 5 分〜 1 2時間、 より好ましくは 1 0分〜 6時間、 吸入 投与する。 吸入は、 一酸化炭素元 （例えば、 一酸化炭素ボンべ又は濃縮装置） に 接続したマスク、 鼻カテーテル、 鼻力ニューレを用いて行うことができる。 また COの飽和溶液 （生理食塩水） を血管から投与することも可能である。  For example, when using gaseous carbon monoxide, adults (60 kg), 0.1 to 300 ppm per day, preferably 10 to 200 ppm, more preferably lOOppm at a concentration of 1 minute to 24 hours, preferably 5 minutes Administer by inhalation for ~ 12 hours, more preferably from 10 minutes to 6 hours. Inhalation can be performed using a mask, nasal catheter, or nasal force neurone connected to a carbon monoxide source (eg, a carbon monoxide cylinder or concentrator). It is also possible to administer a saturated solution of CO (saline) through blood vessels.
また、 一酸化炭素含有錯体、 又は赤血球、 修飾ヘモグロビン、 代謝により CO を放出する化合物及び liposome-encapsulated hemoglobin等に結合した一酸化 炭素 （修飾型一酸化炭素） を使用する場合は、 成人 （60 kg) 、 1 日当たり、 0.1nmol〜1000 ju mol、 好ましくは lnmol〜： 100 μ mol、 より好ましくは lOnmol 〜： lO yu mol で投与する。 protoheme IX 又は protoporphyrin IX の成人 （60 kg) 1 日当たりの投与量は、 0.1〜10000 /i mol/kg、 好ましくは 1〜： ΙΟΟΟ μ mol/kg、 より好ましくは 10〜： 100 micromol/kgである。  When using carbon monoxide-containing complexes, or red blood cells, modified hemoglobin, compounds that release CO through metabolism, and carbon monoxide bound to phospho-encapsulated hemoglobin (modified carbon monoxide), adults (60 kg) ), 0.1 nmol to 1000 ju mol, preferably lnmol to 100 μmol, more preferably lOnmol to lO yu mol per day. Adult dose of protoheme IX or protoporphyrin IX (60 kg) Daily dosage is 0.1-10000 / i mol / kg, preferably 1-: ： μmol / kg, more preferably 10-: 100 micromol / kg .
修飾型一酸化炭素を使用する場合は、 そのまま使用しても良いし、 薬理学的に 許容された担体と組み合わせて製剤化したものを使用しても良い。 また、 protoneme IX 若し fま protoporphyrin IX、 又 fま dopamine 若しく dobutamine を使用する場合も、 そのまま使用しても良いし、 製剤化したものを 使用しても良い。  When using modified carbon monoxide, it may be used as it is, or it may be formulated in combination with a pharmacologically acceptable carrier. When protoneme IX or fma protoporphyrin IX, or fma dopamine or dobutamine is used, it may be used as it is, or it may be used as a preparation.
薬理学的に許容される担体としては、 例えば通常医薬に使用される、 賦形剤、 結合剤、 崩壊剤、 滑沢剤、 潤滑剤、 乳化剤、 着色剤、 矯味矯臭剤、 界面活性剤、 溶解補助剤、 懸濁化剤、 等張剤、 安定化剤、 緩衝剤、 抗酸化剤などを挙げること ができる。 Examples of pharmacologically acceptable carriers include excipients, binders, disintegrants, lubricants, lubricants, emulsifiers, coloring agents, flavoring agents, surfactants, and the like, which are usually used in medicine. Examples include solubilizers, suspending agents, isotonic agents, stabilizers, buffers, and antioxidants.
製剤としては、 錠剤、 散剤、 顆粒剤、 カプセル剤、 シロップ剤などの経口剤、 坐剤、 軟膏剤、 点眼剤、 パップ剤などの外用剤、 又は注射剤を挙げることができ る。  Examples of the preparation may include tablets, powders, granules, capsules, oral preparations such as syrups, suppositories, ointments, eye drops, external preparations such as eye drops, and injections.
' 上記注射剤は、 点滴、 筋注、 皮下注、 静注などの方法で使用することができる。 注射剤は、 上記の薬理学的に許容される担体を適宜組み合わせて、 リボソーム製 剤として製剤化してもよレ、。 また、 本発明は、 一酸化炭素を含むキット （以下、 「本発明のキット」 とも称 する） を提供する。 本発明のキットは、 生体高分子のメチル化の調節もしくはァ セチル化の調節又は細胞の分化誘導に用いるものである。 本発明のキットに含ま れる一酸化炭素は、 特に限定されず、 例えば、 気体の一酸化炭素、 修飾型一酸化 炭素、 又は製剤化した一酸化炭素である。 本発明のキットは、 一酸化炭素の他に、 生体高分子のメチル化もしくはァセチル化の調節又は細胞の分化誘導の実施及び 検出に使用する他の試薬、 例えば、 メチル化検出試薬 （例えば抗メチル化抗体） 、 ァセチル化検出試薬 （例えば抗ァセチル化抗体） 生体高分子又は細胞、 酵素基質 (発色性基質など） 、 酵素反応停止剤を めることができる。 さらに、 本発明の キットには、 各種緩衝液、 滅菌水、 培養容器、 反応容器、 実験操作説明書などを 含めることもできる。 以下に、 実施例を挙げて本発明をより具体的に説明するが、 本発明はこれらに 限定されるものではない。 ， 実施例 1 ： Chromatin immnoprecipitation methodによる DNA修飾の検出 'The above injections can be used by methods such as infusion, intramuscular injection, subcutaneous injection, and intravenous injection. An injection may be formulated as a ribosome preparation by appropriately combining the above pharmacologically acceptable carriers. The present invention also provides a kit containing carbon monoxide (hereinafter also referred to as “kit of the present invention”). The kit of the present invention is used for regulation of methylation or acetylation of biopolymers or induction of cell differentiation. The carbon monoxide contained in the kit of the present invention is not particularly limited, and is, for example, gaseous carbon monoxide, modified carbon monoxide, or formulated carbon monoxide. In addition to carbon monoxide, the kit of the present invention can be used to regulate methylation or acetylation of biopolymers or to induce and detect differentiation of cells, such as methylation detection reagents (for example, antimethylation reagents). Acetylated antibodies), acetylated detection reagents (for example, anti-acetylated antibodies), biopolymers or cells, enzyme substrates (chromogenic substrates, etc.), and enzyme reaction terminators. Furthermore, the kit of the present invention may contain various buffers, sterilized water, culture vessels, reaction vessels, experimental operation instructions, and the like. Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples. Example 1: Detection of DNA modification by Chromatin immnoprecipitation method
<方法 > (図 1 ) <Method> (Figure 1)
COによる細胞內の methionine poolの上昇が遺伝子発現に影響するかを検討 するために、 ヒ ト細胞株 U937の分化マーカ一である CDllaの基本転写調節領 域中のヒス トン H3 の修飾状態をクロマチン免疫沈降 （CMP) 法により調べた。 CD l l は liii球細胞の分化誘導マーカ一であり、 白血病細胞である U937 が単球 マクロファージ系に分化する際に細胞膜上に発現する。 U937 細胞 lx lO7 cells を 10 ng/ml phorbor myristate acetate (PMA)又は CO除放剤 (CO releasing molecule-2 : CORM.2 I [Ru(CO)3Cl2]2 (Ozawa N， Goda N, Makino N， Yamaguchi T， Yoshimura Y, Suematsu M (2002) Leydig cell- derived heme oxygenase- 1 regulates apoptosis of premeiotic germ cells in response to stress. J Clin Invest 109: 457-467.) ) 25 μ mol/L で 48 時間処理した。 細胞を 1% formaldehyde で固定した後、 enzymatic shearingにより 250〜500 bp平均に なるように DNA を断片化し、 anti-dimethyl histone H3 (Lys 9) antibody (upstate # 07-441) 及び anti.acetyl histone H3 (Lys9) antibody (Upstate湖- 352)を用いて免疫沈降を行った。 その後、 reverse-crosslinking及び elution を 施して、 続いて PCRにより遺伝子増幅を行った。 PCRでは、 図 2に示すように、 CD l la遺伝子の上流の約- 200〜+100を増幅した。 CO処理のコントローノレとし ては Ru(DMSO)4Cl2を用いた。 ぐ結果 > In order to examine whether the increase in methionine pool of cell fistula caused by CO affects gene expression, the basic transcriptional regulatory region of CDlla, the differentiation marker of human cell line U937, is examined. The modified state of histone H3 in the region was examined by chromatin immunoprecipitation (CMP) method. CDll is a differentiation-inducing marker for liii sphere cells, and is expressed on the cell membrane when leukemia cell U937 differentiates into monocyte macrophage. U937 cells lx lO 7 cells at 10 ng / ml phorbor myristate acetate (PMA) or CO releasing molecule-2 (CO releasing molecule-2: CORM.2 I [Ru (CO) 3 Cl 2 ] 2 (Ozawa N, Goda N, Makino N, Yamaguchi T, Yoshimura Y, Suematsu M (2002) Leydig cell-derived heme oxygenase-1 regulates apoptosis of premeiotic germ cells in response to stress. J Clin Invest 109: 457-467.)) At 25 μmol / L Processed for 48 hours. After fixing the cells with 1% formaldehyde, the DNA was fragmented by average shearing to an average of 250-500 bp, and anti-dimethyl histone H3 (Lys 9) antibody (upstate # 07-441) and anti.acetyl histone H3 Immunoprecipitation was performed using (Lys9) antibody (Lake Upstate-352). Subsequently, reverse-crosslinking and elution were performed, followed by gene amplification by PCR. In PCR, as shown in FIG. 2, approximately −200 to +100 upstream of the CDlla gene was amplified. Ru (DMSO) 4 Cl 2 was used as the CO control controller. Results>
図 2 に示すよ う に、 CORM-2 を添加する と、 コ ン ト ロ ールである Ru(DMSO)4Cl2処理群に比べて chromatinのヒストン H3の Lysine残基のメチ ノレ化 （MeH3) が低下し （青色枠で囲んだパネル (左から 2列目のパネルの上 2 つ)参照） 、 一方では七ス トン H3 の Lysine残基 （Lys9) のァセチル化が顕著 に増加する （オレンジ色枠で囲んだ'パネル (最右列のパネルの上 2つ)参照） こと が明らかになった。 この変化は、，分化誘導物質である PMAの処理によっても同 様の変化が認められたことから、 CO は白血病細胞の分化誘導を惹起したこと力^ 明らかになった （図 2 ) 。 実施例 2 ： Anti dimethyl-arginine antibod (ADMA) を用いた蛋白質メチ ル化の検出 く方法〉 As shown in Fig. 2, when CORM-2 was added, methinorelation of the Lysine residue of chromatin histone H3 (MeH3) compared to the control Ru (DMSO) 4 Cl 2 treatment group. (See panel in blue frame (top two in the second column from the left)), on the other hand, the acetylation of Lysine residue (Lys9) of 7-ston H3 is markedly increased (orange) It became clear that 'panels surrounded by color frames (see the top two panels in the rightmost column)). This change was also confirmed by the treatment of PMA, a differentiation inducer, and it was revealed that CO induced differentiation induction in leukemia cells (Fig. 2). Example 2: Detection of protein methylation using anti dimethyl-arginine antibod (ADMA) Method>
細胞内の蛋白質のメチル化を検出するために anti-dimethyl-arginine antibody (ADMA)を用いた Western blot analysisを行った。 細胞としてヒ ト胎 児肝芽細胞腫細胞株である HepG2， 副腎由来細胞 PC12 を用い、 それぞれから 蛋白質を抽出し、 15 i gを Western解析に用いて blottingを行った。 CORM-2 (50 μ mol/L 又は ΙΟΟ μ ιηοΙ/L) あるいはそのコン ト ロール化合物である Ru(DMSO)4Cl2 (50 /i mol/L) を細胞培養液に加えて、 6-24時間培養したときの メチル化蛋白質の増加を検出した。 COを加えた際の内因性の CQ生成系である heme oxygenase- 1 (ΗΟ·1) の消長を確認するため、 同じサンプルについて anti-HO- 1 antibody GTS- 1を用いた Western blottingを行った。 ぐ結果 > Western blot analysis using anti-dimethyl-arginine antibody (ADMA) was performed to detect the methylation of intracellular proteins. We used HepG2, a fetal hepatoblastoma cell line, and PC12 derived from the adrenal gland as cells. Proteins were extracted from each cell and blotted using 15 ig for Western analysis. Add CORM-2 (50 μmol / L or ΙΟΟ μ ιηοΙ / L) or its control compound Ru (DMSO) 4 Cl 2 (50 / i mol / L) to the cell culture medium. An increase in methylated protein was detected when incubated for a long time. Western blotting using anti-HO-1 antibody GTS-1 was performed on the same sample to confirm the fate of heme oxygenase-1 (ΗΟ · 1), an endogenous CQ production system when CO was added . Results>
図 3に示すように、 HepG2及び PC12 を CORM-2 で処理すると、 複数の蛋 白質 （異なる分子量の位置） においてメチル化が亢進していることが明らかであ つたため、 COが細胞内蛋白質のメチル化を活性化することが明確に示された。 さらに、 図 3の最下段に示すように、 外因性の CO を添加すると内因性の HO-1 の誘導がかかることが合わせて明らかになった。 すなわち、 CO の外因性 投与は内因性の COを増加させ、 細胞機能制御を相加的に制御する可能性も示さ れた。 実施例 3 ： CO処理によりメチル化修飾を受けるタンパク質の同定  As shown in Fig. 3, when HepG2 and PC12 were treated with CORM-2, it was clear that methylation was increased in multiple proteins (positions of different molecular weights), so CO was an intracellular protein. It was clearly shown to activate methylation. Furthermore, as shown at the bottom of Fig. 3, it was also revealed that the addition of exogenous CO induces endogenous HO-1. In other words, exogenous administration of CO increased endogenous CO, and the possibility of controlling cell function additively was shown. Example 3: Identification of proteins that undergo methylation modification by CO treatment
く方法〉 Method>
CO 処理によりメチル化修飾を受けるタンパク質の同定は、 以下のように行つ た （図 4 ) 。  Proteins that undergo methylation modification by CO treatment were identified as follows (Fig. 4).
まず、 細胞を、 コントロールである Ru(DMSO)4Cl2 (ΙΟΟ μ Μ) 、 又は CO ド ナーである CORM-2 (100 μ Μ) の存在下で 3 0分培養し、 細胞を回収した。 次 に、 回収した細胞から調製した全細胞溶解液 （lGO g/ ゲル） を 2次元電気泳 動し （pH6-ll) 、 分離されたタンパク質を実施例 2で使用した抗ジメチルアル ギニン （ADMA) 抗体を用いて Western blot analysisした。 First, the cells were cultured for 30 minutes in the presence of Ru (DMSO) 4 Cl 2 (ΙΟΟμΜ) as a control or CORM-2 (100 μΜ) as a CO donor, and the cells were collected. Next, whole cell lysate (lGO g / gel) prepared from the collected cells was electrophoresed two-dimensionally (pH 6-ll), and the separated protein was treated with anti-dimethyl alcohol used in Example 2. Western blot analysis was performed using ginin (ADMA) antibody.
Ru(DMSO)4Cl2で処理したサンプルのスポットと、 CORM-2 で処理したサン プルのスポットを比較し、 メチル化の程度に差のあるスポットを切り出した。 切 り出したスポッ トのタンパク質を ト リプシンによりゲル内消化し、 次に MALDI-TOF/MS により質量分析を行った。 質量分析の結果から、 PMF 法'によ 'りタンパク質を同定した。 く結果〉 The spot of the sample treated with Ru (DMSO) 4 Cl 2 was compared with the spot of the sample treated with CORM-2, and spots with different degrees of methylation were cut out. The excised spot protein was digested in gel with trypsin and then subjected to mass spectrometry using MALDI-TOF / MS. From the results of mass spectrometry, proteins were identified by the PMF method. Result>
Western blot analysisの結果、 CO処理により、 スポット 3， 5， 6， 34, 35及び 36 のメチル化がコントロール処理に比べて亢進していることが確認された （図 5 ) 。 これらのスポッ 卜を切り出し、 MALDI-TOF/MS解析し、 PMF法でタン パク質を同定した結果を図 6に示す。 同定の結果、 図 5で CO処理により発現の増強が確認されたスポッ トの中、 ス ポット 3, 5及び 6は transketoraseであり、 スポット 34, 35及び 36は alpha- 6nol&s6であった  Western blot analysis confirmed that methylation at spots 3, 5, 6, 34, 35 and 36 was enhanced by CO treatment compared to control treatment (Fig. 5). Figure 6 shows the results of excision of these spots, MALDI-TOF / MS analysis, and identification of proteins by the PMF method. As a result of identification, among the spots whose expression was enhanced by CO treatment in Fig. 5, spots 3, 5 and 6 were transketorases, and spots 34, 35 and 36 were alpha-6nol & s6.
また、 同様にヒ ト単球由来 U937細胞を 3 0分間 CORM-2又はコントロール である Ru(DMSO)4Cl2処理したときの細胞から、 細胞質画分を分離し、 2次元 電気泳動を行い、 ADMA抗体を用いて Western blot analysisを行った。 その結 果、 CO 処理により、 コントロール処理に比べてメチル化が亢進しているスポッ 卜 （矢印） と脱メチル化が生じているスポッ トが （白抜き矢印） 確認された （図 7 ) 。 Similarly, cytoplasmic fractions were separated from human monocyte-derived U937 cells treated with CORM-2 or control Ru (DMSO) 4 Cl 2 for 30 minutes, and two-dimensional electrophoresis was performed. Western blot analysis was performed using ADMA antibody. As a result, spots with increased methylation (arrows) and spots with demethylation (open arrows) were confirmed by CO treatment (Fig. 7).
本実施例で検討したスポッ トとその同定結果を囱 8にまとめた。 スポッ ト 3，5 及び 6の transketorase、 並びにスポット 34， 35及び 36の alpha-enolaseを線 で囲んだ。 この結果から、 CO は、 タンパク質のメチル化の調節作用を有することが示さ れた。 また、 CO によりメチル化が亢進するタンパク質として、 transketolase 及び alpha-enolaseが同定された 本発明では、 以上の結果から鑑み、 次のような応用例を実施することもできる と考えられる。 The spots examined in this example and their identification results are summarized in Table 8. Spots 3, 5 and 6 transketorase, and spots 34, 35 and 36 alpha-enolase were enclosed in a line. From these results, it was shown that CO has a regulating action on protein methylation. In addition, as a protein whose methylation is enhanced by CO, transketolase In the present invention in which alpha-enolase is identified, it is considered that the following application examples can be implemented in view of the above results.
(1) 白血病細胞や未分化な細胞の形質転換による分化誘導療法  (1) Differentiation induction therapy by transformation of leukemia cells and undifferentiated cells
例えば、 一酸化炭素により、 癌細胞などの未分化な細胞を正常細胞に分化さ せるような分化誘導療法に対して一酸化炭素を使用することが可能である。  For example, carbon monoxide can be used for differentiation-inducing therapy in which undifferentiated cells such as cancer cells are differentiated into normal cells by carbon monoxide.
(2) 炎症反応や移植における拒絶反応の人為的制御  (2) Artificial control of inflammatory reaction and rejection in transplantation
(3) これまでの解析でまだ明らかになつていないメチル化制御を受ける蛋白質及 び遺伝子の特定が行われることを前提とした、 これら生体高分子が担う生物機能 すべての人為的制御 産業上の利用可能性  (3) All the biological functions of these biopolymers under the assumption that proteins and genes subject to methylation control, which have not yet been clarified by previous analysis, will be identified. Availability
本発明により、 生体高分子のメチル化調節剤及び生体高分子のメチル化調節方 法が提供される。 また、 本発明より、 細胞の分化誘導剤及び細胞の分化誘導方法 が提供される。  The present invention provides a biopolymer methylation regulator and a biopolymer methylation regulation method. The present invention also provides a cell differentiation inducer and a cell differentiation induction method.
本発明により、 一酸化炭素が担う生物機能を制御することが可能となるため、 本発明は、 例えば、 炎症反応や移植における拒絶反応の制御に適用することがで さる。  Since the present invention makes it possible to control the biological functions of carbon monoxide, the present invention can be applied to control of inflammatory reactions and rejection in transplantation, for example.

Claims

請 求 の 範 囲 The scope of the claims
一酸化炭素を含有することを特徴とする、 生体高分子のメチル化調節剤。 生体高分子が、 タンパク質又は DNAである、 請求項 1に記載の調節剤。 生体咼分子が、 transketorase又は alpha-enolaseでめる、 言 S求項 1に § 載 の調節剤。 A biopolymer methylation regulator comprising carbon monoxide. The regulator according to claim 1, wherein the biopolymer is a protein or DNA. The regulator according to § S claim 1, wherein the biomolecule is transketorase or alpha-enolase.
生体高分子に一酸化炭素を接触させることを特徴とする、 生体高分子のメ チル化調節方法。 A method for regulating methylation of a biopolymer, which comprises contacting carbon monoxide with the biopolymer.
一酸化炭素を含有することを特徴とする、 細胞の分化誘導剤。 A cell differentiation inducer comprising carbon monoxide.
細胞に一酸化炭素を接触させ、 細胞中のタンパク質の脱メチル化を亢進す ることを特徴とする、 細胞の分化誘導方法。 A method for inducing cell differentiation, comprising bringing a cell into contact with carbon monoxide to enhance demethylation of a protein in the cell.
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JP2016500821A (en) * 2012-10-25 2016-01-14 アソシエーション プール ラ ルシェルシュ セラピューティク アンティ−カンセルースＡｓｓｏｃｉａｔｉｏｎ Ｐｏｕｒ Ｌａ Ｒｅｃｈｅｒｃｈｅ Ｔｈｅｒａｐｅｕｔｉｑｕｅ Ａｎｔｉ−Ｃａｎｃｅｒｅｕｓｅ Methylglyoxal as a cancer marker

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