2000 Fiscal Year Annual Research Report

モノクローナル抗体を用いた遺伝子損傷の高感度検出系の開発とその応用

Research Project

Project/Area Number	11470498
Research Institution	Kanazawa University
Principal Investigator	二階堂修金沢大学, 薬学部, 教授 (60019669)
Co-Investigator(Kenkyū-buntansha)	松永司金沢大学, 薬学部, 助教授 (60192340)
Keywords	モノクローナル抗体 / シクロブタン型ビリミジン二量体 / (6-4)光産物 / ドットブロット解析 / キャピラリー電気泳動 / 酵素標識免疫測定法 / デワー型光産物 / DNA損傷
Research Abstract	紫外線(UVBとUVC)によって誘発される二量体型DNA損傷には、シクロブタン型ピリミジン二量体(CPD)、(6-4)光産物(64P)とデワー型光産物(DwP)が含まれる。これら損傷は細胞死、突然変異とがん誘発に関わることから、その検出は太陽光紫外線中のUVBの影響を検討する上で必要である。本研究では低線量紫外線によって誘発される遺伝子損傷検出を目的として、下記の検出系の樹立を目指した。全ての検出系は我々が樹立したDNA損傷を特異的に認識するモノクローナル抗体を利用した系である。 1.キャピラリー電気泳動法を利用した損傷の高感度検出系の開発抗CPDモノクローナル抗体(TDM-2)を一次抗体とし、二次抗体にはAlexa Fluor標識goat anti-mouse IgG(H+L)を用いた。DNA損傷は損傷と一次・蛍光標識二次抗体の複合体をキャピラリー電気泳動後、488nmレーザー励起蛍光検出器(BioFocus2000:Bio Rad)で検出した。DNAと一次抗体の非特異的結合を抑制するため反応バッファー中の塩濃度を高く設定した。ヒトA549細胞由来のゲノムDNAを分離し低線量紫外線(UVC、<10J/m^2)を照射した後損傷を検出した。その結果、0〜1J/m^2のレベルで紫外線線量に依存して検出複合体量が直線的に増加した。また、検出された複合体のピークが大腸菌由来光回復酵素処理によって低下したことから、損傷に結合した一次・二次抗体複合体であることが確認された。今後は本検出系を用いてO〜1J/m^2のレベルでの損傷の細胞内修復動態の解析を行う。 2.ECL(Enhanced Chemiluminescence)法を用いた増感酵素標識免疫測定法(ELISA)の樹立損傷を高感度に検出することを目的として従来のELISA法を改良した。マイクロプレートにLumiNunc^<TM>を、化学発光基質としてSuperSignal^*,ELISA Femto Maximum Sensitivity Substrate(Pierce,IL)を用いた。その結果、0〜1J/m^2のUVC線量域で損傷の直線的検出が可能となった。従来の方法に比べ10〜20倍程度の検出感度の上昇が見られた。今後は64PとDwPの定量的検出にも本系を適用する予定である。 3.ドットブロット法を利用した損傷検出系の開発 plasmid DNAに各種の紫外線を照射し、種々の損傷修復タンパクで消化後、超螺旋型から開環型への変換を測定して検量線を得た。一方でECLを利用したドットブロット法を結合抗体の検出に利用して各種抗体が結合する損傷数を決定した。その結果、UVAランプの照射でもCPDがDNA中に誘発されること、Dwpは人工太陽光紫外線照射で多く誘発され、CHO細胞ではCPDと同様に遅く修復されることが分かった。従って太陽光紫外線に暴露時の主たる損傷はCPDとDwPとなる可能性があり、それらは共に細胞内に長期残存することから変異源性が高いことが示唆された。

Research Products

(20 results)

All Other

All Publications (20 results)

[Publications] Kobayashi,H.: "Probing the interaction between a high-affinity single-chain Fv and a pyrimidine (6-4)pyrimidone photodimer by site-directed mutagenesis."Biochemistry,. 38(2). 532-539 (1999)
[Publications] Harada,YN.: "Postnatal growth, short life span, and early onset of cellular senescence and subsequent immortalization in mice lacking the xeroderma pigmentosum group G gene."Mol.Cell.Biol.,. 19(3). 2366-2372 (1999)
[Publications] Riou,L.: "The relative expression of mufated XPB genes results in xeroderma pigmentosum/Cockayne's syndrome or trichothiodystrophy cellular phenotypes."Hum.Mol.Genet.,. 8(6). 1125-1133 (1999)
[Publications] Torizawa,T.: "Conformational multiplicity of the antibody combining site of a monoclonal antibody specific for (6-4)photoproduct."J.Mol.Biol.,. 290(3). 731-740 (1999)
[Publications] Ishigaki,Y.: "Development and characterization of a DNA solar dosimeter."J.Photochem.Photobiol.B,. 50(2-3). 184-188 (1999)
[Publications] Negishi,T.: "Somatic cell mutation induced by sunlight in Drosophila."J.Epidemiol.,. 9(6 Suppl). S66-S71 (1999)
[Publications] Ahmed,NU.: "High levels of 8-hydorxyguanosine appear in normal human epidermis after a single dose of ultraviolet radiation."Br.J.Dermatol.,. 140(2). 226-231 (1999)
[Publications] Yokoyama,H.: "Crystal structure of the 64M-2 and 64M-3 antibody Fabs complexed with DNA (6-4) photoproducts."Nucleic Acids Symp.Ser.,. 42. 267-268 (1999)
[Publications] Otoshi,E,: "Respective role of cyclobutane pyrimidine dimers,(6-4)photoproducts, andminor photoproducts in ultraviolet mutagenesis of repair deficient xeroderma pigmentosum A cells."Cancer Res C.,. 60(6). 1729-1735 (2000)
[Publications] Ide,F.: "In vivo detection of ultraviolet photoproducts and their repair in Purkinje cells."Lab.Invest.. 80(4). 465-470 (2000)
[Publications] Horiki,S.: "Protective effects of sunscreening agents on photocarcinogenesis, photoageimg, and DNA damage in XPA gene knockout mice."Arch.Delmatol.Res.. 292(10). 511-518 (2000)
[Publications] Torizawa,T.: "DNA binding mode of the Fab fragment of a monoclonal antibody specific for cyclobutane pyrimidine dimer."Nuclejc Acids Res.,. 28(4). 944-951 (2000)
[Publications] Perdiz,D.,: "Distribution and repair of bipyrimidine photoproducts in solar UV-irradiated mammalian cells, Possible role of Dewar photoproducts in solar mutagenesis."J.Biol.Chem.. 275(35). 26732-26742 (2000)
[Publications] Yokoyama,H.: "Crystal structure of the 64M-2 antibody Fab fragment in complex with a DNA dt(6-4)T photoproduct formed by ultraviolet radiation."J.Mol.Biol.,. 299(3). 711-723 (2000)
[Publications] Cario-Andre,M.: "Studies on epidermis reconstructed with and without melanocytes : melanocytes prevent sunburn cell formation but not appearance of DNA damaged cells in fair-skinned caucasians."J.Invest.Dermatol.. 115(2). 193-199 (2000)
[Publications] Ray,AJ.: "The spectrum of mitochondrial DNA deletions in a ubiquitous marker of ultraviolet radiation exposurein human skin."J.Invest.Dermatol.. 115(4). 674-679 (2000)
[Publications] Budiyanto,A.: "Protective effect of topically applied olive oil against photocarcinogenesis following UVB exposure of mice."Carcinogensis,. 21(11). 2085-2090 (2000)
[Publications] Horiki,S.: "Protective effects of sunscreening agents on photocarcinogenesis, photoaging, and DNA damage in XPA gene knockout mice."Arch.Dermatol.Res.,. 292(10). 511-518 (2000)
[Publications] Tanaka,M.: "Effects of photoreactivation of cyclobutane phrimidine dimers and pyrimidine (6-4) pyrimidine photoproducts on ultraviolet mutagenesis in SOS-in duced repair-deficient Escherichia coli."Mutagenesis,. 16(1). 1-6 (2001)
[Publications] Wakasugi,M.: "DDB stimulates the excision of cyclobutane pyrimidine dimers in vitro in concert with XPA and RPA."J.Biol.Chem.,. (In press). (2001)

2000 Fiscal Year Annual Research Report

モノクローナル抗体を用いた遺伝子損傷の高感度検出系の開発とその応用

Principal Investigator

二階堂 修 金沢大学, 薬学部, 教授 (60019669)

Research Products

[Publications] Kobayashi,H.: "Probing the interaction between a high-affinity single-chain Fv and a pyrimidine (6-4)pyrimidone photodimer by site-directed mutagenesis."Biochemistry,. 38(2). 532-539 (1999)

[Publications] Harada,YN.: "Postnatal growth, short life span, and early onset of cellular senescence and subsequent immortalization in mice lacking the xeroderma pigmentosum group G gene."Mol.Cell.Biol.,. 19(3). 2366-2372 (1999)

[Publications] Riou,L.: "The relative expression of mufated XPB genes results in xeroderma pigmentosum/Cockayne's syndrome or trichothiodystrophy cellular phenotypes."Hum.Mol.Genet.,. 8(6). 1125-1133 (1999)

[Publications] Torizawa,T.: "Conformational multiplicity of the antibody combining site of a monoclonal antibody specific for (6-4)photoproduct."J.Mol.Biol.,. 290(3). 731-740 (1999)

[Publications] Ishigaki,Y.: "Development and characterization of a DNA solar dosimeter."J.Photochem.Photobiol.B,. 50(2-3). 184-188 (1999)

[Publications] Negishi,T.: "Somatic cell mutation induced by sunlight in Drosophila."J.Epidemiol.,. 9(6 Suppl). S66-S71 (1999)

[Publications] Ahmed,NU.: "High levels of 8-hydorxyguanosine appear in normal human epidermis after a single dose of ultraviolet radiation."Br.J.Dermatol.,. 140(2). 226-231 (1999)

[Publications] Yokoyama,H.: "Crystal structure of the 64M-2 and 64M-3 antibody Fabs complexed with DNA (6-4) photoproducts."Nucleic Acids Symp.Ser.,. 42. 267-268 (1999)

[Publications] Otoshi,E,: "Respective role of cyclobutane pyrimidine dimers,(6-4)photoproducts, andminor photoproducts in ultraviolet mutagenesis of repair deficient xeroderma pigmentosum A cells."Cancer Res C.,. 60(6). 1729-1735 (2000)

[Publications] Ide,F.: "In vivo detection of ultraviolet photoproducts and their repair in Purkinje cells."Lab.Invest.. 80(4). 465-470 (2000)

[Publications] Horiki,S.: "Protective effects of sunscreening agents on photocarcinogenesis, photoageimg, and DNA damage in XPA gene knockout mice."Arch.Delmatol.Res.. 292(10). 511-518 (2000)

[Publications] Torizawa,T.: "DNA binding mode of the Fab fragment of a monoclonal antibody specific for cyclobutane pyrimidine dimer."Nuclejc Acids Res.,. 28(4). 944-951 (2000)

[Publications] Perdiz,D.,: "Distribution and repair of bipyrimidine photoproducts in solar UV-irradiated mammalian cells, Possible role of Dewar photoproducts in solar mutagenesis."J.Biol.Chem.. 275(35). 26732-26742 (2000)

[Publications] Yokoyama,H.: "Crystal structure of the 64M-2 antibody Fab fragment in complex with a DNA dt(6-4)T photoproduct formed by ultraviolet radiation."J.Mol.Biol.,. 299(3). 711-723 (2000)

[Publications] Cario-Andre,M.: "Studies on epidermis reconstructed with and without melanocytes : melanocytes prevent sunburn cell formation but not appearance of DNA damaged cells in fair-skinned caucasians."J.Invest.Dermatol.. 115(2). 193-199 (2000)

[Publications] Ray,AJ.: "The spectrum of mitochondrial DNA deletions in a ubiquitous marker of ultraviolet radiation exposurein human skin."J.Invest.Dermatol.. 115(4). 674-679 (2000)

[Publications] Budiyanto,A.: "Protective effect of topically applied olive oil against photocarcinogenesis following UVB exposure of mice."Carcinogensis,. 21(11). 2085-2090 (2000)

[Publications] Horiki,S.: "Protective effects of sunscreening agents on photocarcinogenesis, photoaging, and DNA damage in XPA gene knockout mice."Arch.Dermatol.Res.,. 292(10). 511-518 (2000)

[Publications] Tanaka,M.: "Effects of photoreactivation of cyclobutane phrimidine dimers and pyrimidine (6-4) pyrimidine photoproducts on ultraviolet mutagenesis in SOS-in duced repair-deficient Escherichia coli."Mutagenesis,. 16(1). 1-6 (2001)

[Publications] Wakasugi,M.: "DDB stimulates the excision of cyclobutane pyrimidine dimers in vitro in concert with XPA and RPA."J.Biol.Chem.,. (In press). (2001)

二階堂修金沢大学, 薬学部, 教授 (60019669)