JP5704545B2 - 甲状腺癌の検出方法 - Google Patents
甲状腺癌の検出方法 Download PDFInfo
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- JP5704545B2 JP5704545B2 JP2013147286A JP2013147286A JP5704545B2 JP 5704545 B2 JP5704545 B2 JP 5704545B2 JP 2013147286 A JP2013147286 A JP 2013147286A JP 2013147286 A JP2013147286 A JP 2013147286A JP 5704545 B2 JP5704545 B2 JP 5704545B2
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Images
Description
増幅やホモ欠失といった遺伝子のコピー数変化は、癌化の原因となるがん遺伝子やがん抑制遺伝子を同定する上での有用な目印となる。
好ましくは、前記遺伝子はITCH、AHCY、DYNLRB1、MAP1LC3A、PIGU、TP53IPN2、NCOA6、HMG4L、又はASIP1から選択される少なくとも1つ以上である。
好ましくは、増幅の指標は、正常検体と比較して1.32倍以上である。
好ましくは、遺伝子はITCH遺伝子である。
好ましくは、癌は、甲状腺癌である。
好ましくは、遺伝子の変化を、DNAチップ法、サザンブロット法、ノーザンブロット法、リアルタイムRT−PCR法、FISH法、CGH法、または、アレイCGH法、Bsulfite Sequence法、又はCOBRA法を用いて検出する。
好ましくは、蛋白質の量を免疫組織化学的法により検出する。
好ましくは、検体の悪性度を含めた癌化を検出する。
(1)癌の検出方法
本発明による癌の検出方法は、検体において、1q41、3q28、7q31.2、8p12、8q22.2、8q24.21、11q14.1、17q12、20q11、9p21.3、16q13.2、16q23.1の染色体領域に存在する遺伝子の変化の少なくとも1つ以上を検出することを特徴とする。好ましくは、検出される遺伝子は、ITCH遺伝子である。
本発明の染色体領域および本発明の複数の遺伝子の変異を検出する対象となる甲状腺由来の細胞や甲状腺癌は、検体提供者の生検組織細胞が好適である。
本発明の染色体領域および本発明の複数の遺伝子の増幅や欠失の検出を直接的におこなうことができる代表的な方法として、CGH(Comparative Genomic Hybridization)法とFISH(Fluorescence in situ hybridization)法を挙げることができる。この態様の本検出方法は、本発明の染色体領域および本発明の複数の遺伝子を有するBAC(Bacterial Artificial Chromosome)DNA、YAC(Yeast Artificial Chromosome)DNA、PAC(P1−drived Artificial Chromosome)DNA(以下、BAC DNA等ともいう)を標識し、FISHをおこなうと、本発明の染色体領域および本発明の複数の遺伝子の変異を検出することができる。
さらに検体が、甲状腺由来の組織であることが好ましく、甲状腺癌であることがより好ましい。
本発明によれば、 ITCH、AHCY、DYNLRB1、MAP1LC3A、PIGU、TP53IPN2、NCOA6、HMG4L、又はASIP1から選択される少なくとも1つ以上の遺伝子のsiRNA、アンチセンスオリゴヌクレオチドまたは機能欠失型遺伝子を、インビトロで細胞に導入することを含む細胞の増殖を抑制する方法、並びに上記のsiRNA、shRNA、アンチセンスオリゴヌクレオチドまたは機能欠失型遺伝子を含む細胞増殖抑制剤が提供される。
本発明によればさらに、ITCH、AHCY、DYNLRB1、MAP1LC3A、PIGU、TP53IPN2、NCOA6、HMG4L、又はASIP1から選択される少なくとも1つ以上の遺伝子、または、該遺伝子の発現産物である蛋白質をインビトロで細胞に導入することを含む、細胞の増殖を活性化する方法、並びに上記遺伝子又は蛋白質を含む細胞増殖活性化剤が提供される。
本発明の細胞増殖抑制(抗腫瘍剤)の適用対象となる腫瘍を選別するための検出方法は、ITCH遺伝子の全部又はその一部を含むDNA又はRNAを用いて検体試料中のITCH遺伝子を解析する工程を含む。ここで、ITCH遺伝子の一部とは、ITCH遺伝子の塩基配列のうち、例えば約10〜30個の連続する塩基配列からなるオリゴヌクレオチドを意味する。検体試料としては、腫瘍が疑われる組織切片、血液、リンパ液、喀痰、肺洗浄液、尿、便、組織培養上清などを用いることができる。
本発明の細胞増殖抑制剤(抗腫瘍剤)の適用対象となる腫瘍を選別するための検出方法は、ITCHタンパク質に対する抗体又はその断片を用いて検体試料中のITCHタンパク質の量を解析する工程を含む。
本方法に用いるITCHタンパク質に対する抗体(以下、ITCH抗体という)は、ITCHタンパク質の全部又は一部を抗原として、通常の方法で作製することができる。ITCHタンパク質の一部とは、配列番号2に記載するITCHタンパク質のアミノ酸配列のうち、例えば連続する少なくとも6個のアミノ酸、好ましくは少なくとも約8〜10個のアミノ酸、さらに好ましくは、少なくとも約11〜20個のアミノ酸からなるポリペプチドをいう。抗原とするITCHタンパク質の全部又は一部の調製法は生物学的手法、化学合成手法いずれでもよい。
用いた14種のATC細胞株(KTA−1,KTA−2,KTA−3,KTA−4,ARO,FRO,TTA−1,TTA−2,TTA−3,8305C,8505C,HTC/C3,TCO−1,KHM/5M)は臨床サンプルより樹立した細胞株を使用した。これら細胞株を10%胎児牛血清と100U/mlpenicillin/100μg/mlstreputomycin液で培養をおこなった。原発性甲状腺癌の116の臨床検体は伊藤病院より入手し、各患者の同意をもってかつ同組織の倫理委員会の承認を得て使用した。
未分化甲状腺癌での新規な遺伝子変化を検出するために、上述の14種類のATC細胞株から調製したゲノムDNAを用いてMGC Whole Genome Array−4500(Inazawa J., et al.,Cancer Sci.95,559−563,2004))を使用したCGHアレイ解析をおこなった。なお、対象として甲状腺由来の正常な細胞から抽出したゲノムを使用しCy5で標識した。被検DNAとして未分化甲状腺癌細胞株から調整したゲノムDNAを使用しCy3で標識した。
以上の結果より、表1、2に示す染色体領域の変化を検出することで甲状腺のがん化を検出することが可能と考えられる。
20q11.22の増幅領域の絞込みをおこなうため、RP11−318N1を中心する7つのBACおよび、20p11領域のBACをコントロールプローブとして、定法(Inoue J, Otsuki T, Hirasawa A, et al. Am J Pathol.;165:71−81.,2004)により、FISH解析を行った。
さらにこれらのターゲット遺伝子の中で、より好ましいターゲットを明確にするために、遺伝子の増幅と、発現状態の関係を決定することにした。
8305C,KTA−4のITCHの過剰発現を確認するため、特異抗体を用いたウエスタンブロッキング法により、タンパクの発現を確認した。
具体的には、各細胞をprotease−inhibitor cocktail(Roche Diagnostics)を含むRIPAバッファー(10mM Tris−HCl,150mM NaCl,1mM EDTA,1% sodium deoxycholate,0.1% SDS,1% Triton X−100,pH7.4)にて溶解後、BCA assay(Pierce Chemical)にてタンパク濃度を測定し各30μgをSDS−ポリアクリルアミドゲルにて電気泳動した。これを、difluoride膜に転写し、抗ITCH抗体(Santa Cruz Biotechnology)、コントロールとして抗β−アクチン抗体(Sigma)にて一次検出後、パーオキシダーゼ結合二次抗体にてenhanced electrochemiluminescence system(Amersham)を用い発色、検出した(図1E)。
ATCを含む原発性甲状腺癌のITCHの発現状態を調べるため、109の原発性甲状腺癌検体(ATC 49検体、PTC 25検体、PMC 25検体、甲状腺腫(adenomatous goiters) 10検体)について、ITCH蛋白質の発現レベルを免疫組織化学染色により評価することにした。
ATC細胞増殖に対するITCH過剰発現の効果を調べるため、特異的siRNAによるITCHの発現抑制後の細胞成長試験をおこなった。
ITCH遺伝子に対応するsiRNAは、GGUGACAAAGAGCCAACAAGAG(配列番号19)とデザインし購入(シグマ社)した。また、コントロールのsiRNAとしてルシフェラーゼ遺伝子に対応する CGUACGCGGAAUACUUCGA(配列番号20)を購入(シグマ社)した。合成したsiRNA(10nmol/L)は、Lipofectamine siRNA MAX試薬(インビトロジェン社)を用いる(製品プロトコールで処理)ことで、それぞれのATC細胞株に遺伝子導入された。遺伝子導入後、その効率を実施例3と同様Western blotting法にて解析した。生存細胞の数はwater−soluble tetrazolium salt(WST)アッセイ(Cell counting kit−8;同仁堂)にて測定した。コントロールとして抗β−アクチン抗体を用いた。予想どおりITCHが増幅/過剰発現している8305CおよびKTA−4細胞について、非特異的なsiRNAのコントロールに比べ、24−72h時間後にITCHに特異的なsiRNAにより内在性ITCHタンパクの抑制されていることをウエスタンブロット法により確認した(図3A、B)。
ATC細胞の成長に対するITCHの作用機序を明らかにするため、ITCH特異的siRNAを導入した8305C細胞とコントロール細胞の細胞周期の解析をFACSを用いて行った。
これまでの結果をふまえ、ITCH遺伝子発現を活性化することで、ATC細胞の増殖が促進されるかどうかどうかを検討した。まず、ITCH遺伝子のMycタグを発現する2つのプラスミド(ワイルドタイプ:pCMV−Tag3B−ITCH WT、ユビキチン連結酵素活性を無くしたミュータントタイプ:pCMV−Tag3B−ITCH MUT)を構築した。本プラスミドは、RT−PCRにより増幅したITCHのWTおよびMUTの cDNAをpCMV−3Tag4ベクター(Stratagene社)にMycタグと翻訳フレームがあうように挿入して作製した。対照として、ITCH遺伝子を挿入しない空ベクター(pCMV−Tag3B−mock)を使用した。これらの発現プラスミドを、トランスフェクション試薬であるLipofectamine 2000(Invitrogen)と混合し、TTA−1細胞または8505C細胞へトランスフェクションした。48時間後細胞を回収し、抗Myc抗体(Cell Signaling Technology社)を用いたウエスタンブロットによりITCH蛋白質の発現を確認した(図3D,E)。
(1) アレイCGH法によるスクリーニングから、1q41、3q28,7q31.2、8p12、8q22.2、8q24.21、11q14.1、11q22.2、17q12、20q11、9p21.3、16q13.2、16q23.1の遺伝子領域が、甲状腺癌の新しい癌マーカーとなることを見出した。
(2) その中で、22q11領域、およびそこに含まれる9の遺伝子(ITCH、AHCY、DYNLRB1、MAP1LC3A、PIGU、TP53IPN2、NCOA6、HMG4L、ASIP1)が、より好ましい癌マーカーとなることを見出した。
(3) 14種の未分化甲状腺癌由来細胞におけるDNAの増幅遺伝子のスクリーニングと発現解析データを組み合わせた確認によりITCH遺伝子を特に好ましい、新たな癌マーカーとして同定した。
(4) ITCH遺伝子の発現は甲状腺癌の細胞増殖を促進していることが明らかとなった。
Claims (13)
- 甲状腺由来の組織検体において、ITCH遺伝子の増幅又は発現増大を検出し、増幅又は発現増大が存在する場合に癌化が認められると判断することを含む、甲状腺癌の検出方法。
- 検体の悪性度を含めた癌化を検出する、請求項1に記載の甲状腺癌の検出方法。
- 甲状腺癌が、未分化甲状腺癌である、請求項1又は2に記載の甲状腺癌の検出方法。
- 遺伝子の増幅又は発現増大を、DNAチップ法、サザンブロット法、FISH法、CGH法、または、アレイCGH法、Bsulfite Sequence法、又はCOBRA法を用いて検出する、請求項1から3の何れか1項に記載の甲状腺癌の検出方法。
- 増幅又は発現増大の指標が、正常検体と比較して1.32倍以上である、請求項1から4の何れか1項に記載の甲状腺癌の検出方法。
- 甲状腺由来の組織検体において、ITCH遺伝子から翻訳される蛋白質の量を検出し、蛋白質の量が増大している場合に癌化が認められると判断することを含む、甲状腺癌の検出方法。
- 検体の悪性度を含めた癌化を検出する、請求項6に記載の甲状腺癌の検出方法。
- 甲状腺癌が、未分化甲状腺癌である、請求項6又は7に記載の甲状腺癌の検出方法。
- 蛋白質の量を免疫組織化学的法により検出する、請求項6から8の何れか1項に記載の甲状腺癌の検出方法。
- ITCH遺伝子のsiRNAまたはアンチセンスオリゴヌクレオチドを、インビトロでITCH遺伝子が過剰発現している細胞に導入することを含む、前記細胞の増殖を抑制する方法。
- ITCH遺伝子のsiRNAまたはアンチセンスオリゴヌクレオチドを含む、ITCH遺伝子が過剰発現している細胞の増殖抑制剤。
- ITCH遺伝子を、インビトロで細胞に導入することを含む、細胞の増殖を活性化する方法。
- ITCH遺伝子を含む、細胞増殖活性化剤。
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