JP2010220577A - 食道癌の検出方法及び抑制剤 - Google Patents
食道癌の検出方法及び抑制剤 Download PDFInfo
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Abstract
【解決手段】検体において、1q32−1q41の染色体領域に存在する遺伝子の増幅を少なくとも1つ以上を検出することにより癌化を検出することを含む、癌の検出方法。
【選択図】なし
Description
(1) 検体において、1q32−1q41の染色体領域に存在する遺伝子の増幅を少なくとも1つ以上を検出することにより癌化を検出することを含む、癌の検出方法。
(2) 前記遺伝子がDTL、C1orf75、ATF3、SNFT、NSL1、FLVCR、ANGEL2、SMYD2、PTPN14、又はCENPFから選択される少なくとも1つ以上である、(1)に記載の癌の検出方法。
(3) 増幅の指標が、正常検体と比較して1.32倍以上である、(1)又は(2)に記載の癌の検出方法。
(5) 検体が食道由来の組織である、(1)から(4)の何れかに記載の癌の検出方法。
(6) 癌が食道癌である、(1)から(5)の何れかに記載の癌の検出方法。
(7) 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、(1)〜(6)の何れかに記載の癌の検出方法。
(9) 蛋白質の量を免疫組織化学的法により検出する、(8)に記載の癌の検出方法。
(10) 検体の悪性度を含めた癌化を検出する、(1)から(9)の何れかに記載の癌の検出方法。
(11) SMYD2およびp53の発現を指標に癌化を検出する、(1)から(10)の何れかに記載の癌の検出方法。
(12) SMYD2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含む、細胞増殖抑制剤。
(13) SMYD2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。
(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、1q32−1q41の染色体領域(以下、本発明の染色体領域とも称する)に存在する遺伝子の増幅を少なくとも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、DTL、C1orf75、ATF3、SNFT、NSL1、FLVCR、ANGEL2、SMYD2、PTPN14、又はCENPFから選択される少なくとも1つ以上の遺伝子(以下、本発明の遺伝子とも称する)であり、更に好ましくはSMYD2遺伝子である。また、本発明においては、検体において、DTL、C1orf75、ATF3、SNFT、NSL1、FLVCR、ANGEL2、SMYD2、PTPN14、又はCENPFから選択される少なくとも1つ以上の遺伝子から翻訳される蛋白質の量を検出することによって、癌を検出することもできる。
本発明の染色体領域および本発明の遺伝子の増幅を検出する対象となる食道由来の細胞や食道癌は、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遺伝子の増幅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遺伝子の増幅を検出することができる。具体的には、SMYD2遺伝子を有するBAC DNAとしては、RP11−74E6等を上げることができる。
本発明によれば、DTL、C1orf75、ATF3、SNFT、NSL1、FLVCR、ANGEL2、SMYD2、PTPN14、又はCENPF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、SMYD2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SMYD2遺伝子を解析する工程を含む。ここで、SMYD2遺伝子の一部とは、配列番号1に記載するSMYD2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、SMYD2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SMYD2タンパク質の量を解析する工程を含む。
用いた43種のESCC細胞株(表1)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。
食道癌での新規な遺伝子変化を検出するために、上述の43種類のESCC細胞株から調製したゲノムDNAを用いてCGH data base Japan(http://www.cghtmd.jp/CGHDatabase/)で公開しているESCC細胞株の既知の増幅領域のうち1q32−1q41に位置する約12MBの増幅領域を高密度オリゴアレイ(アジレント244K high−density oligo−array )CGH解析とFISH法でマッピングし、領域内の42候補遺伝子を同定した(図1A)。具体的には、オリゴアレイで評価した微細なコピー数変化のうち、コピー数の最も高い領域では、FISH法でも丸数字1から丸数字4の領域でHomogeneously Staining region、HSRパターンとして検出され、BACの丸数字6 、丸数字8の領域ではコピー数の減少と相関したシグナルパターンの変化を認めた(図1B)。また、この一段高い領域内には22種の遺伝子が含まれる事がわかった(図1A)。
2μg分のDNAをNuclease free waterで希釈し合計20.2μlになるようにサンプルを調節する。次に、1反応あたりNuclease-free water 2.0μ、10xReaction Buffer C 2.6μl、Acetylated BSA(10μg/μl) 0.2μl、AluI(10U/μl)0.5μl、RsaI(10U/μl)0.5μlを加え合計26μlになるようにする。37℃のウオーターバスまたはヒートブロックで2時間インキュベートする。反応終了後、65℃のヒートブロックで20分インキュベートし酵素を失活させる。失活後氷上におく。
アジレントGenomic DNA Labelingキットを利用して、制限酵素消化ゲノムDNAのラベル化を行う。具体的には、ランダムプライマーとExo-Klenowを用いた反応で、Cyanine3-dUTPあるいはCyanine5-dUTPを取り込ませ、ゲノムDNAのラベル化を行う。
Microcorn YM-30 filter units(ミリポア製、製品番号42410)を用いてラベル化DNAの精製及び濃縮を行う。NanoDrop ND-1000(UV-Vis測定器)を使ってラベル化DNAの測定を行う。Cy3-dUTP、Cy5-dUTPの取り込み効率の計算を行う。
ラベル化サンプル(Cy3及びCy5ラベル化DNAの混合)液153μl、Human Cot-1 DNA(1.0mg/ml)50μl、10xBlocking Agent52μl、2xHybridizaton Buffer 260μlを加え、95度で3分間インキュベート、37度30分間のインキュベートを行う。ハイブリダーゼーションチャンバーにアレイスライドをセットし、溶液をアプライする。65度のオーブンのローターで40時間ハイブリダイゼーションさせる。
Agilent Oligo aCGH洗浄バッファー1とAgilent Oligo aCGH洗浄バッファー2で洗浄し、Agilentスキャナーを用いてスキャンニングする。スキャンニング画像をAgilent Feature Extractionソフトウエアを用いて数値化し、CGH analysisソフトウエアを用いて染色体上にコピー数の変化を可視化して表現する。
前述のESCC43株を用いて、実施例1で選択した22種類の遺伝子のmRNAの発現定量解析を行った。定量RT−PCRの発現量のコントロールとして正常食道上皮を用いた。対数増殖期の各細胞よりtotal RNAを採取後、定法にてcDNAを作製した。ABI社のTaqMan Gene Expression Assays (Applied Biosystems)のプロトコールを用いて、各遺伝子に特異的なプライマーでcDNAからquantitative real-time fluorescence detection method (ABI PRISM 7500 sequence detection System; Applied Biosystems, Foster City, CA, USA)でmRNAの発現レベルの測定を行った。
実施例2の10種の遺伝子の各種siRNAを用いてノックダウンを行いcell growth assayを行った。具体的には、増幅株を用いて、Santa Cruzs(Santa Cruz Biotechnology, Inc.)社 、Dharmacon (Lafayette, CO, USA)社またはSigma (Tokyo, Japan)社のsiRNAを用いて解析した。Lipofectamine 2000 (Invitrogen, St. Louis, MO, USA) を用いてsiRNA(Santa Cruzs 10nmol/L, Dharmacon 20nmol/L, Sigma 50nmol/L)添付プロトコールを参考にトランスフェクションした。増殖抑制程度の評価は、WST assay (colorimetric water-soluble tetrazolium salt assay)を用いて行った。
SMYD2の遺伝子の増幅を確認するため、BACのRP11−74E6 (1q41, SMYD2; green)を 、RP11−82D16 (1p36.3, control; red)をコントロールとして、FISH法で解析した。また、解析方法としては、定法(Inoue J, Otsuki T, Hirasawa A, et al. Am J Pathol.;165:71−81.,2004)により行った。
SMYD2の遺伝子のmRNAレベルでの発現亢進を確認するため、ESCC43株での定量的発現解析(real-time RT-PCR)を行った。ABI社のTaqMan Gene Expression Assays (Applied Biosystems)のプロトコールを用いて、SMYD2の遺伝子に特異的なプライマーでcDNAからquantitative real-time fluorescence detection method (ABI PRISM 7500 sequence detection System; Applied Biosystems, Foster City, CA, USA)でmRNAの発現レベルの測定を行った。
ESCC細胞株でのSMYD2遺伝子の過剰発現を確認するため、特異抗体を用いたウェスタン・ブロット法によりタンパクの発現を解析した。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20μg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膜に転写した。特異抗体は、ヒトSMYD2の12個のアミノ酸からなるペプチドを用いて、抗SMYD2ポリクローナル抗体(HPYISEIKQEIESH; Operon Biotechnology, Tokyo, Japan)を作製し、アフィニティ精製後の抗体を用いた。SMYD2の抗体検定はKYSE150株をポジティブコントロールとし、HLE、KYSE510をネガティブコントロールとした。また、KYSE200、KYSE510にpCMV-3tag1A−SMYD2を過剰発現させてFLAGtag抗体或いはSMYD2抗体で検出することでも確認を行った(図13)。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。
ESCC細胞増殖に対するSMYD2発現の過剰効果を調べるため、WST assay (colorimetric water-soluble tetrazolium salt assay)による解析を行った。具体的な実験方法を以下に示す。
ESCC細胞の細胞周期に対するSMYD2の作用機序を明らかにするため、SMYD2高発現のKYSE150、KYSE790、SMYD2低発現株のKYDE220、KYSE200を用いて、SMYD2特異的siRNAを導入した細胞とコントロール細胞の細胞周期をFACS解析で比較した(図7)。
実施例7と同様の方法にて、食道扁平上皮癌細胞株でSMYD2をノックダウンしp53の標的分子が活性化されるかどうかを確認した。
その結果、mRNAレベルで細胞周期停止の指標となるp21が活性化されることがわかった。ウェスタン・ブロット法でも同様に確認された。FACSの結果から、p21が誘導されることでG1/Sアレストを生じていることが明らかとなった(図7)。また、SMYD2高発現株でかつp53mutation(+)のKYSE150や、SaOS2(p53null)でもSMYD2ノックダウンによりp21の発現誘導が認められることから、p53を介さずにp21が誘導され細胞周期を起こすことが明らかとなった。SMYD2は一部でp53とindependentにp21を抑制し、細胞サイクルを活性化して癌細胞増殖に関与していることが明らかとなった(図7、図9及び図14)。
コロニーフォーメーションアッセイにより、SMYD2遺伝子過剰発現系における細胞増殖能を調べた。具体的には、SMYD2低発現株であるKYSE200株、KYSE510株にpCMV-3tag1A-empty vector, pCMV-3tag1A-SMYD2, pCMV-3tag1A-SMYD2 MD(methylation defective mutant of SMYD2)をリポフェクトアミン2000を用いてトランスフェクションし、24時間後に細胞を回収し各プラスミドベクターからの蛋白の発現をWestern blotで確認した。同時に、1x104個/mlでシャーレに細胞をまき、24時間後にG418(Neomycin)でセレクションを開始してコロニー形成能を評価した。
ESCCでのSMYD2の発現状態を調べるため、153の原発性食道癌検体の免疫組織化学染色を行った(図10の上段)。また、治療後の経過日数と生存率の関係を生存曲線(図10の下段)で示した。
上述の153の原発性食道癌検体と臨床病理学的因子との比較検討では、SMYD2蛋白高発現症例では有意に静脈浸潤が陽性で、腫瘍深達度が深く、再発頻度が高かった(表3)。Cox比例ハザードモデルによる多変量解析ではSMYD2が独立した予後因子であることが明らかとなった(表4)。
p53の発現パターンと予後を解析した結果を図11の上段に示す。また、免疫組織化学染色法によるp53タンパクの発現量を解析した結果を図11の下段に示す。
p53タンパクの免疫組織化学染色で陽性または陰性の症例で予後に差を認めなかった(図11)。また、p53の発現とSMYD2発現には相関関係を認めなかった。
免疫組織学的染色によるSMYD2とp53のタンパク発現の有無と、予後の関連の相関解析を行った。
実施例1から14の結果をまとめると以下の通りである。
(1)アレイCGH法によるスクリーニングから、1q32−1q41の遺伝子領域が、食道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、1q32−1q41の染色体領域に含まれるSMYD2遺伝子が、より好ましい癌マーカーとなることを見出した。
(3)SMYD2遺伝子の発現は食道癌の細胞増殖を促進していることが明らかとなった。
Claims (12)
- 検体において、1q32−1q41の染色体領域に存在する遺伝子の増幅を少なくとも1つ以上を検出することにより癌化を検出することを含む、癌の検出方法。
- 前記遺伝子がDTL、C1orf75、ATF3、SNFT、NSL1、FLVCR、ANGEL2、SMYD2、PTPN14、又はCENPFから選択される少なくとも1つ以上である、請求項1に記載の癌の検出方法。
- 増幅の指標が、正常検体と比較して1.32倍以上である、請求項1又は2に記載の癌の検出方法。
- 遺伝子が、SMYD2である、請求項3に記載の癌の検出方法。
- 検体が食道由来の組織である、請求項1から4の何れかに記載の癌の検出方法。
- 癌が食道癌である、請求項1から5の何れかに記載の癌の検出方法。
- 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、請求項1から6の何れかに記載の癌の検出方法。
- 検体において、DTL、C1orf75、ATF3、SNFT、NSL1、FLVCR、ANGEL2、SMYD2、PTPN14、又はCENPFから選択される少なくとも1つ以上の遺伝子から翻訳される蛋白質の量を検出することを含む、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8に記載の癌の検出方法。
- 検体の悪性度を含めた癌化を検出する、請求項1から9の何れかに記載の癌の検出方法。
- SMYD2およびp53の発現を指標に癌化を検出する、請求項1から10の何れかに記載の癌の検出方法。
- SMYD2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含む、細胞増殖抑制剤。
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WO2013080400A1 (en) * | 2011-12-02 | 2013-06-06 | Oncotherapy Science, Inc. | Smyd2 as a target gene for cancer therapy and diagnosis |
JP2015506665A (ja) * | 2011-12-02 | 2015-03-05 | オンコセラピー・サイエンス株式会社 | 癌の治療および診断の標的遺伝子としてのsmyd2 |
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US9090942B2 (en) | 2015-07-28 |
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