JP2004523232A - Means and methods of treatment evaluation - Google Patents

Abstract

The present invention provides a method of determining whether a treatment is effective in altering the state of a group of target cells, such as cancer cells, in an individual. The method includes obtaining a sample from the patient after initiating treatment and determining whether the sample contains an expression product of at least one marker gene. Preferably, the sample is a blood sample. In one aspect, the expression product is expressed by a peripheral blood single cell. The marker gene can be a gene involved in the generation, maintenance and / or damage of blood vessels (angiogenesis). The method of the present invention is well suited to determine within a few days whether a particular treatment for Kaposi's sarcoma is successful. Further, the method is suitable for measuring angiogenesis and / or the presence of cancer cells in a patient.

Description

【００４０】
実施例４
ＫＳ１とＫＳ２ライブラリに比較してカポジ肉腫ＳＡＧＥライブラリＫＳ３とＫＳ４で発現が増加している未知因子を有する遺伝子のＥＳＴ’ｓ由来の同定不可能なタグ（ＴＡＧ’ｓ）を示す分析結果。
【００４１】
タグ（ＴＡＧ）数は、そのライブラリと公開された他のライブラリ間で比較できるようにまず、ライブラリ当たりの、配列決定された１００．０００総ＴＡＧレベルに標準化された。
配列ｃａｔｇは表２の縦列２に示された各タグ（ＴＡＧ）配列に優先して起こる。
【００４２】
【表２】

【００４３】
実施例５
カポジ肉腫の皮膚組織が実施例１に記載されたのと同じ２人のエイズ患者から採取され、その患者からＫＳ３とＫＳ４ライブラリが作成された。両患者とも同性愛の患者でヨーロッパでＨＩＶ−１が流行した初期に感染した。インドネシアで出生した患者１は、ＨＩＶ−１陽性であると１９８２年に証明された。１９８５年２月に組織学的な検査によってカポジ肉腫の診断が確認された。彼は１３ヵ月後に死亡し、検死によって内蔵ＫＳの形態学的変種であることが判明した。患者２は、１９８４年２月アカデミックメディカルセンター（Academic Medical Centre）で進行性ＫＳ皮膚病変を示した。追跡治療中に、ＫＳは腸管、咽頭部、肺、舌、梨状陥凹およびリンパ節へ進行した。１９８６年３月、この患者は死亡し、検死が行われた。上記２名の患者から採取した組織試料は、ＫＳ３とＫＳ４と命名された。
【００４４】
正常成人の胸部皮膚組織が、乳房形成での廃棄組織として採取された（我々の病院の形成手術部から得られた）。３つの胸部整復手術からの単離ＲＮＡは、正常皮膚発現プロファイルのライブラリに使用された。
【００４５】
生検試料の発現プロファイルは、実施例２に記載したようなＳＡＧＥ技術で決定された。我々は４つの生検試料から約４７，０００タグ（ＴＡＧ’ｓ）、ＫＳ３ライブラリから４９，３３５タグ（ＴＡＧ’ｓ）そしてＫＳ４ライブラリから４８，８１４タグ（ＴＡＧ’ｓ）の配列を決定した。タグ（ＴＡＧ）リスト（すなわち、個々のタグ（ＴＡＧ’ｓ）プラス出現回数）はＵＳＡＧＥで利用可能なａｍｃｔ２ｇデータベースで同定された（これはＣＧＡＰ（ＧｅｎＢａｎｋで利用可能）からのＳＡＧＥｍａｐデータベースと比較して進歩したタグ（ＴＡＧ）同定法である）。次に、タグ（ＴＡＧ）リストはＨＴＭプログラムで染色体部位にマッピングされ、同時に特定のタグ（ＴＡＧ）リスト（たとえば血管内皮、公に利用可能）と比較され、ＳＡＧＥｍａｐデータベース（ＨＴＭで「全て」を指定）の全てのタグ（ＴＡＧ）リストと組み合わせて、ＫＳ３とＫＳ４で特に昂進した遺伝子に属しているタグ（ＴＡＧ’ｓ）が同定された（表１）。ヌクレオチド１５はＵＳＡＧＥの原本ジタグ（diTAG）リストから決定された。１５ヌクレオチド（１５nt.）のＴＡＧ配列は、その同定を確認するためにＧｅｎＢａｎｋ（ＢＬＡＳＴ）によって照合された。１５番目のヌクレオチドでの不明確さのゆえにあるいは間違った同定のゆえに、少数のタグ（ＴＡＧ）は排除された。
【００４６】
実施例６
ＫＳ１とＫＳ２ライブラリに比較してカポジ肉腫ＳＡＧＥライブラリＫＳ３とＫＳ４で発現が増加している既知遺伝子由来の同定可能なタグ（ＴＡＧ’ｓ）を示している分析結果。タグ（ＴＡＧ）数は、そのライブラリと公開された他のライブラリ間で比較できるようにまず、ライブラリ当たりの配列決定された１００．０００総タグ（ＴＡＧ）レベルに標準化された。
配列ｃａｔｇは、表３の縦列２に示された各ＴＡＧ配列に優先して起こる。
【００４７】
【表３】

【００４８】
実施例７
ＫＳ１とＫＳ２ライブラリに比較してカポジ肉腫ＳＡＧＥライブラリＫＳ３とＫＳ４で発現が増加している未知因子を有する遺伝子のＥＳＴ’ｓ由来の同定不可能なタグ（ＴＡＧ’ｓ）を示す分析結果。タグ（ＴＡＧ）数は、そのライブラリと公開された他のライブラリ間で比較できるようにまず、ライブラリ当たりの、配列決定された１００．０００総ＴＡＧレベルに標準化された。
配列ｃａｔｇは、表４の縦列２に示された各ＴＡＧ配列に優先して起こる。
【００４９】
【表４】

【００５０】
表３と表４に記載された過剰発現タグ（ＴＡＧ’ｓ）は表１および表２記載のものとそれぞれ同一である。このことは，治療後の発現パターンは正常な発現パターンを有する健常個体の発現パターンに匹敵することを示している。
【００５１】
実施例８
ＲＴ−ＰＣＲに基づく方法を使用して我々は、ＴＡＧ１１（表２）／ＴＡＧ００４（表４）が確かに相違して発現された遺伝子を示していることを決定することができた。ＲＮＡがＫＳ病巣から単離され、最初のストランドｃＤＮＡ合成が、５’Ｍ１３テイル（５’ＣＴＡ ＧＴＴ ＧＴＡ ＡＡＡ ＣＧＡ ＣＧＧ ＣＣＡＧ−（Ｔ）₂₄３’）のオリゴ（ｄＴ）プライマーで試みられた。１０マイクロリットルの全ＲＮＡが使用され、プラスプライマーと５μｌＲＴ−ｍｉｘ （５０ｍＭ Ｔｒｉｓ、ｐＨ８．３、７５ｍＭ ＫＣｌ、３ｍＭ ＭｇＣｌ₂、１０ｍＭ ＤＴＴ）、８０ｍＭ ｄＮＴＰｓと２０ユニットＲＮＡｓｉｎが加えられ、６５℃で３分間インキュベーションされて、氷上で冷却された。ＲＴ反応は５ユニットのＡＭＶ ＲＴを加えて開始され、続いて４２℃で４５分間インキュベートされた。ＰＣＲのために、我々は１９−塩基ＴＡＧ−特異的プライマー（アニ−リング温度を増加させるために、5'NLAIII制限反応サイトとイノシン ヌクレオチドでＳＡＧＥで厳密に同定された１１ ヌクレオチド（nt）からなる）と−２１Ｍ１３プライマーを使用した。ＲＴ混合物（RT−mix）は、１００ngの各プライマー（−２１ｍ１３プライマー：５’ＧＴＡ ＡＡＡ ＣＧＡ ＣＧＧ ＣＣＡ ＧＴ３’と５’ＩＩＩ ＩＩＣ ＡＴＧ ＡＣＣ ＴＣＣ ＡＣＴ ＧＧ３’）、５０ｍＭ Ｔｒｉｓ（ｐＨ８．３）、２０ｍＭ ＫＣｌ、１ミリリットル当たり０．１ｍｇＢＳＡ、dNTPs（各０．１ｍＭ）、２．４ｍＭ ＭｇＣｌ₂および２ユニットＴａｑポリメラーゼを含有する８０μｌＰＣＲ混合物に加えられた。５分間９４℃でインキュベーションの後、反応はサーモサイクラー（９７００パーキン−エルマー（Perkin−Elmer））中で３５サイクルの増幅にかけられた。１サイクルは、９５℃で１分間の変性、５５℃で１分間のアニーリングそして７２℃で２分間の伸長を含む。最後のサイクルの次に７２℃で１０分間のインキュベーションが実施された。
【００５２】
増幅されたフラグメントは、ＡＴプラスミド（インビトロジェン（InvitroGen））にクローン化され続いて挿入物は、アプライドビオシステムズ（Applied Biosystems Inc.）製の染色ターミネーター配列決定キット（dye terminator sequencing kit）を用いて配列が決定された。フラグメントは、１０２塩基対の長さを有しており、フラグメントの配列分析は図１に示す配列を示した。この配列はヒト胎児の心臓から同定されたＥＳＴ配列（ジェンバンク 登録番号（GenBank acc.）ＡＩ２１７５６５およびその他）と同一であり、その結果これは染色体１９上の予想されたエクソンと合致した。新脈管形成との関係は文献未記載であり新規である。
【００５３】
実施例９
タグ配列の同定の確認。
１５ヌクレオチドからなる配列は特定のｍＲＮＡまたは遺伝子を同定するのに十分であろう。タグの独自性を確認するために、我々はＲＴ反応のためのオリゴ２４ｄＴプライマーを用いてＲＴ−ＰＣＲを作製した。タグ配列自身を含有する５’プライマーが２番目のストランド合成に使用された。オリゴ２４ｄＴは、−２１Ｍ１３配列で延長された５’位置に延長された。ＰＣＲ反応とそれに続くＲＴ反応において、−２１Ｍ１３プライマーが、タグ配列を含む５’プライマーとともに増幅に使用される。タグ配列を含む５’プライマーは、このプライマーの結合能力を増大するためにその５’位置に５イノシンで延長された。増幅されたフラグメントの配列は、これがタグ配列で同定された遺伝子であることを確認するために決定することができる。タグを確認するためのＲＴ−ＰＣＲ反応は、タグ配列の発現プロファイルを調製するために使用されたと同一の組織検体で実施された。
【００５４】
手順
実験に使用された共通の緩衝液
１０×ＲＴ緩衝液：
− ５００ｍＭ ＴＲＩＳ、ｐＨ８．３
− ７５０ｍＭ ＫＣＬ
− ３０ｍＭ ＭｇＣｌ₂
− １００ｍＭ ＤＴＴ
１０ｘＰＣＲ緩衝液:
− ２００ｍＭ ＴＲＩＳ ｐＨ８．３
− ５００ｍＭ ＫＣＬ
− １ｍｇ/ｍｌ ＢＳＡ
１ｍｌ トリゾール（ＴＲＩｚｏｌ）試薬（インビトロジェンライフテクノロジ（Invitrogen Life Technologies）カタログ番号１５５９６）が、１０〜１００ｍｇの組織または約１０⁷細胞に即時に加えられる（組織は、ミクロトームで１４μｍの厚さに薄く切り取られる）。
【００５５】
検体の全ＲＮＡ単離は製造者プロトコルに従って、以下の通り実施される：
・クロロフォルム（メルク）０．２ｍｌ添加、そして手で１５秒間試験管を激しく振盪。
・室温で５分間インキュベート。
・４℃で１５分間、１２,０００×ｇ以下で遠心分離。
・無色の上部水層６００μｌを新しい試験管に移す。下部の有機層は赤色であるべき。
・０．５ｍｌイソプロピルアルコール（メルク）を添加し、撹拌。
・室温で５分間インキュベート。
・４℃で１５分間、１２,０００×ｇ以下で遠心分離。
・上澄みを捨て、１ｍｌの８０％エタノールでＲＮＡペッレットをボルテキシング（vortexing）により洗浄し、４℃で５分間、７,５００×ｇ以下で遠心分離し、上澄み液を捨てる。
・ペレットを乾燥するために、５６℃に３分間試験管おき、以下の欄に記載の通りポリＡ⁺ＲＮＡ単離を続ける。
【００５６】
ポリＡ⁺ＲＮＡ単離は、製造者インビトロジェン コーポレーション、カールスバッド、ＵＳＡ カタログ番号Ｋ１５２０）から提供されたミクロ ファ−ストトラック（Micro Fasttrack）２．０ポリＡ⁺ｍＲＮＡ 単離キットのマニュアルに従って実施された。
【００５７】
続いて、単離されたポリＡ⁺ＲＮＡは、ＲＴ−ＰＣＲ反応の分析にインプットとして使用された。ＲＴ−ＰＣＲ反応は、以下の材料の混合物を使用して開始した：
２１Ｍ１３ポリＴプライマー（１００ｎｇ／μｌ） １．２５μｌ
１０ ｘ ＲＴ緩衝液 ２．０μｌ
１００ｍＭ ｄＮＴＰ（ファルマシア） ０．８μｌ
２０Ｕ ＲＮＡｓｉｎ（ロッシュ） ０．３μｌ
ｄＨ₂Ｏ（ベイカー） ０．６５μｌ
・ポリＡ⁺ｍＲＮＡの１０μｌ希釈液をＲＴ−mixの５μｌに加える。
・プライマーをテンプレートにアニーリングするために反応溶液を６５℃で４５分間インキュベートし、その後室温まで冷却する。
・５μｌ １Ｕ／μｌ ＡＭＶ−ＲＴを反応溶液に加え４２℃で４５分間インキュベーションすることによって逆転写を実施する。
・逆転写の後、すぐに混合物を９５℃で５分間インキュベートし反応を停止する。そして，反応混合物を室温まで冷却する。
・ＰＣＲ−mixの８０μｌを各反応混合物（全容量は１００μｌ）に添加。
反応あたりのＰＣＲ−mixは次の通り調製する：
−５’プライマー（１００ｎｇ／μｌ）、表５参照 １．０μｌ
−２１Ｍ１３（１００ｎｇ／μｌ） １．０μｌ
−１０×ＰＣＲ緩衝液 ８．０μｌ
−１００ｍＭ ＭｇＣｌ₂ ２．１μｌ
−Ａｍｐｌｉｔａｑ ５Ｕ／μｌ（パーキンエルマー） ０．４μｌ
−ｄＨ₂Ｏ（ベイカー） ６７．５μｌ
・ＰＣＲ増幅は、９７００ ＤＮＡ サーマルサイクラー（パーキンエルマー）中で以下のプログラムに従い実施した。
−９５℃で５分間
−９５℃で１分間；５５℃で１分間；７２℃で２分間、３５サイクル
−７２℃で１０分間
【００５８】
ＲＴ−ＰＣＲ産物のＴＡ−クローニングは、製造者（インビトロジェン コーポレーション、カールスバッド、（Invitrogen Corporation, Carlsbad） 米国 カタログ番号Ｋ４６００）から提供されたＴＯＰＯ ＴＡクローニング（登録商標）（キットのマニュアルに従って、ｐＣＲ（登録商標）ＩＩ−ＴＯＰＯ（登録商標） ＤｕａｌプロモターベクターとＴＯＰ１０ Ｏｎｅ Ｓｈｏｔ（登録商標）細胞を使用して実施された。
【００５９】
クローンのスクリーニングは、ＳＰ６とＴ７プライマーでＰＣＲを使用して以下のとおり実施された。
【００６０】
・コロニーを５０μｌｄＨ₂Ｏ（ベイカー）に最懸濁する。この細菌懸濁液をＰＣＲ混合物の１０μｌに懸濁する。
・反応あたりのＰＣＲ−混合物は次の通り調製する：
ＳＰ６（１００ｎｇ／μｌ）、 ０．１０μｌ
Ｔ７（１００ｎｇ／μｌ） ０．１０μｌ
１０×ＰＣＲ緩衝液 １．００μｌ
１００ｍＭ ＭｇＣｌ₂ ０．２０μｌ
１００ｍＭ ｄＮＴＰ’ｓ（ファルマシア） ０．０８μｌ
Ａｍｐｌｉｔａｑ ５Ｕ／μｌ（パーキンエルマー） ０．０４μｌ
ｄＨ₂Ｏ（ベイカー） ７．４８μｌ
・ＰＣＲ増幅は、９７００ ＤＮＡ サーマルサイクラー（パーキンエルマー）中で以下のプログラムに従い実施した。
−９５℃で５分間
−９５℃で３０秒間；５５℃で３０秒間；７２℃で１分間、３５サイクル
−７２℃で１０分間
・コロニー−ＰＣＲ産物の５μｌを１．５％アガロース １×ＴＢＥ ゲルにかけ エチジュウム ブロマイド（Ethidium Bromide）で染める。
・挿入物含有（insert−containing）クローンを同定するために、増幅産物をＵＶ−照射装置（ＵＶ−illuminator）上で目に見えるようにする。
【００６１】
挿入物含有クローン（Clones containing insert）は、ＳＰ６とＴ７プラーマ−、およびエイビアイ プリズム ビグ−ダイ ターミネイターサイクル配列決定キット（ABI Prism Big−Dye terminator cycle sequencing kit、パーキンエルマー アプライド ビオシステズ、フォスター市、カルフォルニア、米国（ Perkin Elmer Applied Biosystems, Foster City, Caif USA））を使用して両方向から配列決定された。
【００６２】
【表５】

【００６３】
タグ配列の確認結果：
オリゴ２４ｄＴプライマーと上記のタグを基本としたプライマー（tag−based primer primer）（表５中で指定されたタグ ００４、００７、０１１、０１２、０１４、０１５、０１６、０１７、０２２、０２５，０２９、０３０、０３２、０３６）とのＲＴ−ＰＣＲを使用した配列分析によってこの実施例１４に記載されたプロトコルで分析された１８のタグ配列が確認された。４種のタグ配列（指定されたタグ０１０、０１３、０１８、０１９）はこの方法で確認できなかった。これはおそらく、タグを基本としたプライマーは充分に特異的でないかあるいはｍＲＮＡのポリＡ末尾が充分に長くはなかった事実によるものであった。１つのタグに対してもう一つのもっと特異的な５’プライマーが、−２１Ｍ１３ポリＴ（指定タグ００４）とともにＲＴ−ＰＣＲを実施するためにデザインされた。タグ０１０について、逆転写と増幅を実施するために完全に特異的なプライマーセットがデザインされた。他のタグは特異的なＲＴ−ＰＣＲプライマーセットを使用し、次いで特異的なネスト化プライマーセット（指定タグ０１３、０１８、０１９）でのネスト化ＰＣＲ（Nested PCR）を使用して確認された。全て確認された配列の結果は、以下と、図に一覧として記載されている。ｍＲＮＡ配列中のタグ配列は、もし存在すれば太文字で示されている。
【００６４】
ＴＡＧ００４（ＥＳＴ ＡＩ２１７５６５、ｇｅｎｂａｎｋ番号ＢＥ４６６７２８）：
この実施例のプロトコルで確認された。

（図１）
【００６５】
ＴＡＧ００７（ケラチン１４、ｇｅｎｂａｎｋ番号ＸＭ＿００８５７８）：この実施例のプロトコルで確認された。

（図２）
【００６６】
ＴＡＧ０１０（エフリンＡ２（ephrin A2）、ｇｅｎｂａｎｋ番号ＸＭ＿００２０８８）：
catg−位置上にデザインされた５’プライマー（元のプライマーは表５に示す）でのＲＴ−ＰＣＲでは確認できなかった。おそらく確実にタグを基礎とするプライマー（tag−based primer）が充分特異的でない。エフリンＡ２（Ephrin A2）特異的プライマーが確認のために使用された。タグ配列は、エフリン（Ephrin）特異的ＲＴ−ＰＣＲに含まれていない。

（図３）
【００６７】
ＴＡＧ０１１（デュアルスペシフィシティホスファターゼ（dual specificity phosphatase）、ｇｅｎｂａｎｋ番号ＸＭ＿００３７２０）：この実施例のプロトコルで確認された。

（図４）
【００６８】
ＴＡＧ０１２（ＩＬ１レセプター、タイプ１、ｇｅｎｂａｎｋ番号ＸＭ＿００２６８６）：この実施例のプロトコルで確認された。

（図５）
【００６９】
ＴＡＧ０１３（エフリン Ｂ１（ephrin B1）、ｇｅｎｂａｎｋ番号ＸＭ＿００２５３５、ＢＣ００２５２４）：
catg−位置上にデザインされた５’プライマー（元のプライマーは表５に示す）でのＲＴ−ＰＣＲでは確認できなかった。おそらく確実に、タグを基礎とするプライマーは充分特異的でない。エフリンＢ１（Ephrin B1）特異的プライマーが確認のために使用された。タグ配列は、エフリンＢ１（Ephrin B1）特異的ＲＴ−ＰＣＲフラグメントの３’プライマーに含まれている。ネスト化されたＰＣＲフラグメント（Nested PCR fragment）がここに示されており、タグ配列のちょうど上流部に位置している。

（図６）
【００７０】
ＴＡＧ０１４（ＡＴグループＤ蛋白、ｇｅｎｂａｎｋ番号ＸＭ＿００６１８４、ＡＦ２３０３８８）：
この実施例のプロトコルで確認された。

（図７）
【００７１】
ＴＡＧ０１５（ＴＩＥ１、ｇｅｎｂａｎｋ番号ＸＭ＿００２０３７）：
この実施例のプロトコルで確認された。

（図８）
【００７２】
ＴＡＧ０１６（小さいind.サイトカインＡ１８、ｇｅｎｂａｎｋ番号ＸＭ＿００８４５１、Ｙ１３７１０，ＡＦ１１１１９８）：
この実施例のプロトコルで確認された。

（図９）
【００７３】
ＴＡＧ０１７（補体、１Ｑベータ、ｇｅｎｂａｎｋ番号ＸＭ＿０１０６６６）：この実施例のプロトコルで確認された。

（図１０）
【００７４】
ＴＡＧ０１８ （ガレクチン７（galectin 7）、ｇｅｎｂａｎｋ番号ＮＭ＿００２３０７、Ｕ０６６４３）：
catg−位置上にデザインされた５’プライマー（元のプライマーは表５に示す）でのＲＴ−ＰＣＲでは確認できなかった。おそらく確実に、タグを基礎とするプライマー（tag−based primer）は充分特異的でない。ガレクチン７（galectin 7）特異的プライマーが確認のために使用された。ガレクチン７（galectin 7）特異的プライマーで使用された５’プライマーは、タグ配列を含む。タグ配列は、ガレクチン７（galectin 7）特異的ＲＴ−ＰＣＲフラグメントに含まれている。ネスト化されたＰＣＲフラグメント（Nested PCR fragment）がここに示されており、タグ配列のちょうど下流部に位置している。

（図１１）
【００７５】
ＴＡＧ０１９ （ＦＧＦＲ３、ｇｅｎｂａｎｋ番号ＮＭ＿０２２９６５、ＮＭ＿０００１４２）：
catg−位置上にデザインされた５’プライマー（元のプライマーは表５に示す）でのＲＴ−ＰＣＲでは確認できなかった。おそらく確実に、タグを基礎とするプライマー（tag−based primer）は充分特異的でない。ＦＧＦＲ３特異的プライマーが確認のために使用された。タグ配列は、エフリン（Ephrin）特異的ＲＴ−ＰＣＲに含まれていない。ネスト化されたＰＣＲフラグメント（Nested−PCR fragment）がここに示されており、タグ配列の上流部に位置している。

（図１２）
【００７６】
ＴＡＧ０２２（プソリアシン（Psoriasin）（Ｓ１００ Ａ７））、ｇｅｎｂａｎｋ番号ＸＭ＿０４８１２０）：この実施例のプロトコルで確認された。

（図１３）
【００７７】
ＴＡＧ０２５（ＥＳＴユニジーン（EST Unigene）番号Ｈｓ１７３７８９、ｇｅｎｂａｎｋ番号ＸＭ＿０１８４０４、ＡＬ１３７２６２）：
この実施例のプロトコルで確認された。

（図１４）
【００７８】
ＴＡＧ０２９（ＰＩＧ、ｇｅｎｂａｎｋ番号ＸＭ＿０１１４５３、ＡＪ２５１８３０）：
この実施例のプロトコルで確認された。

（図１５）
【００７９】
ＴＡＧ０３０（ＥＳＴ ユニジーン（EST Unigene）番号Ｈｓ４６９８７、ｇｅｎｂａｎｋ番号ＤＧ１５１１９０、ＢＧ０５７２８９、ＢＥ８５８２７６、ＡＶ６８１７５９、ＢＥ５０３１６９）：
この実施例のプロトコルで確認された。

（図１６）
【００８０】
ＴＡＧ０３２（シアロアドヘシン（SialoAdhesin）、シグレック１（Siglec 1）とも呼ばれる、ｇｅｎｂａｎｋ番号ＸＭ＿０１６２４５）：
この実施例のプロトコルで確認された。

（図１７）
【００８１】
ＴＡＧ０３６（デスモプラキン（Dsmoplakin）、ｇｅｎｂａｎｋ番号ＸＭ＿００４４６３、ＮＭ＿００４４１５、ＡＦ１３９０６５）：
この実施例のプロトコルで確認された。

（図１８）
【００８２】
実施例１０
皮膚検体中のタグ配列の遺伝子発現レベルの測定
新脈管形成のマーカーとしてタグ配列の使用に関する感触を得るために、カポジ肉腫病変のある皮膚検体（５種の異なる検体）とカポジ肉腫病変のない皮膚検体（２種の対照検体）が、タグ配列で同定された遺伝子の発現レベルについて分析された。
【００８３】
手順：
１ｍｌ トリゾール（TRIzol）試薬（インビトロジェン ライフ テクノロジーズ（Invitrogen Life Technologies）カタログ番号１５５９６）が、１０〜１００ｍｇの組織または約１０⁷細胞に即時に加えられる（組織は、ミクロトームで１４μｍの厚さに薄く切り取られる）。
【００８４】
検体の全ＲＮＡの単離は製造者プロトコルに従って、以下の通り実施される：
・クロロフォルム（メルク）０．２ｍｌ添加、そして手で１５秒間試験管を激しくし振盪。
・室温で５分間インキュベートする。
・４℃で１５分間、１２,０００×ｇ以下で遠心分離。
・無色の上部水層６００μｌを新しい試験管に移す。下部の有機層は赤色であるべき。
・０．５ｍｌイソプロピルアルコール（メルク）を添加し、混合。
・室温で１０分間インキュベートする。
・４℃で１５分間、１２,０００×ｇ以下で遠心分離。
・上澄みを捨て、１ｍｌの８０％エタノールでＲＮＡペッレットをボーテクシング（vortexing）により洗浄し、４℃で５分間、７,５００×ｇ以下で遠心分離し、上澄み液を捨てる。
・ペレットを乾燥するために、５６℃に３分間試験管をおき、以下の欄に記載の通りＤＮＡ分解酵素（DNase）処理をする。
【００８５】
全ての単離されたＲＮＡにゲノミック（genomic）ＤＮＡが存在ないことを確認するために、われわれはＤＮＡ分解酵素処理を実施し、ＤＮＡ分解酵素反応にＲＮＡ分解酵素阻害剤（ＲＮａｓｉｎ）を添加することによって、ＤＮアーゼ ＩのＲＮアーゼ活性に対してＲＮＡが保護された。ＤＮＡ分解酵素処理は以下の通り実施された：/
・トリゾール（Trizol）全ＲＮＡ単離の後、ペッレットを８８μｌのｄＨ₂Ｏ中に再懸濁する。
・以下を、順次、ＲＮＡ溶液に加える：
−１０μｌ １０×ＤＮＡ分解酵素緩衝液 （アムビオン（Ambion））
−１μｌ ４０Ｕ／μｌ ＲＮＡ分解酵素阻害剤 （RNasin,ロッシュ）
−１μｌ １０Ｕ／μｌ ＤＮＡ分解酵素 I（DNase I, ロッシュ）/
・３７℃で１時間インキュベートする。
・１００μｌのｄＨ₂Ｏを加えて検体容量を２００μｌに増量する。
・２００μｌの冷フェノール／クロロフォルム ｐＨ８（ＰＣ８）を加える。
・マイクロ遠心分離機中、全速で室温下５分間遠心分離する。
・水性上層を新しいマイクロ遠心分離試験管に移し、順次、以下を加える：
−３μｌグリコーゲン（ロッシュ）
−１００μｌ １０Ｍ 酢酸アンモニウム
−７００μｌ １００％エタノール
・完全に混合し２５，０００×ｇで１５分間４℃で遠心分離し、傾斜し上澄を注ぎだす。
・７００μｌの８０％エタノールを加えて２回洗浄する。完全に混合し２５，０００×ｇで５分間、４℃で遠心分離し、傾斜し上澄を注ぎ出す。
・ペレットを５６℃で５分間乾燥し、１１μｌのｄＨ₂Ｏに再懸濁する。
・１μｌのＲＮＡ単離物を１４０μｌのｄＨ₂Ｏに希釈し、ＯＤ₂₆₀測定によって単離物のＲＮＡ濃度と収率を計算する。ＤＮＡ分解酵素処理された全ＲＮＡの収率は、完全なタグ発現プロファイルの測定には少なくとも１３．５μｇであるべきである。
・濃度５０ｎｇ／μｌのＲＮＡ単離物の溶液を調製する。これは、ＴＡＧ特異的なＲＴ−ＰＣＲのための希釈用のもととなるＲＮＡ溶液である。
【００８６】
ＤＮＡ分解酵素処理された全ＲＮＡのサンプルごとに、１８のＴＡＧ特異的ＲＴ−ＰＣＲ／ネスティド ＰＣＲ（Nested PCR）反応が、ＲＮＡの希釈シリーズで実施された。各検体に対して５つの対照ＲＴ−ＰＣＲが希釈シリーズ、すなわち、ＨＩＶ−１ ＧＡＧ（ＳＫ３９／１４５）、ＧＡＰＤＨ＋ＲＴ（２回）およびＲＴ反応なし（２回）、で実施されるだろう。次の希釈体系は、ＲＴ−ＰＣＲで分析される検体のシリーズの希釈を得るために使用された：
１． ２７０μｌ ５０ｎｇ／μｌＤＮＡ分解酵素処理された全ＲＮＡ。
各プライマーセットのＰＴ−ＰＣＲにおける投入量としての、１０μｌ ５０ｎｇ／μｌ溶液＝５００ｎｇ。
２． ２７μｌ ５０ｎｇ／μｌ全ＲＮＡ １０×希釈＝２７０μｌ ５ｎｇ／μｌ。
各プライマーセットのＰＴ−ＰＣＲにおける投入量としての、１０μｌ ５ｎｇ／μｌ溶液＝５０ｎｇ。
３． ２７μｌ ５ｎｇ／μｌ全ＲＮＡ １０×希釈＝２７０μｌ ０．５ｎｇ／μｌ。
各プライマーセットのＰＴ−ＰＣＲにおける投入量としての、１０μｌ ０．５ｎｇ／μｌ溶液＝５ｎｇ。
４． ２７μｌ ０．５ｎｇ／μｌ全ＲＮＡ １０×希釈＝２７０μｌ ０．０５ｎｇ／μｌ。
各プライマーセットのＰＴ−ＰＣＲにおける投入量としての、１０μｌ ０．０５ｎｇ／μｌ溶液＝０．５ｎｇ。
５． ２７μｌ ０．０５ｎｇ／μｌ全ＲＮＡ １０×希釈＝２７０μｌ ０．００５ｎｇ／μｌ。
各プライマーセットのＰＴ−ＰＣＲにおける投入量としての、１０μｌ ０．００５ｎｇ／μｌ溶液＝０．０５ｎｇ。
６． ２７μｌ ０．００５ｎｇ／μｌ全ＲＮＡ １０×希釈＝２７０μｌ ０．０００５ｎｇ／μｌ。
各プライマーセットのＰＴ−ＰＣＲにおける投入量としての、１０μｌ ０．０００５ｎｇ／μｌ溶液＝０．００５ｎｇ。
７． ２７μｌ ０．０００５ｎｇ／μｌ全ＲＮＡ １０×希釈＝２７０μｌ ０．００００５ｎｇ／μｌ。
各プライマーセットのＰＴ−ＰＣＲにおける投入量としての、１０μｌ ０．００００５ｎｇ／μｌ溶液＝０．０００５ｎｇ。
８． ２７μｌ ０．００００５ｎｇ／μｌ全ＲＮＡ １０×希釈＝２７０μｌ ０．０００００５ｎｇ／μｌ。
各プライマーセットのＰＴ−ＰＣＲにおける投入量としての、１０μｌ ０．０００００５ｎｇ／μｌ溶液＝０．００００５ｎｇ。
【００８７】
ＡＭＶ−ＲＴを用いる希釈の全ＲＮＡシリーズにおけるＴＡＧ特異的ＲＴ−ＰＣＲは以下の通り実施された。希釈シリーズ中のＤＮＡ分解酵素処理全ＲＮＡ上の全てのＴＡＧの逆転写反応は、９６ウエルＰＣＲ板で実施された。それぞれの全ＲＮＡ希釈液の１０μｌがＲＴ ＰＣＲの投入量として使用された。逆転写反応の反応容量は、２０μｌでありｄＮＴＰ、ＭｇＣｌ₂およびＲＮＡ分解酵素阻害剤（RNasin）を含有している。
【００８８】
・反応毎のＲＴ混合物（RT−mix）を次の通り調製する：
３’プライマー（１００ｎｇ／μｌ）表６参照 １．２５μｌ
１０×ＲＴ緩衝液 ２．０μｌ
１００ｍＭ ｄＮＴＰ（ファルマシア） ０．８μｌ
２０ＵＲＮＡｓｉｎ（ロッシュ） ０．３μｌ
ｄＨ₂Ｏ（ベイカー（Ｂａｋｅｒ）） ０．６５μｌ
・全ＲＮＡ希釈液１０μｌをＲＴ混合物（RT−mix）の５μｌに加える。
・プライマーをテンプレートにアニーリングするために反応溶液を６５℃で５分間インキュベートし、その後室温まで冷却する。
・５μｌ １Ｕ／μｌ ＡＭＶ−ＲＴを反応溶液に加え、４２℃で４５分間インキュベーションすることによって逆転写を実施する。
・逆転写の後、すぐに混合物を９５℃で５分間インキュベートし反応を停止する。そして、反応物を室温まで冷却する。
・ＰＣＲ反混合物（PCR−mix）の８０μｌを各反応混合物に添加（全容量は１００μｌ）。
反応あたりのＰＣＲ混合物（PCR−mix）は次の通り調製する：
−５’プライマー（１００ｎｇ／μｌ）、表６参照 １．０μｌ
−１０×ＰＣＲ緩衝液 ８．０μｌ
−１００ｍＭ ＭｇＣｌ₂ １．９μｌ
−Ａｍｐｌｉｔａｑ ５Ｕ／μ ０．４μｌ
−ｄＨ₂Ｏ（ベイカー） ６８．７μｌ
・ＰＣＲ増幅は、９７００ ＤＮＡ サーマルサイクラー（パーキンエルマー）中で以下のプログラムに従い実施される。
−９５℃で５分間
−９５℃で１分間；５５℃で１分間；７２℃で２分間、３５サイクル
−７２℃で１０分間
【００８９】
この最初の増幅に続いて、第２回のネスト化された （nested）増幅が実施された。ＲＴ−ＰＣＲ産物上のＴＡＧ特異的な第２回のネスト化された（nested）ＰＣＲは、以下のとおり実施された：
ネスト化ＰＣＲ混合物（Nested−PCR mix）４５μｌにＴＡＧ ＲＴ−ＰＣＲ産物５μｌを加える。
・反応ごとのネスト化されたＰＣＲ混合物（Nested−PCR mix）を次のように調製する：
５’ネスト化（nested）プライマー（１００ｎｇ／μｌ）、表７参照０．５μｌ
３’ネスト化（nested）プライマー（１００ｎｇ／μｌ）、表７参照０．５μｌ
１０×ＰＣＲ緩衝液 ５．０μｌ
１００ｍＭ ＭｇＣｌ₂ １．２５μｌ
１００ｍＭ ｄＮＴＰ（ファルマシア） ０．４μｌ
Ａｍｐｌｉｔａｑ ５Ｕ／μｌ ０．２μｌ
ｄＨ₂Ｏ（ベイカー） ３７．１５μｌ
タグによって同定された遺伝子の増幅のために使用されたプライマーの組み合わせと増幅されたフラグメントの長さは、表８に示されている。
・ＰＣＲ増幅は、９７００ ＤＮＡ サーマルサイクラー（パーキンエルマー中で以下のプログラムに従い実施される。
−９５℃で５分間
−９５℃で１分間；５５℃で１分間；７２℃で２分間、２５サイクル
−７２℃で１０分間
・ネスト化（nested）ＰＣＲ産物の１０μｌを１．５％アガロース １×ＴＢＥ ゲルにかけ、エチジュウム ブロマイド（Ethidium Bromide）で染める。
・希釈シリーズのＴＡＧ増幅フラグメントをＵＶ−照射装置（ＵＶ−illuminator）上で可視化することで、希釈しても明白なシグナルを示す最高希釈度を決定することにより発現レベルが明らかになる。
【００９０】
【表６】

【００９１】
【表７】

【００９２】
【表８】

【００９３】
結果
タグ配列によって同定された遺伝子の発現レベルの測定結果は、図１９に示されている。データ−は明らかにタグ配列によって同定された遺伝子の数は、正常皮膚に比べてカポジ肉腫病変の皮膚検体において高発現である事を示している：タグ００７、タグ０１０、タグ０１２、タグ０１３、タグ０１４、タグ０１５、タグ０１６、タグ０１７、タグ０２２、タグ０２９、タグ０３０、タグ０３２およびタグ０３６。
【００９４】
実施例１１
末梢血単核細胞（ＰＢＭＣ）検体中でのタグ配列の遺伝子発現レベルの測定
新脈管形成部位、すなわち皮膚中のカポジ肉腫または体内の他の癌からの検体中ではなく、血液（ＰＢＭＣ）から入手可能な検体で、新脈管形成過程のマーカーとしてのタグ配列の使用感触を得るために、５種のタグ同定された遺伝子の発現が、ＰＢＭＣ検体で測定された。ＰＢＭＣ検体は、カポジ肉腫病変のある患者の血液から（４種の異なる検体）とそうでない患者（２種の対照検体）血液由来であり、タグ配列で同定された５種の遺伝子のＰＢＭＣ中での発現について分析された。
【００９５】
この実施例の手法は、皮膚生検のかわりにＰＢＭＣ検体（１検体当たり、約１千万細胞、２５０万から５０００万の範囲）が使用される以外は実施例１０で記載されたものと同一であった。分析される遺伝子は、タグ００７、タグ０１７、タグ０１０、タグ０１３、タグ０１５、タグ０２９およびタグ０３２によって同定された。分析結果は、図２０に示されている。これらのデータ−から、タグ０１５（ＴＩＥ１）とタグ０３２（サリオアドヘシン（Salioadhesin）またはシグレック１（Siglec１））で同定された遺伝子の癌位置での昂進された発現は、血液細胞画分すなわちＰＢＭＣ、と対応していることが明らかである。
【００９６】
結果
タグ００７（ケラチン１４）で皮膚検体（実施例５参照）中で同定された遺伝子の過剰発現は、血液と対応していないことを、このデータ−は明らかに示している。対照的に、この実施例で試験された検体中では、タグ００７によって同定された遺伝子は血液画分で全く発現されていない。このことは、タグ００７を発現している癌細胞は血液中に存在しないことを示している。その結果、血液中のタグ００７の測定は、血液中のこれら癌細胞存在のためのよい標識になるであろう、そして従って、転移を起こし得る循環癌細胞のマーカーになる。
【００９７】
皮膚検体中のタグ０１５（ＴＩＥ１）とタグ０３２（サリオアドヘシン（Salioadhesin）またはシグレック１（Siglec１））によって同定された遺伝子のアップレギュレイションは明らかに血液と相関している。これら２つのタグは、健常人と比較して癌保持患者の血液中で高く発現している。このアップレギュレイションは、典型的な血液細胞中の発現のアップレギュレイションに起因している。
【００９８】
このアップレギュレイションは、血液中でタグ０１５とタグ０３２を発現する癌細胞の存在に帰する事はできない、なぜなら、タグ００７によって同定された遺伝子発現のないことが、上記で説明したよう、血中に癌細胞が存在しないことを示すからである。このことは、血液中のタグ０１５および／またはタグ０３２の発現の測定が体内のどこか他の場所での癌細胞の存在を示唆することを意味する。さらに、抗癌治療および／または抗新脈管形成治療中でのタグ０１５および／またはタグ０３２によって同定された遺伝子の発現の測定は、この治療の有効性をモニターするために使用できる。
【００９９】
結論
タグ０１５（ＴＩＥ１）とタグ０３２（サリオアドヘシン（Ｓalioadhesin）またはシグレック１（Siglec 1））によって癌と血液中で同定された遺伝子の相関したアップレグレーションは、癌の成長の減少を目的とする治療、特に抗新脈管形成性癌治療の有効性をモニターすることを可能にする。このことは、もしこの２つの遺伝子が体内の他の場所での癌中の血管形成のためのＰＢＭＣ中でのマーカーであるとすると、血中のこの２つのマーカーも癌側でのこの血管形成の減少と共に減少するであろうとの推論による。
【０１００】
タグ００７（ケラチン１４）、タグ０１５（ＴＩＥ１）およびタグ０３２（サリオアドヘシン（Ｓalioadhesin）またはシグレック１（Siglec 1））によって同定された遺伝子は、正常個体と比較して癌保有患者の血中で異なった発現をしている。従って、これらの遺伝子は、特にその発現は、その住民の各メンバーで癌の存在の危険に曝されている住民を集団検診するのに使用できる。
【０１０１】
正常組織と癌組織を比べて変化した発現レベルを示す、この研究でタグによって同定された全ての遺伝子、すなわち、タグ００７、タグ０１０、タグ０１２、タグ０１３、タグ０１４、タグ０１５、タグ０１６、タグ０１７、タグ０２２、タグ０２９、タグ０３０、タグ０３２およびタグ０３６は、抗新脈管形成効果を有し癌治療に適用可能な治療薬剤とっての潜在的な標的分子をエンコードしている、そして／またはこれらの遺伝子は、たとえば心臓および冠動脈疾患の治療で血管成長を刺激する治療薬剤の潜在的標的分子になりえる潜在的標的分子をエンコードしている。
【図面の簡単な説明】
【０１０２】
【図１】新脈管形成に含まれる配列。特定の治療後のこの配列の発現変化は、その治療が有効であることを示す。この配列は、ヒト胎児の心臓から同定されたＥＳＴ配列と同一である（ＧｅｎＢａｎｋ登録番号ＡＩ２１７５６５およびその他）、その結果これは染色体１９上の予測されたエクソンと合致する。新脈管形成との関係について今まで記載されたことがない。
【図２】名称とＧｅｎｂａｎｋ番号で同定された配列（ＮＣＢＩデータベース）。他の同定は表１〜４（ユニジーン（Unigene）番号）で見ることができ、これはＮＣＢＩのＳＡＧＥデータベースで見つけることができる。
【図３】名称とＧｅｎｂａｎｋ番号で同定された配列（ＮＣＢＩデータベース）。他の同定は表１〜４（ユニジーン（Unigene）番号）で見ることができ、これはＮＣＢＩのＳＡＧＥデータベースで見つけることができる。
【図４】名称とＧｅｎｂａｎｋ番号で同定された配列（ＮＣＢＩデータベース）。他の同定は表１〜４（ユニジーン（Unigene）番号）で見ることができ、これはＮＣＢＩのＳＡＧＥデータベースで見つけることができる。
【図５】名称とＧｅｎｂａｎｋ番号で同定された配列（ＮＣＢＩデータベース）。他の同定は表１〜４（ユニジーン（Unigene）番号）で見ることができる、これはＮＣＢＩのＳＡＧＥデータベースで見つけることができる。
【図６】名称とＧｅｎｂａｎｋ番号で同定された配列（ＮＣＢＩデータベース）。他の同定は表１〜４（ユニジーン（Unigene）番号）で見ることができ、これはＮＣＢＩのＳＡＧＥデータベースで見つけることができる。
【図７】名称とＧｅｎｂａｎｋ番号で同定された配列（ＮＣＢＩデータベース）。他の同定は表１〜４（ユニジーン（Unigene）番号）で見ることができ、これはＮＣＢＩのＳＡＧＥデータベースで見つけることができる。
【図８】名称とＧｅｎｂａｎｋ番号で同定された配列（ＮＣＢＩデータベース）。他の同定は表１〜４（ユニジーン（Unigene）番号）で見ることができ、これはＮＣＢＩのＳＡＧＥデータベースで見つけることができる。
【図９】名称とＧｅｎｂａｎｋ番号で同定された配列（ＮＣＢＩデータベース）。他の同定は表１〜４（ユニジーン（Unigene）番号）で見ることができ、これはＮＣＢＩのＳＡＧＥデータベースで見つけることができる。
【図１０】名称とＧｅｎｂａｎｋ番号で同定された配列（ＮＣＢＩデータベース）。他の同定は表１〜４（ユニジーン（Unigene）番号）で見ることができ、これはＮＣＢＩのＳＡＧＥデータベースで見つけることができる。
【図１１】名称とＧｅｎｂａｎｋ番号で同定された配列（ＮＣＢＩデータベース）。他の同定は表１〜４（ユニジーン（Unigene）番号）で見ることができ、これはＮＣＢＩのＳＡＧＥデータベースで見つけることができる。
【図１２】名称とＧｅｎｂａｎｋ番号で同定された配列（ＮＣＢＩデータベース）。他の同定は表１〜４（ユニジーン（Unigene）番号）で見ることができ、これはＮＣＢＩのＳＡＧＥデータベースで見つけることができる。
【図１３】名称とＧｅｎｂａｎｋ番号で同定された配列（ＮＣＢＩデータベース）。他の同定は表１〜４（ユニジーン（Unigene）番号）で見ることができ、これはＮＣＢＩのＳＡＧＥデータベースで見つけることができる。
【図１４】名称とＧｅｎｂａｎｋ番号で同定された配列（ＮＣＢＩデータベース）。他の同定は表１〜４（ユニジーン（Unigene）番号）で見ることができ、これはＮＣＢＩのＳＡＧＥデータベースで見つけることができる。
【図１５】名称とＧｅｎｂａｎｋ番号で同定された配列（ＮＣＢＩデータベース）。他の同定は表１〜４（ユニジーン（Unigene）番号）で見ることができ、これはＮＣＢＩのＳＡＧＥデータベースで見つけることができる。
【図１６】名称とＧｅｎｂａｎｋ番号で同定された配列（ＮＣＢＩデータベース）。他の同定は表１〜４（ユニジーン（Unigene）番号）で見ることができ、これはＮＣＢＩのＳＡＧＥデータベースで見つけることができる。
【図１７】名称とＧｅｎｂａｎｋ番号で同定された配列（ＮＣＢＩデータベース）。他の同定は表１〜４（ユニジーン（Unigene）番号）で見ることができ、これはＮＣＢＩのＳＡＧＥデータベースで見つけることができる。
【図１８】名称とＧｅｎｂａｎｋ番号で同定された配列（ＮＣＢＩデータベース）。他の同定は表１〜４（ユニジーン（Unigene）番号）で見ることができ、これはＮＣＢＩのＳＡＧＥデータベースで見つけることができる。
【図１９】カポジ肉腫の皮膚５検体（明るい色の棒線）と２つの対照正常皮膚検体（黒棒線）から単離された全ＲＮＡの５００ｎｇから希釈し始めてもなお陽性を示すログ希釈ファクターとして表示された平均発現レベルと標準偏差。
【図２０】カポジ肉腫患者の４ＰＢＭＣ検体（明るい色の棒線）と２つの対照正常ＰＢＭＣ検体（黒棒線）から単離された全ＲＮＡの５００ｎｇから希釈し始めてもなお陽性をしめすログ希釈ファクターとして表示された平均発現レベルと標準偏差。[0001]
The present invention relates to the field of medicine. In particular, the invention relates to the field of molecular biology and detection methods.
[0002]
Recent advances in knowledge of intracellular molecular changes and techniques for studying these molecular changes have led to improved methods of determining disease states and methods of treatment. Understanding the intrinsic molecular diversity of cancer has, for example, led to a better understanding of the diversity of treatment responses for morphologically similar cancers.
[0003]
Improved pathogenesis impacts the treatment of cancer patients undergoing treatment. A disadvantage of current therapies, however, is that it takes a relatively long time to determine whether the treatment of a patient is actually effective. This delays optimization of dosage and / or treatment regimen. In addition, this, together, slows down the possibility of rehabilitation. For example, treatment optimization is currently only possible when the treatment administered is found to be ineffective by macroscopic analysis of cancer cells in the body. Macroscopic changes usually take weeks to manifest themselves, and instruments that measure such changes are often not ready for use.
[0004]
The present invention provides a method for determining whether a treatment is effective in altering the state of a particular group of target cells in an individual, comprising: obtaining a sample from a patient after the treatment has begun; and Determining whether or not contains the expression product of at least one marker gene. In one embodiment of the invention, the group of target cells comprises cancer cells. A change in state of a group of target cells herein means that at least one property of the group of target cells changes. For example, the number of the target cells may change. Such quantities may increase or decrease. Alternatively, the activity of the target cell may change. The activity may be a copy activity. As another example, the activity can be an activity associated with angiogenesis. Alternatively, it may be an apoptotic activity.
[0005]
It has been discovered that at the molecular level, cancer cells and / or surrounding tissue respond rapidly to effective treatment at the molecular level. This reaction can be detected by measuring the expression product of the marker gene. The expression product of the marker gene indicates a response to the therapy. Marker genes are usually genes that are expressed by the group of target cells, eg, cancer cells and / or surrounding tissues. However, marker genes are also expressed in other non-cancerous cells in the body, such as erythrocytes and / or cardiovascular cells.
[0006]
Alternatively, expression of the marker gene can be initiated when the individual is treated. The marker gene expression product responds to the therapy given to the patient. The response can be a change in the relative amount of the expression product. However, it can also be a change in whether expression products such as RNA and / or proteins are absolutely present or absent.
[0007]
According to the invention, the specimen obtained from the patient comprises at least one of the aforementioned target cells. This is particularly preferred for detecting circulating cancer cells free of cancer and circulating in the patient's blood. Alternatively, the target cells can be non-cancerous cells. In another embodiment of the invention, the analyte does not contain any target cells. However, the specimen can include another non-target cell. The expression or altered expression of the at least one marker gene by the non-target cells is indicative of the status of a particular group of target cells. The non-target cells preferably include peripheral blood mononuclear cells as described below. In yet another aspect, the specimen does not contain any cells. For example, the expression product of a marker gene produced by target cells or non-target cells elsewhere in each individual may also be present at levels detectable in the sample.
[0008]
It is possible to determine whether a treatment is effective for the individual by the method of the present invention. This can be done during the treatment or shortly after the treatment. In this way, for example, the treatment regimen, dose and type can be optimized for each individual patient. The sample is preferably collected within one week of starting treatment. More preferably, the sample is collected within 2 days of starting treatment. By the method of the present invention, the therapeutic effect can be evaluated almost immediately after the start of the treatment. Thus, the method of the present invention provides a good opportunity to determine whether treatment optimization is needed.
[0009]
Marker genes preferably include genes involved in the formation, maintenance and / or damage of blood vessels (angiogenesis). The genes involved in the process of angiogenesis include other receptors, ligands and signal molecules. Cancer cells rely on the growth of new blood vessels to maintain the growth of the cancer mass. On the other hand, blood vessels need to transport nutrients to cancer sites, whereas waste products need to be transported from cancer. In the present invention, it has been shown that the expression products of the genes involved in the formation, maintenance and / or damage of blood vessels, inter alia, respond to anti-cancer therapy. Thus, such genes are highly preferred marker genes of the present invention. In one embodiment, the marker gene comprises a sequence set forth in Table 1 or 2. In other embodiments, the marker gene comprises the sequences shown in FIGS. 1-18, or portions or analogs of these sequences. In a preferred embodiment, the marker comprises a TIE1 sequence, a Salioadhesin or Siglec 1 sequence, the sequence shown in FIG. 8 or 17, or a portion or analog thereof.
[0010]
A change in the level of the expression product of the marker gene indicates whether the treatment is effective or not. For example, the level of the expression product of the marker gene will be high when the treatment is effective, otherwise the level of the expression product of the marker gene will be low. Therefore, preferably, the expression product of the marker gene is quantified. The level of the expression product in the sample changes due to a change in the expression of the marker gene. However, expression product levels can change due to changes in the cell type containing the expression product in the sample, for example, killing of cells at the site in the body from which the sample was taken for treatment. Given that the level of the expression product of the marker gene varies from patient to patient, the method of the present invention further comprises comparing the level of the expression product of the marker gene to a control. Preferably, the control comprises the same type of cancer cells before or without treatment. Preferably, the cancer cells are obtained from the same patient. The difference between the expression product level of the marker gene and the effective treatment is very large. In extreme cases, the level of the expression product varies from detectable to undetectable. In the present invention, a marker gene expressing such a 0: 1 relationship in the expression product is preferable. The 0 to 1 relationship can be used for relatively simple inspection systems. Of course, a 0 to 1 relationship depends on the detection system used to detect the expression product of the marker gene. A very sensitive expression product detection system will normally detect an expression product even if a less sensitive detection system does not detect the expression product. The expression product can be RNA transcribed from the marker gene or a part thereof, or translated protein or a part thereof. Those of ordinary skill in the art will be able to fully design the most appropriate expression detection system to practice this preferred embodiment of the present invention.
[0011]
A portion of an RNA or DNA molecule is defined herein as an RNA or DNA sequence containing at least 50 nucleotides. As used herein, a portion and / or analog of an expression product may have a different detection sensitivity, but a portion and / or portion of the expression product that can be detected using substantially the same type of detection method as the expression product. Defined as analog. An analog of an RNA or DNA molecule is defined herein as an RNA or DNA sequence whose sequence is substantially identical to a particular RNA or DNA sequence. However, nucleotide mutations, substitutions, alterations, additions and / or deletions occur naturally and / or artificially without substantially altering the detection of the analog relative to the detection of the particular RNA or DNA sequence. You may. One of ordinary skill in the art can readily determine whether a submitted RNA or DNA is a particular RNA or DNA analog using techniques known in the art.
[0012]
In a preferred embodiment, said cancer comprises Kaposi's Sarcoma. Kaposi's sarcoma is a proliferative vascular disease and is therefore suitable for identifying marker genes involved in angiogenesis. According to the invention, changes in angiogenic factors are, inter alia, events that occur at the marker as a result of treatment. Kaposi's sarcoma (KS) clinically manifests as a reddish skin lesion.
[0013]
Kaposi's sarcoma is characterized by bluish-red skin nodules that usually develop in the lower limbs, more often in the toes and legs, and slowly increase in size and number and spread to areas closer to the base. Multicentric malignant vascular growth. Cancer has a vascular space in which endothelial connecting pathways and aggregates of various sizes of spindle-shaped cells are intermingled, and often remains confined to the skin and subcutaneous tissues. obtain. Kaposi's sarcoma occurs naturally in Jewish and Italian men in Europe and the United States. Malignant variants in infants are endemic to some parts of Africa. A third type occurs in about 0.04% of kidney transplant patients. There is also a high incidence of AIDS patients. (From Dorland, 27th ed & Hollandet. Al., Cancer Medicine, 3d ed. Pp 2105-7))
[0014]
Kaposi's sarcoma is malignant in HIV-infected individuals. The angiogenic mechanisms that cause damage result from the interaction of viral and cellular gene expression, and it is not well understood what genes are involved and what controls their expression. The proliferation of angiogenesis in KS involves a mechanism that seems to be common to angiogenesis. The major role of Kaposi's sarcoma is clearly explained by the French name of this cancer: angiosarcomatose kaposi). Since angiogenesis plays a major role in Kaposi's sarcoma, measurement of marker genes involved in angiogenesis is very suitable for determining whether treatment for Kaposi's sarcoma is effective.
[0015]
In the present invention, the gene expression pattern of Kaposi's sarcoma is tested by a method called serial analysis of gene expression (SAGE) (Velculescu et. Al., (1995) Science 270; 484-487). Was done. This method allows for quantitative and continuous analysis of large numbers of transcripts. SAGE is based on two principles. First, the short nucleotide sequence tag (TAG) (14 base pairs) contains sufficient information to uniquely identify the transcript if it is isolated from a defined location within the transcript. I have. Second, concatenation of short sequence tags (TAG's) allows efficient analysis of transcripts in a continuous manner by sequencing a wide variety of tags (TAG's) within a single clone.
[0016]
Briefly, in this method, a biotinylated oligo (dT) primer is used for cDNA synthesis from mRNA, and after digestion with a restriction enzyme, the extreme 3 'end (close to the poly-A end) is isolated. It is. The 3 'fragment of this cDNA is linked to a linker and cut with a type II restriction enzyme to release the short sequence (14 base pairs) of the original cDNA. The tag (TAG's) is bound to a ditag (di-TAG's) and amplified by the PCR method. The ditags (di-TAG's) are linked by ligase to form concatamers, and the concatamers are cloned and sequenced. In this way, one sequencing reaction gives information about the distribution of many different RNAs. Finally, calculations are performed on a number of different tags using the required computer software and matched against Genbank tags (TAG's).
[0017]
In another embodiment, the present invention provides the use of a nucleic acid comprising a sequence set forth in Figures 1-18 and / or Tables 1 or 2, or a portion or analog thereof, in a method for detecting the expression product. . Preferably, the nucleic acid comprises a TIE1 sequence, a Salioadhesin or Siglec 1 sequence, the sequence depicted in FIG. 8 or 17, or a portion or analog thereof. Expression of a marker gene in an individual can be detected by measuring whether the nucleic acid, or a portion or analog thereof, hybridizes to a nucleic acid, preferably an RNA, in a sample of the individual. If hybridization occurs, it indicates that the marker gene is expressed in the individual. Of course, as is known to those of ordinary skill in the art, the coding strand of DNA / RNA may hybridize with the complementary strand of the corresponding duplex nucleic acid sequence. it can. Thus, a complementary strand of a particular coding strand is particularly preferred for detecting expression of the coding strand. For example, the complementary strand of the coding strand shown in FIGS. 1-18 and / or Table 1 or 2 is suitable for detecting the expression of a gene containing the coding strand.
[0018]
In yet another embodiment, the present invention relates specifically to proteins encoded by nucleic acids comprising the sequences set forth in FIGS. 1-18 and / or Tables 1 or 2, or portions or analogs thereof. Provided is a use in a method for detecting a binding proteinaceous molecule. Preferably, the proteinaceous molecule is a protein encoded by a nucleic acid comprising a TIE1 sequence, a Salioadhesin or Siglec 1 sequence, the sequence shown in FIG. 8 or 17, or a portion or analog thereof. Has the ability to specifically bind. In another embodiment of the invention, the use is directed to determining the presence of a site of angiogenesis in an individual. In another embodiment of the invention, the use is directed to measuring the presence of cancer cells in the individual. The presence of cancer cells in an individual can be measured because the cancer cells typically express a marker gene that can be detected by an expression product detection method. For example, an antibody or an analog thereof specifically directed to the expression product of the marker gene can be generated. The antibody or analog thereof is suitable for measuring the expression product of the marker gene in a specimen. A sample obtained from an individual to determine the presence of cancer cells in the individual is incubated with the antibody. If the analyte contains the expression product of the marker gene, the antibody will bind. Binding can be demonstrated by techniques known in the art, for example, an enzyme immunoassay (ELISA). If the binding of the antibody is demonstrated, it can be concluded that the analyte contains the expression product of the marker molecule. Since the marker molecule is expressed by a cancer cell, the presence of an expression product of the marker molecule can indicate the presence of the cancer cell in an individual. Of course, there are many alternative techniques for detecting expression products by use of proteinaceous binding molecules, but they are well known in the art and need not be discussed further here. That is, the protein expressed by the cancer cells can be expressed in any of FIGS. 1-18 and / or Tables 1 or 2, e.g., a TIE1 sequence, a Salioadhesin and / or a Siglec 1 sequence, or a portion or analog thereof. It can be detected with a proteinaceous molecule that has specific binding ability to the protein encoded by the containing nucleic acid. Similarly, the presence of an angiogenic site in an individual can be measured by detecting the expression product of a marker gene.
[0019]
In another embodiment of the invention, the use of a nucleic acid comprising the sequence shown in FIGS. 1-18 and / or Table 1 or 2 or comprising the sequence shown in FIGS. 1-18 and / or Table 1 or 2 The use of proteinaceous molecules capable of specifically binding proteins encoded by nucleic acids or portions or analogs thereof is suitable for determining whether treatment is effective for changing the context of a given set of target cells in an individual. It is. In a preferred embodiment, the nucleic acid comprises a TIE1 sequence, a Salioadhesin or Siglec 1 sequence, the sequence shown in FIG. 8 or 17, or a portion or analog thereof. In another preferred embodiment, the proteinaceous molecule is a nucleic acid comprising a TIE1 sequence, a Salioadhesin or Siglec 1 sequence, the sequence shown in FIG. 8 or 17, or a portion or analog thereof. Has the ability to specifically bind to the protein encoded by More preferably, the use is suitable for determining whether a treatment is effective in counteracting cancer cells in an individual. In one embodiment of the invention, the cancer cells comprise Kaposi's sarcoma.
[0020]
In one aspect of the present invention, there is provided the use of a nucleic acid comprising a sequence shown in Table 1 or 2 and / or FIGS. 1-18 as an indicator for angiogenesis. In a preferred embodiment, the invention relates to angiogenesis of a nucleic acid comprising a TIE1 sequence, a Salioadhesin or Siglec 1 sequence, the sequence shown in FIG. 8 or 17, or a portion or analog thereof. Provide use as an indicator. For example, nucleic acids comprising the sequences shown in FIGS. 1-18 and / or Tables 1 or 2 can be used as detection markers for angiogenic processes in the course of regenerative therapy. A change in the expression level of the detectable marker is indicative of active blood vessel growth (ie, angiogenesis) induced by the course of regenerative therapy. In a preferred embodiment, such an application is aimed at generating a new blood supply to organs affected by new blood vessels in the field of heart and coronary artery disease. Similarly, treatment with an anti-angiogenic agent may result in a detection marker such as the TIE1 sequence, Salioadhesin and / or Siglec 1 sequence shown in FIGS. 1-18 and / or Table 1 or 2. Can be monitored by changes in the expression level.
[0021]
In another aspect, the present invention provides the use of a nucleic acid comprising the sequence shown in FIGS. 1-18 and / or Table 1 or 2 as a marker for detecting cancer cells. In yet another embodiment, the invention relates to a protein-like molecule encoded by a nucleic acid comprising a sequence shown in FIGS. 1-18 and / or Tables 1 or 2, or Provided is the use of a protein-like molecule capable of binding to a protein encoded by a nucleic acid comprising a sequence as shown, as a marker for detecting cancer cells.
[0022]
With the method of the invention, it is possible to monitor the special condition of the individual. The presence of a disease, or an ongoing lesion, can be determined by measuring whether an individual's specimen contains an expression product of a marker gene. This also means that if the marker gene is not present in the sample, there is a risk of disease present or disease progression. This is possible for any disease as long as the disease involves altered expression patterns of at least one marker gene. Preferably, the presence of the marker gene in the sample is measured.
[0023]
In addition, the healing process can be tracked. For example, recovery of damaged tissue can be accompanied by an increase in the amount of marker gene expression product over that period. However, recovery of damaged tissue may be accompanied by a decrease in the amount of marker gene expression product over that period. Specimens taken at different time intervals provide information about the amount of expression product produced at different time intervals. The amount of change in a particular expression product found in the sample during that time indicates the amount of tissue produced. Similarly, a decrease in the expression product found in a sample during a particular time period indicates a certain beneficial or harmful process. For example, the process may be involved in disease progression or cure. An important application is in the treatment of heart and coronary artery disease. The method of the present invention is well suited for monitoring the generation of new myocardial tissue.
[0024]
That is, one aspect of the present invention provides a diagnostic method, particularly a method for determining whether an individual contains cancer cells and / or an angiogenic site, collecting a specimen from the individual, and Comprises determining whether or not contains the expression product of at least one marker gene. Preferably, the marker gene comprises Table 1 or 2 and / or Figures 1-18, or a portion or analog thereof. More preferably, the marker gene comprises a TIE1 sequence, a Salioadhesin or Siglec 1 sequence, the sequence shown in FIG. 8 or 17, or a portion or analog thereof. With the method of the present invention, it is possible to detect cancer cells that are free from cancer and present elsewhere in the body. In a preferred embodiment, such a detection method is performed by detecting circulating cancer cells in human blood. These circulating cancer cells can be used for preliminary detection in other parts of the human body, and can also be used to detect the risk of cancer metastasis to other parts of the human body adjacent to the original part of the human body. It is possible. Similarly, the method of the present invention is suitable for measuring a site of neovascularization in an individual. For example, an increased level of angiogenesis is indicative of the presence of cancer cells or is indicative of healing of damaged tissue, such as, for example, recovery of heart and coronary artery disease.
[0025]
Since the angiogenesis process can now be easily monitored by the method of the present invention, it is as easy as determining whether a particular treatment is effective in altering the angiogenesis process. For example, if a particular treatment is effective in disrupting the angiogenesis process, the amount of expression products of marker genes involved in angiogenesis will also decrease over that period. Many agents for treating angiogenesis are known in the art. Accordingly, one embodiment of the present invention provides a method for determining whether a treatment is effective in altering an angiogenic process in an individual, and removing a sample from the individual after initiating the treatment. And determining whether the sample contains at least one marker gene. Preferably, the marker gene includes the sequences shown in Tables 1, 2, and / or FIGS. 1-18, or portions or analogs thereof. More preferably, the marker gene comprises a TIE1 sequence, a Salioadhesin or Siglec 1 sequence, the sequence shown in FIG. 8 or 17, or a portion or analog thereof. In one embodiment, the treatment comprises preventing angiogenesis in the individual. In yet another embodiment, the treatment comprises the use of at least one of the following agents: 2ME2, Angiostatin, Angiozyme, Anti-VEGF (Anti-VEGF) RhuMAb, Apra ( Apra (CT-2584)), Avicine, Benefin, BMS275291, Carboxamidotriazole, CC4047, CC5013, CC7085, CDC801, CGP-41251 (PKC412), CM101, Combretastatin A-4 Prodrug, EMD121974, Endostatin, Flavopiridol, Genistein (GCP), green tea extract, IM-862, Imser, Imm Ther, Interferon alpha, Interleukin-12, A Ressa (ZD1839), Marimastat, Metastat (Col-3), Neovastat, Neotrestat, Octreotide, Paclitaxel, Penicillamine, Photofrin, Photopoint ), PI-88, Prinomastat (AG-3340), PTK787 (ZK22584), RO317453, Solimastat, Squalamin, SU101, SU5416, SU-6668, Suradista (FCE26644) )), Suramin (Metaret, Tetrathiomolybdate, Thalidomide, TNP-470 and / or Vitaxin), however, the skilled person will come up with more and more drugs that can be used during the treatment. This Can.
[0026]
In a preferred embodiment, the sample of the method of the present invention is a blood sample. For example, the site of the angiogenesis process may be cancer or part of the skin, but blood samples are particularly preferred because they are easy to collect. Blood specimens are also often easy to test and do not require expensive and / or special equipment. Quite surprisingly, we believe that the expression of certain marker genes by hematopoietic cells, such as peripheral blood mononuclear cells (PBMC), is a series of changes that occur elsewhere in the individual. Can be displayed. For example, a change in the presence or amount of a marker molecule expression product in PBMC can indicate the presence of cancer cells somewhere in the body. In Example 10, for example, both the TIE1 sequence (Tag 15, Table 3) or the Salioadhesin or Siglec 1 (Tag 32, Table 4) are highly regulated in PBMC cells of skin cancer and Kaposi's sarcoma patients. Is shown. Furthermore, while the keratin 14 sequence is overexpressed in cancer cells, the absence of the expression product of the keratin 14 (Tag 7, Table 3) sequence in the blood sample of the patient indicates that the sample is a cancer sample. Example 8 demonstrates that it is not contaminated with cells. Similarly, expression of certain marker genes by PBMC may provide another diagnostic indicator. The presence or absence of a marker gene expression product in PBMC provides appropriate information about different aspects and / or individual body changes. Preferably, the amount of expression product in non-hematopoietic cells is compared to a control value. Thus, an index is obtained for an increase or decrease in expression in the hematopoietic cells.
[0027]
Thus, in one aspect, the present invention provides a method for determining whether an individual comprises non-hematopoietic cancer cells and / or an angiogenic site, wherein the method comprises comparing hematopoietic cells from the patient to control values. The expression level of the marker gene expression product. Preferably, the marker gene includes a gene involved in angiogenesis. More preferably, the gene comprises the sequence shown in Table 1 or 2 and / or FIGS. 1-18, or a portion or analog thereof. Most preferably, the gene comprises a TIE1 sequence, a Salioadhesin or Siglec 1 sequence, the sequence shown in FIG. 8 or 17, or a portion or analog thereof. In one embodiment of the invention, the hematopoietic cells comprise peripheral blood mononuclear cells.
[0028]
In one aspect, the present invention provides the measurement method of the present invention, wherein the expression product is expressed by PBMC. Preferably, the expression product comprises a TIE1 sequence, a Salioadhesin or Siglec 1 sequence, the sequence shown in FIG. 8 or 17, or a portion or analog thereof. Since these sequences are involved in angiogenesis, the present invention provides for the use of the keratin 14 sequence, TIE1 sequence, Salioadhesin or Siglec 1 sequence, the sequence shown in FIG. The use of a part or analog as an indicator of angiogenesis is provided. Similarly, these sequences are involved in the presence of cancer cells. Accordingly, a cancer in an individual of a PBMC expressing the keratin 14 sequence, TIE1 sequence, Salioadhesin or Siglec 1 sequence, the sequence shown in FIG. 2, 8 or 17, or a portion or analog thereof, Uses for determining the presence of cells are also provided. Further, the present invention also provides an isolated keratin 14 sequence, TIE1 sequence, Salioadhesin or Siglec 1 sequence, FIG. 2, 8, or 17, for use in a diagnostic method. The sequences, or portions or analogs thereof, are provided. The diagnostic method is performed using a diagnostic kit. Accordingly, nucleic acids comprising FIGS. 1-18 and / or Tables 1 or 2 or a portion or analog thereof, and / or proteins capable of specifically binding to the protein encoded by the nucleic acid or a portion or analog thereof Diagnostic kits containing like molecules are also provided herein. Preferably, the kit includes suitable detection means. In one embodiment, the keratin 14 sequence and / or TIE1 sequence, and / or Salioadhesin or Siglec 1 sequence of the invention, and / or the sequence shown in FIG. 2, 8 or 17 Also provided is a diagnostic kit comprising a portion or analog of The diagnostic kit of the present invention is particularly useful for performing the method of the present invention. In another further embodiment, therefore, to determine whether a treatment is effective in altering a particular group of target cell states in an individual and / or altering angiogenic processes in an individual. The use of the diagnostic kit of the present invention is provided. Furthermore, the present invention provides the use of the diagnostic kit of the present invention for determining whether an individual contains cancer cells and / or sites of angiogenesis.
[0029]
The marker gene of the present invention enables screening of a drug against a disease indicated by the marker gene. There are many different methods available for screening for specific drug activity. For example, cells can be incubated with a variety of different drug candidate compounds, and the expression pattern of the marker gene in the cells can be compared before and after exposing the cells to the drug candidate compound. A difference in the specific expression pattern after exposing the cells to a drug candidate compound indicates that the compound is a suitable candidate for drug development. Accordingly, the present invention provides the use of the expression product of a gene comprising the sequence shown in FIGS. 1-18, Table 1 or Table 2 as a drug target.
[0030]
Preferably, the sequence is a Salioadhesin or Siglec 1, TIE1, and / or keratin 14 sequence.
[0031]
Capable of altering the activity of Salioadhesin or Siglec 1, TIE1, keratin 14 and / or expression of Salioadhesin or Siglec 1, TIE1 and / or keratin 14 in cells Also provided here are substances capable of changing The substance is particularly suitable for the preparation of a medicament.
[0032]
The invention is further described in more detail by the following examples, which do not limit the invention in any way.
[0033]
Example
Example 1
In this embodiment, a sample is selected for analyzing an expression profile. A 31 year old man was determined to be HIV-1 seropositive in February 1997. The initial CD4 cell count is 25 × 10⁶/ L. The patient developed mucocutaneous herpes simplex and extrapulmonary cryptococcosis within two months, and was given special medication. HIV-1 RNA load initially increased from 15,000 copies / ml to 33,000 copies / ml in 3 months. Thus, antiviral therapy was started using zidovudine, lamivudine, and indinavir. Shortly after the start of treatment, HIV-1 RNA load dropped below the limit of detection. In November 1997, the patient gradually showed increasing purple skin lesions, which were clinically similar to Kaposi's sarcoma. Diagnosis was confirmed by histological examination of the lesions. Early in chemotherapy (intravenous bleomycin, vincristine, and adriamycin), KS advanced to approximately 150 skin lesions. The rest period during the course of chemotherapy was 3 weeks and was discontinued after the fifth course. Several lesions disappeared after 3 weeks of treatment, and after one year gradually approached full recovery.
[0034]
Several biopsies were performed during chemotherapy. The first biopsy was taken 24 hours after starting chemotherapy (designated KS1) and the second biopsy was taken 48 hours later (designated KS2). All biopsies were snap frozen in liquid nitrogen immediately after surgical removal and stored at -80 ° C. The diagnosis of Kaposi's sarcoma was confirmed histologically.
[0035]
Control SAGE libraries KS3 and KS4 were made from frozen material collected at necropsy from two AIDS patients with Kaposi's sarcoma who died in 1986 without any form of chemotherapy or antiviral treatment.
[0036]
Example 2
The expression profile of the biopsy specimen was determined using the SAGE technique. All biopsies were cut with a microtome into 15-20 μm sections and transferred to tubes containing TRIzol. RNA isolation was performed with TRIzol according to the manufacturer's instructions. Poly (A) RNA is micro-fast track^TM(Micro-FastTrack^TM) Obtained using the 2.0 mRNA isolation kit. The preparation of the cDNA and the subsequent steps were performed as described by Velculescu. Preliminary testing of the sequencing results was performed using software specifically designed for SAGE by the Academic Medical Center, Bioinformatics Laboratory, Amsterdam (van Kampen et al., USAGE: A web-based approach to SAGE data analysis, Bioinfomaticcs, in press). The library was also analyzed with the Human Transcriptome Map (HTM), a program developed by AMC that maps TAGs to human chromosomes (Caron et al., Human Transome Map is highly expressed in chromosomal domains) Clarification of the clustering of the selected genes.
[0037]
We have approximately 47,000 tags (TAG's) from four biopsy materials, 47,298 tags (TAG's) from the KS1 library, 46,671 from the KS2 library, and 49,335 tags (TAG's) from the KS3 library. ) And the sequence of the 48,814 tags (TAG's) from the KS4 library. The tag (TAG) list (ie, individual TAG's plus the number of occurrences) was compared to each other by USAGE, and the tag (TAG) sequence with the highest count was identified in the amct2g database available at USAGE (this Is an advanced TAG identification method compared to the CGAP (available in GenBank) SAGEmap database)). Next, the tag (TAG) list is mapped to chromosomal positions in the HTM program, and simultaneously compared to a specific TAG list (eg, vascular endothelium, publicly available), and all of the SAGEmap database (HTM specifies “all”) In combination with TAG of, a tag (TAG's) belonging to a gene particularly enhanced in KS3 and KS4 was identified (Table 1). Nucleotide 15 was determined from USAGE's original diTAG list. The TAG sequence of 15 nucleotides (15 nt.) Was checked with GenBank (BLAST) to confirm its identity. A small number of tags (TAG's) were excluded due to ambiguity at the 15th nucleotide or due to incorrect identification.
[0038]
Example 3
Analysis results showing identifiable tags (TAG's) derived from known genes whose expression is increased in Kaposi's sarcoma SAGE libraries KS3 and KS4 compared to KS1 and KS2 libraries. Tag (TAG) numbers were first normalized to 100.000 total tag (TAG) levels sequenced per library so that comparisons could be made between the library and other published libraries.
The sequence catg occurs in preference to each tag (TAG) sequence shown in column 2 of Table 1.
[0039]
[Table 1]

[0040]
Example 4
The analysis result which shows the unidentifiable tag (TAG's) derived from EST's of the gene which has an unknown factor whose expression is increasing in Kaposi's sarcoma SAGE library KS3 and KS4 compared with KS1 and KS2 libraries.
[0041]
Tag (TAG) numbers were first normalized to 100.000 total TAG levels sequenced per library so that comparisons could be made between the library and other published libraries.
The sequence catg occurs in preference to each tag (TAG) sequence shown in column 2 of Table 2.
[0042]
[Table 2]

[0043]
Example 5
Kaposi's sarcoma skin tissue was collected from the same two AIDS patients as described in Example 1, and KS3 and KS4 libraries were generated from the patients. Both patients were homosexual and infected early in the HIV-1 epidemic in Europe. Patient 1 born in Indonesia was proven in 1982 to be HIV-1 positive. Histologic examination confirmed the diagnosis of Kaposi's sarcoma in February 1985. He died 13 months later and autopsy revealed a morphological variant of visceral KS. Patient 2 had a progressive KS skin lesion at the Academic Medical Center in February 1984. During follow-up treatment, KS progressed to the intestine, pharynx, lungs, tongue, piriform depression and lymph nodes. In March 1986, the patient died and was necropsied. Tissue samples collected from the two patients were named KS3 and KS4.
[0044]
Normal adult thoracic skin tissue was harvested as discarded tissue in mammoplasty (obtained from plastic surgery in our hospital). Isolated RNA from three thoracic reduction operations was used for a library of normal skin expression profiles.
[0045]
The expression profile of the biopsy sample was determined with the SAGE technique as described in Example 2. We sequenced about 47,000 tags (TAG's) from four biopsy samples, 49,335 tags (TAG's) from the KS3 library and 48,814 tags (TAG's) from the KS4 library. The tag (TAG) list (ie, individual tags (TAG's) plus the number of occurrences) was identified in the amct2g database available at USAGE (this is compared to the SAGEmap database from CGAP (available in GenBank)). Advanced tag (TAG) identification method). The tag (TAG) list is then mapped to a chromosomal site by the HTM program, and simultaneously compared to a specific tag (TAG) list (eg, vascular endothelium, publicly available), and the SAGEmap database (specify “all” in the HTM) In combination with the list of all tags (TAG) in (1), tags (TAG's) belonging to genes that were particularly enhanced in KS3 and KS4 were identified (Table 1). Nucleotide 15 was determined from USAGE's original diTAG list. The TAG sequence of 15 nucleotides (15 nt.) Was checked by GenBank (BLAST) to confirm its identity. Due to ambiguity at the 15th nucleotide or due to incorrect identification, a small number of tags (TAG) were excluded.
[0046]
Example 6
Analysis results showing identifiable tags (TAG's) derived from known genes whose expression is increased in Kaposi's sarcoma SAGE libraries KS3 and KS4 compared to KS1 and KS2 libraries. Tag (TAG) numbers were first normalized to 100.000 total tag (TAG) levels sequenced per library so that comparisons could be made between the library and other published libraries.
The sequence catg occurs in preference to each TAG sequence shown in column 2 of Table 3.
[0047]
[Table 3]

[0048]
Example 7
The analysis result which shows the unidentifiable tag (TAG's) derived from EST's of the gene which has an unknown factor whose expression is increasing in Kaposi's sarcoma SAGE library KS3 and KS4 compared with KS1 and KS2 libraries. Tag (TAG) numbers were first normalized to 100.000 total TAG levels sequenced per library so that comparisons could be made between the library and other published libraries.
The sequence catg occurs in preference to each TAG sequence shown in column 2 of Table 4.
[0049]
[Table 4]

[0050]
The overexpression tags (TAG's) described in Tables 3 and 4 are the same as those described in Tables 1 and 2, respectively. This indicates that the expression pattern after treatment is comparable to that of a healthy individual having a normal expression pattern.
[0051]
Example 8
Using a method based on RT-PCR, we could determine that TAG11 (Table 2) / TAG004 (Table 4) did indeed represent differentially expressed genes. RNA was isolated from KS foci and the first strand cDNA synthesis was performed at the 5'M13 tail (5'CTA GTT GTA AAA CGA CGG CCAG- (T)_{twenty four}3 ') with an oligo (dT) primer. Ten microliters of total RNA was used, plus primer and 5 μl RT-mix (50 mM Tris, pH 8.3, 75 mM KCl, 3 mM MgCl_Two, 10 mM DTT), 80 mM dNTPs and 20 units RNAsin were added, incubated at 65 ° C. for 3 minutes and cooled on ice. The RT reaction was started by adding 5 units of AMV RT, followed by incubation at 42 ° C. for 45 minutes. For PCR, we used a 19-base TAG-specific primer (11 nucleotides (nt) strictly identified by SAGE at the 5 'NLA III restriction reaction site and inosine nucleotides to increase the annealing temperature). ) And -21M13 primers were used. The RT mixture (RT-mix) was prepared with 100 ng of each primer (-21 m13 primer: 5 ′ GTA AAA CGA CGG CCA GT3 ′ and 5′III IIIC ATG ACC TCC ACT GG3 ′), 50 mM Tris (pH 8.3), 20 mM KCl 0.1 mg BSA, dNTPs (0.1 mM each), 2.4 mM MgCl per milliliter_TwoAnd 2 units Taq polymerase were added to the 80 μl PCR mix. After incubation at 94 ° C. for 5 minutes, the reaction was subjected to 35 cycles of amplification in a thermocycler (9700 Perkin-Elmer). One cycle involves denaturation at 95 ° C. for 1 minute, annealing at 55 ° C. for 1 minute, and extension at 72 ° C. for 2 minutes. A 10 minute incubation at 72 ° C. was performed following the last cycle.
[0052]
The amplified fragment was cloned into an AT plasmid (InvitroGen) and the insert was sequenced using a dye terminator sequencing kit from Applied Biosystems Inc. Was decided. The fragment had a length of 102 base pairs and sequence analysis of the fragment showed the sequence shown in FIG. This sequence was identical to the EST sequence identified from human fetal heart (GenBank acc. AI217565 and others), which was consistent with the expected exon on chromosome 19. The relationship with angiogenesis is not described in the literature and is novel.
[0053]
Example 9
Confirmation of tag sequence identification.
A sequence of 15 nucleotides will be sufficient to identify a particular mRNA or gene. To confirm the uniqueness of the tags, we generated RT-PCR using oligo 24dT primers for the RT reaction. A 5 'primer containing the tag sequence itself was used for the second strand synthesis. Oligo 24dT was extended to the 5 'position extended by the -21M13 sequence. In the PCR reaction followed by the RT reaction, the -21M13 primer is used for amplification with the 5 'primer containing the tag sequence. The 5 'primer containing the tag sequence was extended with 5 inosine at its 5' position to increase the binding capacity of the primer. The sequence of the amplified fragment can be determined to confirm that this is the gene identified in the tag sequence. The RT-PCR reaction to confirm the tag was performed on the same tissue specimen used to prepare the expression profile of the tag sequence.
[0054]
procedure
Common buffers used in experiments
10 × RT buffer:
-500 mM TRIS, pH 8.3
-750 mM KCL
-30 mM MgCl_Two
-100 mM DTT
10x PCR buffer:
-200 mM TRIS pH 8.3
-500 mM KCL
-1 mg / ml BSA
1 ml of TRIzol reagent (Invitrogen Life Technologies cat. No. 15596) contains 10-100 mg of tissue or about 10⁷Immediately added to the cells (the tissue is sliced with a microtome to a thickness of 14 μm).
[0055]
Specimen total RNA isolation is performed according to the manufacturer's protocol as follows:
Add 0.2 ml chloroform (Merck) and shake the tube vigorously by hand for 15 seconds.
-Incubate for 5 minutes at room temperature.
Centrifuge at 12,000 xg or less at 4 ° C for 15 minutes.
• Transfer 600 μl of the colorless upper aqueous layer to a new tube. The lower organic layer should be red.
-Add 0.5 ml isopropyl alcohol (Merck) and stir.
-Incubate for 5 minutes at room temperature.
Centrifuge at 12,000 xg or less at 4 ° C for 15 minutes.
Discard the supernatant, wash the RNA pellet with 1 ml of 80% ethanol by vortexing, centrifuge at 7,500 xg or less at 4 ° C for 5 minutes, and discard the supernatant.
To keep the pellet dry, place the test tube at 56 ° C. for 3 minutes and mix poly A as described in the following column.⁺Continue RNA isolation.
[0056]
Poly A⁺RNA isolation was performed using Micro Fasttrack 2.0 poly A provided by the manufacturer Invitrogen Corporation, Carlsbad, USA, catalog number K1520).⁺The procedure was performed according to the manual of the mRNA isolation kit.
[0057]
Subsequently, the isolated poly A⁺RNA was used as input for analysis of the RT-PCR reaction. The RT-PCR reaction was started using a mixture of the following materials:
1.25 μl of 21M13 poly T primer (100 ng / μl)
2.0 x 10 x RT buffer
0.8 mM 100 mM dNTP (Pharmacia)
20U RNAsin (Roche) 0.3 μl
dH_TwoO (baker) 0.65 μl
・ Poly A⁺Add 10 μl dilution of mRNA to 5 μl of RT-mix.
Incubate the reaction solution at 65 ° C. for 45 minutes to anneal the primer to the template, then cool to room temperature.
Perform reverse transcription by adding 5 μl 1 U / μl AMV-RT to the reaction solution and incubating at 42 ° C. for 45 minutes.
-Immediately after reverse transcription, incubate the mixture at 95 ° C for 5 minutes to stop the reaction. Then, the reaction mixture is cooled to room temperature.
Add 80 μl of PCR-mix to each reaction mixture (100 μl total volume).
The PCR-mix per reaction is prepared as follows:
−5 ′ primer (100 ng / μl), see Table 5 1.0 μl
-21M13 (100 ng / μl) 1.0 μl
8.0 μl of -10 × PCR buffer
-100 mM MgCl_Two                              2.1 μl
-Amplitaq 5 U / μl (Perkin Elmer) 0.4 μl
-DH_TwoO (baker) 67.5 μl
-PCR amplification was carried out in a 9700 DNA thermal cycler (PerkinElmer) according to the following program.
5 minutes at -95 ° C
35 cycles of 1 minute at -95 ° C; 1 minute at 55 ° C; 2 minutes at 72 ° C.
10 minutes at -72 ° C
[0058]
TA-cloning of the RT-PCR product was performed using the TOPO TA Cloning® (pCR (registered) according to the kit manual provided by the manufacturer (Invitrogen Corporation, Carlsbad, USA, catalog number K4600). (TM) II-TOPO (R) Dual Promoter Vector and TOP10 One Shot (R) cells.
[0059]
Screening of clones was performed as follows using PCR with SP6 and T7 primers.
[0060]
-50 μldH of colonies_TwoResuspend in O (Baker). This bacterial suspension is suspended in 10 μl of the PCR mixture.
• Prepare the PCR-mix per reaction as follows:
SP6 (100 ng / μl), 0.10 μl
T7 (100 ng / μl) 0.10 μl
1.00 μl of 10 × PCR buffer
100 mM MgCl_Two                                0.20μl
0.08 μl of 100 mM dNTP's (Pharmacia)
Amplitaq 5U / μl (PerkinElmer) 0.04μl
dH_TwoO (baker) 7.48 μl
-PCR amplification was carried out in a 9700 DNA thermal cycler (PerkinElmer) according to the following program.
5 minutes at -95 ° C
35 cycles at -95 ° C for 30 seconds; 55 ° C for 30 seconds; 72 ° C for 1 minute.
10 minutes at -72 ° C
• Colony-5 μl of PCR product is run on a 1.5% agarose 1 × TBE gel and stained with Ethidium Bromide.
Make the amplification product visible on a UV-illuminator to identify insert-containing clones.
[0061]
Clones containing inserts were cloned from SP6 and T7 primers, and ABI Prism Big-Dye terminator cycle sequencing kit, PerkinElmer Applied Biosystems, Foster City, California, USA (Perkin Elmer Applied Biosystems, Foster City, Caif USA)).
[0062]
[Table 5]

[0063]
Confirmation result of tag sequence:
An oligo 24dT primer and a tag-based primer primer described above (tags 004, 007, 011, 012, 014, 015, 016, 017, 022, 025, 029, designated in Table 5) 030, 032, 036) confirmed the 18 tag sequences analyzed by the protocol described in this Example 14 using RT-PCR. Four types of tag sequences (designated tags 010, 013, 018, 019) could not be confirmed by this method. This was probably due to the fact that the tag-based primer was not specific enough or that the poly A tail of the mRNA was not long enough. Another more specific 5 'primer for one tag was designed to perform RT-PCR with -21M13 poly T (designated tag 004). For tag 010, a completely specific primer set was designed to perform reverse transcription and amplification. Other tags were identified using a specific RT-PCR primer set, followed by nested PCR with specific nested primer sets (designated tags 013, 018, 019). The results of all confirmed sequences are listed below and in the figures. Tag sequences in the mRNA sequence, if present, are shown in bold.
[0064]
TAG004 (EST AI217565, genbank number BE466728):
This was confirmed with the protocol of this example.

(Fig. 1)
[0065]
TAG007 (Keratin 14, genbank number XM — 008578): Confirmed with the protocol of this example.

(Fig. 2)
[0066]
TAG010 (ephrin A2, genbank number XM_002088):
RT-PCR with a 5 'primer designed on the catg-position (original primers are shown in Table 5) could not be confirmed. Possibly, tag-based primers are not sufficiently specific. Ephrin A2 specific primers were used for confirmation. The tag sequence is not included in the Ephrin-specific RT-PCR.

(Fig. 3)
[0067]
TAG011 (dual specificity phosphatase, genbank number XM_003720): Confirmed by the protocol of this example.

(FIG. 4)
[0068]
TAG012 (IL1 receptor, type 1, genbank number XM_002686): Confirmed with the protocol of this example.

(FIG. 5)
[0069]
TAG013 (ephrin B1; genbank number XM_002535, BC002524):
RT-PCR with the 5 'primer designed on the catg-position (original primers are shown in Table 5) could not be confirmed. Perhaps certainly, tag-based primers are not specific enough. Ephrin B1 specific primers were used for confirmation. The tag sequence is contained in the 3 'primer of the Ephrin B1 specific RT-PCR fragment. A nested PCR fragment is shown here and is located just upstream of the tag sequence.

(FIG. 6)
[0070]
TAG014 (AT group D protein, genbank number XM_006184, AF230388):
This was confirmed with the protocol of this example.

(FIG. 7)
[0071]
TAG015 (TIE1, genbank number XM_002037):
This was confirmed with the protocol of this example.

(FIG. 8)
[0072]
TAG016 (small ind. Cytokine A18, genbank number XM_008451, Y13710, AF111198):
This was confirmed with the protocol of this example.

(FIG. 9)
[0073]
TAG017 (complement, 1Q beta, genbank number XM_010666): Confirmed with the protocol of this example.

(FIG. 10)
[0074]
TAG018 (galectin 7, genbank number NM_002307, U06643):
RT-PCR with the 5 'primer designed on the catg-position (original primers are shown in Table 5) could not be confirmed. Perhaps certainly, tag-based primers are not specific enough. Galectin 7 specific primers were used for confirmation. The 5 'primer used in the galectin 7 specific primer contains a tag sequence. The tag sequence is contained in a galectin 7-specific RT-PCR fragment. A nested PCR fragment is shown here and is located just downstream of the tag sequence.

(FIG. 11)
[0075]
TAG019 (FGFR3, genbank number NM_022965, NM_000142):
RT-PCR with the 5 'primer designed on the catg-position (original primers are shown in Table 5) could not be confirmed. Perhaps certainly, tag-based primers are not specific enough. FGFR3 specific primers were used for confirmation. The tag sequence is not included in the Ephrin-specific RT-PCR. A nested-PCR fragment is shown here and is located upstream of the tag sequence.

(FIG. 12)
[0076]
TAG022 (Psoriasin (S100 A7)), genbank number XM_048120: Confirmed with the protocol of this example.

(FIG. 13)
[0077]
TAG025 (EST Unigene number Hs173789, genbank number XM_018404, AL137262):
This was confirmed with the protocol of this example.

(FIG. 14)
[0078]
TAG029 (PIG, genbank number XM_011453, AJ251830):
This was confirmed with the protocol of this example.

(FIG. 15)
[0079]
TAG030 (EST Unigene number Hs46987, genbank number DG151190, BG057289, BE858276, AV681759, BE503169):
This was confirmed with the protocol of this example.

(FIG. 16)
[0080]
TAG032 (SialoAdhesin, also called Siglec 1, genbank number XM_016245):
This was confirmed with the protocol of this example.

(FIG. 17)
[0081]
TAG036 (Dsmoplakin, genbank number XM_004463, NM_004415, AF139906):
This was confirmed with the protocol of this example.

(FIG. 18)
[0082]
Example 10
Measurement of gene expression level of tag sequence in skin samples
To obtain a feel for the use of the tag sequence as a marker of angiogenesis, skin samples with Kaposi's sarcoma lesions (5 different samples) and skin samples without Kaposi's sarcoma lesions (2 control samples) The genes identified in the sequence were analyzed for expression levels.
[0083]
procedure:
One ml of TRIzol reagent (Invitrogen Life Technologies cat. No. 15596) contains 10-100 mg of tissue or about 10⁷Immediately added to the cells (the tissue is sliced with a microtome to a thickness of 14 μm).
[0084]
Isolation of total RNA from the sample is performed according to the manufacturer's protocol as follows:
-Add 0.2 ml of chloroform (Merck) and shake the tube vigorously by hand for 15 seconds.
-Incubate at room temperature for 5 minutes.
Centrifuge at 12,000 xg or less at 4 ° C for 15 minutes.
• Transfer 600 μl of the colorless upper aqueous layer to a new tube. The lower organic layer should be red.
-Add 0.5 ml isopropyl alcohol (Merck) and mix.
-Incubate for 10 minutes at room temperature.
Centrifuge at 12,000 xg or less at 4 ° C for 15 minutes.
Discard the supernatant, wash the RNA pellet with 1 ml of 80% ethanol by vortexing, centrifuge at 4 ° C. for 5 minutes at 7,500 × g or less, and discard the supernatant.
-To dry the pellet, place the test tube at 56 ° C for 3 minutes and treat it with DNase as described in the following column.
[0085]
To confirm the absence of genomic DNA in all isolated RNAs, we perform a DNase treatment and add an RNase inhibitor (RNasin) to the DNase reaction. Protected the RNA against the DNase I RNase activity. DNase treatment was performed as follows:
• After isolation of Trizol total RNA, pipette 88 μl dH_TwoResuspend in O.
Add the following sequentially to the RNA solution:
-10 μl 10 × DNase buffer (Ambion)
-1 μl 40 U / μl RNase inhibitor (RNasin, Roche)
-1 μl 10 U / μl DNAse I (DNase I, Roche) /
Incubate at 37 ° C for 1 hour.
100 μl dH_TwoAdd O to increase the sample volume to 200 μl.
Add 200 μl of cold phenol / chloroform pH8 (PC8).
Centrifuge at full speed for 5 minutes at room temperature in a microcentrifuge.
• Transfer the aqueous upper layer to a new microcentrifuge tube and add, in order:
-3 μl glycogen (Roche)
-100 μl 10 M ammonium acetate
-700 μl 100% ethanol
-Mix thoroughly, centrifuge at 25,000 xg for 15 minutes at 4 ° C, decant and pour off supernatant.
Wash twice with 700 μl of 80% ethanol. Mix thoroughly, centrifuge at 25,000 × g for 5 minutes at 4 ° C., decant and pour off supernatant.
Dry the pellet at 56 ° C. for 5 minutes and add 11 μl dH_TwoResuspend in O.
1 μl of RNA isolate to 140 μl of dH_TwoDiluted to O, OD₂₆₀Calculate the RNA concentration and yield of the isolate by measurement. The yield of DNase-treated total RNA should be at least 13.5 μg for determination of the complete tag expression profile.
Prepare a solution of RNA isolate at a concentration of 50 ng / μl. This is the base RNA solution for dilution for TAG-specific RT-PCR.
[0086]
For each sample of DNase-treated total RNA, 18 TAG-specific RT-PCR / Nested PCR reactions were performed in a dilution series of RNA. Five control RT-PCRs for each sample will be performed in a dilution series: HIV-1 GAG (SK39 / 145), GAPDH + RT (2 times) and no RT reaction (2 times). The following dilution scheme was used to obtain dilutions of the series of samples analyzed by RT-PCR:
1. 270 μl 50 ng / μl total RNA treated with DNAse.
10 μl 50 ng / μl solution = 500 ng as input amount in PT-PCR of each primer set.
2. 27 μl 50 ng / μl total RNA 10 × dilution = 270 μl 5 ng / μl.
10 μl 5 ng / μl solution = 50 ng as input amount in PT-PCR of each primer set.
3. 27 μl 5 ng / μl total RNA 10 × dilution = 270 μl 0.5 ng / μl.
10 μl 0.5 ng / μl solution = 5 ng as input amount in PT-PCR of each primer set.
4. 27 μl 0.5 ng / μl total RNA 10 × dilution = 270 μl 0.05 ng / μl.
10 μl 0.05 ng / μl solution = 0.5 ng as input amount in PT-PCR of each primer set.
5. 27 μl 0.05 ng / μl total RNA 10 × dilution = 270 μl 0.005 ng / μl.
10 μl 0.005 ng / μl solution = 0.05 ng as input amount in PT-PCR of each primer set.
6. 27 μl 0.005 ng / μl total RNA 10 × dilution = 270 μl 0.0005 ng / μl.
10 µl 0.0005 ng / µl solution = 0.005 ng as input amount in PT-PCR of each primer set.
7. 27 μl 0.0005 ng / μl total RNA 10 × dilution = 270 μl 0.00005 ng / μl.
10 μl 0.00005 ng / μl solution = 0.0005 ng as the input amount in PT-PCR of each primer set.
8. 27 μl 0.00005 ng / μl total RNA 10 × dilution = 270 μl 0.000005 ng / μl.
10 μl 0.000005 ng / μl solution = 0.00005 ng as input amount in PT-PCR of each primer set.
[0087]
TAG-specific RT-PCR in a diluted total RNA series using AMV-RT was performed as follows. Reverse transcription reactions of all TAGs on DNase-treated total RNA in the dilution series were performed in 96-well PCR plates. 10 μl of each total RNA dilution was used as input for RT PCR. The reaction volume of the reverse transcription reaction was 20 μl, and dNTP, MgCl_TwoAnd an RNase inhibitor (RNasin).
[0088]
• Prepare the RT mixture for each reaction (RT-mix) as follows:
3 'primer (100 ng / μl) See Table 6 1.25 μl
2.0 × 10 × RT buffer
0.8 mM 100 mM dNTP (Pharmacia)
20 U RNAsin (Roche) 0.3 μl
dH_TwoO (Baker) 0.65 μl
Add 10 μl of total RNA dilution to 5 μl of RT mix (RT-mix).
Incubate the reaction solution at 65 ° C. for 5 minutes to anneal the primer to the template, then cool to room temperature.
Perform reverse transcription by adding 5 μl 1 U / μl AMV-RT to the reaction solution and incubating at 42 ° C. for 45 minutes.
-Immediately after reverse transcription, incubate the mixture at 95 ° C for 5 minutes to stop the reaction. Then, the reaction is cooled to room temperature.
Add 80 μl of PCR anti-mix (PCR-mix) to each reaction mixture (total volume 100 μl).
The PCR mixture per reaction (PCR-mix) is prepared as follows:
−5 ′ primer (100 ng / μl), see Table 6 1.0 μl
8.0 μl of -10 × PCR buffer
-100 mM MgCl_Two                            1.9 μl
-Amplitaq 5U / μ 0.4μl
-DH_TwoO (Baker) 68.7 μl
-PCR amplification is performed in a 9700 DNA thermal cycler (PerkinElmer) according to the following program.
5 minutes at -95 ° C
35 cycles of 1 minute at -95 ° C; 1 minute at 55 ° C; 2 minutes at 72 ° C.
10 minutes at -72 ° C
[0089]
Following this initial amplification, a second round of nested amplification was performed. A second round of TAG-specific nested PCR on the RT-PCR product was performed as follows:
Add 5 μl of the TAG RT-PCR product to 45 μl of the nested-PCR mix.
• Prepare a nested-PCR mix for each reaction as follows:
5 'nested primer (100 ng / μl), see Table 7 0.5 μl
3 'nested primer (100 ng / μl), see Table 7 0.5 μl
5.0 μl of 10 × PCR buffer
100 mM MgCl_Two                             1.25 μl
0.4 mM 100 mM dNTP (Pharmacia)
Amplitaq 5U / μl 0.2μl
dH_TwoO (baker) 37.15 μl
The primer combinations and the lengths of the amplified fragments used for amplification of the gene identified by the tag are shown in Table 8.
-PCR amplification is performed according to the following program in a 9700 DNA thermal cycler (Perkin Elmer).
5 minutes at -95 ° C
25 cycles of 1 minute at -95 ° C; 1 minute at 55 ° C; 2 minutes at 72 ° C.
10 minutes at -72 ° C
• Run 10 μl of the nested PCR product on a 1.5% agarose 1 × TBE gel and stain with Ethidium Bromide.
Visualization of the TAG amplified fragments of the dilution series on a UV-illuminator reveals the expression level by determining the highest dilution that shows a clear signal even when diluted.
[0090]
[Table 6]

[0091]
[Table 7]

[0092]
[Table 8]

[0093]
result
The results of measuring the expression level of the gene identified by the tag sequence are shown in FIG. The data clearly shows that the number of genes identified by the tag sequence is more highly expressed in skin specimens of Kaposi's sarcoma lesion compared to normal skin: tag 007, tag 010, tag 012, tag 013, Tags 014, 015, 016, 017, 022, 029, 030, 032, and 036.
[0094]
Example 11
Measurement of gene expression level of tag sequence in peripheral blood mononuclear cell (PBMC) specimen
Feel the use of the tag sequence as a marker of the angiogenesis process in samples available from the blood (PBMC), but not in angiogenesis sites, ie, Kaposi's sarcoma in the skin or other cancers in the body To obtain the expression of five tag-identified genes was measured in PBMC samples. PBMC samples were derived from the blood of patients with Kaposi's sarcoma lesion (4 different samples) and not (2 control samples), and were included in PBMCs of 5 genes identified by the tag sequence. Was analyzed for expression.
[0095]
The procedure of this example is the same as that described in Example 10, except that a PBMC specimen (about 10 million cells per sample, in the range of 2.5 to 50 million) is used instead of a skin biopsy Met. The genes analyzed were identified by tag 007, tag 017, tag 010, tag 013, tag 015, tag 029 and tag 032. The analysis result is shown in FIG. From these data, it can be seen that the enhanced expression at the cancer site of the genes identified with tag 015 (TIE1) and tag 032 (Salioadhesin or Siglec 1) was expressed in the blood cell fraction, ie, PBMC. It is clear that they correspond.
[0096]
result
This data clearly shows that overexpression of the gene identified in skin samples (see Example 5) with tag 007 (keratin 14) does not correspond to blood. In contrast, in the samples tested in this example, the gene identified by tag 007 is not expressed at all in the blood fraction. This indicates that cancer cells expressing tag 007 are not present in the blood. As a result, measurement of tag 007 in blood would be a good label for the presence of these cancer cells in the blood, and thus would be a marker for circulating cancer cells that could undergo metastasis.
[0097]
Up-regulation of the genes identified by tag 015 (TIE1) and tag 032 (Salioadhesin or Siglec 1) in skin specimens clearly correlates with blood. These two tags are highly expressed in the blood of cancer-bearing patients as compared to healthy individuals. This upregulation is due to the upregulation of expression in typical blood cells.
[0098]
This upregulation cannot be attributed to the presence of cancer cells expressing tag 015 and tag 032 in the blood because the absence of the gene expression identified by tag 007, as explained above, This is because it indicates that no cancer cells are present. This means that measuring the expression of tag 015 and / or tag 032 in the blood indicates the presence of cancer cells elsewhere in the body. In addition, measuring the expression of a gene identified by tag 015 and / or tag 032 during anti-cancer and / or anti-angiogenic treatment can be used to monitor the efficacy of this treatment.
[0099]
Conclusion
Correlated up-regulation of genes identified in cancer and blood by tags 015 (TIE1) and tag 032 (Salioadhesin or Siglec 1) may be used in treatments aimed at reducing cancer growth, In particular, it allows to monitor the effectiveness of anti-angiogenic cancer treatment. This means that if the two genes are markers in PBMC for angiogenesis in cancer elsewhere in the body, the two markers in the blood will also cause this angiogenesis in the cancer side. It will be inferred that it will decrease with the decrease of.
[0100]
Genes identified by tag 007 (keratin 14), tag 015 (TIE1) and tag 032 (Salioadhesin or Siglec 1) differed in the blood of cancer-bearing patients compared to normal individuals. Has been expressed. Thus, these genes, in particular, the expression of which can be used for mass screening of populations at risk for the presence of cancer in each member of the population.
[0101]
All genes identified by tags in this study that showed altered expression levels compared to normal and cancer tissues: tag007, tag010, tag012, tag013, tag014, tag015, tag016, Tag 017, Tag 022, Tag 029, Tag 030, Tag 032 and Tag 036 encode potential target molecules as therapeutic agents with anti-angiogenic effects and applicable to cancer treatment. And / or these genes encode potential target molecules that can be potential target molecules for therapeutic agents that stimulate vascular growth, for example, in the treatment of heart and coronary artery disease.
[Brief description of the drawings]
[0102]
FIG. 1. Sequences involved in angiogenesis. Changes in the expression of this sequence after a particular treatment indicate that the treatment is efficacious. This sequence is identical to the EST sequence identified from human fetal heart (GenBank accession number AI217565 and others) so that it is consistent with the predicted exon on chromosome 19. The relationship with angiogenesis has never been described.
FIG. 2: Sequence identified by name and Genbank number (NCBI database). Other identifications can be found in Tables 1-4 (Unigene numbers), which can be found in NCBI's SAGE database.
FIG. 3 shows a sequence identified by a name and a Genbank number (NCBI database). Other identifications can be found in Tables 1-4 (Unigene numbers), which can be found in NCBI's SAGE database.
FIG. 4: Sequences identified by name and Genbank number (NCBI database). Other identifications can be found in Tables 1-4 (Unigene numbers), which can be found in NCBI's SAGE database.
FIG. 5: Sequence identified by name and Genbank number (NCBI database). Other identities can be found in Tables 1-4 (Unigene numbers), which can be found in NCBI's SAGE database.
FIG. 6: Sequence identified by name and Genbank number (NCBI database). Other identifications can be found in Tables 1-4 (Unigene numbers), which can be found in NCBI's SAGE database.
FIG. 7: Sequence identified by name and Genbank number (NCBI database). Other identifications can be found in Tables 1-4 (Unigene numbers), which can be found in NCBI's SAGE database.
FIG. 8: Sequence identified by name and Genbank number (NCBI database). Other identifications can be found in Tables 1-4 (Unigene numbers), which can be found in NCBI's SAGE database.
FIG. 9: Sequence identified by name and Genbank number (NCBI database). Other identifications can be found in Tables 1-4 (Unigene numbers), which can be found in NCBI's SAGE database.
FIG. 10: Sequence identified by name and Genbank number (NCBI database). Other identifications can be found in Tables 1-4 (Unigene numbers), which can be found in NCBI's SAGE database.
FIG. 11: Sequence identified by name and Genbank number (NCBI database). Other identifications can be found in Tables 1-4 (Unigene numbers), which can be found in NCBI's SAGE database.
FIG. 12: Sequence identified by name and Genbank number (NCBI database). Other identifications can be found in Tables 1-4 (Unigene numbers), which can be found in NCBI's SAGE database.
FIG. 13: Sequence identified by name and Genbank number (NCBI database). Other identifications can be found in Tables 1-4 (Unigene numbers), which can be found in NCBI's SAGE database.
FIG. 14: Sequence identified by name and Genbank number (NCBI database). Other identifications can be found in Tables 1-4 (Unigene numbers), which can be found in NCBI's SAGE database.
FIG. 15: Sequence identified by name and Genbank number (NCBI database). Other identifications can be found in Tables 1-4 (Unigene numbers), which can be found in NCBI's SAGE database.
FIG. 16: Sequence identified by name and Genbank number (NCBI database). Other identifications can be found in Tables 1-4 (Unigene numbers), which can be found in NCBI's SAGE database.
FIG. 17: Sequence identified by name and Genbank number (NCBI database). Other identifications can be found in Tables 1-4 (Unigene numbers), which can be found in NCBI's SAGE database.
FIG. 18: Sequence identified by name and Genbank number (NCBI database). Other identifications can be found in Tables 1-4 (Unigene numbers), which can be found in NCBI's SAGE database.
FIG. 19: Log dilution factor that remains positive even when starting to dilute from 500 ng of total RNA isolated from five Kaposi's sarcoma skin samples (light colored bars) and two control normal skin samples (black bars). Mean expression level and standard deviation, indicated as.
FIG. 20: A log dilution factor that remains positive after initiating dilution from 500 ng of total RNA isolated from 4 PBMC samples (light colored bars) and two control normal PBMC samples (black bars) of a Kaposi's sarcoma patient Mean expression level and standard deviation, indicated as.

Claims (41)

A method of determining whether a treatment is effective in altering a particular group of target cell states in an individual, comprising:
A method comprising: obtaining a sample from the individual after initiation of treatment; and determining whether the sample contains an expression product of at least one marker gene. The method of claim 1, wherein the target cells include cancer cells. 3. The method according to claim 1 or claim 2, wherein the specimen comprises at least one target cell. The method according to any one of claims 1 to 3, wherein the specimen is collected within one week of starting the treatment. The method according to any one of claims 1 to 4, wherein the specimen is collected within 2 days of the start of the treatment. The method according to any one of claims 1 to 5, wherein the marker gene comprises a gene involved in blood vessel generation, maintenance and / or damage. The method according to any one of claims 1 to 6, wherein the marker gene comprises a sequence shown in Table 1 or 2. The method according to any one of claims 1 to 7, wherein the marker gene comprises the sequence shown in FIGS. 1 to 18, or a part thereof, or an analog of the sequence. The method according to any one of claims 1 to 8, wherein the expression of the marker gene is quantified. 10. The method according to any one of claims 1 to 9, further comprising comparing the expression of the marker gene with a control. 11. The method according to any one of claims 2 to 10, wherein the cancer comprises Kaposi's sarcoma. Use of a nucleic acid comprising a sequence shown in FIGS. 1 to 18 and / or Table 1 or 2 or a part or analog thereof in a method for detecting an expression product. Use of a protein-like molecule having the ability to specifically bind to a protein encoded by a nucleic acid comprising the sequence shown in FIGS. 1-18 and / or Table 1 or 2 or a portion or analog thereof in a detection method. 14. Use according to claim 12 or claim 13 for measuring the presence of cancer cells in an individual. 14. Use according to claim 12 or claim 13 for determining the presence of an angiogenic site in an individual. 14. Use according to claim 12 or claim 13 for determining whether a treatment is effective in altering a particular group of target cell status in an individual. 17. Use according to any one of claims 12 to 16 for determining whether a treatment is effective in counteracting cancer in an individual. 18. Use according to claim 14 or claim 17, wherein the cancer comprises Kaposi's sarcoma. A method for determining whether an individual comprises cancer cells and / or angiogenesis sites,
Collecting a specimen from the individual; and measuring whether the specimen contains an expression product of at least one marker gene. 20. The method of claim 19, wherein the marker gene comprises a sequence shown in Table 1 or 2 and / or Figures 1-18, or a portion or analog thereof. A method for determining whether an individual comprises non-hematopoietic cancer cells and / or an angiogenic site,
Determining whether the hematopoietic cells from the patient contain an altered amount of a marker gene expression product as compared to a control value. 22. The method according to claim 21, wherein the marker gene comprises a gene involved in angiogenesis. 23. The method according to claim 21 or claim 22, wherein the marker gene comprises the sequence shown in Table 1 or 2 and / or Figures 1 to 18, or a part or analog thereof. 24. The method according to any one of claims 21 to 23, wherein the hematopoietic cells comprise peripheral blood mononuclear cells. A method of determining whether a treatment is effective in altering an angiogenic process in an individual, comprising:
Collecting a sample from the individual after initiating the treatment; and determining whether the sample contains an expression product of at least one marker gene. 26. The method of claim 25, wherein treating comprises preventing angiogenesis in the patient. 27. The method according to claim 25 or claim 26, wherein the marker gene comprises the sequence shown in Table 1, Table 2 or Figures 1 to 18, or a part or analog thereof. Drugs with at least the following treatments:
2ME2, angiostatin, angiozyme, angiozyme, anti-VEGF (Anti-VEGF) RhuMAb, apra (Apra (CT-2584)), avicin (Avicine), benefin (Benefin), BMS275291, carboxamidotriazole, CC4047, CC5013, CC7085, CDC801, CGP-41251 (PKC412), CM101, Combretastatin A-4 prodrug, EMD121974, Endostatin, Flavopiridol (Flavopiridol), Genistein (Genistein (GCP)) ), Green tea extract, IM-862, Imm Ther, interferon alfa, interleukin-12, Iressa (ZD1839), marimastat (Marimastat), metastat (Costat) -3)), Neovastat, Octreotide, Paclitaxel, Penicillamine, Photofrin, Photopoint, PI-88, Prinomastat (AG-3340)) PTK787 (ZK22584), RO317453, Solimastat, Squalamin, SU101, SU5416, SU-6668, Surradista (FCE26644), Suramin (Metaret, Tetrathiomolybdate) Tetrathiomolybdate), thalidomide, TNP-470, Vitaxin
28. The method according to any one of claims 25 to 27, wherein at least one of the following is used. The method according to any one of claims 1 to 11, 19 to 20, or 23 to 26, wherein the specimen is a blood specimen. The method according to any one of claims 1 to 11, 19 to 20, or 24 to 27, wherein the specimen comprises peripheral blood mononuclear cells. 20. The expression product comprises a TIE1 sequence, a Salioadhesin or Siglec 1 sequence, the sequence shown in FIG. 8 or 17, or a part or analog thereof. 31. The method according to any one of claims 30 to 30. An indication of angiogenesis of PBMCs in which the keratin 14 sequence, TIE1 sequence, Salioadhesin or Siglec 1 sequence, the sequence shown in FIG. 2, 8 or 17 or a part or analog thereof are expressed Use as. PBMC expressing the keratin 14 sequence, TIE1 sequence, Salioadhesin or Siglec 1 sequence, the sequence shown in FIG. 2, 8 or 17, or a portion or analog thereof, in a cancer cell in an individual. Use to determine presence. An isolated keratin 14 sequence, TIE1 sequence, Salioadhesin or Siglec 1 sequence, a sequence shown in FIG. 2, 8 or 17, or a portion or analog thereof, for use in a diagnostic method. 1-18 and / or Table 1 or 2 or a nucleic acid comprising a portion or analog thereof, and / or a protein-like molecule capable of specifically binding to a protein encoded by the nucleic acid or a portion or analog thereof A diagnostic kit comprising: Characterized in that it comprises the keratin 14 sequence and / or TIE1 sequence, and / or the Salioadhesin or Siglec 1 sequence, and / or the sequence shown in FIG. 2, 8 or 17, or a part or analog thereof. The diagnostic kit according to claim 35, wherein 37. The method of claim 35 or claim 36 for determining whether a treatment is effective in altering a particular group of target cell states in an individual and / or altering angiogenic processes in the individual. Use of diagnostic kits. 37. Use of a diagnostic kit according to claim 35 or claim 36 for determining whether an individual contains cancer cells or an angiogenic site. Use of a gene expression product comprising the sequence shown in Figures 1-18, Table 1 or Table 2 as a drug target. Capable of altering the activity of Salioadhesin or Siglec 1, TIE1, keratin 14 and / or expressing Salioadhesin or Siglec 1, TIE1 and / or keratin 14 in cells A substance that has the ability to change Use of a substance according to claim 40 for the preparation of a medicament.

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