JP2004049122A - Gene exhibiting disease type-specific change in expression in various types of cancer, and utilization thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for utilizing a gene specific to various types of cancer. <P>SOLUTION: Hybridization is carried out on slides obtained by preparing RNAs from 38 cell strains derived from lung cancer, 3 normal fibroblast strains, 43 clinical specimens of the lung cancer and 6 normal pulmonary tissues, labeling them with a fluorescent material, and immobilizing 7685 kinds of gene fragments thereon. Usual hierarchical clustering is applied thereto, and the whole cell strains and clinical specimens are separated to each different cluster. The gene expression profile influenced by each type of diseases is considered to be obtained by removing genes commonly highly expressing or lowly expressing in the cultured cell strains or the clinical specimens from the analysis, and a filtering method enabling the removal is newly considered and carried out. As a result, the gene exhibiting disease type-specific expression profile can be identified. The gene is considered to be useful in diagnosis and therapy of each type of the cancer. <P>COPYRIGHT: (C)2004,JPO

Description

【００７８】
【表２】

【００７９】
臨床検体は（財）癌研究会癌研究所にて患者のｉｎｆｏｒｍｅｄ　ｃｏｎｓｅｎｔおよび倫理委員会の承認のもとで外科的摘除時に採取したものである。レファレンスサンプルとしては可能な限り多くの遺伝子について情報が得られる事を考慮して、２０％の正常肺組織にＮＣＩ−Ｈ２２６、ＮＣＩ−Ｈ５２２、ＥＫＶＸ、ＮＣＩ−Ｈ４６０、Ｌｕ−１３４Ａ、ＭＲＣ５、ＳＱ５、Ｈ２３、ＰＣ１４、ＭＳ１の１０種の細胞株を夫々８％ずつ加えたものを用いた。全ての細胞株はＲＮＡｌａｔｅｒ　（Ａｍｂｉｏｎ社製）を用いて細胞浮遊液とした。臨床検体はＲＮＡの分解を最小限にすべく採取後液体窒素にて瞬時に凍結せしめた。Ｉｓｏｇｅｎ（ニッポンジーン社製）を用いてＲＮＡを抽出後、Ｌｕｏ，　Ｌ．等の方法（Ｎａｔ．　Ｍｅｄ．　（１９９９）　５　：　１１７−１２２）に従ってｃＲＮＡ合成を行った。プローブ合成はＨｕｇｈｅｓ，　ＴＲ等の方法（Ｎａｔ．　Ｂｉｏｔｅｃｈｎｏｌ．（２００１）　１９　：　３４２−３４７）に従って２ｕｇのｃＲＮＡを鋳型として行った。レファレンスサンプルはＣｙ３にてテストサンプルはＣｙ５にて蛍光標識した。ハイブリダイゼーションとその後の洗浄はＨｅｇｄｅ，　Ｐ．等の方法（前出）に従った。ハイブリダイズしたシグナルの測定と定量はＳｃａｎｎａｒｒａｙ　４０００スキャナーとＱｕａｎｔａｒｒａｙ　（何れもＰａｃｋａｒｄ　Ｂｉｏｓｃｉｅｎｃｅ社製）を用いて行った。
【００８０】
［実施例３］　データ解析
全てのｃＲＮＡサンプルはｄｕｐｌｉｃａｔｅにて蛍光標識を行った。標識後のプローブを７６８５個のＤＮＡスポットがｄｕｐｌｉｃａｔｅで貼り付けてあるスライドにハイブリダイズさせる事により、個々の遺伝子について４回の独立した測定を行った。スポットした７６８５　ｅｌｅｍｅｎｔ中２５２はｃｏｎｔｒｏｌとして加えたもの（Ｌｕｃｉｆｅｒａｓｅ、Ａｌｕ等）で残る７４３３のうち６３８４は５６２２個のＵｎｉＧｅｎｅクラスターに相当し、１０４９はＵｎｉＧｅｎｅクラスターに一致しない事から全体で６６７１個のユニークな遺伝子がスポットされている事になる。さらに初期の検討結果から、このうち２８５は全ての臨床検体・細胞株を含めた異なる検体間で同様な発現パターンを示した事から以後の解析から除外した。この初期フィルタリングの結果、６３８６のユニークな遺伝子が以下の解析の対象として残った（図１）。１回のアレイ実験内でのデータの標準化はｌｏｗｅｓｓ　ｎｏｒｍａｌｉｚａｔｉｏｎ法（Ｎｕｃｌｅｉｃ　Ａｃｉｄ　Ｒｅｓ．　（２００２），　３０　：　ｅ１５）に準拠し、標準化されたシグナル強度をレファレンスサンプルのシグナル強度で割る事により値を得た後に、Ｔｓｅｎｇ等のプログラム（Ｎｕｃｌｅｉｃ　Ａｃｉｄ　Ｒｅｓ．（２００１），　２９　：　２５４９−２５５７）を用いてＣＶ値が９６％信頼区間から外れるデータをｏｕｔｌｉｅｒとして以後の解析から除外した。得られたデータセットはＧｅｎｅＳｐｒｉｎｇ（Ｓｉｌｉｃｏｎ　Ｇｅｎｅｔｉｃｓ社製）を用いてクラスター解析、視覚化を行った（図２）。腫瘍臨床検体と細胞株のデータの直接比較をした場合に異なる病型に関わらず臨床検体に共通して高い発現を示す遺伝子、逆に細胞株に共通して高い発現を示す遺伝子が認められた。これらの遺伝子を選別する目的でＢｉｎａｒｙ　Ｓｅａｒｃｈ　Ａｌｇｏｒｉｔｈｍを新たに考えた。すなはち、個々の遺伝子について全てのサンプルでのシグナルの平均値を求めこれより高い値を示すものに”１”、低い値を示すものに”０”の値を与える事により個々のサンプルのプロファイルをＢｉｎａｒｙ　Ｓｔｒｉｎｇｓとして表現した。これをｉｄｅａｌ　ｓｔａｔｅ（臨床検体で全て”１”／細胞株で全て”０”あるいはその逆）のｓｔｒｉｎｇと比較して６６％以上のサンプルで”１”の場合、”ｏｖｅｒｅｘｐｒｅｓｓｅｄ”、６６％以上のサンプルで”０”の場合、”ｕｎｄｅｒｅｘｐｒｅｓｓｅｄ”とした。この処理により６３８６のユニークな遺伝子中２２１８が臨床検体で細胞株と比較して発現が高い（１３１３）あるいは低い（９０５）ものとして選別された（図３）。さらにこれら２２１８遺伝子のクラスタリングの結果を目で見て確認した結果、２９９の遺伝子は臨床検体と細胞株間で際立った発現の差を認めなかった事から残る１９１９を以下の検討からは除去する事とした（図４）。全てのフィルタリングの結果最終的に４２４０の遺伝子が解析の対象として残った。Ｈｉｅｒａｒｃｈｉｃａｌ　ｃｌｕｓｔｅｒｉｎｇの結果（図５、図６）、９０の検体（肺癌臨床検体４３、正常肺臨床検体６、肺癌細胞株３８、正常胎児繊維芽細胞株３）は４つの大きなｂｒａｎｃｈに分かれた（表３）。
【００８１】
【表３】

ＳＣＬＣは小細胞性肺癌、ＳＣＣは扁平上皮癌、ＮＳＣＬＣは非小細胞性肺癌、ＡＣは腺癌、ＬＣＣは大細胞性癌を示す。また、＊は、非小細胞性肺癌由来細胞株（病型分類不明）を示す。
【００８２】
Ｂｒａｎｃｈ　１は殆ど全ての小細胞性肺癌由来細胞株と全ての小細胞性肺癌とカルチノイドの臨床検体を含む。Ｂｒａｎｃｈ　２には扁平上皮癌の臨床検体と細胞株が多く含まれ、Ｂｒａｎｃｈ　３は細胞株のｂｒａｎｃｈで胎児由来正常繊維芽細胞株は全てこのｂｒａｎｃｈに含まれる。Ｂｒａｎｃｈ　４には全ての腺癌、大細胞癌と正常肺の臨床検体が含まれる。２株の非小細胞性肺癌由来細胞株（病型分類不明）は上記４つのｂｒａｎｃｈからは独立したｂｒａｎｃｈを形成している。Ｂｉｎａｒｙ　Ｓｅａｒｃｈ　Ａｌｇｏｒｉｔｈｍを適用して、細胞株全般に固有の遺伝子発現プロファイルおよび臨床検体全般に固有の遺伝子発現プロファイルを除去する事により、それまで困難であった臨床検体と細胞株を直接比較する事が可能となった。特に小細胞性肺癌由来細胞株の大部分（１１／１３）と扁平上皮癌由来細胞株の一部（４／８）は夫々の細胞株が由来する病型の臨床検体クラスターに帰属された事からこれらの細胞株はもとの腫瘍の形質をよく保持していると考えられた。腺癌については今回実施したフィルタリング・クラスタリングでは正常肺組織との分離が不充分でかつ腺癌由来細胞株の帰属は得られなかった（０／１１）。この理由として、腺癌臨床検体が異なる分化度の症例からなっており、正常肺組織の混入度が高い為正常肺組織とのクラスター分離が不充分であったと考えられる一方、腺癌由来細胞株は由来する癌組織中の最も未分化な細胞が株化の過程で選択された事によると考えられた。
【００８３】
以下に、各種病型肺癌で病型特異的に発現変化を示す遺伝子一覧を示す。
（１）正常肺組織で高発現していて癌化で低下するもの（順に配列番号：１〜１７に示す）
【表４】

【００８４】
（２）小細胞性肺癌で高発現を示すもの（順に配列番号：１８〜６１に示す）
【表５】

【００８５】
（３）扁平上皮癌で高発現を示すもの（順に配列番号：６２〜１０７に示す）
【表６】

【００８６】
（４）カルチノイドで高発現を示すもの（順に配列番号：１０８〜１１７に示す）
【表７】

【００８７】
（５）腺癌および正常肺組織で高発現を示すもの（順に配列番号：１１８〜１２９に示す）
【表８】

【００８８】
【発明の効果】
本発明によって、病型特異的な癌の検査や治療が可能になるものと期待される。例えば、本発明者らが見出した各種病型癌に特異的なＤＮＡを利用することで、癌の早期検査や病型検査が可能になる。また、該ＤＮＡのプロモーターを利用することで、病型特異的な癌の遺伝子治療が可能になる。さらに、該ＤＮＡの発現プロファイルを指標として抗癌活性を有する試料のスクリーニングが可能になる。
【００８９】
【配列表】

【図面の簡単な説明】
【図１】６３８６の遺伝子の２次元階層的クラスター化の樹状図を示す写真である。タイプに応じて着色された標本が、左側に示され、赤はＡＣ、緑はＳＣＣ、紫は正常、青はＳＣＬＣ、黒はＬＣＣ；　オレンジ色はカルチノイド、茶色は線維芽細胞、ピンクはＮＳＬＣを表わしている。各欄は、特定の遺伝子を表わす。正方形は、４つの複製にわたる対数平均発現比率に従って着色されている。基準標本との関係において、赤は１より大きい発現比率（過剰発現）、緑は１未満（過小発現）、黒はほぼ１に等しい比率（発現変化無し）を表わしている。
【図２】本文中に記述されているように統計的にろ過することによって得られた細胞系統中の５２５５の遺伝子の２次元階層的クラスター化の樹状図を示す写真である。タイプに応じて着色された標本が左側に示され、赤はＡＣ、緑はＳＣＣ、青はＳＣＬＣ、茶色は線維芽細胞、ピンクはＮＳＬＣを表わしている。各欄は、特定の遺伝子を表わす。正方形は４つの複製にわたる対数平均発現比率に従って着色されている。基準標本との関係において、赤は１より大きい発現比率（過剰発現）、緑は１未満（過小発現）、黒はほぼ１に等しい比率（発現変化無し）を表わしている。
【図３】腫瘍及び細胞系統の間で示差的に調節された遺伝子の同定を示す図および写真である。（Ａ）新鮮な標本に比べ細胞系統標本内で一般に過剰発現された９０５の遺伝子及び（Ｂ）新鮮な標本に比べて細胞系統標本中で一般に過小発現された１３１３の遺伝子についての全ての標本にわたる発現変化。（Ｃ）新鮮な標本と細胞系統標本の間で示差的に調節されている１９１９の遺伝子の２次元階層的クラスター化。左側の樹状図は、細胞系統標本と新鮮な標本のクラスター化を示す。分岐色は、図１に示されたとおりである。上部樹状図は、遺伝子のクラスター化を示している。
【図４】新鮮な標本と細胞系統標本の間で示差的に調節されるＢｉｎａｒｙ　Ｓｅａｒｃｈ　Ａｌｇｏｒｉｔｈｍによって選択され２２１８の遺伝子の２次元階層的クラスター化の樹状図を示す写真である。左側の樹状図は、細胞系統標本と新鮮な標本のクラスター化を示す。上部樹状図は、遺伝子のクラスター化を示す。新鮮な標本中でアップレギュレートされた遺伝子は右に向かってクラスター化している状態で示され、細胞系統標本中でアップレギュレートされた遺伝子は、左に向かってクラスター化している状態で示されている。主要データセットに内含させるのに、新鮮な標本と細胞系統標本の間で著しい対比を示さない遺伝子分岐（合計２９９）が選定された。
【図５】新鮮な標本又は細胞系統標本内の一般的に調節された遺伝子についてろ過した後の４２４０の遺伝子の削減されたデータセットの樹状図を示す写真である。標本は、図１の場合と同様に着色されている。左側に示されたグループは、特定の癌腫タイプの全く異なるクラスターを表わす。ｃｌ：細胞系統標本；　ｆｒ：新鮮な腫瘍標本。
【図６】全く異なる発現パターンを示す、図５からの８つの選択された遺伝子クラスターの全ての標本にわたる発現プロファイルを示す写真である。図５からの樹状図及び強調されたクラスターは、参考として底部に沿って及び個々の遺伝子クラスターの間に示されている。（Ａ）カルチノイド内でアップレギュレートされた遺伝子。（Ｂ）正常組織内でアップレギュレートされた遺伝子。（Ｃ）ＡＣ及びＬＣＣ内でアップレギュレートされた遺伝子。（Ｄ〜Ｅ）ＳＣＣ内でアップレギュレートされた遺伝子。（Ｆ〜Ｈ）ＳＣＬＣ内でアップレギュレートされた遺伝子。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to genes specific to various types of cancer and uses thereof.
[0002]
[Prior art]
Lung cancer is the world's leading cause of cancer death for both men and women, and the number is growing. In Japan alone, 50,000 lives are lost to lung cancer each year. Lung cancers are pathologically classified into small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) according to their morphology, and the latter is further squamous cell carcinoma (squamous). It is classified into cell carcinoma (SCC), adenocarcinoma (adenocarcinoma: AC), and large cell carcinoma (large cell carcinoma: LCC). Since individually classified lung cancers differ in malignancy and responsiveness to treatment, accurate diagnosis of the type is extremely important in determining treatment guidelines. In particular, small cell lung cancer, when diagnosed, is often not a candidate for surgical resection because it is often accompanied by metastasis, and chemotherapy and radiotherapy are usually selected. In general, small cell lung cancer has good responsiveness to chemotherapy and a tumor shrinkage effect, but is often accompanied by recurrence, and the relapsed cancer cells acquire multidrug resistance and are resistant to treatment. On the other hand, in the case of non-small cell lung cancer, if there is no localized and obvious distant metastasis, surgical resection is the only option, and chemotherapy is performed for the purpose of preventing postoperative recurrence. Particularly, in the case of adenocarcinoma, those with different degrees of differentiation are known from morphological observation, and poorly differentiated ones generally have poor prognosis.
[0003]
Conventionally, the diagnosis of a disease type has been performed mainly on morphological observation of a biopsy specimen. However, morphological observation requires judgment by a skilled person, and there is room for subjective factors in the judgment. In addition, since the treatment policy of cancer greatly differs depending on the disease type, accurate and objective diagnosis of the disease type is extremely important, but such a diagnosis method has not been known.
[0004]
[Problems to be solved by the invention]
The present invention has been made in view of such a situation, and an object of the present invention is to find a gene specific to various types of cancers and to provide a method for using the gene.
[0005]
[Means for Solving the Problems]
The present inventors performed gene expression profile analysis for each type of disease in order to find genes specific to various types of cancer. Specifically, RNA was prepared from 38 cases of lung cancer-derived cell lines, 3 cases of normal fibroblast cell lines, 43 cases of clinical specimens of lung cancer, and 6 cases of normal lung tissues. Hybridization was performed on the slides. As a result of the usual hierarchical clustering, all cell lines and clinical specimens were separated into different clusters. Cell line clusters are thought to be formed by gene expression common to cell lines due to factors specific to the culture system, while clinical sample clusters are strongly influenced by normal tissue mixed in tumor samples. Was done. Therefore, by excluding from the analysis those genes that are highly expressed or decreased in expression in these cultured cell lines or clinical samples, it is thought that a gene expression profile that reflects each disease type can be obtained. (Binary String Search) is newly considered. By this filtering method, genes whose expression was commonly increased in cell lines or clinical samples could be excluded from the analysis, and genes showing a disease type-specific expression profile could be identified. The identified gene is considered to be useful in diagnosis and treatment of various types of cancer.
[0006]
That is, the present invention
[1] a method for detecting cancer, comprising the step of measuring the expression level of the DNA described in the following (a) or (b) or a polypeptide encoded from the DNA:
(A) DNA containing the coding region of the nucleotide sequence of any of SEQ ID NOs: 1 to 119
(B) a DNA that hybridizes under stringent conditions to a DNA consisting of the nucleotide sequence of any of SEQ ID NOs: 1 to 119;
[2] The inspection method according to [1], including the following steps (a) to (c):
(A) Step of preparing a polypeptide sample from a subject
(B) a step of measuring the amount of the polypeptide according to [1] contained in the polypeptide sample
(C) comparing the measured amount of polypeptide with a control
[3] The inspection method according to [1], including the following steps (a) to (c):
(A) Step of preparing RNA sample from subject
(B) a step of measuring the amount of RNA encoding the polypeptide according to [1] contained in the RNA sample
(C) comparing the measured amount of RNA with a control
[4] The inspection method according to [1], including the following steps (a) to (c):
(A) Step of preparing cDNA sample from subject
(B) a step of measuring the amount of cDNA encoding the polypeptide according to [1] contained in the cDNA sample
(C) comparing the measured amount of cDNA with a control
[5] The inspection method according to [1], including the following steps (a) to (d):
(A) (i) a cRNA or cDNA sample prepared from a subject, and
(Ii) a step of providing a substrate on which a nucleotide probe that hybridizes with the DNA according to [1] is immobilized;
(B) contacting the cRNA or cDNA sample of step (a) (i) with the substrate of step (a) (ii)
(C) detecting the level of hybridization between the cRNA sample or the cDNA sample and the nucleotide probe immobilized on the substrate to reduce the expression level of the DNA according to [1] contained in the cRNA sample or the cDNA sample; Measurement process
(D) comparing the measured DNA expression level with a control
[6] a method for examining cancer, comprising a step of measuring the expression level of a plurality of DNAs or a polypeptide encoded from the DNAs of the DNA according to [1];
[7] The inspection method according to [6], including the following steps (a) to (d):
(A) (i) a cRNA or cDNA sample prepared from a subject, and
(Ii) a step of providing a substrate on which nucleotide probes that respectively hybridize with a plurality of DNAs among the DNAs according to [1] are fixed.
(B) contacting the cRNA or cDNA sample of step (a) (i) with the substrate of step (a) (ii)
(C) detecting the intensity of hybridization between the cRNA sample or cDNA sample and the nucleotide probe immobilized on the substrate to obtain a plurality of DNAs of [1] contained in the cRNA sample or cDNA sample; Step of measuring the expression level of DNA
(D) comparing the measured expression level with a control
[8] a test agent for cancer, comprising an oligonucleotide which hybridizes to the DNA of [1] and has an oligonucleotide length of at least 15 nucleotides;
[9] a test agent for cancer, comprising an antibody that binds to the polypeptide of [1];
[10] the test agent of [8] or [9], wherein the cancer is lung cancer;
[11] the test agent of [10], wherein the lung cancer is a small cell lung cancer;
[12] the test agent of [10], wherein the lung cancer is squamous cell carcinoma, adenocarcinoma, or carcinoid;
[13] a DNA in which a DNA encoding a cytotoxic protein is operably linked to the 3 'side of the promoter region of the DNA according to [1],
[14] a vector into which the DNA of [13] has been inserted,
[15] the vector of [14], which is for gene therapy;
[16] a transformed cell carrying the vector of [14] or [15],
[17] a method for treating cancer, comprising a step of administering the vector according to [15];
[18] a method for evaluating the presence or absence of anticancer activity in a test sample,
(A) contacting a test sample with a cancer cell;
(B) (i) a cRNA or cDNA sample prepared from a cancer cell contacted with a test sample, and
(Ii) a step of providing a substrate on which nucleotide probes that respectively hybridize with a plurality of DNAs among the DNAs according to [1] are fixed.
(C) contacting the cRNA or cDNA sample of step (a) (i) with the substrate of step (a) (ii)
(D) detecting the intensity of hybridization between the cRNA sample or cDNA sample and the nucleotide probe immobilized on the substrate to obtain a plurality of DNAs of [1] contained in the cRNA sample or cDNA sample; Step of measuring the expression level of DNA
A method wherein the test sample is determined to have anti-cancer activity when the expression level is restored to the expression level of the DNA in a control as compared to when the test sample is not contacted,
[19] a method for screening a sample having anticancer activity, comprising the following steps (a) and (b):
(A) a step of evaluating the presence or absence of anticancer activity in a plurality of test samples by the evaluation method described in [18].
(B) selecting a sample evaluated to have anti-cancer activity from a plurality of test samples; A method for producing a pharmaceutical composition having anticancer activity,
[21] the method of any one of [1] to [7], [17] to [20], wherein the cancer is lung cancer;
[22] the method of [21], wherein the lung cancer is a small cell lung cancer;
[23] the method of [21], wherein the lung cancer is a squamous cell carcinoma, an adenocarcinoma, or a carcinoid;
Is provided.
[0007]
BEST MODE FOR CARRYING OUT THE INVENTION
The present invention provides a cancer test method (diagnosis method) using DNA specifically expressed in various types of cancer as an index. Examples of DNAs specifically expressed in various types of cancers are described in SEQ ID NOs: 1 to 119.
[0008]
DNAs specifically expressed in various disease-type cancers in the present invention include DNAs functionally equivalent to the above-mentioned DNAs. Here, “functionally equivalent” means that the polypeptide encoded by the target DNA is an organism equivalent to the polypeptide encoded by the DNA consisting of the nucleotide sequence of any of SEQ ID NOs: 1 to 119. It has a biological function and a biochemical function.
[0009]
Other methods well known to those skilled in the art for preparing DNA encoding a polypeptide functionally equivalent to a polypeptide include hybridization techniques (Sambrook, J et al., Molecular Cloning 2nd ed., 9. 47-9.58, Cold Spring Harbor Lab. Press, 1989). That is, it is a well-known technique for a person skilled in the art to isolate a DNA highly homologous to the nucleotide sequence described in any of SEQ ID NOs: 1 to 119 or a part thereof.
[0010]
In the present invention, as the DNA functionally equivalent to any of SEQ ID NOs: 1 to 119, for example, mutations derived from human, monkey, mouse, rat, guinea pig, pig, cow, sheep, goat, etc. Examples include, but are not limited to, body, allele, variant, homolog, and the like.
[0011]
Hybridization conditions for isolating a DNA functionally equivalent to the DNA described in any of SEQ ID NOs: 1 to 119 can be appropriately selected by those skilled in the art. Hybridization conditions include, for example, low stringency conditions. The low stringent conditions are, for example, conditions of 42 ° C., 5 × SSC, 0.1% SDS in washing after hybridization, preferably 50 ° C., 5 × SSC, 0.1% SDS. is there. More preferable hybridization conditions include high stringency conditions. High stringency conditions are, for example, conditions of 65 ° C., 0.1 × SSC and 0.1% SDS. Under these conditions, it can be expected that DNA with higher homology can be efficiently obtained as the temperature is increased. However, a plurality of factors such as temperature and salt concentration can be considered as factors affecting the stringency of hybridization, and those skilled in the art can realize the same stringency by appropriately selecting these factors. .
[0012]
Further, a gene amplification method using a primer synthesized based on the base sequence information described in any of SEQ ID NOs: 1 to 119, for example, a polymerase chain reaction (PCR) method, It is also possible to isolate DNA functionally equivalent to any of the DNAs described above.
[0013]
DNAs functionally equivalent to the DNAs of any of SEQ ID NOs: 1 to 119 isolated by these hybridization techniques or gene amplification techniques usually have high homology at the amino acid sequence level. High homology usually refers to at least 50% or more identity, preferably 75% or more identity, more preferably 85% or more identity, more preferably 95% or more identity at the amino acid level. .
[0014]
The identity of an amino acid sequence or a base sequence can be determined by the algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993). Based on this algorithm, programs called BLASTN and BLASTX have been developed (Altschul et al. J. Mol. Biol. 215: 403-410, 1990). When a base sequence is analyzed by BLASTN based on BLAST, parameters are set, for example, to score = 100 and wordlength = 12. When analyzing an amino acid sequence by BLASTX based on BLAST, parameters are set, for example, to score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known (http://www.ncbi.nlm.nih.gov.).
[0015]
Cancer (preferably lung cancer) can be tested by using such DNA expression as an index. For example, the expression of DNA described in any of SEQ ID NOs: 1 to 17 (DNA described in Table 4 in Examples) or a DNA functionally equivalent to the DNA is reduced, or any of SEQ ID NOs: 18 to 119 Lung cancer can be determined by using as an index the DNA described in C. (DNA described in Tables 5 to 8 in the Examples) or a DNA functionally equivalent to the DNA. In addition, small cell lung cancer is determined using the DNA of any of SEQ ID NOs: 18 to 61 (the DNA described in Table 5 in the Examples) or an increase in the expression of DNA functionally equivalent to the DNA as an index. It is possible. In addition, the determination of squamous cell carcinoma is performed by using the DNA of any of SEQ ID NOs: 62 to 107 (the DNA described in Table 6 in the Examples) or an increase in the expression of DNA functionally equivalent to the DNA as an index. Is possible. In addition, a carcinoid can be determined using the DNA of any one of SEQ ID NOs: 108 to 117 (the DNA described in Table 7 in the Examples) or an increase in the expression of DNA functionally equivalent to the DNA as an index. It is. Adenocarcinoma can be determined using the DNA of SEQ ID NO: 118 or 119 (the DNA described in Table 8 in the Examples) or an increase in the expression of DNA functionally equivalent to the DNA as an index. .
[0016]
As the test method in the present invention, a test method including a step of measuring the expression level of the DNA of the present invention or the polypeptide encoded from the DNA, or a method comprising the steps of: A test method including a step of measuring the expression level of the encoded polypeptide. Such a test method can also be used in monitoring the effect of cancer treatment. Here, the expression level of the DNA usually means the expression level of RNA encoded from the DNA, but the level of cDNA or cRNA converted from the RNA is also included in the above “expression level of DNA”. Is done.
[0017]
Hereinafter, specific embodiments of the inspection method of the present invention will be described, but the inspection method of the present invention is not limited to those methods. In the test of the present invention, the control means a non-affected patient of cancer.
[0018]
In one embodiment of the test method including the step of measuring the expression level of the DNA of the present invention or the polypeptide encoded by the DNA, first, a polypeptide sample is prepared from a subject. In the present invention, examples of the polypeptide sample from the subject include, for example, a polypeptide prepared by a method well known to those skilled in the art from blood, urine, saliva, tissue or cells collected by biopsy or surgery of the subject. A sample.
[0019]
Next, in the method of the present invention, the amount of the polypeptide encoded from the DNA of the present invention contained in the polypeptide sample is measured. The measured amount of polypeptide is then compared to a control.
[0020]
Such methods include those well known to those skilled in the art, for example, enzyme-linked immunosorbent assay (ELISA), double monoclonal antibody sandwich immunoassay (US Pat. No. 4,376,110), monoclonal polyclonal antibody sandwich assay (Wide et al., Edited by Kirkham and Hunter, "Radioimmunoassay", E. and S. Livingstone, Edinburgh, (1970)), immunofluorescence, Western blotting, dot blotting, immunoprecipitation, protein chip. (Protein nucleic acid enzyme Vol. 47 No. 5 (2002), protein nucleic acid enzyme Vol. 47 No. 8 (2002)), two-dimensional electrophoresis, SDS polyacrylamide electrophoresis Law including but the inspection process is not limited thereto.
[0021]
Specific embodiments of the ELISA method among the above methods are described below, but the present invention is not limited to this method. In the ELISA method, a target fusion protein is adsorbed on the surface of a microtiter well. The remaining protein-binding sites on the surface are then buffered in bovine serum albumin (BSA), heat-inactivated normal goat serum (NGS), or BLOTTO (fat-free dry milk containing preservatives, salts and antifoams). Liquid). The wells are then incubated with a sample suspected of containing the specific antibody. The sample may be applied undiluted or more often diluted in a buffer that usually contains small amounts (0.1-5.0% by weight) of proteins such as BSA, NGS, or BLOTTO. it can. After incubation for a time sufficient for specific binding to occur, the wells are washed to remove unbound protein and then incubated with an anti-species-specific immunoglobulin antibody labeled with a reporter group. I do. The reporter group can be selected from a variety of enzymes, such as horseradish peroxidase, β-galactosidase, alkaline phosphatase, and glucose oxidase. After a period of time sufficient for specific binding to occur, the wells are then washed again to remove unbound complex and to add the enzyme substrate. The color is developed and the optical density of the contents of the wells is determined visually or by instrument.
[0022]
In the present invention, for example, four secreted proteins highly expressed in squamous cell carcinoma, W49672 (wingless-type MMTV integration site family, member 5A (WNT5A)), T62075 (prothrombin), and AA12587o angi by the above-mentioned method. 2 (ANGPT2)) and AA936767 (heparin-binding growth factor binding protein (HBP17)), an early test for lung squamous cell carcinoma can be performed.
[0023]
In another embodiment of the test method of the present invention, first, an RNA sample is prepared from a subject. Next, the amount of RNA encoding the polypeptide of the present invention contained in the RNA sample is measured. The measured amount of RNA is then compared to a control. In another embodiment, first, a cDNA sample is prepared from a subject. Next, the amount of cDNA encoding the polypeptide of the present invention contained in the cDNA sample is measured. The measured amount of cDNA is then compared to a control. Examples of such a method include a method well known to those skilled in the art, for example, a Northern blotting method, an RT-PCR method, and the like.
[0024]
As another aspect of the test method of the present invention, first, a cRNA sample or a cDNA sample prepared from a subject and a substrate on which a nucleotide probe that hybridizes with the DNA of the present invention are provided are provided. Next, the cRNA sample or the cDNA sample is brought into contact with the substrate. Next, the expression level of the DNA of the present invention contained in the cRNA sample or cDNA sample is measured by detecting the intensity of hybridization between the cRNA sample or cDNA sample and the nucleotide probe immobilized on the substrate. Further, the measured expression level of the DNA is compared with a control. Examples of such a method include a microarray method (SNP gene polymorphism strategy, Kenichi Matsubara and Yoshiyuki Sakaki, Nakayama Shoten, p. 128-135).
[0025]
Preparation of a cRNA sample or a cDNA sample from a subject can be performed, for example, by using mRNA extracted from a biopsy of the subject as a template and by a method well known to those skilled in the art. For example, mRNA is isolated from a cancer-affected site of a subject. mRNA can be isolated by known methods, for example, guanidine ultracentrifugation (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299), and AGPC method (Chomczynski, P. and Sacchi, N. et al. , Anal. Biochem. (1987) 162, 156-159) and the like, and the mRNA is purified from the total RNA using an mRNA Purification Kit (Pharmacia) and the like. Alternatively, mRNA can be directly prepared by using QuickPrep mRNA Purification Kit (Pharmacia). CDNA is synthesized from the obtained mRNA using reverse transcriptase. cDNA can also be synthesized using AMV Reverse Transcriptase First-Strand cDNA Synthesis Kit (Seikagaku Corporation) or the like. The synthesis of cRNA can be performed by the method described in Examples. The prepared cRNA sample or cDNA sample can be labeled, if necessary, for detection by a method well known to those skilled in the art.
[0026]
In the present invention, “substrate” means a plate-like material to which nucleotides can be fixed. In the present invention, nucleotides include oligonucleotides and polynucleotides. The substrate of the present invention is not particularly limited as long as it can fix nucleotides, but a substrate generally used in DNA array technology can be suitably used.
[0027]
Generally, DNA arrays are composed of thousands of nucleotides printed on a substrate at high density. Usually, these DNAs are printed on the surface of a non-porous substrate. The surface of the substrate is typically glass, but a permeable membrane, such as a nitrocellulose membrane, can be used.
[0028]
In the present invention, an array based on oligonucleotides developed by Affymetrix can be exemplified as a method for immobilizing (arraying) nucleotides. In an array of oligonucleotides, the oligonucleotides are usually synthesized in situ. For example, a method of in situ synthesis of oligonucleotides using a photolithographic technique (Affymetrix) or the like is already known, and such a technique can be used for producing the substrate of the present invention.
[0029]
The nucleotide probe immobilized on the substrate is not particularly limited as long as it specifically hybridizes with the DNA of the present invention. The term "specifically hybridizes" as used herein means a normal hybridization condition, preferably a stringent hybridization condition (for example, Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, The conditions described in the second edition 1989) means that no significant cross-hybridization occurs with DNA other than the DNA of the present invention. As long as specific hybridization is possible, it is not necessary to be completely complementary to the nucleotide sequence of the DNA to be detected.
[0030]
The nucleotides of the present invention include oligonucleotides and cDNAs. The length of the nucleotide probe bound to the substrate is usually 100 to 4000 bases when immobilizing cDNA, preferably 200 to 4000 bases, and more preferably 500 to 4000 bases. When the oligonucleotide is immobilized, it is usually 15 to 500 bases, preferably 30 to 200 bases, and more preferably 50 to 200 bases.
[0031]
In the present invention, a method for detecting the presence or absence or the intensity of the hybridization between the cRNA sample or the cDNA sample and the nucleotide probe immobilized on the substrate can be performed, for example, by reading a fluorescent signal with a scanner or the like.
[0032]
In the above method, when the expression level of the DNA of the present invention is significantly increased or decreased as compared to the control, the subject is suspected of developing cancer in the future (high risk), or It is determined that the subject has cancer.
[0033]
Further, as one embodiment of the test method including a step of measuring the expression level of a plurality of DNAs or a polypeptide encoded from the DNAs of the DNAs of the present invention, the method of the present invention uses a microarray method. A method of analyzing the expression profile of a plurality of DNAs is included. First, a cRNA sample or a cDNA sample prepared from a subject and a substrate on which a nucleotide probe that hybridizes to a plurality of DNAs of the DNA of the present invention are provided. Next, the cRNA sample or the cDNA sample is brought into contact with the substrate. Next, by detecting the intensity of hybridization between the cRNA sample or cDNA sample and the nucleotide probe immobilized on the substrate, the expression levels of a plurality of DNAs of the DNA of the present invention contained in the cRNA sample or cDNA sample are detected. Is measured. In addition, the measured expression level is compared with a control.
[0034]
The nucleotide probe immobilized on the substrate is not particularly limited as long as it is a nucleotide capable of specifically hybridizing with a plurality of DNAs in the present invention. The number of types of nucleotide probes to be immobilized on the substrate is determined according to the DNA of the present invention whose expression is to be detected in the present invention. For example, when it is desired to detect the expression of 10 DNAs of the present invention, nucleotide probes that specifically hybridize with each of the 10 DNAs are immobilized on a substrate. At that time, the nucleotide probe corresponding to each DNA is not necessarily limited to one kind, and may be a mixture of plural kinds of nucleotide probes having complementarity with an arbitrary region of the DNA of the present invention to be detected. .
[0035]
In the above method, when the expression levels (expression profiles) of a plurality of DNAs of the DNA of the present invention are significantly changed as compared with the control, the subject is suspected of developing cancer in the future (risk High) or cancer.
[0036]
The present invention also provides a test agent for use in the cancer test method of the present invention. Examples of such a test agent include a test agent containing an antibody that binds to the polypeptide encoded from the DNA of the present invention.
[0037]
To prepare an antibody that binds to the above polypeptide, first, the DNA of the present invention is inserted into a known expression vector system, and a host cell is transformed with the vector. The polypeptide or its fragment to be obtained may be obtained by a known method, and these may be used as a sensitizing antigen.
[0038]
The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with a parent cell used for cell fusion. , Lagomorphs, primates are used.
[0039]
Immunization of an animal with a sensitizing antigen is performed according to a known method. As a general method, the sensitizing antigen is injected intraperitoneally or subcutaneously into a mammal. Then, it is confirmed by a conventional method that the desired antibody level in the serum increases.
[0040]
In order to obtain a polyclonal antibody against the polypeptide encoded by the DNA of the present invention, after confirming that the level of the desired antibody in the serum has increased, the blood of the mammal sensitized with the antigen is removed. The serum is separated from this blood by a known method. As the polyclonal antibody, serum containing the polyclonal antibody may be used, or if necessary, a fraction containing the polyclonal antibody may be further isolated from the serum and used. For example, by using an affinity column to which the protein of the present invention is coupled, a fraction that recognizes only the protein of the present invention is obtained, and the fraction is further purified by using a protein A or protein G column. And immunoglobulin G or M can be prepared.
[0041]
To obtain a monoclonal antibody, after confirming that the level of the desired antibody is increased in the serum of a mammal sensitized with the antigen, immune cells may be removed from the mammal and subjected to cell fusion. At this time, preferred immune cells used for cell fusion include spleen cells. The other parent cell to be fused with the immunocyte is preferably a mammalian myeloma cell, more preferably a myeloma cell that has acquired the properties for selecting fused cells by a drug.
[0042]
The cell fusion of the immune cells and myeloma cells is basically performed by a known method, for example, the method of Milstein et al. (Galfred, G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46). It can be performed according to it.
[0043]
Hybridomas obtained by cell fusion are selected by culturing them in a normal selective culture solution, for example, a HAT culture solution (a culture solution containing hypoxanthine, aminopterin and thymidine). Culturing in the HAT culture solution is carried out for a period of time sufficient to kill cells (non-fused cells) other than the target hybridoma, usually for several days to several weeks. Next, a conventional limiting dilution method is performed to screen and clone a hybridoma producing the desired antibody.
[0044]
Further, in addition to obtaining the hybridoma by immunizing an animal other than a human with an antigen, a human lymphocyte, for example, a human lymphocyte infected with an EB virus is sensitized in vitro with a protein, a protein-expressing cell or a lysate thereof, and sensitized. Lymphocytes can be fused with human-derived myeloma cells having permanent division ability, for example, U266, to obtain a hybridoma that produces a desired human antibody having protein binding activity (Japanese Patent Application Laid-Open No. 63-17688). .
[0045]
Subsequently, the obtained hybridoma was transplanted into a mouse intraperitoneal cavity, ascites was recovered from the mouse, and the obtained monoclonal antibody was subjected to, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, and the present invention. It can be prepared by purifying with an affinity column to which a polypeptide encoded from DNA is coupled.
[0046]
The monoclonal antibody thus obtained can also be obtained as a recombinant antibody produced using a gene recombination technique (for example, Borrebaeck, CAK and Larrrick, JW, THERAPEUTIC). MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990). Recombinant antibodies are produced by cloning DNA encoding the same from immune cells such as hybridomas or sensitized lymphocytes that produce the antibody, incorporating the DNA into an appropriate vector, and introducing this into a host.
[0047]
Further, the antibody may be an antibody fragment or a modified antibody thereof as long as it binds to the polypeptide encoded from the DNA of the present invention. For example, antibody fragments include Fab, F (ab ') ₂ , Fv, or a single chain Fv (scFv) in which an Fv of an H chain and an L chain are linked by an appropriate linker (Huston, JS et al., Proc. Natl. Acad. Sci. U.S.A. 1988) 85, 5879-5883). Specifically, an antibody is treated with an enzyme, for example, papain or pepsin, to generate an antibody fragment, or a gene encoding these antibody fragment is constructed and introduced into an expression vector. (E.g., Co, MS et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, A.H., Methods Enzymol. (1989) 178, 476). Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-us.Ru; . Methods Enzymol (1986) 121, 663-669;. Bird, R. E. and Walker, B. W., Trends Biotechnol (1991) 9, see 132-137).
[0048]
As the modified antibody, an antibody bound to various molecules such as polyethylene glycol (PEG) can be used. The “antibody” of the present invention also includes these modified antibodies. Such a modified antibody can be obtained by subjecting the obtained antibody to chemical modification. These methods are already established in this field.
[0049]
In addition, an antibody against the polypeptide encoded by the DNA of the present invention can be prepared by using a known technique to obtain a chimeric antibody comprising a variable region derived from a non-human antibody and a constant region derived from a human antibody or a CDR derived from a non-human antibody (complementary) A sex-determining region), a human antibody-derived FR (framework region) and a constant region can be obtained as a humanized antibody.
[0050]
The antibody obtained as described above can be purified to homogeneity. The separation and purification of the antibody used in the present invention may be performed by the separation and purification methods used for ordinary proteins. For example, if appropriate selection and combination of chromatography columns such as affinity chromatography, filters, ultrafiltration, salting-out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, etc., antibodies can be separated and purified. (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988), but are not limited thereto. The concentration of the antibody obtained as described above can be measured by absorbance measurement or enzyme-linked immunosorbent assay (ELISA).
[0051]
Columns used for affinity chromatography include a protein A column and a protein G column. For example, as a column using a protein A column, Hyper D, POROS, Sepharose F.R. F. (Pharmacia) and the like.
[0052]
Examples of chromatography other than affinity chromatography include ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These chromatographys can be performed using liquid phase chromatography such as HPLC and FPLC.
[0053]
The antibody thus obtained can be used not only for the above-described test method, but also for a cancer test method, which performs imaging targeting at a polypeptide encoded from the DNA of the present invention. Can also be used. In one embodiment of the method, an antibody that binds to the above polypeptide is prepared. The antibody is then labeled with a positron emitting isotope. Next, the labeled antibody is administered to the subject. The radioactivity is then detected by positron emission tomography. The detected radioactivity is then compared to a control.
[0054]
In particular, detection of micrometastases and determination of the degree of cancer invasion are important factors when applying surgery for non-small cell lung cancer, and can be performed by imaging targeting specific cell surface markers. Such an imaging technique can also be used for the purpose of evaluating responsiveness to treatment including small cell lung cancer.
[0055]
The test agent of the present invention also includes a test agent which hybridizes to the DNA of the present invention and contains an oligonucleotide having a chain length of at least 15 nucleotides. The oligonucleotide specifically hybridizes to the DNA of the present invention. The oligonucleotide need not be completely complementary to the DNA of the present invention, as long as specific hybridization is possible.
[0056]
Examples of the test agent containing an oligonucleotide that hybridizes to the DNA of the present invention and has a chain length of at least 15 nucleotides include a probe usable in the above-described test method of the present invention (in the form of a substrate to which the probe is immobilized, And primers. When the above-mentioned oligonucleotide is used as a primer, its length is usually 15 bp to 100 bp, preferably 17 bp to 30 bp. The primer is not particularly limited as long as it can amplify at least a part of the DNA of the present invention.
[0057]
When the oligonucleotide is used as a probe, the probe is not particularly limited as long as it specifically hybridizes to the DNA of the present invention. The probe may be a synthetic oligonucleotide and usually has a chain length of at least 15 bp.
[0058]
The oligonucleotide of the present invention can be produced by, for example, a commercially available oligonucleotide synthesizer. The probe can also be prepared as a double-stranded DNA fragment obtained by restriction enzyme treatment or the like.
[0059]
When the oligonucleotide of the present invention is used as a probe, it is preferable to appropriately label and use it. As a method for labeling, the T4 polynucleotide kinase is used to label the 5 ′ end of the oligonucleotide. ³² A method of labeling by phosphorylation with P, and using a DNA polymerase such as Klenow enzyme, using a random hexamer oligonucleotide or the like as a primer ³² A method of incorporating a substrate base labeled with an isotope such as P, a fluorescent dye, or biotin (eg, a random prime method) can be exemplified.
[0060]
The test agent of the present invention includes, besides oligonucleotides and antibodies as active ingredients, for example, sterile water, physiological saline, vegetable oil, surfactant, lipid, solubilizing agent, buffer, protein stabilizer (BSA or gelatin) And the like, and a preservative and the like may be mixed as necessary.
[0061]
The present invention also provides a DNA in which a DNA encoding a cytotoxic protein is operably linked to the 3 ′ side of the promoter region of the DNA of the present invention. Such DNA is a useful DNA that can be used as an anticancer agent.
[0062]
In the present invention, “functionally linked” means that the expression of a DNA encoding a cytotoxic protein is induced by binding of a transcription factor to a promoter region of the DNA of the present invention. Means that the promoter region of the DNA and the DNA encoding the cytotoxic protein are linked. Therefore, even when the DNA encoding the cytotoxic protein binds to another gene and forms a fusion protein with another gene product, the transcription factor binds to the promoter region of the DNA of the present invention. If the expression of the fusion protein is induced by doing so, it is included in the meaning of “functionally linked”.
[0063]
Examples of the cytotoxic protein in the present invention include diphtheria toxin and HSV-derived thymidine kinase, but are not limited thereto, and any protein that directly or indirectly damages cells and has a lethal effect. , Included in the cytotoxic protein of the present invention.
[0064]
The promoter region of the DNA of the present invention is extracted from, for example, publicly available genomic sequence data, and a corresponding region (for example, a region of about 3 kb from the 5 ′ end of mRNA, including, but not limited to) is extracted by PCR. It can be obtained by amplification. By linking a DNA encoding a cytotoxic protein to the 3 ′ side of the obtained promoter region by a method well known to those skilled in the art, a cytotoxic protein is added to the 3 ′ side of the promoter region of the DNA of the present invention. DNA can be constructed in which the encoding DNA is operably linked. Further, the constructed DNA can be incorporated into an appropriate vector and amplified in a host cell such as Escherichia coli.
[0065]
As a method for expressing a DNA in which a DNA encoding a cytotoxic protein is operably linked to the 3 ′ side of the promoter region of the DNA of the present invention in vivo in an animal body including a human, the DNA is converted into an appropriate vector. Incorporation, for example, a method of introducing into a living body by a retrovirus method, a liposome method, a cationic liposome method, an adenovirus method, and the like. This makes it possible to perform gene therapy for cancer. Examples of the vector used include, but are not limited to, an adenovirus vector (for example, pAdexlcw) and a retrovirus vector (for example, pZIPneo). General genetic operations such as insertion of the DNA of the present invention into a vector can be performed according to a conventional method (Molecular Cloning, 5.61-5.63). Administration into a living body may be by an ex vivo method or an in vivo method.
[0066]
The present invention provides a method for evaluating the presence or absence of anticancer activity in a test sample and a method for screening a sample having anticancer activity.
[0067]
The method for evaluating the presence or absence of anticancer activity in a test sample is a method using the expression profile of a plurality of DNAs of the DNA of the present invention as an index. As such a method, first, a test sample is brought into contact with cancer cells. Next, a cRNA sample or a cDNA sample prepared from a cancer cell brought into contact with a test sample, and a substrate on which a nucleotide probe that hybridizes with a plurality of DNAs of the DNA of the present invention, respectively, are provided. Next, the cRNA sample or the cDNA sample is brought into contact with the substrate. Next, by detecting the intensity of hybridization between the cRNA sample or cDNA sample and the nucleotide probe immobilized on the substrate, the expression levels of a plurality of DNAs of the DNA of the present invention contained in the cRNA sample or cDNA sample are detected. Is measured.
[0068]
The “test sample” in the method of the present invention is not particularly limited. For example, a natural compound, an organic compound, an inorganic compound, a protein, a single compound such as a peptide, and a compound library, an expression product of a gene library Cell extract, cell culture supernatant, fermented microorganism product, marine organism extract, plant extract, prokaryotic cell extract, eukaryotic single cell extract or animal cell extract. The test sample can be appropriately labeled and used as needed. Examples of the label include a radiolabel, a fluorescent label, and the like. The “plurality of test samples” is not particularly limited, and includes, for example, a mixture of a plurality of these test samples in addition to the above test samples.
[0069]
In the method of the present invention, examples of the cancer cells include cells in cancer tissues and cultured cell lines obtained by culturing cells collected from cancer tissues. In addition, the cancer cell in the method of the present invention is not particularly limited as long as it is an organism-derived cancer cell suitable for identifying a therapeutic agent for human cancer. Such an organism is preferably a mammal (eg, a model mammal of a human disease such as a rat or mouse, a model mammal into which a human cancer has been transplanted), and most preferably a human.
[0070]
In the present invention, the method of “contacting a test sample with a cancer cell” includes not only the case where the test sample is directly contacted with the cancer cell but also the case where the test sample is indirectly contacted with the cancer cell (in this case, for example, (Using cells from an organism inoculated with the test sample).
[0071]
For example, treatment of a cancer cell with a test sample can be performed using, for example, Scherf et al. (U Scherf, DT Ross, M Waltham, LH Smith, JK Lee, L Tanabe, KW Kohn, W C Reinhold, for isolated cells). , T G Myers, D T Andrews, D A Scudiero, M B Eisen, E A Sausville, Y Pommier, D Botstein, P O Brown & J N Weinstein, A gene expression database for the molecular pharmacology of cancer. Nat Genet. 2000 , 24. 236-244). In the case of a living individual, it can be carried out by administering the compound orally or parenterally.
[0072]
In the present invention, the expression levels of a plurality of DNAs of the DNA of the present invention when the test sample is treated have been restored to the expression levels of the DNAs in the control (from the cancer cell-specific expression profile, the cancer has become cancerous). Test sample is determined to have anticancer activity.
[0073]
Further, the present invention provides a method for efficiently screening a sample having anticancer activity. In this method, the presence or absence of anticancer activity is evaluated for a plurality of test samples using the above evaluation method, and a sample evaluated to have anticancer activity is selected.
[0074]
Further, the present invention provides a method for producing a pharmaceutical composition having anticancer activity. In the production method, a sample evaluated to have anticancer activity by the above screening method is mixed with a pharmaceutically acceptable carrier. Such carriers include, for example, surfactants, excipients, colorants, flavors, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binders, disintegrants, lubricants, fluidity Examples include an accelerator, a flavoring agent, and the like, but are not limited thereto, and other conventional carriers can be appropriately used. Specifically, light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride, Polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts and the like can be mentioned.
[0075]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
[Example 1] Preparation of array slide
Of the 7885 genes affixed to the slide glass, 6734 is an IMAGE clone purchased from Research Genetics, 699 is derived from a Chugai-owned clone that is known or supposed to be involved in cancer, and the remaining 252 is added as a negative control. (Alu sequence, luciferase, etc.) were excluded from the data analysis. Slides were created by Hegde, P.E. (Cancer Res. (2001) 61: 7792-7797), and 7683 genes were attached to one slide using a Microgrid II (manufactured by BioRobotics).
[0076]
[Example 2] Preparation of probe, hybridization and measurement
Thirty-eight human lung cancer-derived cell lines, three normal human fibroblast cell lines (Table 1), 43 clinical specimens of various types of lung cancer, and 6 normal lung tissues (Table 2) were used as materials.
[0077]
[Table 1]

[0078]
[Table 2]

[0079]
The clinical specimens were collected at the time of surgical excision at the Cancer Research Institute Cancer Institute with the patient's informant and approval from the Ethics Committee. Considering that information can be obtained for as many genes as possible as reference samples, NCI-H226, NCI-H522, EKVX, NCI-H460, Lu-134A, MRC5, SQ5, H23, PC14, and MS1 were used, each of which was added with 8% of each of 10 cell lines. All cell lines were used as cell suspensions using RNAlater (Ambion). Clinical samples were snap frozen in liquid nitrogen after collection to minimize RNA degradation. After extracting RNA using Isogen (Nippon Gene), Luo, L. et al. CRNA synthesis was performed according to the method of Nat. Med. (1999) 5: 117-122. Probe synthesis was performed using 2 ug of cRNA as a template according to the method of Hughes, TR, etc. (Nat. Biotechnol. (2001) 19: 342-347). The reference sample was labeled with Cy3 and the test sample was labeled with Cy5. Hybridization and subsequent washing are described in Hegde, P .; Etc. (supra). The measurement and quantification of the hybridized signal were carried out using a Scannerray 4000 scanner and Quantarray (both manufactured by Packard Bioscience).
[0080]
[Example 3] Data analysis
All cRNA samples were fluorescently labeled by duplicate. The probe after labeling was hybridized to a slide on which 7865 DNA spots were affixed in duplicate, and four independent measurements were performed for each gene. Of the 7885 elements spotted, 252 was added as a control (Luciferase, Alu, etc.), and 6384 of the remaining 7433 corresponded to 5622 UniGene clusters, and 1049 did not correspond to the UniGene clusters, and thus 6671 unique totals were obtained. Genes are spotted. Furthermore, from the results of the initial examination, 285 of them showed similar expression patterns among different specimens including all clinical specimens and cell lines, and were excluded from the subsequent analysis. As a result of this initial filtering, 6386 unique genes remained as targets for the following analysis (FIG. 1). The standardization of data within one array experiment is based on the low normalization method (Nucleic Acid Res. (2002), 30: e15), and a value is obtained by dividing the standardized signal intensity by the signal intensity of the reference sample. After that, using a program such as Tseng (Nucleic Acid Res. (2001), 29: 2549-2557), data whose CV value deviated from the 96% confidence interval was excluded as an outlier from subsequent analysis. The obtained data set was subjected to cluster analysis and visualization using GeneSpring (manufactured by Silicon Genetics) (FIG. 2). When directly comparing data from tumor clinical specimens to cell lines, genes that were highly expressed in clinical specimens regardless of different disease types, and conversely, genes that were highly expressed in cell lines were observed . For the purpose of selecting these genes, Binary Search Algorithm was newly considered. That is, for each gene, the average value of the signal in all samples is obtained, and a higher value is given "1" and a lower value is given "0". The profile was expressed as Binary Strings. Compared to the string of ideal state (all “1” in clinical specimen / all “0” in cell line or vice versa), if over 1% in 66% or more samples, “overexpressed”, 66% or more In the case of "0" in the sample, it was set to "underexpressed". As a result of this treatment, 2218 of the 6386 unique genes were selected as those having higher (1313) or lower (905) expression in clinical samples as compared to the cell lines (FIG. 3). Further, the results of clustering of these 2218 genes were visually confirmed, and as a result, 219 genes, which did not show a remarkable difference in expression between the clinical sample and the cell line, were removed from the following examination. (FIG. 4). As a result of all filtering, 4240 genes were finally left for analysis. The results of Hierarchical clustering (FIGS. 5 and 6) and 90 specimens (lung cancer clinical specimen 43, normal lung clinical specimen 6, lung cancer cell line 38, normal fetal fibroblast cell line 3) were divided into four large branches (Table). 3).
[0081]
[Table 3]

SCLC indicates small cell lung cancer, SCC indicates squamous cell carcinoma, NSCLC indicates non-small cell lung cancer, AC indicates adenocarcinoma, and LCC indicates large cell carcinoma. * Indicates a non-small cell lung cancer-derived cell line (disease type unknown).
[0082]
Branch 1 includes almost all small cell lung cancer-derived cell lines and all small cell lung cancer and carcinoid clinical specimens. Branch 2 contains many clinical specimens and cell lines of squamous cell carcinoma. Branch 3 is a cell line branch, and all fetal-derived normal fibroblast cell lines are included in this branch. Branch 4 includes all adenocarcinoma, large cell carcinoma and normal lung clinical specimens. Two non-small cell lung cancer-derived cell lines (type unknown) form a branch independent of the above four branches. By applying the Binary Search Algorithm to remove gene expression profiles specific to cell lines and gene samples in general to clinical samples, it is possible to directly compare previously difficult clinical samples with cell lines. It has become possible. In particular, most of the cell lines derived from small cell lung cancer (11/13) and some of the cell lines derived from squamous cell carcinoma (4/8) were assigned to the clinical specimen cluster of the disease type derived from each cell line. Therefore, it was considered that these cell lines well retained the characteristics of the original tumor. With respect to adenocarcinoma, the filtering and clustering performed this time was insufficiently separated from normal lung tissue, and no attribution of an adenocarcinoma-derived cell line was obtained (0/11). The reason for this is that the adenocarcinoma clinical specimens consisted of cases with different degrees of differentiation, and the degree of contamination with normal lung tissues was high. This was thought to be due to the fact that the most undifferentiated cells in the derived cancer tissue were selected during the cell lineage process.
[0083]
The following is a list of genes showing expression changes specific to disease types in various types of lung cancer.
(1) Highly expressed in normal lung tissue and reduced by canceration (sequences shown in SEQ ID NOs: 1 to 17)
[Table 4]

[0084]
(2) High expression in small cell lung cancer (shown in order of SEQ ID NOs: 18 to 61)
[Table 5]

[0085]
(3) Highly expressed squamous cell carcinoma (shown in order of SEQ ID NOs: 62 to 107)
[Table 6]

[0086]
(4) Carcinoids that exhibit high expression (shown in order of SEQ ID NOs: 108 to 117)
[Table 7]

[0087]
(5) Highly expressed in adenocarcinoma and normal lung tissue (sequences shown in SEQ ID NOs: 118 to 129)
[Table 8]

[0088]
【The invention's effect】
The present invention is expected to enable examination and treatment of type-specific cancer. For example, by using DNAs specific to various types of cancer found by the present inventors, early examination of cancer and examination of disease types become possible. In addition, the use of the promoter of the DNA makes it possible to carry out gene therapy for a type-specific cancer. Further, it becomes possible to screen a sample having anticancer activity using the expression profile of the DNA as an index.
[0089]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a photograph showing a tree diagram of two-dimensional hierarchical clustering of 6386 genes. Specimens colored according to type are shown on the left, red for AC, green for SCC, purple for normal, blue for SCLC, black for LCC; orange for carcinoid, brown for fibroblasts, pink for NSLC. It represents. Each column represents a particular gene. The squares are colored according to the log average expression ratio over the four replicates. In relation to the reference specimen, red represents an expression ratio greater than 1 (overexpression), green represents less than 1 (underexpression), and black represents a ratio approximately equal to 1 (no change in expression).
FIG. 2 is a photograph showing a dendrogram of a two-dimensional hierarchical clustering of 5255 genes in a cell line obtained by statistical filtration as described in the text. Specimens colored according to type are shown on the left, red for AC, green for SCC, blue for SCLC, brown for fibroblasts, pink for NSLC. Each column represents a particular gene. The squares are colored according to the log average expression ratio over the four replicates. In relation to the reference specimen, red represents an expression ratio greater than 1 (overexpression), green represents less than 1 (underexpression), and black represents a ratio approximately equal to 1 (no change in expression).
FIG. 3 is a diagram and photograph showing the identification of differentially regulated genes between tumor and cell line. (A) across all specimens for 905 genes that were generally overexpressed in cell line specimens compared to fresh specimens and (B) 1313 genes that were commonly underexpressed in cell line specimens compared to fresh specimens Expression changes. (C) Two-dimensional hierarchical clustering of 1919 genes that are differentially regulated between fresh and cell line specimens. The dendrogram on the left shows the clustering of cell line specimens and fresh specimens. The branch colors are as shown in FIG. The upper dendrogram shows the clustering of the genes.
FIG. 4 is a photograph showing a dendrogram of a two-dimensional hierarchical clustering of 2218 genes selected by the Binary Search Algorithm, which is differentially regulated between fresh and cell line specimens. The dendrogram on the left shows the clustering of cell line specimens and fresh specimens. The upper dendrogram shows the clustering of the genes. Genes up-regulated in fresh specimens are shown clustered to the right, and genes up-regulated in cell line specimens are shown clustered to the left. ing. Gene branches that did not show a significant contrast between fresh and cell line specimens were selected for inclusion in the main data set (299 total).
FIG. 5 is a photograph showing a dendrogram of a reduced dataset of 4240 genes after filtering for commonly regulated genes in fresh or cell line samples. The specimen is colored as in FIG. The groups shown on the left represent completely different clusters of specific carcinoma types. cl: cell line specimen; fr: fresh tumor specimen.
FIG. 6 is a photograph showing the expression profile across all specimens of the eight selected gene clusters from FIG. 5, showing completely different expression patterns. The dendrogram and the highlighted clusters from FIG. 5 are shown along the bottom and between the individual gene clusters for reference. (A) Gene up-regulated in carcinoids. (B) Genes up-regulated in normal tissues. (C) Genes up-regulated in AC and LCC. (DE) Genes up-regulated in SCC. (FH) Genes up-regulated in SCLC.

Claims (23)

A method for examining cancer, comprising a step of measuring the expression level of the DNA described in the following (a) or (b) or a polypeptide encoded from the DNA.
(A) DNA containing the coding region of the nucleotide sequence of any of SEQ ID NOs: 1 to 119
(B) a DNA that hybridizes under stringent conditions to a DNA consisting of the nucleotide sequence of any of SEQ ID NOs: 1 to 119; The inspection method according to claim 1, comprising the following steps (a) to (c).
(A) a step of preparing a polypeptide sample from a subject; (b) a step of measuring the amount of the polypeptide according to claim 1 contained in the polypeptide sample; and (c) measuring the amount of the measured polypeptide. Process to compare with control The inspection method according to claim 1, comprising the following steps (a) to (c).
(A) a step of preparing an RNA sample from a subject; (b) a step of measuring the amount of RNA encoding the polypeptide of claim 1 contained in the RNA sample; and (c) measuring the amount of the measured RNA. Process to compare with control The inspection method according to claim 1, comprising the following steps (a) to (c).
(A) a step of preparing a cDNA sample from a subject; (b) a step of measuring the amount of cDNA encoding the polypeptide according to claim 1 contained in the cDNA sample; and (c) measuring the amount of the measured cDNA. Process to compare with control The inspection method according to claim 1, comprising the following steps (a) to (d).
(A) providing (i) a cRNA or cDNA sample prepared from a subject, and (ii) a substrate on which a nucleotide probe that hybridizes with the DNA according to claim 1 is immobilized; a) contacting the cRNA sample or cDNA sample of (i) with the substrate of step (a) (ii) (c) measuring the hybridization intensity of the cRNA sample or cDNA sample with the nucleotide probe immobilized on the substrate; The step of measuring the expression level of the DNA according to claim 1, which is contained in the cRNA sample or the cDNA sample by detection, and (d) comparing the measured expression level of the DNA with a control. A method for examining cancer, comprising a step of measuring the expression level of a plurality of DNAs or a polypeptide encoded from the DNAs according to claim 1. The inspection method according to claim 6, comprising the following steps (a) to (d).
(A) (i) a cRNA or cDNA sample prepared from a subject; and (ii) a substrate on which a nucleotide probe that hybridizes with a plurality of DNAs among the DNAs according to claim 1 is fixed. Step (b) contacting the cRNA sample or cDNA sample of step (a) (i) with the substrate of step (a) (ii) (c) contacting the cRNA sample or cDNA sample with a nucleotide probe immobilized on the substrate (D) measuring the expression level of a plurality of DNAs among the DNAs according to claim 1 contained in the cRNA sample or cDNA sample by detecting the hybridization intensity of (c). Process to compare A test agent for cancer, comprising an oligonucleotide that hybridizes to the DNA according to claim 1 and has a chain length of at least 15 nucleotides. A test agent for cancer, comprising an antibody that binds to the polypeptide according to claim 1. The test agent according to claim 8 or 9, wherein the cancer is lung cancer. The test agent according to claim 10, wherein the lung cancer is small cell lung cancer. The test agent according to claim 10, wherein the lung cancer is squamous cell carcinoma, adenocarcinoma, or carcinoid. A DNA in which a DNA encoding a cytotoxic protein is operably linked to the 3 'side of the promoter region of the DNA according to claim 1. A vector into which the DNA according to claim 13 has been inserted. The vector according to claim 14, which is for gene therapy. A transformed cell carrying the vector according to claim 14. A method for treating cancer, comprising a step of administering the vector according to claim 15. In a test sample, a method for evaluating the presence or absence of anticancer activity,
(A) contacting a test sample with a cancer cell;
(B) (i) a cRNA sample or a cDNA sample prepared from a cancer cell contacted with a test sample, and (ii) a nucleotide probe that hybridizes with a plurality of DNAs among the DNAs according to claim 1. (C) contacting the cRNA sample or cDNA sample of steps (a) and (i) with the substrate of step (a) and (ii); and (d) fixing the cRNA sample or cDNA sample to the substrate. Measuring the expression level of a plurality of DNAs among the DNAs according to claim 1 contained in said cRNA sample or cDNA sample by detecting the intensity of hybridization with said nucleotide probe, wherein said expression level is determined. When the test sample has recovered to the level of expression of the DNA in the control as compared to when the test sample is not contacted, the test sample has an anticancer activity. Method is determined to have. A method for screening a sample having anticancer activity, comprising the following steps (a) and (b):
(A) a step of evaluating the presence or absence of anticancer activity in a plurality of test samples by the evaluation method according to claim 18; (b) selecting a sample evaluated to have anticancer activity from the plurality of test samples. Process 20. A method for producing a pharmaceutical composition having anticancer activity, further comprising the step of mixing a sample evaluated to have anticancer activity with a pharmaceutically acceptable carrier. The method according to any one of claims 1 to 7, 17 to 20, wherein the cancer is lung cancer. 22. The method of claim 21, wherein the lung cancer is a small cell lung cancer. 22. The method according to claim 21, wherein the lung cancer is squamous cell carcinoma, adenocarcinoma, or carcinoid.

Images