JPWO2004061102A1 - Cell cycle regulatory proteins and uses thereof - Google Patents

Abstract

An object of the present invention is to provide a protein involved in human HCC, a gene encoding the protein, and a pharmaceutical use. The present invention provides a protein having a novel cell cycle regulatory activity (FRM protein) and a nucleic acid molecule encoding the protein. Furthermore, the method comprises a method of regulating the cell cycle comprising changing (enhancing or inhibiting) the biological activity of the cell cycle regulatory protein, and a substance having an activity of changing (enhancing or inhibiting) the biological activity of the cell cycle regulatory protein. And a pharmaceutical composition as a therapeutic or prophylactic agent for liver disease (particularly liver cancer) or renal disease.

Description

  The present invention relates to a novel human cell cycle regulatory protein, a polynucleotide encoding the same, a production method and use thereof.

Hepatocellular carcinoma (HCC) is one of the most common cancers in humans, and its occurrence is known to be deeply related to infection with hepatitis B or C virus, subsequent inflammation, liver regeneration, etc. There are many unclear points in the molecular mechanism of development. The present inventors have already found a rat HP33 protein closely related to rat HCC and specifically expressed in the liver and kidney, and that this protein is overexpressed in the regeneration process of rat HCC cells and liver. In addition, it has been reported that it is localized in the vicinity of the centrosome of microtubules in the cell, and the localization varies depending on the cell cycle (WO99 / 000495, Tamura et al. Journal of Cell Science 112, 1353-1364). (1999)). In addition, HP33 protein is highly homologous to bovine N-acyltransferase, and it has been reported that the expression level of mammalian acyltransferase family changes in liver regeneration and liver cancer, but the mechanism at the molecular level is unknown. It was. In addition, rat HP33 and human homologous proteins corresponding to bovine N-acyltransferase were not known at all.
If we can identify a protein that is specifically expressed or increased in human HCC and the gene encoding that protein and elucidate the function of that protein, we will be able to elucidate the mechanism of cancer development and develop pharmaceuticals for cancer treatment. Can be expected to be useful. On the other hand, in recent years, many ESTs, which are cDNA fragments, have been registered in databases, and DNA sequences encoding human homologous proteins have been searched based on sequence information in other mammals. It has also been suggested that the gene searched based only on this is likely to be a pseudogene, and it is difficult to determine whether it actually encodes a human homolog protein and to elucidate its function.

As a result of intensive studies in view of the above circumstances, the inventors of the present invention conducted a TBLASTN database search using the amino acid sequence of rat HP33 protein, and found that a human gene having a base sequence homology of 63% (GenBank accession no. AB013093). ) And named it “FRM”. Amplification of a DNA fragment of FRM by PCR and screening of a human liver cDNA library using the fragment led to the acquisition of several clones. . As a result of analyzing the sequence using these clones, the entire nucleotide sequence of the cDNA described in SEQ ID NO: 1 was obtained. This confirmed that both rat HP33 and human FRM cDNAs consisted of 888 bp nucleotides, encoded 295 amino acids, and that both DNAs had significantly high homology (identity 63%, similarity) 74%). Furthermore, the expression analysis of FRM mRNA in human organs by Northern blotting confirmed that the human FRM gene was transcribed only in the liver and kidney in the same manner as the rat HP33 gene, and the human FRM protein The human FRM protein was found to be overexpressed in HCC cells in the same manner as the rat HP33 protein by an immunohistological technique using an antibody that specifically recognizes human FRM protein. It was confirmed that it is a human homologue protein of HP33 protein. Based on the above findings, the present inventors have completed the present invention.
That is, the present invention provides (1) a polynucleotide encoding a cell cycle regulatory protein according to any one of the following (a) to (d): (a) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2. (B) a polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids are added, deleted, substituted and / or inserted in the amino acid sequence set forth in SEQ ID NO: 2; DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1, (d) a polynucleotide that hybridizes with the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 under stringent conditions; (2) (a) to ( c) the cell cycle regulatory protein or salt thereof according to any one of the above: (a) a protein comprising the amino acid sequence represented by SEQ ID NO: 2, (b) SEQ ID NO: A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by (c), and is encoded by a DNA comprising the base sequence described in SEQ ID NO: 1. (3) a partial peptide of the cell cycle regulatory protein according to (2) or a salt thereof; (4) a recombinant vector into which the polynucleotide according to (1) is inserted; (5) the (4) A transformant transformed with the recombinant vector according to). (6) The cell cycle regulatory protein or salt thereof according to (2), wherein the transformant according to (5) is cultured to produce the protein according to (2). (7) an antibody against the cell cycle regulatory protein according to (2) above or the partial peptide according to (3) above or a salt thereof; (8) using the antibody according to (7) above ( (9) a method for quantifying the cell cycle regulatory protein or salt thereof according to (2); (9) a cell cycle regulation method comprising enhancing or inhibiting the biological activity of the cell cycle regulatory protein or salt thereof according to (2); (10) The method according to (9) above, which enhances or inhibits the biological activity of the cell cycle regulatory protein or salt thereof according to (2) above in G _{2 to} M phase cells; 11) The cell is a cancer cell. (9) the method according to (9) or (10); (12) by increasing or decreasing the intracellular content of the cell cycle regulatory protein or salt thereof according to (2) above, The method according to any one of (9) to (11) above, characterized by enhancing or inhibiting biological activity; (13) characterized by combining the use of an anticancer agent, (9) The method according to any one of (12); (14) Use of the anticancer agent increases the endogenous amount of the cell cycle regulatory protein or salt thereof according to (2). Or the method according to (13), which is simultaneous with the means for decreasing, or before and after the means at a certain time interval; (15) the anticancer agent is taxol or a salt thereof; (13) or (14) (16) The method according to any one of (9) to (11), which comprises enhancing or suppressing the expression of a polynucleotide encoding the protein according to (2) above; 17) The method according to (12), comprising suppressing the biological activity of the protein by RNA interference targeting the polynucleotide encoding the cell cycle regulatory protein according to (2). (18) The RNA molecule used for RNA interference is a double-stranded RNA-DNA chimera molecule consisting of the base sequences set forth in SEQ ID NO: 4 and SEQ ID NO: 5, or the sequence described in SEQ ID NO: 6 and SEQ ID NO: 7. The method according to (17) above, which is a double-stranded RNA-DNA chimeric molecule having the base sequence described in Double-stranded RNA-DNA chimeric molecule comprising a base sequence (19) A double-stranded RNA molecule or double-stranded RNA-DNA chimeric molecule having a length of 19 to 25 bases, which has an action of mediating RNA interference of the mRNA encoding the cell cycle regulatory protein according to (2) above; (20) The double-stranded RNA-DNA chimera according to (19), comprising the base sequence described in SEQ ID NO: 4 and SEQ ID NO: 5, or the base sequence described in SEQ ID NO: 6 and SEQ ID NO: 7. (21) A method of inducing cell death comprising the use of the method according to any one of (9) to (18); (22) the cell cycle regulatory protein or salt thereof according to (2); A pharmaceutical composition comprising a substance that enhances or inhibits biological activity; (23) the substance is (a) the polynucleotide according to (1), (b) the protein according to (2) or a salt thereof; , (C) (3 (D) the composition according to (22), which is the partial peptide or salt thereof according to (7), or the antibody according to (7); (24) a therapeutic or preventive agent for liver disease or renal disease; A composition according to (22) above; (25) a composition according to (24) above wherein the liver disease is liver cancer; (26) for the manufacture of a therapeutic / prophylactic agent for liver disease or kidney disease; Use of the cell cycle regulatory protein or salt thereof according to (2) above or the partial peptide of the cell cycle regulatory protein according to (3) above or salt thereof; (27) Therapeutically effective of the composition according to (22) above (28) the cell cycle regulatory protein according to (2) above or the partial peptide according to (3) above, or a method for their treatment, which comprises administering an amount to a patient; detect mRNA or (29) the polynucleotide according to (1) encoding the cell cycle regulatory protein, the cell cycle regulatory protein or salt thereof according to (2), Or a method for screening a compound having a cell cycle regulatory activity or a salt thereof using the partial peptide of claim 3 or a salt thereof; (30) (a) expressing the cell cycle regulatory protein of (2) above A compound having a cell cycle-regulating activity, or a salt thereof, comprising contacting a test sample with a cell to be tested, and (b) detecting and measuring a change in biological activity of the protein or measuring cell death of the cell (31) The polynucleotide according to (1), encoding the cell cycle regulatory protein, the cell cycle regulatory protein according to (2) or Or a kit for screening a substance having cell cycle regulatory activity using the partial peptide or salt thereof according to (3) above; (32) the method according to (29) or (30) above or The present invention relates to a compound having a cell cycle regulatory activity or a salt thereof, which can be isolated by the kit according to (31).
As a result of intensive studies using human FRM protein and rat HP33 protein, the present inventors have found the following with respect to the protein.
First, in order to search for the mechanism of organ-specific expression and overexpression in HCC, the genomic DNA sequence of FRM protein was analyzed, and several transcription factor binding region candidates were found in the promoter region. Thus, as a result of preparing a reporter plasmid in which each candidate region was deleted and introducing it into human hepatoma cell HepG2, it was revealed that Sp1 was required as an Sp1 binding region and a transcription factor for FRM protein expression.
Subsequently, as a result of analysis using Ensembl Human Genome Server after analysis by the FISH method, it was confirmed that the FRM gene was located in the 11q13.2 region of chromosome 11.
It has already been reported that Sp1 is overexpressed in HCC cells (Yang, H. et al., Faseb J (2001) 15 (9): 1507-1516, Xu, XR. Et al., Proc Natl. Acad Sci USA (2001) 98 (26): 15089-94), and the 11q13.2 region of chromosome 11 is known to be amplified in various cancer diseases, particularly HCC and breast cancer (Gergely, F.,). et al., Proc Natl Acad Sci USA (2000) 97 (26): 14352-7, Raff, JW et al., Trends Cell Biol (2002) 12 (5): 222-5). From these facts, it became clear that the overexpression of FRM protein in HCC originates from two different mechanisms, namely transcriptional control and gene amplification, and the correlation between overexpression of FRM protein and HCC was shown. Such a specific expression mechanism has not been elucidated for the rat HP33 protein and bovine N-acyltransferase.
In addition, using a normal rat-derived RL-34 cell line, a rat HP33 overexpressing strain and an expression-suppressing strain were prepared, and tubulin, DNA and HP33 protein were stained and observed. It was confirmed that not only the FRM protein but also tubulin disappeared for those whose expression was suppressed.
Next, when a rat-derived liver cancer cell line in which the expression of HP33 protein was suppressed by introduction of an HP33 antisense expression plasmid and a normal rat-derived liver cancer cell line were transplanted into rats, HP33 expression was suppressed. In the individual, it was confirmed that the growth of the tumor was significantly suppressed as compared with the individual whose expression was not suppressed.
Subsequently, by comparing the sensitivity of Taxol in the HP33 overexpressing strain and the RL-34 cell line by the FACS method, even if a very small amount of taxol is added to the extent that no effect is seen in the RL-34 cell line. In the HP33 overexpressing strain, it was confirmed that cells in the G1 phase shift to the G2 / M phase. From this, it can be said that the co-action of HP33 overexpression and taxol advances the death of the cells.
Furthermore, when the state of DNA and microtubules during cell division in FRM protein expression-suppressed strains and FRM protein over-expressing strains was observed with a fluorescence microscope, it was found that the FRM protein expression-suppressed strain cells were more probable than other cells. It was also confirmed that abnormal formation of microtubules occurred at a significantly high probability.
In human hepatoma cell lines, when the expression of human FRM protein in the cells was suppressed by the siRNA method (RNA interference), it was shown that apoptosis was induced in the cells.

FIG. 1 shows the results of FRM mRNA expression analysis by Northern blotting.
FIG. 2 shows the experimental results of the cross-reaction of anti-rat HP33 antibody and human FRM protein.
FIG. 3 shows the results of an immunohistochemical test in which the expression of FRM protein in HCC cells was analyzed using an anti-rat HP33 antibody.
FIG. 4 is a schematic diagram showing various promoter / luciferase reporter systems lacking the 5 ′ end.
FIG. 5 shows a comparison of luciferase activity in HepG2 cells and Hela cells.
FIG. 6 shows a comparison of luciferase activity in HepG2 cells and Hela cells with and without overexpression of Sp1.
FIG. 7 shows the results of analysis by the FISH method that confirmed the localization of the FRM gene on the chromosome.
FIG. 8 shows the results of staining FRM protein, tubulin and DNA in a rat HP33 overexpression strain and an expression suppression strain.
FIG. 9 shows changes in tumor weight when the expression of the HP33 gene is suppressed or not in the rat-derived liver cancer cell line.
FIG. 10 shows the results of a taxol sensitivity test in an FRM overexpressing strain.
FIG. 11 shows a state of abnormal microtubule formation during cell division in an FRM expression-suppressed strain.

本発明者らは、ヒトＦＲＭ蛋白質及びラットＨＰ３３蛋白質は、肝臓および腎臓において特異的に発現すること、ＨＣＣにおいて過剰発現していること、癌細胞においてその発現を抑制すると腫瘍の増殖が抑制されること、細胞内において過剰発現させた上で、式

で表されるタキソール（Ｔａｘｏｌ（Ｐａｃｌｉｔａｘｅｌ；
（２Ｒ，５Ｒ，７Ｓ，１０Ｒ，１３Ｓ）−１０，２０−Ｂｉｓ（ａｃｅｔｏｘｙ）−２−ｂｅｎｚｏｙｌｏｘｙ−１，７−ｄｉｈｙｄｒｏｘｙ−９−ｏｘｏ−５，２０−ｅｐｏｘｙｔａｘ−１１−ｅｎ−１３−ｙｌ
（３Ｓ）−３−ｂｅｎｚｏｙｌａｍｉｎｏ−３−ｐｈｅｎｙｌ−Ｄ−ｌａｃｔａｔｅ））またはその塩を添加するとその細胞の死が誘導されること、アンチセンス法により細胞内での発現が抑制されるとその細胞のアポトーシスが誘導されること、アンチセンス法により細胞内での発現が抑制されるとその細胞で微小管形成の異常が発生する確率が高くなること等を見出した。
本発明にかかる「細胞周期調節方法」は、各種抗癌剤との併用される方法であってもよい。前記抗癌剤として好適なのは、タキソールまたはその塩である。抗癌剤の使用は、ＦＲＭ蛋白質の生物活性を亢進または阻害する手段を用いるのと同時であってもよいし、一定時間間隔をおいて前記手段の前または後の使用であってもよい。ＦＲＭ蛋白質の細胞内濃度を変化させたり、ＦＲＭ蛋白質の生物活性を亢進または抑制する物質は、医薬の有効成分として有用である。ＦＲＭ蛋白質の細胞内の濃度が増加または減少するとアポトーシスが誘導されることから、ＦＲＭ蛋白質の生理学的作用を阻害する作用を有する抗体、化合物又はＦＲＭ蛋白質をコードするポリヌクレオチドに対するアンチセンスオリゴヌクレオチドを投与することにより、アポトーシス抑制による疾患の予防及び／又は治療等に寄与することができる。より具体的には、ＦＲＭ蛋白質は肝臓及び腎臓で特異的に発現していることから、これらの臓器の細胞内におけるＦＲＭ蛋白質濃度を減少させることにより、当該細胞のアポトーシスを誘導することができる。アポトーシス抑制による疾患とは、例えば各種癌疾患や自己免疫疾患などが挙げられ、最も好適には肝細胞癌（肝がん）に有効であると期待することができる。その他、ＦＲＭ蛋白質を患者の細胞内に投与したり、ＦＲＭ蛋白質をコードするポリヌクレオチドを患者に投与して発現させたり、対象となる細胞にＦＲＭ蛋白質をコードするポリヌクレオチドを導入して発現させた後に、この細胞を患者の体内に移植したりして、当該細胞のアポトーシスを抑制することができ、アポトーシス亢進による疾患の予防及び／又は治療に有用であると期待することもできる。細胞のアポトーシスを抑制するためには、本発明にかかるＦＲＭ蛋白質の機能を亢進する抗体や化合物を当該細胞に投与することもできる。アポトーシス亢進による疾患とは、例えば、エイズや肝炎などのウィルス感染症や、アルツハイマー病などの神経変性疾患などが挙げられる。
本発明にかかる「医薬組成物」とは、細胞周期調節蛋白質（ＦＲＭ）またはその塩の生物活性を亢進または阻害する物質を含んでなる医薬組成物である。該物質は、ＦＲＭ蛋白質の生物活性を亢進または阻害する活性を有するものである限りにおいて得に限定されず、ＦＲＭ蛋白質をコードする遺伝子の発現を亢進または抑制する物質、ＦＲＭ内在量を増加させる物質、ＦＲＭ蛋白質の活性を亢進または阻害する作用を有する物質等が含まれる。これらの物質の具体例としては、例えばＦＲＭ蛋白質をコードするポリヌクレオチド、ＦＲＭ蛋白質またはその塩、ＦＲＭ部分ペプチドまたはその塩、ＦＲＭ蛋白質の抗体、ＦＲＭ蛋白質のアゴニスト・アンタゴニスト等があげられる。即ち、前記医薬組成物の有効成分物質は、ＦＲＭ蛋白質に直接または間接的に作用して、ＦＲＭの生物活性を変化させるものであればよい。
本発明にかかる医薬組成物の用途は特に限定されないが、とりわけ肝疾患または腎疾患の治療剤または予防剤として有用である。前記肝疾患と腎疾患の例は特に限定されず、医学的にこれらの疾患に分類される全ての疾患を含む。肝疾患として特に有効なのは肝がんである。
本発明にかかる医薬組成物を治療剤又は予防剤として使用する場合は、通常の手段に従って製剤化することができる。例えば、本発明の医薬組成物は薬理的に許容しうる担体と均一に混合して使用することができる。前期担体は、投与に望ましい製剤の形態に応じて、広い範囲の形態をとることができ、経口的に又は注射により投与しうる単位服用形態にあることが望ましい。懸濁剤及びシロップ剤のような経口液体調整物は、水、ショ糖、ソルビトール、果糖等の糖類、ポリエチレングリコール等のグリコール類、ゴマ油、オリーブ油、大豆油当の油類、アキルパラヒドロキシベンゾエート等の防腐剤、ストロベリー・フレーバー、ペパーミント・フレーバーなどのフレーバー類を使用することができる。散剤、丸剤、カプセル及び錠剤は、乳糖、ブドウ糖、ショ糖、マンニトール等の賦形剤、デンプン、アルギン酸ソーダ等の崩壊剤、マグネシウムステアレート、タルク等の滑沢剤、ポリビニルアルコール、ヒドロキシプロピルセルロース、ゼラチン等の結合剤、脂肪酸エステル等の表面活性剤、グリセリン等の可塑剤等を用いて製造できる。また、注射用の溶液は、塩溶液、グルコース溶液、又は塩水とグルコース溶液の混合物からなる担体を用いて調整することができる。
又本発明のポリヌクレオチドを予防剤や治療剤として使用する場合には、本発明のポリヌクレオチドを単独で、またはレトロウィルスベクター、アデノウィルスベクターなどの適当なベクターに挿入した後、公知の手段によって投与することができる。本発明のポリヌクレオチドは、単独で、あるいは摂取促進のための補助剤とともに、遺伝子銃やハイドロゲルカテーテルのようなカテーテルによって投与できる。
本発明にかかる「スクリーニング方法」とは、細胞周期調節蛋白質（ＦＲＭ）をコードするポリヌクレオチド、ＦＲＭもしくはその塩、または、ＦＲＭの部分ペプチドもしくはその塩を用いた、細胞周期調節活性を有する化合物またはその塩のスクリーニング方法を意味する。該スクリーニング方法としては、例えば、（ａ）ＦＲＭ蛋白質を発現する細胞に被験試料を接触させ；（ｂ）前記蛋白質の生物活性の変化を検出し測定するか、または、当該細胞の細胞死を測定することを含む、細胞周期調節活性を有する化合物またはその塩のスクリーニング方法があげられる。スクリーニングに用いるＦＲＭ蛋白質は、ヒトの細胞から抽出されたものでもよく、遺伝子組換えにより得られたものでもよく、前記方法で得られた部分ペプチドであってもよい。
他のスクリーニング法の例として、ＦＲＭ蛋白質に結合する蛋白質のスクリーニング方法として例えばウェスタンブロッティング法が挙げられる。被検試料としてある細胞の全蛋白質をＳＤＳ（Ｓｏｄｉｕｍ ｄｏｄｅｃｙｌ ｓｕｌｆａｔｅ）を含む緩衝液で溶解抽出したのち、ＳＤＳ−アクリルアミドゲル電気泳動にかけ、分子量に従って分離展開する。次にゲル中に展開された蛋白質をニトロセルロース膜に電気転写する。そして標識したＦＲＭ蛋白質をこの膜に接触させ、この標識を検出することにより、ＦＲＭ蛋白質に結合する蛋白質のバンドを検出することができる。標識の方法としては、ラジオアイソトープを利用する方法、蛍光を利用する方法、ＦＲＭ蛋白質と標識となる蛋白質の融合蛋白質を利用する方法などがある。また、ＦＲＭ蛋白質自体を標識せず、本発明にかかる抗体を標識して、ニトロセルロース膜上の蛋白質に結合したＦＲＭ蛋白質を検出する方法も可能である。
本発明にかかるＦＲＭ蛋白質に結合する低分子化合物のスクリーニング方法としては、例えばＦＲＭ蛋白質をビオチンで標識し、ビオチンとアビジンの反応を利用してＦＲＭ蛋白質を基質上に固定し、これに低分子化合物を含む被検試料を接触させる。この方法の場合、ＦＲＭ蛋白質と低分子化合物の結合は、低分子化合物を予め標識しておくことで検出することも可能であり、例えば表面プラズモン共鳴を利用したシステムや質量分析計を利用して標識をせずに結合を検出することも可能である。
また、ＦＲＭ蛋白質をアフィニティーカラム内に固定し、被検試料をこのカラム内に通し、カラムに特異的に結合した化合物を精製することによっても、ＦＲＭ蛋白質に結合する化合物を得ることができる。
ＦＲＭ蛋白質に結合する活性、又はＦＲＭ蛋白質の生物学的活性を変化させる活性を有する化合物は、天然由来であっても、人工的に合成された化合物であってもよい。
本発明にかかるスクリーニング用キットとは、本発明にかかるスクリーニング方法の実施に用いるためのキットであり、即ち、細胞周期調節蛋白質（ＦＲＭ）をコードするポリヌクレオチド、ＦＲＭ蛋白質もしくはその塩、または、ＦＲＭ蛋白質の部分ペプチドもしくはその塩を用いた、細胞周期調節活性を有する物質のスクリーニング用キットをいう。
本発明にかかるスクリーニング方法またはスクリーニング用キットにより単離される化合物またはその塩は、細胞周期調節活性を有する化合物またはその塩である。前記化合物またはその塩は、ＦＲＭ蛋白質の生物活性を亢進または抑制する活性を有する。
本発明はまた、細胞内におけるＦＲＭ蛋白質の量を調節することを含む各種癌疾患、自己免疫疾患、ウィルス感染症若しくは神経変性疾患の治療方法または予防方法を提供する。本発明者らは、既に述べたように、ラット肝臓癌細胞株を移植したラットにおいてアンチセンス法によりＨＰ３３蛋白質の発現を抑制したところ、腫瘍細胞の増殖が抑制されることを確認した。また、ＲＮＡｉ法により細胞内におけるＦＲＭ蛋白質の発現を抑制したところ、当該細胞のアポトーシスが誘導されることを見出した。さらに、細胞内でＨＰ３３蛋白質を過剰発現させ、同時にＴａｘｏｌを添加すると、当該細胞の死が誘導されることを確認した。以上より、細胞内におけるＦＲＭ蛋白質量を調節することにより、アポトーシス関連疾患、好ましくは癌疾患、さらに好ましくは肝臓癌の治療又は予防をすることができると期待される。細胞内におけるＦＲＭ蛋白質量の調節は、本発明にかかる医薬組成物の治療上有効量を患者に投与することによりおこなうことができる。
また、本発明は、ＦＲＭ蛋白質若しくは部分ペプチド又はその塩をマーカーとして肝臓癌又は腎臓癌を診断する方法も提供する。上述のように、ＦＲＭ蛋白質はＨＣＣ細胞において過剰発現しているので、ある細胞ないおけるＦＲＭ蛋白質や、本発明のポリヌクレオチド、好ましくはＦＲＭ蛋白質をコードするｍＲＮＡを検出及び／又は定量し、過剰発現しているかどうかを確認することで、その細胞がＨＣＣに侵されているかどうかの診断をすることができる。ポリヌクレオチドの検出及び定量には公知の方法を使用することができ、例えば、ＦＲＭ蛋白質をコードするＤＮＡをプローブとした競合ハイブリダイゼーション法や、ドットブロット法、ノーザンブロット法、ＲＮアーゼプロテクションアッセイ法、ＲＴ（Ｒｅｖｅｒｓｅ Ｔｒａｎｓｃｒｉｐｔｉｏｎ）−ＰＣＲ法などの発現解析方法を用いることができる。ＦＲＭ蛋白質の検出及び定量も公知の方法を使用することができる。例えば本発明の抗体（フラグメントを含む。）を使用して、適当な標識を用いて検出又は定量する方法のほか、各種質量分析法を用いてＦＲＭ蛋白質と等しい質量の蛋白質量を計測し、これを健常者の平均値を比較する方法、電気泳動によりＦＲＭ蛋白質を検出する方法などを用いることができる。
以下、本発明を実施例により具体的に説明するが、本発明はこれら実施例に制限されるものではない。
［実施例１］ ヒトＦＲＭｃＤＮＡの単離、及び配列解析
ＴＢＬＡＳＴＮデータベース検索により、ラットＨＰ３３蛋白質のアミノ酸配列をコードする塩基配列と相同性の高い（６３％）ヒト遺伝子（ＧｅｎＢａｎｋ ａｃｃｅｓｓｉｏｎ ｎｏ．ＡＢ０１３０９３）を見出した。この遺伝子配列の断片をＰＣＲにより増幅し、ヒト肝臓ｃＤＮＡライブラリーのスクリーニングに用い、いくつかのクローンを得た。これらのクローンの配列を解析した結果、ヒトｃＤＮＡが８８８ｂｐのヌクレオチドからなり、２９５のアミノ酸をコードすることが確認された。ｃＤＮＡの塩基配列を配列番号：１に、アミノ酸配列を配列番号：２に示す。ヒトＦＲＭ蛋白質の分子量は３４ｋＤと計算された。
［実施例２］ ノーザンブロット法による、ＦＲＭｍＲＮＡの発現解析
Ｒａｎｄｏｍ Ｐｒｉｍｅｄ ＤＮＡ Ｌａｂｅｌｉｎｇ Ｋｉｔ（Ｒｏｃｈｅ Ｄｉａｇｎｏｓｔｉｃｓ社）を用いてヒトＦＲＭ蛋白質のｃＤＮＡを放射能ラベルし、プローブとした（１×１０^６ｃｐｍ／ｍｌ）。仕様書に従って、２ｕｇ／ｌａｎｅのポリ（Ａ）＋ＲＮＡがブロットされたＭｕｌｔｉｐｌｅ Ｔｉｓｓｕｅ Ｎｏｒｔｈｅｒｎ（ＭＴＮ）ｍｅｍｂｒａｎｅ（Ｃｌｏｎｔｅｃｈ社）に前記プローブをハイブリダイズさせ、オートラジオグラフィーにより検出した。結果を図１に示す。
［実施例３］ ＦＲＭ抗体を用いたＨＣＣ細胞におけるヒトＦＲＭ蛋白質の発現解析
３−１．ＦＲＭ及びＨＰ３３抗体の交差反応の確認
ｐＥＴ−３ａ発現ベクターに、Ｎ末端にＦｌａｇタグ及びＨｉｓｔｉｄｉｎｅタグを有するＨＰ３３及びＦＲＭのコード領域を挿入し、ＩＰＴＧ誘導により、ラットＨＰ３３及びヒトＦＲＭ蛋白質をＥ．Ｃｏｌｉ ＢＬ２１（ＤＥ３）ｐＬｙｓＳ株を用いて過剰発現させた。Ｅ．Ｃｏｌｉの菌体を超音波処理のにより破壊し、Ｎｉ−ＮＴＡアガロース（Ｑｉａｇｅｎ社）によって上清をアフィニティ精製した。得られた組換え蛋白質５０ｎｇを１２．５％ＳＤＳ−ＰＡＧＥで分離後、ＰＶＤＦ膜（Ｍｉｌｌｉｐｏｒｅ社）に転写した。ＨｉＴｒａｐ ＮＨＳ−ａｃｔｉｖａｔｅｄ Ｃｏｌｕｍｎ（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ）にヒトＦＲＭ蛋白質を固定して抗ラットＨＰ３３抗体をアフィニティー精製し、３０００倍に希釈した。蛋白質はＡｌｋａｌｉｎｅ ｐｈｏｓｐｈａｔａｓｅ ｃｏｎｊｕｇａｔｅ ｓｕｂｓｔｒａｔｅ ｋｉｔ（ＢｉｏＲａｄ）のプロトコルにしたがって検出した。結果を図２に示す。
３−２．ＨＣＣ細胞におけるＦＲＭ蛋白質の過剰発現の検出
ヒトＦＲＭ蛋白質の免疫組織化学検査は、ＨＣＣ組織片を用いて、ＳａｓａｋｉらによるＡｖｉｄｉｎ−ｂｉｏｔｉｎ ｐｅｒｏｘｉｄａｓｅ ｃｏｍｐｌｅｘ（ＡＢＣ）法に従って、標準的な手法により行った。組織片はアフィニティー精製された抗ラットＨＰ３３抗体とインキュベーションした。結果を図３に示す。上段は健常者、下段がＨＣＣ患者から得た組織片である。
［実施例４］ ＦＲＭのゲノムＤＮＡ配列解析とプロモーターの研究、ＦＩＳＨ、及び臓器特異的発現とＨＣＣにおける過剰発現機構
４−１．ヒトＦＲＭのゲノムＤＮＡクローンの単離
４−２．プロモーター／レポーター・プラスミドの構築
ＦＲＭゲノムＰ１クローンをテンプレートとして、ＦＲＭ遺伝子の−２９３２の下流、＋１０２の上流部分のＤＮＡ断片（ｐＢＳ−２９３２）をＰＣＲにより作成し、ｐＢｌｕｅｓｃｒｉｐｔ ＩＩ ＳＫ＋のＥｃｏＲＶ部位にサブクローニングした。−８２２から＋１０２までの配列を配列番号：３に示す。そして、このｐＢＳ−２９３２をテンプレートとして、５’末端を欠損した種々のプロモーター／ルシフェラーゼ・レポーター系（ｐＬｕｃ−２９３２からｐＬｕｃ＋１）を構築し、ルシフェラーゼ発現ベクターのｐＧＬ３−Ｂａｓｉｃ（Ｐｒｏｍｅｇａ社）のＳｍａｌ部位にサブクローニングした。プロモータの欠損の種類を図４に示す。図中、それぞれの数字は５’末端を表す。ｐＬｕｃ−２５５−Ｓｐ１はＡｐａｌ部位からＥｃｏＲＩ部位（Ｓｐ１様モチーフを含む）を欠損することにより作成した。全てのプラスミドは配列を確認した。
４−３．細胞培養、遺伝子導入、ルシフェラーゼ・アッセイ
ＨｅｐＧ２細胞は１０％ＦＣＳ、１００ｕＭ ＮＥＡＡ溶液（Ｇｉｂｃｏ社）及び抗生物質を含むＭＥＭ培地（Ｇｉｂｃｏ社）で、ＨｅＬａ細胞とＲＬ３４細胞は、１０％ＦＣＳと抗生物質を含むＤＭＥＭ培地（Ｇｉｂｃｏ社）で、ＦＡＡ−ＨＴＣ１細胞は１０％ＦＣＳ、２ｍＭ Ｌ−Ｇｌｕｔａｍｉｎｅ及び抗生物質とともにＷｉｌｌｉａｍｓ Ｅ培地（Ｇｉｂｃｏ社）で培養した。ルシフェラーゼ・アッセイには、３０％ｃｏｎｆｌｕｅｎｃｅの培養細胞を２４穴プレート（Ｆａｌｃｏｎ社）に用意し、３５０ｎｇ／ｗｅｌｌのｐＧＬ３−Ｂａｓｉｃ由来のレポーター遺伝子と、コントロールとして５ｎｇ／ｗｅｌｌのｐＲＬ−ＴＫ（Ｐｒｏｍｅｇａ社）をｃｏｔｒａｎｓｆｅｃｔｉｏｎさせた。Ｓｐ１を用いたルシフェラーゼ・アッセイに関しては、Ｓｐ１／ｐｃＤＮＡ３．１（＋）を５０ｎｇ／ｗｅｌｌ、同様にｃｏｔｒａｎｓｆｅｃｔｉｏｎさせた。遺伝子の導入にはＬｉｐｏｆｅｃｔａｍｉｎｅ Ｐｌｕｓ（Ｇｉｂｃｏ社）を用いた。４８から６０時間後、Ｄｕａｌ Ｌｕｃｉｆｅｒａｓｅ Ｋｉｔ（Ｐｒｏｍｅｇａ社）とＴＤ２０ ｌｕｍｉｎｏｍｅｔｅｒ（Ｐｒｏｍｅｇａ社）を使用してルシフェラーゼ活性を測定し、コントロールのｐＲＬ−ＴＫ活性を基準として標準化した。結果を図５及び図６に表す。
４−４．Ｆｌｕｏｒｅｓｃｅｎｃｅ ｉｎ ｓｉｔｕ ｈｙｂｒｉｄｉｚａｔｉｏｎ（ＦＩＳＨ）
ヒトの血液からリンパ球を単離して解析した。Ｇｉｂｃｏ ＢＲＬ ＢｉｏＮｉｃｋ ｌａｂｅｌｉｎｇ ｋｉｔを使用し、ｄＡＴＰ存在下でＰ１クローン由来のヒトＦＲＭ遺伝子断片をビオチン化してハイブリダイゼーションのプローブとした。ＦＩＳＨ法は、Ｈｅｎｇら（１９９２）の方法及びＨｅｎｇとＴｓｕｉ（１９９３）による方法に従った。染色体はＤＡＰＩにより染色された。結果を図７に示す。
［実施例５］ ＨＰ３３過剰発現株及び発現抑制株におけるチューブリンとＦＲＭ蛋白質の挙動
５−１．ＨＰ３３過剰発現株の作成
ラットＨＰ３３のｃＤＮＡを用いてラクトース依存性動物細胞発現用ＨＰ３３発現プラスミドを作成し、正常ラット由来のＲＬ−３４細胞株にリポフェクトアミンを用いて遺伝子導入を行った。遺伝子導入後２日目に、４００ｕｇ／ｍｌのハイグロマイシン、４００ｕｇ／ｍｌのジェネテシンが入った培地に取替え、培養を行った。その後ＨＰ３３を発現させるため５ｍＭ ＩＰＴＧを添加してＨＰ３３の発現を行った。
５−２．ＨＰ３３の発現抑制株の作成
テトラサイクリン依存性動物細胞発現用ＨＰ３３アンチセンス発現プラスミドを作成し、テトラサイクリン活性化因子ベクターとＧ４１８耐性遺伝子ベクターと共にリポフェクトアミン（Ｉｎｖｉｔｒｏｇｅｎ社）を用いてＲＬ３４株に導入した。導入後２日目に４００ｕｇ／ｍｌのジェネテシン、１５ｕｇ／ｍｌのテトラサイクリンが入った培地に取替え、培養を行い、Ｇ４１８耐性の細胞株を分離した。ＨＰ３３の発現抑制はテトラサイクリンを含まない培地で培養することにより行った。
５−３．チューブリン、ＨＰ３３蛋白質、ＤＮＡの染色
ＲＬ−３４細胞をカバーグラス上で４％パラホルムアルデヒド、５ｍＭ ＥＧＴＡ、２ｍＭ ＭｇＣｌ２入りリン酸ナトリウム緩衝液にて８分間固定後、Ｔｒｉｔｏｎ Ｘ−１００にて５分間膜透過処理を行い、１％ＢＳＡにて１時間ブロッキング反応を行った。ブロッキング後、１％ＢＳＡにて２０００倍希釈した抗α−チューブリン抗体、２００倍希釈した抗ＨＰ３３抗体を用いて１時間反応を行い、その後１００倍希釈したＦＩＴＣ標識ロバ抗ウサギＩｇＧ、Ｔｅｘａｓ Ｒｅｄ標識ヒツジ抗マウスＩｇＧにて１時間染色を行った。最後に１ｕｇ／ｍｌのＤＡＰＩにてＤＮＡを染色し観察に供した。結果を図８に示す。
［実施例６］ ラット由来肝臓癌細胞株ＦＡＡ−ＨＴＣ１におけるＨＰ３３遺伝子の発現抑制と腫瘍増殖抑制効果
Ｗｉｓｔｅｒ Ｒａｔにラット由来肝臓ガン細胞株ＦＡＡ−ＨＴＣ１（ＦＡＡ）、ＨＰ３３発現抑制株（α１、α１４）を１０^６個経腹投与を行い、２ヵ月後の腫瘍増殖の様子を観察した。ＨＰ３３発現抑制株はテトラサイクリン依存性動物細胞発現用ＨＰ３３アンチセンス発現プラスミド（ｐＴｅｔ−ＨＰ３３）を作成し、親株であるＦＡＡ細胞株にリポフェクトアミンを用い遺伝子導入を行った。遺伝子導入後、２日目に４００μｇ／ｍｌのジェネテシン１５ｕｇ／ｍｌのテトラサイクリンが入った培地に取替え、培養を行いＧ４１８耐性の細胞株を分離した。ＨＰ３３の発現抑制はテトラサイクリンを含まない培地で培養することにより行った。結果を図９に示す。グラフはラット体重における腫瘍重量の割合である（ｎ＝３）。ＦＲＭ蛋白質の発現を抑制したラットでは、腫瘍重量の増加がほとんど見られなかった。
［実施例７］ ＦＲＭ過剰発現株におけるＴａｘｏｌ感受性
培養フラスコ中のＲＬ−３４、ファーム過剰発現株（それぞれタキソール０ ｏｒ ０．１ｕＭ含むＤＭＥＭ培地で２４時間培養）をトリプシン／ＥＤＴＡにて３７℃、３分処理し、血清を含むＤＭＥＭ培地で反応を停止、細胞浮遊液を得、その後、冷ＰＢＳ（ＦＡＣＳ用ＰＢＳ）にて３回洗浄し、最終的に３ｍｌのＰＢＳに懸濁した。この懸濁液に７ｍｌの−２０℃エタノールを激しく攪拌しながら滴下し、細胞を固定した。固定した細胞は−２０℃で一晩固定し、その後、ＰＢＳで２回洗浄した後、０．５ｍｇ／ｍｌのＲＮＡｓｅを含むＰＢＳ５００ｕｌに懸濁し、プロピディウムイオダイド（ＰＩ）を細胞浮遊液中に加え、室温にて１０分間反応させる。この溶液を、ＦＡＣＳ解析に用いた。結果を図１０に示す。図中、ピークが二つ見られるものについては、左のピークはＧ１期にある細胞、右のピークはＧ２／Ｍ期にある細胞を示す。過剰発現株にＴａｘｏｌを添加したものはＧ１期の細胞が極端に減少し、Ｇ２／Ｍ期の細胞が増大している。
［実施例８］ ＦＲＭ発現抑制株における細胞分裂時の微小管形成異常
ＲＬ−３４細胞株、ファーム過剰発現株、ファーム発現抑制株での細胞分裂時のＤＮＡ、微小管の様子を蛍光顕微鏡にて観察した。細胞をカバーグラス上で４％パラホルムアルデヒド、５ｍＭ ＥＧＴＡ、２ｍＭ ＭｇＣｌ２入り燐酸ナトリウム緩衝液にて８分間固定後、Ｔｒｉｔｏｎ Ｘ−１００にて５分間膜透過処理を行ない、１％ＢＳＡにて１時間ブロッキング反応を行なった。ブロッキング後、１％ＢＳＡにて２００倍希釈した抗微小管抗体を用いて１時間反応を行いその後２０倍希釈したＦＩＴＣ標識ヤギ抗マウスＩｇＧ、１時間染色を行った。最後に１μｇ／ｍｌの濃度のＤＡＰＩにてＤＮＡを染色し観察に供した。結果を図１１に示す。上段のパネルはファーム蛋白発現抑制株のＤＮＡ、微小管を示す。パネル内右下にはＲＬ−３４細胞の様子を示す。下段グラフはそれぞれの細胞をスライドグラス上にて培養し、微小管染色を行った後の微小管形成異常があった細胞の顕微鏡下にて計数し、全細胞数に対する異常細胞の割合を示す。
［実施例９］ ｓｉＲＮＡ法を用いたヒトＦＲＭの発現抑制によるアポトーシスの誘導
（１）ヒト肝癌細胞株ＨｅｐＧ２におけるＦＲＭ遺伝子発現抑制の影響
ヒト肝癌細胞株ＨｅｐＧ２を１ｘ１０^４個の濃度にて６穴培養皿で培養を行った。培養２日目に２００ｎＭの濃度のＦＲＭ遺伝子に相補的な２本鎖ＲＮＡ−ＤＮＡキメラを培養中に添加し、遺伝子発現抑制を行った。用いたＦＲＭ遺伝子に相補的な２本鎖ＲＮＡ−ＤＮＡキメラの配列として、以下に示す配列番号：４と配列番号：５とに記載の塩基配列からなる２本鎖、または配列番号：６と配列番号：７とに記載の塩基配列からなる２本鎖を用いた。また、対照群には、配列番号：３０と配列番号３１とに記載のランダムな塩基配列からなる２本鎖ＲＮＡ−ＤＮＡキメラを用いた。

上記オリゴヌクレオチドを培養中に添加後、２４ｈ，４８ｈ，７２ｈの細胞の様子を示す。
結果：ＦＲＭ遺伝子に相補的な２本鎖ＲＮＡ−ＤＮＡキメラ添加群では経日的な細胞の減少が観察されるが、対象群では細胞の減少は観察されなかった。
（２）ＦＲＭ遺伝子発現抑制したヒト肝癌細胞株ＨｅｐＧ２における細胞死の機序
ヒト肝癌細胞株ＨｅｐＧ２を１ｘ１０^４個の濃度にて６穴培養皿で培養を行った。培養２日目に２００ｎＭの濃度のＦＲＭ遺伝子に相補的な２本鎖ＲＮＡ−ＤＮＡキメラを培養中に添加し、遺伝子発現抑制を行った。用いたＦＲＭ遺伝子に相補的な２本鎖ＲＮＡ−ＤＮＡキメラの配列は以下の配列を用いた。また、対照群にはランダムな塩基配列の２本鎖ＲＮＡ−ＤＮＡキメラを用いた。

上記オリゴヌクレオチドを培養中に添加後、４８ｈ，７２ｈで細胞を回収し、細胞より核を抽出し、ＤＮＡの断片化をアガロース電気泳動にて観察した。核の抽出にはＡｐｏｐｔｏｔｉｃ ＤＮＡ−Ｌａｄｄｅｒ ｋｉｔ（ＲｏｃｈｅＤｉａｇｎｏｓｔｉｃｓ社）を用いた。
結果：ＦＲＭ遺伝子に相補的な２本鎖ＲＮＡ−ＤＮＡキメラ添加群ではＤＮＡの断片化が観察されるが、対象群ではＤＮＡの断片化は観察されなかった。この断片化はアポトーシスを起こした細胞に特徴的に見られるが、ＦＲＭ遺伝子の発現を抑制したヒト肝癌細胞株ＨｅｐＧ２ではアポトーシスにより細胞が死んでいくことを示す。Hereinafter, the present invention will be described in detail with reference to the meanings of symbols, terms, and the like described in the present specification and embodiments of the present invention.
The base sequence of the isolated FRM cDNA according to the present invention is shown in SEQ ID NO: 1, and the amino acid sequence of the FRM protein is shown in SEQ ID NO: 2.
The polynucleotide concerning this invention contains the polynucleotide which codes the protein which consists of an amino acid sequence of sequence number: 2. Any polynucleotide may be used as long as it includes the base sequence encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, for example, DNA, RNA such as mRNA, etc. Alternatively, it may be double stranded. Double stranded includes double stranded DNA, double stranded RNA and RNA / DNA hybrids. In the case of a single strand, it may be a sense strand or an antisense strand. Moreover, as long as the sequence is included in the present invention, those derived from cells and tissues, those synthesized and amplified are also included in the present invention.
The polynucleotide according to the present invention comprises an amino acid sequence in which one or more amino acids are added, deleted, substituted, and / or inserted in the amino acid sequence shown in SEQ ID NO: 2, and the sequence shown in SEQ ID NO: 2 A polynucleotide encoding a protein functionally equivalent to a protein consisting of an amino acid sequence is also included. The “protein” means a protein having the same biological activity as the FRM protein. Such a polynucleotide can be obtained by introducing a mutation into the DNA described in SEQ ID NO: 1. As such a “method for introducing a mutation”, for example, a site-specific mutagenesis method can be used. According to this method, a gene to be mutated is first incorporated into a part of a vector such as M13 phage to obtain a single-stranded circular phage DNA, which is designed by changing some bases so as to introduce a mutation therein. The hybridized oligonucleotides are hybridized and then repaired with DNA polymerase and DNA ligase to obtain double-stranded circular DNA partially mismatched. When this is infected with Escherichia coli or the like and cultured, when the double-stranded recombinant M13 is replicated, the mutant and non-mutant strains are separated and become single-stranded and enter individual phage particles. Therefore, by selecting only the strand into which the mutation has been introduced, a sequence having a desired mutation at a specific site can be obtained. In addition, a method in which a sticky end is generated using an appropriate restriction enzyme and ligated with DNA ligase as it is to cause deletion of several bases, or amplified by PCR under conditions where errors are likely to occur, is made into a certain region. Mutations can be introduced using a known method such as a random mutation method, a Kunkel method, an Eckstein method, or a similar method. Moreover, not only a polynucleotide in which a mutation is artificially introduced, but also a polynucleotide having a mutation naturally induced in a cell is a protein functionally equivalent to the protein consisting of the amino acid sequence described in SEQ ID NO: 2. As long as it is coded, it is included in the present invention.
The polynucleotide according to the present invention also includes DNA containing the base sequence set forth in SEQ ID NO: 1. Examples of the DNA containing the base sequence described in SEQ ID NO: 1 include DNA encoding a fusion protein containing a protein encoded by the base sequence described in SEQ ID NO: 1. Such DNA can be obtained by ligating DNA containing the nucleotide sequence set forth in SEQ ID NO: 1 and DNA encoding the protein to be fused so that the frames match. For example, a FRM protein having a His tag can be obtained by ligating and expressing seven Histidine-encoding polynucleotides at either end of the base sequence set forth in SEQ ID NO: 1. Therefore, NTA (Nitrotropic) acid) column and can be eluted with a buffer containing 0-400 mM imidazole.
The polynucleotide according to the present invention also includes a polynucleotide that hybridizes under stringent conditions with a DNA comprising the base sequence set forth in SEQ ID NO: 1. Although the stringent conditions can be selected as appropriate, the conditions such as 1 × SSC, 0.1% SDS, 60 ° C. can be preferably used. Stringency can be controlled by the concentration and temperature of SSC, and polynucleotides having higher complementarity can be obtained by selecting highly stringent conditions. The hybridizing polynucleotide may be DNA or RNA.
The FRM protein according to the present invention is a human homolog protein of rat HP33 protein, and so long as the amino acid sequence is included in the FRM protein, a protein derived from a living body and a synthesized protein are also included in the present invention. The FRM protein can be obtained by purifying, for example, from a human hepatocyte or kidney cell homogenate by an ordinary protein purification method, and an expression vector into which the polynucleotide of the present invention is inserted is introduced into a suitable host as described below. You can also get it. It can also be produced by a cell-free protein synthesis method known per se.
The amino acid sequence of the protein in the present specification is in accordance with a normal peptide notation, and the left end is the N-terminus and the right end is the C-terminus. The C-terminus of the protein of the present invention is usually carboxyl group (—COOH) or carboxylate (—COO). ⁻ ) But the C-terminus is an amide (-CONH ₂ ) Or esterified (-COOR) is also included in the present invention. Here, R of the ester may be, for example, an alkyl group having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, which may be linear or branched and saturated. May also contain one or more double bonds and may have one or more functional groups. In addition, those in which the N-terminal is protected with an acyl group such as formyl group and acetyl group, and those which are pyroglutamine oxidized are also included in the present invention. Substituents on the side chains of amino acids other than the N-terminal and C-terminal are amidated or esterified, protected by a formyl group, acetyl group, etc., or glycoproteins to which sugar chains are bound Complex proteins are also included in the present invention.
In addition to the protein consisting of the amino acid sequence represented by SEQ ID NO: 2, the FRM protein according to the present invention has one or more amino acids added, deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 2. Alternatively, it includes a protein consisting of an inserted amino acid sequence. The protein can be produced by introducing a mutation into DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 2. Specifically, for example, a transformation in which a desired mutation is introduced into a specific site of the base sequence described in SEQ ID NO: 1 using the above-described site-specific mutagenesis method and transformed with a vector into which this DNA has been inserted The target protein can be expressed by culturing the body.
The “protein encoded by the DNA comprising the base sequence described in SEQ ID NO: 1” according to the present invention is, for example, a DNA comprising the base sequence described in SEQ ID NO: 1 and a DNA encoding another protein linked. It means the entire protein encoded by DNA. Such a protein includes a fusion protein containing an FRM protein, a transformant transformed with an expression vector into which a DNA containing the base sequence described in SEQ ID NO: 1 has been inserted, and the expressed protein Is recovered. The protein to be fused is not particularly limited, but maltose binding protein (MBP), glutathione S transferase (GST) and the like are preferable for purification, and may be an oligopeptide such as a His tag. A fusion protein with a fluorescent protein such as GFP is useful for detection.
The “salt” in the present invention is not particularly limited as long as it is a pharmacologically acceptable salt. For example, a salt with an inorganic acid such as hydrochloride, phosphate, hydrobromide, sulfate, etc. Or organic such as acetate, formate, propionate, fumarate, maleate, succinate, tartrate, citrate, malate, benzoate, methanesulfonate, benzenesulfonate Examples include salts with acids.
The present invention also includes a partial peptide of the FRM protein. Examples of the “partial peptide” include a peptide corresponding to a site where the FRM protein binds to its physiological ligand. By using such a partial peptide, it is possible to antagonistically inhibit the FRM protein from binding to its ligand, and it can also be used to screen for compounds that can bind to the binding site of the FRM protein. The partial peptide of the FRM protein can be obtained by a genetic engineering technique, or can be synthesized by a known solid phase or liquid phase peptide synthesis method, and the FRM protein is cleaved with an appropriate peptidase or cyanogen bromide. Can also be obtained.
The recombinant vector according to the present invention means a recombinant vector into which the polynucleotide of the present invention is inserted, and there is no limitation as long as the polynucleotide of the present invention can be expressed in a host cell. Either a phage or a cosmid may be used. Plasmids include those derived from gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, gram-positive bacteria such as Bacillus subtilis, Staphylococcus, and yeast. These plasmids are selected to be compatible with the host. Moreover, the stability and usefulness of a plasmid can be improved by appropriately inserting a drug resistance gene or a gene having auxotrophy as a selection marker. Examples of the phage include a λ phage system and an M13 phage system often used when Escherichia coli is used as a host, regardless of whether the phage is a lytic phage or a lysogenic phage. When animal cells are used as hosts, vectors using SV40, papilloma virus, vaccinia virus, retrovirus, baculovirus and other viral DNA are used as vectors. In addition, a promoter sequence, an enhancer sequence, a Shine-Dalgarno sequence, a signal sequence, a poly A signal, and the like suitable for the host can be appropriately selected and inserted into the vector so that the FRM protein is efficiently expressed in the selected host. .
The transformant according to the present invention includes a transformant transformed with the recombinant vector of the present invention. The transformant of the present invention can be obtained by selecting a host suitable for the vector into which the DNA of the present invention has been inserted and introducing the vector into this host. Examples of the host include Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, Gram-positive bacteria such as Bacillus subtilis, actinomycetes, yeast, filamentous fungi, animal and plant culture cells, and insect culture cells. In the case of an animal cell, it may be a cell derived from a healthy animal (eg, rat-derived RL-34) or a diseased animal (eg, liver cancer rat-derived FAA-HTC1). The method using animal cells as a host also includes a method for obtaining a transgenic animal having an innate gene by introducing a gene into a fertilized egg of the animal and obtaining a protein produced by the animal. It is. In addition, a method for introducing a gene into Drosophila using a transposable element called a P element and a method for obtaining a protein produced by silkworm from a body fluid by infecting silkworm with a baculovirus are also included.
As a vector introduction method, when the host is Escherichia coli, the calcium chloride method, when Bacillus subtilis is the host, the competent cell method or the lithium acetate method, Bacillus subtilis, actinomycetes, yeast, etc., the protoplast method, As a method widely used for animal and plant cells, yeast, bacteria, etc., it can be appropriately selected from a calcium phosphate coprecipitation method, an electroporation method, a method of forming a complex with a polymer such as DEAE-dextran and polybrene. Lipofectamine (Invitrogen) that forms a complex with a cationic lipid liposome can also be used.
The present invention also includes a method for producing an FRM protein or a salt thereof, which comprises culturing the transformant of the present invention to produce an FRM protein or a salt thereof. The culture of the transformant can be selected depending on the characteristics of the host cell, the characteristics of the expressed protein, the characteristics of the promoter, and the like. For example, a known medium such as MEM medium, DMEM medium, Williams E medium (Gibco) is used. can do. For example, when a plasmid having antibiotic resistance is used, the expression of the target protein can be regulated by adding the antibiotic to the medium. When a lactose-dependent plasmid is used, IPTG is added to the medium. Addition can induce expression.
The FRM protein expressed by culturing the transformant thus obtained can be purified using a conventional protein purification method. Depending on the expression system used, various chromatographies, ultrafiltration methods, and the like can be used. , Salting out, osmotic shock method, sonication and the like can be combined for purification. Examples of the chromatography method include ion exchange chromatography performed in an aqueous solution, gel filtration, hydrophobic chromatography, affinity chromatography, and reverse phase chromatography using an organic solvent. The present invention also includes proteins purified using these purification methods.
Further, the expressed protein can be modified or partially removed with a suitable protein modifying enzyme before or after purification, and these modified proteins are also included in the present invention. . Examples of protein modifying enzymes include trypsin, chymotrypsin, protein kinase and glucosidase. A person skilled in the art can easily change the expressed protein to a salt if it is released, or to a released state if it is obtained as a salt.
The antibody according to the present invention may be any antibody that specifically binds to the FRM protein, and may be a polyclonal antibody or a monoclonal antibody. In addition to antibodies obtained by immunizing mammals other than humans with this protein, human antibodies and antibodies obtained by genetic recombination are also included in the present invention as long as they specifically bind to the FRM protein of the present invention. . The antibody according to the present invention can be produced by a method known per se or a method analogous thereto, and typical methods are exemplified below. Polyclonal antibodies immunize mammals, preferably rodents such as mice and rats, primates such as rabbits, monkeys, etc. using FRM protein, obtain serum, and use this serum as FRM protein or partial peptide Can be obtained by purification through a fixed affinity column. Monoclonal antibodies are also used to immunize mammals first, fuse antibody-producing cells obtained from these animals with highly proliferative myeloma cells, isolate individual fused cells, and test the desired antibody-producing ability. Thus, cells that produce only one type of antibody molecule that specifically reacts with one epitope of the antigen can be selected and cultured by culturing the cells. A monoclonal antibody can also be obtained by a genetic engineering method by inserting a DNA encoding the amino acid sequence of a monoclonal antibody into a vector, introducing the vector into a host, and producing the vector. By immunizing a transgenic animal into which a human antibody gene has been introduced, human antibodies can also be produced, and the cDNA encoding the antigen-binding site of the mouse monoclonal antibody and the gene encoding the constant region of the human IgG gene are linked. It is also possible to produce chimeric antibody molecules by introducing them into B lymphocytes, and these antibodies are also included in the present invention. The antibody of the present invention may be a fragment or a modified antibody as long as it specifically recognizes and binds to the FRM protein. Examples of the antibody fragment include F (ab), F (ab ′) 2, Fc fragment, or single chain Fv, and the modified antibody includes, for example, an antibody bound to a compound such as polyethylene glycol. Can be mentioned. The antibody of the present invention can be used for purification, detection and quantification of FRM protein, and can be an agonist or antagonist when it has an action of enhancing or suppressing the biological activity of FRM protein.
The present invention also includes a method for quantifying FRM protein using the antibody of the present invention. Specifically, the FRM protein can be detected and / or quantified by labeling the antibody (including fragment) of the present invention by an appropriate method and detecting and / or quantifying the label. The labeling method includes a method using a radioisotope or a stable isotope, and a method using a substituent that labels the end of an antibody molecule with a fluorescent group or a substituent having absorption in the ultraviolet or visible region, There is a method of measuring enzyme activity by enzyme labeling. The antibody to be labeled may be the antibody of the present invention or a secondary antibody that recognizes the antibody of the present invention as an antigen.
The “cell cycle regulation method” according to the present invention refers to a method of controlling cell division by regulating the cell cycle of the cell by changing, ie enhancing or inhibiting, the biological activity of the FRM protein in the cell. means. The term “enhancing or inhibiting biological activity” means that the biological activity of FRM is changed by acting directly or indirectly on FRM protein. For example, (1) the gene encoding FRM protein Overexpressing or suppressing expression of FRM protein by enhancing or suppressing expression (for example, enhancing or suppressing protein expression by controlling transcription of a polynucleotide encoding the protein); (2) Adding FRM protein, etc. (3) To increase or decrease the activity of FRM by directly or indirectly interacting with the FRM protein, and the like. Specific examples of substances useful for such “enhancement or inhibition” include, for example, polynucleotides encoding FRM protein, double-stranded RNA having RNA interference, FRM protein or salt thereof, FRM partial peptide or salt thereof, Examples thereof include FRM protein antibodies, agonists / antagonists of FRM proteins, and other substances having affinity for FRM proteins. For example, cell death (apoptosis) can be induced in the used cells by using the “cell cycle regulation method”.
The “cell cycle regulation method” according to the present invention is preferably useful in proliferating cells, that is, cells in the process of cell division. ₂ It is most useful for cells in any stage from stage to stage M. The target cells may be normal cells or cancer cells / tumor cells, and when used for cancer cells, anticancer effects / antitumor effects can be obtained.
The double-stranded RNA or double-stranded RNA-DNA chimeric molecule used in the RNAi (or siRNA) method can be obtained as long as it has an effect of inactivating the gene encoding the FRM protein, ie, suppressing the expression of the gene. Although not limited thereto, it is preferably a double-stranded RNA molecule or a double-stranded RNA-DNA chimeric molecule having a length of 19 to 25 bases, more preferably a double strand having a length of 19 to 23 bases, most preferably 23 bases A double-stranded RNA molecule or a double-stranded RNA-DNA chimeric molecule. As an example, a double-stranded RNA molecule having the following base sequence as a target DNA can be mentioned. Such an RNA molecule has one of the following double-stranded DNA as a sense strand and the other as a complement thereof. The strand, ie the antisense strand to the sense sequence.

The present inventors have shown that human FRM protein and rat HP33 protein are specifically expressed in the liver and kidney, overexpressed in HCC, and suppression of tumor cell growth suppresses tumor growth. Overexpression in the cell

Taxol represented by (Taxol (Paclitaxel;
(2R, 5R, 7S, 10R, 13S) -10,20-Bis (acetoxy) -2-benzoyloxy-1,7-dihydroxy-9-oxo-5,20-epoxytax-11-en-13-yl
(3S) -3-benzoylamino-3-phenyl-D-lactate)) or a salt thereof induces the death of the cell, and when the expression in the cell is suppressed by the antisense method, It has been found that apoptosis is induced, and that when the expression in the cell is suppressed by the antisense method, the probability of occurrence of abnormal microtubule formation in the cell increases.
The “cell cycle regulation method” according to the present invention may be a method used in combination with various anticancer agents. Taxol or a salt thereof is suitable as the anticancer agent. The use of the anticancer agent may be performed simultaneously with the use of a means for enhancing or inhibiting the biological activity of the FRM protein, or may be used before or after the above means at a certain time interval. A substance that changes the intracellular concentration of FRM protein or enhances or suppresses the biological activity of FRM protein is useful as an active ingredient of a medicine. Since apoptosis is induced when the intracellular concentration of the FRM protein increases or decreases, an antibody, a compound having an action of inhibiting the physiological action of the FRM protein, or an antisense oligonucleotide to the polynucleotide encoding the FRM protein is administered. By doing so, it is possible to contribute to prevention and / or treatment of diseases by suppressing apoptosis. More specifically, since the FRM protein is specifically expressed in the liver and kidney, apoptosis of the cell can be induced by decreasing the FRM protein concentration in the cells of these organs. Examples of the diseases caused by apoptosis inhibition include various cancer diseases and autoimmune diseases, and it can be expected to be most effective for hepatocellular carcinoma (liver cancer). In addition, the FRM protein was administered into the patient's cells, the polynucleotide encoding the FRM protein was administered to the patient and expressed, or the target cell was introduced and expressed with the polynucleotide encoding the FRM protein. Later, the cells can be transplanted into the body of a patient to suppress apoptosis of the cells, and can be expected to be useful for prevention and / or treatment of diseases caused by increased apoptosis. In order to suppress cell apoptosis, an antibody or compound that enhances the function of the FRM protein according to the present invention can be administered to the cell. Examples of diseases caused by increased apoptosis include viral infections such as AIDS and hepatitis, and neurodegenerative diseases such as Alzheimer's disease.
The “pharmaceutical composition” according to the present invention is a pharmaceutical composition comprising a substance that enhances or inhibits the biological activity of a cell cycle regulatory protein (FRM) or a salt thereof. The substance is not particularly limited as long as it has an activity to enhance or inhibit the biological activity of the FRM protein, and a substance that enhances or suppresses the expression of the gene encoding the FRM protein, or a substance that increases the FRM internal amount. And substances having an action of enhancing or inhibiting the activity of the FRM protein. Specific examples of these substances include, for example, polynucleotides encoding FRM proteins, FRM proteins or salts thereof, FRM partial peptides or salts thereof, FRM protein antibodies, agonists / antagonists of FRM proteins, and the like. That is, the active ingredient substance of the said pharmaceutical composition should just change the biological activity of FRM by acting directly or indirectly on FRM protein.
The use of the pharmaceutical composition according to the present invention is not particularly limited, but is particularly useful as a therapeutic or prophylactic agent for liver disease or renal disease. Examples of the liver disease and kidney disease are not particularly limited, and include all diseases that are medically classified into these diseases. Liver cancer is particularly effective as a liver disease.
When using the pharmaceutical composition concerning this invention as a therapeutic agent or a preventive agent, it can formulate according to a normal means. For example, the pharmaceutical composition of the present invention can be used by uniformly mixing with a pharmacologically acceptable carrier. The initial carrier may take a wide variety of forms depending on the form of preparation desired for administration, and is preferably in unit dosage form that can be administered orally or by injection. Oral liquid preparations such as suspensions and syrups include sugars such as water, sucrose, sorbitol and fructose, glycols such as polyethylene glycol, oils such as sesame oil, olive oil, soybean oil, and aquilparahydroxybenzoate. And other flavors such as strawberry flavor and peppermint flavor can be used. Powders, pills, capsules and tablets are excipients such as lactose, glucose, sucrose and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl alcohol, hydroxypropylcellulose And a binder such as gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin. A solution for injection can be prepared using a carrier made of a salt solution, a glucose solution, or a mixture of salt water and a glucose solution.
When the polynucleotide of the present invention is used as a prophylactic or therapeutic agent, the polynucleotide of the present invention alone or after being inserted into an appropriate vector such as a retrovirus vector or adenovirus vector, is administered by known means. can do. The polynucleotide of the present invention can be administered alone or together with an auxiliary agent for promoting intake by a catheter such as a gene gun or a hydrogel catheter.
The “screening method” according to the present invention refers to a compound having a cell cycle regulatory activity using a polynucleotide encoding a cell cycle regulatory protein (FRM), FRM or a salt thereof, or a partial peptide of FRM or a salt thereof, or It means a screening method for the salt. Examples of the screening method include: (a) contacting a test sample with a cell expressing FRM protein; (b) detecting and measuring a change in biological activity of the protein, or measuring cell death of the cell. And a method for screening a compound having a cell cycle regulatory activity or a salt thereof. The FRM protein used for screening may be extracted from human cells, may be obtained by gene recombination, or may be a partial peptide obtained by the above method.
Examples of other screening methods include, for example, Western blotting as a screening method for proteins that bind to the FRM protein. The total protein of a cell as a test sample is dissolved and extracted with a buffer solution containing SDS (Sodium dodecyl sulfate), then subjected to SDS-acrylamide gel electrophoresis, and separated and developed according to the molecular weight. Next, the protein developed in the gel is electrotransferred to a nitrocellulose membrane. Then, the labeled FRM protein is brought into contact with the membrane, and the label of the protein that binds to the FRM protein can be detected by detecting the label. Examples of the labeling method include a method using a radioisotope, a method using fluorescence, and a method using a fusion protein of an FRM protein and a protein to be labeled. Further, a method of detecting the FRM protein bound to the protein on the nitrocellulose membrane by labeling the antibody according to the present invention without labeling the FRM protein itself is also possible.
As a screening method for a low molecular weight compound that binds to the FRM protein according to the present invention, for example, the FRM protein is labeled with biotin, the reaction of biotin and avidin is used to immobilize the FRM protein on a substrate, and the low molecular weight compound is attached thereto. A test sample containing is brought into contact. In this method, the binding between the FRM protein and the low molecular weight compound can be detected by labeling the low molecular weight compound in advance. For example, a system using a surface plasmon resonance or a mass spectrometer is used. It is also possible to detect binding without labeling.
A compound that binds to the FRM protein can also be obtained by immobilizing the FRM protein in an affinity column, passing a test sample through the column, and purifying the compound that specifically binds to the column.
The compound having the activity of binding to the FRM protein or the activity of changing the biological activity of the FRM protein may be a naturally derived compound or an artificially synthesized compound.
The screening kit according to the present invention is a kit for use in carrying out the screening method according to the present invention, that is, a polynucleotide encoding a cell cycle regulatory protein (FRM), an FRM protein or a salt thereof, or FRM. A kit for screening a substance having a cell cycle regulatory activity using a partial peptide of a protein or a salt thereof.
The compound or salt thereof isolated by the screening method or screening kit according to the present invention is a compound having cell cycle regulatory activity or a salt thereof. The compound or a salt thereof has an activity of enhancing or suppressing the biological activity of the FRM protein.
The present invention also provides a method for treating or preventing various cancer diseases, autoimmune diseases, viral infections or neurodegenerative diseases, which comprises regulating the amount of FRM protein in cells. As described above, the present inventors confirmed that the growth of tumor cells was suppressed when the expression of HP33 protein was suppressed by an antisense method in rats transplanted with a rat liver cancer cell line. Moreover, when the expression of FRM protein in a cell was suppressed by RNAi method, it discovered that the apoptosis of the said cell was induced | guided | derived. Furthermore, when HP33 protein was overexpressed in the cells and Taxol was added at the same time, it was confirmed that death of the cells was induced. From the above, it is expected that apoptosis-related diseases, preferably cancer diseases, more preferably liver cancer can be treated or prevented by regulating the amount of FRM protein in cells. The regulation of the amount of FRM protein in cells can be performed by administering a therapeutically effective amount of the pharmaceutical composition according to the present invention to a patient.
The present invention also provides a method for diagnosing liver cancer or kidney cancer using FRM protein or partial peptide or a salt thereof as a marker. As described above, since the FRM protein is overexpressed in HCC cells, the FRM protein in a certain cell and the polynucleotide of the present invention, preferably mRNA encoding the FRM protein, are detected and / or quantified, and overexpressed. It is possible to diagnose whether the cell is invaded by HCC by confirming whether or not the cell is in contact with HCC. Known methods can be used for detection and quantification of polynucleotides, such as competitive hybridization using DNA encoding FRM protein as a probe, dot blotting, Northern blotting, RNase protection assay, An expression analysis method such as RT (Reverse Transcription) -PCR can be used. Known methods can also be used for detection and quantification of FRM protein. For example, in addition to a method of detecting or quantifying using an antibody of the present invention (including a fragment) using an appropriate label, various mass spectrometry methods are used to measure the amount of protein equal to the FRM protein. A method for comparing average values of healthy subjects, a method for detecting FRM protein by electrophoresis, and the like can be used.
EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.
[Example 1] Isolation and sequence analysis of human FRM cDNA
By searching the TBLASTN database, a human gene (GenBank accession no. AB013093) having a high homology (63%) with the nucleotide sequence encoding the amino acid sequence of rat HP33 protein was found. A fragment of this gene sequence was amplified by PCR and used to screen a human liver cDNA library to obtain several clones. As a result of analyzing the sequences of these clones, it was confirmed that the human cDNA consists of 888 bp nucleotides and encodes 295 amino acids. The nucleotide sequence of cDNA is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. The molecular weight of the human FRM protein was calculated to be 34 kD.
[Example 2] Expression analysis of FRM mRNA by Northern blotting
Using a Random Primed DNA Labeling Kit (Roche Diagnostics), the human FRM protein cDNA was radiolabeled to give a probe (1 × 10 ⁶ cpm / ml). According to the specifications, the probe was hybridized to Multiple Tissue Northern (MTN) membrane (Clontech) blotted with 2 ug / lane poly (A) + RNA, and detected by autoradiography. The results are shown in FIG.
[Example 3] Expression analysis of human FRM protein in HCC cells using FRM antibody
3-1. Confirmation of cross-reactivity of FRM and HP33 antibodies
The HP33 and FRM coding regions having a Flag tag and a Histidine tag at the N-terminus were inserted into the pET-3a expression vector, and rat HP33 and human FRM proteins were transformed into E. coli by IPTG induction. Overexpression was performed using the Coli BL21 (DE3) pLysS strain. E. Coli cells were disrupted by sonication, and the supernatant was affinity purified with Ni-NTA agarose (Qiagen). 50 ng of the obtained recombinant protein was separated by 12.5% SDS-PAGE and then transferred to a PVDF membrane (Millipore). Human FRM protein was immobilized on HiTrap NHS-activated Column (Amersham Pharmacia Biotech), and the anti-rat HP33 antibody was affinity purified and diluted 3000 times. The protein was detected according to the protocol of Alkaline phosphatase conjugate substrate (BioRad). The results are shown in FIG.
3-2. Detection of FRM protein overexpression in HCC cells
Immunohistochemical examination of human FRM protein was performed by standard techniques using HCC tissue pieces according to the Avidin-biotin peroxidase complex (ABC) method by Sasaki et al. Tissue pieces were incubated with affinity purified anti-rat HP33 antibody. The results are shown in FIG. The upper part is a healthy person and the lower part is a tissue piece obtained from an HCC patient.
[Example 4] Genomic DNA sequence analysis of FRM and promoter study, FISH, organ-specific expression and overexpression mechanism in HCC
4-1. Isolation of human FRM genomic DNA clones
4-2. Construction of promoter / reporter plasmid
Using the FRM genome P1 clone as a template, a DNA fragment (pBS-2932) downstream of −2932 and upstream of +102 of the FRM gene was prepared by PCR, and subcloned into the EcoRV site of pBluescript II SK +. The sequence from −822 to +102 is shown in SEQ ID NO: 3. Then, using this pBS-2932 as a template, various promoter / luciferase reporter systems (pLuc-2932 to pLuc + 1) lacking the 5 ′ end were constructed, and the luciferase expression vector pGL3-Basic (Promega) was placed in the Smal site. Subcloned. The types of promoter deficiency are shown in FIG. In the figure, each number represents the 5 ′ end. pLuc-255-Sp1 was created by deleting the EcoRI site (including the Sp1-like motif) from the Apal site. All plasmids were sequence verified.
4-3. Cell culture, gene transfer, luciferase assay
HepG2 cells are 10% FCS, 100 uM NEAA solution (Gibco) and MEM medium (Gibco) containing antibiotics, and HeLa and RL34 cells are DMEM medium (Gibco) containing 10% FCS and antibiotics. FAA-HTC1 cells were cultured in Williams E medium (Gibco) with 10% FCS, 2 mM L-Glutamine and antibiotics. For the luciferase assay, cultured cells of 30% confluence were prepared in 24-well plates (Falcon), 350 ng / well of pGL3-Basic reporter gene, and 5 ng / well of pRL-TK (Promega) as a control. Was cotransfected. For the luciferase assay using Sp1, Sp1 / pcDNA3.1 (+) was cotransfected in the same manner at 50 ng / well. Lipofectamine Plus (Gibco) was used for gene introduction. After 48 to 60 hours, luciferase activity was measured using Dual Luciferase Kit (Promega) and TD20 luminometer (Promega) and standardized based on the control pRL-TK activity. The results are shown in FIGS.
4-4. Fluorescence in situ hybridization (FISH)
Lymphocytes were isolated and analyzed from human blood. Using Gibco BRL BioNick labeling kit, the human FRM gene fragment derived from the P1 clone was biotinylated in the presence of dATP to obtain a hybridization probe. The FISH method followed the method of Heng et al. (1992) and the method of Heng and Tsui (1993). Chromosomes were stained with DAPI. The results are shown in FIG.
[Example 5] Behavior of tubulin and FRM protein in HP33 overexpression strain and expression suppression strain
5-1. Creation of HP33 overexpression strain
An HP33 expression plasmid for expression of lactose-dependent animal cells was prepared using the rat HP33 cDNA, and gene transfer was performed using lipofectamine into a normal rat-derived RL-34 cell line. On the second day after the gene transfer, the medium was replaced with a medium containing 400 ug / ml hygromycin and 400 ug / ml geneticin, and cultured. Thereafter, in order to express HP33, 5 mM IPTG was added to express HP33.
5-2. Creation of strains that suppress the expression of HP33
An HP33 antisense expression plasmid for tetracycline-dependent animal cell expression was prepared and introduced into the RL34 strain using Lipofectamine (Invitrogen) together with a tetracycline activator vector and a G418 resistance gene vector. On day 2 after the introduction, the medium was replaced with a medium containing 400 ug / ml geneticin and 15 ug / ml tetracycline, and cultured to isolate a G418-resistant cell line. The suppression of HP33 expression was performed by culturing in a medium not containing tetracycline.
5-3. Tubulin, HP33 protein, DNA staining
RL-34 cells were fixed on a cover glass with 4% paraformaldehyde, 5 mM EGTA, 2 mM MgCl 2 -containing sodium phosphate buffer for 8 minutes, and then subjected to membrane permeabilization with Triton X-100 for 5 minutes to obtain 1% BSA. The blocking reaction was performed for 1 hour. After blocking, reaction was performed for 1 hour using anti-α-tubulin antibody diluted 2000 times with 1% BSA, anti-HP33 antibody diluted 200 times, and then FITC-labeled donkey anti-rabbit IgG, Texas Red-labeled 100-fold diluted. Staining was performed with sheep anti-mouse IgG for 1 hour. Finally, the DNA was stained with 1 ug / ml DAPI for observation. The results are shown in FIG.
[Example 6] Inhibition of HP33 gene expression and tumor growth in rat-derived liver cancer cell line FAA-HTC1
10 rat-derived liver cancer cell lines FAA-HTC1 (FAA) and HP33 expression-suppressed strains (α1, α14) ⁶ Individual transperitoneal administration was performed, and the state of tumor growth after 2 months was observed. The HP33 expression-suppressed strain was prepared as an HP33 antisense expression plasmid (pTet-HP33) for expression of tetracycline-dependent animal cells, and the gene was introduced into the parent FAA cell line using lipofectamine. On the second day after gene introduction, the medium was replaced with a medium containing 400 μg / ml geneticin 15 ug / ml tetracycline, and cultured to isolate a G418-resistant cell line. The suppression of HP33 expression was performed by culturing in a medium not containing tetracycline. The results are shown in FIG. The graph shows the ratio of tumor weight to rat body weight (n = 3). In rats where the expression of FRM protein was suppressed, almost no increase in tumor weight was observed.
[Example 7] Taxol sensitivity in FRM overexpressing strains
RL-34 in a culture flask and a farm overexpressing strain (each cultured in DMEM medium containing Taxol 0 or 0.1 uM for 24 hours) were treated with trypsin / EDTA at 37 ° C. for 3 minutes, and reacted with DMEM medium containing serum. After stopping, the cell suspension was obtained, then washed 3 times with cold PBS (PBS for FACS), and finally suspended in 3 ml of PBS. To this suspension, 7 ml of -20 ° C ethanol was added dropwise with vigorous stirring to fix the cells. The fixed cells were fixed overnight at −20 ° C., then washed twice with PBS, suspended in 500 ul of PBS containing 0.5 mg / ml RNAse, and propidium iodide (PI) was suspended in the cell suspension. And allowed to react for 10 minutes at room temperature. This solution was used for FACS analysis. The results are shown in FIG. In the figure, for the two peaks, the left peak indicates cells in the G1 phase, and the right peak indicates cells in the G2 / M phase. When Taxol is added to the overexpressing strain, cells in the G1 phase are extremely decreased, and cells in the G2 / M phase are increased.
[Example 8] Abnormal microtubule formation during cell division in an FRM expression-suppressed strain
The state of DNA and microtubules during cell division in the RL-34 cell line, the farm overexpression strain, and the farm expression suppression strain were observed with a fluorescence microscope. The cells were fixed on a cover glass with 4% paraformaldehyde, 5 mM EGTA, 2 mM MgCl 2 in sodium phosphate buffer for 8 minutes, then subjected to membrane permeabilization with Triton X-100 for 5 minutes, and blocked with 1% BSA for 1 hour. Reaction was performed. After blocking, reaction was performed for 1 hour using an anti-microtubule antibody diluted 200-fold with 1% BSA, and then 20-fold diluted FITC-labeled goat anti-mouse IgG was stained for 1 hour. Finally, the DNA was stained with DAPI at a concentration of 1 μg / ml for observation. The results are shown in FIG. The upper panel shows DNA and microtubules of a farm protein expression-suppressed strain. The state of RL-34 cells is shown in the lower right of the panel. The lower graph shows the ratio of abnormal cells to the total number of cells after culturing each cell on a slide glass and counting under a microscope of cells with abnormal microtubule formation after microtubule staining.
[Example 9] Induction of apoptosis by suppressing expression of human FRM using siRNA method
(1) Influence of FRM gene expression suppression in human liver cancer cell line HepG2
1 × 10 human liver cancer cell line HepG2 ⁴ Culturing was performed in 6-well culture dishes at individual concentrations. On the second day of culture, a double-stranded RNA-DNA chimera complementary to the FRM gene at a concentration of 200 nM was added to the culture to suppress gene expression. As a double stranded RNA-DNA chimera sequence complementary to the FRM gene used, a double strand consisting of the base sequences described in SEQ ID NO: 4 and SEQ ID NO: 5 shown below, or SEQ ID NO: 6 and sequence The double strand which consists of a base sequence as described in No. 7 was used. For the control group, a double-stranded RNA-DNA chimera consisting of random base sequences described in SEQ ID NO: 30 and SEQ ID NO: 31 was used.

After addition of the above-mentioned oligonucleotide during culturing, the appearance of 24h, 48h, 72h cells is shown.
Results: In the double-stranded RNA-DNA chimera addition group complementary to the FRM gene, a decrease in cells over time was observed, but no decrease in cells was observed in the control group.
(2) Mechanism of cell death in human hepatoma cell line HepG2 in which FRM gene expression is suppressed
1 × 10 human liver cancer cell line HepG2 ⁴ Culturing was performed in 6-well culture dishes at individual concentrations. On the second day of culture, a double-stranded RNA-DNA chimera complementary to the FRM gene at a concentration of 200 nM was added to the culture to suppress gene expression. The following sequence was used as the sequence of the double-stranded RNA-DNA chimera complementary to the FRM gene used. In addition, a double-stranded RNA-DNA chimera having a random base sequence was used for the control group.

After the oligonucleotide was added to the culture, the cells were collected at 48 h and 72 h, the nuclei were extracted from the cells, and DNA fragmentation was observed by agarose electrophoresis. Apoptotic DNA-Ladder kit (Roche Diagnostics) was used for nuclear extraction.
Results: DNA fragmentation was observed in the double-stranded RNA-DNA chimera addition group complementary to the FRM gene, but no DNA fragmentation was observed in the target group. This fragmentation is characteristically observed in apoptotic cells, but in human hepatoma cell line HepG2 in which the expression of the FRM gene is suppressed, it is shown that cells die due to apoptosis.

  According to the present invention, a novel human cell cycle regulatory protein (FRM protein), a gene encoding the same, and a cell cycle regulatory method using the protein could be provided. By using the cell cycle regulation method according to the present invention, cell death can be induced, and various diseases based on cell death can be treated and prevented. FRM proteins are closely involved in human HCC and cell apoptosis, etc., and are used to elucidate the mechanism of cancer development and develop pharmaceuticals for apoptosis-related diseases using antibodies that specifically recognize FRM proteins and FRM proteins. Screening and the like can be easily performed.

[Sequence Listing]

Claims (32)

The polynucleotide according to any one of (a) to (d) below, which encodes a cell cycle regulatory protein.
(A) a polynucleotide encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2.
(B) a polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids are added, deleted, substituted and / or inserted in the amino acid sequence of SEQ ID NO: 2.
(C) DNA comprising the base sequence set forth in SEQ ID NO: 1.
(D) A polynucleotide that hybridizes with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 under stringent conditions. The cell cycle regulatory protein or salt thereof according to any of (a) to (c) below.
(A) A protein comprising the amino acid sequence represented by SEQ ID NO: 2.
(B) A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 2.
(C) a protein encoded by a DNA comprising the base sequence set forth in SEQ ID NO: 1. A partial peptide of the cell cycle regulatory protein according to claim 2 or a salt thereof. A recombinant vector into which the polynucleotide according to claim 1 is inserted. A transformant transformed with the recombinant vector according to claim 4. The method for producing a cell cycle regulatory protein or a salt thereof according to claim 2, wherein the transformant according to claim 5 is cultured to produce the protein according to claim 2. An antibody to the cell cycle regulatory protein according to claim 2, the partial peptide according to claim 3, or a salt thereof. A method for quantifying the cell cycle regulatory protein or salt thereof according to claim 2 using the antibody according to claim 7. A method for regulating the cell cycle, comprising enhancing or inhibiting the biological activity of the cell cycle regulatory protein or salt thereof according to claim 2. In cells of G 2 _~M period, characterized by enhancing or inhibiting the cell cycle regulatory proteins or biological activity of the salt thereof according to claim 2, method of claim 9. The method according to claim 9 or 10, wherein the cell is a cancer cell. The biological activity of the protein or a salt thereof is enhanced or inhibited by increasing or decreasing the endogenous amount of the cell cycle regulatory protein or the salt thereof according to claim 2 in the cell. 11. The method according to any one of 11 above. The method according to any one of claims 9 to 12, wherein the method comprises combining the use of anticancer agents. The use of the anticancer agent is simultaneous with the means for increasing or decreasing the endogenous amount of the cell cycle regulatory protein or salt thereof according to claim 2, or at regular intervals. 14. The method of claim 13 before and after. The method according to claim 13 or 14, wherein the anticancer agent is taxol or a salt thereof. The method according to any one of claims 9 to 11, comprising enhancing or suppressing the expression of a polynucleotide encoding the protein of claim 2. 13. The method of claim 12, comprising inhibiting the biological activity of the protein by RNA interference targeting a polynucleotide encoding the cell cycle regulatory protein of claim 2. The RNA molecule used for RNA interference consists of a double-stranded RNA-DNA chimera molecule consisting of the base sequences set forth in SEQ ID NO: 4 and SEQ ID NO: 5, or the base sequences set forth in SEQ ID NO: 6 and SEQ ID NO: 7. The method according to claim 17, which is a double-stranded RNA-DNA chimeric molecule. A double-stranded RNA molecule or a double-stranded RNA-DNA chimeric molecule having a length of 19 to 25 bases, which has an action of mediating RNA interference of mRNA encoding the cell cycle regulatory protein according to claim 2. The double-stranded RNA-DNA chimeric molecule according to claim 19, which consists of the base sequence described in SEQ ID NO: 4 and SEQ ID NO: 5, or the base sequence described in SEQ ID NO: 6 and SEQ ID NO: 7. A method for inducing cell death comprising the use of the method according to any one of claims 9 to 18. A pharmaceutical composition comprising a substance that enhances or inhibits the biological activity of the cell cycle regulatory protein or salt thereof according to claim 2. The substance is (a) a polynucleotide according to claim 1, (b) a protein according to claim 2, or a salt thereof, (c) a partial peptide according to claim 3, or a salt thereof, or (d). 23. The composition of claim 22, which is the antibody of claim 7. The composition according to claim 22, which is a therapeutic agent or a preventive agent for liver disease or renal disease. 25. The composition of claim 24, wherein the liver disease is liver cancer. Use of the cell cycle regulatory protein according to claim 2 or a salt thereof or the partial peptide of the cell cycle regulatory protein according to claim 3 or a salt thereof for the manufacture of a therapeutic / preventive agent for liver disease or renal disease. A method for treating or preventing a liver disease or a renal disease, comprising administering a therapeutically effective amount of the composition according to claim 22 to a patient. A method for diagnosing liver disease or kidney disease, comprising detecting or quantifying the cell cycle regulatory protein according to claim 2, the partial peptide according to claim 3, or mRNA thereof. Cell cycle regulation using the polynucleotide according to claim 1, encoding the cell cycle regulation protein, the cell cycle regulation protein according to claim 2, or a salt thereof, or the partial peptide or salt thereof according to claim 3. A screening method for a compound having activity or a salt thereof. (A) contacting a test sample with a cell expressing the cell cycle regulatory protein according to claim 2;
(B) A screening method for a compound having a cell cycle regulatory activity or a salt thereof, which comprises detecting and measuring a change in biological activity of the protein or measuring cell death of the cell. Cell cycle regulation using the polynucleotide according to claim 1, encoding the cell cycle regulation protein, the cell cycle regulation protein according to claim 2, or a salt thereof, or the partial peptide or salt thereof according to claim 3. A kit for screening for a substance having activity. A compound having a cell cycle regulatory activity or a salt thereof, which can be isolated by the method according to claim 29 or 30, or the kit according to claim 31.

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