新規の遺伝性腎癌ラット"Nihonラット"に関する病理学的および遺伝子生物学的研究

[背景]　ヒトの腎癌は、腎に発生する原発性腫瘍の中でもっとも多く、約90%を占めており、すべての癌の約2%を占めるといわれている。米国においては、癌による死亡の原因として6番目に多く、成人における悪性腫瘍の3%を占めている。これまでに、ヒトの腎癌の発症と進展に関与する遺伝子として、VHL(von Hippel-Lindau 病)遺伝子、c-Met遺伝子、FH(fumarate hydratase)遺伝子、Tsc1(Tuberous sclerosis)遺伝子やTsc2遺伝子が明らかにされている。腎癌の発症メカニズムの解明や遺伝子治療などの開発にあたっては、ラット等の疾患モデルを用いた研究が不可欠であるが、現在までのところ腎癌の自然発症ラットモデルは、1954年ノルウェーのEkerによって見出されたEkerラットのみであり、さらなる疾患モデル動物の開発が待たれるところである。　著者らは毒性試験のために飼育していたSprague-Dawley(SD)系ラットの中に腎癌を自然発症するものを偶然見出し、この癌が遺伝性であることを確認し、"Nihonラット"と命名し、さらに腎癌発症の病因について遺伝子生物学的研究を行った。　本論文では、まず"Nihonラット"の発見の経緯、繁殖・維持過程で得られた病理学的、遺伝学的知見を述べ、次に腎癌の病因候補遺伝子(Nihon遺伝子)を探索するための染色体地図の作成とポジショナル・クローニング(位置的候補遺伝子探索)法による染色体領域の特定、さらに特定した染色体領域における、腎癌の有力な病因性候補遺伝子の一つと考えられるBHDホモログ遺伝子の単離と変異の解析について記述し、最後に本ラットの自然史について詳細な病理学的観察を行い、遺伝性腎癌モデル動物としての有用性を考察した。[第1章]"Nihonラット"における腎癌病変のメンデル遺伝　著者らは、同一の繁殖場から購入した7～16週齢の若齢SD系ラット群の343例中15例に腎癌の発生を見出し、その後、同一の繁殖場から雌2例の元親を得て大日本製薬安全性研究所の施設内で繁殖・交配を開始した。繁殖・交配の成績から本ラットにおける腎細胞癌は、メンデルの法則に従った単一遺伝子の変異による常染色体優性遺伝形式をとるものと考えられ、本ラットを遺伝性腎癌モデル動物として"Nihonラット"と命名した。これまでに報告されている遺伝性腎癌モデルラットは、1954年Ekerにより見出されたEkerラットで、1995年に腎癌病因遺伝子としてTsc2遺伝子が単離・同定されている。そこで、"Nihonラット"とEkerラットにおける腎癌の組織表現型と遺伝子変異の特徴を比較した。その結果、"Nihonラット"の腎細胞癌がclear cell優位の組織像を示すのに対して、Ekerラットのそれはnon-clear cellにより構成されるものであった。また、"Nihonラット"では腎臓における変異尿細管、変異過形成、腺腫および腺癌といった多段階的な病変発生が、Ekerラットと比較していずれも早期に起こるという特徴を有していた。さらに、遺伝子変異の解析では、Ekerラットに認められるTsc2遺伝子の変異はなく、ヒト腎癌の原因遺伝子として同定されているTsc1、Tsc2、VHL遺伝子およびc-Met遺伝子にも変異は見出せなかった。　以上のように"Nihonラット"は、腎癌の組織像や遺伝子変異においてEkerラットとは異なることから、新規の腎癌モデルラットであることが強く示唆された。(Jpn. J. Cancer Res., 91, 1096-1099, 2000.)[第2章]"Nihonラット"の腎癌発症に関与する"Nihon遺伝子"探索のための染色体地図の作成　"Nihon遺伝子"を探索するための最初のステップとして、BNラットを用いた113例の戻し交配ラットを作製し、ラット全ゲノムをカバーする既知の遺伝子多型マーカーを用い遺伝的リンケージ解析を実施した。まず、ゲノムワイドスクリーニングを行い、染色体の平均10～20cM断片をカバーする各多型マイクロサテライトマーカーの遺伝子型と、病的形質を呈した個体数(陽性個体数)とのassociation studyをχ2乗検定し、その値の高いマーカーがある染色体を選び、その候補染色体上のマーカーを基点として、その近傍にある病因遺伝子にできるだけ近いマーカーを求めた。　その結果、"Nihon遺伝子"は、ラット染色体10番に存在しているinterleukin-3(IL3)遺伝子、lethal(2)giant larvae(Llgl1)およびmyosin heavy chain, embryonic skeletal muscle(MYHSE)遺伝子のそれぞれから4.4cM、0.9cMおよび5.3cM離れた位置に座位していた。すなわち、"Nihon遺伝子"は、ラット染色体10番の遠位部にあり、IL3遺伝子とLlgl1遺伝子の間に局在していた。(Jpn. J. Cancer Res., 92, 1147-1149, 2001.)[第3章]"Nihonラット"の腎癌発症に関与する"Nihon遺伝子"の単離　第2章においては、ラット染色体10番における"Nihon遺伝子"の詳細な位置特定は不可能であった。ヒトではIL3遺伝子、Llgl1遺伝子、MYHSE遺伝子はそれぞれ5番長腕23-31、17番短腕11.2、17番短腕13.1に局在しているが、この時点では、ラット染色体10番に対応するヒト染色体が5番か17番か判明しておらず、この領域の既知の遺伝子の中に、腎癌の発生に関連する遺伝子は認められていなかった。しかし、本実験と同一時期に、腎癌を高いリスクで発生するヒトのBirt-Hogg-Dubé(BHD)症候群の未知の病因遺伝子(BHD遺伝子)がヒト染色体17番短腕11.2あるいは17番短腕12－長腕11.2に存在することが報告された。そこで本章では、ヒト染色体17番短腕11.2に相同するラット染色体10番の領域における"Nihon遺伝子"の局在とラットBHDホモログ遺伝子の同定を試み、さらにその変異について検索した。　その結果、"Nihonラット"において、染色体10番にBHDホモログ遺伝子の存在をラットで初めて確認し、さらにこの遺伝子に一塩基挿入を原因とする生殖細胞系列の変異を見出した。BHDホモログ遺伝子におけるこの一塩基挿入は、フレームシフト変異を生じ、読み枠内に終止コドンを生じさせた。その結果、本BHDホモログ遺伝子の変異では大半のアミノ酸が欠損していた。また、ホモ変異接合体のラットは胎児期の早期に致死に至ることも確認した。さらに、"Nihonラット"の11例中10例の腎癌細胞にBHD遺伝子のLOH(ヘテロ接合体の消失)が認められ、LOHを認めなかった1例においても点変異(ナンセンス変異)を見出した。　以上の結果から、"Nihon遺伝子"はBHDホモログ遺伝子と極めて近い関係にあり、本ラットにおける腎癌の発生は癌抑制遺伝子におけるKnudsonの2ヒット説を実証するものであると考えられた。(Proc. Natl. Acad. Sci. USA, 101, 2023-2027, 2004.)[第4章]"Nihonラット"の自然史　本章では、"Nihonラット"における腎癌以外の腫瘍性病変発生の有無、さらにNihonラット特有の病変を見出すために"Nihonラット"を長期飼育し、全身組織を病理組織学的に検討した。　その結果、"Nihonラット"における腎癌の発症は病理組織学的に変異尿細管から、変異過形成、腺腫、腺癌へと多段階的に進展し浸透率は100%であった。腎癌の組織像はclear cellが優位であったが、clear/acidophilic ellとpapillary basophilic cell typeもみられた。腎癌以外には子宮内膜のclear cell過形成/腺腫、若齢時における心筋横紋筋腫、さらに腎癌内の異所性骨形成、まれに唾液腺の線条部円柱上皮のclear cell化を認めた。　今回確認された腎癌以外の病変は、SD系ラットを含め、ラットでの報告はまれ、あるいは未報告であり、"Nihonラット"に特有の病変であり、BHD遺伝子の変異に伴い、あるいは関連して発生したものと考えられた。ヒトBHD症候群では毛包の過誤腫、腎腫瘍および自然気胸・肺シストを好発するが、"Nihonラット"とBHD症候群の共通の表現型は腎癌のみであった。(Curr. Mol. Med., 4, 887-893, 2004)[結論]　"Nihonラット"の腎癌は、メンデルの法則に従った常染色体優性遺伝形式をとり、その組織像はEkerラットのそれよりもヒトに類似し、また病変の発生がより早期に認められる特徴を持つ。Nihonラットの腎癌の病因候補遺伝子"Nihon遺伝子"は、ラット染色体10番の遠位部に局在し、その遺伝子としてBHDホモログ遺伝子が有力である。NihonラットのBHDホモログ遺伝子には一塩基挿入による生殖細胞系列の変異が存在し、本ラットの腎癌細胞にはヒトと同様にBHDの遺伝子のLOH(ヘテロ接合体の消失)が認められることから、"Nihonラット"はKnudsonの2ヒット説を実証するモデルであると考えられる。　以上のことから、"Nihonラット"はヒトBHD症候群の動物モデルとして、また、BHD遺伝子機能や腎癌の発生とがん抑制遺伝子の関連について、有用な情報を提供するものと考えられ、ヒトの癌化機構の解明や遺伝子治療の開発に大きく寄与するものと考えられる。Background An adult onset malignancy arising from the epithelial cells of the renal nephron is almost renal cell carcinoma(RCC). RCC accounts for 2 % of all cancers. RCC is the sixth leading cause of cancer deaths in the United States and accounts for 3 % of adult malignancies. To data, kidney cancer-related genes were identified by positional cloning and candidate gene approach: von Hipel-Lindau disease (VHL gene), papillary renal cell carcinoma (MET protooncogene), the Krebs cycle enzyme fumarate hydratase (FH gene), tuberous sclerosis (TSC1 and TSC2 gene). To search for renal carcinogenesis and gene therapy, research by the animal model is required. Until recently, the hereditary renal carcinoma model was only the Eker rat, which was first described in the rat by Eker in 1954 in Oslo. The Eker rat model of hereditary renal carcinoma was the first example of a Mendelian dominantly inherited predisposition to a specific cancer in an experimental model, and has been contributing to the elucidation of renal carcinogenesis. Recently, the author and collaborators found a second hereditary RCC model in the Sprague-Dawley (SD) rat, in Japan in 2000. The author and collaborators have named this novel RC model the Nihon rat and performed the gene biological study. First, the author and collaborators described the origin, transmission mode, and phenotypic and molecular features of Nihon rat in this study. Next, the author and collaborators performed a genetic linkage analysis to the Nihon mutation, as a first step toward its identification and narrowed the Nihon locus to a region of the rat chromosome 10 homologous with human chromosome 17p11.2, and the author and collaborators identified a rat BHD homologue, mutations in which predispose to the renal cancer phenotype in the Nihon rat. Finally, the natural history in the Nihon rat was conducted to characterize RCC and extra-renal lesions histologically, and the author discussed a valuable experimental tool for functional studies related to renal carcinogenesis.Chapter 1."Nihon rat" Model of a Mendelian Inherited Renal Cell Carcinoma Bilateral, multicentric renal tubule tumors were found in a rat colony of the Sprague-Dawley strain. The renal tubule tumors were found in 15 out of 343 rats during 5 toxicity studies during the safety evaluation. These rats had all obtained from the same supplier, and the age of the rats ranged from 7 to 16 weeks at termination of the treatment period in each of the studies. After then, the supplier kindly provided 2 female founder rats and a carrier female rat was used in mating with a normal male SD rat. From the mating and pedigree, this animal model is an example of a Mendelian dominantly inherited predisposition for development RCC, and named the "Nihon rat". Until recently, the hereditary renal carcinoma model was only the Eker rat, which was first described in the rat by Eker in 1954 in Oslo. In 1995, Hino and others isolated and identified a germ line mutation in the rat homologous to the human tuberous sclerosis gene (TSC2). When the characteristic features of phenotype and genotype of the Nihon rat and the Eker rat is compared, the Nihon rat characteristically shows clear cell type RCs histologically, whereas the Eker rat dose not develop clear cell type RCs. The heterozygous Nihon rat typically develops RCs through multiple stages from early preneoplastic lesions (e.g., altered renal tubules and atypical hyperplasia) to carcinoma, and occurs at an early stage rather than those of the Eker rat. Besides, Southern blot, northern blot and SSCP analyses have not revealed any change in the Tsc1, Tsc2, VHL, and c-Met genes. In conclusion, the Nihon rat appears to be a novel hereditary renal cell carcinoma model, phenotypically distinct from the Eker rat, and with no mutation in the Tsc2 gene.Chapter 2.Chromosomal Mapping of the Predisposing Gene, "Nihon gene", in the Nihon rat The author performed a genetic linkage analysis of the Nihon rat using 113 backcross animals, as a first step toward its identification. Rat DNA markers ("MAP PAIRS"), covering whole rat chromosomes, were purchased from Research Genetics, Inc. First, the author checked 121 DNA markers covering rat chromosomes, then the author chose DNA markers clearly showing polymorphism between Nihon (SD) and BN strains and started to screen. One marker (D10Rat27) showed the smallest recombination fraction among DNA markers. Therefore, the author focused and narrowed the Nihon locus to the rat chromosome 10. In conclusion, the predisposing inherited gene in the Nihon rat was mapped to the rat chromosome 10 between interleukin-3 (IL3) (human 5q23-31) and lethal (2) giant larvae (LLGL1) (human 17p11.2)/myosin heavy chain, embryonic skeletal muscle (MYHSE) (human 17p13.1) loci, away from 4.4 centimorgans (cM) distal and 0.9 cM/5.3 cM proximal, respectively.Chapter 3.Isolation of the Predisposing Gene, "Nihon gene", in the Nihon rat The predisposing inherited gene in the Nihon rat was mapped to rat chromosome 10 between Il3 (human 5q23-31) and Llgl1 (human 17p11.2)/Myhse (human 17p13.1) loci, 4.4 centimorgans (cM) distal and 0.9 cM/5.3 cM proximal, respectively. At this time, the author did not know the human chromosome to which it corresponded (e.g., human chromosome 5 or 17) and the predisposing gene associated with renal cancer had not located in this locus. However, it was noted that the predisposing gene of the Birt-Hogg-Dubé (BHD) syndrome associated with renal cancer had been mapped to human chromosome 17p11.2 or 17p12-q11.2. In this chapter, the author narrowed the Nihon locus to a region of the rat chromosome 10 homologous with human chromosome 17p11.2, and the author identified a rat BHD homologous, mutations in which predispose to the renal cancer phenotype in the Nihon rat. From the result, complete concordance of segregation between putative rat BHD homologue (Bhd) and renal phenotype of the Nihon rat was found. Thus, rat Bhd was localized on rat chromosome 10 and tightly linked to the causative gene of the Nihon rat. In addition, the author described a germ-line mutation in Birt-Hogg-Dubé (Bhd) (human 17p11.2) caused by the insertion of a single nucleotide in the Nihon rat, resulting in a frame shift and producing a stop codon 26 amino acids downstream. The resulting mutant protein is lacking the vast majority of the normal rat Bhd (folliculin) sequence. The author found that the homozygous mutants condition was lethal at an early stage of fetal life in the rat. The author detected a high frequency of loss of heterozygosity (LOH) in primary RCs (10/11) at the Bhd locus and found a point mutation (nonsense) in one LOH-negative case. As mentioned above, the author identified a rat BHD homologue, "Nihon gene". These results indicate that the loss of folliculin function by a Knudson "two-hit" mechanism is a critical step for renal carcinogenesis in the Nihon rat.Chapter 4.The Natural History of the Nihon rat This chapter was conducted to characterize extra-renal lesions of the Nihon rat, and to compare the phenotypes with those in the human BHD syndrome, histopathologically. The heterozygous Nihon rat typically develops RCC through multiple stages from early preneoplastic lesions (e.g., altered renal tubules and atypical hyperplasia) to carcinoma, and penetrance for this Nihon gene was virtually complete. Histologically, the phenotype of RCs in the Nihon rats was clear cell type predominant, the phenotype that is most common in humans. In addition, the component showed clear/acidophilic cell and papillary basophilic cell type. Investigation of extra-renal primary lesions occurring in Nihon rats revealed clear cell hyperplasia/adenoma of the endometrium, cardiac rhabdomyomatosis at a young age, heterotopic ossification within renal cell carcinoma, and clear cell change of the epithelium of striated portions of salivary glands. To the best of our knowledge, these lesions are extremely uncommon lesions in Sprague-Dawley rats or other strains of rats and are thus clearly identifiable with the Nihon familial syndrome. Human BHD syndrome, originally described by Birt, Hogg and Dubé in 1977, is a rare inherited autosomal genodermatosis characterized by benign tumors of the hair follicle, and is associated with renal neoplasia, lung cysts, and spontaneous pneumothorax. At this time, the author have not detected in the Nihon rat skin tumor, lung cysts or spontaneous pneumothorax.Conclusion The Nihon rat is a model of a Mendelian dominantly inherited predisposition for development of RCs, which are predominantly of the clear cell type, and develop from earlier preneoplastic lesions than the Eker rat. The author performed a genetic linkage analysis of the Nihon rat using 113 backcross animals, and found that the Nihon mutation was tightly linked to genes, which are located on the distal part of rat chromosome 10. Finally, the author identified a germ line mutation in the Birt-Hogg-Dubé gene (Bhd) rat chromosome 10, human chromosome 17p11.2 caused by the insertion of a single nucleotide in the rat gene sequence, resulting in a frame shift and producing a stop codon 26 amino acids downstream. Thus, the Nihon rat should be a valuable experimental tool for functional studies related to renal carcinogenesis and a novel tumor suppressor gene BHD, and the Nihon rat will contribute to search for renal carcinogenesis and gene therapy.博士(獣医学)麻布大

Serine 62 is a phosphorylation site in folliculin, the Birt–Hogg–Dubé gene product

AbstractRecently, it was reported that the product of Birt–Hogg–Dubé syndrome gene (folliculin, FLCN) is directly phosphorylated by 5′-AMP-activated protein kinase (AMPK). In this study, we identified serine 62 (Ser62) as a phosphorylation site in FLCN and generated an anti-phospho-Ser62-FLCN antibody. Our analysis suggests that Ser62 phosphorylation is indirectly up-regulated by AMPK and that another residue is directly phosphorylated by AMPK. By binding with FLCN-interacting proteins (FNIP1 and FNIP2/FNIPL), Ser62 phosphorylation is increased. A phospho-mimic mutation at Ser62 enhanced the formation of the FLCN–AMPK complex. These results suggest that function(s) of FLCN–AMPK–FNIP complex is regulated by Ser62 phosphorylation.Structured summaryMINT-7298145, MINT-7298166: Flcn (uniprotkb:Q76JQ2) physically interacts (MI:0915) with AMPK alpha 1 (uniprotkb:P54645) by anti tag coimmunoprecipitation (MI:0007)MINT-7298267: AMPK alpha 1 (uniprotkb:Q13131) phosphorylates (MI:0217) tsc2 (uniprotkb:P49816) by protein kinase assay (MI:0424)MINT-7298182: FNIP1 (uniprotkb:Q8TF40) physically interacts (MI:0915) with Flcn (uniprotkb:Q76JQ2) by anti tag coimmunoprecipitation (MI:0007)MINT-7298132: AMPK alpha 1 (uniprotkb:Q13131) phosphorylates (MI:0217) Flcn (uniprotkb:Q76JQ2) by protein kinase assay (MI:0424)MINT-7298229: FNIPL (uniprotkb:Q9P278) physically interacts (MI:0915) with Flcn (uniprotkb:Q76JQ2) by anti tag coimmunoprecipitation (MI:0007

A germ-line insertion in the Birt–Hogg–Dubé (BHD) gene gives rise to the Nihon rat model of inherited renal cancer

A rat model of hereditary renal carcinoma (RC) was found in a rat colony of the Sprague–Dawley strain in Japan and named the “Nihon” rat. In heterozygotes, RCs, predominantly the clear cell type, develop from early preneoplastic lesions, which began to appear as early as 3 weeks of age, to adenocarcinomas by the age of 6 months. The Nihon rat is an example of a Mendelian dominantly inherited predisposition for development of RCs like the Eker (Tsc2 gene mutant) rat. We have previously shown that the Nihon mutation was tightly linked to genes that are located on the distal part of rat chromosome 10. The order of the genes is the Eker (Tsc2 gene (human 16p13.3)–Il3 gene–Nihon gene–Llgl1 locus– Myhse gene. We now describe a germ-line mutation in the Birt–Hogg–Dubé gene (Bhd) (human 17p11.2) caused by the insertion of a single nucleotide in the Nihon rat, resulting in a frameshift and producing a stop codon 26 aa downstream. We found that the homozygous mutant condition was lethal at an early stage of fetal life in the rat. We detected a high frequency of loss of heterozygosity (LOH) in primary RCs (10/11) at the Bhd locus and found a point mutation (nonsense) in one LOH-negative case, fitting Knudson's “two-hit” model. The Nihon rat may therefore provide insights into a tumor-suppressor gene that is related to renal carcinogenesis and an animal model of human BHD syndrome

Ouverture de ‘Global Networks and Local Development-1’

The global competitive landscapes of innovation and imitation have significantly changed the relative position of many Nation-States and the business relations between global networks and local firms.The US large corporations have lost their historical leadership in innovation. As a matter of fact US in the past had ruled the diffusion of innovations and the 'block' of imitations, but now they are looking for a new role in the control of the innovation and creative imitation processes, without any engagement in the local development.In addition, the main European countries (such as Germany, UK and the Russia) lost their leadership in innovation, although they played a leading role in the social and economic development of last century closed markets.At the same time, global markets have expanded the market power of corporations based in countries with high investments in innovation (e.g. the Japanese firms) or focused on creative imitation (e.g. the South Korea and Taiwan corporations)