VHL tumor suppressor gene : Identification its function for cell growth inhibition and cell ular apoptosis for kidne cancer to develop new modality of treatment

VHL抑癌基因：鉴定其对肾癌细胞生长抑制和细胞凋亡的功能，以开发新的治疗方式

• 批准号: 09470348
• 负责人: SHUIN Taro
• 金额: $ 7.94万
• 依托单位: Kochi Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09470348/
• 关键词: von Hippel-Lindau (VHL) disease; actin fiber; vinculin; atypical protein kinase C・(aPKC・); atypical protein kinase C・(aPKC); VHL癌抑制遺伝子; 細胞接着; 胚細胞変異; atypical protein kinase cλ; von Hippel-Lindau病; VHL遺伝子; 癌抑制遺伝子; actin fiber; protein kinase C

Our purpose in this grant is to identify specific proteins which affect tumor growth or invasion that are controlled by VHL tumor suppressor protein (pVHL) with cell biolgy and molecular biological methods. We first examined morphological change in 99VHL, lung cancer cell lines in which pVHL is specially inducible to 100-fold by ponasterone treatment. In the experiment for the induction of pVHL in that cell lines, almost of the cells are flattened and the cell adhesion ability to the cell culture dish is highly increased. The specific proteins for cellular adhesion, vinculin is increased in the adherent side of cell membrane. The total amount of cellular vinculin itself is not changed. The actin finbers are well polymerized at the same time with the movement of vinculin to the adherent membrane. The cell motitilty is dramatically decreased with these changes. According to these results, the function of pVHL is to reorganize vinculin to the adherent side of membranem, to polymerize acti … More n fiber and finally to decrease cell motility.We next examined the characteristics of germline mutation of the VHL gene in the whole world. We detected 4 mutation-clustered regions, that are the end of the exonl, the junction of the exon 1 and 2, the middle portion of exon 2 and the first part of exon 3.The first part of exon 3 is well known as the binding domain of pVHL with Elongin C.It is highly possible that other 3 regions have important function. As the results of several experiments, the 2^<nd> clustered region of mutation coinsides with the binding domain of pVHL with atyipcal protein kinase lambda (aPKC・・). This resion is located at the beta-domain of pVHL (amine acid 114 to 122). Since the essential function of pVHL is ubiqutination and final degradation of short-lived proteins, that bind the beta domain, one of the important candidate protein for function of pVHL is aPKC・・・ Since aPKC・・・・ has ・anti-apoptotic function and promote cell growth, pVHL is working to degrade aPKC・・・ and induce cellular apoptosis.Our purpose in this grant is to identify specific proteins which affect tumor growth or invasion that are controlled by VIIL tumor suppressor protein (pVHL) with cell biolgy and molecular biological methods. We first examined morphological change in 99VHL.lung cancer cell lintes in which pVHL is specially inducible to 100-fold by ponasterone treatment. In the experiment for the induction of pVHL in that cell lines, almost of the cells are flattened and the cell adhesion ability to the cell culture dish is higltly increased. The specific proteins for cellular adhesion, vinculin is increased in the adherent side of cell membrane. The total amount of cellular vinculin itself is not changed. The actin finbers are well polymerized at the same time with the movement of vinculin to the adherent membrane. The cell motitilty is dramatically decreased with these changes. According to these results, the function of pVHL is to reorganize vinculin to the adherent side of membranem, to polymerize actin fiber and finally to decrease cell motility.As a summary, it is shown that the direct effect of pVHL is possible aPKC・・ degradation and indirect effect is to reorganize vinculin, to polymerize actin fiber and finally to reduce cell motility. Less

本课题的目的是利用细胞生物学和分子生物学的方法，研究VHL肿瘤抑制蛋白（pVHL）调控的影响肿瘤生长或侵袭的特异性蛋白。我们首先研究了99VHL的形态学变化，99VHL是肺癌细胞系，其中pVHL被ponasterone处理特异性诱导至100倍。在该细胞系中诱导pVHL的实验中，几乎所有的细胞都变平，并且细胞对细胞培养皿的粘附能力高度增加。粘着斑蛋白是细胞粘附的特异性蛋白质，在细胞膜的粘附侧增加。细胞粘着斑蛋白本身的总量不变。在黏着斑蛋白向粘附膜移动的同时，肌动蛋白纤维聚合良好。细胞运动性随着这些变化而显著降低。根据这些结果，pVHL的功能是将黏着斑蛋白重组到膜的粘附侧，并使黏着斑蛋白粘附到细胞膜上，从而使细胞膜上的粘附细胞粘附到细胞膜上。 ...更多信息 我们接下来研究了全世界VHL基因的种系突变特征。在pVHL基因中检测到4个突变聚集区，分别是外显子1的末端、外显子1和2的连接处、外显子2的中间部分和外显子3的第一部分。外显子3的第一部分是已知的pVHL与Elongin C的结合域，其他3个区域很可能具有重要的功能。实验结果表明，突变的第2<nd>簇区域与pVHL的非典型蛋白激酶λ（aPKC··）结合域一致。该受体位于pVHL的β结构域（氨基酸114至122）。由于pVHL的基本功能是结合β结构域的短寿命蛋白的泛素化和最终降解，因此pVHL功能的重要候选蛋白之一是aPKC···由于aPKC···具有抗凋亡功能并促进细胞生长，pVHL致力于降解aPKC···本研究的目的是鉴定受肿瘤抑制蛋白（pVHL）调控的影响肿瘤生长或侵袭的特异性蛋白用细胞生物学和分子生物学的方法。我们首先研究了99VHL肺癌细胞株的形态学变化，其中pVHL被ponasterone处理特异性诱导至100倍。在诱导pVHL的实验中，大部分细胞扁平化，细胞对细胞培养皿的粘附能力大大增强。粘着斑蛋白是细胞粘附的特异性蛋白质，在细胞膜的粘附侧增加。细胞粘着斑蛋白本身的总量不变。在黏着斑蛋白向粘附膜移动的同时，肌动蛋白纤维聚合良好。细胞运动性随着这些变化而显著降低。结果表明，pVHL的作用是重组vinculin，使其向膜的贴壁面移动，并使其向肌动蛋白纤维移动，从而降低细胞的运动性，pVHL的直接作用可能是降解aPKC··，间接作用是重组vinculin，使其向肌动蛋白纤维移动，从而降低细胞的运动性。少

项目成果

Okuda H, Hirai S, Shuin T.et al: "Direct interaction of the beta-domain of VHL tumor suppressor protein with the regulatory domain of atypical PKC isotypes."Biochem Biophys Res Commun. 263. 491-497 (1999)

Okuda H、Hirai S、Shuin T.等人：“VHL 肿瘤抑制蛋白的 β 结构域与非典型 PKC 同种型的调节结构域的直接相互作用。”Biochem Biophys Res Commun。

Shuin T: "Germline and somatic mutations in von Hippel-Lindan disease gene and its significance in the development of kidney cancer"Contrib Nephrol. 128. 1-10 (1999)

Shuin T：“von Hippel-Lindan 疾病基因的种系和体细胞突变及其在肾癌发展中的意义”Contrib Nephrol。

Masaya Baba, Syu-ichi Hirai, Taro Shuin, Shigeo Ohno et al.: "Tumor suppressor protein VHL is induced at high density and mediates contact inhibition of cell growth"Oncogene. (in press). (2000)

Masaya Baba、Syu-ichi Hirai、Taro Shuin、Shigeo Ohno 等人：“肿瘤抑制蛋白 VHL 在高密度下被诱导并介导细胞生长的接触抑制”癌基因。

Okuda H: "Direct interaction of the beta-domain of VHL tumor suppressor protein with the regulatory domain of atypical PKC isotypes"Biochem Biophys Res Commun. 263(2). 491-497 (1999)

Okuda H：“VHL 肿瘤抑制蛋白的 β 结构域与非典型 PKC 同种型的调节结构域的直接相互作用”Biochem Biophys Res Commun。

Masahiro Yao,et al.: "Molecular genetics of the kidney cancers;implication of the VHL tumor suppressor gene." Gann Monograph on Cancer Research. 46. 205-214 (1999)

Masahiro Yao 等人：“肾癌的分子遗传学；VHL 抑癌基因的意义”。