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Functional analysis of tuberin by using animal models of tumor suppressor Tsc2-mutant.

利用抑癌基因 Tsc2 突变体动物模型对马铃薯蛋白进行功能分析。

基本信息

批准号：
14580804
负责人：
KOBAYASHI Toshiyuki
金额：
$ 2.24万
依托单位：
Japanese Foundation for Cancer Research
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14580804/
关键词：
Tsc2 tuberin knockout mouse transgenic Eker rat renal tumor tuberous sclerosis S6 kinase Erc mesothelin

项目摘要

Expression vector for Tsc2 (tuberous sclerosis 2 gene) cDNA was introduced in Tsc2-deficient mouse renal tumor (RT) cell line (MKOC1.-277) and stable lines expressing tuberin (T2 cells) were established. Phosphorylation of p70 S6 kinase (S6K) was suppressed in T2 cells as compared with control cell lines with empty vector. Tumorigenic potential of RC cells in the nude mouse was suppressed by tuberin expression. Administration of rapamycin, a mTOR inhibitor, also blocked tumorigenesis of RC cells. These results suggest that mTOR〜S6K pathway has some role in tumorigenesis caused by Tsc2-mutation. Interestingly, expression of Erc mRNA (mesothelin gene homologue) was also suppressed in T2 cells. In contrast, rapamycin treatment of parental MKOC1-277 cells did not change expression level of Erc mRNA. These results suggest that Erc is regulated by downstream signaling pathway of tuberin, which is independent of mTOR〜S6K. This novel tuberin-regulated pathway may be a new candidate for therapeutic molecular target for tuberous sclerosis. To search for pathways regulating Erc expression, Hela cells were treated with various stimuli or chemical inhibitors and activators of signal transduction pathways and analyzed by northern and western blot analyses. In the case of serum starvation, amino-terminal half of Erc protein was increased, probably by protease-mediated cleavage. Thus, expression and function of Erc protein may be regulated by post-translational level in relation with cell growth. In this study, search for candidates of tuberin-binding protein was also performed. Gamma-adaptin and myomegalin were identified by two-hybrid analysis using carboxy-terminal region of tuberin containing Rapt-GAP homology domain as a bait.

将Tsc 2（结节性硬化症2基因）cDNA的表达载体导入Tsc 2缺陷型小鼠肾肿瘤（RT）细胞系（MKOC 1.-MKOC 2）中。277)并建立了表达块茎蛋白的稳定细胞系（T2细胞）。与空载体的对照细胞系相比，T2细胞中p70 S6激酶（S6 K）的磷酸化被抑制。结球蛋白表达可抑制RC细胞在裸鼠体内的成瘤潜能。给予雷帕霉素（一种mTOR抑制剂）也阻断了RC细胞的肿瘤发生。这些结果提示mTOR β S6 K通路在Tsc 2-突变引起的肿瘤发生中具有一定的作用。有趣的是，Erc mRNA（间皮素基因同源物）的表达在T2细胞中也被抑制。与此相反，雷帕霉素处理亲本MKOC 1 -277细胞没有改变Erc mRNA的表达水平。这些结果表明Erc受tuberin下游信号通路的调控，而不依赖于mTOR β 6 K。这种新型的结核菌素调节途径可能是结节性硬化症治疗分子靶点的新候选者。为了寻找调节Erc表达的途径，用各种刺激物或信号转导途径的化学抑制剂和活化剂处理Hela细胞，并通过北方和Western印迹分析进行分析。在血清饥饿的情况下，氨基末端的Erc蛋白的一半增加，可能是由蛋白酶介导的切割。因此，Erc蛋白的表达和功能可能受到与细胞生长相关的翻译后水平的调节。在这项研究中，还进行了候选的tuberin结合蛋白的搜索。以含有Rapt-GAP同源结构域的tuberin羧基端为诱饵，通过双杂交技术鉴定了gamma-adaptin和myomegalin。