Subcellular localization and control of activity of telomerase

由于端粒酶活性在原代细胞中几乎检测不到，但在癌细胞中却能清楚地检测到，因此端粒酶被认为是癌症治疗的有力候选靶点。在本财政年度，我们集中精力进行了几项实验，包括重组人端粒酶复合物的纯化和表征，从稳定表达FLAG-hTERT的HeLa细胞系中回收的核仁素与hTERT特异性相互作用的生物学功能（Murakami），通过hTERT永生化的人原代细胞的分析（本田），以及在细胞增殖和分化中起关键作用的受体型酪氨酸激酶（Yoshioka）。实验的主要结论如下：1）部分纯化的端粒酶活性复合物由复合物I（约680 kDa）和复合物II（约400 kDa）两种不同的复合物组成，它们与可从共表达FLAG-hTERT和hTERC的昆虫细胞中回收的复合物非常相似。的com ...更多信息 复合物I不含Hsp 90并且对Hsp 90抑制剂有抗性，而复合物II含有Hsp 90并且对Hsp 90抑制剂敏感。复合物II中的人TEAT在Vim和环中孵育期间相当不稳定。在配合物I中没有观察到这种不稳定的性质。由于特异性蛋白酶体抑制剂MG 132而非阴性对照MG 133稳定了复合物Z中的hTERT，这强烈表明复合物If中的hTERT处于蛋白酶体控制下的降解途径中。人端粒酶的两种不同的复杂性似乎暗示着两种不同的活性端粒酶实体在不同的亚细胞定位或其不同的功能。2)我们建立了稳定表达hTERT的人原代成纤维细胞（BJ细胞）系，比较了MERE永生化的RI细胞和KJ细胞的表达谱，发现与细胞周期和细胞增殖有关的一些基因在永生化的131-hTERT细胞中的表达明显高于原代细胞。3)核仁素（Nucleolin）是hTERT的特异性相互作用伙伴之一，可与丙型肝炎病毒（Hepatitis C Virus，HCV）NS 5 B结合，参与HCV的RNA依赖性复制。我们评估了核仁素和NS 5 B的特异性相互作用在HCV复制中的作用。HCV亚复制子系统已被应用于解决这一问题。我们发现，在HepG 2 ml/s的瞬时HCV复制系统中，含有丙氨酸取代突变或NS 5 B内两个关键序列之一突变的HCV亚复制子根本不能复制，这两个关键序列都是核仁结合所必需的。尽管野生型复制子可以支持有效的HCV。通过引入核仁素的瞬时RNA干扰，使核仁素的表达下调一半至三分之一，使用HCV亚复制子系统明显减少HCV复制。总之，核仁素和NS 5 B的特异性相互作用对HCV复制至关重要。少

Since telomerase activity is barely detectable in primary cells but clearly detected in cancerous cells, telomerase has been considered to be a strong candidate of the targets to cancer treatment In the fiscal year, we concentrated in several experiments including purification and characterization of recombinant human telomerase complex, and biological function of the specific interaction of nucleolin and hTERT recovered from a HeLa cell line stably expressing FLAG-hTERT (Murakami), analyses of human primary cells immortalized by hTERT (Honda), and the tyrosine kinases of receptor type which play critical odes in cell proliferation and differentiation (Yoshioka). The followings are the main conclusions of the experiments.1) The partially purified active telomerase complexes comprise two different complexities, complex I (around 680 kDa), and complex II (around 400 kDa), that are quite alike to those that can be recovered firm the insect cells co-expressing FLAG-hTERT and hTERC. The com … More plex I does not contain Hsp90 and is resistant to Hsp90 inhibitors, whereas complex II contains Hsp90 and is sensitive to Hsp90 inhibitors. Human TEAT in the complex II is rather unstable during incubation in vim and in ring. Such unstable property was not observed with the complex I. Since MG132, a specific proteasome inhibitor, but not MG133, a negative control, stabilized hTERT in the complex Z strongly suggesting that hTERT in the complex If is under degradation pathway under the control of proteasome. The two different complexities of human telomerase seem to imply two different entities of active telomerase in different subcellular localization or its different functions. 2) We have established a cell line of the human primary fibroblasts(BJ cells) stably expressing hTERT, and we compared expression profiles of RI cells and the KJ cells immortalized by MERE Some genes involving in cell cycle progression and cell proliferation were evidently expressed higher in the immortalized 131-hTERT than these in in the primary cells. 3) Nucleolin, one of the specific interacting partners of hTERT can bind to Hepatitis C Virus (HCV) NS5B, RNA-dependent RNA replication of HCV. We evaluated the role of the specific interaction of nucleolin and NS5B in HCV replication. The HCV subreplicon system has been applied for the address We found that the HCV subreplicons harboring alanine substitution mutations or mutation at the residue(s) either one of two critical sequences within NS5B both of which are necessary for the nucleolin-binding could not replicate at all in a transient HCV replication system in HepG2 ml/s., although the wild replicon could support efficient HCV. By introducing transiently RNAi of nucleolin, that down regulated expression of nucleolin by half to one third, reduced HCV evidently reduced HCV replication using the HCV subreplicon system. Taken together, the specific interaction of nucleolin and NS5B is critical for HCV replication. Less