Functional analysis of a noveloncoprotein, gankyrin, isolated from hepatpmas, and its clinical application

我们分离了新发现的在肝细胞癌中过表达的致癌蛋白gankyrin。Gankyrin的过表达引起NIH/3 T3细胞的细胞转化，并导致Rb的磷酸化和E2 F-1的活化增加。此外，Gankyrin在体外和体内加速Rb的降解。这些结果表明，Gankyrin参与Rb途径，其过表达通过使Rb失活和不稳定而促进肝癌发生（Nature Medicine，2000）（Alcohol Clin Exp Res，2001）。通过与CDK 4激酶结合，gankyrin促进Rb的磷酸化。这种结合与p16竞争结合CDK 4并抵消p16 INK 4a的抑制功能。我们证明Gankyrin与19 S调节亚基的组分S6 ATP酶（Rpt 3）瞬时相互作用（J Biol Chem，2002）。除Rb外，某些肿瘤抑制因子的另一种产物在gankyrin过表达的情况下容易降解。本品与Gankyrin在体内外均无相互作用。然而，Gankyrin减弱了该产物的转录后修饰（磷酸化、乙酰化等）及其细胞内亚定位。我们尝试使用酵母双杂交方法鉴定Gankyrin的其他结合伴侣。一种候选物通过Gankyrin过表达抑制锚定非依赖性细胞生长。尽管gankyrin和该蛋白之间的相关性的机制和重要性尚未阐明，该蛋白的一部分诱导gankyrin过表达细胞系的促凋亡活性。此外，为了在用于人类基因治疗的肝癌细胞系中实现高转基因表达，我们研究了哪些启动子区域参与其中（Gene therapy，2002）。我们将研究这些肽是否能特异性地对抗Gankyrin的致癌功能。

We have isolated the newly discovered oncogenic protein gankyrin overexpressed in hepatocellular carinoma. Overexpression of gankyrin caused cellular transformation in NIH/3T3 cells, and led to increased phosphorylation of Rb and activation of E2F-1. Furthermore, gankyrin accelerated the degradation of Rb in vitro and in vivo. These results suggest that gankyrin is involved in Rb pathway and its overexpression contributes to hepatocarcinogenesis by inactivating and destabilizing Rb (Nature Medicine, 2000) (Alcohol Clin Exp Res, 2001). By binding with CDK4 kinase, gankyrin facilitates the phosphorylation of Rb. This binding competes with pl6 binding to CDK4 and counteracts the inhibitory function of p16INK4a. We demonstrate that gankyrin intetacts with S6 ATPase (Rpt3), a component of 19S regulatory subunit transiently (J Biol Chem, 2002). Besides Rb, another product of some tumor suppressor is readily degraded in case of gankyrin overexpression. This product doed not interaet with gankyrin in vitro or in vivo. Nevertheless, gankyrin attenuaees the posttranscriptional modification (phosphorylation, acetylation, and so on) of this product, and its intracellular sublocalization. We attempted to identify the other binding partners of gankyrin, using the yeast two hybrid method. 0ne candidate inhibits the anchorage independent cell growth by gankyrin overexpression. Whereas the mechanism and importance of the correlation between gankyrin and this protein are yet to be elucidated, a part of this protein induces the proapoptotic activity to gankyrin-overexpressing cell lines. Furthermore, to achieve high transgene expression in hepatoma cell lines for human gene therapy, we investigated which promoter regions are involved in it (Gene therapy, 2002). We will investigate whether these peptides counteract the oncogenic function of gankyrin specifically.