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高表达的致癌蛋白Gankyrin。Gankyrin过表达导致NIH/3T3细胞转化，并导致Rb磷酸化增加和E2F-1激活。此外，甘草苷在体外和体内均能加速Rb的降解。这些结果表明，Gankyrin参与了Rb途径，其过表达通过使Rb失活和不稳定而导致肝癌的发生(自然医学，2000)(Alcool Clin Exp，2001)。甘草蛋白通过与CDK4激酶结合，促进Rb的磷酸化。这种结合与pL6与CDK4的结合竞争，并抵消p16INK4a的抑制功能。我们证明了Gankyrin与S6 ATPase(Rpt3)瞬时相互作用，S6 ATPase是19S调节亚基的一个组成部分(J Biol Chem，2002)。除Rb外，某些肿瘤抑制因子的另一种产物在gankyrin过表达的情况下很容易被降解。该产品在体外和体内均不与甘草蛋白发生相互作用。然而，甘草蛋白减弱了该产物的转录后修饰(磷酸化、乙酰化等)及其细胞内的亚区块化。我们尝试用酵母双杂交的方法来鉴定甘草蛋白的其他结合伙伴。One候选蛋白通过过表达gankyrin抑制锚定非依赖性细胞的生长。虽然Gankyrin与该蛋白相关的机制和重要性尚不清楚，但该蛋白的一部分可诱导过表达Gankyrin的细胞系的促凋亡活性。此外，为了实现转基因在人肝癌细胞系中的高表达，我们研究了哪些启动子区域参与了这一过程(基因治疗，2002)。我们将研究这些多肽是否特异性地中和甘结蛋白的致癌功能。

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.