华人生化学家RNAi方法破解修复谜团

　　

生物通报道：虽然目前有关DNA损伤应答的检测点激酶1（checkpoint kinase 1，Chk1)的功能已经得到了很好的理解，但是其在正常细胞增殖循环过程中扮演的角色仍然是一个谜团。来自普渡大学生物化学与癌症研究中心，以及哈佛大学分子与细胞生物学的研究人员利用RNA干扰（RNAi）技术特异性敲除了Chk1确认了HeLa细胞基因缺失表现型，发现Chk1对于正常细胞增殖和存活的重要意义，这一研究成果公布在8月8日PNAS杂志上。

完成课题的包括Xiaoqi Liu及其研究员Jiabin Tang等人。

检验点激酶1 (checkpoint kinase 1, Chk1)是一种进化保守的蛋白激酶，是细胞检验点的转导因子。当电离辐射、紫外线等引起细胞DNA损伤或者DNA复制叉停滞时Chk1活化，诱导细胞产生细胞周期阻滞、DNA修复或细胞凋亡等特征。

研究人员通过vector-based RNAi方法发现了Chk1在正常细胞增殖和存活过程中的必要作用，也同时发现在Chk1缺失的细胞中G₂/M期显著的细胞周期停滞和大量的细胞凋亡现象。除此之外，siRNA细胞同步实验也进一步揭示了Chk1缺陷将导致细胞中期的阻滞。另外研究人员还发现Chk1是polo-like激酶1（polo-like kinase 1，简称Plk1，参与有丝分裂调控的重要分子）的一个负调控因子，这些发现对于研究DNA损伤修复以及癌症治疗研究都具有重要的意义。
（生物通：张迪）

原文：
Published online before print July 27, 2006, 10.1073/pnas.0604987103 
PNAS | August 8, 2006 | vol. 103 | no. 32 | 11964-11969 
Checkpoint kinase 1 (Chk1) is required for mitotic progression through negative regulation of polo-like kinase 1 (Plk1) 
[Abstract]

附：

Xiaoqi Liu Assistant Professor of Biochemistry 233C Hansen, [phone] Ph.D. Washington State University, 1999 [e-mail] Investigators: Hongchang Li, Jiabin Tang Area: Roles of Polo-like kinase 1 and its interacting proteins in cell proliferation and carcinogenesis

It is now widely accepted that cancer arises at least partly due to the perturbation of normal cell cycle progression, in which reversible protein phosphorylation plays an important regulatory role. In addition to the well-documented cyclin-dependent kinases, the Polo-like kinase (Plk) family has emerged as a key player in many cell cycle related events. Genetic and biochemical experiments with several different organisms have demonstrated that polo kinases are involved in many aspects of mitosis, such as mitotic entry, sister chromatids separation and cytokinesis. To investigate the function of Plk1 in mammalian cells in more detail, we recently reported the phenotype of cultured cells after Plk1 depletion using RNA interference (RNAi). Plk1 depletion in cancer cells induces G2/M arrest, followed by apoptosis. A close correlation between mammalian Plk1 expression and carcinogenesis was recently documented. Overexpression of Plk1 was observed in various human tumors, such as melanomas, non-small cell lung cancer, etc. Constitutive expression of Plk1 in normal cells causes oncogenic focus formation and induces tumor growth in nude mice. Thus, inhibition of Plk1 function may be an important application for cancer therapy. Because inhibition of cell proliferation and reaction of apoptosis are basic principles in anticancer therapy, it will be intriguing to test the effect of Plk1 depletion in normal primary cells. A lentivirus-based RNAi approach is currently being used to deplete Plk1 in several types of normal cells. Using a yeast two-hybrid system, we have identified a battery of potential Plk1-interacting proteins, and the significance of these interactions during cell cycle progression will be analyzed.