Tang Jiabin, Erikson Raymond L, Liu Xiaoqi
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
Proc Natl Acad Sci U S A. 2006 Aug 8;103(32):11964-9. doi: 10.1073/pnas.0604987103. Epub 2006 Jul 27.
Although the essential function of checkpoint kinase 1 (Chk1) in DNA damage response has been well established, the role of Chk1 in normal cell cycle progression is unclear. By using RNAi to specifically deplete Chk1, we determined loss-of-function phenotypes in HeLa cells. A vector-based RNAi approach showed that Chk1 is required for normal cell proliferation and survival, inasmuch as a dramatic cell-cycle arrest at G(2)/M phase and massive apoptosis were observed in Chk1-deficient cells. Coupling of siRNA with cell synchronization further revealed that Chk1 depletion leads to metaphase block, as indicated by various mitotic markers. Neither bipolar spindle formation nor centrosome functions were affected by Chk1 depletion; however, the depleted cells exhibited chromosome misalignment during metaphase, chromosome lagging during anaphase, and kinetochore defects within the regions of misaligned/lagging chromosomes. Moreover, we showed that Chk1 is a negative regulator of polo-like kinase 1 (Plk1), in either the absence or presence of DNA damage. Finally, Chk1 depletion leads to the activation of the spindle checkpoint because codepletion of spindle checkpoint proteins rescues the Chk1 depletion-induced mitotic arrest.
尽管关卡激酶1（Chk1）在DNA损伤应答中的基本功能已得到充分证实，但其在正常细胞周期进程中的作用尚不清楚。通过使用RNA干扰技术特异性地敲低Chk1，我们确定了HeLa细胞中的功能缺失表型。基于载体的RNA干扰方法表明，Chk1是正常细胞增殖和存活所必需的，因为在Chk1缺陷细胞中观察到了显著的G(2)/M期细胞周期阻滞和大量细胞凋亡。将小干扰RNA（siRNA）与细胞同步化相结合进一步表明，Chk1的敲低导致中期阻滞，各种有丝分裂标记物表明了这一点。Chk1的敲低既不影响双极纺锤体的形成，也不影响中心体功能；然而，敲低后的细胞在中期表现出染色体排列紊乱，在后期表现出染色体滞后，并且在排列紊乱/滞后染色体区域内存在动粒缺陷。此外，我们表明，无论是否存在DNA损伤，Chk1都是polo样激酶1（Plk1）的负调节因子。最后，Chk1的敲低导致纺锤体检查点的激活，因为纺锤体检查点蛋白的共敲低可挽救Chk1敲低诱导的有丝分裂阻滞。
