Effects of PP4 suppression on the proliferation of MCF7 cells  

作　　者：NING Lifeng LONG Zhitao HUANG Xiuqing SUN Lingling SANG Jianli 

机构地区：[1]The Key Laboratory for Cell Proliferation and Regulation Biology of Ministry of Education, College of Life Science, Beijing Normal University, Beijing 100875

出　　处：《Chinese Science Bulletin》2006年第19期2335-2341,共7页

基　　金：This work was supported by the National Natural Science Foundation of China （Grant No. 30270663）.

摘　　要：PP4, one of the few protein phosphata- ses associated with centrosome in cells of many species such as Drosophila, C. elegans and mam- mals, plays an essential role in the regulation of cen- trosome functions in Drosophila and C. elegans. In order to explore the role of PP4 in mammalian cells, full-length PP4 gene was obtained by RT-PCR from MCF7 cell total RNA and inserted into eukaryotic expression vector pEGFP-C1. The resultant con- struct pEGFP-C1-PP4 was transfected into MCF7 cells and immunostaining was carried out to confirm the centrosome localization of PP4. Then we re- versely subcloned a non-conserved domain of PP4 into pXJ41 to construct an anti-sense vector pXJ41- as-PP4. By transfecting pXJ41-as-PP4 into MCF7 cells and screening with G418, we obtained a stable cell line in which PP4 expression was stably sup- pressed. The cell line was analyzed on cell mor- phology, cytoskeleton structure, growth characteris- tics and the mitosis process. It was found that the proliferation rate decreased and serum-dependence increased in PP4-suppressed cells. Furthermore, flow cytometry and mitotic index analysis showed that G2/M transition was prolonged. PP4 suppression resulted in abnormal interphase microtubule, forma- tion of multipolar spindles and an increase in per- centage of multinuclear cells. These results sug- gested that PP4 is required for centrosome function in mammalian cells.PP4, one of the few protein phosphatases associated with centrosome in cells of many species such as Drosophila, C. elegans and mammals, plays an essential role in the regulation of centrosome functions in Drosophila and C. elegans. In order to explore the role of PP4 in mammalian cells, full-length PP4 gene was obtained by RT-PCR from MCF7 cell total RNA and inserted into eukaryotic expression vector pEGFP-C1. The resultant construct pEGFP-C1-PP4 was transfected into MCF7 cells and immunostaining was carried out to confirm the centrosome localization of PP4. Then we reversely subcloned a non-conserved domain of PP4 into pXJ41 to construct an anti-sense vector pXJ41- as-PP4. By transfecting pXJ41-as-PP4 into MCF7 cells and screening with G418, we obtained a stable cell line in which PP4 expression was stably suppressed. The cell line was analyzed on cell morphology, cytoskeleton structure, growth characteristics and the mitosis process. It was found that the proliferation rate decreased and serum-dependence increased in PP4-suppressed cells. Furthermore, flow cytometry and mitotic index analysis showed that G2/M transition was prolonged. PP4 suppression resulted in abnormal interphase microtubule, formation of multipolar spindles and an increase in percentage of multinuclear cells. These results suggested that PP4 is required for centrosome function in mammalian cells.

关 键 词：细胞周期 MCF7 PP4 蛋白质磷酸酶 微管 中心体 有丝分裂 反义RNA 

分 类 号：Q813[生物学—生物工程]