Stacey D W, Hitomi M, Kanovsky M, Gan L, Johnson E M
Department of Molecular Biology, Lerner Research Institute, Cleveland Clinic Foundation, Ohio 44195, USA.
Oncogene. 1999 Jul 22;18(29):4254-61. doi: 10.1038/sj.onc.1202795.
Levels of Pur alpha, a protein implicated in control of both DNA replication and gene transcription, fluctuate during the cell cycle, being lowest in early S phase and highest just after mitosis. Here we have employed a new video time-lapse technique enabling us to determine the cell cycle position of each cell in an asynchronous culture at a given time and to ask whether introduction of Pur alpha protein at specific times can affect cell cycle progression. Approximately 80% of all NIH3T3 cells injected with Pur alpha were inhibited from passing through mitosis. Cells injected with Pur alpha during S or G2 phases were efficiently blocked with a 4N (G2 phase) DNA level, as determined by quantitative DNA photometry of individual cells. Of the cells injected with Pur alpha during G1 phase, 40% experienced a rapid cell death characterized by extreme cellular fragmentation. Of those G1 injected cells which remained viable, approximately equal numbers were arrested with either 2N or 4N DNA levels. Cells arrested by Pur alpha in G2 phase grew to cover a large surface area. These results link fluctuations in Pur alpha levels to aspects of cell cycle control.
Pur alpha是一种与DNA复制和基因转录控制均有关的蛋白质，其水平在细胞周期中波动，在S期早期最低，在有丝分裂刚结束后最高。在此，我们采用了一种新的视频延时技术，该技术使我们能够确定给定时间异步培养物中每个细胞的细胞周期位置，并探究在特定时间引入Pur alpha蛋白是否会影响细胞周期进程。注射了Pur alpha的所有NIH3T3细胞中，约80%被阻止进入有丝分裂。通过对单个细胞进行定量DNA光度测定确定，在S期或G2期注射Pur alpha的细胞被有效地阻滞在4N（G2期）DNA水平。在G1期注射Pur alpha的细胞中，40%经历了以极端细胞破碎为特征的快速细胞死亡。在那些仍存活的G1期注射细胞中，约有相等数量的细胞被阻滞在2N或4N DNA水平。在G2期被Pur alpha阻滞的细胞生长并覆盖了很大的表面积。这些结果将Pur alpha水平的波动与细胞周期控制的各个方面联系起来。
