Cellular Ras and Cyclin D1 Are Required during Different Cell Cycle Periods in Cycling NIH 3T3 Cells
Cellular Ras and Cyclin D1 Are Required during Different Cell Cycle Periods in Cycling NIH 3T3 Cells
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DOI：
10.1128/mcb.19.7.4623
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发表时间：
1999-07
影响因子：
5.3
通讯作者：
M. Hitomi;D. Stacey
中科院分区：
文献类型：
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作者：
M. Hitomi;D. Stacey
摘要采用新技术来确定增殖的NIH 3T3细胞在细胞周期中何时需要细胞Ras和细胞周期蛋白D1。为了比较，在静止细胞中，所有四种细胞周期进程的抑制剂测试（抗Ras，抗细胞周期蛋白D1，血清去除，放线菌酮）成为无效的G1期基本上相同的点，约4小时前开始的DNA合成。为了将这些研究扩展到循环细胞，使用延时方法来确定在抑制剂处理时异步培养物中单个细胞的大致细胞周期位置，然后确定抑制剂对受体细胞的影响。通过这种方法，抗Ras抗体仅在前一次有丝分裂之前，S期开始前几小时引入细胞时才有效抑制进入S期。另一方面，抗细胞周期蛋白D1是一种有效的抑制剂，直到DNA合成开始之前。放线菌酮治疗，如抗细胞周期蛋白D1微量注射，是抑制整个G1期（持续4至5小时，在这些细胞）。最后，血清去除只在有丝分裂后的第一个小时内阻止进入S期。动力学分析和一种新的双标记技术被用来确认Ras，细胞周期蛋白D1，放线菌酮的细胞周期要求的差异。这些研究表明，在静止和循环NIH 3T3细胞之间的促有丝分裂信号转导的根本差异，并揭示了在增殖NIH 3T3细胞的细胞周期进程所需的信号事件的序列。
ABSTRACT Novel techniques were used to determine when in the cell cycle of proliferating NIH 3T3 cells cellular Ras and cyclin D1 are required. For comparison, in quiescent cells, all four of the inhibitors of cell cycle progression tested (anti-Ras, anti-cyclin D1, serum removal, and cycloheximide) became ineffective at essentially the same point in G1 phase, approximately 4 h prior to the beginning of DNA synthesis. To extend these studies to cycling cells, a time-lapse approach was used to determine the approximate cell cycle position of individual cells in an asynchronous culture at the time of inhibitor treatment and then to determine the effects of the inhibitor upon recipient cells. With this approach, anti-Ras antibody efficiently inhibited entry into S phase only when introduced into cells prior to the preceding mitosis, several hours before the beginning of S phase. Anti-cyclin D1, on the other hand, was an efficient inhibitor when introduced up until just before the initiation of DNA synthesis. Cycloheximide treatment, like anti-cyclin D1 microinjection, was inhibitory throughout G1 phase (which lasts a total of 4 to 5 h in these cells). Finally, serum removal blocked entry into S phase only during the first hour following mitosis. Kinetic analysis and a novel dual-labeling technique were used to confirm the differences in cell cycle requirements for Ras, cyclin D1, and cycloheximide. These studies demonstrate a fundamental difference in mitogenic signal transduction between quiescent and cycling NIH 3T3 cells and reveal a sequence of signaling events required for cell cycle progression in proliferating NIH 3T3 cells.