Cellular ras and cyclin D1 are required during different cell cycle periods in cycling NIH 3T3 cells

Novel techniques were used to determine when in the cell cycle of prolifera ting NIH 3T3 cells cellular Ras and cyclin D1 are required. For comparison, in quiescent cells, all four of the inhibitors of cell cycle progression t ested (anti-Ras, anti cyclin D1, serum removal, and cycloheximide) became i neffective at essentially the same point in G(1) phase, approximately 4 h p rior to the beginning of DNA synthesis. To extend these studies to cycling cells, a time-lapse approach was used to determine the approximate cell cyc le position of individual cells in an asynchronous culture at the time of i nhibitor treatment and then to determine the effects of the inhibitor upon recipient cells. With this approach, anti-Ras antibody efficiently inhibite d 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 b efore the initiation of DNA synthesis. Cycloheximide treatment, like anti-c yclin D1 microinjection, was inhibitory throughout G(1) phase (which lasts a total of 4 to 5 h in these cells). Finally, serum removal blocked entry i nto 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 studi es demonstrate a fundamental difference in mitogenic signal transduction be tween quiescent and cycling MH 3T3 cells and reveal a sequence of signaling events required for cell cycle progression in proliferating NIH 3T3 cells.