Cyclin D1 production in cycling cells depends on Ras in a cell-cycle-specific manner

BACKGROUND Cellular Ras and cyclin D1 are required at similar times of the cell cycle in quiescent NIH3T3 cells that have been induced to proliferate, but not in the case of cycling NIH3T3 cells. In asynchronous cultures, Ras activity has been found to be required only during G2 phase to promote passage through the entire upcoming cell cycle, whereas cyclin D1 is required through G1 phase until DNA synthesis begins. To explain these results in molecular terms, we propose a model whereby continuous cell cycle progression in NIH3T3 cells requires cellular Ras activity to promote the synthesis of cyclin D1 during G2 phase. Cyclin D1 expression then continues through G1 phase independently of Ras activity, and drives the G1-S phase transition. RESULTS We found high levels of cyclin D1 expression during the G2, M and G1 phases of the cell cycle in cycling NIH3T3 cells, using quantitative fluorescent antibody measurements of individual cells. By microinjecting anti-Ras antibody, we found that the induction of cyclin D1 expression beginning in G2 phase was dependent on Ras activity. Consistent with our model, cyclin D1 expression during G1 phase was particularly stable following neutralization of cellular Ras. Finally, ectopic expression of cyclin D1 largely overcame the requirement for cellular Ras activity during the continuous proliferation of cycling NIH3T3 cells. CONCLUSIONS Ras-dependent induction of cyclin D1 expression beginning in G2 phase is critical for continuous cell cycle progression in NIH3T3 cells.