Elongation of G1 phase by transient exposure of rat 3Y1 fibroblasts to caffeine during the previous and present generations.

When density-arrested rat 3Y1 fibroblasts were stimulated to enter S phase by seeding sparsely in fresh medium, caffeine inhibited this stimulation. When compared at the doses that gave the same levels of the inhibition of entry into S phase, caffeine inhibited protein synthesis to a far lesser extent than cycloheximide. This indicates that caffeine affects some event(s) specific to entry into S phase rather than general protein synthesis. When cells synchronized at early S phase were exposed to caffeine, progression of S and G2 phases was prolonged by only 1 h (from 6 h to 7 h). However, after removal of caffeine at mitosis, the G1 phase was prolonged for 5 h (from 11 h to 16 h). These results are consistent with our model that the initiation of S phase is regulated throughout the period between the adjacent S phases. When cells were incubated with normal medium containing serum during the S and G2 periods, a subsequent 6-h pulse exposure to caffeine caused prolongation of G1 phase for 7 h (from 11 h to 18 h). On the other hand, when cells were incubated in the absence of serum during these periods, the prolongation was only 2 h (from 16 h to 18 h). Similarly, entry into S phase was prolonged only 2 h, when a 6-h pulse exposure to caffeine was given immediately after release from density arrest or serum-deprivation arrest. These results indicate the involvement of the relaxation process, which is not affected by caffeine, when serum-deprived cells or density-arrested cells restore the process prerequisite for entry into S phase.