We have discovered and report here a means of separating a mitotic "subcycle" from the Gj-and S-phase events of the mammalian cell cycle. Time-lapse videomicroscopy of Syrian hamster fibroblast (BHK) cells revealed that caffeine could induce multiple entries into mitosis while cells were blocked in DNA synthesis. As with normal mitoses, the abundance of mitosis-specific phosphoproteins was coupled with the condensation of chromatin. The BHK temperaturesensitive mutant tsBN2 also completed multiple entries into mitosis while arrested during DNA replication and raised to the restrictive temperature. Periodic mitotic events occurred even when BHK cells were exposed to low concentrations of serum or cycloheximide, conditions that prevent the cycling of BHK cells by blocking their entry into S phase. These results suggest that an oscillation governing the activation and inactivation of mitotic factors can be generated in mammalian cells and uncoupled from the G, and DNA replication events of the normal cell cycle. This system will be useful for examining the molecular nature of mitotic factors.The mammalian cell cycle is classically viewed as an interdependent sequence of biochemical events. The completion of DNA replication has therefore been considered a prerequisite for the initiation of mitosis (1). This view was supported by the studies of Rao and Johnson (2), which showed that mitosis was delayed in G2-phase cells that were fused with S-phase cells. Both nuclei underwent mitosis simultaneously, but only after the S-phase nucleus had completed DNA replication. Two examples of alterations in the temporal sequence of cell cycle events have been reported in mammalian cells. Chromosome condensation, nuclear envelope breakdown, and other mitotic events occur at the restrictive temperature in the BHK temperature-sensitive mutant tsBN2 (3) and in normal BHK cells that have been arrested in S phase and exposed to caffeine (4, 5). These studies showed that one final mitosis can be uncoupled from the completion of DNA replication. Both exposure to caffeine and suppression of DNA replication are required for this premature induction of mitosis in normal BHK cells (4). Experiments with inhibitors suggest that these conditions permit the accumulation of mitosis-inducing mRNA(s) and their protein(s) (5). The present report extends these observations by providing evidence that mitotic events can be made to occur periodically, independent of Gj-and S-phase events. We propose that an oscillator controlling the entry into and exit from mitosis can be uncoupled from the normal cell cycle and examine its regulation during growth-inhibiting conditions.
MATERIALS AND METHODSCell Culture and Synchronization. Syrian Hamster fibroblast (BHK) cells were grown and synchronized in early S phase as described (4). Briefly, cells were plated at 1 X 105 cells per 60-mm dish and grown at 370C in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum for 24 hr. Synchrony in early S phase was achieved by incubation in isoleuc...