Figure 3.

REF-52 cells are inhibited from entering S phase and do not proliferate following tetraploidization induced by exposure to DCB. (A) REF-52 cells, released from mitotic synchronization into DCB for 5 h, and then released into drug-free medium for 19 h, did not enter S phase by 24 h after mitosis, as determined by BrdU incorporation measured by two-dimensional flow cytometry. By contrast, controls released into drug-free medium for 24 h following mitotic synchronization reentered S phase and incorporated BrdU. (B) REF-52 cells made tetraploid by 5 h treatment with DCB following mitotic synchronization showed >95% inhibition of S phase entry through 67 h following release from DCB, relative to controls, measured 24 h following release from mitotic synchronization. Inhibition of S phase was quantitated for 10,000 cells each from two-dimensional FACS histograms to obtain the number of cells incorporating BrdU. (C) Counts of cell number show that REF-52 cells did not proliferate following tetraploidization induced by treatment with DCB. Cells were sychronized in mitosis, selectively detached, and then released from mitotic synchronization into me dium containing 10 μM DCB for 5 h. Cells were then fixed (0 time) or were released in drug-free medium for up to 96 h. Cells were fixed at the time points indicated and cell numbers counted using a hemacytometer. By contrast, mitotically synchronized cells replated in drug-free medium resumed proliferation. Each point is an average of eight counts, and error bars represent SDs.