Heterogeneity in the HeLa Cell Cycle Response to UVC Analyzed by the BrdUrd Two-Parameter Method

Using pulse, pulse-chase, and continuous bromodeoxyuridine (BrdUrd) labeling schedules, the effects of 11 Jm -2 of UVC on cell cycling of HeLa cultures were analyzed. The fine resolution of the bivariate DNA distribution method allowed precise quantitation of the grossly perturbed cell distribution with >90% of cells accumulated in S phase 14-17 h postirradiation. By pulse-chase, it was determined that the transit of cells through S was extended more than fourfold. Cells irradiated in G 2 + M were slowed threefold, while the G 1 compartment was fully emptied in only 2-3 h longer than the normal duration. Egress of cells from G 1 immediately post-UVC was slowed for the first 4 h, but thereafter emptying occurred at the normal speed. These G 1 → S cells incorporated BrdUrd at the control rate and were seen as a "crest" on the bivariate dot plot, moving with much greater speed through S than did cells irradiated in S, thus producing a heterogeneous population of cells engaged in repair and semiconservative replication at different rates. UVC irradiation inhibited late S phase BrdUrd incorporation more than early S, but hydroxyuren only inhibited the incorporation into the crest of G 1 → S cells. Several novel features of the control, sham-irradiated cultures were elucidated. G 1 phase cell transit was a linear function with time. S phase cell transit was not uniform, mainly due to cells accumulating to nearly twice the expected frequency at the beginning of the first quarter of S, due to slower rates of DNA synthesis. The heterogeneity revealed in these studies influenced the response to UVC in all phases of the cell cycle.