HETEROGENEITY IN THE HELA-CELL CYCLE RESPONSE TO UVC ANALYZED BY THE BRDURD 2-PARAMETER METHOD

Using pulse, pulse-chase, and continuous bromodeoxyuridine (BrdUrd) la beling schedules, the effects of 11 Jm(-2) of UVC on cell cycling of H eLa cultures were analyzed. The fine resolution of the bivariate DNA d istribution method allowed precise quantitation of the grossly perturb ed cell distribution with >90% of cells accumulated in S phase 14-17 h postirradiation. By pulse-chase, it was determined that the transit o f 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 ther eafter emptying occurred at the normal speed. These G(1) --> S cells i ncorporated 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 differe nt rates. UVC irradiation inhibited late S phase BrdUrd incorporation more than early S, but hydroxyurea only inhibited the incorporation in to the crest of G(1) --> S cells. Several novel features of the contro l, sham-irradiated cultures were elucidated. G(1) phase cell transit w as a linear function with time. S phase cell transit was not uniform, mainly due to cells accumulating to nearly twice the expected frequenc y at the beginning of the first quarter of S, due to slower rates of D NA synthesis. The heterogeneity revealed in these studies influenced t he response to UVC in all phases of the cell cycle. (C) 1994 Academic Press,Inc.