FLOW CYTOMETRIC BRDURD-PULSE-CHASE STUDY OF HEAT-INDUCED CELL-CYCLE PROGRESSION DELAYS

The flow cytometric, bromodeoxyuridine (BrdUrd)-pulse-chase method was extended by analysing five kinetic parameters to study perturbed cell progression through the cell cycle. The method was used to analyse th e cell-cycle perturbations induced by heat shock. Exponentially growin g, asynchronous Chinese hamster ovary (CHO) cells were pulse labelled with BrdUrd and simultaneously heated at 43-degrees-C for 5, 10 or 15 min. The cells were then incubated in a BrdUrd-free medium and, at var ious times thereafter, were prepared for flow cytometry. Five compartm ents (BrdUrd-labelled divided and undivided, and unlabelled G1, G1S, a nd G2) were defined in the resulting dual-parameter histograms. The fr action of cells and the mean DNA content, when appropriate, were calcu lated for each compartment. The rates of cell-cycle progression were a ssessed as time-dependent changes in the fraction of cells in a given compartment and/or the relative DNA content of cells within a given co mpartment. Linear regression analysis of the data revealed two distinc t modes of alteration in cell progression: 1 a delay in cell transit ( either out of or into a given compartment), and 2 a decrease in the ra te of cell transit. Hyperthermia produced a delay in the exit of cells from the G1 compartment of approximately 16 min per minute of heat at 43-degrees-C with no threshold. In contrast, the delay in the exit of cells from all other compartments showed a threshold of from 3 to 5 m in at 43-degrees-C. Above this threshold the delay in exit of cells fr om the BrdUrd-labelled, undivided compartment was approximately 25 min per minute of heat at 43-degrees-C. The more complex dose-response fu nction of this latter compartment may reflect the fact that it include s two cell-cycle phases, S and G2 + M. The decrease in the rate of tra nsit out of G2 for cells heated in G2 was significantly larger than th at for any other compartment, consistent with previous studies, which showed a G2 accumulation following hyperthermia. These results indicat e that heat exposure induces very complex alterations in cell-cycle pr ogression and that this flow cytometric method offers a straightforwar d approach for observing such alterations.