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 the cell-cycle perturbations induced by heat shock. Exponentially growing, 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 various times thereafter, were prepared for flow cytometry. Five compartments (BrdUrd-labelled divided and undivided, and unlabelled G1, G1S, and G2) were defined in the resulting dual-parameter histograms. The fraction of cells and the mean DNA content, when appropriate, were calculated for each compartment. The rates of cell-cycle progression were assessed as time-dependent changes in the fraction of cells in a given compartment and/or the relative DNA content of cells within a given compartment. Linear regression analysis of the data revealed two distinct 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 rate 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 min at 43 degrees C. Above this threshold the delay in exit of cells from the BrdUrd-labelled, undivided compartment was approximately 25 min per minute of heat at 43 degrees C. The more complex dose-response function of this latter compartment may reflect the fact that it includes two cell-cycle phases, S and G2 + M. The decrease in the rate of transit out of G2 for cells heated in G2 was significantly larger than that for any other compartment, consistent with previous studies, which showed a G2 accumulation following hyperthermia. These results indicate that heat exposure induces very complex alterations in cell-cycle progression and that this flow cytometric method offers a straightforward approach for observing such alterations.
Higashikubo
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R
Section of Cancer Biology, Washington University Medical Center, St Louis, MO 63108.
White
R A
RA
Roti Roti
J L
JL
D000818:Animals; D001973:Bromodeoxyuridine; D016466:CHO Cells; D002453:Cell Cycle; D006224:Cricetinae; D004247:DNA / Q000032:analysis; D005434:Flow Cytometry; D006358:Hot Temperature; D066298:In Vitro Techniques
D004247:DNA; D001973:Bromodeoxyuridine