Method for kinetic analysis of drug‐induced cell cycle perturbations

Abstract

A method is described for quantitative study of the flux of cells through the cell cycle phases in in vitro systems perturbed by chemicals, such as chemotherapeutic agents. The method utilizes cell count and the flow cytometric technique of bromodeoxyuridine (BrdUrd) labeling, according to an optimized strategy. Cells are exposed to BrdUrd during the last minutes of drug treatment and fixed for analysis at 0,1/3T,, 2/3T,, T,, and T, + TG1 recovery times, where T,, TG1, T, are the mean durations of phases S and G1 and of the whole cycle of control cells. As an example of application of the proposed procedure, a kinetic study of the effect of l-(2-chloroethyl)-l-nitrosourea (CNU) on the L1210 cell cycle is described. Simple data analysis, requiring only a pocket calculator, showed that cells in phases G1 and G2M at the end of a 1 h treatment with 1 pg/ml CNU were fully able to leave these phases but were destined to remain blocked in the following G2M phase (G1 for a minority of them). We also found that cells initially in S phase were slightly delayed in completing their S phase and that 50% of them remained temporarily blocked in the subsequent G2M phase, irrespective of their position in the S phase.

Measuring the complexity of cell cycle arrest and killing of drugs: Kinetics of phase-specific effects induced by Taxol

“…Because BrdUrd replaces thymidine during DNA synthesis, BrdUrd pulse labeling at the time of treatment catches S-phase cells at that stage (13,14). Analysis 7 h after treatment detects their movement through the S-phase and the outflow of unlabeled G 1 and G 2 M cells.…”

Kinetic Heterogeneity of an Experimental Tumour Revealed by BrdUrd Incorporation and Mathematical Modelling

“…For the flow cytometric analysis according to the DNABrdUrd method, the fixed cells were stained with propidium iodide (PI) and antiBrdUrd fluoresceinated antibody, as described before (Ubezio et al, 1991). Briefly, each sample of ethanol-fixed cell suspension was centrifuged and incubated with 3N HCl for 20 min, to obtain partially denatured DNA.…”

Cell cycle analysis by flow cytometry: Principles and applications

“…Shorter incubations with BrdUrd permit to follow cellular evolution by using the anti-BrdUrd antibody method: progression in the cycle of labelled and unlabelled cells allows to identify the proliferative fraction, to estimate cycle phase durations, particularly S, and to appreciate cellular dispersion after BrdUrd incorporation. Analysis can be done with synchronous or asynchronous populations, in vitro or in vivo [129,155].…”