How the Change from FLM to FACS Affected Our Understanding of the G1-Phase of the Cell Cycle

Abstract

The frequency of labeled mitoses (FLM) method for analyzing cell-cycle phases necessitates a determination of cell-cycle interdivision times and the absolute lengths of the cell-cycle phases. The change to flow sorting (FACS) analysis, a simpler, less labor intensive, and more rapid method, eliminated determinations of absolute phase times, yielding only percents of cells exhibiting particular DNA contents. Without an interdivision time value, conversion of these fractions into absolute phase lengths is not possible. This change in methodology has led to an alteration in how the cell cycle is viewed. The FLM method allowed the conclusion that G 1 -phase variability resulted from constancy of S and G 2 phase lengths. In contrast, with FACS analysis, slow growing cells exhibiting a large fraction of cells with a G 1 -phase amount of DNA appeared to be "arrested in G 1 phase". The loss of absolute phase length determinations has therefore led to the proposals of G 1 -phase arrest, G 1 -phase controls, restriction points, and G 0 phase. It is suggested that these G 1 -phase controls and phenomena require a critical reevaluation in the light of an alternative cell-cycle model that does not require or postulate such G 1 -phase controls.After the original identification of cell-cycle phases by Howard and Pelc in the early 1950s, the next two decades brought forth a large number of measurements of cell-cycle phase lengths using the method of frequency of labeled mitoses (FLM). FLM is essentially the method use by Howard and Pelc. 1,2 The FLM method is illustrated in Figure 1. Because this method is not now in common use, it is of interest to explain the method and the type of results obtained by this method. For FLM, growing cells are pulse-labeled with tritiated thymidine. After removal of label, growing cells are sampled at intervals and subjected to autoradiography. The fraction of mitotic cells that are radioactive is determined. As shown in Figure 1, there is an initial period with no labeled mitoses. This period gives the length, in absolute time, of the G 2 phase. Then there is a rise in labeled mitoses, a plateau, and a fall in labeled mitoses. The time interval of these labeled mitoses indicates the S-phase length. The G 1 phase length is determined from the G 1 + G 2 period-the period between the decrease in labeled mitoses and the subsequent increase in labeled mitoses-by subtracting the initially determined G 2 phase time. The total interdivision time (IDT) is determined by the time between the rise times of labeled mitoses.The major generalization to come out of a large number of FLM analyses in the

Control and maintenance of mammalian cell size

“…The model of the cell cycle presented here explains many experimental results without postulating checkpoints, G1-phase events, restriction points, or similar phenomena. Experimental support for these ideas [28,29] and the application of these ideas to other problems of cell growth and differentiation [3,13-15,26,28-35] have been published. These ideas have also been reviewed [36-39].…”

Experimental reconsideration of the utility of serum starvation as a method for synchronizing mammalian cells

“…The change in cell-cycle analysis from methods that give absolute times of the phases (such as with frequency of labeled mitoses (FLM)) to methods that merely determine fractions of cells with different DNA contents (FACS) has led to the loss measurements of the time that cells are in different phases (Cooper, 2003a). We postulate that when cells are placed in low concentrations of serum the cells are not totally arrested in growth but grow slowly with a much longer interdivision time.…”

Microarray analysis of gene expression during the cell cycle

“…Whole-culture methods (also called "batch" methods or "forcing" methods) are those that take an entire culture of growing cells, and produce a synchronized culture from all cells. The use of whole-culture methods for synchronization has been challenged on theoretical [2-7] and experimental grounds [8-11]. In summary, it is proposed that whole-culture methods cannot synchronize cells.…”

“…This alternative view of the cell cycle takes issue with such well-accepted ideas as the existence of a G0 phase, G1-phase arrest points, the restriction point, G1-phase specifically expressed genes, and related aspects of cell-cycle progression [2-4,6,7,9,13-21]. Most important for the analysis of synchronization experiments, this alternative view takes issue with the ability of whole-culture methods to synchronize cells.…”

“…Despite hundreds or even thousands of papers that use whole-culture methods to synchronize cells, available evidence [8-11] and theoretical considerations [2-7] indicate that these methods cannot synchronize cells. We will point out where whole-culture synchronization has been used.…”