A MULTIPARAMETER APPROACH TO CELL CYCLE ANALYSIS AS A STANDARD TOOL IN ONCOLOGY DRUG DISCOVERY - Flow Cytometry in Drug Discovery and Development - page 100

on other events happening at the sampling time. Some examples include cyclin-
dependent proteins and associated phosphoproteins.
Several analytical packages are available to do cell cycle analysis [(Flowjo (Tree
Star) (Tree Star), Modfit (Verity), Multicycle (Phoenix Flow Systems)], all
of which use more or less modified versions of the Watson or Dean Jett Fox
algorithms [9, 10]. Figure 5.4 is a Flowjo analytical example of one algorithm that
demonstrates the importance of excluding the clumps defined previously, which
otherwise will distort the data.
The results presented in this chapter will be derived using Flowjo package. We are
not going to imply that the different algorithms may impact analysis of data except to
say that, as with any algorithm, they are based on some assumptions. For example,
while the Watson pragmatic is expected to have an overestimation of the S phase, the
Dean Jett Fox has an underestimation of the S phase (Figure 5.5). Nevertheless, they
both give us tools that only require us to standardize the assays where they need to be
applied and chose one over the other, almost as any arbitrary measurement tool would
require. Regardless of the choice, it is important to stay with the same algorithm
throughout the whole experiment or series of experiments to standardize the system.
5.2.3.1 AStandardCell Cycle Profile As previously described, a cell cycle profile
is a histogram representation of the distribution of DNA content of individual cells in a
growing cell population. Figure 5.1 shows that three major subpopulations can be
found in a standard DNAprofile consisting of two peaks and a valley, as determined by
their DNA content. The first peak represents cells at rest (G 0 phase) and/or cells
preparing to engage into division (G 1 phase), both of which have the same DNA
content (2N, where N represents the haploid chromosome number). The second
population (the valley, S phase) represents cells that have engaged in DNA replication
and their DNA content is higher than the G 0 /G 1 peak (higher than 2N and lower than
4N). And the third population includes cells that have doubled the DNA content but
not yet engaged in mitosis (4N,G 2 phase) and the cells already engaged in the
preliminary steps of mitosis (4N, M phase).
This overall profile also shows some trailing cells at lower DNAcontent thanG 0 /G 1
cells (sub-G 1 ). The sub-G 1 population contains those cells that had less DNA in the
process of apoptosis or are vesicles containing some DNA resulting from a necrotic
event. This profile also shows the presence of cells with higher DNA content than the
G 2 /M peak that identifies a cell population with higher content of DNA per cell than
tetraploid cells.
5.2.3.2 Beyond the Standard Profile The standard profile, as defined above, is
subject to pronounced variability. This variability can be attributed to the doubling
times of specific cell populations where cells that have shorter doubling times (faster
division rate) will have a profile different from that of cells with longer doubling times
(slower division rates), as shown in Figure 5.3. However, alterations of a specific
phase traverse time can also account for this kind of variability.
Another important feature of this distribution is that there is a direct correlation
between the frequency distribution of the cell populations at each phase of the cell