Biomedical Engineering Reference
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progression of a growing cell population. We are going to only mention here two
examples of biomarkers as main examples. The first example is the addition of
analogues of thymidine, such as the derivatives BrdU or EdU [11, 12]. Incorporation
of these nucleotides into DNA of cells can be readily detected by FACS using other
fluorophore-associated probes. The second example is the evaluation of cyclins,
because these proteins participate in the movements of cells along the cycle either in
their native forms or as cascades of phosphorylation-dependent events in a series or
interactions through regulatory proteins [13, 14].
Identification of Phase Traverse Times: The BrdU/EdU Approach When analyzing
cell cycle phase traverse, specific time markers that become incorporated into the
DNA during duplication are required. The sole tool used for many years to do this
evaluation by flow cytometry has been BrdU, a modified analogue of thymidine
(bromodeoxyuridine). BrdU is incorporated in cellular DNA and only cells that have
entered S phase or passed through S phase will be susceptible to being stained with an
anti-BrdU-specific fluorescent antibody (see Figure 5.7a-c). Only recently has EdU
become another analytical tool where the uridine has a different modification [15].
This modification allows the staining of cells that incorporated this marker using a
milder technique that avoids the harsh steps required by BrdU. Briefly, we have two
methodologies that work in a comparable way to identify DNA synthesis. Both use
analogues of Uridine that are incorporated into the DNAwith the same efficiency but,
the BrdUmethod requires DNAdenaturation in order to expose the BrdU for detection
by anti-BrdU antibodies, whereas, with the EdU method, direct staining is possible
using a diffusible azide-fluorophore conjugate which binds EdU in the DNAwithout
denaturation. Several algorithms can be applied to the cells containing these
analogues to determine the number of cells that have passed through S phase and
calculate the traverse times from the beginning of DNA synthesis to the completion of
a duplication cycle [16].
Identification of Phase Driver Molecules in the Control of Cell Cycle Traverse Times
(Cyclins and Phosphoproteins) Several proteins have been identified that control
the movements of cells across the cell cycle, and these are mainly the cyclins and
cyclin-dependent kinases [17]. For the purpose of this chapter, we are going to name
only a few as characteristic identifiers under special stress conditions when analyzing
compounds interfering with the progression of cell proliferation. Figure 5.8 illustrates
this point. Cells, as control or after camptothecin treatment, were analyzed for cyclin
expression, as previously reported by Traganos et al. [18].
5.3 SPECIFIC ACTIVITIES EVALUATED
With the approaches defined in Section 5.2.3, we now have a set of tools with which to
address specific activities that can be followedwhen compounds interact at the level of
a proliferating cell. Thus, these activities can be used to monitor the impact of those
compounds on proliferation distributions across the cell cycle. The main advantages
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