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Cell Cycle: Synchronization
at Various Stages
Sharon Tate, Hutchison/MRC Research Centre, Cambridge, UK
Introductory article
Article Contents
. Overview of the Eukaryotic Cell Cycle
. Synchronization Methods
. Future Directions
Paul Ko Ferrigno, Hutchison/MRC Research Centre, Cambridge, UK
. Conclusions
Progression through, and exit from, the cell cycle are tightly coupled processes that are key
to the development and health of multicellular organisms. Synchronization is a technique
commonly used to enrich cell populations at a single cell-cycle stage, enabling the study of
cell-cycle-specific regulation.
Overview of the Eukaryotic Cell Cycle
The four phases of the eukaryotic cell cycle
The eukaryotic cell cycle can be divided into four distinct
phases, G1, S, G2 and M (Figure 1). G1 and G2 are growth
phases, preparing the cells for the subsequent phases of
DNA synthesis (S phase) and division (mitotic or M phase)
of the cell cycle. It is imperative for the survival of the
doi: 10.1038/npg.els.0002570
daughter cells produced by division that each phase of the
cell cycle occurs in the correct sequence, is completed before
the next begins, and that each phase is faithfully carried out.
Crucially, three events that must be fully completed in each
cell cycle are: (1) duplication of all the genomic DNA, once
and only once; (2) faithful segregation of one complete set
of chromosomes to each daughter cell; (3) separation of
daughter cells with equal distribution of their organelles.
G0
M
G1
Restriction
point
G2
S
(a)
Mitosis
Prophase
Metaphase
Anaphase
Telophase
Cytokinesis
(b)
Figure 1 (a) Cells in G1 may decide to exit the cell cycle to enter a quiescent G0 phase or pass the restriction point, progressing through the cell cycle. Cells
duplicate their DNA before entering mitosis. (b) Cells in mitosis undergo a series of distinct morphological changes. In prophase, chromatin condenses
forming visible chromosomes, centrosomes begin to form the mitotic spindle. During metaphase, chromosomes attach to each pole of the mitotic spindle
by connecting their kinetochores to microtubules. In anaphase, the spindle poles begin to move apart separating the chromosomes. Chromosome
separation is completed in telophase and cells are separated into two daughters by cytokinesis.
ENCYCLOPEDIA OF LIFE SCIENCES & 2005, John Wiley & Sons, Ltd. www.els.net
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Cell Cycle: Synchronization at Various Stages
Master controllers of the cell cycle
Ordered progression through the cell cycle is controlled by
temporal and sequential production and degradation of
cyclins. Cyclins activate cyclin-dependent kinases (CDKs).
CDKs regulate cell cycle events by phosphorylating and
activating or deactivating effector proteins.
Cell-cycle checkpoints
Molecular mechanisms known as checkpoints are in place
throughout the cell cycle, checking that events are taking
place with the utmost accuracy, maintaining the integrity
of the cell. Many synchronization methods work by activating cell-cycle checkpoints to arrest cell-cycle progression. The most studied checkpoints regulate G1/S and
G2/M, preventing entry into these phases until conditions
are optimal, but mechanisms are in place throughout the
cycle to arrest cells should problems arise. For example, the
spindle checkpoint monitors the alignment of chromosomes on the mitotic spindle prior to their separation into
the two daughter cells, and can detect if any chromosome
kinetochores are not attached to the spindle. Should this
happen, a signal feeds back into the mitotic machinery,
arresting mitosis until all kinetochores are attached and
chromosomes aligned.
Synchronizing cells
Cell populations divide asynchronously. Thus, at any
given moment, a dish of tissue culture cells will comprise
individual cells that are in each phase of the cell cycle.
Current technologies do not allow us to perform biochemical analysis on individual cells, so the study of specific
phases or transient events in the cell cycle requires that we
enrich the population in cells transiting a specific phase.
This can be achieved by synchronizing cells at a particular
point in the cell cycle, so that the population cycles in phase
when released from the block.
Deciding when to induce a cell-cycle arrest is probably
the biggest consideration for researchers. A number of
considerations when deciding on how cells will be synchronized are: (1) the number of cells required for the
experimental investigation; (2) the phase in which cells are
to be synchronized; (3) whether to use a chemical or an
alternative method.
The question of which phase to synchronize in is usually
dictated by the experiment. As a general rule it is best to
synchronize in a phase before the one you are interested in
studying, as cells do not maintain a high degree of synchrony over several rounds of division. If a large number of
cells are required chemical synchronization is probably the
most desirable method to use. Chemicals are often inexpensive and work quickly. However, chemicals may have
more than one target in the cell not all of which are known
and they can also be disruptive to the point of inducing
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apoptosis. Such disadvantages are not encountered by
nonchemical methods, but these may require expensive
equipment such as a flow cytometer.
Synchronization Methods
Synchronization methods fall into two categories: physical
methods, such as mitotic shake off or flow cytometry; and
chemical methods, where drug-like inhibitors prevent cells
from progressing beyond a given point in the cycle. Flow
cytometry is a general method that can be used to sort cells
into populations based on size and deoxyribonucleic acid
(DNA) content. It is thus possible, for example, to obtain
large cells with 1C DNA that correspond to a G1 population, and distinguish them from large cells with 2C DNA
(post-S and, pre-M phase) and small cells with 1C DNA
(post-M phase, early G1 cells). Given large populations of
cells, it is possible to obtain relatively pure populations. By
contrast, chemical synchronization methods work by reversibly arresting the cell cycle at a certain point. Cells not
yet at this point in the cell cycle will continue to cycle until
they reach the arrest point and then stop.
Synchronizing cells in G0/G1
Nonchemical methods for synchronizing cells in G0/G1
include serum and amino acid starvation. Serum starvation
was one of the earliest methods used. Cells in culture, that
are isolated from their natural environment, depend upon a
rich supply of growth factors for their survival and proliferation. These growth factors are generally supplied in the
form of blood serum drawn from pregnant cattle. Similarly,
cell growth requires an ample supply of amino acids in the
growth medium. Withdrawal of serum or amino acids
from cells will thus lead them to withdraw from the cell cycle
(see Figure 1). Restoring complete medium should then
allow cells to reenter the cell cycle in synchrony. Such nonchemical methods are apparently straightforward. However, long periods of withdrawal of serum or amino acids
can reduce synchrony in a population when cells are released. In addition, some cell types do not respond to
serum starvation, and transformed cells in particular may
respond to amino acid starvation by death or partial differentiation.
Chemical inhibitors (Table 1) offer greater confidence
that cells will arrest uniformly, and knowledge of the molecular target of the chemical ensures that the experimenter
does not affect the outcome of the experiment by using a
treatment that affects the process he or she is trying to
study.
Lovastatin
Lovastatin arrests cells in G0/G1 by inhibiting 3-hydroxy3-methyl glutaryl coenzyme A (HMG-CoA) reductase and
Cell Cycle: Synchronization at Various Stages
Table 1 Summary of cell-cycle inhibitors most commonly used, the cell-cycle phase they inhibit, mechanism of inhibition and
working concentrations found in the literature
Inhibitor
Cell-cycle phase
Mode of action
Working concentration
Lovastatin
Mimosine
Tunicamycin
Thymidine
Hydroxyurea
Aphidicolin
Nocodazole
Colchicine
Colcimid
Taxol
Vinblastine
Roscovitine
Merbarone
VM-26
G1
G1/S
G1
S
S
S
G2/M
G2/M
G2/M
G2/M
M
HMG-CoA reductase
Ribonuclease reductase
Inhibits glucosylation
dNTP synthesis
Ribonucleoside reductase
DNA polymerase a
Destabilizes microtubules
Destabilizes microtubules
Destabilizes microtubules
Stabilizes microtubules
Microtubule aggregation
CDKs
Topoisomerase II
Topoisomerase II
10–40 mM
100–1200 mM
0.5–6 mg mL21
2 mM
1 mM
1 mg mL21
10–400 ng mL21
100 nM
0.3 mg mL21
1 nm ! 10 nM
1.6 nm
16 mM
5 mM
1–10 mM
G2
G2
therefore cholesterol synthesis. Lovastatin treatment has
been shown to decrease CDK2 activity, by causing the release of the CDK2 inhibitors p21 and p27 from CDK4–
cyclinD complex, inhibiting entry into S phase. Alternatively, low concentrations of lovastatin can induce a G2/M
arrest. A further caveat is that sustained incubation with
lovastatin can induce apoptosis.
Synchronizing cells in S phase
Inhibitors of S phase commonly act by reducing the
cellular pool of deoxynucleotide triphosphate (dNTP)s or
their incorporation into growing DNA strands.
Thymidine block
A procedure known as ‘thymidine block’ is the oldest
method used for synchronizing cells in S phase. Thymidine
acts primarily through inhibiting conversion of cytidine-5’diphosphate into deoxycytidine-5’-diphosphate. Growth
inhibition by the addition of excess thymidine to the culture medium can also involve feedback regulation of several other pyrimidine biosynthetic enzymes. The caveat
here is that excessive levels of thymidine have been shown
to cause chromosomal damage in some cell types.
A double thymidine block, where cells are arrested in S
phase by thymidine and then released for a short period
before reintroducing thymidine is often used alone or in
conjunction with other cell-cycle inhibitors to increase the
degree of synchrony in a population of cells.
Mimosine
Mimosine is a rare plant amino acid derived from Mimosa,
which arrests cells in late G1 or G1/S phase and there is still
some debate over the precise arrest point. Mimosine is
thought to inhibit ribonucleotide reductase by chelating
metal ions required by the enzyme. Mimosine is a less potent inhibitor than other chemicals, and if levels are not
sufficiently high, cells can still progress through S phase
albeit at a slower rate.
Hydroxyurea
Hydroxyurea inactivates ribonucleotide reductase by
forming a nitroxide-free radical that binds and inhibits
the enzyme active site, thus preventing conversion of ribonucleotides into deoxyribonucleotides.
Hydroxyurea treatment can also lead to the formation of
hydrogen peroxide and nitric oxide, which can cause sitespecific DNA damage, stalling DNA replication forks, and
thus S phase through the activation of a DNA-damage
checkpoint. This means that cells treated with hydroxyurea
will arrest in S phase, but may consist of a mixed population of cells with different histories. Other potential
drawbacks of using hydroxyurea is that low concentrations
give rise to only partial synchrony and high concentrations
have been shown to be toxic to S phase cells.
Aphidicolin
Aphidicolin is potentially a more efficient inhibitor of S
phase than hydroxyurea and it has not been shown to be
toxic to cells. It acts by preventing DNA polymerase a from
being able to bind dNTPs, without blocking the activity of
DNA polymerases b and g. Aphidicolin does not have an
effect on the levels of dNTPs. In fact, dNTP levels continue
to increase in the presence of aphidicolin, so that once the
block is released DNA synthesis resumes immediately.
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Cell Cycle: Synchronization at Various Stages
Synchronizing cells in G2/M
Mitotic cells can be enriched by mitotic shake off, which
simply involves shaking the tissue culture dish so that the
rounded mitotic cells, which have reduced cell contacts
with the culture dish, float off into the medium. A caveat
here is that apoptotic cells also round up and float off, and
will severely complicate experimental analysis if they are
not removed.
The mitotic spindle is the defining feature of the mitotic
cell, and is appropriately enough a key target for chemical
inhibitors.
Colchicine, colcemid and nocodazole
Drugs that depolymerize mitotic spindles include colchicine, colcemid and nocodazole. Colchicine and colcemid are natural compounds isolated from plants, while
nocodazole is a synthetic variant. All three compounds
arrest cells in mitosis. However, if cells are arrested for
extended periods, abnormalities are seen upon release:
some cells return straight to interphase, while others form
multiple spindles or enter apoptosis without dividing their
chromosomes. Nocodazole is experimentally the most
practical of the three reagents, being the most readily reversible and causing the least side effects. Colchicine is the
most detrimental agent, preventing cells from producing a
functional spindle upon release, while release from colcemid allows far fewer cells to form functional spindles, with
very slow kinetics compared to nocodazole.
Taxol
Taxol (used in cancer chemotherapy as Paclitaxel) is a
natural compound that binds microtubules, but not unpolymerized tubulin dimers (Figure 2), at a site distinct from
other microtubule targeting drugs. Treatment with taxol
stabilizes microtubules, suppressing dynamic changes such
as growing and shortening of the microtubules.
Taxol along with many other cell cycle drugs, has been
used at a wide range of concentrations in the literature
(1 nM!100 mM; see Table 1). This in turn has led to a broad
spectrum of often contradictory cellular and molecular effects observed in treated cells. Clinically relevant concentrations (5 to 200 nM) are considered to be most
appropriate for arresting cells in mitosis and inducing
apoptosis.
Future Directions
Chemical inhibitors have allowed great progress in our
understanding of the molecular biology of the cell cycle.
However, these drugs have limitations, the most important
being that they are blunt tools that may arrest cells in a
particular phase of the cell cycle, but their multiple effects
often prevent a clean interpretation of the data.
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Figure 2 a- and b-tubulin are the basic subunits of microtubules.
a-tubulin is bound by stable guanosine triphosphate (GTP), b-tubulin by
unstable GTP, which can be hydrolysed to GDP. a- and b-tubulin
monomers form a stable heterodimer. Heterodimers make longitudinal
contacts forming a protofilament. Thirteen protofilaments make lateral
contacts generating a microtubule cylinder. If the plus (+) end of the
microtubule is GTP bound (on the b-tubulin subunits), additional tubulin
dimers can be added, once a dimer is incorporated the GTP on b-tubulin is
hydrolysed to GDP. If GTP bound tubulin dimer concentration is low, GTP
in the microtubule is hydrolysed. GDP microtubule caps are unstable
leading to depolymerization of the microtubule, microtubules peel apart to
release tubulin subunits.
Methods to synchronize cells in a more specific manner,
by specifically targeting a single molecule would be greatly
advantageous. One technology that may allow us to
achieve this is ribonucleic acid interference (RNAi). Messenger RNA transcribed from a specific gene is targeted by
short interfering oligonucleotides for degradation, preventing its translation into protein. Using RNAi to target
individual cell-cycle regulators or effectors may allow us to
begin to dissect the cell cycle with a fine scalpel. For RNAi
technology to be employed would involve targeting gene
products unambiguously involved in moving the cell from
one phase to another. The greatest limitation to using
RNAi technology is that stable protein products that could
persist from one cell cycle to the next would remain fully
functional in the cell. Thus, proteins that are degraded
during each cell cycle would be good targets to choose.
Cyclins A and B, and other substrates of the anaphase
Cell Cycle: Synchronization at Various Stages
promoting complex/cyclosome (APC/C) or the cullins are
obvious targets as they are synthesized and degraded once
per cell cycle and are involved in driving the cell through its
different stages.
Generating chemical inhibitors that specifically target
and inhibit certain cyclin–CDK interactions may lead to a
more clear-cut synchronization being accomplished. The
cell division cycle (CDC)2 inhibitor Roscovitine showed
early promise, but it has been shown also to inhibit CDK2
and CDK5, and so lacks the required specificity to arrest
cells in a specific phase. Cyclins and CDKs present difficulties as targets for inhibition due to the high degree of
structural and functional homology, but CDK inhibitors
are being actively sought in the biotech industry.
Conclusions
Synchronization is a technique commonly used to enrich
cell populations at a single stage in the cell cycle, thus
facilitating the study of cell cycle specific regulation. Cells
respond to chemical treatment by signalling to cell-cycle
checkpoints, which arrest cells, preventing further progression. The biggest drawbacks to all the methods described, but in particular chemical ones, are lack of
specificity and toxicity to the cell. Many chemicals have
been shown to arrest cells in multiple phases. These problems can usually be overcome by optimizing the concentration and duration of the cell-cycle block for each cell
type used. Generally, low concentration coupled with short
incubation allows cells to arrest without being compromised. As with every technique in experimental biology,
it is crucial to include appropriate controls to assess the
effect synchronization may be having on the experimental
readout.
Further Reading
Blagosklonny MV and Fojo T (1999) Molecular effects of paclitaxel:
myths and reality (a critical review). International Journal of Cancer 83:
151–156.
Hammond EM, Green SL and Giaccia AJ (2003) Comparison of hypoxia-induced replication arrest with hydroxyurea and aphidicolininduced arrest. Mutation Research 532: 205–213.
Jordan MA, Thrower D and Wilson L (1992) Effects of vinblastine,
podophyllotoxin and nocodazole on mitotic spindles: implications for
the role of microtubule dynamics in mitosis. Journal of Cell Science
102: 401–416.
Kufe DW, Egan EM, Rosowsky A, Ensminger W and Frei E (1980)
Thymidine arrest and synchrony of cellular growth in vivo. Cancer
Treatment Reports 64: 1307–1317.
Lodish H, Baltimore D, Berk A et al. (1995) Molecular Cell Biology, 3rd
edn. New York: Scientific American Books.
Martinez-Botas J, Ferruelo AJ, Suarez Y et al. (2001) Dose-dependent
effects of lovastatin on cell cycle progression. Distinct requirement of
cholesterol and non-sterol mevalonate derivatives. Biochimica et
Biophysica Acta 1532: 185–194.
Murray A and Hunt T (1993) The Cell Cycle: an Introduction, 1st edn.
Oxford: Oxford University Press.
Orr GA, Verdier-Pinard P, McDaid H and Horwitz SB (2003)
Mechanisms of taxol resistance related to microtubules. Oncogene
22: 7280–7295.
Pedrali-Noy G, Spadari S, Miller-Faures A et al. (1980) Synchronization
of HeLa cell cultures by inhibition of DNA polymerase alpha with
aphidicolin. Nucleic Acids Research 25: 377–387.
Zieve GW, Turnbull D, Mullins M and McIntosh R (1980) Production
of large numbers of mitotic mammalian cells by use of the reversible
microtubule inhibitor nococdazole. Experimental Cell Research 126:
397–405.
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