Cell Cycle Checkpoint Control Protocols

345 Pages·2010·5.295 MB·English

by  Howard B. Lieberman

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Methods to Induce Cell Cycle Checkpoints 3
1
Methods to Induce Cell Cycle Checkpoints
Howard B. Lieberman and Kevin M. Hopkins
1. Introduction
The way cells respond to radiation or chemical exposure that damages
deoxyribonucleic acid (DNA) is important because induced lesions left
unrepaired, or those that are misrepaired, can lead to mutation, cancer, or
lethality. Prokaryotic and eukaryotic cells have evolved mechanisms that repair
damaged DNA directly, such as nucleotide excision repair, base excision repair,
homology-based recombinational repair, or nonhomologous end joining, which
promote survival and reduce potential deleterious effects (1). However, at least
eukaryotic cells also have cell cycle checkpoints capable of sensing DNA dam-
age or blocks in DNA replication, signaling the cell cycle machinery, and
causing transient delays in progression at specific phases of the cell cycle
(2; see ref. 3 for a review). A related but more primitive system may exist in
prokaryotes (4–7). These delays are thought to provide cells with extra time for
mending DNA lesions before entry into critical phases of the cell cycle, such
as S or M, events that could be lethal with damaged DNA.
The precise mechanisms by which checkpoints function are under intensive
investigation, and details of the molecular events involved are being pursued
vigorously. This is owing not only to the complexity and the intellectually and
technically challenging aspects of the process (see ref. 3 for a review) but also
to the relevance of these pathways to the stabilization of the genome and car-
cinogenesis (8). Nevertheless, it is clear that checkpoint mechanisms are very
sensitive and can be induced by the presence of relatively small amounts of
DNA damage. For example, in the yeast Saccharomyces cerevisiae, as little as
a single double-strand break in DNA can cause a delay in cell cycle progres-
sion (9,10). One important aspect of studying cell cycle checkpoint mecha-
nisms is an understanding of how to induce the process.
From: Methods in Molecular Biology, vol. 241: Cell Cycle Checkpoint Control Protocols
Edited by: H. B. Lieberman © Humana Press Inc., Totowa, NJ
3
4 Lieberman and Hopkins
This chapter focuses on the application of radiations, such as gamma rays and
ultraviolet (UV) light, that are capable of causing DNA damage, and thus leading
to the induction of cell cycle checkpoints. Certain chemicals, or the use of tem-
perature-sensitive mutants to disrupt DNA replication, are also used routinely to
induce checkpoints, but related protocols are not described in this chapter.
Gamma rays cause primarily single- and double-strand breaks in DNA but can
infrequently induce nitrogenous base damage as well. In contrast, UV light
(i.e., 254 nm) causes a preponderance of bulky lesions, such as pyrimidine
dimers, although single-base damage and strand breaks are a smaller part of the
array of lesions that can be produced. Regulation of cell cycle checkpoints
induced by ionizing radiation versus UV light is mediated by overlapping but not
identical genetic elements (11–13). Although the protocols described in this
chapter concern the treatment of mammalian cells, the same general principles
can apply to irradiation of yeast and other types of nonmammalian cells as well.
2. Materials
2.1. Supplies
1. Cells: Any mammalian cell type is appropriate for exposure to gamma rays, but
those that can grow attached to a Petri dish surface (glass slide or any other open
surface) as a monolayer, such as fibroblasts, are ideal for UV-related experi-
ments because this nonionizing radiation does not efficiently penetrate medium
or reach one cell “shielded” by another.
2. Growth medium: standard mammalian medium appropriate for the cells of inter-
est (i.e., Dulbecco’s modified Eagle’s medium [DMEM], Roswell Park Memo-
rial Institute-1640 [RPMI-1640], McCoy’s, etc.), available commercially from
several companies: Atlanta Biologicals (Norcross, GA), Invitrogen (Carlsbad,
CA), Mediatech (Herndon, VA), Sigma-Aldrich (St. Louis, MO), Specialty
Media (Phillipsburg, NJ).
3. Sterile phosphate buffered saline (PBS) made up as 0.144 g/L KH PO , 9 gm/L
2 4
NaCl, 0.795 g/L Na HPO ·7H O in distilled H O, pH adjusted to 7.0 and auto-
2 4 2 2
claved, or purchased commercially from Atlanta Biologicals (Norcross, GA)
Invitrogen (Carlsbad, CA), Mediatech (Herndon, VA), Sigma-Aldrich (St. Louis,
MO), Specialty Media (Phillipsburg, NJ).
4. Petri dishes or flasks (see Note 1): Any size and shape Petri dish, multiwell dish,
or flask will be fine, and it should be chosen based on the number of cells needed
to irradiate, as well as any particular requirements posttreatment. A large selec-
tion of tissue-culture ware is available from numerous commercial suppliers
(e.g., BD Falcon (Bedford, MA), Corning (Corning, NY), Nunc [Naperville, IL]).
2.2. Equipment
2.2.1. Source of Ionizing Radiation
Several different types of equipment are used, and various manufacturers
provide the needed sources. The following are some examples:
Methods to Induce Cell Cycle Checkpoints 5
1. X-rays: Siemens Stabilipan (Siemens, Iselin, NJ)
2. Gamma rays: Based on the decay of 60Co, such as a Gammacell 220 (Nordion,
Alberta, Canada) for a high dose rate, or based on the decay of 137Cs, such as a
Gammacell 40 (Nordion, Alberta, Canada) for a lower dose rate (see Notes 2 and 3).
3. Source of UV light: Usually a germicidal bulb is used to produce 254 nm UV
light as an inducer of cell cycle checkpoints (see Fig. 1 and Note 4 for details).
4. Voltage stabilizer: Constant Voltage Transformer, Catalog number 30806 (Sola
Electric, Chicago, IL; see Note 5).
5. UV meter and probe (Model UVX Digital Radiometer, Probe Model UVX-25,
UVP Inc., Upland, CA; see Note 6).
3. Methods
3.1. Preparation of Cells
1. All procedures involving cell culturing should follow standard sterile techniques
and optimum conditions for growth of the specific cells of interest.
2. For cell cycle studies, our laboratory has routinely employed mouse embryonic
stem cells, so their use will serve as an example. Other mammalian cells can
easily be adapted, with modifications, to essentially the same procedures.
3. Cells are grown in DMEM.
4. Add 0.1 mM nonessential amino acids.
5. Add 1 mM sodium pyruvate.
6. Add 10–4 M β-mercaptoethanol.
7. Add 2 mM L-glutamine.
8. Add 15% fetal bovine serum (FBS) ES cell qualified (heat inactivated, 56°C,
30 min).
9. Add 50 µg/mL penicillin.
10. Add 50 µg/mL streptomycin.
11. Add 1000 U/mL Leukemia Inhibitory Factor (LIF).
12. The cells are seeded into 6 well or 10-cm dishes at a concentration of 1 × 105 cells
per mL or 1 × 106 cells per mL, respectively.
13. Cells should be plated and allowed to attach as well as grow for 1 d prior to
irradiation.
14. At this same time, an equal number of cells and dishes should be prepared to
provide conditioned medium for the experimental cells postirradiation.
15. Control cells should be prepared separately from the cells that will be irradiated
if multiwell dishes are being used.
16. Cells are grown in a 37°C incubator with a 5% CO humidified atmosphere.
2
17. At the time of irradiation, cells should be actively growing and in log phase.
18. Cells should not be confluent at the time of irradiation, unless studies on a quies-
cent population are specifically planned.
19. In addition, for UV-light-related experiments, cells should be plated at least
0.25 in. from the perimeter of the Petri dishes because the lip can interfere with
exposure of cells in the vicinity.
6 Lieberman and Hopkins
3.2. Exposure to Ionizing Radiation
1. To expose cells to gamma rays, dishes or flasks are transferred from the 37°C
incubator to the irradiator.
2. The instructions that accompany each machine should then be followed to ensure
accurate and safe operation (see Note 7).
3. When the irradiation is completed, the dishes are removed from the chamber and
transferred back to the 37°C incubator for further incubation (see Note 8).
3.3. Exposure to UV Light (254 nm)
1. The UV light apparatus must be turned on for at least 10 min prior to the irradia-
tion of cells. This will ensure that the UV light is emitted at a stable, constant
dose rate, and the chamber is sterilized.
2. The dose of UV light can be determined by using a radiometer, in conjunction
with the appropriate probe for detecting 254 nm wavelength light. We typically
expose cells at a dose rate of 1.0 J/m2 (see Note 9).
3. Before exposing cells to UV light, the cell growth medium needs to be removed.
This is achieved by either aspiration or pipetting.
4. The cells are then washed twice with sterile PBS to remove residual medium.
The PBS must be completely removed before exposing the cells to UV light.
5. Place the covered dishes in the UV chamber, making sure that the dishes will be
directly underneath the UV bulb. Remove the lids from the dishes, close the
chamber door, then simultaneously fully pull open the shutter and start timing
the exposure.
6. When the appropriate time has been reached, push the shutter to the completely
closed position. Open the chamber door, replace the lids, then remove the dishes
from the chamber.
7. Immediately add conditioned medium to the irradiated cells equivalent to the
amount of medium present prior to irradiation.
8. The dishes should then be returned to their appropriate incubating apparatus.
9. This wavelength of light is carcinogenic and cataractogenic. Therefore, proper
precautions should be taken to avoid investigator exposure (see Note 10).
10. Furthermore, manipulations during and soon after irradiation should be performed
in very dim light or under yellow lights to ensure exposure occurs without the
neutralizing effects of repair by photoreactivation (if potentially active in the cells
being exposed) or photorepair (1). These repair processes usually need intense light
for proper function, so even a dimly lit room should be appropriate for avoiding
unwanted repair by these activities that could reduce a checkpoint inducing DNA
damage signal. Mammalian cells, in general though, have weak photoreactivation
capability. This coupled with the usual presence of other more active repair mecha-
nisms makes this issue, however, essentially not a significant concern.
11. For mammalian cells (or yeast and other microorganisms for that matter) that
must be in liquid culture, resuspend in a minimum amount of PBS or sterile water
if cells will remain viable, then irradiate while swirling the liquid to optimize for
even exposure of samples. Circular movement of the dishes to cause swirling can
Methods to Induce Cell Cycle Checkpoints 7
be performed manually or by use of an electric gyrating platform available
commercially (Lab Rotator Model 1304, Lab-Line Instruments, Melrose Park,
IL). If performed manually, remember to follow the precautions outlined in
Note 10.
4. Notes
1. Gamma rays and X-rays are highly energetic and can penetrate as well as
pass through cells, Petri dishes, and flasks. UV light cannot pass through these
objects efficiently. Therefore, for UV irradiation, cells should be plated onto Petri
dishes such that the lids can be removed for proper exposure.
2. Gamma rays and X-rays are both forms of ionizing radiation, with slightly differ-
ent energies. However, they produce essentially comparable biological effects
when applied at the same doses and similar dose rates.
3. Although we use equipment manufactured by Siemens and Nordion, as listed,
comparable devices are available from other commercial sources, such as Shep-
herd Model 280, JL Shepherd & Associates, San Fernando, CA.
4. The main component for production of UV light is a germicidal bulb capable of
emitting 254 nm UV light (Model X-15B, bulb number 34000801, UVP Inc.,
Upland, CA). An apparatus illustrated in Fig. 1 is most convenient for exposing
cells to UV, but other perhaps simpler systems are just as valid.
5. It is important to have a stable, constant voltage delivered to the UV light fixture.
This will ensure a uniform, constant, reproducible dose rate during the exposure
of samples.
6. The dose rate emitted from a germicidal bulb usually remains fairly constant for
many years. However, when a new bulb is first set up, a UV meter should be used
to determine the dose rate, and this parameter should be checked periodically.
Be sure to use a probe for the meter that is capable of measuring 254 nm UV
light, as probes are available for detecting different wavelengths of light.
7. Consult the manufacturer of the equipment, as well as the local Radiation Safety
Department, to ensure that the equipment is monitored, maintained, and used
properly.
8. Dose and dose rate are important parameters to consider when using gamma rays
to induce cell cycle checkpoints. We typically expose mammalian cells to
between 8 and 20 gy (800 to 2000 rads) of gamma rays, although even lower
doses may be sufficient to induce a cell cycle checkpoint or the desired effect.
Even though the high dose range kills 99.99% of the cells, we use this high dose
when long-term viability is not an issue. This dose is fine when using flow
cytometry to study delays in cell cycle progression, within 24 h posttreatment,
because even this high dose range will not immediately kill cells and will allow
them to cycle long enough to be able to express a transient delay. This high dose
is also reasonable if cell extracts will be isolated, and intact reproductive capac-
ity is not a relevant issue. Some published papers have reported the use of doses
as high as 50 or more gy, but usually such levels are not necessary to observe a
cell cycle effect. We typically use a dose rate of approximately 1 gy/min
8 Lieberman and Hopkins
Fig. 1. Source of 254 nm UV light. (A) Photograph of UV light box closed. (B) Same
light box opened with Petri dishes inside. (C) (next page) Diagram of light box depict-
ing dimensions and side view. Germicidal bulb serving as the source of UV is on top
of makeshift shutter system. A voltage stabilizer connecting light fixture to an A/C
socket is also presented. The inside walls are painted black, and black material is used
for the bottom surface as well. This reduces reflection of light.
8
Methods to Induce Cell Cycle Checkpoints 9
Fig. 1. (continued).
(For yeast, we typically use a Gammacell 220 irradiator with a dose rate of
30 gy/min.) Higher dose rates are probably fine, but significantly lower dose
rates should be avoided. The problem involves DNA repair and the elimination
of the potential checkpoint-inducing signal. Low dose rates will allow repair to
occur efficiently, resulting in the rapid removal of damage and, thus, the cell
cycle checkpoint signal. If equipment constraints will only allow the application
of ionizing radiation at low dose rates, cells can be kept on ice during exposure.
However, this is not ideal because such incubation can by itself potentially per-
turb cell cycle kinetics and add an additional experimental variable that should
really be avoided.
9. Dose rate can be altered by changing the distance between the germicidal bulb
and the sample. The dose rate changes as the inverse square of the distance, such
that for example if the distance between the sample and the bulb is halved, then
the dose rate increases fourfold.
10. Do not look directly or indirectly at the light emitted from the bulb. Wear a long-
sleeved shirt or a lab coat. Protective eyewear would also be helpful.
Acknowledgments
The authors are grateful to Mr. Gary Johnson for building the UV light box
and helping prepare Fig. 1. Research related to this chapter is supported by
NIH grants GM52493 and CA89816.
10 Lieberman and Hopkins
References
1. Friedberg, E. C., Walker, G. C., and Siede, W. (1995) DNA Repair and Mutagen-
esis. ASM Press, Washington, DC.
2. Hartwell, L. H. and Weinert, T. A. (1989) Checkpoints: controls that ensure the
order of cell cycle events. Science 246, 629–634.
3. Nyberg, K. A., Michelson, R. J., Putnam, C. W., and Weinert, T. A. (2002) Toward
maintaining the genome: DNA damage and replication checkpoints. Annu. Rev.
Genet. 36, 617–656.
4. Bridges, B. A. (1995) Are there DNA damage checkpoints in E. coli? Bioessays
17, 63–70.
5. Autret, S., Levine, A., Holland, I. B., and Seror, S. J. (1997) Cell cycle check-
points in bacteria. Biochimie 79, 549–554.
6. Opperman, T., Murli, S., Smith, B. T., and Walker, G. C. (1999) A model for a
umuDC-dependent prokaryotic DNA damage checkpoint. Proc. Natl. Acad. Sci.
USA 96, 9218–9223.
7. Sutton, M. D., Smith, B. T., Godoy, V. G., and Walker, G. C. (2000) The SOS
response: recent insights into umuDC-dependent mutagenesis and DNA damage
tolerance. Annu. Rev. Genet. 34, 479–497.
8. Hartwell, L. H., and Kastan, M. B. (1994) Cell cycle control and cancer. Science
266, 1821–1828.
9. Garvik, B., Carson, M., and Hartwell, L. (1995) Single-stranded DNA arising at
telomeres in cdc13 mutants may constitute a specific signal for the RAD9 check-
point. Mol. Cell. Biol. 15, 6128–6138.
10. Toczyski, D. P., Galgoczy, D. J., and Hartwell, L. H. (1997) CDC5 and CKII
control adaptation to the yeast DNA damage checkpoint. Cell 90, 1097–1106.
11. Canman, C. E., Wolff, A. C., Chen, C. Y., Fornace, A. J., Jr, and Kastan, M. B.
(1994) The p53-dependent G1 cell cycle checkpoint pathway and ataxia-telang-
iectasia. Cancer Res. 54, 5054–5058.
12. Meyer, K. M., Hess, S. M., Tlsty, T. D., and Leadon, S. A. (1999) Human mam-
mary epithelial cells exhibit a differential p53-mediated response following expo-
sure to ionizing radiation or UV light. Oncogene 18, 5795–5805.
13. Kim, S. T., Xu, B., and Kastan, M. B. (2002) Involvement of the cohesin protein,
Smc1, in Atm-dependent and independent responses to DNA damage. Genes Dev.
16, 560–570.
Methods for Synchronizing Mammalian Cells 11
2
Methods for Synchronizing Mammalian Cells
Michael H. Fox
1. Introduction
When studying cell cycle checkpoints, it is often very useful to have
large numbers of cells that are synchronized in various stages of the cell
cycle. A variety of methods have been developed to obtain synchronous (or
partially synchronous) cells, all of which have some drawbacks. Many cell
types that attach to plastic culture dishes round up in mitosis and can then be
dislodged by agitation. This mitotic shake-off method, originally discovered
by Terasima and Tolmach (1), is useful for cells synchronized in metaphase,
which on plating into culture dishes move into G1 phase in a synchronous
manner. A drawback to the mitotic shake-off method is that only a small per-
centage (2–4%) of cells are in mitosis at any given time, so the yield is very
small. Also, cells rapidly become asynchronous as they progress through G1
phase, so the synchronization in S phase and especially G2 phase is not very
good. The first limitation can be overcome by plating multiple T150 flasks with
cells, using roller bottles, or blocking cells in mitosis by inhibitors such as Colcemid
or nocodazole (2). Mitotic cells that are collected can be held on ice for an hour or
so while multiple collections are done to obtain larger numbers of cells.
To obtain more highly synchronous populations of cells in S phase, the mitotic
shake-off procedure can be combined with the use of deoxyribonucleic acid
(DNA) synthesis inhibitors, such as hydroxyurea (HU) or aphidicolin (APH),
to block cells at the G1/S border (but probably past the G1 checkpoint). APH
inhibits DNA polymerase α (3–5), whereas HU inhibits the enzyme ribonucleo-
tide reductase (6), though it may operate by other mechanisms also (7). On release
from the block, cells move in a highly synchronized fashion through S phase
and into G2 phase (8). In terms of number of synchronized cells, this method
has the same limitation as discussed above, because the starting cell population
From: Methods in Molecular Biology, vol. 241: Cell Cycle Checkpoint Control Protocols
Edited by: H. B. Lieberman © Humana Press Inc., Totowa, NJ
11