cell cycle checkpoint control protocols

Methods in Molecular Biology TM Methods in Molecular Biology TM Edited by Howard B. Lieberman Cell Cycle Checkpoint Control Protocols VOLUME 241 Edited by Howard B. Lieberman Cell Cycle Checkpoint Control Protocols Methods to Induce Cell Cycle Checkpoints 3 3 From: Methods in Molecular Biology, vol. 241: Cell Cycle Checkpoint Control Protocols Edited by: H. B. Lieberman © Humana Press Inc., Totowa, NJ 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. 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 2 PO 4 , 9 gm/L NaCl, 0.795 g/L Na 2 HPO 4 ·7H 2 O in distilled H 2 O, pH adjusted to 7.0 and auto- 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 60 Co, such as a Gammacell 220 (Nordion, Alberta, Canada) for a high dose rate, or based on the decay of 137 Cs, 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 × 10 5 cells per mL or 1 × 10 6 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 2 humidified atmosphere. 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/m 2 (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 (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. Fig. 1. (continued). 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 11 From: Methods in Molecular Biology, vol. 241: Cell Cycle Checkpoint Control Protocols Edited by: H. B. Lieberman © Humana Press Inc., Totowa, NJ 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 [...]... cytometry with these unique features is irreplaceable for monitoring the cell cycle status and its regulation Cell cycle checkpoint genes are key elements in cell cycle regulation Checkpoint gene mutation can lead to defects in one or more cell cycle checkpoint controls, which can then result in cell death or cancer Many of the cell cycle checkpoint genes are tumor suppressors, such as p53, ataxia-telangiectasia... the cell cycle of mammalian cells Flow cytometry can measure the deoxyribonucleic acid (DNA) content of individual cells at a rate of several thousand cells per second and thus conveniently reveals the distribution of cells through the cell cycle The DNA-content distribution of a typical exponentially growing cell population is composed of two peaks (cells in G1/G0 and G2/M phases) and a valley of cells... cell size is correlated with cell cycle stage, these cells are synchronized with respect to their position in the cycle Centrifugal elutriation has been applied, with variable degrees of success, to the separation of hemopoietic cells, mouse tumor cells, testicular cells, and a variety of other specialized cells as well as lymphoblastoid cells in particular phases of the cell cycle The capacity of the... (5,6) In mammalian cells, the cell cycle checkpoint controls that can be analyzed by flow cytometry are G1 arrest, suppression of DNA replication, and ATMdependent as well as independent G2 arrest Exposure to a genotoxic agent can activate some or all of the checkpoints The flow cytometry methods to analyze From: Methods in Molecular Biology, vol 241: Cell Cycle Checkpoint Control Protocols Edited by:... G2/M-phase cells have twice the amount of DNA as G1/G0-phase cells, and S-phase cells contain varying amounts of DNA between that found in G1 and G2 cells Most flow-cytometric methods of cell cycle analysis cannot distinguish between G1 and G0 cells or G2 and M cells, so they are grouped together as G1/G0 and G2/M However, there are flowcytometric methods that can distinguish four or even all five cell cycle. .. flask will depend on cell type The number of plates needed will depend on how many cells need to be synchronized 4 Because about 3–4% of cells are in mitosis at any given time, the number of synchronized cells needed will govern how many flasks and how many cells are needed The time for incubation to increase cell numbers will also depend on the cycle time for the cells CHO cells have a cycle time of 12–14... of cells, simultaneously in various phases of the cell cycle, from exponentially growing cell populations Centrifugal elutriation described for the first time by Lindahl (1) is used to enrich cells in different phases of the cell cycle simultaneously with minimum changes in conditions during cell culture Centrifugal elutriation can be used to obtain samples of uniformly sized cells, and because cell. .. Suspend cells in 1-mL PBS containing 50-µg/mL PI and 40-U/mL RNase A, and stain at 4°C for at least 30 min 5 Use a 488-nm argon laser line to excite PI and measure fluorescence at wavelengths >600 nm (see Note 3) Measure at least 10,000 cells 6 Determine the G1 -checkpoint status of cell lines by inspection, quantification, or both of the cell cycle distribution A cell line with normal cell cycle checkpoint. .. cycle checkpoint control will have an increased number of cells in G1 phase and a decreased number of cells in S phase compared to unirradiated cells at about 12 h after irradiation (see Fig 2 and Note 2) At the same time-point, cell populations with a G1 -checkpoint defect contain fewer cells in G1 phase and significantly more cells in S phase than normal cells Numbers of G1/G0, S, and G2/M cells can be... G1-phase checkpoint control in normal cells but not in AT cells Incubation in a CO2 incubator at 37°C for a few hours (usually 6 h or longer) after irradiation reveals G1-phase checkpoint activity in normal cells: S-phase cells during irradiation have moved out of S phase, and new G1 cells have not been able to enter S phase, and number of early S-phase cells (gated area) is reduced to very low levels AT cells . by Howard B. Lieberman Cell Cycle Checkpoint Control Protocols VOLUME 241 Edited by Howard B. Lieberman Cell Cycle Checkpoint Control Protocols Methods to Induce Cell Cycle Checkpoints 3 3 From:. in Molecular Biology, vol. 241: Cell Cycle Checkpoint Control Protocols Edited by: H. B. Lieberman © Humana Press Inc., Totowa, NJ 1 Methods to Induce Cell Cycle Checkpoints Howard B. Lieberman. 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