HeLa细胞同步化

Chapter 10

Synchronization of HeLa Cells

Hoi Tang Ma and Randy Y.C. Poon

Abstract

HeLa is one of the oldest and most commonly used cell lines in biomedical research. Owing to the ease of which they can be effectively synchronized by various methods, HeLa cells have been used extensively for studies of the cell cycle. Here we describe several protocols for synchronization of HeLa cells from different phases of the cell cycle. Synchronization in G 1phase can be achieved with the HMG-CoA reductase inhibitor lovastatin, S phase with a double thymidine block procedure, and G 2phase with the CDK inhibitor RO3306. Cells can also be enriched in mitosis by treating with nocodazole and mechanical shake-off. Release of the cells from these blocks enables researchers to follow gene expression and other events through the cell cycle. We also describe several protocols, including flowcytometry, BrdU labeling, immunoblotting, and time-lapse microscopy, for validating the synchrony of the cells and monitoring the progression of the cell cycle after release.

Key words:Cell cycle, cyclin, cyclin-dependent kinases, flowcytometry, synchronization.

1. Introduction

HeLa is one of the oldest and most commonly used cell lines in biomedical research. The cell line was originally derived from human cervical carcinoma taken from an individual named Henri-etta Lacks (1). Due to the presence of the human papillomaviruses E6and E7proteins, proper control of both cell cycle checkpoints and apoptosis is impaired (2). Partly due of this, HeLa cells are relatively easy to be synchronized by many methods, making them good model systems for studying cell cycle regulation. In addition to looking at individual gene products, whole genome approaches have been performed using synchronized HeLa cells, including microarray analysis of gene expression (3, 4), miRNA expression G. Banfalvi (ed.),Cell Cycle Synchronization , Methods in Molecular Biology 761,

DOI 10.1007/978-1-61779-182-6_10,Springer Science+BusinessMedia, LLC 2011151

152Ma and Poon

patterns (5), as well as proteomic analysis of protein modi-fications(6).

Synchronization involves the isolation of cells in specificcell cycle phases based on either physical properties or perturbation of cell cycle progression with biochemical constraints. Methods based on physical characteristics have the advantage that cells are not exposed to pharmacological agents. For example, centrifugal elutriation can be used to separate cells from different points of the cell cycle based on cell size (7, 8). A major limitation of this method is that specially designated equipments are required.

Several chemicals are effective for synchronization because they are able to reversibly inhibit unique steps of the cell cycle. Releasing the blockade allows the population to progress syn-chronously into the cell cycle. Although these synchronizations are relatively easy to perform, a caveat is that gene expression and post-translational modificationsmay be altered after blocking the cell cycle, making them very different from that of the unper-turbed cell cycle. Another limitation of synchronization using chemicals is that while synchrony is good immediately after the time of release, it deteriorates progressively at later time points. Therefore, experiments should ideally be designed to use more than one synchronization methods from different parts of the cell cycle.

We describe below protocols for blocking HeLa cells in G 1phase, S phase, G 2phase, or mitosis, and for releasing them syn-chronously into the cell cycle. Unlike cells such as fibroblasts,HeLa cells cannot be synchronized at G 0with methods based on serum starvation or contact inhibition.

To trap HeLa cells in S phase, inhibitors of DNA synthesis including thymidine, aphidicolin, and hydroxyurea are frequently used. High concentration of thymidine interrupts the deoxynu-cleotide metabolism pathway, thereby halting DNA replication. As treatment with thymidine arrests cells throughout S phase, a double thymidine block procedure (whichinvolves releasing cells from a firstthymidine block before trapping them with a second thymidine block) is generally used to induce a more synchronized early S phase blockade.

Cyclin-dependent kinase 1(CDK1)is the key engine that drive cells from G 2phase into mitosis. Accordingly, inhibition of CDK1activity with a specificinhibitor called RO3306blocks cells in G 2phase (9). As RO3306is a reversible inhibitor, the cells can then be released synchronously into mitosis when the drug is washed out.

In HeLa cells, mitosis typically only lasts for 40–60min. But cells can be trapped in mitosis by the continued activation of the spindle-assembly checkpoint. The checkpoint is activated by unattached kinetochores or the absence of tension between the paired kinetochores. Hence spindle poisons such as nocodazole

Synchronization of HeLa Cells 153

(whichprevents microtubule assembly) can activate the check-point and trap cells in a prometaphase-like state. Several critical factors should be considered when using nocodazole to synchro-nize HeLa cells. As nocodazole displays a relatively high cyto-toxic activity, it is used in combination with other synchronization methods (suchas the double thymidine block described here) to minimize the incubation time. Furthermore, as nocodazole-blocked cells can exit mitosis precociously by mitotic slippage, the synchronization protocol also relies on the isolation of mitotic cells based on their physical properties (byusing mechanical shake-off). In fact, mechanical shake-off is one of the oldest syn-chronization procedure devised for mammalian cells (10).

Finally, the method described here for synchronizing HeLa cells in G 1phase is based on lovastatin. Lovastatin is an inhibitor of HMG-CoA reductase (11, 12), an enzyme which catalyzes the conversion of HMG-CoA to mevalonate. Cells are released from the lovastatin-mediated blockade by the removal of lovastatin and addition of mevalonic acid (mevalonate).

An important aspect of synchronization experiments is to val-idate the degree of synchronization and to monitor the progres-sion of cells through the cell cycle. Here we describe protocols for analyzing the cell cycle by flowcytometry after propidium iodide staining. This provides basic information about the DNA con-tents of the cell population after synchronization. A more accu-rate method of cell cycle analysis based on BrdU labeling and flowcytometry is also detailed below. Biochemically, cell-free extracts can be prepared and the periodic fluctuationof cell cycle mark-ers can be analyzed by immunoblotting. Finally, finedetails of progression through mitosis can be monitored using time-lapse microscopy.

2. Materials

2.1. Stock Solutions

and Reagents 1. BrdU:10mM in H 2O (Note 1).

2. BrdU antibody (DAKO,Glostrup, Denmark).

3. Cell lysis buffer:50mM Tris–HCl(pH7.5), 250mM

NaCl, 5mM EDTA, and 50mM NaF, and 0.2%NP40. Add fresh:1mM PMSF, 1μg/mlleupeptin, 2μg/mlaprotinin, 10μg/mlsoybean trypsin inhibitor, 15μg/mlbenzamidine, 10μg/mlchymostatin, and 10μg/mlpep-statin.

4. Deoxycytidine:240mM in H 2O.

154Ma and Poon

2.2. Cell Culture

2.3. Equipments 5. FITC-conjugated rabbit anti-mouse immunoglobulins (DAKO).6. Lovastatin (Mevinolin):10mM (Note 2). 7. Mevalonic acid (Sigma):0.5M (Note 3). 8. Nocodazole (Sigma):5mg/mlin DMSO (Note 1). 9. PBS (phosphate-bufferedsaline):137mM NaCl, 2.7mM KCl, 10mM sodium phosphate dibasic, and 2mM potas-sium phosphate monobasic, pH 7.4. 10. PBST:PBS with 0.5%Tween 20and 0.05%w/vBSA. 11. PI/RNaseA solution:40μg/mlpropidium iodide and 40μg/mlRNase A in TE (makefresh). 12. Propidium iodide (Sigma):4mg/mlin H 2O (Note 1). 13. RNase A:10mg/mlin 0.01M NaOAc (pH5.2); heat to 100◦C for 15min to remove DNase; then add 0.1volume of 1M Tris–HCl(pH7.4). 14. RO3306(Alexis,SanDiego, CA, USA):10mM in DMSO (Note 4). 15. SDS sample buffer:10%w/vSDS, 1M Tris–HCl(pH6.8), 50%v/vglycerol, and bromophenol blue (totaste). Add 50μl/ml2-mercaptoethanol before use. 16. TE:10mM Tris–HCl(pH7.5) and 0.1mM EDTA. 17. Thymidine:100mM in DMEM (Note 5). All solutions and equipment coming into contact with the cells must be sterile. Proper sterile technique should be used accord-ingly:1. HeLa cells (AmericanType Culture Collection, Manassas, VA, USA). Cells are grown in a humidifiedincubator at 37◦C in 5%CO 2. 2. HeLa cells stably expressing histone H2B-GFP or similar cell lines for live cell imaging. 3. Growth medium:Dulbecco’sModifiedEagle Medium (DMEM)containing 10%heat-inactivated calf serum and 30U/mlpenicillin–streptomycin.4. Trypsin (0.25%with EDTA). 5. Tissue culture plates and standard tissue culture consum-ables. 1. Standard tissue culture facility.

2. Centrifuge that can accommodate 15and 50ml centrifuge

tubes.

3. Microcentrifuge that can reach 16,000×g at 4◦C.

3. Methods

3.1. Synchronization from Early S:Double

Thymidine Block

3.2. Synchronization

from G 2:RO3306Synchronization of HeLa Cells 1554. Flow cytometer equipped with 488nm laser. 5. Inverted fluorescencewide-fieldmicroscope equipped con-trolled environment chamber and camera for time-lapse analysis. 1. Grow HeLa cells in 100-mm plates with 10ml growth medium to ∼40%confluency(see Note 6). 2. Add 200μl of 100mM thymidine (2mM finalconcentration). 3. Incubate for 14h. 4. Aspirate the medium and wash the cells twice with 10ml PBS. 5. Add 10ml growth medium supplemented with 24μM deoxycytidine. 6. Incubate for 9h. 7. Add 200μl of 100mM thymidine. 8. Incubate for 14h. 9. Aspirate the medium and wash the cells twice with 10ml PBS. 10. Add 10ml growth medium supplemented with 24μM deoxycytidine and return the cells to the incubator. 11. Harvest the cells at different time points for analysis. Typically, cells are harvested every 2or 3h for up to 24h. This should cover at least one cell cycle. As significantloss of synchrony occurs after one cell cycle, it is not very meaningful to follow the cells with longer time points (see Note 7). Here we describe a method that involves firstblocking the cells with a double thymidine block procedure before releasing them

into a RO3306block. Alternatively, asynchronously growing cells can be treated directly with RO3306for 16–20h. The main chal-lenge is that cells can escape the G 2arrest and undergo genome reduplication if they are exposed to RO3306for a long period of time (13):

1. Synchronize cells at early S phase with the double thymidine block procedure (Section 3.1).

2. After release from the second thymidine block, incubate the cells for 2h.

3. Add RO3306to 10μM finalconcentration (see Note 8).

156Ma and Poon

4. Incubate for 10h.

3.3. Synchronization from Mitosis:Nocodazole

3.4. Synchronization

from G 1:Lovastatin 5. Aspirate the medium and wash the cells twice with 10ml PBS. 6. Add 10ml of growth medium. 7. Harvest the cells at different time points for analysis. Cells treated with RO3306are trapped in late G 2phase. As cells rapidly enter mitosis after release from the block, this synchro-nization procedure is best suited for studying entry and exit of mitosis. After release from the block, the cells can be harvested every 15min for up to 4h. Progression through mitosis can also be tracked with time-lapse microscopy (seebelow). While it is possible to treat asynchronously growing HeLa cells with nocodazole directly, the yield and purity of the mitotic pop-ulation are rather low. On the one hand, many cells remain in interphase if the nocodazole treatment is too short. On the other hand, cells may undergo mitotic slippage and apoptosis following a long nocodazole treatment. In the method described here, cells were firstsynchronized with a double thymidine block procedure before releasing into the nocodazole block. 1. Synchronize cells at early S phase with the double thymi-dine block procedure (Section 3.1). 2. After release from the second thymidine block, allow the cells to grow for 2h. 3. Add nocodazole to a finalconcentration of 0.1μg/ml.4. Incubate for 10h. 5. Collect the mitotic cells by mechanical shake-off and trans-fer the medium to a centrifuge tube (see Note 9). 6. Add 10ml PBS to the plate and repeat the shake-off procedure. 7. Combine the PBS with the medium and pellet the cells by centrifugation. 8. Wash the cell pellet twice with 10ml growth medium by resuspension and centrifugation. 9. Resuspend the cell pellet with 10ml growth medium and put onto a plate. 10. Harvest the cells at different time points for analysis. 1. Grow HeLa cells in 100-mm plates in 10ml of growth medium to ∼50%confluency.

2. Add 20μM of lovastatin.

3. Allow the cells to grow for 24h.

4. Aspirate the medium and wash the cells twice with 10ml

of PBS.

3.5. Assessment of

Synchronization:

Flow Cytometry

(PropidiumIodide)

3.6. Assessment of

Synchronization:

Flow Cytometry

(BrdU)Synchronization of HeLa Cells 1575. Add 10ml fresh growth medium supplemented with 6mM mevalonic acid. 6. Harvest the cells at different time points for analysis. The position of the synchronized cell cycle can be determined by the DNA content of the cells. While G 1cells contain two copies of the halpoid genome (2N), cells in G 2and mitosis contain four copies (4N). After staining with propidium iodide, the amount of DNA in cells can be quantifiedwith flowcytometry:1. Collect medium to a 15-ml centrifugation tube. 2. Wash the plates with 2ml PBS and combine with the medium. 3. Add 2ml

min. 4. Add back the medium. Dislodge cells from the plate by pipetting up and down. 5. Collect the cells by centrifugation at 1.5krpm for 5min. 6. Wash the cells twice with 10ml of ice-cold PBS containing 1%calf serum by resuspension and centrifugation. 7. Resuspend the cell pellet with the residue buffer (∼0.1ml) (see Note 10). 8. Add 1ml cold 80%ethanol dropwise with continuous vortexing. 9. Keep the cells on ice for 15min (fixedcells can then be stored indefinitelyat 4◦C). 10. Centrifuge the cells at 1.5krpm for 5min. Drain the pellet thoroughly. 11. Resuspend the pellet in 0.5ml PI/RNaseA solution. 12. Incubate at 37◦C for 30min. 13. Analyze with flowcytometry (see Note 11). The DNA contents of G 1cells (2N)can readily be distinguished from those in G 2/M(4N) by propidium iodide staining and flowcytometry. However, the DNA contents of G 1and G 2/Mcells

overlap with a significantportion of S phase cells. Cells in early S phase contain DNA contents indistinguishable from G 1cells. Likewise, cells in late S phase contain similar amount of DNA as G 2/Mcells. Although several computer algorithms are avail-able to estimate the S phase population from the DNA distri-bution profile,they at best provide a good approximation. Their use is particularly limiting for synchronized cells. The BrdU label-ing method described here provides more precise information on the percentage of cells in G 1, S, and G 2/M.BrdU (5-bromo-2-deoxyuridine) is a thymidine analogue that can be incorporated into newly synthesized DNA. If a brief pulse of BrdU is used,

158Ma and Poon

3.7. Assessment of

Synchronization:

Cyclins only S phase cells will be labeled. The BrdU-positive cells are then detected by antibodies against BrdU:1. Add 10μM BrdU at 30min before harvesting cells at each time point. 2. Harvest and fixcells as described in Section 3.5Steps 1–9.3. Collect the cells by centrifugation at 1.5krpm for 5min. 4. Wash the cells twice with 10ml PBS by resuspension and centrifugation. Remove all supernatant. 5. Add 500μl of freshly diluted 2M HCl. 6. Incubate at 25◦C for 20min. 7. Wash the cells twice with 10ml PBS and once with 10ml PBST by resuspension and centrifugation. 8. Resuspend the cell pellet with the residue buffer (∼0.1ml). 9. Add 2μl anti-BrdU antibody. 10. Incubate at 25◦C for 30min. 11. Wash twice with 10ml PBST by resuspension and centrifu-gation. 12. Resuspend the cell pellet in the residue buffer (∼0.1ml). 13. Add 2.5μl of FITC-conjugated rabbit anti-mouse immunoglobulins. 14. Incubate at 25◦C for 30min. 15. Wash the cells once with 10ml PBST by resuspension and centrifugation. 16. Stain the cells with propidium iodide as described in Section 3.5Steps 10–12.17. Analyze with bivariate flowcytometry. Another way to evaluate the synchrony of cells is through the detection of proteins that vary periodically during the cell cycle. Given that cyclins are components of the engine that drives the

cell cycle, we are using this as an example. Cyclin E1accumu-lates during G 1and decreases during S phase. In contrast, cyclin A2increases during S phase and is destroyed during mitosis. The accumulation and destruction of cyclin B1are slightly later than cyclin A2:

1. Harvest cells as described in Section 3.5Steps 1–6.

2. Resuspend the cells with 1ml PBS and transfer to a

microfuge tube.

3. Centrifuge at 16,000×g for 1min.

4. Aspirate the PBS and store the microfuge tube at –80◦C

until all the samples are ready.

5. Add ∼2pellet volume of cell lysis buffer into the microfuge

tube. Vortex to mix.

Synchronization of HeLa Cells 159

6. Incubate on ice for 30min.

7. Centrifuge at 16,000×g at 4◦C for 30min.

8. Transfer the supernatant to a new tube.

9. Measure the protein concentration of the lysates. Dilute to

1mg/mlwith SDS sample buffer (see Note 12).

10. Run the samples on SDS-PAGE and analyze by

immunoblotting with specificantibodies against cyclin A2, cyclin B1, and cyclin E1(see Note 13).

3.8. Assessment of

Synchronization:

Time-Lapse

Microscopy As they have the same DNA contents, cells in G 2and mitosis can-not be distinguished by flowcytometry after propidium iodide staining. To differentiate these two populations, mitotic markers such as phosphorylated histone H3Ser10can be analyzed. Anti-

bodies that specificallyrecognize phosphorylated form histone H3Ser10are commercially available and can be used in conjunc-tion with either immunoblotting or flowcytometry.

Another method for monitoring mitosis is based on micro-scopic analysis of the chromosomes. Here we describe a method using time-lapse microscopy, thereby allowing the tracking indi-vidual cells into and out of mitosis after RO3306synchronization. For this purpose, HeLa cells expressing GFP (greenfluorescentprotein)-tagged histone H2B are used in the following method:

1. Synchronize cells in G 2with RO3306as described in

Section 3.2. An extra plate is needed to set aside for the time-lapse microscopy.

2. Setup the fluorescencemicroscope and equilibrate the

growth chamber with 5%CO 2at 37◦C (see Note 14).

3. After release from the RO3306block, place the plate imme-

diately into the growth chamber.

4. Focus the microscope at the optical plane of the chromatin.

As the cells are going to round up during mitosis, it is not a good idea to focus the images based on the bright field.

5. Images are taken every 3min for 2–4h (see Note 15).

4. Notes

1. Mutagen. Handle with care and use gloves.

2. Inactive lactone form of mevinolin is activated by dissolving

52mg in 1.04ml EtOH. Add 813μl of 1M NaOH and then neutralized with 1M HCl to pH 7.2. Bring the solu-tion to 13ml with H 2O to make a 10mM stock solution. Store at –20◦C. It has been reported that in vitro activa-tion of mevinolin lactone may not be necessary (12, 14).

160Ma and Poon

In that case, simply dissolve 52mg of mevinolin in 13ml 70%EtOH.

3. Dissolve 1g of mevanlonic acid lactone in 3.5ml of EtOH.

Add 4.2ml of 1M NaOH. Bring the solution to 15.4ml with H 2O to make a 0.5M stock solution.

4. RO3306is sensitive to light and freeze–thawcycle. We

keep the stocks in small aliquots wrapped with aluminum foils at –80◦C.

5. Dissolve thymidine and filtersterile to make the 100mM

stock solution. Incubation at 37◦C may help to solubilize the thymidine.

6. The synchronization procedures described in these pro-

tocols are for using 100-mm plates. Cells obtained from one 100-mm plate at each time point should be sufficientfor both flowcytometry analysis and immunoblotting. The procedures can be scaled up proportionally.

7. It is possible to break up a 24-h experiment into two

independent sessions. Alternatively, it is possible for two researchers to work in shifts to harvest the cells at differ-ent time points. However, we found that the best results are obtained when all the cells are harvested by the same researcher.

8. For HeLa cells, CDK1but not other CDKs is inhib-

ited with 10μM of RO3306(9, 13). The exact concen-tration of RO3306used should be determined for each stock.

9. The basis of synchronization by nocodazole treatment is

that mitotic cells are rounded and attach less well to the plate than cells in interphase. It is possible to collect the mitotic cells by blasting them off with the medium using a pipette. Alternatively, shakers that hold plates and flaskscan be used securely. It is also possible to hold the plates on a vortex and shake for 20s with the highest setting. In any case, the cells should be examined under a light microscope before and after the mechanical shake-off to ensure that most of the mitotic cells are detached.

10. It is crucial to resuspend the cells very well before adding

ethanol to avoid crumbing.

11. As cells from different phases of the cell cycle may be miss-

ing in the synchronized population, it is a good idea to firstuse asynchronously growing cells to setup the DNA profile.

12. Many reagents are available for measuring the concentra-

tion of the lysates. We use BCA protein assay reagent from Pierce (Rockford,IL, USA) using BSA as standards.

Synchronization of HeLa Cells 161

13. Cyclins are readily detectable in HeLa cells using com-mercially available monoclonal antibodies:cyclin A2(E23),cyclin B1(V152),and cyclin E2(HE12).14. We use a TE2000E-PFS inverted fluorescentmicro-scope (Nikon,Tokyo, Japan) equipped with a SPOT BOOST TM EMCCD camera (DiagnosticInstrument, Ster-ling Heights, MI, USA) and a INU-NI-F1temperature, humidity, and CO 2control system (TokaiHit, Shizuoka, Japan). Data acquisition and analysis are carried out using the Metamorph software (MolecularDevices, Downing-town, PA, USA). 15. A critical parameter in every time-lapse microscopy exper-iment is photobleaching and UV damage to the cells. The exposure time should be minimized.

References

1. Skloot, R. (2010)The immortal life of Hen-rietta Lacks. New York:Random House. 2. McLaughlin-Drubin, M. E., and Munger, K.

(2009)Oncogenic activities of human papil-lomaviruses. Virus Res. 143, 195–208.

3. Chaudhry, M. A., Chodosh, L. A., McKenna,

W. G., and Muschel, R. J. (2002)Gene expression profilingof HeLa cells in G1or G2phases. Oncogene 21, 1934–1942.

4. Whitfield,M. L., Sherlock, G., Saldanha,

A. J., Murray, J. I., Ball, C. A., Alexander, K. E. et al. (2002)Identificationof genes periodically expressed in the human cell cycle and their expression in tumors. Mol. Biol. Cell 13, 1977–2000.

5. Zhou, J. Y., Ma, W. L., Liang, S., Zeng, Y.,

Shi, R., Yu, H. L., et al. (2009)Analysis of microRNA expression profilesduring the cell cycle in synchronized HeLa cells. BMB Rep. 42, 593–598.

6. Chen, X., Simon, E. S., Xiang, Y.,

Kachman, M., Andrews, P . C., and Wang, Y. (2010)Quantitative proteomics analysis of cell cycle-regulated Golgi disassem-bly and reassembly. J. Biol. Chem. 285, 7197–7207.

7. Wahl, A. F., and Donaldson, K. L. (2001)

Centrifugal elutriation to obtain synchronous populations of cells. Curr. Protoc. Cell Biol. Chapter 8, Unit 8.5.

8. Banfalvi, G. (2008)Cell cycle synchroniza-tion of animal cells and nuclei by centrifugal elutriation. Nat. Protoc. 3, 663–673.

9. Vassilev, L. T., Tovar, C., Chen, S.,

Knezevic, D., Zhao, X., Sun, H., et al. (2006)Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1. Proc. Natl. Acad. Sci. USA 103, 10660–10665.

10. Terasima, T., and Tolmach, L. J. (1963)

Growth and nucleic acid synthesis in syn-chronously dividing populations of HeLa cells. Exp. Cell Res. 30, 344–362.

11. Keyomarsi, K., Sandoval, L., Band, V., and

Pardee, A. B. (1991)Synchronization of tumor and normal cells from G1to multi-ple cell cycles by lovastatin. Cancer Res. 51, 3602–3609.

12. Javanmoghadam-Kamrani, S., and Keyo-marsi, K. (2008)Synchronization of the cell cycle using lovastatin. Cell Cycle 7, 2434–2440.

13. Ma, H. T., Tsang, Y. H., Marxer, M.,

and Poon, R. Y. C. (2009)Cyclin A2-cyclin-dependent kinase 2cooperates with the PLK1-SCFbeta-TrCP1-EMI1-anaphase-promoting complex/cyclosomeaxis to pro-mote genome reduplication in the absence of mitosis. Mol. Cell Biol. 29, 6500–6514.

14. Mikulski, S. M., Viera, A., Darzynkiewicz, Z.,

and Shogen, K. (1992)Synergism between a novel amphibian oocyte ribonuclease and lovastatin in inducing cytostatic and cyto-toxic effects in human lung and pancre-atic carcinoma cell lines. Br. J. Cancer 66, 304–310.

Chapter 10

Synchronization of HeLa Cells

Hoi Tang Ma and Randy Y.C. Poon

Abstract

HeLa is one of the oldest and most commonly used cell lines in biomedical research. Owing to the ease of which they can be effectively synchronized by various methods, HeLa cells have been used extensively for studies of the cell cycle. Here we describe several protocols for synchronization of HeLa cells from different phases of the cell cycle. Synchronization in G 1phase can be achieved with the HMG-CoA reductase inhibitor lovastatin, S phase with a double thymidine block procedure, and G 2phase with the CDK inhibitor RO3306. Cells can also be enriched in mitosis by treating with nocodazole and mechanical shake-off. Release of the cells from these blocks enables researchers to follow gene expression and other events through the cell cycle. We also describe several protocols, including flowcytometry, BrdU labeling, immunoblotting, and time-lapse microscopy, for validating the synchrony of the cells and monitoring the progression of the cell cycle after release.

Key words:Cell cycle, cyclin, cyclin-dependent kinases, flowcytometry, synchronization.

1. Introduction

HeLa is one of the oldest and most commonly used cell lines in biomedical research. The cell line was originally derived from human cervical carcinoma taken from an individual named Henri-etta Lacks (1). Due to the presence of the human papillomaviruses E6and E7proteins, proper control of both cell cycle checkpoints and apoptosis is impaired (2). Partly due of this, HeLa cells are relatively easy to be synchronized by many methods, making them good model systems for studying cell cycle regulation. In addition to looking at individual gene products, whole genome approaches have been performed using synchronized HeLa cells, including microarray analysis of gene expression (3, 4), miRNA expression G. Banfalvi (ed.),Cell Cycle Synchronization , Methods in Molecular Biology 761,

DOI 10.1007/978-1-61779-182-6_10,Springer Science+BusinessMedia, LLC 2011151

152Ma and Poon

patterns (5), as well as proteomic analysis of protein modi-fications(6).

Synchronization involves the isolation of cells in specificcell cycle phases based on either physical properties or perturbation of cell cycle progression with biochemical constraints. Methods based on physical characteristics have the advantage that cells are not exposed to pharmacological agents. For example, centrifugal elutriation can be used to separate cells from different points of the cell cycle based on cell size (7, 8). A major limitation of this method is that specially designated equipments are required.

Several chemicals are effective for synchronization because they are able to reversibly inhibit unique steps of the cell cycle. Releasing the blockade allows the population to progress syn-chronously into the cell cycle. Although these synchronizations are relatively easy to perform, a caveat is that gene expression and post-translational modificationsmay be altered after blocking the cell cycle, making them very different from that of the unper-turbed cell cycle. Another limitation of synchronization using chemicals is that while synchrony is good immediately after the time of release, it deteriorates progressively at later time points. Therefore, experiments should ideally be designed to use more than one synchronization methods from different parts of the cell cycle.

We describe below protocols for blocking HeLa cells in G 1phase, S phase, G 2phase, or mitosis, and for releasing them syn-chronously into the cell cycle. Unlike cells such as fibroblasts,HeLa cells cannot be synchronized at G 0with methods based on serum starvation or contact inhibition.

To trap HeLa cells in S phase, inhibitors of DNA synthesis including thymidine, aphidicolin, and hydroxyurea are frequently used. High concentration of thymidine interrupts the deoxynu-cleotide metabolism pathway, thereby halting DNA replication. As treatment with thymidine arrests cells throughout S phase, a double thymidine block procedure (whichinvolves releasing cells from a firstthymidine block before trapping them with a second thymidine block) is generally used to induce a more synchronized early S phase blockade.

Cyclin-dependent kinase 1(CDK1)is the key engine that drive cells from G 2phase into mitosis. Accordingly, inhibition of CDK1activity with a specificinhibitor called RO3306blocks cells in G 2phase (9). As RO3306is a reversible inhibitor, the cells can then be released synchronously into mitosis when the drug is washed out.

In HeLa cells, mitosis typically only lasts for 40–60min. But cells can be trapped in mitosis by the continued activation of the spindle-assembly checkpoint. The checkpoint is activated by unattached kinetochores or the absence of tension between the paired kinetochores. Hence spindle poisons such as nocodazole

Synchronization of HeLa Cells 153

(whichprevents microtubule assembly) can activate the check-point and trap cells in a prometaphase-like state. Several critical factors should be considered when using nocodazole to synchro-nize HeLa cells. As nocodazole displays a relatively high cyto-toxic activity, it is used in combination with other synchronization methods (suchas the double thymidine block described here) to minimize the incubation time. Furthermore, as nocodazole-blocked cells can exit mitosis precociously by mitotic slippage, the synchronization protocol also relies on the isolation of mitotic cells based on their physical properties (byusing mechanical shake-off). In fact, mechanical shake-off is one of the oldest syn-chronization procedure devised for mammalian cells (10).

Finally, the method described here for synchronizing HeLa cells in G 1phase is based on lovastatin. Lovastatin is an inhibitor of HMG-CoA reductase (11, 12), an enzyme which catalyzes the conversion of HMG-CoA to mevalonate. Cells are released from the lovastatin-mediated blockade by the removal of lovastatin and addition of mevalonic acid (mevalonate).

An important aspect of synchronization experiments is to val-idate the degree of synchronization and to monitor the progres-sion of cells through the cell cycle. Here we describe protocols for analyzing the cell cycle by flowcytometry after propidium iodide staining. This provides basic information about the DNA con-tents of the cell population after synchronization. A more accu-rate method of cell cycle analysis based on BrdU labeling and flowcytometry is also detailed below. Biochemically, cell-free extracts can be prepared and the periodic fluctuationof cell cycle mark-ers can be analyzed by immunoblotting. Finally, finedetails of progression through mitosis can be monitored using time-lapse microscopy.

2. Materials

2.1. Stock Solutions

and Reagents 1. BrdU:10mM in H 2O (Note 1).

2. BrdU antibody (DAKO,Glostrup, Denmark).

3. Cell lysis buffer:50mM Tris–HCl(pH7.5), 250mM

NaCl, 5mM EDTA, and 50mM NaF, and 0.2%NP40. Add fresh:1mM PMSF, 1μg/mlleupeptin, 2μg/mlaprotinin, 10μg/mlsoybean trypsin inhibitor, 15μg/mlbenzamidine, 10μg/mlchymostatin, and 10μg/mlpep-statin.

4. Deoxycytidine:240mM in H 2O.

154Ma and Poon

2.2. Cell Culture

2.3. Equipments 5. FITC-conjugated rabbit anti-mouse immunoglobulins (DAKO).6. Lovastatin (Mevinolin):10mM (Note 2). 7. Mevalonic acid (Sigma):0.5M (Note 3). 8. Nocodazole (Sigma):5mg/mlin DMSO (Note 1). 9. PBS (phosphate-bufferedsaline):137mM NaCl, 2.7mM KCl, 10mM sodium phosphate dibasic, and 2mM potas-sium phosphate monobasic, pH 7.4. 10. PBST:PBS with 0.5%Tween 20and 0.05%w/vBSA. 11. PI/RNaseA solution:40μg/mlpropidium iodide and 40μg/mlRNase A in TE (makefresh). 12. Propidium iodide (Sigma):4mg/mlin H 2O (Note 1). 13. RNase A:10mg/mlin 0.01M NaOAc (pH5.2); heat to 100◦C for 15min to remove DNase; then add 0.1volume of 1M Tris–HCl(pH7.4). 14. RO3306(Alexis,SanDiego, CA, USA):10mM in DMSO (Note 4). 15. SDS sample buffer:10%w/vSDS, 1M Tris–HCl(pH6.8), 50%v/vglycerol, and bromophenol blue (totaste). Add 50μl/ml2-mercaptoethanol before use. 16. TE:10mM Tris–HCl(pH7.5) and 0.1mM EDTA. 17. Thymidine:100mM in DMEM (Note 5). All solutions and equipment coming into contact with the cells must be sterile. Proper sterile technique should be used accord-ingly:1. HeLa cells (AmericanType Culture Collection, Manassas, VA, USA). Cells are grown in a humidifiedincubator at 37◦C in 5%CO 2. 2. HeLa cells stably expressing histone H2B-GFP or similar cell lines for live cell imaging. 3. Growth medium:Dulbecco’sModifiedEagle Medium (DMEM)containing 10%heat-inactivated calf serum and 30U/mlpenicillin–streptomycin.4. Trypsin (0.25%with EDTA). 5. Tissue culture plates and standard tissue culture consum-ables. 1. Standard tissue culture facility.

2. Centrifuge that can accommodate 15and 50ml centrifuge

tubes.

3. Microcentrifuge that can reach 16,000×g at 4◦C.

3. Methods

3.1. Synchronization from Early S:Double

Thymidine Block

3.2. Synchronization

from G 2:RO3306Synchronization of HeLa Cells 1554. Flow cytometer equipped with 488nm laser. 5. Inverted fluorescencewide-fieldmicroscope equipped con-trolled environment chamber and camera for time-lapse analysis. 1. Grow HeLa cells in 100-mm plates with 10ml growth medium to ∼40%confluency(see Note 6). 2. Add 200μl of 100mM thymidine (2mM finalconcentration). 3. Incubate for 14h. 4. Aspirate the medium and wash the cells twice with 10ml PBS. 5. Add 10ml growth medium supplemented with 24μM deoxycytidine. 6. Incubate for 9h. 7. Add 200μl of 100mM thymidine. 8. Incubate for 14h. 9. Aspirate the medium and wash the cells twice with 10ml PBS. 10. Add 10ml growth medium supplemented with 24μM deoxycytidine and return the cells to the incubator. 11. Harvest the cells at different time points for analysis. Typically, cells are harvested every 2or 3h for up to 24h. This should cover at least one cell cycle. As significantloss of synchrony occurs after one cell cycle, it is not very meaningful to follow the cells with longer time points (see Note 7). Here we describe a method that involves firstblocking the cells with a double thymidine block procedure before releasing them

into a RO3306block. Alternatively, asynchronously growing cells can be treated directly with RO3306for 16–20h. The main chal-lenge is that cells can escape the G 2arrest and undergo genome reduplication if they are exposed to RO3306for a long period of time (13):

1. Synchronize cells at early S phase with the double thymidine block procedure (Section 3.1).

2. After release from the second thymidine block, incubate the cells for 2h.

3. Add RO3306to 10μM finalconcentration (see Note 8).

156Ma and Poon

4. Incubate for 10h.

3.3. Synchronization from Mitosis:Nocodazole

3.4. Synchronization

from G 1:Lovastatin 5. Aspirate the medium and wash the cells twice with 10ml PBS. 6. Add 10ml of growth medium. 7. Harvest the cells at different time points for analysis. Cells treated with RO3306are trapped in late G 2phase. As cells rapidly enter mitosis after release from the block, this synchro-nization procedure is best suited for studying entry and exit of mitosis. After release from the block, the cells can be harvested every 15min for up to 4h. Progression through mitosis can also be tracked with time-lapse microscopy (seebelow). While it is possible to treat asynchronously growing HeLa cells with nocodazole directly, the yield and purity of the mitotic pop-ulation are rather low. On the one hand, many cells remain in interphase if the nocodazole treatment is too short. On the other hand, cells may undergo mitotic slippage and apoptosis following a long nocodazole treatment. In the method described here, cells were firstsynchronized with a double thymidine block procedure before releasing into the nocodazole block. 1. Synchronize cells at early S phase with the double thymi-dine block procedure (Section 3.1). 2. After release from the second thymidine block, allow the cells to grow for 2h. 3. Add nocodazole to a finalconcentration of 0.1μg/ml.4. Incubate for 10h. 5. Collect the mitotic cells by mechanical shake-off and trans-fer the medium to a centrifuge tube (see Note 9). 6. Add 10ml PBS to the plate and repeat the shake-off procedure. 7. Combine the PBS with the medium and pellet the cells by centrifugation. 8. Wash the cell pellet twice with 10ml growth medium by resuspension and centrifugation. 9. Resuspend the cell pellet with 10ml growth medium and put onto a plate. 10. Harvest the cells at different time points for analysis. 1. Grow HeLa cells in 100-mm plates in 10ml of growth medium to ∼50%confluency.

2. Add 20μM of lovastatin.

3. Allow the cells to grow for 24h.

4. Aspirate the medium and wash the cells twice with 10ml

of PBS.

3.5. Assessment of

Synchronization:

Flow Cytometry

(PropidiumIodide)

3.6. Assessment of

Synchronization:

Flow Cytometry

(BrdU)Synchronization of HeLa Cells 1575. Add 10ml fresh growth medium supplemented with 6mM mevalonic acid. 6. Harvest the cells at different time points for analysis. The position of the synchronized cell cycle can be determined by the DNA content of the cells. While G 1cells contain two copies of the halpoid genome (2N), cells in G 2and mitosis contain four copies (4N). After staining with propidium iodide, the amount of DNA in cells can be quantifiedwith flowcytometry:1. Collect medium to a 15-ml centrifugation tube. 2. Wash the plates with 2ml PBS and combine with the medium. 3. Add 2ml

min. 4. Add back the medium. Dislodge cells from the plate by pipetting up and down. 5. Collect the cells by centrifugation at 1.5krpm for 5min. 6. Wash the cells twice with 10ml of ice-cold PBS containing 1%calf serum by resuspension and centrifugation. 7. Resuspend the cell pellet with the residue buffer (∼0.1ml) (see Note 10). 8. Add 1ml cold 80%ethanol dropwise with continuous vortexing. 9. Keep the cells on ice for 15min (fixedcells can then be stored indefinitelyat 4◦C). 10. Centrifuge the cells at 1.5krpm for 5min. Drain the pellet thoroughly. 11. Resuspend the pellet in 0.5ml PI/RNaseA solution. 12. Incubate at 37◦C for 30min. 13. Analyze with flowcytometry (see Note 11). The DNA contents of G 1cells (2N)can readily be distinguished from those in G 2/M(4N) by propidium iodide staining and flowcytometry. However, the DNA contents of G 1and G 2/Mcells

overlap with a significantportion of S phase cells. Cells in early S phase contain DNA contents indistinguishable from G 1cells. Likewise, cells in late S phase contain similar amount of DNA as G 2/Mcells. Although several computer algorithms are avail-able to estimate the S phase population from the DNA distri-bution profile,they at best provide a good approximation. Their use is particularly limiting for synchronized cells. The BrdU label-ing method described here provides more precise information on the percentage of cells in G 1, S, and G 2/M.BrdU (5-bromo-2-deoxyuridine) is a thymidine analogue that can be incorporated into newly synthesized DNA. If a brief pulse of BrdU is used,

158Ma and Poon

3.7. Assessment of

Synchronization:

Cyclins only S phase cells will be labeled. The BrdU-positive cells are then detected by antibodies against BrdU:1. Add 10μM BrdU at 30min before harvesting cells at each time point. 2. Harvest and fixcells as described in Section 3.5Steps 1–9.3. Collect the cells by centrifugation at 1.5krpm for 5min. 4. Wash the cells twice with 10ml PBS by resuspension and centrifugation. Remove all supernatant. 5. Add 500μl of freshly diluted 2M HCl. 6. Incubate at 25◦C for 20min. 7. Wash the cells twice with 10ml PBS and once with 10ml PBST by resuspension and centrifugation. 8. Resuspend the cell pellet with the residue buffer (∼0.1ml). 9. Add 2μl anti-BrdU antibody. 10. Incubate at 25◦C for 30min. 11. Wash twice with 10ml PBST by resuspension and centrifu-gation. 12. Resuspend the cell pellet in the residue buffer (∼0.1ml). 13. Add 2.5μl of FITC-conjugated rabbit anti-mouse immunoglobulins. 14. Incubate at 25◦C for 30min. 15. Wash the cells once with 10ml PBST by resuspension and centrifugation. 16. Stain the cells with propidium iodide as described in Section 3.5Steps 10–12.17. Analyze with bivariate flowcytometry. Another way to evaluate the synchrony of cells is through the detection of proteins that vary periodically during the cell cycle. Given that cyclins are components of the engine that drives the

cell cycle, we are using this as an example. Cyclin E1accumu-lates during G 1and decreases during S phase. In contrast, cyclin A2increases during S phase and is destroyed during mitosis. The accumulation and destruction of cyclin B1are slightly later than cyclin A2:

1. Harvest cells as described in Section 3.5Steps 1–6.

2. Resuspend the cells with 1ml PBS and transfer to a

microfuge tube.

3. Centrifuge at 16,000×g for 1min.

4. Aspirate the PBS and store the microfuge tube at –80◦C

until all the samples are ready.

5. Add ∼2pellet volume of cell lysis buffer into the microfuge

tube. Vortex to mix.

Synchronization of HeLa Cells 159

6. Incubate on ice for 30min.

7. Centrifuge at 16,000×g at 4◦C for 30min.

8. Transfer the supernatant to a new tube.

9. Measure the protein concentration of the lysates. Dilute to

1mg/mlwith SDS sample buffer (see Note 12).

10. Run the samples on SDS-PAGE and analyze by

immunoblotting with specificantibodies against cyclin A2, cyclin B1, and cyclin E1(see Note 13).

3.8. Assessment of

Synchronization:

Time-Lapse

Microscopy As they have the same DNA contents, cells in G 2and mitosis can-not be distinguished by flowcytometry after propidium iodide staining. To differentiate these two populations, mitotic markers such as phosphorylated histone H3Ser10can be analyzed. Anti-

bodies that specificallyrecognize phosphorylated form histone H3Ser10are commercially available and can be used in conjunc-tion with either immunoblotting or flowcytometry.

Another method for monitoring mitosis is based on micro-scopic analysis of the chromosomes. Here we describe a method using time-lapse microscopy, thereby allowing the tracking indi-vidual cells into and out of mitosis after RO3306synchronization. For this purpose, HeLa cells expressing GFP (greenfluorescentprotein)-tagged histone H2B are used in the following method:

1. Synchronize cells in G 2with RO3306as described in

Section 3.2. An extra plate is needed to set aside for the time-lapse microscopy.

2. Setup the fluorescencemicroscope and equilibrate the

growth chamber with 5%CO 2at 37◦C (see Note 14).

3. After release from the RO3306block, place the plate imme-

diately into the growth chamber.

4. Focus the microscope at the optical plane of the chromatin.

As the cells are going to round up during mitosis, it is not a good idea to focus the images based on the bright field.

5. Images are taken every 3min for 2–4h (see Note 15).

4. Notes

1. Mutagen. Handle with care and use gloves.

2. Inactive lactone form of mevinolin is activated by dissolving

52mg in 1.04ml EtOH. Add 813μl of 1M NaOH and then neutralized with 1M HCl to pH 7.2. Bring the solu-tion to 13ml with H 2O to make a 10mM stock solution. Store at –20◦C. It has been reported that in vitro activa-tion of mevinolin lactone may not be necessary (12, 14).

160Ma and Poon

In that case, simply dissolve 52mg of mevinolin in 13ml 70%EtOH.

3. Dissolve 1g of mevanlonic acid lactone in 3.5ml of EtOH.

Add 4.2ml of 1M NaOH. Bring the solution to 15.4ml with H 2O to make a 0.5M stock solution.

4. RO3306is sensitive to light and freeze–thawcycle. We

keep the stocks in small aliquots wrapped with aluminum foils at –80◦C.

5. Dissolve thymidine and filtersterile to make the 100mM

stock solution. Incubation at 37◦C may help to solubilize the thymidine.

6. The synchronization procedures described in these pro-

tocols are for using 100-mm plates. Cells obtained from one 100-mm plate at each time point should be sufficientfor both flowcytometry analysis and immunoblotting. The procedures can be scaled up proportionally.

7. It is possible to break up a 24-h experiment into two

independent sessions. Alternatively, it is possible for two researchers to work in shifts to harvest the cells at differ-ent time points. However, we found that the best results are obtained when all the cells are harvested by the same researcher.

8. For HeLa cells, CDK1but not other CDKs is inhib-

ited with 10μM of RO3306(9, 13). The exact concen-tration of RO3306used should be determined for each stock.

9. The basis of synchronization by nocodazole treatment is

that mitotic cells are rounded and attach less well to the plate than cells in interphase. It is possible to collect the mitotic cells by blasting them off with the medium using a pipette. Alternatively, shakers that hold plates and flaskscan be used securely. It is also possible to hold the plates on a vortex and shake for 20s with the highest setting. In any case, the cells should be examined under a light microscope before and after the mechanical shake-off to ensure that most of the mitotic cells are detached.

10. It is crucial to resuspend the cells very well before adding

ethanol to avoid crumbing.

11. As cells from different phases of the cell cycle may be miss-

ing in the synchronized population, it is a good idea to firstuse asynchronously growing cells to setup the DNA profile.

12. Many reagents are available for measuring the concentra-

tion of the lysates. We use BCA protein assay reagent from Pierce (Rockford,IL, USA) using BSA as standards.

Synchronization of HeLa Cells 161

13. Cyclins are readily detectable in HeLa cells using com-mercially available monoclonal antibodies:cyclin A2(E23),cyclin B1(V152),and cyclin E2(HE12).14. We use a TE2000E-PFS inverted fluorescentmicro-scope (Nikon,Tokyo, Japan) equipped with a SPOT BOOST TM EMCCD camera (DiagnosticInstrument, Ster-ling Heights, MI, USA) and a INU-NI-F1temperature, humidity, and CO 2control system (TokaiHit, Shizuoka, Japan). Data acquisition and analysis are carried out using the Metamorph software (MolecularDevices, Downing-town, PA, USA). 15. A critical parameter in every time-lapse microscopy exper-iment is photobleaching and UV damage to the cells. The exposure time should be minimized.

References

1. Skloot, R. (2010)The immortal life of Hen-rietta Lacks. New York:Random House. 2. McLaughlin-Drubin, M. E., and Munger, K.

(2009)Oncogenic activities of human papil-lomaviruses. Virus Res. 143, 195–208.

3. Chaudhry, M. A., Chodosh, L. A., McKenna,

W. G., and Muschel, R. J. (2002)Gene expression profilingof HeLa cells in G1or G2phases. Oncogene 21, 1934–1942.

4. Whitfield,M. L., Sherlock, G., Saldanha,

A. J., Murray, J. I., Ball, C. A., Alexander, K. E. et al. (2002)Identificationof genes periodically expressed in the human cell cycle and their expression in tumors. Mol. Biol. Cell 13, 1977–2000.

5. Zhou, J. Y., Ma, W. L., Liang, S., Zeng, Y.,

Shi, R., Yu, H. L., et al. (2009)Analysis of microRNA expression profilesduring the cell cycle in synchronized HeLa cells. BMB Rep. 42, 593–598.

6. Chen, X., Simon, E. S., Xiang, Y.,

Kachman, M., Andrews, P . C., and Wang, Y. (2010)Quantitative proteomics analysis of cell cycle-regulated Golgi disassem-bly and reassembly. J. Biol. Chem. 285, 7197–7207.

7. Wahl, A. F., and Donaldson, K. L. (2001)

Centrifugal elutriation to obtain synchronous populations of cells. Curr. Protoc. Cell Biol. Chapter 8, Unit 8.5.

8. Banfalvi, G. (2008)Cell cycle synchroniza-tion of animal cells and nuclei by centrifugal elutriation. Nat. Protoc. 3, 663–673.

9. Vassilev, L. T., Tovar, C., Chen, S.,

Knezevic, D., Zhao, X., Sun, H., et al. (2006)Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1. Proc. Natl. Acad. Sci. USA 103, 10660–10665.

10. Terasima, T., and Tolmach, L. J. (1963)

Growth and nucleic acid synthesis in syn-chronously dividing populations of HeLa cells. Exp. Cell Res. 30, 344–362.

11. Keyomarsi, K., Sandoval, L., Band, V., and

Pardee, A. B. (1991)Synchronization of tumor and normal cells from G1to multi-ple cell cycles by lovastatin. Cancer Res. 51, 3602–3609.

12. Javanmoghadam-Kamrani, S., and Keyo-marsi, K. (2008)Synchronization of the cell cycle using lovastatin. Cell Cycle 7, 2434–2440.

13. Ma, H. T., Tsang, Y. H., Marxer, M.,

and Poon, R. Y. C. (2009)Cyclin A2-cyclin-dependent kinase 2cooperates with the PLK1-SCFbeta-TrCP1-EMI1-anaphase-promoting complex/cyclosomeaxis to pro-mote genome reduplication in the absence of mitosis. Mol. Cell Biol. 29, 6500–6514.

14. Mikulski, S. M., Viera, A., Darzynkiewicz, Z.,

and Shogen, K. (1992)Synergism between a novel amphibian oocyte ribonuclease and lovastatin in inducing cytostatic and cyto-toxic effects in human lung and pancre-atic carcinoma cell lines. Br. J. Cancer 66, 304–310.


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