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Cell Synchronization by Double Thymidine Block    

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Original research article

A brief version of this protocol appeared in:
The Journal of Clinical Investigation
Jan 2018

Abstract

Cell synchronization is widely used in studying mechanisms involves in regulation of cell cycle progression. Through synchronization, cells at distinct cell cycle stage could be obtained. Thymidine is a DNA synthesis inhibitor that can arrest cell at G1/S boundary, prior to DNA replication. Here, we present the protocol to synchronize cells at G1/S boundary by using double thymidine block. After release into normal medium, cell population at distinct cell cycle phase could be collected at different time points.

Keywords: Cell synchronization, Cell cycle, Thymidine, DNA synthesis, DNA replication

Background

Cell cycle and cell division lie at the heart of cell biology. To build multicellular organism, cell duplication is necessary to generate specialized cells, which can execute particular function. The normal cell cycle is composed of interphase (G1, S and G2 phase) and mitotic (M) phase (Rodríguez-Ubreva et al., 2010; Léger et al., 2016). During interphase, the genetic materials are duplicated and make everything ready for mitosis. Whereas, during mitotic phase, the duplicated chromosomes are segregated and distributed into daughter cells (Sakaue-Sawano et al., 2008).

To precisely preserve genetic information, cell cycle progression must be tightly regulated. Cyclin/CDK complexes control the cell cycle progression through rapidly promoting activities at their respective stages, and are quickly inactivated when their stages are completed (Graña and Reddy, 1995).

Cell synchronization is particularly useful for investigating a cell-cycle regulated event. Using different methods, cells could be synchronized at different cell cycle stage. Treatment of nocodazole, which is an inhibitor of microtubule formation, could synchronize cells at G2/M phase (Ho et al., 2001), while, hydroxyurea, a dNTP synthesis inhibitor, synchronize cells at early S phase (Koç et al., 2004). As an Inhibitor of DNA synthesis (Schvartzman et al., 1984), thymidine can arrest cell at G1/S boundary. Here, we describe a detail method to synchronize cells at G1/S boundary by thymidine (Chen et al., 2018).

Materials and Reagents

  1. 10 cm culture dish (Corning, catalog number: 430167 )
  2. Gloves (VWR International, catalog number: 82026 )
  3. Protective clothing (VWR International, catalog number: 414004-444 )
  4. Eyewear (VWR International, catalog number: 89187-984 )
  5. Human tumor cell lines: H1299 (ATCC, catalog number: ATCC® CRL-5803TM )
  6. Dulbecco's Modified Eagle's Medium (DMEM) (high glucose with L-glutamine) (Corning, catalog number: 10-013-CV )
  7. Phosphate-Buffered Saline (PBS) (Corning, catalog number: 21-040-CV )
  8. Fetal bovine serum (FBS) (ATLANTA BIOLOGICALS, catalog number: S11150 )
  9. Thymidine (Sigma-Aldrich, catalog number: T9250 )
  10. Propidium Iodide (PI) (Thermo Fisher Scientific, catalog number: P3566 )
  11. Antibodies
    1. Anti-Cyclin A (Abcam, catalog number: ab38 )
    2. Anti-Cyclin D (Santa Cruz Biotechnology, catalog number: sc-753 )
    3. Anti-β-Actin (Santa Cruz Biotechnology, catalog number: sc-58673 )
  12. Tris-HCl, pH 8.0 (Thermo Fisher Scientific, catalog number: 15568025 )
  13. NaCl (Sigma-Aldrich, catalog number: S9888 )
  14. NP-40 (Abcam, catalog number: ab142227 )
  15. EDTA (Thermo Fisher Scientific, catalog number: 15576028 )
  16. β-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  17. EBC cell lysis buffer (see Recipes)
  18. Electrophoresis running buffer (see Recipes)
  19. Transfer buffer (see Recipes)

Equipment

  1. Cell culture incubator (VWR International, model: 98000-368 )
  2. Flow cytometry system (BD, model: FACSLyric )
  3. X-RAY Film processor (Konica Minolta Healthcare Americas, model: SRX-101A )

Procedure

  1. Plate H1299 cells at 20-30% confluence in a 10 cm culture dish (2 x 106-3 x 106 cells per dish) containing 10 ml of Roswell Park Memorial Institute (RPMI) 1640 Medium supplemented with 10% Fetal Bovine Serum (FBS).
  2. Incubate cells at 37 °C overnight.
  3. Add thymidine to a final concentration of 2 mM.
  4. Culture cells in a tissue culture incubator at 37 °C for 18 h.
  5. Remove thymidine by washing cells through addition of 10 ml pre-warmed 1x PBS and discard PBS.
  6. Add 10 ml of pre-warmed fresh medium and incubate for 9 h in a tissue culture incubator at 37 °C.
  7. Add second round of thymidine to a final concentration of 2 mM.
  8. Culture cells at the tissue culture incubator for another 18 h at 37 °C.
  9. Cells are now in G1/S boundary.
  10. Release cells by washing with pre-warmed 1x PBS and incubating cells in pre-warmed fresh media. Cells are collected at 0, 2, 6, 8, 10, 12, 14, 24 h for analysis of cell cycle by DNA staining using PI, or analysis of protein by Western blot using cyclin A, cyclin D and β-Actin antibodies (Figure 1).


    Figure 1. G1/S phase synchronized H1299 cells enter into normal cell cycle progression after release into fresh medium. A. Cell cycle profiles at indicated time points after release following double thymidine block. B. Expression levels of Cyclin A, Cyclin B and β-actin in cells at indicated time points after release.

Data analysis

Cell cycle was analyzed by flow cytometry with Flowjo software (Figure 1A). Cyclin A, Cyclin B and β-actin were detected by Western blotting (Figure 1B). Data are the representative of three independent experiments.

Notes

  1. Dissolve thymidine in PBS and make 100 mM stock solution.
  2. The time points for distinct cell cycle phase are dependent on the cell cycle progression time of different cell lines.
  3. Propidium Iodide (PI) is a mutagen. Gloves, protective clothing, and eyewear should be worn.

Recipes

  1. EBC cell lysis buffer
    50 mM Tris-HCl pH 7.6-8.0
    120 mM NaCl
    0.5% NP-40
    1 mM EDTA
    1 mM Na3VO4
    50 mM NaF
    1 mM β-Mercaptoethanol
  2. Electrophoresis running buffer
    25 mM Tris-HCl pH 8.3
    192 mM glycine
    0.1% SDS
  3. Transfer buffer
    25 mM Tris-HCl pH 8.3
    192 mM glycine
    10% methanol
  4. Cell culture medium
    Roswell Park Memorial Institute (RPMI) 1640 Medium
    10% Fetal Bovine Serum (FBS)

Acknowledgments

This work was supported by NIH/NCI grants R01CA193828, R01CA136534 and R01CA200905 (to X. Deng), by the Winship Research Pathology and Integrated Cellular Imaging shared resource and the Emory Comprehensive Glycomics Core (ECGC) supported by the Winship Cancer Institute of Emory University (P30CAJ 38292), by the Winship Fashion a Cure Research Scholar Award (to X. Deng), a philanthropic award provided by the Winship, and by the Winship Endowment Fund (to XD). This protocol was adapted from our previous work (Chen et al., 2018).

Competing interests

The authors have declared that no conflict of interest exists.

References

  1. Chen, G., Magis, A. T., Xu, K., Park, D., Yu, D. S., Owonikoko, T. K., Sica, G. L., Satola, S. W., Ramalingam, S. S., Curran, W. J., Doetsch, P. W. and Deng, X. (2018). Targeting Mcl-1 enhances DNA replication stress sensitivity to cancer therapy. J Clin Invest 128(1): 500-516.
  2. Graña, X. and Reddy, E. P. (1995). Cell cycle control in mammalian cells: role of cyclins, cyclin dependent kinases (CDKs), growth suppressor genes and cyclin-dependent kinase inhibitors (CKIs). Oncogene 11(2): 211-219.
  3. Ho, Y. S., Duh, J. S., Jeng, J. H., Wang, Y. J., Liang, Y. C., Lin, C. H., Tseng, C. J., Yu, C. F., Chen, R. J. and Lin, J. K. (2001). Griseofulvin potentiates antitumorigenesis effects of nocodazole through induction of apoptosis and G2/M cell cycle arrest in human colorectal cancer cells. Int J Cancer 91(3): 393-401.
  4. Koç, A., Wheeler, L. J., Mathews, C. K. and Merrill, G. F. (2004). Hydroxyurea arrests DNA replication by a mechanism that preserves basal dNTP pools. J Biol Chem 279(1): 223-230.
  5. Léger, K., Hopp, A. K., Fey, M. and Hottiger, M. O. (2016). ARTD1 regulates cyclin E expression and consequently cell-cycle re-entry and G1/S progression in T24 bladder carcinoma cells. Cell Cycle 15(15): 2042-2052.
  6. Rodríguez-Ubreva, F. J., Cariaga-Martinez, A. E., Cortés, M. A., Romero-De Pablos, M., Ropero, S., López-Ruiz, P. and Colás, B. (2010). Knockdown of protein tyrosine phosphatase SHP-1 inhibits G1/S progression in prostate cancer cells through the regulation of components of the cell-cycle machinery. Oncogene 29(3): 345-355.
  7. Sakaue-Sawano, A., Kurokawa, H., Morimura, T., Hanyu, A., Hama, H., Osawa, H., Kashiwagi, S., Fukami, K., Miyata, T., Miyoshi, H., Imamura, T., Ogawa, M., Masai, H. and Miyawaki, A. (2008). Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell 132(3): 487-498.
  8. Schvartzman, J. B., Krimer, D. B. and Van't Hof, J. (1984). The effects of different thymidine concentrations on DNA replication in pea-root cells synchronized by a protracted 5-fluorodeoxyuridine treatment. Exp Cell Res 150(2): 379-389.
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Chen, G. and Deng, X. (2018). Cell Synchronization by Double Thymidine Block. Bio-protocol 8(17): e2994. DOI: 10.21769/BioProtoc.2994.
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