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Cell Synchronization by Inhibitors of DNA Replication Induces Replication Stress and DNA Damage Response: Analysis by Flow Cytometry

  • Zbigniew DarzynkiewiczEmail author
  • H. Dorota Halicka
  • Hong Zhao
  • Monika Podhorecka
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 761)

Abstract

Cell synchronization is often achieved by inhibition of DNA replication. The cells cultured in the presence of such inhibitors as hydroxyurea, aphidicolin, or thymidine become arrested at the entrance to S phase and upon release from the block they synchronously progress through S, G2, and M. We recently reported that exposure of cells to these inhibitors at concentrations commonly used to synchronize cell populations led to phosphorylation of histone H2AX on Ser139 (induction of γH2AX) through activation of ataxia telangiectasia mutated and Rad3-related protein kinase (ATR). These findings imply that the induction of DNA replication stress by these inhibitors activates the DNA damage response signaling pathways and caution about interpreting data obtained with use of cells synchronized such way as representing unperturbed cells. The protocol presented in this chapter describes the methodology of assessment of phosphorylation of histone H2AX-Ser139, ATM/ATR substrate on Ser/Thr at SQ/TQ cluster domains as well as ataxia telangiectasia mutated (ATM) protein kinase in cells treated with inhibitors of DNA replication. Phosphorylation of these proteins is detected in individual cell immunocytochemically with phospho-specific antibody (Ab) and measured by flow cytometry. Concurrent measurement of cellular DNA content and phosphorylated proteins followed by multiparameter cytometric analysis allows one to correlate extent of their phosphorylation with cell cycle phase.

Key words

DNA repair DNA double-strand breaks flow cytometry apoptosis DNA fragmentation G1/S boundary 

Notes

Acknowledgment

This project is supported by NCI CA RO1 28 704.

References

  1. 1.
    Grdina, D. J., Meistrich, M. L., Meyn, R. E., Johnson, T. S., and White, R. A. (1987) Cell synchrony techniques. A comparison of methods. In: Techniques in Cell Cycle Analysis, Gray, J. W. and Darzynkiewicz, Z. (eds). Humana Press Inc, Clifton, NJ, pp. 367–402.CrossRefGoogle Scholar
  2. 2.
    Davis, P. K., Ho, A., and Dowdy, S. F. (2001) Biological methods for cell-cycle synchronization of mammalian cells. BioTechniques 30, 1322–1331.PubMedGoogle Scholar
  3. 3.
    Merril, G. F. (1998) Cell synchronization. Methods Cell Biol. 57, 229–249.CrossRefGoogle Scholar
  4. 4.
    Amon, A. (2002) Synchronization procedures. Methods Enzymol. 351, 457–467.PubMedCrossRefGoogle Scholar
  5. 5.
    Terasima, T., and Tolmach, L. J. (1963) Growth and nucleic acid synthesis in synchronously dividing populations of HeLa cells. Exp. Cell Res. 30, 344–362.PubMedCrossRefGoogle Scholar
  6. 6.
    Edward, K. L., Van Ert, M. N., Thornton, M., and Helmstetter, C. E. (2004) Cyclin mRNA stability does not vary during the cell cycle. Cell Cycle 3, 1057–1060.Google Scholar
  7. 7.
    Thornton, M., Edward, K. L., and Helmstetter, C. E. (2002) Production of minimally disturbed synchronous cultures of hematopoetic cells. BioTechniques 32, 1098–10100.PubMedGoogle Scholar
  8. 8.
    Huang, X., Dai, W., and Darzynkiewicz, Z. (2005) Enforced adhesion of hematopoietic cells to culture dish induces endomitosis and polyploidy. Cell Cycle 4, 801–805.PubMedCrossRefGoogle Scholar
  9. 9.
    Jakóbisiak, M., Bruno, S., Skierski, J., and Darzynkiewicz, Z. (1991) The cell cycle specific effects of lovastatin. Proc. Natl. Acad. Sci. USA 88, 3628–3632.PubMedCrossRefGoogle Scholar
  10. 10.
    Crissman, H. A., Gadbois, D. M., Tobey, R. A., and Bradbury, E. M. (1991) Transformed mammalian cells are deficient in kinase-mediated progression through the G1 phase of the cell cycle. Proc. Natl. Acad. Sci. USA 88, 7580–7585.PubMedCrossRefGoogle Scholar
  11. 11.
    Bruno, S., Ardelt, B., Skierski, J. S., Traganos, F., and Darzynkiewicz, Z. (1992) Different effects of staurosporine, an inhibitor of protein kinases, on the cell cycle and chromatin structure of normal and leukemic lymphocytes. Cancer Res. 52, 470–476.PubMedGoogle Scholar
  12. 12.
    Bruno, S., Traganos, F., and Darzynkiewicz, Z. (1996) Cell cycle synchronizing properties of staurosporine. Methods Cell Sci. 18, 99–107.CrossRefGoogle Scholar
  13. 13.
    Griffin, M. J. (1976) Synchronization of some human cell strains by serum and calcium starvation. In Vitro 12, 393–400.PubMedCrossRefGoogle Scholar
  14. 14.
    Tobey, R. A., and Crissman, H. A. (1972) Use of flow microfluorimetry in detailed analysis of effects of chemical agents on cell cycle progression. Cancer Res. 32, 2726–2731.PubMedGoogle Scholar
  15. 15.
    Holley, R. W., and Kiernan, M. A. (1968) “Contact inhibition” of cell division in 3T3 cells. Proc. Natl. Acad. Sci. USA 60, 300–305.PubMedCrossRefGoogle Scholar
  16. 16.
    Pardee, A. B., and Keyomarsi, K. (1992) Modification of cell proliferation with inhibitors. Curr. Opin. Cell Biol. 4, 186–191.PubMedCrossRefGoogle Scholar
  17. 17.
    Mitchell, B. F., and Tupper, J. T. (1977) Synchronization of mouse 3T3 and SV40 3T3 cell by way of centrifugal elutriation. Exp. Cell Res. 106, 351–355.PubMedCrossRefGoogle Scholar
  18. 18.
    Banfalvi, G. (2008) Cell cycle synchronization of animal cells and nuclei by centrifugal elutriation. Nat. Protoc. 3, 663–673.PubMedCrossRefGoogle Scholar
  19. 19.
    Cymerman, U., and Beer, J. B. (1980) Some problems in using density gradient centrifugation for synchronization of L5178Y-S cells. Neoplasma 27, 429–436.PubMedGoogle Scholar
  20. 20.
    Juan, G., Hernando, E., and Cordon-Cardo, C. (2002) Separation of live cells in different phases of the cell cycle for gene expression analysis. Cytometry 49, 170–175.PubMedCrossRefGoogle Scholar
  21. 21.
    Zhao, H., Traganos, F., Dobrucki, J., Wlodkowic, D., and Darzynkiewicz Z. (2009) Induction of DNA damage response by the supravital probes of nucleic acids. Cytometry A 75A, 510–519.CrossRefGoogle Scholar
  22. 22.
    Zhang, X., Chen, J., Davis, B., and Kiechle, F. (1999) Hoechst 33342 induces apoptosis in HL-60 cells and inhibits topoisomerase I in vivo. Arch. Pathol. Lab. Med. 123, 921–927.PubMedGoogle Scholar
  23. 23.
    Mohorko, N., Kregar-Velikonja, N., Repovs, G., Gorensek, M., and Bresjanac, M. (2005) An in vitro study of Hoechst 33342 redistribution and its effects on cell viability. Hum. Exp. Toxicol. 24, 573–580.PubMedCrossRefGoogle Scholar
  24. 24.
    Samake, S., and Smith, L. C. (1996) Synchronization of cell division in eight-cell bovine embryos produced in vitro: effects of nocodazole. Mol. Reprod. Dev. 44, 486–492.PubMedCrossRefGoogle Scholar
  25. 25.
    Harper, J. V. (2005) Synchronization of cell populations in G1/S and G2/M phases of the cell cycle. Methods Mol. Biol. 296, 157–165.PubMedGoogle Scholar
  26. 26.
    Darzynkiewicz, Z., Crissman, H., Traganos, F., and Stainkamp, J. (1982) Cell heterogeneity during the cell cycle. J. Cell Physiol. 112, 465–474.CrossRefGoogle Scholar
  27. 27.
    Urbani, L., Sherwood, S. W., and Schimke, R. T. (1995) Dissociation of nuclear and cytoplasmic cell cycle progression by drugs employed in cell synchronization. Exp. Cell Res. 219, 159–168.PubMedCrossRefGoogle Scholar
  28. 28.
    Chou, L. F., and Chou, W. G. (1999) DNA-end binding activity of Ku in synchronized cells. Cell Biol. Int. 23, 663–670.PubMedCrossRefGoogle Scholar
  29. 29.
    Piotrowska, K., Modlinski, J. A., Korwin-Kossakowski, M., and Karasiewicz, J. (2000) Effects of preactivation of ooplasts or synchronization of blastomere nuclei in G1 on preimplantation development of rabbit serial nuclear transfer embryos. Biol. Reprod. 63, 677–682.PubMedCrossRefGoogle Scholar
  30. 30.
    Tobey, R. A., Oishi, N., and Crissman, H. A. (1990) Cell cycle synchronization: reversible induction of G2 synchrony in cultured rodent and human diploid fibroblasts. Proc. Natl. Acad. Sci. USA 87, 5104–5109.PubMedCrossRefGoogle Scholar
  31. 31.
    Vogel, W., Schempp, W., and Sigwarth, I. (1978) Comparison of thymidine, fluorodeoxyuridine, hydroxyurea, and methotrexate blocking at the G1/S phase transition of the cell cycle, studied by replication patterns. Hum. Genet. 45, 193–198.PubMedCrossRefGoogle Scholar
  32. 32.
    Fox, M. H., Read, R. A., and Bedford, J. S. (1987) Comparison of synchronized Chinese hamster ovary cells obtained by mitotic shake-off, hydroxyurea, aphidicolin, or methotrexate. Cytometry 8, 315–320.PubMedCrossRefGoogle Scholar
  33. 33.
    Matherly, L. H., Schuetz, J. D., Westin, E., and Goldman, I. D. (1989) A method for the synchronization of cultured cells with aphidicolin: application to the large-scale synchronization of L1210 cells and the study of the cell cycle regulation of thymidylate synthase and dihydrofolate reductase. Anal. Biochem. 182, 338–345.PubMedCrossRefGoogle Scholar
  34. 34.
    Kues, W. A., Anger, M., Carnawath, J. W., Paul, D., Motlik, J., and Niemann, H. (2000) Cell cycle synchronization of porcine fetal fibroblasts: effects of serum deprivation and reversible cell cycle inhibitors. Biol. Reprod. 62, 412–419.PubMedCrossRefGoogle Scholar
  35. 35.
    Cohen, L. S., and Studzinski, G. P. (1967) Correlation between cell enlargement and nucleic acid and protein content of HeLa cells in unbalanced growth produced by inhibitors of DNA synthesis. J. Cell Physiol. 69, 331–339.PubMedCrossRefGoogle Scholar
  36. 36.
    Gong, J., Traganos, F., and Darzynkiewicz, Z. (1995) Growth imbalance and altered expression of cyclins B1, A. E and D3 in MOLT-4 cells synchronized in the cell cycle by inhibitors of DNA replication. Cell Growth Differ. 6, 1485–1492.PubMedGoogle Scholar
  37. 37.
    Kurose, A., Tanaka, T., Huang, X., Traganos, F., Dai, W., and Darzynkiewicz, Z. (2006) Effects of hydroxyurea and aphidicolin on phosphorylation of ATM on Ser 1981 and histone H2AX on Ser 139 in relation to cell cycle phase and induction of apoptosis. Cytometry A 69A, 212–221.CrossRefGoogle Scholar
  38. 38.
    Kurose, A., Tanaka, T., Huang, X., Traganos, F., and Darzynkiewicz, Z. (2006) Synchronization in the cell cycle by inhibitors of DNA replication induces histone H2AX phosphorylation, an indication of DNA damage. Cell Prolif. 39, 231–240.PubMedCrossRefGoogle Scholar
  39. 39.
    Rogakou, E. P., Pilch, D. R., Orr, A. H., Ivanova, V. S., and Bonner, W. M. (1998) DNA double-stranded breaks induce histone H2AX phosphorylation on serine139. J. Biol. Chem. 273, 5858–5868.PubMedCrossRefGoogle Scholar
  40. 40.
    Sedelnikova, O. A., Rogakou, E. P., Panuytin, I. G., and Bonner, W. (2002) Quantitative detection of 125IUdr-induced DNA double-strand breaks with γ-H2AX antibody. Radiation Res. 158, 486–494.PubMedCrossRefGoogle Scholar
  41. 41.
    Mah, L.-J., El-Osta, A., and Karagiannis, T. C. (2010) GammaH2AX: a sensitive molecular marker of DNA damage and repair. Leukemia 24, 679–686.PubMedCrossRefGoogle Scholar
  42. 42.
    Lavin, M. F., and Kozlov, S. (2007) ATM activation and DNA damage response. Cell Cycle 6, 931–942.PubMedCrossRefGoogle Scholar
  43. 43.
    Zhao, H., Traganos, F., and Darzynkiewicz, Z. (2010) Kinetics of the UV-induced DNA damage response in relation to cell cycle phase. Correlation with DNA replication. Cytometry A 77A, 285–293.Google Scholar
  44. 44.
    Tanaka, T., Huang, X., Halicka, H. D., Zhao, H., Traganos, F., Albino, A. P., Dai, W., and Darzynkiewicz, Z. (2007) Cytometry of ATM activation and histone H2AX phosphorylation to estimate extent of DNA damage induced by exogenous agents. Cytometry A 71A, 648–661.CrossRefGoogle Scholar
  45. 45.
    MacPhail, S. H., Banath, J. P., Yu, T. Y., Chu, E. H., Lambur, H., and Olive, P. L. (2003) Expression of phosphorylated histone H2AX in cultured cell lines following exposure to X-rays. Int. J. Radiat. Biol. 79, 351–358.PubMedCrossRefGoogle Scholar
  46. 46.
    Zhao, H., Traganos, F., and Darzynkiewicz, Z. (2008) Kinetics of histone H2AX phosphorylation and Chk2 activation in A549 cells treated with topotecan and mitoxantrone in relation to the cell cycle phase. Cytometry A 73A, 480–489.CrossRefGoogle Scholar
  47. 47.
    Kurose, A., Tanaka, T., Huang, X., Halicka, H. D., Traganos, F., Dai, W., and Darzynkiewicz, Z. (2005) Assessment of ATM phosphorylation on Ser-1981 induced by DNA topoisomerase I and II inhibitors in relation to Ser-139-histone H2AX phosphorylation, cell cycle phase and apoptosis. Cytometry A 68A, 1–9.CrossRefGoogle Scholar
  48. 48.
    Traven, A., and Heierhorst, J. (2005) SQ/TQ cluster domains: concentrated ATM/ATR kinase phosphorylation site regions in DNA-damage-response proteins. BioEssays 27, 397–407.PubMedCrossRefGoogle Scholar
  49. 49.
    Yajima, H., Lee, K. J., Zhang, S., Kobayashi, J., and Chen, B. P. (2009) DNA double-strand break formation upon UV-induced replication stress activates ATM and DNA-PKcs kinases. J. Mol. Biol. 385, 800–810.PubMedCrossRefGoogle Scholar
  50. 50.
    Tanaka, T., Halicka, H. D., Huang, X., Traganos, F., and Darzynkiewicz, Z. (2006) Constitutive histone H2AX phosphorylation and ATM activation, the reporters of DNA damage by endogenous oxidants. Cell Cycle 5, 1940–1945.PubMedCrossRefGoogle Scholar
  51. 51.
    Zhao, H., Tanaka, T., Halicka, H. D., Traganos, F., Zarebski, M., Dobrucki, J., and Darzynkiewicz, Z. (2007) Cytometric assessment of DNA damage by exogenous and endogenous oxidants reports the aging-related processes. Cytometry A 71A, 905–914.CrossRefGoogle Scholar
  52. 52.
    Kajstura, M., Halicka, H. D., Pryjma, J., and Darzynkiewicz, Z. (2007) Discontinuous fragmentation of nuclear DNA during apoptosis revealed by discrete “sub-G1” peaks on DNA content histograms. Cytometry A 71A, 125–131.CrossRefGoogle Scholar
  53. 53.
    Tanaka, T., Kurose, A., Huang, X., Dai, W., and Darzynkiewicz, Z. (2006) ATM kinase activation and histone H2AX phosphorylation as indicators of DNA damage by DNA topoisomerase I inhibitor topotecan and during apoptosis. Cell Prolif. 39, 49–60.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Zbigniew Darzynkiewicz
    • 1
    Email author
  • H. Dorota Halicka
    • 1
  • Hong Zhao
    • 1
  • Monika Podhorecka
    • 2
  1. 1.Department of PathologyBrander Cancer Research Institute, New York Medical CollegeValhallaUSA
  2. 2.Department of Hemato-Oncology and Bone Marrow TransplantationMedical UniversityLublinPoland

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