Ionizing Radiation-Induced DNA Strand Breaks and γ-H2AX Foci in Cells Exposed to Nitric Oxide

  • Kai RothkammEmail author
  • Susanne Burdak-Rothkamm
Part of the Methods in Molecular Biology book series (MIMB, volume 704)


A number of studies have demonstrated that nitric oxide enhances radiosensitivity of anoxic and hypoxic cells in vitro and in vivo, and some evidence points to a role for DNA damage and repair in this phenomenon. We have recently observed that nitric oxide enhances the formation of DNA single- and double-strand breaks following ionising irradiation, measured by the alkaline comet assay and immunofluorescence microscopy for γ-H2AX .

Key words

Nitric oxide DNA strand break γ-H2AX single cell gel electrophoresis ionising radiation 



The methods described here were employed in a recent Cancer Research UK-funded study at the Gray Cancer Institute under Peter Wardman’s leadership, with support from Mick Woodcock, Lisa Folkes and Peter Johnston.


  1. 1.
    Wardman, P. (2007) Chemical radiosensitizers for use in radiotherapy. Clin Oncol (R Coll Radiol) 19, 397–417.CrossRefGoogle Scholar
  2. 2.
    Hirst, D. G., Robson, T. (2007) Nitrosative stress in cancer therapy. Front Biosci 12, 3406–3418.PubMedCrossRefGoogle Scholar
  3. 3.
    Xu, W., Liu, L. Z., Loizidou, M., Ahmed, M., Charles, I. G. (2002) The role of nitric oxide in cancer. Cell Res 12, 311–320.PubMedCrossRefGoogle Scholar
  4. 4.
    Xu, W., Liu, L., Smith, G. C., Charles, L. G. (2000) Nitric oxide upregulates expression of DNA-PKcs to protect cells from DNA-damaging anti-tumour agents. Nat Cell Biol 2, 339–345.PubMedCrossRefGoogle Scholar
  5. 5.
    Cook, T., Wang, Z., Alber, S., Liu, K., Watkins, S. C., Vodovotz, Y., Billiar, T. R., Blumberg, D. (2004) Nitric oxide and ionizing radiation synergistically promote apoptosis and growth inhibition of cancer by activating p53. Cancer Res 64, 8015–8021.PubMedCrossRefGoogle Scholar
  6. 6.
    Chien, Y., Bau, D., Jan, K. (2004) Nitric oxide inhibits DNA-adduct excision in nucleotide excision repair. Free Radic Biol Med 36, 1011–1017.PubMedCrossRefGoogle Scholar
  7. 7.
    Shao, C., Folkard, M., Michael, B. D., Prise, K. M. (2004) Targeted cytoplasmic irradiation induces bystander responses. Proc Natl Acad Sci USA 101, 13495–13500.PubMedCrossRefGoogle Scholar
  8. 8.
    Leach, J. K., Black, S. M., Schmidt-Ullrich, R. K., Mikkelsen, R. B. (2002) Activation of constitutive nitric-oxide synthase activity is an early signaling event induced by ionizing radiation. J Biol Chem 277, 15400–15406.PubMedCrossRefGoogle Scholar
  9. 9.
    Wardman, P., Rothkamm, K., Folkes, L. K., Woodcock, M., Johnston, P. J. (2007) Radiosensitization by nitric oxide at low radiation doses. Radiat Res 167, 475–484.PubMedCrossRefGoogle Scholar
  10. 10.
    Rothkamm, K., Krüger, I., Thompson, L. H., Löbrich, M. (2003) Pathways of DNA double-strand break repair during the mammalian cell cycle. Mol Cell Biol 23, 5706–5715.PubMedCrossRefGoogle Scholar
  11. 11.
    Rogakou, E., Boon, C., Redon, C., Bonner, W. (1999) Megabase chromatin domains involved in DNA double-strand breaks in vivo. J Cell Biol 146, 905–916.PubMedCrossRefGoogle Scholar
  12. 12.
    McKenna, D. J., McKeown, S. R., McKelvey-Martin, V. J. (2008) Potential use of the comet assay in the clinical management of cancer. Mutagenesis 23, 183–190.PubMedCrossRefGoogle Scholar
  13. 13.
    Rothkamm, K., Horn, S. (2009) Gamma-H2AX as protein biomarker for radiation exposure. Ann Ist Super Sanita 45, 265–271.PubMedGoogle Scholar
  14. 14.
    Qvarnstrom, O. F., Simonsson, M., Johansson, K. A., Nyman, J., Turesson, I. (2004) DNA double strand break quantification in skin biopsies. Radiother Oncol 72, 311–317.PubMedCrossRefGoogle Scholar
  15. 15.
    Barber, P. R., Locke, R. J., Pierce, G. P., Rothkamm, K., Vojnovic, B. (2007) Gamma-H2AX foci counting: image processing and control software for high content screening. Proc SPIE 6441, M1–M10.Google Scholar
  16. 16.
    Costes, S. V., Boissiere, A., Ravani, S., Romano, R., Parvin, B., Barcellos-Hoff, M. H. (2006) Imaging features that discriminate between foci induced by high- and low-LET radiation in human fibroblasts. Radiat Res 165, 505–515.PubMedCrossRefGoogle Scholar
  17. 17.
    Bocker, W., Iliakis, G. (2006) Computational methods for analysis of foci: validation for radiation-induced gamma-H2AX foci in human cells. Radiat Res 165, 113–124.PubMedCrossRefGoogle Scholar
  18. 18.
    Lovell, D. P., Omori, T. (2008) Statistical issues in the use of the comet assay. Mutagenesis 23, 171–182.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Health Protection Agency Centre for Radiation, Chemical & Environmental HazardsOxonUK
  2. 2.Histopathology DepartmentStoke Mandeville HospitalAylesburyUK
  3. 3.Radiation Protection DivisionHealth Protection AgencyDidcotUK

Personalised recommendations