Comet sensitivity in assessing DNA damage and repair in different cell cycle stages.

The comet assay is a sensitive tool for estimation of DNA damage and repair at the cellular level, requiring only a very small number of cells. In comparing the levels of damage or repair in different cell samples, it is possible that small experimental effects could be confounded by different cell cycle states in the samples examined, if sensitivity to DNA damage, and repair capacity, varies with the cell cycle. We assessed this by arresting HeLa cells in various cell cycle stages and then exposing them to ionizing radiation. Unirradiated cells demonstrated significant differences in strand break levels measured by the comet assay (predominantly single-strand breaks) at different cell cycle stages, increasing from G(1) into S and falling again in G(2). Over and above this variation in endogenous strand break levels, a significant difference in susceptibility to breaks induced by 3.5 Gy ionizing radiation was also evident in different cell cycle phases. Levels of induced DNA damage fluctuate throughout the cycle, with cells in G(1) showing slightly lower levels of damage than an asynchronous population. Damage increases as cells progress through S phase before falling again towards the end of S phase and reaching lowest levels in M phase. The results from repair experiments (where cells were allowed to repair for 10 min after exposure to ionizing radiation) also showed differences throughout the cell cycle with G(1)-phase cells apparently being the most efficient at repair and M-phase cells the least efficient. We suggest, therefore, that in experiments where small differences in DNA damage and repair are to be investigated with the comet assay, it may be desirable to arrest cells in a specific stage of the cell cycle or to allow for differential cycle distribution.

[1]  V. Bohr,et al.  Gene-specific and strand-specific DNA repair in the G1 and G2 phases of the cell cycle , 1995, Molecular and cellular biology.

[2]  J. Rice.  Mathematical Statistics and Data Analysis , 1988 .

[3]  P. Lansdorp,et al.  Mouse but not human embryonic stem cells are deficient in rejoining of ionizing radiation-induced DNA double-strand breaks. , 2008, DNA repair.

[4]  J. Ward,et al.  The yield of DNA double-strand breaks produced intracellularly by ionizing radiation: a review. , 1990, International journal of radiation biology.

[5]  E. Dikomey,et al.  Human and rodent cell lines showing no differences in the induction but differing in the repair kinetics of radiation‐induced DNA base damage , 2004, International journal of radiation biology.

[6]  D. McArt,et al.  Systematic random sampling of the comet assay. , 2009, Mutagenesis.

[7]  Marco Foiani,et al.  Regulation of DNA repair throughout the cell cycle , 2008, Nature Reviews Molecular Cell Biology.

[8]  J. Hain,et al.  Increased G2 chromosomal radiosensitivity in cancer patients: the role of cdk1/cyclin-B activity level in the mechanisms involved. , 2000, International journal of radiation biology.

[9]  Karen H. Almeida,et al.  A unified view of base excision repair: lesion-dependent protein complexes regulated by post-translational modification. , 2007, DNA repair.

[10]  G. Kraft,et al.  The influence of radiation quality on the formation of DNA breaks. , 1989, Advances in space research : the official journal of the Committee on Space Research.

[11]  R. Wood,et al.  UV-light-induced mutations in synchronous CHO cells. , 1980, Mutation research.

[12]  P. Olive,et al.  Phosphorylation of histone H2AX as a measure of radiosensitivity. , 2004, International journal of radiation oncology, biology, physics.

[13]  P. Olive,et al.  Heterogeneity in DNA damage using the comet assay , 2005, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[14]  G. Almouzni,et al.  When repair meets chromatin , 2002, EMBO reports.

[15]  P. Olive,et al.  Detection of subpopulations resistant to DNA-damaging agents in spheroids and murine tumours. , 1997, Mutation research.

[16]  H. McNulty,et al.  Global DNA and p53 region-specific hypomethylation in human colonic cells is induced by folate depletion and reversed by folate supplementation. , 2006, The Journal of nutrition.

[17]  Shiaw-Yih Lin,et al.  DNA damage and breast cancer. , 2011, World journal of clinical oncology.

[18]  Takamitsu A Kato,et al.  Comparison of the induction and disappearance of DNA double strand breaks and gamma-H2AX foci after irradiation of chromosomes in G1-phase or in condensed metaphase cells. , 2008, Mutation research.

[19]  S. Graubmann,et al.  Induction and repair of DNA strand breaks in CHO-cells irradiated in various phases of the cycle. , 1983, International journal of radiation biology and related studies in physics, chemistry, and medicine.

[20]  M. Smerdon,et al.  DNA repair and the role of chromatin structure. , 1991, Current opinion in cell biology.

[21]  C. J. Grant.  Chromosome aberrations and the mitotic cycle in Trillium root tips after x-irradiation. , 1965, Mutation research.

[22]  Jiri Bartek,et al.  Cell-cycle checkpoints and cancer , 2004, Nature.

[23]  Steffen Loft,et al.  Variation in assessment of oxidatively damaged DNA in mononuclear blood cells by the comet assay with visual scoring. , 2008, Mutagenesis.

[24]  U. Plappert,et al.  Does physical activity induce DNA damage? , 1994, Mutagenesis.

[25]  A. Collins,et al.  The comet assay for DNA damage and repair , 2004, Molecular biotechnology.

[26]  G. Wasson,et al.  The use of the comet assay in the study of human nutrition and cancer. , 2008, Mutagenesis.

[27]  E. Friedberg,et al.  Influence of DNA repair deficiencies on the UV sensitivity of yeast cells in different cell cycle stages. , 1990, Mutation research.

[28]  K. Lohman,et al.  DNA damage and breast cancer risk. , 2003, Carcinogenesis.

[29]  John D. Potter,et al.  Flow cytometric analysis of the cell cycle phase specificity of DNA damage induced by radiation, hydrogen peroxide and doxorubicin. , 2002, Carcinogenesis.

[30]  A. Collins,et al.  DNA damage in synchronized HeLa cells irradiated with ultraviolet. , 1979, Biophysical journal.

[31]  P. Olive,et al.  Detection of DNA double-strand breaks through the cell cycle after exposure to X-rays, bleomycin, etoposide and 125IdUrd. , 1993, International journal of radiation biology.

[32]  P. Hornsby.  Replicative senescence of human and mouse cells in culture: significance for aging research , 2003, Mechanisms of Ageing and Development.

[33]  P. Olive,et al.  DNA double-strand breaks measured in individual cells subjected to gel electrophoresis. , 1991, Cancer research.

[34]  B. Tzang,et al.  Function and sequence analyses of tumor suppressor gene p53 of CHO.K1 cells. , 1999, DNA and cell biology.

[35]  G. McKerr,et al.  The bromodeoxyuridine comet assay: detection of maturation of recently replicated DNA in individual cells. , 1999, Cancer research.

[36]  A. Collins,et al.  Effects of DNA replication inhibitors on UV excision repair in synchronised human cells. , 1982, Nucleic acids research.

[37]  R. Hagen,et al.  Nicotine induces DNA damage in human salivary glands. , 2009, Toxicology letters.

[38]  A. Collins,et al.  The comet assay in human biomonitoring: gene-environment interactions. , 2008, Mutagenesis.

[39]  N. Oleinick,et al.  Gamma radiation as a probe of chromatin structure: damage to and repair of active chromatin in the metaphase chromosome. , 1984, Radiation research.