Abstract
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.
Highlights
DNA is under constant threat of damage from both exogenous sources in the environment, such as solar radiation, and endogenous agents, such as oxygen free radicals [1], and it is essential that cells resolve this damage through DNA repair mechanisms
Levels of endogenous strand breaks were measured at different stages of the cell cycle in HeLa cells with % tail DNA ranging from 4.3% in G2/M to 10.7% in S3 (Figure 1A)
Analysis by analysis of variance (ANOVA) showed that there was a significant difference in the basal levels of DNA damage in different cell cycle stages (P, 0.001)
Summary
DNA is under constant threat of damage from both exogenous sources in the environment, such as solar radiation, and endogenous agents, such as oxygen free radicals [1], and it is essential that cells resolve this damage through DNA repair mechanisms. The amount of DNA damage and the efficiency with which the cell deals with this damage may vary throughout the cell cycle [2]. This may be due to a number of factors including changes in chromatin conformation that may make the DNA more susceptible to assault and reduce levels of repair [3]. In the case of ionizing radiation, the DNA damage induced includes single-strand breaks (SSBs) and double-strand breaks (DSBs) that, if not accurately repaired, can lead to cell death or chromosomal instability. In S and G2 phases of the cell cycle, DSBs are generally repaired by HR, but in G1 phase, NHEJ is more commonly used in account of the lack of the homologous sister chromatid [2]
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