Induction and rejoining of radiation-induced DNA single-strand breaks: “tail moment” as a function of position in the cell cycle

doi:10.1016/0921-8777(93)90010-E

Mutation Research/DNA Repair

Volume 294, Issue 3, October 1993, Pages 275-283

Mutation Research/DN...

https://doi.org/10.1016/0921-8777(93)90010-E Get rights and content

Abstract

Previous results using a variety of methods have shown no significant difference in induction or rejoining of radiation-induced DNA single-strand breaks (SSBs) as a function of cell-cycle position. However, concurrent of DNA damage and cell-cycle position measured using the alkaline comet assay indicates cycle-dependent differences in “tail momen” which has been shown to be a measure of SSBs. Unirradiated S phase cells showed a significantly higher tail moment presumably as a result of the presence of active replication sites. The time required to repair half of the damage appeared to be consistently shorter for G₁ cells than for cells in other phases of the cell cycle (3.7 min versus approximately 5 min). Although early kinetics of rejoining (within 5 min) was identical for CHO, V79 and repair-deficient TK6 cells, TK6 cells subsequently repaired fewer breaks than did CHO or V79 cells. S phase TK6 cells rejoined SSBs more slowly than cells in the other phases; differences in rejoining rates could not be explained by the presence of a subset of slowly repairing TK6 cells. We concluide that the comet assay detects subtle differences in the tail moment when cells in different phases of the cell cycle are irradiated and allowed to repair damage. The biological importance of these observations remains to be determined.

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Next, we analyzed the cell cycle profiles of hESC in the different conditions. Okazaki fragments also can be misinterpreted as DNA damage using the COMET assay (Olive and Banáth, 1993), so an increase of cells in S phase could have influenced the interpretation of the results described above. However, the S-phase fraction remained constant for all conditions tested, ruling out this bias (Figure 3B; individual profiles are listed in Figure S2).
Human embryonic stem cells (hESC) show great promise for clinical and research applications, but their well-known proneness to genomic instability hampers the development to their full potential. Here, we demonstrate that medium acidification linked to culture density is the main cause of DNA damage and genomic alterations in hESC grown on feeder layers, and this even in the short time span of a single passage. In line with this, we show that increasing the frequency of the medium refreshments minimizes the levels of DNA damage and genetic instability. Also, we show that cells cultured on laminin-521 do not present this increase in DNA damage when grown at high density, although the (long-term) impact on their genomic stability remains to be elucidated. Our results explain the high levels of genome instability observed over the years by many laboratories worldwide, and show that the development of optimal culture conditions is key to solving this problem.
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Overall chromatin loop organization of the mutants was assessed and compared with that of the progenitor wild type strain by Chromatin Comet Assay (ChCA). Representative ChCA images are shown in Fig. 3A. Chromatin Comet Assay data quantification was done by automatic measurement of the parameter R by means of the TriTek CometScoreTM freeware v1.5 software (Georgieva et al., 2012; Olive and Banath, 1993; Olive et al., 1990). The parameter R allows quantification of general chromatin loop lengths in the nuclei (Georgieva et al., 2012; Georgieva et al., 2008).
Chromatin structure promotes important epigenetic mechanisms that regulate cellular fate by organizing, preserving and controlling the way by which the genetic information works. Our understanding of chromatin and its functions is sparse and not yet well defined. The uncertainty comes from the complexity of chromatin and is induced by the existence of a large number of nuclear proteins that influence it. The intricate interaction among all these structural and functional nuclear proteins has been under extensive study in the recent years. Here, we show that Saccharomyces cerevisiae linker histone physically interacts with Arp4p (actin-related protein 4) which is a key subunit of three chromatin modifying complexes – INO80, SWR1 and NuA4. A single – point mutation in the actin – fold domain of Arp4p together with the knock-out of the gene for the linker histone in S. cerevisiae severely abrogates cellular and nuclear morphology and leads to complete disorganizing of the higher levels of chromatin organization.
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Image analysis was performed using the Komet software (Kinetics Imaging) on 100 randomly selected cells (50 cells each of two replicate slides). DNA damage was quantified by the increase of Olive Tail Moment (OTM, arbitrary unit) (Olive and Banáth, 1993). A nonlinear regression analysis was performed on the normalized distribution frequencies using a χ2 function with TableCurve 2D®.
To evaluate the genotoxic risk that contaminated sediment could constitute for benthic organisms, three contaminated (VA, VC and VN) and one uncontaminated (RN) sediment samples were collected in the Berre lagoon (France). Potentially bioavailable contaminants in sediments were obtained using sediment extraction with synthetic seawater adjusted to pH 4 or pH 6, simulating the range of pH prevailing in the digestive tract of benthic organisms. The genotoxic activities of these extracts were evaluated by three short-term bioassays: the Salmonella mutagenicity test using the Salmonella typhimurium strain TA102, the alkaline comet assay and the micronucleus assay on the Chinese Hamster Ovary cells CHO-K1. Results of the Salmonella mutagenicity assay detected a mutagenic response for RN extract at pH 6, and for VA extract at pH 4. Results of the comet and micronucleus assays detected low genotoxic/clastogenic activities for VA and VC extracts at pH 6 and higher activities for RN, VA and VC extracts at pH 4. To identify if metals (Al, Fe, Mn, As, Cd, Co, Cr, Cu, Hg, Ni, Pb and Zn) were involved in these genotoxic activities, their concentrations were determined in the extracts, and their speciation was assessed by thermodynamic calculations. Results showed that extracts from sites VA, VC and VN generally presented the highest trace metal contents for both extractants, while the site RN presented lower trace metal contents but the highest Fe and Mn contents. Thermodynamic calculations indicated that Fe, Mn, As and in a lower extend Co, Ni and Zn were mainly present under free forms in extracts, and were consequently, more likely able to induce a genotoxic effect. Results globally showed no correspondence between free metal contents and genotoxic activities. They suggested that these positive results could be due to uncharacterized compounds, acting as direct genotoxic agents or enhancing the genotoxic properties of analyzed metals.
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The r parameter: The parameter r was defined by the distance [in arbitrary units] between the center of mass of the comet head and the center of mass of the comet tail according to Olive et al. [28]. Tail moment (TM): Tail moment (TM) was calculated by multiplying the percentage of DNA in the tail—IODtail (integrated optical density of the tail) by the parameter r [29]. DNA percentage in the tail was calculated as a part of the whole DNA content in the comet, which was taken as 100%.
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Olive et al. (1992) have utilized this advantage and introduced a new technique for measuring DNA damage at different positions of the cell cycle, basing on the tail moment versus the comet fluorescence bivariate analysis. Later, this technique has been utilized to measure the effects of ionizing radiation on DNA damage at different positions of the cell cycle (Olive and Banath, 1995,1993a,1993b). The results of these studies revealed a lower tail moment and DNA migration from S-phase cells as compared to G1 and G2-cells.
The aim of the study was to develop a protocol for both estimating cell cycle position and the level of ionizing radiation-induced DNA dsb using the neutral comet assay. Using DNA histograms, cell cycle positions were determined for human dermal fibroblasts. The tail intensity was used to estimate the level of DNA damage induced by X-rays, at different positions of the cell cycle. The results of tail intensity versus DNA content bivariate analysis of exponentially growing cells showed a remarkable decrease in tail intensity with transition of cells from G1 to S-phase and increases slightly with transition to G2/M phase. This effect is observed at all doses including unirradiated cells, indicating that the effect is not caused by X-rays and the comet assay based on the current tail parameters is not relevant to measure DNA damage at various stages of the cell cycle. The results of dose response curves showed a linear decrease in the comet fluorescence with the X-ray dose. This observation provides a basis for estimating the fraction of damaged DNA, based on the fluorescence decrement induced by ionizing radiation. The results of this new approach showed a linear increase in DNA damage with dose, at various stages of the cell cycle, with rates, which vary in the following order G0>G2/M>S/G1 cells.
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Induction and rejoining of radiation-induced DNA single-strand breaks: “tail moment” as a function of position in the cell cycle

Abstract

J. Biol. Chem.

Mutation Res.

Biochem. Biophys. Res. Commun.

Exp. Cell Res.

Radiation-induced single-strand breaks in DNA determined by rate of alkaline strand separation and hydroxylapatite chromatography: An alternative to velocity sedimentation

Int. J. Radiat. Biol.

The regulation of chromosome replication

J. Pathol.

Survival and cycle-progression delay of human lymphoma cells in vitro exposed to VP-16-213

Cancer Treat. Rep.

The Radiobiology of Cultured Mammalain Cells

DNA double-strand break rejoining deficiency in TK6 and other B lymphoblast cell lines

Radiat. Res.

DNA single-strand rejoining in two pairs of cell-lines showing the same and different sensitivities to X-rays

Int. J. Radiat. Biol.