Cell inactivation and DNA single- and double-strand breaks in cultured mammalian cells irradiated by a thermal neutron beam

The effects on the cellular viability and induction and repair kinetics of DNA strand breaks in HeLa cells were examined after exposure to a thermal neutron beam and compared with those after gamma-irradiation. The thermal neutron survival curve had no initial shoulder. The relative biological effectiveness (r.b.e.) value of the neutron beam was determined to be 2.2 for cell killing (ratio of D0 values), 1.8 and 0.89 for single strand breakage (ssb) by alkaline sedimentation and alkaline elution respectively, and for double strand breakage (dsb) 2.6 by neutral elution. No difference was observed between thermal neutrons and gamma-rays in the repair kinetics of ssb and dsb. It is suggested that the effect induced by the intracellular nuclear reaction, 14N(n,p)14C is mainly responsible for the high r.b.e. values observed.     

Repair of DNA single-strand and double-strand breaks in the Chinese hamster xrs 5 mutant cell line as determined by DNA unwinding

The DNA unwinding technique has been used to measure the induction and repair of DNA strand breaks by X-rays in the X-ray-sensitive (xrs 5) mutant and its parent CHO K1 line of Chinese hamster cells. Results show that frequency of induction of DNA strand breaks was the same for both cell lines. The repair of single-strand breaks was found to be slightly slower in xrs 5 over the first 20 min after X-ray exposure, but the level of repair of ssb reached after an incubation of 1h following X-ray exposure in xrs 5 was the same as in CHO K1. Our results also show that the rate of repair of DNA double-strand breaks in xrs 5 cells was clearly slower than that in CHO K1, supporting the conclusion of Kemp et al. (1984) who used the neutral elution technique, that xrs 5 is defective in the repair pathway of DNA double-strand breaks.     

Rejoining of double strand breaks in normal human and ataxia-telangiectasia fibroblasts after exposure to 60Co gamma-rays, 241Am alpha-particles or bleomycin

The rejoining of DNA double strand breaks (dsb) induced by 60Co gamma-rays, 241Am alpha-particles or bleomycin was measured by neutral filter elution. In agreement with their colony-forming ability, ataxia-telangiectasia cells (AT2BE) and normal fibroblasts exhibited similar dsb rejoining capacity following alpha-irradiation, but showed marked differences in the rejoining kinetics of dsb induced by gamma-rays or bleomycin.     

Elevated levels of DNA double-strand breaks (dsb) in restriction endonuclease-treated xrs5 cells correlate with the reduced capacity to repair dsb.

Recently we have reported the kinetics of DNA double-strand breaks (dsb) induced in electroporated mammalian (CHO) cells that had been treated with the restriction endonuclease PvuII, as measured by the filter elution assay at the non-denaturing pH of 9.6. A gradual accumulation of dsb was observed over a 24-h incubation period following the restriction endonuclease (RE) treatment and this was attributed to a competition between incision of the DNA by PvuII and dsb repair. In order to test this 'competition' hypothesis we have carried out similar experiments in the radiosensitive xrs5 mutant cell line, which has been shown to be deficient in dsb repair. The levels of dsb monitored by the non-denaturing filter elution assay in the xrs5 cell line treated with PvuII was found to be 3-4 times higher than that found for the wild-type CHO K1 cell line. Levels of dsb were also significantly raised in xrs5 cells treated with BamHI, as compared with the background levels observed in the CHO line. These data lend strong support to the competition hypothesis of simultaneous incision and repair of RE-induced dsb.     

Neutral filter elution detects only limited inhibition of double-strand break repair by 9-beta-D-arabinofuranosyladenine

Bradley and Kohn (1979) showed that the neutral filter elution technique detects DNA double-strand breaks (dsb), yet there is still some uncertainty regarding the interpretation of results obtained with this technique (viz. the curvilinear dose-response curve and the rapid repair kinetics). In this report we have investigated the effect of the nucleoside analogue 9-beta-D-arabinofuranosyladenine (ara A), a known inhibitor of dsb repair, on the DNA repair in X-irradiated Ehrlich ascites tumour cells as measured by this technique. We have compared the effect of ara A on repair in these cells with results previously obtained with the same cell line and using the neutral velocity sedimentation and DNA unwinding techniques (Bl?cher, 1982; Bryant and Bl?cher, 1982). Our results suggest that the lesions measured by the neutral elution technique are different from those measured by neutral velocity sedimentation or long-term repair measured by DNA unwinding.     

2-Chlorodeoxyadenosine inhibits the repair of DNA double-strand breaks and does not inhibit the repair of DNA single-strand breaks in X-irradiated Chinese hamster V79 cells.

The exposure of log-phase Chinese hamster V79 cells to 2-chlorodeoxyadenosine (CdA) for 3 h after X irradiation enhanced the lethal effects of X-rays in a concentration-dependent manner. The enhancement of the killing efficiency of X-rays by CdA was mainly observed in the reduction of quasi-threshold doses (Dq) of the dose-response curves. When the ability of CdA to inhibit the repair of X-ray-induced double- and single-strand breaks (dsb and ssb) of DNA was investigated by neutral- and alkaline-filter elution techniques, respectively, it was observed that 90% of dsb were rejoined in the absence of CdA within 30 min after X irradiation and 15-40% of dsb rejoining was suppressed by co-incubation of the cells with 5-10 microM of CdA for 3 h after X irradiation, whereas almost 100% of ssb were rejoined within 15 min regardless of the presence or absence of CdA. From these results it was concluded that CdA interfered exclusively with the repair of DNA dsb in X-irradiated Chinese hamster V79 cells and thereby increased the lethality of X-rays.     

125I-induced DNA double strand breaks: use in calibration of the neutral filter elution technique and comparison with X-ray induced breaks

The neutral filter elution assay, for measurement of DNA double strand breakage, has been calibrated using mouse L cells and Chinese hamster V79 cells labelled with [125I]dUrd and then held at liquid nitrogen temperature to accumulate decays. The basis of the calibration is the observation that each 125I decay, occurring in DNA, produces a DNA double strand break. Linear relationships between 125I decays per cell and lethal lesions per cell (minus natural logarithm survival) and the level of elution, were found. Using the calibration data, it was calculated that the yield of DNA double strand breaks after X-irradiation of both cell types was from 6 to 9 X 10(-12) DNA double strand breaks per Gy per dalton of DNA, for doses greater than 6 Gy. Neutral filter elution and survival data for X-irradiated and 125I-labelled cells suggested that the relationships between lethal lesions and DNA double strand breakage were significantly different for both cell types. An attempt was made to study the repair kinetics for 125I-induced DNA double strand breaks, but was frustrated by the rapid DNA degradation which occurs in cells that have been killed by the freezing-thawing process.     

Single-strand and double-strand deoxyribonucleic acid breaks produced by several bleomycin analogues

Production of single-strand breaks (ssb) and double-strand breaks (dsb) of PM2 phage DNA by several structurally related bleomycin (BLM) analogues was studied by gel electrophoresis. BLM A2 and BLM B2 produced a comparable extent of dsb. In various experiments, BLM A2 and BLM B2, at 22-41 ng/mL, degraded 50% of the form I DNA into 33-38% form II and 12-17% form III DNA. BLM B1' produced ssb and dsb at a ratio similar to that of BLM A2, but both at a rate less than half that of BLM A2. Phleomycin (PLM) D1 induced an equivalent amount of ssb but only one-eighth of dsb induced by BLM B2. The relatively lower extent dsb production for PLM D1 was observed either in borate buffer (pH 9.5) or in Tris-HCl buffer (pH 7.5) and in the presence or absence of exogenous Fe(II). Deamido-BLM A2 produced ssb to an extent approximately half that of BLM A2 and dsb to less than one-eighth that of BLM A2. The following conclusions were drawn. (1) BLM analogues produced ssb and dsb to different extents and ratios. (2) The ratio of dsb to ssb varied depending on the analogue, indicating a lack of a direct correlation between ssb and dsb. (3) The extent of ssb and dsb was affected by modifications on both the C- and N-terminal half-molecules of BLM: modification of either the N-terminal amide or the bithiazole greatly reduced dsb, whereas changes in structure or charge in the C-terminal amine affected ssb and dsb to a similar extent.     

Differences in accumulation of blunt- and cohesive-ended double-strand breaks generated by restriction endonucleases in electroporated CHO cells.

Restriction endonucleases (RE) have been used in cytogenetic studies to mimic the DNA double-strand break (dsb)-inducing action of radiation. In the experiments presented here, we have treated electroporated CHO cells with RE and have measured the resulting dsb using the filter elution technique under non-denaturing conditions (pH 9.6). PvuII, which generates blunt-ended dsb, gave rise to a significant number of measurable dsb. The frequency of the dsb induced by PvuII is shown to increase over a 3-12-h post-treatment incubation period, which implies that the RE is active in the cell for a considerable length of time. We postulate that the accumulation of dsb reflects a competition between enzymatic incision and repair of the DNA. The presence of araA, a known inhibitor of DNA synthesis, did not affect the frequency of PvuII-induced breaks indicating a lack of an inhibitory effect of araA on the repair of RE-induced dsb. Two RE which cause cohesive-ended dsb, namely BamHI and EcoRI, were found to be ineffective in giving rise to measurable dsb. Our interpretation of this is that for cohesive-ended dsb (caused by BamHI and EcoRI) the rate at which these breaks are rejoined matches or exceeds the rate of enzymatic incision and hence no dsb were observed. In the case of PvuII, the possibly slower rate of repair of blunt-ended termini would on this hypothesis result in the observed net accumulation of dsb.     

Enhanced strand break induction of DNA by resonant metal-innershell photoabsorption

We determined the number of single and double strand breaks (ssb and dsb) in a DNA-chloroterpyridine platinum complex induced by resonant photoabsorption in the L(III) innershell of a platinum atom. The number of ssb and dsb were measured in supercoiled plasmids (AG30) versus the chloroterpyridine platinum concentration, i.e., the ratio of intercalated molecules to the number of phosphate sites in DNA. A significant increase in the number of ssb and dsb was observed when the DNA contained intercalated molecules. This technique is an efficient way to induce ssb and dsb triggered by the atomic Auger effect.     

On the nature and origin of DNA strand breaks in elongating spermatids

Transient DNA strand breaks are generated in the whole population of elongating spermatids and are perfectly coincident with histone H4 hyperacetylation at chromatin-remodeling steps. Given the limited DNA repair capacity of elongating spermatids, chromatin remodeling may present a threat to genetic integrity of the male gamete. The nature of the DNA strand breakage, the enzymes involved, and the role of H4 hyperacetylation in the process must be determined to further investigate the potential mutagenic consequences of this important transition. We used the metachromatic dye acridine orange in combination with fluorescence-activated cell sorting to achieve separation of spermatids according to their condensation state. Using single-cell electrophoresis (comet assay), in both alkaline and neutral conditions, we demonstrated that double-stranded breaks account for most of the DNA fragmentation observed in purified elongating spermatids. DNA strand breaks were generated in round spermatids as a result of de novo histone hyperacetylation induced by trichostatin A, whereas an increase in endogenous DNA strand breaks was observed in elongating spermatids. Using a short-term culture of testicular cells, we demonstrated that DNA strand breaks in spermatids were abolished on incubation with two functionally different topoisomerase II inhibitors. Hence, topoisomerase II appears as the unique enzyme responsible for the transient double-stranded breaks in elongating spermatids but depends on histone hyperacetylation for its activity.     

Coping with DNA double strand breaks

The repair of DNA double strand breaks (dsb) is important for maintaining the physical and genetic integrity of the genome. Moreover, in humans it is associated with the prevention of diseases such as immune deficiencies and cancer. This review briefly explores the fundamental strategies for repairing dsb, examines how cells maximize the fidelity of dsb repair in the cell cycle and discusses the requirements for dsb repair in the context of chromatin.     

Determination of G-values for single and double strand break induction in plasmid DNA using agarose gel electrophoresis and a curve-fitting procedure

Covalently closed circular double-stranded DNA (CC) of native plasmids was used to determine the yield of single strand breaks (ssb) and double strand breaks (dsb) as a consequence of X-irradiation. One ssb transforms DNA of the CC form to the nicked circular form (NC), whereas one dsb produced either directly or from random coincidence of single strand breaks transforms DNA of the CC as well as of the NC form to linear DNA molecules (LI form). Plasmids with more than one dsb are cleaved to linear fragments. DNA (30-800 micrograms/ml) was irradiated in air-saturated sodium phosphate buffer. The different forms of DNA were separated by gel electrophoresis and their amounts measured fluorometrically using ethidium bromide. Large linear DNA fragments with the same electrophoretic mobility as the LI form were considered by using a curve-fitting procedure. From the quantitative changes of each conformation D37 values of ssb and dsb were calculated as a function of the DNA concentration. Finally G-values were calculated by competition plots. The following yields were determined: Gssb 3.4 X 10E-8 molJ-1, and Gdsb 3.3 X 10E-10 molJ-1. Gdsb refers only to those dsb produced directly. Yields are related to strand breaks without further treatment by heat or alkali.     

Reduction of intracellular glutathione content and radiosensitivity

The intracellular glutathione (GSH) content of HeLa, CHO and V79 cells was reduced by incubating the cells in growth medium containing buthionine sulphoximine or diethyl maleate (DEM). Clonogenicity, single-strand DNA breaks (ssb) and double-strand DNA breaks (dsb) were used as criteria for radiation-induced damage after X- or gamma-irradiation. In survival experiments, DEM gave a slightly larger sensitization although it gave a smaller reduction of the intracellular GSH. In general, sensitization was larger for dsb than for ssb, also the reduction of the o.e.r. was generally larger for dsb than for ssb. This may be due to the higher dose rate in case of dsb experiments resulting in a higher rate of radiochemical oxygen consumption. In general, no effect was found on post-irradiation repair of ssb and dsb.     

Delayed repair of DNA single-strand breaks does not increase cytogenetic damage

DNA damage and cytogenetic effects of ionizing radiation were investigated in Chinese hamster ovary (CHO) cells and unstimulated human peripheral blood lymphocytes. DNA damage and repair were analysed by alkaline elution under conditions that predominantly measured DNA single-strand breaks (ssb). X-radiation (2.5 Gy) induced ssb in both CHO cells and unstimulated lymphocytes, and the breaks were repaired within 30 and 90 min, respectively. This rapid repair was delayed by the poly(ADP-ribose) polymerase inhibitor, 3-aminobenzamide (3AB). The cytogenetic effects of the 3AB-induced delay in DNA repair were examined by analysing sister chromatid exchange (SCE) frequency in CHO cells and fragmentation of prematurely condensed chromosomes (PCC) in unstimulated human lymphocytes after 2.5 Gy of X-rays. Although 3AB delayed the rejoining of DNA ssb, this delay did not result in increased cytogenetic damage manifested as either SCE or fragmentation of PCC. These results indicate that the rapidly rejoining DNA ssb are not important in the production of chromosome damage.     

Alkaline step elution analysis of gamma-ray induced DNA strand breaks and repair in diploid yeast

Gamma-ray induction of DNA strand breaks and their repair was analysed in the diploid yeast strain D7 (Saccharomyces cerevisiae) by means of the alkaline step elution technique. A dose-dependent increase of DNA strand breakage was observed in the dose range 25-2000 Gy corresponding to 100 and 0.01 per cent survival. When, after exposure to gamma-irradiation, the cells were incubated for 2 h in liquid growth medium, the elution profiles reached the pattern of unirradiated controls, thus indicating the restoration of cellular DNA due to repair. The alkaline step elution analysis is found to be a useful and reproducible technique for studying the induction of DNA strand breaks and repair in yeast. In comparison with other current methods, such as alkaline sucrose gradients and DNA unwinding, this method appears to be more rapid, versatile and easier to handle.     

Effects of trypsin on X-ray-induced cell killing, chromosome abnormalities and kinetics of DNA repair in mammalian cells

When cells are trypsinized before irradiation a potentiation of X-ray damage may occur. This is known as the 'trypsin effect'. Potentiation of X-ray damage on cell killing was seen in V79 Chinese hamster cells but was marginal in Chinese hamster ovary (CHO K1) cells and not evident in murine Ehrlich ascites tumour (EAT) cells. Trypsinization did however increase the number of X-ray-induced chromosomal abnormalities in all 3 lines. To investigate the possibility that trypsin acts by digestion of proteins in chromatin, further experiments were performed to monitor DNA damage and repair. Induction of DNA breaks by X-rays was unaffected by trypsin but trypsinized EAT (suspension) cells repaired single-strand breaks (ssb) less rapidly than controls indicating an inhibitory effect of trypsin on ssb repair. However double-strand break (dsb) repair was unaffected by trypsin. It was also found that the EDTA solution in which the trypsin was dissolved also contributes to the inhibition of dsb repair. The results show that trypsinization can enhance X-ray-induced cell killing, chromosomal damage and DNA repair, the effect varying between cell lines.     

Filter elution assays for DNA damage: practical and mechanistic significance of the DNA in the filter support wash.

The alkaline and neutral (or nondenaturing) filter elution assays are popular methods for the measurement of DNA strand breakage and its repair in eukaryotic cells. In both alkaline and neutral elution, it is recommended practice to wash the filter support after removal of the filter and to analyze the DNA recovered by this procedure together with that remaining on the filter as uneluted DNA, although it is not obvious why the DNA in the filter support wash should be so interpreted. We have observed that the sum of the DNA on the filter and that recovered in the filter support wash is approximately constant when the pH of the alkaline filter elution assay for total strand breaks is increased from 12.1 to 12.6, whereas the fraction on the filter itself is markedly smaller at the higher pH. This behavior characterized DNA elution from undamaged cells, as well as from cells treated with various DNA-damaging agents. These findings are consistent with the "tug-of-war" mechanism that has been proposed for alkaline elution, but are inconsistent with the simplest mechanism of the "sieve" class. In the neutral filter elution assay for double-strand breaks, by contrast, the distribution of DNA between the filter and the filter support wash is pH-independent. This suggests that single- and double-stranded DNA segments traverse a filter by different physical mechanisms. Our observations underscore the importance of carrying out the filter support wash and the analysis of the DNA it contains as uneluted DNA in alkaline elution, while indicating that a different analysis of this DNA might be appropriate for neutral elution.     

The level of induced DNA double-strand breaks does not correlate with cell killing in X-irradiated mitotic and G1-phase CHO cells

The neutral (pH 9.6) filter elution technique was used to evaluate DNA damage induced in CHO cells irradiated at mitosis or in G1-phase under various incubation and postirradiation treatment conditions. Mitotic and G1/S border cells were more sensitive to radiation than G1 cells with respect to cell killing, but showed similar (G1/S) or lower (M) DNA elution dose--response curves. Similar cell survival and DNA/elution dose--response curves were obtained with plateau-phase cultures containing mainly G1-cells, as well as with G1 cells obtained after division of mitotic cells in either fresh or conditioned medium. However, survival of plateau-phase cells could be modified substantially by delayed-plating or postirradiation treatment with araA. These results, together with previously published observations, indicate that induction of DNA dsb cannot be invoked as an explanation for the variations in radiosensitivity observed through the cycle, or as an explanation for the formation of the survival curve shoulder. It is proposed that repair and fixation of radiation-induced DNA damage, expressed at the cell survival level as repair and fixation of alpha-PLD, are responsible for these effects.     

Differences in repair profiles of interstitial telomeric sites between normal and DNA double-strand break repair deficient Chinese hamster cells

Interstitial Telomeric Repeat Sequence (ITRS) blocks are recognized as hot spots for spontaneous and ionizing radiation-induced chromosome breakage and recombination. Background and ionizing radiation-induced DNA breaks in large blocks of ITRS from Chinese hamster cell lines were analyzed using the DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) procedure. Our results indicate an extremely alkali-sensitivity of ITRS. Furthermore, it appears that ITRS blocks exhibit a particular chromatin structure, being enriched in short unpaired DNA segments. These segments could be liable to severe topological stress in highly compacted areas of the genome resulting in their spontaneous fragility and thus explaining their alkali-sensitivity. The induction and repair kinetics of DNA single-strand breaks (ssb) and DNA double-strand breaks (dsb) induced by ionizing radiation were assessed by DBD-FISH on neutral comets using Chinese hamster cells deficient in either DNA-PKcs or Rad51C. Our results indicate that the initial rejoining rate of dsb within ITRS is slower than that in the whole genome, in wild-type cells, demonstrating an intragenomic heterogeneity in dsb repair. Interestingly, in the absence of DNA-PKcs activity, the rejoining rate of dsb within ITRS is not modified, unlike in the whole genome. This was also found in the case of Rad51C mutant cells. Our results suggest the possibility that different DNA sequences or chromatin organizations may be targeted by specific dsb repair pathways. Furthermore, it appears that additional unknown dsb repair pathways may be operational in mammalian cells.