Lack of interference of DNA single-strand breaks with the measurement of double-strand breaks in mammalian cells using the neutral filter elution assay.

In this study, the effect of DNA single strand breaks (ssb) on the neutral (pH 9.6) filter elution of DNA from Chinese hamster ovary (CHO K1) cells containing DNA double strand breaks (dsb) was investigated. Protein associated ssb were induced by the inhibition of DNA topoisomerase I with camptothecin (cpt). Protein associated dsb were introduced by treating cells with the DNA topoisomerase II poison; etoposide (VP-16). Protein associated ssb and dsb were converted to ssb and dsb by proteinase K present in the lysis solution. In some experiments dsb were generated by the restriction endonuclease Pvu II. It was found that elution of DNA in the presence and absence of ssb was similar under neutral conditions. This finding is consistent with the view that the fast component of the bi-phasic repair kinetics observed in irradiated mammalian cells with the neutral filter elution technique is not attributable to the interference of ssb with the measurement of dsb, and thus suggests that the two components of repair observed with the neutral filter elution elution technique may represent two different types of dsb or modes of repair of dsb.     

Possible correlations between cell killing, chromosome damage and DNA repair after X-irradiation

To gain information about the possible pathway from primary DNA damage to cell killing via the formation of chromosome aberrations, we have examined the effects of the DNA synthesis inhibitor ara A on survival, on the occurrence of chromosome abnormalities and on the repair of DNA strand breaks. Our results are not inconsistent with the idea that the increased expression or 'fixation' of PLD measured after treatment with ara A is a reflection of an increase in the formation of chromosome damage comprising both exchange type and deletion type aberrations. These aberrations may arise from unrepaired or misrepaired dsb in the DNA. Treatment of irradiated cells with ara A results in a larger number of residual dsb which may be partly the reason for the increase in the frequency of acentric chromosome fragments. The reasons for the increase also in the frequency of exchange aberrations in the presence of ara A are not known but one possibility is that the probability of interaction between two dsb remains high during treatment with ara A due to the strong inhibition of dsb repair, whereas in untreated controls this probability decreases steeply with time after irradiation.     

An effect of elevated postirradiation pH on the yield of double-strand breaks in DNA from irradiated bacterial cells

Exposure of DNA isolated from irradiated cells of Escherichia coli to a pH of 9.6 caused a marked increase in the yield of double-strand breaks (dsb). The dsb were measured by sedimentation analysis of E. coli chromosomal DNA using neutral sucrose gradients. After incubation for 4 hr at 37 degrees C and pH 9.6 the dsb yields were 95% and 71% higher than when incubation was at pH 7.0 for irradiation under oxic and anoxic conditions, respectively. This effect was not apparent when dsb were induced enzymatically and it was linearly related to radiation dose. After oxic irradiation, the increase in dsb at pH 9.6 was consistent with first-order kinetics over greater than 2 half-lives (t1/2 = 1.6 hr at 37 degrees C). The effect of elevated pH was largely additive to a previously reported increase in dsb yield caused by ethanol. It is proposed that the effects of elevated pH and of ethanol revealed the presence in intracellularly irradiated DNA of previously unidentified sites where both strands of the DNA were damaged as a result of single radiation events. The possible nature of the proposed sites and the relevance of these findings to the "neutral" elution technique are discussed.     

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.     

DNA conformation of Chinese hamster V79 cells and sensitivity to ionizing radiation

Chinese hamster V79 cells grown for 20 h in suspension culture form small clusters of cells (spheroids) which are more resistant to killing by ionizing radiation than V79 cells grown as monolayers. This resistance appears to be due to the greater capacity of cells grown in contact to repair radiation damage. Attempts to relate this "contact effect" to differences in DNA susceptibility or DNA repair capacity have provided conflicting results. Two techniques, alkaline sucrose gradient sedimentation and alkaline elution, show no difference in the amounts of radiation-induced DNA single-strand breakage or its repair between suspension or monolayer cells. However, using the alkali-unwinding assay, the rate of DNA unwinding is much slower for suspension cells than for monolayer cells. Interestingly, a decrease in salt concentration or in pH of the unwinding solution eliminates these differences in DNA unwinding kinetics. A fourth assay, sedimentation of nucleoids on neutral sucrose gradients, also shows a significant decrease in radiation damage produced in suspension compared to monolayer cultures. It is believed that this assay measures differences in DNA conformation (supercoiling) as well as differences in DNA strand breakage. We conclude from these four assays that the same number of DNA strand breaks/Gy is produced in monolayer and spheroid cells. However, changes in DNA conformation or packaging occur when cells are grown as spheroids, and these changes are responsible for reducing DNA damage by ionizing radiation.     

Relationship between double strand break rejoining and G2 block formation in V79 cells

Summary Caffeine and hypertonicity affect the survival of-irradiated Chinese Hamster V79 cells in different ways: while caffeine reduces the shoulder of the dose effect curve, hypertonic treatment mainly affects its final slope suggesting that different types of damage and (or) repair mechanisms are involved. Rejoining of DNA double strand breaks (dsb), as measured by neutral filter elution technique, exhibits a fast and a slow component, indicating that dsb rejoining consists of two different processes. Hypertonicity causes a temporary inhibition of the fast rejoining step but has no effect on the overall rejoining efficiency. Thus, it appears that its sensitizing effect on survival is not correlated with impaired dsb rejoining. Caffeine was found to inhibit the rejoining of dsb even after 6 h but the length of G2 phase was normal. By contrast, hypertonically treated cells are blocked in G2 but rejoining of dsb was normal. From these results we conclude that successful rejoining of part of the dsb involves arresting the cells reversibly in G2.Dedicated to Prof. G. Hotz on his 60^th birthday     

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 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.     

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.     

Quiescent human peripheral blood lymphocytes do not contain a sizable amount of preexistent DNA single-strand breaks

Sedimentation of nucleoids through neutral sucrose density gradients has shown that nucleoids isolated from phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes (PBL) sediment faster than nucleoids derived from quiescent lymphocytes, which was attributed to rejoining of DNA single-strand breaks (SSB) present in the resting cells (A.P. Johnstone, and G.T. Williams (1982) Nature (London) 300, 368). We isolated PBL from donors and determined the amount of SSB in nonradiolabeled, untreated resting and PHA-stimulated cells by applying the alkaline filter elution technique. Calibration was based on dose-dependent induction of SSB by 60Co-gamma-radiation. Quiescent cells did not contain a sizable amount of SSB. Mitogen-stimulated cells showed equally low amounts of SSB per cell. The present study indicates that the interpretation of the results obtained with the nucleoid sedimentation technique concerning the supposed rejoining of SSB in PHA-stimulated human lymphocytes is incorrect. Other, equally sensitive, techniques such as alkaline filter elution appear to be preferable for studies on DNA damage and repair.     

The relationship between radiation-induced DNA double-strand breaks and cell kill in hamster V79 fibroblasts irradiated with 250 kVp X-rays, 2.3 MeV neutrons or 238Pu alpha-particles

Using the neutral filter elution technique, the induction of DNA double-strand breaks (dsb) has been measured in 250 kVp X-irradiated V79-379A Chinese hamster cells irradiated under air or nitrogen. The dose-effect curves for induced dsb were curvilinear, mirroring cell survival curves, such that there was an approximately linear relationship between induced dsb and lethal lesions (-In (cell survival)) which was independent of oxygen. With cells irradiated with 2.3 MeV neutrons or 238Pu alpha-particles the correlations between lethal events and dsb, although also approximately linear, do not match those for X-rays. With neutrons there is approximately a 2.5-fold reduction in the level of dsb induction per lethal event. Thus either the apparently linear relationships found are spurious, and there is no general correlation between induced dsb and lethal effect, or there are qualitative differences between neutron, alpha-particle and X-ray induced dsb that give them differing probabilities of cell kill.     

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.     

The level of induced DNA double-strand breakage correlates with cell killing after X-irradiation

The neutral filter elution technique has been used to examine the relationship between X-ray-induced DNA double-strand breakage (dsb) and lethal lesions. The ratios of the different lesions produced by X-irradiation were varied by irradiation in the presence of different radiomodifiers, and in each case the same linear relationship between lethal lesions and induced DNA dsb was found. This relationship also held for cells given a hyperthermic treatment before irradiation. It is concluded that DNA dsb is probably the lethal lesion induced by ionizing radiation.     

9-beta-D-arabinofuranosyladenine increases the frequency of X-ray induced chromosome abnormalities in mammalian cells

Treatment of X-irradiated stationary Ehrlich ascites tumour cells with the DNA synthesis inhibitor beta-ara A (120 mumol/l, 30 min before and for 7 hours after irradiation) is shown to lead to a large increase in the incidence of anaphase chromosome abnormalities (anaphase bridges and fragments) at the first mitosis following irradiation. This increase is similar to the increase in cell killing observed for this cell line when treated with beta-ara A under the same conditions (Iliakis 1980). The results suggest that the increased frequency of chromosome abnormalities caused by beta-ara A may result not only from the inhibition of DNA double strand break repair, leading to additional unrepaired d.s.b. (Bryant and Bl?cher 1982) and chromosome deletions, but also from an increase in the frequency of misrepair of d.s.b. leading to exchange aberrations.     

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.     

The repair of double-strand DNA breaks correlates with radiosensitivity of L5178Y-S and L5178Y-R cells

To better understand the basis for the difference in radiosensitivity between the variant murine leukemic lymphoblast cell lines L5178Y-R (resistant) and L5178Y-S (sensitive), the production and repair of DNA damage after X irradiation were measured by the DNA alkaline and neutral elution techniques. The initial yield of single-strand DNA breaks and the rates of their repair were found to be the same in both cell lines by the DNA alkaline elution technique. Using the technique of neutral DNA elution, L5178Y-S cells exhibited slightly increased double-strand breakage immediately after irradiation, most significantly at lower doses (i.e., less than 10 Gy). Nevertheless, even at doses that yielded equal initial double-strand breakage of both cell lines, the survival of L5178Y-S cells was significantly less than that of L5178Y-R cells. When the technique of neutral DNA elution was employed to measure the kinetics of DNA double-strand break repair, both cell lines exhibited biphasic fast and slow components of repair. However, the double-strand repair rate was much lower in the radiosensitive L5178Y-S cells than in the L5178Y-R cells (T1/2 of 60 vs 16 min). This difference was more pronounced in the fast-repair component. These results suggest that the repair of double-strand DNA breaks is an important factor determining the radiosensitivity of L5178Y cells.     

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.     

Variation through the cell cycle in the dose-response of DNA neutral filter elution in X-irradiated synchronous CHO-cells

Dose-response curves for DNA neutral (pH 9.6) filter elution were obtained with synchronized CHO cells exposed to X-rays at various phases of the cell cycle. The dose response was similar in synchronized and plateau-phase G1 cells, as well as in cells that were arrested at the G1/S border using aphidicolin; it flattened as cells progressed into S phase and reached a minimum in the middle of this phase. An increase in DNA elution dose response, to values only slightly lower than those obtained with G1 cells, was observed as cells entered G2 phase. Significant alterations in the sedimentation properties of the DNA during S phase were also observed in Ehrlich ascites tumor cells using the neutral sucrose gradient centrifugation technique. A significant proportion of the DNA from S cells irradiated with 10 Gy sedimented at speeds (350S-700S) well above the maximum sedimentation speed expected for free sedimenting DNA molecules (Smax = 350S), indicating the formation of a DNA complex. DNA from G1, G1/S, or G2 + M cells sedimented as expected for free sedimenting molecules. These results indicate significant alterations in the physicochemical properties of the DNA--probably caused by DNA replication-associated alterations in DNA structure and chromatin conformation--as cells enter S phase, and are invoked to explain the observed variation in DNA elution dose response throughout the cycle. It is proposed that the formation of a complex DNA structure, resistant to the proteolytic enzymes and detergents used, affected the elution characteristics of the DNA and gave rise to the observed curvilinear DNA elution dose-response curves, as well as to the fluctuations in elution characteristics observed throughout the cell cycle.     

DNA damage in mouse and rat liver by caprolactam and benzoin, evaluated with three different methods

Benzoin and caprolactam were examined for their capability of inducing alkaline DNA fragmentation in mouse and rat liver DNA after treatment in vivo. Three different methods were used. With the alkaline elution technique we measured an effect presumably related to the conformation of the DNA coil. With a viscometric and a fluorometric unwinding method we measured an effect presumably related to the number of unwinding points in DNA. For both compounds only the alkaline elution technique was clearly positive. The results suggest that both caprolactam and benzoin can induce an important change in the conformation of the DNA coil without inducing true breaks in DNA.     

Repair of DNA double-strand breaks in colcemid-arrested mitotic Chinese hamster cells

Chinese hamster V79 cells blocked in mitosis were irradiated with 60Co gamma-rays and incubated for repair in the presence of colcemid. DNA strand breaks were measured using neutral sucrose gradient centrifugation or the alkaline unwinding technique. It was found that mitotic cells repair DNA double-strand breaks (as well as single-strand breaks) efficiently, with a rate similar to exponentially growing asynchronous cells. It is argued that the dense packing of the chromatin in the mitotic chromosome makes a recombinational repair mechanism unlikely.