The effect of post-irradiation anisotonic treatment on cell survival and repair of DNA damage

V79 379A cells were irradiated and then exposed to anisotonic PBS for 20 min. This enhanced the radiation effect resulting from the fixation of potentially lethal damage. The induction of DNA single- and double-strand breaks is not increased by this treatment. Anisotonic treatment delayed the onset of repair of DNA damage. However when cells were returned to normal medium, they repaired the damage to a similar extent as cells not exposed to the anisotonic treatment. We suggest that the fixation of damage by post-irradiation anisotonic treatment is mediated through an increased probability of misrepair of DNA damage due to the delay in the onset of repair. This is supported by the observation that there is a reduced effect of post-irradiation anisotonic treatment on cells that have a markedly reduced ability to repair double-strand breaks.     

Reduction of Intracellular Glutathione Content and Radiosensitivity

Summary

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 γ-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.     

Differential DNA double strand break fixation dependence on poly(ADP-ribosylation) in L5178Y and CHO cells

PURPOSE: To investigate the role of poly(ADP-ribosylation) in DNA double-strand break repair and fixation in murine lymphoma L5178Y (LY) sublines, LY-R and LY-S, and a pair of Chinese hamster ovary lines: wild-type and mutant xrs6 cells, that have differences in repair competence and degree of radiosensitization with poly(ADP-ribosylation) inhibitors. MATERIALS AND METHODS: Cells (asynchronous, logarithmic phase) were pre-incubated with 2 mM aminobenzamide at 37 or 25 degrees C, X-irradiated with 10 Gy and allowed to repair DNA breaks for 15, 60 and 120 min at 37 or 25 degrees C. The remaining double-strand break were estimated by the neutral comet assay. RESULTS: At 37 degrees C, no effect of AB treatment on the repair kinetics was observed either in xrs6 or Chinese hamster ovary (wild-type) cells. In contrast, aminobenzamide decreased the repair of double-strand break in the LY-S line but not the LY-R line, in agreement with the previously observed radiosensitization of LY cells by poly(ADP-ribosylation) inhibition. However, double-strand break rejoining in the repair competent cell lines, Chinese hamster ovary and LY-R, also was affected by aminobenzamide when the post-irradiation incubation was carried out at 25 degrees C. Analysis of these results together with earlier data on LY-S cells have been interpreted in terms of Radford's model of radiation damage fixation. CONCLUSION: The reported results indicate that poly(ADP-ribosylation) can be an important modulator of the conversion of DNA damage to lethal events.     

Induction and Repair of DNA Strand Breaks in Cultured Mammalian Cells Following Fast Neutron Irradiation

Summary

Induction and repair of DNA breaks following irradiation with NIRS cyclotron neutrons were studied in cultured mammalian cells (L5178Y) in comparison to those following γ-rays.

The yield of the total single-strand breaks, 3′OH terminals and sites susceptible to S₁ endonuclease following fast neutrons was found to be approximately 50 per cent of that following γ-irradiation. On the other hand, the yield of double-strand breaks was slightly higher after fast neutrons than after γ-rays. The percentage of the total single-strand breaks remaining unrejoined at 3 hours after post-irradiation incubation was found to be distinctly higher after the fast neutrons than after γ-rays. The neutron-induced damage appears to carry a higher proportion of alkali-labile lesions compared to γ-rays.

It was concluded that the increase in the yield of double-strand breaks and of unrejoinable breaks is responsible for a high r.b.e. of the cyclotron neutrons.     

Inhibition of repair of X-ray-induced DNA double-strand breaks in human lymphocytes exposed to sodium butyrate

Purpose : Sodium butyrate is known to inhibit histone deacetylase enzymes and to enhance the frequencies of X-ray-induced dicentrics and rings in human lymphocytes. In this study an investigation was made of the mechanisms underlying this enhancement by assessing the effect of sodium butyrate on the extent of X-ray-induced DNA damage and its repair in human peripheral blood lymphocytes. Methods and materials : Unstimulated G 0 lymphocytes were pre-treated for 24h with sodium butyrate at a final concentration of 5 mM, irradiated with different doses of X-rays and then analysed for different endpoints either immediately or after different repair periods. The frequencies of DNA strand breaks were determined biochemically using nucleoid sedimentation, alkaline elution and immunochemical analysis as well as cytogenetically using the premature chromosome condensation (PCC) technique. Results : The results show that sodium butyrate pre-treatment does not lead to a significant increase of DNA double- or single-strand breaks nor to an increase of alkali labile base damage in G 0 lymphocytes. Moreover, sodium butyrate treatment had no effect on the initial frequency of chromosome breaks. However, PCC analysis clearly showed that the presence of sodium butyrate post-irradiation severely inhibited DNA double-strand break (DSB) repair, which most likely accounts for the increase in X-ray-induced chromosome aberrations. Conclusions : Sodium butyrate treatment leading to changes in histone acetylation and increased accessibility of chromatin had no effect on the initial levels of X-ray-induced DNA damage. However, sodium butyrate may affect either the chromatin configuration or the enzymatic activities that play a key role in the repair of DSB.     

Correlation between non-repairable DNA lesions and fixation of cell damage by hypertonic solutions in Chinese hamster cells

In Chinese hamster HA-1 cells, killing induced by gamma-rays was enhanced by post-irradiation treatment with hypertonic solution (0.5 mol/l NaCl in phosphate buffered saline, pH 7.2) for 20 min. The initial DNA double-strand breaks (dsb) induced by gamma-rays were repaired during post-irradiation treatment with hypertonic solution. However, hypertonic treatment following gamma-irradiation enhanced the frequency of non-repairable dsb, as compared with the frequency after incubation at 37 degrees C following gamma-irradiation. Hypertonic treatment did not affect the initial half-time for rejoining of dsb. Hypertonic treatment did not enhance cell killing, nor did it enhance the non-repairable dsb when the irradiated cells were incubated at 37 degrees C for 2 h. These results suggest that fixation of gamma-ray-induced potentially lethal damage by hypertonic treatment results from inhibition of the rejoining of dsb.     

Cellular Responses to Ionizing Radiation: Effects of Interrupting DNA Repair with Chemical Agents

Summary

This review is concerned with the influence of different classes of chemical agents on cellular repair of DNA damage induced by ionizing radiation. Single-strand break rejoining is little affected by inhibitors of DNA synthesis; however, such inhibitors do lead to a persistence of double-strand breaks in the DNA, and this correlates with an enhancement of chromosome aberrations and cell killing. Experiments with antagonists of topoisomerase II suggest an intriguing role for this DNA unwinding enzyme in double-strand break repair. Interference with poly(ADP-ribose) synthesis, by means of the inhibitor 3-aminobenzamide, does not have a clear-cut effect on recovery from ionizing radiation damage. Various substances (for example, caffeine and trypsin) affect DNA repair via a modulation of the cell cycle, altering the time available to the cell for repairing potentially lethal DNA damage before such damage is ‘fixed’ by the process of DNA replication. Finally, disturbing cellular energy metabolism, and depressing the level of ATP, can inhibit the repair of radiation damage.     

Repair of DNA strand breaks at hyperthermic temperatures in Chinese hamster ovary cells.

The repair of DNA double-strand breaks was measured by pH 7.2 filter elution in cells incubated at 25-45 degrees C either before or after X-irradiation. Exposure to 45 degrees C for 15 minutes immediately prior to X-irradiation significantly increased both the half-time for DNA double-strand break closure and the number of DNA double-strand breaks remaining in nuclear DNA 180 minutes after irradiation. Exposure to temperatures between 41 and 45 degrees C immediately after X-irradiation accelerated DNA double-strand break closure and resulted in no increase in the number of DNA double-strand breaks remaining in the cell's genome 180 minutes after irradiation. The results indicate either that the radiosensitization produced by the administration of hyperthermic temperatures before and after irradiation result from two characteristically different molecular mechanisms, or that neither the rate of DNA strand break closure nor the number of DNA strand breaks remaining in nuclear DNA after irradiation accurately predict hyperthermic radiosensitization. These conclusions assume that no DNA strand breaks are below the resolution of this DNA damage assay and that a comparison between cytotoxicity and DNA repair after exposure to high radiation doses is valid.     

Survival of Saccharomyces Cerevisiae after Treatment with the Restriction Endonuclease Alu I

Summary

Treatment of yeast cells proficient in the repair of radiation damage (Saccharomyces cervisiae) with the restriction endonuclease Alu I leads to a positive dose—effect relationship between inactivation level and enzyme concentration. The data suggest an uptake of the active restriction enzyme into the cells and a relationship between induction of DNA double-strand breaks and cell killing.     

Inhibition of repair of X-ray-induced DNA double-strand breaks in human lymphocytes exposed to sodium butyrate

PURPOSE: Sodium butyrate is known to inhibit histone deacetylase enzymes and to enhance the frequencies of X-ray-induced dicentrics and rings in human lymphocytes. In this study an investigation was made of the mechanisms underlying this enhancement by assessing the effect of sodium butyrate on the extent of X-ray-induced DNA damage and its repair in human peripheral blood lymphocytes. METHODS AND MATERIALS: Unstimulated G0 lymphocytes were pretreated for 24h with sodium butyrate at a final concentration of 5 mM, irradiated with different doses of X-rays and then analysed for different endpoints either immediately or after different repair periods. The frequencies of DNA strand breaks were determined biochemically using nucleoid sedimentation, alkaline elution and immunochemical analysis as well as cytogenetically using the premature chromosome condensation (PCC) technique. RESULTS: The results show that sodium butyrate pretreatment does not lead to a significant increase of DNA double- or single-strand breaks nor to an increase of alkali labile base damage in G0 lymphocytes. Moreover, sodium butyrate treatment had no effect on the initial frequency of chromosome breaks. However, PCC analysis clearly showed that the presence of sodium butyrate post-irradiation severely inhibited DNA double-strand break (DSB) repair, which most likely accounts for the increase in X-ray-induced chromosome aberrations. CONCLUSIONS: Sodium butyrate treatment leading to changes in histone acetylation and increased accessibility of chromatin had no effect on the initial levels of X-ray-induced DNA damage. However, sodium butyrate may affect either the chromatin configuration or the enzymatic activities that play a key role in the repair of DSB.     

Radiation-induced Potentially Lethal Damage: DNA Lesions Susceptible to Fixation

Summary

The various postirradiation incubation conditions reported to uncover potentially lethal damage (PLD) induced by ionizing radiation are outlined and critically discussed. The process of damage fixation is the most characteristic determinant in distinguishing between PLD and other forms of damage (lethal or non-lethal). The results compiled indicate the induction of two forms of PLD (termed alpha-and beta-PLD). Evidence is presented that repair and fixation of alpha-PLD may underlie the variation in radiosensitivity observed through the cycle. Beta-PLD appears to be sensitive only to postirradiation treatment in anisotonic salt solutions. Results obtained at the DNA and chromosome level, under conditions allowing repair or causing fixation of PLD, are reviewed and combined together to devise a qualitative model that outlines a possible sequence of events from damage fixation at the DNA level, to damage fixation at the chromosome level and, ultimately, to cell death. It is suggested that damage uncovered at the cellular level as potentially lethal, comprises DNA dsb (single, pairs or groups) and that fixation is mediated by forces transmitted to the double helix through alteration (local or general) in chromatin conformation. Changes in chromatin conformation are caused either as a result of the cell's progression through the cycle or in response to a postirradiation treatment. The fixation process leads to the induction of chromosome aberrations. The validity of the concept of PLD in in vivo systems is shown, and the possible importance of PLD repair in radiation therapy is reviewed. The concept of PLD is compared to the concept of sublethal damage, and the possibility that similar molecular lesions underlie both types of damage is discussed.     

Transient DNA Double-Strand Breakage in 4-Hydroperoxyifosfamide-Treated Mammalian Cells in Vitro Does Not Interact with the Rejoining of Radiation-Induced Double-Strand Breaks

BACKGROUND: 4-Hydroxyifosfamide is the primary metabolite in vivo of the bifunctional alkylating cytostatic ifosfamide. DNA interstrand cross-linking induced by bifunctional alkylators may be repaired through an intermediate with unligated repair patches on both strands which should uncover analytically as DNA double-strand breaks and allow to measure the rejoining kinetic of this repair intermediate. Additionally, the combined effects of drug and radiation treatment on rejoining of double-strand breaks was investigated with two different mammalian cell lines. MATERIAL AND METHODS: V79 (rodent fibroblasts) and Widr (human colon carcinoma) cells were treated for 2 hours with 4-hydroperoxyifosfamide which rapidly decays to 4-hydoxyifosfamide in aqueous solution or were exposed in combination with ionizing radiation followed by incubation for repair with or without the drug. DNA double-strand breakage was measured by pulsed-field electrophoresis. RESULTS: The 2 hours 4-hydroperoxyifosfamide treatment (30 micrograms/ml) resulted in a pronounced DNA fragmentation that, 2-4 hours after drug removal, declined with an estimated half-live of about 4 hours for both cell lines. When the cells were additionally irradiated with 10 Gy given in the middle of drug exposure, the residual fragmentation after 12 or 24 hours incubation for repair was only marginally increased, roughly corresponding to the respective value after radiation, alone. A continuous drug exposure of 6 hours (at 10 micrograms/ml) resulted in a fragmentation that was independent of a preirradiation with a high dose of 30 Gy, immediately before drug addition. CONCLUSIONS: The present data support the idea that unligated/unrejoined double-stranded DNA ends are generated during the repair of lesions from bifunctional alkylators. The rate of subsequent rejoining is in the order of magnitude of the slow rejoining of radiation-induced double-strand breaks. Processing of double-stranded DNA damage from either 4-hydroperoxyifosfamid or radiation exposure is apparently unaffected in combined treatments.     

Time-lapse Microscopy and DNA Double-strand Breakage of Chinese Hamster Cells Under Conditions Promoting or Preventing PLD Repair after Irradiation with 60Co γ Rays

Summary

We have confirmed previous time-lapse microscopic observations (Suzuki 1985) using Chinese hamster hai and V79 cells. The proportion of non-dividing to dividing cells was the same under conditions of potentially lethal damage (PLD) repair and non-PLD repair after irradiation with ⁶⁰Co γ-rays. This finding suggested that the radiation-induced damage to cellular DNA was similarly repaired so that cells undergo a first division to the same extent under both sets of conditions. In fact, direct measurement of double-strand breaks (dsb) in DNA from the two cell lines by the neutral elution technique showed no differences either in the initial amount of damage or in the time-course under conditions promoting or preventing PLD repair. These results indicate that PLD repair (i.e. an increase in cell survival) cannot be simply explained by a difference in the repair of dsb, but it can perhaps be explained by assuming that DNA damage is repaired with either fewer or more errors in the presence or absence of PLD repair respectively.     

Production and Repair of Chromosome Damage in an X-ray Sensitive CHO Mutant Visualized and Analysed in Interphase Using the Technique of Premature Chromosome Condensation

Summary

Production and repair of chromosome damage were studied in interphase xrs-5 cells by means of premature chromosome condensation (PCC). The results obtained were compared with those previously reported for CHO cells. Production of chromosome damage per unit of absorbed radiation dose was in xrs-5 cells larger by a factor of 2·6 than in CHO cells (5·2 breaks per cell per Gy). Changes in chromatin structure, associated with the radiation-sensitive phenotype of xrs-5 cells, that increase the probability of conversion of a DNA double-strand break (dsb) to a chromosome break are invoked to explain this effect. Repair of chromosome breaks as measured in plateau-phase G₁ cells was deficient in xrs-5 cells and the number of residual chromosome breaks was practically identical to the number of lethal lesions calculated from survival data. This observation suggests that non-repaired chromosome breaks are likely to be manifestations of lethal events in the cell. The yield of ring chromosomes scored after a few hours of repair was higher by a factor of three in xrs-5 compared with CHO cells. This increase in ring formation suggests an increase in the probability of misrepair of chromosome damage that may stem either from the reduced ability of xrs-5 cells to repair dsb, or from the higher production of chromosome fragments observed per cell and per Gy.     

A ‘Repair Model’ of Cell Survival Gives the Approximation: S = Exp − (αD + βD2)

Summary

Using exponentially growing Chinese hamster V79 cells we studied the effect of anisotonic phosphate-buffered saline (PBS) treatment on the frequency of mutation following X-irradiation. Induction of mutants was studied at the hypoxanthine-guanine phosphoribosyl transferase locus (HGPRT). When cells were X-irradiated and immediately followed by 0·5 m NaCl/PBS treatment for 20 min at 37°C, cell survival was significantly decreased and a concomitant increase in the number of mutants was observed, indicating that postirradiation treatment with anisotonic salt solution caused conversions of potentially lethal damage and potentially mutagenic damage to lethal and mutagenic damage, respectively. The repair kinetics of potentially mutagenic damage expressible by 0·5 m NaCl/PBS after 5·86 Gy of X-rays were similar to those of potentially lethal damage. The repair of both types of damage was essentially complete within 40 min after X-irradiation.     

Efficiency of radiation-induced base lesion excision and the order of enzymatic treatment

Purpose: To clarify whether initial base excision repair processes at clustered DNA damage sites comprising multiple base lesions affect subsequent excision processes via the formation of additional strand breaks by glycosylase and apurinic/apyrimidinic (AP) endonuclease base excision enzymes.

Materials and methods: Plasmid DNA (pUC18) as a model DNA molecule was exposed to high-linear-energy-transfer (LET) ionizing radiation (He^2+?or C^6+?ions) or low-LET ionizing radiation (X-rays) under various conditions to produce varied radical-scavenging effects. pUC18 was then treated sequentially or simultaneously with two bacterial base excision enzymes (glycosylases), namely, endonuclease III and formamidopyrimidine-DNA glycosylase, which convert pyrimidine (or abasic [AP] site) and purine (or AP site) lesions to single-strand breaks (SSB), respectively. Yields of additional SSB or double-strand breaks (DSB) as digestion products were examined after changing the order of enzymatic treatment.

Results: There were few differences among the enzymatic treatments, indicating that treatment order did not affect the final yields of additional SSB or DSB formed by glycosylase activity. This suggests that of the total damage, the fraction of clustered damage sites with a persistent base lesion dependent on the order of glycosylase treatment was insignificant if present.

Conclusion: Base lesion clusters induced by high- or low-LET radiation appear three or more base pairs apart, and are promptly converted to a DSB by glycosylase, regardless of the order of enzymatic treatment.     

Increased sensitivity to sparsely ionizing radiation due to excessive base excision in clustered DNA damage sites in Escherichia coli

PURPOSE: In order to clarify the cellular processing and repair mechanisms for radiation-induced clustered DNA damage, we examined the correlation between the levels of DNA glycosylases and the sensitivity to ionizing radiation in Escherichia coli. MATERIALS AND METHODS: The lethal effects of gamma-rays, X-rays, alpha-particles and H2O2 were determined in E. coli with different levels of DNA glycosylases. The formation of double-strand breaks by post-irradiation treatment with DNA glycosylase was assayed with gamma-irradiated plasmid DNA in vitro. RESULTS: An E. coli mutM nth nei triple mutant was less sensitive to the lethal effect of sparsely ionizing radiation (gamma-rays and X-rays) than the wild-type strain. Overproduction of MutM (8-oxoguanine-DNA glycosylase), Nth (endonuclease III) and Nei (endonulease VIII) increased the sensitivity to gamma-rays, whereas it did not affect the sensitivity to alpha-particles. Increased sensitivity to gamma-rays also occurred in E. coli overproducing human 8-oxoguanine-DNA glycosylase (hOgg1). Treatment of gamma-irradiated plasmid DNA with purified MutM converted the covalently closed circular to the linear form of the DNA. On the other hand, overproduction of MutM conferred resistance to H2O2 on the E. coli mutM nth nei mutant. CONCLUSIONS: The levels of DNA glycosylases affect the sensitivity of E. coli to gamma-rays and X-rays. Excessive excision by DNA glycosylases converts nearly opposite base damage in clustered DNA damage to double-strand breaks, which are potentially lethal.     

Repair of Potentially Lethal Damage by Introduction of T4 DNA Ligase in Eucaryotic Cells

Summary

The bacterial enzyme PvuII, which generates blunt-ended DNA double-strand breaks, and T4 DNA ligase, which seals adjacent DNA fragments in coupling to ATP cleavage, were introduced in mouse C3H10T1/2 fibroblasts using osmolytic shock of pinocytic vesicles. Cells were then assayed for their clonogenic ability. In agreement with previous studies by others, we find that the PvuII restriction endonuclease simulates ionizing radiation effects by causing a dose-dependent loss of reproductive capacity. Here we show that the concomitant treatment with DNA ligase considerably increases cell survival. Survival curves were shown to be dependent on the ligase enzyme dose and on ATP concentration in the hypertonic medium. We conclude that T4 DNA ligase is able to repair some of the potentially lethal damage produced by restriction endonucleases in eucaryotic cells.     

Effectiveness of 1·5 keV Aluminium K and 0·3 keV Carbon K Characteristic X-rays at Inducing DNA Double-strand Breaks in Yeast Cells

Summary

Induction of DNA double-strand breaks in diploid wild-type yeast cells, and inactivation of diploid mutant cells (rad54-3) unable to repair DNA double-strand breaks, were studied with aluminium K (1·5 keV) and carbon K (0·278 keV) characteristic X-rays. The induction of DNA double-strand breaks was found to increase linearly with absorbed dose for both characteristic X-rays. Carbon K X-rays were more effective than aluminium K X-rays. Relative to ⁶⁰Co γ-rays the r.b.e-values for the induction of DNA double-strand breaks were found to be 3·8 and 2·2 for carbon K and aluminium K X-rays respectively. The survival curves of the rad54-3 mutant cells were exponential for both ultrasoft X-rays. For inactivation of rad54-3 mutant cells, the r.b.e.-values relative to ⁶⁰Co γ-rays were 2·6 and 2·4 for carbon K and aluminium K X-rays, respectively. The DNA double-strand break data obtained with aluminium K and carbon K X-rays are in agreement with the data obtained for gene mutation, chromosome aberrations and inactivation of mammalian cells, suggesting that DNA double-strand breaks are the possible molecular lesions leading to these effects     

Detection of DNA Damage in Cells Exposed to Ionizing Radiation by Use of Anti-single-stranded DNA Monoclonal Antibody

Summary

An immunochemical method has been developed for quantitative detection of DNA damage in mammalian cells. The method is based on the binding of a monoclonal antibody to single-stranded DNA. The clone producing this antibody (D1B) was obtained as a by-product from fusion of mouse myeloma cells with spleen cells isolated from a mouse immunized with chemically modified DNA. The technique is based upon the determination of the percentage single-strandedness resulting from the time-dependent partial unwinding of cellular DNA under alkaline conditions. Single- and double-strand DNA breaks, or lesions converted into such breaks in alkaline medium, form initiation points for the unwinding. The extent of unwinding from these points under defined conditions is a measure of the number of such sites. The method is rapid, does not require radioactive labelling of DNA or physical separation of single- from double-stranded molecules, is sufficiently sensitive to detect damage induced by 1 Gy of ionizing radiation and needs only small numbers of cells. The usefulness of the technique was demonstrated in a study of the induction of DNA damage and its repair in cultured Chinese hamster cells and in human white blood cells after exposure to ⁶⁰Co-γ-rays, and in white blood cells and bone marrow cells of X-irradiated mice. A dose-related DNA unwinding was observed and repair of DNA lesions was observed up to 60 min after irradiation.