DNA DSB Research Articles

Protection of DNA from radiation-induced double-strand breaks: influence of replication and nuclear proteins

Purpose : To study the protective effect of histone and non-histone proteins on double-strand break (dsb) induction in replicating S-phase DNA as well as bulk DNA of plateau phase human tumour cells. Materials and methods : Induction of dsb was studied in two human adenocarcinoma cell lines: Colo320HSR and MCF-7. To assess the influence of chromatin structure on radiation-induced DNA dsb, different nuclear preparations of cells, either continuously labelled with 14 C or pulse labelled with 3 H, were assessed by pulsed-field gel electrophoresis (PFGE). Results and conclusions : Stepwise removal of DNA-bound proteins from the chromatin increased the amount of radiation-induced dsb in both cell lines. However, the protective effect of DNA-associated proteins on dsb induction was significantly reduced in DNA of replicating S-phase cells compared with bulk DNA of plateau phase cells. These data show that proteins associated with the DNA have a different protective effect on radiation-induced dsb, rendering replicating DNA with open chromatin structure more sensitive to dsb induction by ionizing radiation.

Repair of DNA double-strand breaks and radiosensitivity to killing in an isogenic group of P53 mutant cell lines

Purpose: Accumulation of the p53 protein can result in G 1 arrest that may facilitate DNA repair, or alternatively, it may lead to apoptosis. Mutations that alter p53’s ability to mediate these responses are expected to alter cell radiosensitivity to killing. However, the relationship between p53 status and cell radiosensitivity has proven to be complex. Several studies have suggested that p53 mutations are associated with increased radioresistance to killing, while others have shown no such correlation. These differences may be derived from the fact that different mutations of p53 exert different effects on cell radiosensitivity. Methods and Materials: To address this question, we examined a group of isogenic cell lines that express different “hot spot” mutant forms of p53. These cells were generated from human osteosarcoma (SAOS) cells, a p53 null cell line, by transfection with vectors expressing different p53 mutants. Vectors with the following p53 mutations were utilized: 143Ala, 175His, 248Try, 273His, and 281Gly. As controls, we used the original SAOS cells and cells transfected with the vector alone. Results were compared to those obtained with a cell line expressing wild-type p53 (wt p53). Radiosensitivity to killing was determined in the exponential phase of growth by measuring loss of colony-forming ability. Induction and repair of DNA double-strand breaks (dsb) was measured in irradiated cells using pulsed-field gel electrophoresis. Apoptosis was assessed using morphologic evaluation of DAPI-stained cells after treatment either with radiation or paclitaxel. Results: Transfected SAOS-2 cell lines expressed a mutant form of p53 that could not be induced by radiation, and which was transcriptionally inactive. Among the 7 cell lines studied, we observed no difference in cellular radiosensitivity to killing ( p = NS). When examining DNA repair, no difference in either the induction or repair of DNA dsb was noted in any of the cell lines studied ( p = NS). Also, induction of apoptosis, either after exposure to radiation or paclitaxel, was low, and similar in all cell lines ( p = NS). Non-isogenic cells expressing wt p53 were more radioresistant to killing by radiation, but showed similar kinetics of dsb rejoining. Conclusion: The results suggest that expression of different p53 mutants does not alter the yields of radiation-induced dsb, or the ability of cells to repair this type of lesion. In addition, the same p53 mutants do not affect cellular radiosensitivity to killing, or the induction of apoptosis after exposure to radiation or paclitaxel.

Ataxia Telangiectasia-mutated Gene Product Inhibits DNA Damage-induced Apoptosis via Ceramide Synthase

DNA double-stranded breaks (dsb) activate surveillance systems that identify DNA damage and either initiate repair or signal cell death. Failure of cells to undergo appropriate death in response to DNA damage leads to misrepair, mutations, and neoplastic transformation. Pathways linking DNA dsb to reproductive or apoptotic death are virtually unknown. Here we report that metabolic incorporation of 125I-labeled 5-iodo-2'deoxyuridine, which produces DNA dsb, signaled de novo ceramide synthesis by post-translational activation of ceramide synthase (CS) and apoptosis. CS activation was obligatory, since fumonisin B1, a fungal pathogen that acts as a specific CS inhibitor, abrogated DNA damage-induced death. X-irradiation yielded similar results. Furthermore, inhibition of apoptosis using the peptide caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone did not affect CS activation, indicating this event is not a consequence of induction of apoptosis. ATM, the gene mutated in ataxia telangiectasia, is a member of the phosphatidylinositol 3-kinase family that constitutes the DNA damage surveillance/repair system. Epstein-Barr virus-immortalized B cell lines from six ataxia telangiectasia patients with different mutations exhibited radiation-induced CS activation, ceramide generation, and apoptosis, whereas three lines from normal patients failed to manifest these responses. Stable transfection of wild type ATM cDNA reversed these events, whereas antisense inactivation of ataxia telangiectasia-mutated gene product in normal B cells conferred the ataxia telangiectasia phenotype. We propose that one of the functions of ataxia telangiectasia-mutated gene product is to constrain activation of CS, thereby regulating DNA damage-induced apoptosis.

Fibroblast radiosensitivity measured using the comet DNA-damage assay correlates with clonogenic survival parameters.

A study was made of the neutral comet assay as a potential method for measuring normal cell radiosensitivity. Eleven fibroblast strains were studied comprising nine derived from vaginal biopsies from pretreatment cervical cancer patients and two strains from radiosensitive individuals. DNA double strand break (dsbs) dose–response curves for both initial and residual (20-h repair time) damage were obtained over the dose range 0–240 Gy, with slopes varying 3.2 and 8-fold respectively. Clonogenic cell survival parameters were available for all the cell strains following both high- and low-dose rate irradiation. There were no correlations between the dose–response slope of the initial level of DNA dsbs and parameters that mainly describe the initial portion of clonogenic radiation survival curves (SF 2, α, D̄-). A significant correlation (r = –0.63, P = 0.04) was found between the extent of residual DNA dsbs and clonogenicity for all 11 fibroblast strains. The parameter showing the highest correlation with fibroblast cell killing (D-) for the nine normal fibroblasts alone was the ratio of initial/residual DNA dsb dose–response slope (r = 0.80, P = < 0.01). A significant correlation (r = –0.67, P = 0.03) with clonogenic radiosensitivity was also found for all 11 cell strains when using the ratio of initial/residual DNA dsb damage at a single dose of 180 Gy. This study shows that fibroblast radiosensitivity measured using the neutral comet assay correlates with clonogenic radiation survival parameters, and therefore may have potential value in predictive testing of normal tissue radiosensitivity. © 1999 Cancer Research Campaign

Radiation-induced DNA double-strand breaks and rejoining in malignant glioma cells

Purpose : This study was performed to standardize experimental conditions for the quantification by pulsed-field gel electrophoresis (PFGE) of radiation-induced DNA double-strand breaks (dsb) and rejoining in a human malignant brain tumour xenograft model. Materials and methods : Because no correlation was found between DNA dsb induction or rejoining and clinical radiation response for six fresh glioblastoma (GBM) specimens, assay conditions were examined in detail. SF-767 human GBM cells were implanted into the flanks of athymic mice. Resulting tumours were irradiated in vivo, dissociated mechanically or using an enzyme cocktail, and assayed for DNA dsb induction and repair. In other experiments, excised tumour portions were irradiated and allowed to repair either as chunks (50mm3 pieces), as minced tumour (1mm3 pieces), or as single-cell suspensions. Finally, the effect of holding excised tumours in vitro for times of up to 72h before irradiation and assay for DNA dsb and cell survival was studied. Results and conclusions : The method of tumour dissociation had no effect on results; however, both the configuration of specimens during irradiation and the in vitro maintenance time markedly affected the experimental outcome. Chunks irradiated in vitro had DNA dsb results that were very similar to those obtained when tumours were irradiated in situ, while minced pieces or single-cell suspensions resulted in steeper dose-response curves. When tumour chunks were maintained at 4 C in medium, DNA dsb induction was not affected for 24h and DNA dsb rejoining remained constant for 48h but then decreased. Cell survival, however, decreased continually during the 72h in vitro maintenance time.

A comparison of the radiosensitivity of relaxed and supercoiled plasmid DNA

Purpose: The aim of the work was to compare critically the radiosensitivity of the supercoiled and relaxed forms of a plasmid DNA system commonly used in DNA damage assays. Materials and methods: The yields of single- (ssb) and double-strand breaks (dsb) in pBR322 DNA over a range of scavenging capacities were measured in the presence of Tris after irradiation with a single pulse of 400keV electrons. The response was compared with DNA that had been given a preliminary dose of gamma -rays such that an average of one ssb per molecule was present. Results: The yields of dsb were found to be enhanced in the preirradiated DNA when Tris was present during irradiation at concentrations varying between 10 and 100mmoldm-3 with a maximal enhancement ratio of 1.6 at 80mmoldm-3 Tris. This increased yield was not observed when both the pre-irradiation and the experimental doses were given by gamma -irradiation. The increased response was not found in DNA that had been enzymatically relaxed by the introduction of a nick in each molecule using the enzyme gpII. Conclusions: The observations suggest that the conformation per se does not influence the radiosensitivity. The enhanced yields observed in the gamma -ray pre-irradiated DNA with pulsed irradiation appear to be a consequence of the high dose rate used. A proposed explanation is that the combination of high dose levels (1500Gy) and short irradiation times (5ns) would allow some 'spurs' to overlap and it is proposed that the enhanced yields of dsb are related to this.

DNA-PK activation by ionizing radiation-induced DNA single-strand breaks

Purpose : To assess the ability of 60Co gamma-radiation-induced plasmid DNA single-strand breaks (gamma-ssb) to activate the DNA-dependent protein kinase (DNA-PK) in vitro. Materials and methods : Plasmid DNA was gamma-irradiated under aerobic conditions to yield 0-6 gamma -ssb and 0.1 double-strand breaks (dsb) per plasmid molecule. The irradiated DNA was used to stimulate DNA-PK in crude HF19 fibroblast nuclear extracts and/or purified HeLa cell DNA-PK protein, and the activation compared with that obtained with a single enzymatically generated plasmid DNA ssb (GpII endonuclease) or dsb (Eco RI endonuclease). Results: gamma-Irradiated plasmid DNA activates DNA-PK in both crude and purified preparations and the kinase activity increases linearly with dose. As significant DNA-PK activation was detectable using irradiated plasmids which contain 0.1dsb/molecule, it was concluded that this activation is due to gamma-ssb. However, using purified DNA-PK, this activation is relatively weak as 3 gamma -ssb is equivalent to one GpII-generated DNA ssb or one end of an Eco RI-generated dsb in DNA-PK assays. Conclusions: As gamma-ssb are in a 20-fold excess of gamma-dsb in vivo for low LET radiation, gamma-ssb may contribute significantly to DNAPK signalling of gamma-radiation-induced DNA damage in vivo.

DNA double-strand break repair, DNA-PK, and DNA ligases in two human squamous carcinoma cell lines with different radiosensitivity

Purpose: Variation in sensitivity to radiotherapy among tumors has been related to the capacity of cells to repair radiation-induced DNA double-strand breaks (DSBs). DNA-dependent protein kinase (DNA-PK) and DNA ligases may affect DNA dsb rejoining. This study was performed to compare rate of rejoining of radiation-induced DSBs, DNA-PK, and DNA ligase activities in two human squamous carcinoma cell lines with different sensitivity to ionizing radiation. Methods and Materials: Cell survival of two human squamous carcinoma cell lines, UM-SCC-1 and UM-SCC-14A, was determined by an in vitro clonogenic assay. DSB rejoining was studied using pulsed field gel electrophoresis (PFGE). DNA-PK activity was determined using BIOTRAK DNA-PK enzyme assay system (Amersham). DNA ligase activity in crude cell extracts was measured using [5′- 33P] Poly (dA)·(oligo (dT) as a substrate. Proteolytic degradation of proteins was analyzed by means of Western blotting. Results: Applying the commonly used linear-quadratic equation to describe cell survival, S = e -α D-β D 2 , the two cell lines roughly have the same α value (∼0.40 Gy -1) whereas the β value was considerably higher in UM-SCC-14A (0.067 Gy -2 ± 0.007 Gy -2 [SEM]) as compared to UM-SCC-1 (0.013 Gy -2 ± 0.004 Gy -2 [SEM]). Furthermore, UM-SCC-1 was more proficient in rejoining of X-ray-induced DSBs as compared to UM-SCC-14A as quantified by PFGE. The constitutive level of DNA-PK activity was 1.6 times higher in UM-SCC-1 as compared to UM-SCC-14A ( p < 0.05). The constitutive level of DNA ligase activity was similar in the two cell lines. Conclusions: The results suggest that the proficiency in rejoining of DSBs is associated with DNA-PK activity but not with total DNA ligase activity.

The phosphatidylinositol 3-kinase inhibitor wortmannin sensitizes quiescent but not proliferating MG-63 human osteosarcoma cells to radiation

Recent genetic and biochemical studies indicate that DNA-dependent protein kinase (DNA-PK) plays an important role in DNA double-strand break (dsb) repair and V(D)J recombination. Since the catalytic subunit of DNA-PK (DNA-PKcs) has high sequence homology with phosphatidylinositol 3-kinase (PI 3-kinase), we examined the effect of wortmannin, a specific inhibitor of PI 3-kinase, on the survival of human tumor cells after X-irradiation. The present study demonstrates that wortmannin at 20 μM is an effective radiosensitizer of quiescent (Q), but not proliferating (P) cells. In addition, the rejoining of DNA dsb is significantly inhibited in Q, but not in P cells. Finally, we found that Q cell extracts have approximately five-fold less DNA-PK activity than those of P cells. After a 2 h exposure to wortmannin, the DNA-PK activity of Q cell extracts was considerably lower than that of P cells. This can explain why wortmannin sensitizes Q, but not P cells to radiation.

Overview of the Proceedings: From Loose Loops to Sticky Ends

a chromatid break is obviously not a simple matter, since the latter can involve the loss of up to 20 Mbp, and evidence from some radiosensitive sublines suggests that secondary chromatin configuration influences conversion of DNA damage to chromosome breaks. These and other questions were addressed at a meeting held in Montreal in June 1997 which brought together experts in DNA damage and repair, chromatin structure and cytogenetics to delineate the route from a DNA lesion to a dead cell. The topics discussed moved through increasing levels of complexity from damage distribution at the molecular level to nucleoprotein structure to the organization of nuclear domains. Several presentations described investigation of the genomic distribution of DNA lesions. In one of these (M-E. Mirault, Universiti Laval), a single-strand break (SSB)-specific S1 nuclease was used as a probe. Similar cleavage patterns were found with a variety of chemical agents and with ionizing radiation with preferential localization of S1 cleavage in matrix-associated DNA. In another study (A. I. Kassis, Harvard Medical School), 1251 was used to assess the effects of OH radicals produced in proximity to DNA. Results were consistent with DSBs being produced by a single-event mechanism when a high radical density was generated close to naked DNA, while for higher-order chromatin both production of DSBs and toxicity appeared to be largely indirect in nature. The genomic distribution of naturally occurring DNA DSBs was examined using a technique in which illegitimate integration of a transgene in murine fibroblast DNA was localized by fluorescence in situ hybridization (FISH) analysis (G. Dellaire and P. Chartrand, Institut du Cancer de Montrial). Results indicated that illegitimate integration occurs randomly at megabase resolution in murine fibroblasts and, since the initiation of illegitimate integration would require a DNA DSB, the corollary is that DSBs occur randomly within the murine genome at megabase resolution. An important aspect of the organization of the eukaryotic chromosome which was addressed by several speakers at this meeting is the arrangement of DNA supercoils into looped domains anchored at intervals of 30-60 kbp to the nuclear matrix through matrix attachment region (MAR)-specific sequences (J. L. Roti Roti, Washington University, St. Louis; J. L. Schwartz, University of Washington, Seattle). These structures create independent domains of torsional stress in chromatin which are important for spatial control of DNA replication, gene transcription and DNA repair. Based on data obtained with assays that use high-salt extraction of nuclei (nucleoids), it is hypothesized that there are two categories of looped domains: those with loose (protease-sensitive) attachments to the nuclear matrix and those which are tightly attached to the nuclear matrix and are resistant to disruption. In the first case breaks can affect rewinding both in the loop where they occur and in adjacent loops, while in the second case breaks affect rewinding only in the loop where they occur and the adjacent loops are insulated from damage. In one subgroup of cell lines, differences in radiosensitivity could be explained in terms of the ability of anchoring regions to isolate DNA domains from changes in DNA supercoiling, whereas in other cell lines rewinding and radiation resistance are associated with a subset of specific proteins located at the anchoring regions. In another study (P. J. Johnston, British Columbia Cancer Research Centre), selective examination of DSBs occurring as multiples within looped DNA structures revealed identical subsets of DSBs repairing with slow kinetics through the DNA-dependent protein kinase (PK)/Ku pathway. Correction of the results to account for differences in the rate of elution of cells actively replicating DNA gave a looped domain size of 3.0 Mbp for all cells examined. It was suggested that this is a fundamental unit of chromatin structure which may play an important part in the processing of DNA DSBs. 1Selected papers from this meeting will appear in this and future issues of Radiation Research.

Radiosensitization of mouse sarcoma cells by fludarabine (F-ara-A) or gemcitabine (dFdC), two nucleoside analogues, is not mediated by an increased induction or a repair inhibition of DNA doublestrand breaks as measured by pulsed-field gel electrophoresis

Purpose: To investigate the effect of fludarabine (F-ara-A) and gemcitabine (dFdC), two radiosensitizing nucleoside analogues, on the induction and repair of DNA dsb after ionizing radiation. Materials and methods: Radiosensitization of mouse sarcoma SA-NH and FSA cells was studied using a clonogenic assay. Cell survival curves were fitted with the linear-quadratic model. DNA dsbs were detected by pulsed-field gel electrophoresis under neutral conditions. Results: F-ara-A (100 mu moldm-3 for 1 h prior to irradiation) induced a substantial radiosensitization in SA-NH cells with a dose modification factor of 2.0 for a surviving fraction of 0.5. In a FSA mouse sarcoma cell line, dFdC (5 mu moldm-3 for 3 h prior to irradiation) induced a modest radiosensitization with a DMF of 1.2 for a surviving fraction of 0.5. Under similar experimental conditions, neither F-ara-A nor dFdC altered the yield of radiation-induced DNA dsbs in the dose range of 0-40 Gy. After a single dose of 25 Gy (SA-NH cells) or 40 Gy (FSA cells), neither the kinetics of repair nor the amount of residual damage was affected by F-ara-A or dFdC. Conclusions: For experimental conditions under which radiosensitization was observed, neither the induction nor the repair of DNA dsbs after ionizing radiation were affected by F-ara-A or dFdC.

Repair of DNA double-strand breaks and cell killing by charged particles

It has been suggested that it is not simple double-strand breaks (dsb) but the non-reparable breaks which correlate well with the high biological effectiveness of high LET radiations for cell killing (Kelland et al., 1988; Radford, 1986). We have compared the effects of charged particles on cell death in 3 pairs of cell lines which are normal or defective in the repair of DNA dsbs. For the cell lines SL3-147, M10, and SX10 which are deficient in DNA dsb repair, RBE values were close to unity for cell killing induced by charged particles with linear energy transfer (LET) up to 200 keV/μm and were even smaller than unity for the LET region greater than 300 keV/μm. The inactivation cross section (ICS) increased with LET for all 3 pairs. The ICS of dsb repair deficient mutants was always larger than that of their parents for all the LET ranges, but with increasing LET the difference in ICS between the mutant and its parent became smaller. Since a small difference in ICS remained at LET of about 300 keV/μm, dsb repair may still take place at this high LET, even if its role is apparently small. These results suggest that the DNA repair system does not play a major role in protection against the attack of high LET radiations and that a main cause of cell death is non-reparable dsb which are produced at a higher yield compared with low LET radiations. No correlation was observed between DNA content or nuclear area and ICS.

Modelling DNA damage induced by different energy photons and tritium beta-particles

Purpose: To model the production of single- and double-strand breaks (ssb and dsb) in DNA by ionizing radiations. To compare the predicted effectiveness of different energy photon radiations and tritium beta-particles. Materials and methods : Modelling is carried out by Monte Carlo and includes consideration of direct energy depositions in DNA molecules, the production of species, their diffusion and interactions with each other and DNA. Computer-generated electron tracks in liquid water are used to model energy deposition and to derive the initial positions of chemical species. Atomistic representation of the DNA in B form with a first hydration shell is used. Photon radiations in the energy range 70keV-1 MeV and tritium beta-particles are considered. Results: A tentative increase for dsb yield has been predicted for 70keV photons and tritium compared with 137Cs. This increase is more pronounced for complex dsb. Double-strand breaks are much more prone compared with ssb to combine with additional strand breaks and base damage, which contributes to break complexity. At least half of DNA breaks are hydroxyl radical mediated. Conclusions: The developed model makes predictions compatible with features of available experimental data. Break complexity has to be addressed in biophysical modelling when the relative effectiveness of radiations in DNA damage is studied. Obtained data strongly argue against the dominance of direct radiation action in DNA damage in the cellular environment predicted by some theoretical studies.

G2 chromatid breaks in murine SCID cells.

The G2 chromosomal radiosensitivity of murine SCID (severe combined immunodeficient) and normal fibroblasts has been investigated. We have also investigated the G2 response of these cell lines to the restriction endonuclease PvuII. We show that chromatid breaks are induced linearly with radiation dose in both cell lines and SCID cells are approximately 1.6 times as radiosensitive as normal murine fibroblasts when tested using a G2 assay with a 2 h sampling time. The disappearance of chromatid breaks with time after irradiation was first order with a half-time of approximately 1.5 h in both cell lines. Thus, although SCID cells are deficient in the rejoining of double-strand breaks (dsb), they show similar kinetics of disappearance of chromatid breaks with time as normal CB17 cells, indicating that the 'rejoining' of chromatid breaks does not reflect dsb repair. When CB17 and SCID cells were treated with PvuII, which generates dsb in cellular DNA in the presence of streptolysin O (as a porating agent), approximately 3 times more chromatid breaks were observed in SCID than CB17 cells. We conclude that SCID cells convert a higher number of dsb into chromatid breaks than do CB17 cells. The conversion process is interpreted in terms of the recently proposed 'signal' model, whereby a signal, resulting from a single dsb, triggers the cell to make a recombinational exchange which, if incomplete, gives rise to a visible chromatid break. In terms of the signal model, elevated conversion of dsb into chromatid breaks results from altered signalling and the disappearance of chromatid breaks with time following irradiation represents the completion of recombinational exchanges rather than repair of dsb.

Evidence for a hypoxic fixation reaction leading to the induction of ssb and dsb in irradiated DNA

Purpose: To measure hypoxic chemical fixation processes of radiation damage in both isolated plasmid DNA and in GSHdepleted E. coli cells. Materials and methods: Plasmid pBR322 DNA was irradiated with a single 5 ns pulse of 400 keV electrons under hypoxic conditions. At pre-set times, immediately before or after the electron pulse, the chamber containing the DNA was exposed to a high-pressure shot of hydrogen sulphide (H2S) gas. Results: DNA irradiated before contact with the H2S pulse was more sensitive to the production of both single strand breaks (ssb) and double strand breaks (dsb) than DNA irradiated after the addition of H2S. The post-irradiation protection of DNA by H2S was time-dependent, having first-order rate constants of 21 s-1 for ssb and 10 s-1 for dsb. Conclusions: This is the first direct kinetic evidence for the involvement of a hypoxic fixation reaction in the production of DNA damage by ionizing radiation. It indicates that long-lived radical damage is induced in DNA which, even at times of 20-50 ms after irradiation, can be chemically repaired, or rescued, by the addition of a thiol agent. This reaction may partially explain the predicted decrease in oxygen enhancement ratio (OER) with linear energy transfer (LET) on the basis of the increased clustering of radicals produced on the DNA by tracks of ionizing radiation. As radical multiplicity increases with LET there is a greater chance that some of the radicals will become fixed in the absence of oxygen leading to an increased probability of damage under hypoxia and a reduction in the OER.

In vitro rejoining of DNA double strand breaks: a comparison of genomic-DNA with plasmid-DNA-based assays

Purpose: To evaluate potential similarities between the enzymatic activities required to rejoin DNA double strand breaks (dsb) in an in vitro assay based on genomic DNA and an in vitro assay based on plasmid DNA. Because the latter assay is simpler and faster, it should be preferred for the characterization of repair factors if both assays are found to probe for the same activities. If, however, the enzymatic requirements for dsb rejoining are different between the two assays, both should be used as they are likely to play complementary roles in the characterization of repair factors. Materials and methods : A cell-free assay has been used, developed to study rejoining DNA dsb induced by radiation in 'naked' DNA prepared from agarose embedded cells using an extract of HeLa cells as a source of enzymes. Also employed was an in vitro assay using the ligation of linearized plasmid DNA to model dsb rejoining. Results : Evidence is presented that, under the conditions employed, different sets of activities are involved in the ligation of linearized plasmid DNA and in the rejoining of dsb in 'naked' genomic DNA. Optimal rejoining of dsb induced in genomic DNA is observed with cytoplasmic cell extract at 37 C, whereas optimal ligation of plasmid DNA is observed with nuclear extract at 25 C. Rejoining of dsb in genomic DNA comes to a near halt at 14 C, but plasmid DNA ligation proceeds at significant rates at this temperature. Furthermore, the activities required for the rejoining of dsb induced in genomic DNA are partly stable to heating at 50 C for 1 h, whereas activities required for the ligation of plasmid DNA are completely inactivated by a similar treatment. Both reactions require ATP for optimal performance, and in both, DNA joining is inhibited at high ATP concentrations. Conclusions : These observations indicate that the plasmid-and the genomic-DNA-based assays probe for, at least partly, different sets of activities and therefore are expected to play complementary roles in the purification and characterization of activities involved in dsb rejoining.

A review of dsb induction data for varying quality radiations

Purpose: This short review summarizes the data obtained with various techniques for measuring the yields of double strand breaks (dsb) produced by particle radiations of differing linear energy transfer (LET) in order to obtain relative biological effectiveness (RBE) values. Results and conclusions : Studies aimed at understanding the interactions of different types of radiation with cellular DNA have monitored the yields of DNA dsb versus radiation quality. Several techniques have been used to measure dsb yields in mammalian cells, and these include: neutral sedimentation gradients, filter elution and more recently pulsed field gel electrophoresis techniques (PFGE). Recent developments in PFGE have allowed the measurement of both the yields and the distribution of breaks within the genome, which go part of the way to explaining the RBE values close to 1 0 previously measured using other approaches with various radiation qualities. It is clear that future studies to determine the effectiveness of radiations of differing LET must use techniques that determine both yields and distributions of dsb, and assays need to be developed to allow these measurements at biologically relevant doses.

SYBR Green I staining of pulsed field agarose gels is a sensitive and inexpensive way of quantitating DNA double-strand breaks in mammalian cells.

Pulsed field gel electrophoresis (PFGE) is widely used to measure DNA double strand breaks (dsb). The DNA of cultured cells can be prelabelled with radioactivity, which helps greatly in detection and quantitation of DNA dsb. However, this approach cannot be used with non-cycling cells from biopsy material. We describe a method which uses SYBR Green I to stain DNA in dried agarose gels. DNA is detected and analysed using readily available camera equipment and image analysis software. This method is as sensitive as [3H]thymidine prelabelling of cells and allows DNA dsb to be measured simply and economically in non-cycling cells.

The contribution of cytotoxicity to DNA-effects in the single cell gel test (comet assay)

We evaluated genotoxic and cytotoxic effects of the three non-mutagenic and non-carcinogenic compounds p-nitrophenol, d-menthol and sodium N-lauroyl sarcosine which have previously been shown to induce DNA double strand breaks (DNA dsb) secondary to induced cytotoxicity. We tested wheter genotoxic effects in the alkaline single cell gel test (comet assay) may be confounded by cytotoxicity-induced DNA dsb. Cell viability was determined at the end of the treatment using the fluorescein diacetate/ethidium bromide-assay and plating efficiency was used as an indicator of long-term survivability. Experiments with V79 Chinese hamster cells and human white blood cells revealed negative results in the comet assay despite strong cytotoxic effects. However, cells with extremely fragmented DNA (‘clouds’) occured but were excluded from the evaluation under the principle that they represent dead cells. We also noticed a significant loss of cells at cytotoxic concentrations that might be attributed to the induction of highly fragmented DNA which is lost during electrophoresis. Since the comet assay allows the determination of DNA effects on the single cell level, a confounding effect of cytotoxicity on test results can be avoided.

Correlation between slowly repairable double-strand breaks and thermal radiosensitization in the human HeLa S3 cell line

. The effect of heat on double-strand breaks (dsb) repair was compared with thermal radiosensitization using HeLa S3 cells. Cells were exposed to a combined treatment of X-irradiation followed by heat (44 C, 0.5 h) separated by time intervals up to 8 h. DNA dsb were measured by PFGE and survival by the colony forming assay. In non-heated HeLa S3 cells repair of dsb was biphasic with the majority of breaks being repaired fast with a half-time of 14 min and only a minority were repaired slowly with a half-time of 130 min. Heat applied immediately after irradiation was found to cause an increase in both half-times but mainly to result in an increased fraction of slowly repairable dsb. The latter effect was shown to result from the formation of additional dsb. The number of additional dsb declined when irradiation and heat were separated by an interval at 37 C with a half-time of 120 30 min. This half-time was similar to the half-time of 100 20 min found for the loss of thermal radiosensitization studied for the same protocol. Both processes were recently found also to correlate in CHO cells but occurred much faster in rodent cells than in the human HeLa S3 cells used in the current study. These results show that in human cells, unlike previously suggested on the basis of rodent cells, thermal radiosensitization is still a substantial contributor to the killing efficacy of a combined treatment, even when irradiation and heat are separated by a time interval of 4 h.