Analysis of radiation-induced DNA double-strand breaks misrepair is not compromized by broken DNA in human fibroblasts

It has been suggested that the technique for measuring repair fidelity of radiation-induced DNA double-strand breaks (DSBs) using Southern blotting and hybridization to defined regions of the genome could be compromised by broken or poorly-digested DNA. Since misrepair of DNA DSBs is an important aspect of radiation-induced chromosome aberrations, mutations, and cell killing, we checked for such a supposition in non-transformed human fibroblasts. DSB misrepair was assessed in a NotI-cleavable DNA fragment of 3.2 Mbp located on the long arm of chromosome 21 and detected by D21S1 probe. We hypothesized that the suggested DNA degradation, whether spurious in nature or the results of irradiation-induced phenomena such as apoptosis and/or necrosis, should be detectable with or without NotI restriction enzyme treatment. When the DNA embedded in agarose plugs was separated by electrophoresis without prior NotI restriction, no significant difference was observed in the relative amount of migrating DNA between the control (no irradiation) and 24 h of repair following 80 Gy irradiation. Furthermore, only about 10% of the total signal was located below the 3.2 Mbp band. This suggests that the amount of DNA fragmentation due to biological (apoptosis or necrosis) or technical processes was negligible. The Tunel assay supported these results, as there was little to no apoptosis detectable in these fibroblasts up to 24 h after irradiation. We conclude that in primary human fibroblasts, the NotI method for measuring radiation-induced misrepair is not compromised by DNA degradation.     

Repair of radiation-induced DNA double-strand breaks is dependent upon radiation quality and the structural complexity of double-strand breaks

Mammalian cells primarily repair DSBs by nonhomologous end joining (NHEJ). To assess the ability of human cells to mediate end joining of complex DSBs such as those produced by chemicals, oxidative events, or high- and low-LET radiation, we employed an in vitro double-strand break repair assay using plasmid DNA linearized by these various agents. We found that human HeLa cell extracts support end joining of complex DSBs and form multimeric plasmid products from substrates produced by the radiomimetic drug bleomycin, 60Co gamma rays, and the effects of 125I decay in DNA. End joining was found to be dependent on the type of DSB-damaging agent, and it decreased as the cytotoxicity of the DSB-inducing agent increased. In addition to the inhibitory effects of DSB end-group structures on repair, NHEJ was found to be strongly inhibited by lesions proximal to DSB ends. The initial repair rate for complex non-ligatable bleomycin-induced DSBs was sixfold less than that of similarly configured (blunt-ended) but less complex (ligatable) restriction enzyme-induced DSBs. Repair of DSBs produced by gamma rays was 15-fold less efficient than repair of restriction enzyme-induced DSBs. Repair of the DSBs produced by 125I was near the lower limit of detection in our assay and was at least twofold lower than that of gamma-ray-induced DSBs. In addition, DSB ends produced by 125I were shown to be blocked by 3'-nucleotide fragments: the removal of these by E. coli endonuclease IV permitted ligation.     

Induction and repair of radiation-induced DNA double-strand breaks in human fibroblasts are not affected by terminal differentiation

It was studied for human skin fibroblasts, whether the induction or repair of DNA double-strand breaks (dsb) depend on the differentiation status. These studies were performed (a) with a fibroblast strain (HSF1) kept in progenitor state (mitotic fibroblasts, MF) or triggered to premature terminal differentiation (postmitotic fibrocytes, PMF) by exposure to mitomycin C or (b) with 20 fibroblast strains differing intrinsically in their differentiation status. The differentiation status was quantified by determining the fraction of postmitotic fibrocytes by light microscopy. DNA dsb were measured by constant-field gel electrophoresis, and the fraction of apoptotic cells by comet assay. MF and PMF cultures of HSF1 cells were irradiated with X-ray doses up to 160 Gy, and dsb were measured either immediately after irradiation or after a repair incubation of 4 or 24 h. There were a difference neither in the number of initial nor residual dsb. PMF cultures, however, showed a slightly higher number of dsb already present in non-irradiated cells, which was measured to result from a small fraction of 5% apoptotic cells. The 20 analysed fibroblast strains showed a substantial variation in the fraction of postmitotic fibrocytes (9-51%) as well as in the number of dsb remaining at 24 h after irradiation (1.9-4.9%), but there was no correlation between these two parameters. These data demonstrate that for fibroblasts the terminal differentiation has an effect neither on the induction nor the repair of radiation-induced dsb. This result indicates that the variation in dsb-repair capacity previously observed for fibroblast strains and which was considered to be the main cause for the variation in the cellular radiosensitivity, cannot be ascribed to differences in the differentiation status.     

Repair of HZE-particle-induced DNA double-strand breaks in normal human fibroblasts

DNA damage generated by high-energy and high-Z (HZE) particles is more skewed toward multiply damaged sites or clustered DNA damage than damage induced by low-linear energy transfer (LET) X and gamma rays. Clustered DNA damage includes abasic sites, base damages and single- (SSBs) and double-strand breaks (DSBs). This complex DNA damage is difficult to repair and may require coordinated recruitment of multiple DNA repair factors. As a consequence of the production of irreparable clustered lesions, a greater biological effectiveness is observed for HZE-particle radiation than for low-LET radiation. To understand how the inability of cells to rejoin DSBs contributes to the greater biological effectiveness of HZE particles, the kinetics of DSB rejoining and cell survival after exposure of normal human skin fibroblasts to a spectrum of HZE particles was examined. Using gamma-H2AX as a surrogate marker for DSB formation and rejoining, the ability of cells to rejoin DSBs was found to decrease with increasing Z; specifically, iron-ion-induced DSBs were repaired at a rate similar to those induced by silicon ions, oxygen ions and gamma radiation, but a larger fraction of iron-ion-induced damage was irreparable. Furthermore, both DNA-PKcs (DSB repair factor) and 53BP1 (DSB sensing protein) co-localized with gamma-H2AX along the track of dense ionization produced by iron and silicon ions and their focus dissolution kinetics was similar to that of gamma-H2AX. Spatial co-localization analysis showed that unlike gamma-H2AX and 53BP1, phosphorylated DNA-PKcs was localized only at very specific regions, presumably representing the sites of DSBs within the tracks. Examination of cell survival by clonogenic assay indicated that cell killing was greater for iron ions than for silicon and oxygen ions and gamma rays. Collectively, these data demonstrate that the inability of cells to rejoin DSBs within clustered DNA lesions likely contributes to the greater biological effectiveness of HZE particles.     

Efficient rejoining of radiation-induced DNA double-strand breaks in centromeric DNA of human cells

Although major efforts in elucidating different DNA double-strand break (DSB) repair pathways and their contribution to accurate repair or misrepair have been made, little is known about the influence of chromatin structure on the fidelity of DSB repair. Here, the repair of ionizing radiation-induced DSBs was investigated in heterochromatic centromeric regions of human cells in comparison with other genomic locations. A hybridization assay was applied that allows the quantification of correct DSB rejoining events in specific genomic regions by measuring reconstitution of large restriction fragments. We show for two primary fibroblast lines (MRC-5 and 180BR) and an epithelial tumor cell line that restriction fragment reconstitution is considerably more efficient in the centromere than in average genomic locations. Importantly, however, DNA ligase IV-deficient 180BR cells show, compared with repair-proficient MRC-5 cells, impaired restriction fragment reconstitution both in average DNA and in the centromere. Thus, the efficient repair of DSBs in centromeric DNA is dependent on functional non-homologous end joining. It is proposed that the condensed chromatin state in the centromere limits the mobility of break ends and leads to enhanced restriction fragment reconstitution by increasing the probability for rejoining correct break ends.     

Imaging features that discriminate between foci induced by high- and low-LET radiation in human fibroblasts

In this study, we investigated the formation of radiation-induced foci in normal human fibroblasts exposed to X rays or 130 keV/mum nitrogen ions using antibodies to phosphorylated protein kinase ataxia telangiectasia mutated (ATMp) and histone H2AX (gamma-H2AX). High-content automatic image analysis was used to quantify the immunofluorescence of radiation-induced foci. The size of radiation-induced foci increased for both proteins over a 2-h period after nitrogen-ion irradiation, while the size of radiation-induced foci did not change after exposure to low-LET radiation. The number of radiation-induced ATMp foci showed a more rapid rise and greater frequency after X-ray exposure and was resolved more rapidly such that the frequency of radiation-induced foci decreased by 90% compared to 60% after exposure to high-LET radiation 2 h after 30 cGy. In contrast, the kinetics of radiation-induced gamma-H2AX focus formation was similar for high- and low-LET radiation in that it reached a plateau early and remained constant for up to 2 h. High-resolution 3D images of radiation-induced gamma-H2AX foci and dosimetry computation suggest that multiple double-strand breaks from nitrogen ions are encompassed within large nuclear domains of 4.4 Mbp. Our work shows that the size and frequency of radiation-induced foci vary as a function of radiation quality, dose, time and protein target. Thus, even though double-strand breaks and radiation-induced foci are correlated, the dynamic nature of both contradicts their accepted equivalence for low doses of different radiation qualities.     

Modeling the repair of radiation-induced DNA double-strand breaks

A problem often overlooked in the study of the repair of radiation-induced DNA double-strand breads (DSBs) is the question of what the status of a regular site is in the DNA duplex immediately after a radiation treatment. Here, we suggest a mixed repair mechanism which consists of a gradual process and an instantaneous process. A comparison of the present kinetic model with those which have appeared in the literature shows that the former is a generalization of the latter. We have shown that different repair mechanisms may lead to equivalent mathematical representations. Therefore, care must be taken in interpreting the repair mechanism on the basis of the experimentally observed transient number of DSBs.     

mBAND analysis for high- and low-LET radiation-induced chromosome aberrations: a review

During long-term space travel or cancer therapy, humans are exposed to high linear energy transfer (LET) energetic heavy ions. High-LET radiation is much more effective than low-LET radiation in causing various biological effects, including cell inactivation, genetic mutations, cataracts and cancer induction. Most of these biological endpoints are closely related to chromosomal damage, and cytogenetic damage can be utilized as a biomarker for radiation insults. Epidemiological data, mainly from survivors of the atomic bomb detonations in Japan, have enabled risk estimation from low-LET radiation exposures. The identification of a cytogenetic signature that distinguishes high- from low-LET exposure remains a long-term goal in radiobiology. Recently developed fluorescence in situ hybridization (FISH)-painting methodologies have revealed unique endpoints related to radiation quality. Heavy-ions induce a high fraction of complex-type exchanges, and possibly unique chromosome rearrangements. This review will concentrate on recent data obtained with multicolor banding in situ hybridization (mBAND) methods in mammalian cells exposed to low- and high-LET radiations. Chromosome analysis with mBAND technique allows detection of both inter- and intrachromosomal exchanges, and also distribution of the breakpoints of aberrations.     

Enhancement of radiation-induced DNA double-strand breaks and micronuclei in human colon carcinoma cells by N-methylformamide

The differentiation-inducing agent N-methylformamide (NMF) enhances the sensitivity of some cell lines to ionizing radiation. To elucidate the mechanism of NMF-mediated radiosensitization, we examined the effects of this agent on gamma-ray-induced DNA double-strand breaks and micronuclei in two cell lines, clone A (human colon carcinoma) and HCA-1 (murine hepatocarcinoma). Both cell lines form a better differentiated phenotype upon exposure to NMF, yet only clone A is radiosensitized. The neutral (pH 9.6) elution assay was used to evaluate the effects of this maturational agent on radiation-induced double-strand breaks in these cell lines. Exposure of HCA-1 cells to NMF had no effect on the level of DNA double-strand breaks induced by gamma rays. In clone A cells, however, exposure to NMF enhanced the initial formation of gamma-ray-induced double-strand breaks at each dose tested. The repair of double-strand breaks in both cell lines was not influenced by NMF. As a measure of chromosome fragmentation after irradiation, we evaluated micronuclei using the cytokinesis block method. Exposure to NMF had no effect on radiation-induced micronuclei formation in HCA-1 cells yet significantly enhanced the frequency of micronuclei induced by radiation in clone A cells. In clone A cells, the increases in radiation-induced double-strand breaks and micronuclei as a function of NMF exposure time reached maximums by approximately 72 h. These data suggest that NMF-mediated radiosensitization is the result of an increase in the initial level of radiation-induced DNA double-strand breaks.     

Persistence of DNA double-strand breaks in normal human cells induced by radiation-induced bystander effect

Our previous study suggested that the DNA double-strand breaks (DSBs) induced by very low X-ray doses are largely due to bystander effects. The aim of this study was to verify whether DSBs created by radiation-induced bystander effects are likely to be repaired. We examined the generation of DSBs in cells by enumeration of phosphorylated ataxia telangiectasia mutated (ATM) foci, which are correlated with DSB repair, in normal human fibroblast cells (MRC-5) after X irradiation at doses ranging from 1 to 1000 mGy. At 24 h after irradiation, 100% (1.2 mGy), 58% (20 mGy), 12% (200 mGy) and 8.5% (1000 mGy) of the initial number of phosphorylated ATM foci were detected. The number of phosphorylated ATM foci in MRC-5 cells treated with lindane, an inhibitor of radiation-induced bystander effects, prior to X irradiation was assessed; phosphorylated ATM foci were not observed at 5 h (20 mGy) or 24 h (200 mGy) postirradiation. We also counted the number of phosphorylated ATM foci in MRC-5 cells cocultured with MRC-5 cells irradiated with 20 mGy. After 48 h of coculture, 81% of the initial numbers of phosphorylated ATM foci remained. These findings suggest that DSBs induced by the radiation-induced bystander effect persist for long periods, whereas DSBs induced by direct radiation effects are repaired relatively quickly.     

Time course solutions of the Sax-Markov binary eurejoining/misrejoining model of DNA double-strand breaks

The Sax-Markov binary eurejoining/misrejoining (SMBE) model is a stochastic representation of Sax’s breakage-and-reunion mechanism of misrejoining DNA double-strand breaks (DSBs). In this model, to approximate DSB misrejoining probabilities that decrease with increasing distance, the nucleus is treated as a collection ofη isolated nuclear subvolumes called sites; DSB free ends within the same site interact with a probability that is independent of distance, and DSB free ends within different sites never interact. In our previous work, SMBE steady-state solutions were used to estimateη from a combination of high-dose PFGE (pulsed-field gel electrophoresis) data and moderate-dose chromosomal aberration data. Here, analytic SMBE transient solutions (i.e., time courses of DSBs and misrejoinings) are derived and used to estimateη from various sets of misrejoining DSB kinetic data. The time courses are multiexponentials with rate constantsκ, 6κ, 15κ, ... j(2j–1)κ corresponding to different nuclear site states and not different types of DSBs. For example, theκ component corresponds to nuclear sites with two DSB free ends and thus only one possible rejoining interaction, and the 6κ component corresponds to sites with four DSB free ends and thus six (four choose two) potential rejoining interactions – four of these six potential interactions lead to a final state of two misrejoinings and the other two of six lead to a final state of correct repair (unrejoinable DSBs are not represented in the SMBE model). The SMBE time course solutions provide site number estimates that fall in the range ofη≈10–100 for premature chromosome condensation (PCC) data andη≈1000 for PFGE data. Received: 23 December 1999 / Accepted: 1 July 2000     

Mathematical models of the generation of radiation-induced DNA double-strand breaks

The double-strand break (dsb) is one of the most critical lesions leading to a variety of radiobiological effects. In this paper, we reconsider the previously constructed and generally accepted mathematical models for dsb generation, and give a concrete mathematical basis for the generation of dsbs and the calculation of the number of induced dsbs, under the assumption of randomness in the break location in DNA and in the number of breaks. Using these models based on the Poisson distribution and the binomial distribution, we calculate the dose dependence of dsb generation. We deduced from our models that the dose dependence of the number of dsbs is described approximately as a quadratic form in both distribution models where dsb generation is accounted for by two ssbs. Previously reported experimental data on the dsb generation in phage DNA was found to be in good agreement with our models. Though the widely used model, the linear quadratic (LQ) model or the molecular theory of dsb formation based on the Poisson distribution, also gives the quadratic term, in spite of rough estimates or some mathematical incompleteness, a marked feature of our formulation is the absence of a parameter like the $\beta $ in the quadratic term that requires experimental data to determine. Thus in this study we provide mathematical validity to the generally accepted models of the number of dsb.     

Measurement of radiation-induced DNA double-strand breaks by pulsed-field gel electrophoresis

We have examined the use of pulsed-field gel electrophoresis (PFGE) to measure DNA double-strand breaks induced in CHO cells by ionizing radiation. The PFGE assay provides a simple method for the measurement of DNA double-strand breaks for doses as low as 3-4 Gy ionizing radiation, and appears applicable for the measurement of damage produced by any agent producing double-strand breaks. The conditions of transverse alternating field electrophoresis determined both the sensitivity of the assay and the ability to resolve DNA fragments with different sizes. For example, with 0.8% agarose and a 1-min pulse time at 250 V for 18 h of electrophoresis, 0.39% of the DNA per gray migrated into the gel, and only molecules less than 1500 kb could be resolved. With 0.56% agarose and a 60-min pulse time at 40 V for 6 days of electrophoresis, 0.55-0.90% of the DNA per gray migrated into the gel, and molecules between 1500 and 7000 kb could be resolved.     

Cohesin phosphorylation and mobility of SMC1 at ionizing radiation-induced DNA double-strand breaks in human cells

Cohesin, a hetero-tetrameric complex of SMC1, SMC3, Rad21 and Scc3, associates with chromatin after mitosis and holds sister chromatids together following DNA replication. Following DNA damage, cohesin accumulates at and promotes the repair of DNA double-strand breaks. In addition, phosphorylation of the SMC1/3 subunits contributes to DNA damage-induced cell cycle checkpoint regulation. The aim of this study was to determine the regulation and consequences of SMC1/3 phosphorylation as part of the cohesin complex. We show here that the ATM-dependent phosphorylation of SMC1 and SMC3 is mediated by H2AX, 53BP1 and MDC1. Depletion of RAD21 abolishes these phosphorylations, indicating that only the fully assembled complex is phosphorylated. Comparison of wild type SMC1 and SMC1S966A in fluorescence recovery after photo-bleaching experiments shows that phosphorylation of SMC1 is required for an increased mobility after DNA damage in G2-phase cells, suggesting that ATM-dependent phosphorylation facilitates mobilization of the cohesin complex after DNA damage.     

A kinetic analysis of the repair of radiation-induced DNA double-strand breaks

The repair of radiation-induced DNA double-strand breaks (DSBs) is analyzed kinetically. It is assumed that a fraction of the damaged sites in the DNA duplex are irreparable. The kinetic model takes the effect of radiation dose into account. The analysis of the available experimental data reveals that, although the number of irreparable DSBs is a quadratic function of radiation dose, the normalized number of irreparable DSBs correlates linearly with this variable.     

A stochastic analysis of the repair of radiation-induced DNA double-strand breaks

A three-state stochastic model is described for the repair of radiation-induced double-strand breaks (DSBs) in DNA. If irradiated, a site or region in DNA is assumed to be in a potentially damaged state; this site may either become permanently damaged or be repaired after a certain period of time. The result of the analysis of the available experimental data reveals that the present two-parameter model is capable of interpreting the rapid decrease in the number of DSBs in the initial period, which cannot be predicted by previously proposed models. The stochastic analysis yields not only the temporal variation of the mean of the number of DSBs but also its variance, and therefore is a generalization of the conventional deterministic models.     

Decreased repair of radiation-induced DNA double-strand breaks with cellular differentiation.

Although the majority of mammalian cells in situ are terminally differentiated, most DNA repair studies have used proliferating cells. In an attempt to understand better the relationship between differentiation and DNA repair, we have used the murine 3T3-T proadipocyte cell line. In this model system, proliferating (stem) cells undergo growth arrest (GD cells) and subsequently terminally differentiate into adipocytes when exposed to media containing platelet-depleted human plasma. Pulsed-field gel electrophoresis was used to evaluate the induction and repair of DNA double-strand breaks (DSBs) after ionizing radiation. The levels of radiation-induced DSBs in GD and terminally differentiated cells were similar, but in both cases greater than those found in stem cells at each radiation dose tested (0 to 40 Gy); these differences appear to be due to growth arrest in G1 phase. DNA DSBs were repaired with biphasic kinetics for each cell type. For terminally differentiated cells 25% of DNA DSBs remained unrejoined compared with < 10% for GD and stem cells after a repair time of 4 h. These data indicate that terminal differentiation of 3T3-T cells is associated with a reduction in the repair of ionizing radiation-induced DNA DSBs.     

Inhibition of repair of radiation-induced DNA double-strand breaks by nickel and arsenite

The effect of arsenite or nickel on the repair of DNA double-strand breaks (DSBs) was studied in gamma-irradiated Chinese hamster ovary cells using pulsed-field gel electrophoresis. After treatment with nickel chloride or arsenite for 2 h, cells were irradiated with gamma rays at a dose of 40 Gy, and the numbers of DNA DSBs were measured immediately after irradiation as well as at 30 min postirradiation. Both arsenite and nickel(II) inhibited repair of DNA DSBs in a concentration-dependent manner; 0.08 mM arsenite significantly inhibited the rejoining of DSBs, while 76 mM nickel was necessary to observe a clear inhibition. The mean lethal concentrations for the arsenite and nickel(II) treatments were approximately 0.12 and 13 mM, respectively. This indicates that the inhibition of repair by arsenite occurred at a concentration at which appreciable cell survival occurred, but that nickel(II) inhibited repair only at cytotoxic concentrations at which the cells lost their proliferative ability. These novel observations provide insight into the mechanisms underlying the effects of combined exposure to arsenite and ionizing radiation in our environment.     

Rejoining of DNA double-strand breaks in Ku80-deficient mouse fibroblasts

The role of Ku80 in the repair of DNA double-strand breaks (DSBs) was examined in fibroblasts derived from a Ku80 knockout mouse model described by Nussenzweig et al. (Nature 382, 551-555, 1996). Primary fibroblasts from Ku80+/+ and Ku80-/- mice were immortalized by transfection with plasmids containing either the human MYC proto-oncogene or the Simian virus 40 (SV40) T antigen and were used to measure induction and rejoining of DSBs after exposure to ionizing radiation. The number of DSBs in the cells was quantified by either asymmetric field-inversion gel electrophoresis (AFIGE) or clamped homogeneous electrical-field gel electrophoresis (CHEF). The latter method was introduced for a more reliable quantification of repair even when DNA degradation occurs in a fraction of the irradiated cell population during the postirradiation incubation time. The results confirm that Ku80-deficient mouse fibroblasts are sensitive to ionizing radiation and demonstrate that the increased radiosensitivity may result from a deficiency in DSB rejoining. The results further indicate that unless techniques are employed that allow for distinction between DNA degradation and DNA repair, erroneous conclusions may be drawn regarding the potential of cells to repair DSBs.