Martin Kühne

A Double-Strand Break Repair Defect in ATM-Deficient Cells Contributes to Radiosensitivity

The ATM protein, which is mutated in individuals with ataxia telangiectasia (AT), is central to cell cycle checkpoint responses initiated by DNA double-strand breaks (DSBs). ATM’s role in DSB repair is currently unclear as is the basis underlying the radiosensitivity of AT cells. We applied immunofluorescence detection of γ-H2AX nuclear foci and pulsed-field gel electrophoresis to quantify the repair of DSBs after X-ray doses between 0.02 and 80 Gy in confluence-arrested primary human fibroblasts from normal individuals and patients with mutations in ATM and DNA ligase IV, a core component of the nonhomologous end-joining (NHEJ) repair pathway. Cells with hypomorphic mutations in DNA ligase IV exhibit a substantial repair defect up to 24 h after treatment but continue to repair for several days and finally reach a level of unrepaired DSBs similar to that of wild-type cells. Additionally, the repair defect in NHEJ mutants is dose dependent. ATM-deficient cells, in contrast, repair the majority of DSBs with normal kinetics but fail to repair a subset of breaks, irrespective of the initial number of lesions induced. Significantly, after biologically relevant radiation doses and/or long repair times, the repair defect in AT cells is more pronounced than that of NHEJ mutants and correlates with radiosensitivity. NHEJ-defective cells analyzed for survival following delayed plating after irradiation show substantial recovery while AT cells fail to show any recovery. These data argue that the DSB repair defect underlies a significant component of the radiosensitivity of AT cells.

DNA Double-Strand Break Repair of Blood Lymphocytes and Normal Tissues Analysed in a Preclinical Mouse Model: Implications for Radiosensitivity Testing

Purpose: Radiotherapy is an effective cancer treatment, but a few patients suffer severe radiation toxicities in neighboring normal tissues. There is increasing evidence that the variable susceptibility to radiation toxicities is caused by the individual genetic predisposition, by subtle mutations, or polymorphisms in genes involved in cellular responses to ionizing radiation. Double-strand breaks (DSB) are the most deleterious form of radiation-induced DNA damage, and DSB repair deficiencies lead to pronounced radiosensitivity. Using a preclinical mouse model, the highly sensitive γH2AX-foci approach was tested to verify even subtle, genetically determined DSB repair deficiencies known to be associated with increased normal tissue radiosensitivity. Experimental Design: By enumerating γH2AX-foci in blood lymphocytes and normal tissues (brain, lung, heart, and intestine), the induction and repair of DSBs after irradiation with therapeutic doses (0.1-2 Gy) was investigated in repair-proficient and repair-deficient mouse strains in vivo and blood samples irradiated ex vivo. Results: γH2AX-foci analysis allowed to verify the different DSB repair deficiencies; even slight impairments caused by single polymorphisms were detected similarly in both blood lymphocytes and solid tissues, indicating that DSB repair measured in lymphocytes is valid for different and complex organs. Moreover, γH2AX-foci analysis of blood samples irradiated ex vivo was found to reflect repair kinetics measured in vivo and, thus, give reliable information about the individual DSB repair capacity. Conclusions: γH2AX analysis of blood and tissue samples allows to detect even minor genetically defined DSB repair deficiencies, affecting normal tissue radiosensitivity. Future studies will have to evaluate the clinical potential to identify patients more susceptible to radiation toxicities before radiotherapy.

Joining of correct and incorrect DNA double-strand break ends in normal human and ataxia telangiectasia fibroblasts

Chromosomal aberrations are believed to result from the incorrect joining of DNA double‐strand breaks (DSBs). In an attempt to investigate induction and rejoining quality of DSBs following ionizing radiation exposure in specific genomic locations of mammalian DNA, an experimental approach based on Southern hybridization of single‐copy probes to NotI restriction fragments was developed. Induction of DSBs is measured from the decrease of the band intensity representing the unbroken restriction fragment. An increase in intensity of the hybridization band following repair incubation determines reconstitution of the original restriction fragment and thus rejoining of correct DNA ends. We investigated the dose dependence of DSB misrejoining using X‐ray doses of 5, 10, 20, 40, and 80 Gy and provide evidence that the number of misrejoined DSBs exceeds, for the same doses used, the number of cytogenetically visible aberrations by an order of magnitude, reflecting the higher resolution of our assay. Induction of DSBs and joining of correct and incorrect break ends were further investigated in cells from a patient with the cancer‐prone disease ataxia telangiectasia (AT) and in heterozygous AT cells. We found, compared to normal cells, identical induction rates and identical kinetics for joining correct ends following an 80‐Gy X‐ray exposure. After 5 and 10 Gy, however, AT homozygotes showed a 50% elevation in the proportion of breaks that are not correctly rejoined. These data indicate a defect in the accuracy of DSB rejoining in AT cells that may account for radiation sensitivity and the occurrence of the high level of chromosomal aberrations observed in AT cells. Genes Chromosomes Cancer 27:59–68, 2000.

No dose-dependence of DNA double-strand break misrejoining following α-particle irradiation

Purpose : To investigate whether an explanation for the high effectiveness of densely ionizing radiation with regard to complex biological endpoints can be derived from measurements of radiation-induced double-strand break (DSB) misrejoining. Materials and methods : Misrejoining of radiation-induced DSB in normal human fibroblasts was determined by comparing hybridization analysis of large restriction fragments as a measure for correct rejoining, with results from a conventional pulsed-field gel electrophoresis technique (FAR) that measures total DSB rejoining. In order to investigate DSB misrejoining at doses for which chromosome aberration data are available, a dose fractionation protocol was applied so that the number of DSB at any given timepoint was low but the cumulative amount of misrejoined DSB sufficient for detection and precise quantitation. Results and conclusion : After an acute 80 Gy α -particle exposure and a repair incubation period of 24 h, 50% of all initially induced DSB were misrejoined, in agreement with data obtained for X-rays. X-irrradiation with 16 x 5 Gy, 8 x 10 Gy, 4 x 20 Gy, or 2 x 40 Gy and repair incubation of 24 h following each individual dose fraction was recently reported to yield misrejoining frequencies that strongly decrease with increasing fractionation (Löbrich et al. 2000; Genes, Chromosomes and Cancer, 27, 59-68). In the present study, constant misrejoining frequencies of 50% were observed after α -particle exposure with the same fractionation protocol. This difference between α -particles and X-rays is in accordance with the high biological effectiveness of densely ionizing radiation and provides a direct link between misrejoining of DSB and cytologically visible exchange aberrations. Further evidence suggests that if the same dose range is compared, the number of misrejoined DSB exceeds the number of microscopically visible aberrations by an order of magnitude for both radiation types, probably reflecting the high resolution of the hybridization approach compared with cytological techniques.Purpose : To investigate whether an explanation for the high effectiveness of densely ionizing radiation with regard to complex biological endpoints can be derived from measurements of radiation-induced double-strand break (DSB) misrejoining. Materials and methods : Misrejoining of radiation-induced DSB in normal human fibroblasts was determined by comparing hybridization analysis of large restriction fragments as a measure for correct rejoining, with results from a conventional pulsed-field gel electrophoresis technique (FAR) that measures total DSB rejoining. In order to investigate DSB misrejoining at doses for which chromosome aberration data are available, a dose fractionation protocol was applied so that the number of DSB at any given timepoint was low but the cumulative amount of misrejoined DSB sufficient for detection and precise quantitation. Results and conclusion : After an acute 80 Gy α -particle exposure and a repair incubation period of 24 h, 50% of all initially induced DSB were misrejoine...

DNA Repair Alterations in Children With Pediatric Malignancies: Novel Opportunities to Identify Patients at Risk for High-Grade Toxicities

PURPOSE To evaluate, in a pilot study, the phosphorylated H2AX (γH2AX) foci approach for identifying patients with double-strand break (DSB) repair deficiencies, who may overreact to DNA-damaging cancer therapy. METHODS AND MATERIALS The DSB repair capacity of children with solid cancers was analyzed compared with that of age-matched control children and correlated with treatment-related normal-tissue responses (n = 47). Double-strand break repair was investigated by counting γH2AX foci in blood lymphocytes at defined time points after irradiation of blood samples. RESULTS Whereas all healthy control children exhibited proficient DSB repair, 3 children with tumors revealed clearly impaired DSB repair capacities, and 2 of these repair-deficient children developed life-threatening or even lethal normal-tissue toxicities. The underlying mutations affecting regulatory factors involved in DNA repair pathways were identified. Moreover, significant differences in mean DSB repair capacity were observed between children with tumors and control children, suggesting that childhood cancer is based on genetic alterations affecting DSB repair function. CONCLUSIONS Double-strand break repair alteration in children may predispose to cancer formation and may affect childrens susceptibility to normal-tissue toxicities. Phosphorylated H2AX analysis of blood samples allows one to detect DSB repair deficiencies and thus enables identification of children at risk for high-grade toxicities.

THE IMPACT OF INDIVIDUAL IN VIVO REPAIR OF DNA DOUBLE-STRAND BREAKS ON ORAL MUCOSITIS IN ADJUVANT RADIOTHERAPY OF HEAD-AND-NECK CANCER

PURPOSE To evaluate the impact of individual in vivo DNA double-strand break (DSB) repair capacity on the incidence of severe oral mucositis in patients with head-and-neck cancer undergoing adjuvant radiotherapy (RT) or radiochemotherapy (RCT). PATIENTS AND METHODS Thirty-one patients with resected head-and-neck cancer undergoing adjuvant RT or RCT were examined. Patients underwent RT of the primary tumor site and locoregional lymph nodes with a total dose of 60-66 Gy (single dose 2 Gy, five fractions per week). Chemotherapy consisted of two cycles of cisplatin and 5-fluorouracil. To assess DSB repair, γ-H2AX foci in blood lymphocytes were quantified before and 0.5 h, 2.5 h, 5 h, and 24 h after in vivo radiation exposure (the first fraction of RT). World Health Organization scores for oral mucositis were documented weekly and correlated with DSB repair. RESULTS Sixteen patients received RT alone; 15 patients received RCT. In patients who developed Grade≥3 mucositis (n=18) the amount of unrepaired DSBs 24 h after radiation exposure and DSB repair half-times did not differ significantly from patients with Grade≤2 mucositis (n=13). Patients with a proportion of unrepaired DSBs after 24 h higher than the mean value + one standard deviation had an increased incidence of severe oral mucositis. CONCLUSIONS Evaluation of in vivo DSB repair by determination of γ-H2AX foci loss is feasible in clinical practice and allows identification of patients with impaired DSB repair. The incidence of oral mucositis is not closely correlated with DSB repair under the evaluated conditions.

DNA Double-Strand Break Misrejoining after Exposure of Primary Human Fibroblasts to CK Characteristic X Rays, 29 kVp X Rays and 60Co γ Rays

Abstract Kühne, M., Urban, G., Frankenberg, D. and Löbrich, M. DNA Double-Strand Break Misrejoining after Exposure of Primary Human Fibroblasts to CK Characteristic X Rays, 29 kVp X Rays and 60Co γ Rays. Radiat. Res. 164, 669–676 (2005). The efficiency of ionizing photon radiation for inducing mutations, chromosome aberrations, neoplastic cell transformation, and cell killing depends on the photon energy. We investigated the induction and rejoining of DNA double-strand breaks (DSBs) as possible contributors for the varying efficiencies of different photon energies. A specialized pulsed-field gel electrophoresis assay based on Southern hybridization of single Mbp genomic restriction fragments was employed to assess DSB induction and rejoining by quantifying the restriction fragment band. Unrejoined and misrejoined DSBs were determined in dose fractionation protocols using doses per fraction of 2.2 and 4.4 Gy for CK characteristic X rays, 4 and 8 Gy for 29 kVp X rays, and 5, 10 and 20 Gy for 60Co γ rays. DSB induction by CK characteristic X rays was about twofold higher than for 60Co γ rays, whereas 29 kVp X rays showed only marginally elevated levels of induced DSBs compared with 60Co γ rays (a factor of 1.15). Compared with these modest variations in DSB induction, the variations in the levels of unrejoined and misrejoined DSBs were more significant. Our results suggest that differences in the fidelity of DSB rejoining together with the different efficiencies for induction of DSBs can explain the varying biological effectiveness of different photon energies.

Comments on the Paper “No Detectable Misrejoining in Double-Minute Chromosomes” by Nevaldine et al. (Radiat. Res. 152, 154–159, 1999)

In a recent paper by Nevaldine et al. (1), DNA double-strand break (DSB) misrejoining after exposure to ionizing radiation was analyzed in double-minute chromosomes. The authors reported that elimination of broken DNA fragments by a pre-electrophoresis approach also eliminates misrejoined DNA. Based on their experimental results, Nevaldine et al. concluded that measurements of misrejoining using pulsed-field gel electrophoresis, rare-cutting restriction endonucleases, and Southern hybridization are likely to be compromised by artifacts such as apoptosis or necrosis since these processes generate broken DNA which might be more resistant to cleavage by rare-cutting restriction endonucleases sensitive to methylation. The authors further concluded that the key assumptions and consequently the conclusions of our DSB misrejoining measurements first published in ref. (2) are questionable. A DSB rejoining study involving hybridization detection of large restriction fragments measures reconstitution of the intact fragment and hence quantifies the rejoining of correct DNA ends. Only by comparing results from this approach with results from a conventional electrophoresis assay that detects residual DNA fragments (such as the FAR assay) is it possible to quantify the misrejoining frequency. The key assumption in our analysis is that breaks that have not been rejoined to reconstitute the intact restriction fragment either have been misrejoined or remain unrejoined. By applying the FAR assay, unrejoined DSBs, including those which might have been generated by enzymatic processes during the incubation period, are quantified and subtracted from the class of breaks not rejoined to correct DNA ends. Consequently, our measurements of misrejoining already take degradation of DNA as a possible source of artifacts into consideration. Removing broken DNA fragments by preelectrophoresis prior to analysis with hybridization corresponds to a clever application of the same procedure in one experimental step without the need to use two separate gels. Nevaldine et al. did not detect any misrejoining after applying the correct procedure, which involved subtracting broken fragments by preelectrophoresis prior to the misrejoining analysis. This might seem surprising at first, since our studies clearly show that a large proportion of DSBs are misrejoined after doses as high as 100 Gy. However, there are both conceptual and technical problems with the experiments of Nevaldine et al. To detect misrejoining events inside a restriction fragment of limited length, a sufficient number of DSBs must be induced within the fragment. The sizes of the restriction fragments that were analyzed in their experiments were quite small, ranging from 280 to 550 kbp, so that X-ray doses of 50 and 100 Gy induce much less than 1 break per fragment. Since only a fraction of breaks are misrejoined, even a system with multiple copies of the fragments, which will increase the overall signal in the blot, would require complete restriction digestion and clean hybridization signals without background hybridization for a reliable detection of misrejoining. Neither of these requirements were accomplished in the work of Nevaldine et al., which exhibited high and variable background in the blots. That the target size was much too small and the experimental quality far from sufficient for DSB measurements is also documented by the fact that the intensity of the intact restriction fragment did not decrease but rather increased for an irradiated sample without repair (see their Table 1, no pre-electrophoresis of samples). Since that is the case, it is not possible to draw conclusions about DSB misrejoining based on the reported experiments.