γH2AX Assay Research Articles

Evaluation of the co-genotoxic effects of 1800 MHz GSM radiofrequency exposure and a chemical mutagen in cultured human cells

Abstract We investigated the effect of a 1800 MHz radiofrequency GSM signal combined with a known chemical mutagen (4-nitroquinoline-N-oxide: 4NQO) on human THP1 cells. Comet and γ-H2AX assays were used to assess DNA damage. No heating of the cell cultures was noted during exposure (2 h). The exposure of cells to electromagnetic fields with SARs of 2 to 16 W/kg did not increase the DNA damage induced by 4NQO, whereas the number of DNA strand breaks increased with a temperature increase of at least 4 °C. In conclusion, no co-genotoxic effect of radiofrequency was found at levels of exposure that did not induce heating.

ErbB2 expression through heterodimerization with erbB1 is necessary for ionizing radiation- but not EGF-induced activation of Akt survival pathway

Purpose ErbB1-dependent Akt phosphorylation improves post-irradiation cellular survival. In the present study, we investigated the contribution of erbB2 as a heterodimerization partner of erbB1 in activation of Akt survival signaling after irradiation or EGF treatment. Materials and methods Pattern of receptor dimerization and protein phosphorylation were investigated by Western and immunoblotting as well as immunoprecipitation techniques. Residual DNA double-strand breaks (DNA-DSB) and clonogenic activity were analyzed by γH2AX and standard clonogenic assay. To knocked erbB2 expression siRNA was used. Results In lung carcinoma cell lines A549 and H661, the erbB1-tyrosine kinase (TK) inhibitor erlotinib blocked EGF as well as ionizing radiation (IR)-induced Akt and DNA-PKcs phosphorylation. Targeting Akt and erbB1 induced cellular radiation sensitivity while, the erbB2-TK inhibitor AG825 neither affected phosphorylation of Akt and DNA-PKcs nor induced radiosensitization. ErbB2-siRNA and the anti-erbB2 antibody trastuzumab blocked IR-induced, but not EGF-stimulated Akt phosphorylation and impaired the repair of DNA-DSB. Likewise, IR but not EGF enhanced erbB1/erbB2 heterodimerization and resulted in the release of phosphorylated erbB2 cleavage products p135 and p95. Trastuzumab prevented radiation-induced formation of an active erbB1/erbB2 heterodimer and increased cellular radiation sensitivity. ErbB1- but not erbB2-TK inhibition stabilized erbB2 (p185) through preventing its cleavage. Conclusions The data indicates that ErbB2 through heterodimerization with erbB1 is necessary for the activation of Akt signaling following irradiation but not following EGF treatment.

Development of a robotically-based automated biodosimetry tool for high-throughput radiological triage

To provide the best opportunities for life-saving interventions in the event of a radiological or nuclear threat, there is an urgent need to improve the speed and efficiency of biodosimetric assays for triage and therapy. A rapid automated biodosimetric system used to assess thousands of individual radiation exposure doses is helpful to curb mass panic, and to conserve limited medical resources. This paper presents the development of a new robotically-based automated biodosimetry tool (RABiT). The RABiT is capable of automating two mature biodosimetry assays: the micronucleus and γ-H2AX assay. The design considerations guiding the hardware and software architecture are presented with focus on ease of implementation, methods of communication and need for real-time control versus soft time control cycles. Advanced technological developments for the RABiT including multipurpose gripper, non-contact laser cutting, automated biology protocols and transferring of the samples to a transparent substrate and high-speed multiple camera imaging are described in detail. The evaluation results show that the RABiT prototype has a throughput of 5,859 samples in an 18-hour duty cycle.

Chromosome Instability Associates with Radiation and Doxorubicin Resistance in Triple Negative Breast Cancer Cell Lines.

Abstract BackgroundTriple negative (TN) breast cancers often have a worse prognosis, despite receiving appropriate chemotherapy and radiation treatment. Thus, one of the most pressing issues in breast cancer research is investigating the molecular basis for the inherent resistance of TN cells to standard of care treatment. Chromosome instability strongly associates with chemo-resistance and poor prognosis in breast cancer. We hypothesized that genetic heterogeneity imparted by chromosome instability provides the basis for chemotherapy and radiotherapy resistance in TN cells.MethodsFour TN cell lines (HS578, HCC1806, MB231, MB468) and a benign breast tissue line (MCF10) were treated with radiation or doxorubicin. The effects of the treatments were evaluated by the colony forming unit (CFU), MTT survival, Ki-67 immuno-histochemistry (IHC), caspase-3 IHC, micronuclei, and γ-H2AX assays.ResultsRadiation and Chemotherapy Dose Responses: Table I reveals the data from the CFU and MTT assays after treatment with radiation at 0, 1, 2, 4, 6, 8 Gy or doxorubicin at 0.5, 1, 2 μg/ml, respectively. The analyses revealed a range of sensitivities to radiation and doxorubicin, where HS578 was more sensitive to both treatments.Table I. Relative radio- and chemo-resistance of TN cell linesCell TypeTD 50 (Gy)IC50 (μg/ml)HS5781.60.31HCC18061.80.86MB4681.90.95MB2311.91.05MCF101.80.32 Apoptosis and Proliferation: Because the basis for chemo-and radio-resistance of TN cells may be uncontrolled proliferation frequencies and/or decreased apoptosis, proliferation assays (assessed by Ki-67 IHC), as well as apoptotic indexes (i.e. cleaved caspase 3) were performed. The Ki-67 assay showed that 2 Gy reduced proliferation by 13% in the HS578 line, but by only 2% with the MB231cells.After receiving 2 Gy, there was a wide range of apoptotic activity among the TN cell lines. The largest percentage of apoptosis occurred with the MB468 cells at 19% vs the lowest apoptotic activity seen with the HCC1806 line, 7%. Importantly, apoptotic activity was ≤ 7% when these cell lines were treated with 1 μg/ml of doxorubicin.Chromosome instability: The micronuclei and the γ-H2AX assays showed that 3 of the 4 TN cell lines harbored active genomic instability in the absence of doxorubicin or irradiation. The unstable TN cell lines had 15-30% γ-H2AX activity and 20-40% micronuclei formation, whereas the MCF10 and HCC1806 cells had approximately 11% γ-H2AX activity and 10% micronuclei present. To assess the molecular markers contributing to chromosome instability, we performed a real time PCR screen, as well as Western blot analyses of the cyclins, cyclin-dependent inhibitors and other signaling molecules that may contribute to chromosome instability. All cell lines that had high frequencies of chromosome instability had increased levels of cyclin D1, whereas a subset also de-regulated cyclin A.ConclusionOur results show that radio- and chemo- resistance in TN breast cancer cells closely associates with a de-regulated cell cycle and active chromosome instability. We conclude that the molecular basis for chromosome instability, chemotherapy and radiation resistance may be secondary to the unregulated expression of cyclins D1 and A. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1143.

Phase I Study of Sequence-Selective Minor Groove DNA Binding Agent SJG-136 in Patients with Advanced Solid Tumors

This phase I dose-escalation study was undertaken to establish the maximum tolerated dose of the sequence-selective minor groove DNA binding agent SJG-136 in patients with advanced solid tumors. The study also investigated antitumor activity and provided pharmacokinetic and pharmacodynamic data. Sixteen patients were assigned sequentially to escalating doses of SJG-136 (15-240 microg/m(2)) given as a 10-minute i.v. infusion every 21 days. The dose was subsequently reduced in incremental steps to 45 microg/m(2) due to unexpected toxicity. The maximum tolerated dose of SJG-136 was 45 microg/m(2). The main drug-related adverse event was vascular leak syndrome (VLS) characterized by hypoalbuminemia, pleural effusions, ascites, and peripheral edema. Other unexpected adverse events included elevated liver function tests and fatigue. The VLS and liver toxicity had delayed onset and increased in severity with subsequent cycles. Disease stabilization was achieved for >6 weeks in 10 patients; in 2 patients this was maintained for >12 weeks. There was no evidence of DNA interstrand cross-linking in human blood lymphocytes with the use of the comet assay. Evidence of DNA interaction in lymphocytes and tumor cells was shown through a sensitive gamma-H2AX assay. SJG-136 had linear pharmacokinetics across the dose range tested. SJG-136 was associated with dose-limiting VLS and hepatotoxicity when administered by short injection every 21 days. DNA damage was noted, at all dose levels studied, in circulating lymphocytes. The etiology of the observed toxicities is unclear and is the subject of further preclinical research. Alternative clinical dosing strategies are being evaluated.

DNA and chromosomal damage in response to intermittent extremely low-frequency magnetic fields

Considerable controversy still exists as to whether electric and magnetic fields (MF) at extremely low frequencies are genotoxic to humans. The aim of this study was to test the ability of alternating magnetic fields to induce DNA and chromosomal damage in primary human fibroblasts. Single- and double-strand breaks were quantified using the alkaline comet assay and the γH2AX-foci assay, respectively. Chromosomal damage was assayed for unstable aberrations, sister chromatid exchange and micronuclei. Cells were exposed to switching fields – 5 min on, 10 min off – for 15 h over the range 50–1000 μT. Exposure to ionizing radiation was used as a positive-effect calibration. In this study two separate MF exposure systems were used. One was based on a custom-built solenoid coil system and the other on a commercial system almost identical to that used in previous studies by the EU REFLEX programme. With neither system could DNA damage or chromosomal damage be detected as a result of exposure of fibroblasts to switching MF. The sensitive γH2AX assay could also not detect significant DNA damage in the MF-exposed fibroblasts, although the minimum threshold for this assay was equivalent to an X-ray dose of 0.025 Gy. Therefore, with comparable MF parameters employed, this study could not confirm previous studies reporting significant effects for both the alkaline and neutral comet assays and chromosomal aberration induction.

Sulforaphane induces DNA double strand breaks predominantly repaired by homologous recombination pathway in human cancer cells

Cytotoxicity and DNA double strand breaks (DSBs) were studied in HeLa cells treated with sulforaphane (SFN), a well-known chemo-preventive agent. Cell survival was impaired by SFN in a concentration and treatment time-dependent manner. Both constant field gel electrophoresis (CFGE) and γ-H2AX assay unambiguously indicated formation of DSBs by SFN, reflecting the cell survival data. These DSBs were predominantly processed by homologous recombination repair (HRR), judging from the SFN concentration-dependent manner of Rad51 foci formation. On the other hand, the phosphorylation of DNA-PKcs, a key non-homologous end joining (NHEJ) protein, was not observed by SFN treatment, suggesting that NHEJ may not be involved in DSBs induced by this chemical. G2/M arrest by SFN, a typical response for cells exposed to ionizing radiation was also observed. Our new data indicate the clear induction of DSBs by SFN and a useful anti-tumor aspect of SFN through the induction of DNA DSBs.

Relationship Between Induction of Phosphorylated H2AX and Survival in Breast Cancer Cells Exposed to 111In-DTPA-hEGF

The Auger electron-emitting radiopharmaceutical 111In-diethylenetriaminepentaacetic acid human epidermal growth factor (111In-DTPA-hEGF) binds the epidermal growth factor receptor (EGFR), is internalized, and translocates to the nucleus. The purpose of this study was to investigate the relationship between EGFR expression, DNA damage, and cytotoxicity in cells exposed to 111In-DTPA-hEGF. Breast cancer cell lines with a range of EGFR expression levels were exposed to 111In-DTPA-hEGF or gamma-radiation. The cell lines (followed by number of EGFR per cell in parentheses) were MDA-MB-468 (1.3 x 10(6)), MDA-MB-231 (1.3 x 10(5)), and MCF-7 (1.5 x 10(4)). The proportion of radioactivity partitioning into the nucleus was measured by cell fractionation. DNA double-strand breaks were evaluated using the gamma-H2AX assay. Clonogenic survival assays were used to measure cytotoxicity. All data are presented as mean +/- SD. The amount of 111In-DTPA-hEGF that translocated to the nucleus (in mBq/nucleus) in MDA-MB-468, MDA-MB-231, and MCF-7 cells incubated with 111In-DTPA-hEGF (5.2 MBq/mL, 43 nM) for 20 h was 131 +/- 6, 8.1 +/- 0.1, and 1.1 +/- 0.9, respectively. The number of gamma-H2AX foci per nucleus was 35 +/- 15, 19 +/- 10, and 1.7 +/- 0.3, respectively. A reduction in the surviving fraction (SF) in MDA-MB-468 (0.013 +/- 0.001) and MDA-MB-231 (0.5 +/- 0.1) but not in MCF-7 cells after exposure to 111In-DTPA-hEGF (5.2 MBq/mL, 43 nM) for 20 h has been demonstrated. The SF of MDA-MB-468 cells after exposure to DTPA-EGF (43 nM) and 111In-acetate (5.2 MBq/mL) for 20 h was 0.5 +/- 0.1 and 0.53 +/- 0.05, respectively. MDA-MB-468 was the most sensitive of the cell lines to gamma-irradiation, with an SF after 2 Gy of 0.45 +/- 0.04, compared with 0.7 +/- 0.1 and 0.8 +/- 0.1 for MCF-7 and MDA-MB-231, respectively. The number of gamma-H2AX foci per nucleus in MDA-MB-468 cells correlated with the concentration, specific activity, and incubation time of 111In-DTPA-hEGF. DNA damage caused by 111In-DTPA-hEGF correlates with the EGFR expression level of the exposed cells and with concentration, specific activity, and incubation time of 111In-DTPA-hEGF. The gamma-H2AX assay may be a useful biomarker to predict and monitor the outcome of treatment with 111In-DTPA-hEGF.

Processing of clustered DNA damage in human breast cancer cells MCF-7 with partial DNA-PKcs deficiency

Complex DNA damage such as double strand breaks (DSBs) and non-DSB bistranded oxidative clustered DNA lesions (OCDL) (two or more DNA lesions within a short DNA fragment of 1–10 bp on opposing DNA strands) are considered the hallmark of ionizing radiation. Clustered DNA lesions are hypothesized to be repair-resistant lesions challenging the repair mechanisms of the cell. The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays an important role during the processing of DSBs. To evaluate the role of DNA-PKcs in the processing of complex DNA damage in human MCF-7 breast cancer cells we used small interfering RNAs (siRNAs) to target the silencing of the gene Prkdc coding for DNA-PKcs. MCF-7 cells with knockdown DNA-PKcs expression showed a marked decrease in their efficiency to process DSBs and OCDL after exposure to radiotherapy-relevant γ ray doses. For the detection and measurement of complex DSBs and OCDL, we used the γ-H2AX assay and an adaptation of pulsed field gel electrophoresis with Escherichia coli repair enzymes as DNA damage probes. An accumulation of all types of DNA damage was detected for the siRNA-treated MCF-7 cells compared to controls. These findings point to the important role of DNA-PKcs in the processing of complex DNA damage and its potential association with breast cancer development.

Involvement of Oxidative Metabolism and Membrane Signaling in Bystander Response: A Microbeam Study.

Charged particles microbeam facilities are a unique tool that allows targeting of single cells and analysis of the induced damage on a cell-by-cell basis. The ability of these tools to discriminate between hit and non-hit cells, and to deliver a precise dose is of particular interest in the investigation of various phenomena related to low dose radiation responses and more particularly in bystander effects. Since spring 2004, the choice was made to develop a device specifically designed and dedicated to radiobiology studies, with for imperative to adapt the device to the ideal conditions for cell culture. Today the single-cell microbeam system is operational: it can deliver 10 individual alpha particles (3 MeV) to selected cell nuclei in a precisely known proportion of cells in a population. The investigated endpoint in radiobiology studies is the identification of signaling pathways implicated in the appearance of non targeted effects. Here, the induction of bystander effect was studied by assessing DNA double-strand breaks (DSB) formation with the γH2AX and 53BP1 foci formation assay. Then, a comparative kinetics was established between these foci formation assay and reactive oxygen species (ROS) generation. This kinetics was accomplished in a range of time going from 30 minutes till 24 hours post-irradiation. By this way, we showed the presence of ROS in the hit cells as well as in the bystander cells. This was noticed till 2 hours post irradiation and correlated in observation of foci in both populations of cells. Moreover, alpha particles induced generation of ROS in the hit cells by a mitochondria-dependent way. This ROS generation was partly involved in a process of radio-induced apoptosis (24h post-irradiation) in the hit cells. However, no apoptosis was detected in bystander cells. On the other hand, the cell membrane seemed to play a decisive role in the signaling leading to the generation of ROS in the hit and bystander cells. Indeed, the use of filipine, which destabilized the rafts of cholesterol in the membrane, annihilated the ROS generation in these two populations of cells. We thus support the hypothesis that ROS, generated by mitochondria as well as cell membrane, belongs to signaling pathways directly involved in bystander effect.

Comparison of the induction and disappearance of DNA double strand breaks and γ-H2AX foci after irradiation of chromosomes in G1-phase or in condensed metaphase cells

The induction and disappearance of DNA double strand breaks (DSBs) after irradiation of G1 and mitotic cells were compared with the γ-H2AX foci assay and a gel electrophoresis assay. This is to determine whether cell cycle related changes in chromatin structure might influence the γ-H2AX assay which depends on extensive phosphorylation and dephosphorylation of the H2AX histone variant surrounding DSBs. The disappearance of γ-H2AX foci after irradiation was much slower for mitotic than for G1 cells. On the other hand, no difference was seen for the gel electrophoresis assay. Our data may suggest the limited accessibility of dephosphorylation enzyme in irradiated metaphase cells or trapped γ-H2AX in condensed chromatin.

Induction and processing of complex DNA damage in human breast cancer cells MCF-7 and nonmalignant MCF-10A cells

Oxidatively induced stress and DNA damage have been associated with various human pathophysiological conditions, including cancer and aging. Complex DNA damage such as double-strand breaks (DSBs) and non-DSB bistranded oxidatively induced clustered DNA lesions (OCDL) (two or more DNA lesions within a short DNA fragment of 1–10 bp on opposing DNA strands) are hypothesized to be repair-resistant lesions challenging the repair mechanisms of the cell. To evaluate the induction and processing of complex DNA damage in breast cancer cells exposed to radiotherapy-relevant γ-ray doses, we measured single-strand breaks (SSBs), DSBs, and OCDL in MCF-7 and HCC1937 malignant cells as well as MCF-10A nonmalignant human breast cells. For the detection and measurement of SSBs, DSBs, and OCDL, we used the alkaline single-cell gel electrophoresis, γ-H2AX assay, and an adaptation of pulsed-field gel electrophoresis with E. coli repair enzymes as DNA damage probes. Increased levels for most types of DNA damage were detected in MCF-7 cells while the processing of DSBs and OCDL was deficient in these cells compared to MCF-10A cells. Furthermore, the total antioxidant capacity of MCF-7 cells was lower compared to their nonmalignant counterparts. These findings point to the important role of complex DNA damage in breast cancer and its potential association with breast cancer development especially in the case of deficient BRCA1 expression.

Epidermal Growth Factor Receptor Inhibition Modulates the Nuclear Localization and Cytotoxicity of the Auger Electron Emitting Radiopharmaceutical 111In-DTPA Human Epidermal Growth Factor

(111)In-DTPA-human epidermal growth factor ((111)In-DTPA-hEGF [DTPA is diethylenetriaminepentaacetic acid]) is an Auger electron-emitting radiopharmaceutical that targets EGF receptor (EGFR)-positive cancer. The purpose of this study was to determine the effect of EGFR inhibition by gefitinib on the internalization, nuclear translocation, and cytotoxicity of (111)In-DTPA-hEGF in EGFR-overexpressing MDA-MB-468 human breast cancer cells. Western blot analysis was used to determine the optimum concentration of gefitinib to abolish EGFR activation. Internalization and nuclear translocation of fluorescein isothiocyanate-labeled hEGF were evaluated by confocal microscopy in MDA-MB-468 cells (1.3 x 10(6) EGFRs/cell) in the presence or absence of 1 microM gefitinib. The proportion of radioactivity partitioning into the cytoplasm and nucleus of MDA-MB-468 cells after incubation with (111)In-DTPA-hEGF for 24 h at 37 degrees C in the presence or absence of 1 microM gefitinib was measured by cell fractionation. DNA double-strand breaks caused by (111)In were quantified using the gamma-H2AX assay, and radiation-absorbed doses were estimated. Clonogenic survival assays were used to measure the cytotoxicity of (111)In-DTPA-hEGF alone or in combination with gefitinib. Gefitinib (1 microM) completely abolished EGFR phosphorylation in MDA-MB-468 cells. Internalization and nuclear translocation of fluorescein isothiocyanate-labeled EGF were not diminished in gefitinib-treated cells compared with controls. The proportion of internalized (111)In that localized in the nucleus was statistically significantly greater when (111)In-DTPA-hEGF was combined with gefitinib compared with (111)In-DTPA-hEGF alone (mean +/- SD: 26.0% +/- 5.5% vs. 14.6% +/- 4.0%, respectively; P < 0.05). Induction of gamma-H2AX foci was greater in MDA-MB-468 cells that were treated with (111)In-DTPA-hEGF (250 ng/mL, 1.5 MBq/mL) plus gefitinib (1 microM ) compared with those treated with (111)In-DTPA-hEGF alone (mean +/- SD: 35 +/- 4 vs. 24 +/- 5 foci per nucleus, respectively). In clonogenic assays, a significant reduction in the surviving fraction was observed when (111)In-DTPA-hEGF (5 ng/mL, 6 MBq/microg) was combined with gefitinib (1 microM ) compared with (111)In-DTPA-hEGF alone (42.9% +/- 5.7% vs. 22.9% +/- 3.6%, respectively; P < 0.01). The efficacy of (111)In-DTPA-hEGF depends on internalization and nuclear uptake of the radionuclide. Nuclear uptake, DNA damage, and cytotoxicity are enhanced when (111)In-DTPA-hEGF is combined with gefitinib. These results suggest a potential therapeutic role for peptide receptor radionuclide therapy in combination with tyrosine kinase inhibitors.

A defect in DNA double strand break processing in cells from unaffected parents of retinoblastoma patients and other apparently normal humans

Cells from unaffected parents of retinoblastoma (RB) patients were previously shown to be hypersensitive to radiation induced G 1 arrest and cell killing [1]. The hypersensitivity was similar to that reported for cells from ATM heterozygotes. The latter was consistent with a mild DNA DSB rejoining defect which we demonstrated using a γ-H2AX focus assay after low dose-rate (LDR) irradiation of non-cycling G 0 cells [2,3]. Since neither parent carried the mutant RB allele of the RB heterozygous probands, these results suggested the possibility of an enhanced germline mutation rate, perhaps resulting from some mild defect in genome maintenance. We therefore examined levels of γ-H2AX foci for cells from these RB parents in this G 0 LDR assay, which reflects the non-homologous end joining (NHEJ) capacity of cells and in a G 2/M assay, which reflects additional contributions from other G 2-related damage processing systems. For several of the cell strains parallel radiosensitivity comparisons were made for cell killing and for G 2 chromosomal radiosensitivities. G 0 cells from the RB parents were clearly hypersensitive both in the LDR γ-H2AX assay, and for cell killing. In addition, cultured fibroblasts from 6 of 15 apparently normal individuals in this study (and one of six in a previous study) were also hypersensitive in the same assays. In the G 2/M γ-H2AX assay, the relative sensitivities were similar to those seen in the low dose-rate G 0 assay and tracked with chromosomal radiosensitivity, but some differences were observed.

γ-H2AX Foci after Low-Dose-Rate Irradiation RevealAtmHaploinsufficiency in Mice

We have investigated the use of the gamma-H2AX assay, reflecting the presence of DNA double-strand breaks (DSBs), as a possible means for identifying individuals who may be intermediate with respect to the extremes of hyper-radiosensitivity phenotypes. In this case, cells were studied from mice that were normal (Atm+/+), heterozygous (Atm+/-), or homozygous recessive (Atm-/-) for a truncating mutation in the Atm gene. After single acute (high-dose-rate) exposures, differences in mean numbers of gamma-H2AX foci per cell between samples from Atm+/+ and Atm-/- mice were clear at nearly all sampling times, but at no sampling time was there a clear distinction for cells from Atm+/+ and Atm+/- mice. In contrast, under conditions of low-dose-rate irradiation at 10 cGy/h, appreciable differences in the levels of gamma-H2AX foci per cell were observed in synchronized G1 cells derived from Atm+/- mice relative to cells from Atm+/+ mice. The levels were intermediate between those for cells from Atm+/+ and Atm-/- mice. After 24 h exposure at this dose rate, measurements in cells from four different mice for each genotype yielded mean frequencies of foci per cell of 1.77 +/- 0.13 (SEM) for Atm+/+ cells, 4.75 +/- 0.20 for the Atm+/- cells, and 11.10 +/- 0.33 for the Atm-/-cells. The distributions of foci per G1 cell were not significantly different from Poisson. To the extent that variations in sensitivity with respect to gamma-H2AX focus formation reflect variations in radiosensitivity for biological effects of concern, such as carcinogenesis, and that similar differences are seen for other genetic DNA DSB processing defects in general, this assay may provide a relatively straightforward means for distinguishing individuals who may be mildly hypersensitive to radiation such as we observed for Atm heterozygous mice.

Facilitated detection of chromosome break and repair at low levels of ionizing radiation by addition of wortmannin to G 1-type PCC fusion incubation

We have measured rejoining kinetics of chromosome breaks using a modified cell fusion-based premature chromosome condensation (PCC) technique in confluent cultures of normal human fibroblasts irradiated at low doses of X-rays. In order to enhance the sensitivity of the fusion-based PCC assay, we added a DNA double strand break (DSB) repair inhibitor wortmannin during the incubation period for PCC/fusion process resulting in a significantly higher yield of G 1-type chromosome breaks. The initial number of chromosome breaks (without repair) gave a linear dose response with about 10 breaks per cell per Gy which is about two times higher than the value with the conventional G 1-type PCC method. The chromosome rejoining kinetics at 0.5 and 2.0 Gy X-rays reveal a bi-phasic curve with both a fast and a slow component. The fast component (0–30 min) is nearly identical for both doses, but the slow component for 2 Gy kinetics is much slower than that for 0.5 Gy, indicating that the process occurring during this period may be crucial for the ultimate fate of irradiated cells. The chromosome rejoining kinetics obtained here is similar to that of other methods of detecting DNA DSB repair such as the γH2AX assay. Our chromosome repair assay is useful for evaluating the accuracy of other assays measuring DNA DSB repair at doses equal or less than 0.5 Gy of ionizing radiation.