Christophe Badie

Hypersensitivity of ataxia telangiectasia fibroblasts to ionizing radiation is associated with a repair deficiency of DNA double-strand breaks.

We have studied the intrinsic radiosensitivity, repair of potentially lethal damage (PLD) and the repair rate of radiation-induced DNA double-strand breaks (DSB) in 11 non-transformed human fibroblast cell lines, four of which were homozygous for the A-T mutation and two that were heterozygous (A-TH). All the experiments were done on cells in plateau phase of growth (97-99% of cells in G0/G1). With a dose of 30 Gy delivered at 4 degrees C, the A-T cell lines had faster repair rates of up to 6 h, after which the repair curve crossed that of the control so that the residual damage at 24 h was higher in the A-T cells. Irradiation at 37 degrees C at low dose rate 1 cGy.min-1) produced even more marked differences between the A-T cells and controls: the residual DSB level was always higher in A-T cells than controls at doses of 5-40 Gy, due to defective repair of a small fraction of DSB in A-T cells. The two protocols showed DSB repair rates for the A-TH cell lines that were intermediate between those of the A-T and control cells. There was a quantitative relationship between the residual DSB after irradiation at 37 degrees C and the intrinsic radiosensitivity, and with the extent of PLD repair. There were very few apoptotic cells in the non-transformed control and A-T cell line, both before and after irradiation. In combination, these result support the contention that the defective repair of DSB is a mechanism of the hypersensitivity linked to the A-T mutation.

Induction and Rejoining of DNA Double-Strand Breaks and Interphase Chromosome Breaks after Exposure to X Rays in One Normal and Two Hypersensitive Human Fibroblast Cell Lines

The aim of this work was to measure simultaneously and in a quantitative manner double-strand breaks (DSBs), interphase chromosome breaks and cell lethality either immediately after irradiation, or at various times thereafter (up to 24 h), in cells of three nontransformed human fibroblast cell lines of widely different intrinsic radiosensitivity. We wished to assess initial damage, repair kinetics and residual damage at the DNA and the chromosome level, and to correlate these parameters with cell killing. We employed HF19 cells, a normal fibroblast cell line, AT2 cells, a radiosensitive cell line from a patient suffering from ataxia telangiectasia (AT), and 180BR cells, a radiosensitive cell line from a patient with no clinical symptoms of AT. AT2 and 180BR cells, in addition to being radiosensitive, also display a reduced ability to repair potentially lethal damage compared to HF19 cells. The yield of DSBs, as measured by pulsed-field gel electrophoresis, is similar in all three cell lines (slopes correspond to 1.6-1.7% Gy-1 of DNA-associated radioactivity released from the gel well into the lane). In contrast, residual DSBs measured 24 h after irradiation are almost zero for HF19 cells (0.1% confidence interval = 0-1.4%), but are 12.5% (+/- 2.3%) and 43.8% (+/- 1.2%) of those measured immediately after irradiation in AT2 and 180BR cells, respectively. Residual interphase chromosome breaks are 11.6% (+/- 1.6%), 29.7% (+/- 5.7%) and 41.4% (+/- 2.2%) of those measured immediately after irradiation in HF19, AT2 and 180BR cells, respectively. Neither the initial yield of DSBs nor that of excess interphase chromosome breaks can explain the differences in radiosensitivity between the three cell lines; however, there is a correlation between residual DSBs, rate of DSB rejoining at 24 h, residual interphase chromosome breaks on the one hand and cell survival on the other hand.

Gene expression following ionising radiation: identification of biomarkers for dose estimation and prediction of individual response.

Purpose: To establish a panel of highly radiation responsive genes suitable for biological dosimetry and to explore inter-individual variation in response to ionising radiation exposure. Materials and methods: Analysis of gene expression in response to radiation was carried out using three independent techniques (Microarray, Multiplex Quantitative Real-Time Polymerase Chain Reaction (MQRT- PCR) and nCounter® Analysis System) in human dividing lymphocytes in culture and peripheral blood leukocytes exposed ex vivo from the same donors. Results: Variations in transcriptional response to exposure to ionising radiation analysed by microarray allowed the identification of genes which can be measured accurately using MQRT PCR and another technique allowing direct count of mRNA copies. We have identified genes which are consistently up-regulated following exposure to 2 or 4 Gy of X-rays at different time points, for all individuals in blood and cultured lymphocytes. Down-regulated genes including cyclins, centromeric and mitotic checkpoint genes, particularly those associated with chromosome instability and cancer could be detected in dividing lymphocytes only. Conclusions: The data provide evidence that there are a number of genes which seem suitable for biological dosimetry using peripheral blood, including sestrin 1 (SESN1), growth arrest and DNA damage inducible 45 alpha (GADD45A), cyclin-dependent kinase inhibitor 1A (CDKN1A), cyclin G1 (CCNG1), ferredoxin reductase (FDXR), p53 up-regulated mediator of apoptosis (BBC3) and Mdm2 p53 binding protein homolog (MDM2). These biomarkers could potentially be used for triage after large-scale radiological incidents and for monitoring radiation exposure during radiotherapy.

Radiation Leukemogenesis in Mice : Loss of PU. 1 on Chromosome 2 in CBA and C57BL/6 Mice after Irradiation with 1 GeV/nucleon 56Fe Ions, X Rays or γ Rays. Part I. Experimental Observations

Abstract Peng, Y., Brown, N., Finnon, R., Warner, C. L., Liu, X., Genik, P. C., Callan, M. A., Ray, F. A., Borak, T. B., Badie, C., Bouffler, S. D., Ullrich, R. L., Bedford, J. S. and Weil, M. M. Radiation Leukemogenesis in Mice: Loss of PU.1 on Chromosome 2 in CBA and C57BL/6 Mice after Irradiation with 1 GeV/nucleon 56Fe Ions, X Rays or γ Rays. Part I. Experimental Observations. Radiat. Res. 171, 474–483 (2009). Since deletion of the PU.1 gene on chromosome 2 is a crucial acute myeloid leukemia (AML) initiating step in the mouse model, we quantified PU.1 deleted cells in the bone marrow of γ-, X- and 56Fe-ion-irradiated mice at various times postirradiation. Although 56Fe ions were initially some two to three times more effective than X or γ rays in inducing PU.1 deletions, by 1 month postirradiation, the proportions of cells with PU.1 deletions were similar for the HZE particles and the sparsely ionizing radiations. These results indicate that while 56Fe ions are more effective in inducing PU.1 deletions, they are also more effective in causing collateral damage that removes hit cells from the bone marrow. After X, γ or 56Fe-ion irradiation, AML-resistant C57BL/6 mice have fewer cells with PU.1 deletions than CBA mice, and those cells do not persist in the bone marrow of the C57B6/6 mice. Our findings suggest that quantification of PU.1 deleted bone marrow cells 1 month postirradiation can be used as surrogate for the incidence of radiation-induced AML measured in large-scale mouse studies. If so, PU.1 loss could be used to systematically assess the potential leukemogenic effects of other ions and energies in the space radiation environment.

Aberrant CDKN1A transcriptional response associates with abnormal sensitivity to radiation treatment

Normal tissue reactions to radiation therapy vary in severity among patients and cannot be accurately predicted, limiting treatment doses. The existence of heritable radiosensitivity syndromes suggests that normal tissue reaction severity is determined, at least in part, by genetic factors and these may be revealed by differences in gene expression. To test this hypothesis, peripheral blood lymphocyte cultures from 22 breast cancer patients with either minimal (11) or very severe acute skin reactions (11) have been used to analyse gene expression. Basal and post-irradiation expression of four radiation-responsive genes (CDKN1A, GADD45A, CCNB1, and BBC3) was determined by quantitative real-time PCR in T-cell cultures established from the two patient groups before radiotherapy. Relative expression levels of BBC3, CCNB1, and GADD45A 2 h following 2 Gy X-rays did not discriminate between groups. However, post-irradiation expression response was significantly reduced for CDKN1A (P<0.002) in severe reactors compared to normal. Prediction of reaction severity of ∼91% of individuals sampled was achieved using this end point. Analysis of TP53 Arg72Pro and CDKN1A Ser31Arg single nucleotide polymorphisms did not show any significant association with reaction sensitivity. Although these results require confirmation and extension, this study demonstrates the possibility of predicting the severity of acute skin radiation toxicity in simple tests.

Comparison of Established and Emerging Biodosimetry Assays

Rapid biodosimetry tools are required to assist with triage in the case of a large-scale radiation incident. Here, we aimed to determine the dose-assessment accuracy of the well-established dicentric chromosome assay (DCA) and cytokinesis-block micronucleus assay (CBMN) in comparison to the emerging γ-H2AX foci and gene expression assays for triage mode biodosimetry and radiation injury assessment. Coded blood samples exposed to 10 X-ray doses (240 kVp, 1 Gy/min) of up to 6.4 Gy were sent to participants for dose estimation. Report times were documented for each laboratory and assay. The mean absolute difference (MAD) of estimated doses relative to the true doses was calculated. We also merged doses into binary dose categories of clinical relevance and examined accuracy, sensitivity and specificity of the assays. Dose estimates were reported by the first laboratories within 0.3–0.4 days of receipt of samples for the γ-H2AX and gene expression assays compared to 2.4 and 4 days for the DCA and CBMN assays, respectively. Irrespective of the assay we found a 2.5–4-fold variation of interlaboratory accuracy per assay and lowest MAD values for the DCA assay (0.16 Gy) followed by CBMN (0.34 Gy), gene expression (0.34 Gy) and γ-H2AX (0.45 Gy) foci assay. Binary categories of dose estimates could be discriminated with equal efficiency for all assays, but at doses ≥1.5 Gy a 10% decrease in efficiency was observed for the foci assay, which was still comparable to the CBMN assay. In conclusion, the DCA has been confirmed as the gold standard biodosimetry method, but in situations where speed and throughput are more important than ultimate accuracy, the emerging rapid molecular assays have the potential to become useful triage tools.

Mutational signatures of ionizing radiation in second malignancies

Ionizing radiation is a potent carcinogen, inducing cancer through DNA damage. The signatures of mutations arising in human tissues following in vivo exposure to ionizing radiation have not been documented. Here, we searched for signatures of ionizing radiation in 12 radiation-associated second malignancies of different tumour types. Two signatures of somatic mutation characterize ionizing radiation exposure irrespective of tumour type. Compared with 319 radiation-naive tumours, radiation-associated tumours carry a median extra 201 deletions genome-wide, sized 1–100 base pairs often with microhomology at the junction. Unlike deletions of radiation-naive tumours, these show no variation in density across the genome or correlation with sequence context, replication timing or chromatin structure. Furthermore, we observe a significant increase in balanced inversions in radiation-associated tumours. Both small deletions and inversions generate driver mutations. Thus, ionizing radiation generates distinctive mutational signatures that explain its carcinogenic potential.

High and low dose responses of transcriptional biomarkers in ex vivo X-irradiated human blood

Abstract Purpose: Modifications of gene expression following ionizing radiation (IR) exposure of cells in vitro and in vivo are well documented. However, little is known about the dose-responses of transcriptionally responsive genes, especially at low doses. In this study, we investigated these dose-responses and assessed inter-individual variability. Materials and methods: High dose (0.5–4 Gy) and low dose (5–100 mGy) gene expression responses at 2 h and 24 h using 13 biomarkers transcriptionally regulated through the DNA damage response by the tumor suppressor p53 were investigated. Inter-individual variation was also examined. Results: High dose-response curves were best constructed using a polynomial fit while the low dose-response curves used a linear fit with linear R2 values of 0.841–0.985. Individual variation was evident in the high and low dose ranges. The FDXR, DDB2 high dose gene combination produced a mean dose estimate of 0.7 Gy for 1 Gy irradiated ‘unknown’ samples (95% CIs of 0.3–1.1 Gy) and 1.4 Gy for 2 Gy exposure (95% CIs of 0.6–2.1 Gy). The FDXR, DDB2, CCNG1 low dose gene combination estimated 98 mGy (95% CIs of 27–169 mGy) for 100 mGy exposure. Conclusions: These findings identify genes that fulfill some of the requirements of a good exposure biomarker even at low doses, such as sensitivity, reproducibility and simple proportionality with dose.

A new model describing the curves for repair of both DNA double-strand breaks and chromosome damage

A review of reports dealing with fittings of the data for repair of DNA double-strand breaks (DSBs) and excess chromosome fragments (ECFs) shows that several models are used to fit the repair curves. Since DSBs and ECFs are correlated, it is worth developing a model describing both phenomena. The curve-fitting models used most extensively, the two repair half-times model for DSBs and the monoexponential plus residual model for ECFs, appear to be too inflexible to describe the repair curves for both DSBs and ECFs. We have therefore developed a new concept based on a variable repair half-time. According to this concept, the repair curve is continuously bending and dependent on time and probably reflects a continuous spectrum of damage repairability. The fits of the curves for DSB repair to the variable repair half-time and the variable repair half-time plus residual models were compared to those obtained with the two half-times plus residual and two half-times models. Similarly, the fits of the curves for ECF repair to the variable repair half-time and variable half-time plus residual models were compared to that obtained with the monoexponential plus residual model. The quality of fit and the dependence of adjustable parameters on the portion of the curve fitted were used as comparison criteria. We found that: (a) It is useful to postulate the existence of a residual term for unrepairable lesions, regardless of the model adopted. (b) With the two cell lines tested (a normal and a hypersensitive one), data for both DSBs and ECFs are best fitted to the variable repair half-time plus residual model, whatever the repair time range.

Correlation of in vitro lymphocyte radiosensitivity and gene expression with late normal tissue reactions following curative radiotherapy for breast cancer

BACKGROUND AND PURPOSE Identification of mechanisms of late normal tissue responses to curative radiotherapy that discriminate individuals with marked or mild responses would aid response prediction. This study aimed to identify differences in gene expression, apoptosis, residual DNA double strand breaks and chromosomal damage after in vitro irradiation of lymphocytes in a series of patients with marked (31 cases) or mild (28 controls) late adverse reaction to adjuvant breast radiotherapy. MATERIALS AND METHODS Gene expression arrays, residual γH2AX, apoptosis, G2 chromosomal radiosensitivity and G0 micronucleus assay were used to compare case and control lymphocyte radiation responses. RESULTS Five hundred and thirty genes were up-regulated and 819 down-regulated by ionising radiation. Irradiated samples were identified with an overall cross-validated error rate of 3.4%. Prediction analyses to classify cases and controls using unirradiated (0Gy), irradiated (4Gy) or radiation response (4-0Gy) expression profiles correctly identified samples with, respectively, 25%, 22% or 18.5% error rates. Significant inter-sample variation was observed for all cellular endpoints but cases and controls could not be distinguished. CONCLUSIONS Variation in lymphocyte radiosensitivity does not necessarily correlate with normal tissue response to radiotherapy. Gene expression analysis can predict of radiation exposure and may in the future help prediction of normal tissue radiosensitivity.