Transcriptional responses to ionising radiation for biological dosimetry purposes

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University LondonExposures to ionising radiation resulting from natural sources or medical diagnostics are generally very low. In contrast, exposures to therapeutic radiation, although, they are often partial exposures, represent much higher doses. Similar levels of exposure may also occur as a consequence of a radiological accident, where it would be necessary to quickly separate individuals requiring urgent medical attention from the “worried-well”. The well-established biodosimetry techniques based on cytogenetics, particularly scoring dicentric chromosomes, are accurate and sensitive, yet, they are unsuitable for mass screening due to limited capacity and the time required for providing dose estimates. Measuring gene expression changes following radiation exposure was suggested over a decade ago to be an alternative method for dose estimation, as it is a quick, sensitive and suitable technique for high throughput application. The qPCR protocol was extensively optimised for increased reproducibility and sensitivity in order to be suitable for biodosimetry purposes. Radiation-responsive transcripts were identified and characterised in terms of temporal- and dose-responses. Finally, candidate transcripts were validated in human blood irradiated ex vivo and in vivo in blood samples obtained from cancer patients undergoing radiotherapy treatment. The data generated here serve as a proof of principle that qPCR-based gene expression assays can be used for radiation biodosimetry purposes to aid classical cytogenetics tools in an event of mass causality.Public Health Englan

Combining CDKN1A gene expression and genome-wide SNPs in a twin cohort to gain insight into the heritability of individual radiosensitivity

Individual variability in response to radiation exposure is recognised and has often been reported as important in treatment planning. Despite many efforts to identify biomarkers allowing the identification of radiation sensitive patients, it is not yet possible to distinguish them with certainty before the beginning of the radiotherapy treatment. A comprehensive analysis of genome-wide single-nucleotide polymorphisms (SNPs) and a transcriptional response to ionising radiation exposure in twins have the potential to identify such an individual. In the present work, we investigated SNP profile and CDKN1A gene expression in blood T lymphocytes from 130 healthy Caucasians with a complex level of individual kinship (unrelated, mono- or dizygotic twins). It was found that genetic variation accounts for 66% (95% CI 37-82%) of CDKN1A transcriptional response to radiation exposure. We developed a novel integrative multi-kinship strategy allowing investigating the role of genome-wide polymorphisms in transcriptomic radiation response, and it revealed that rs205543 (ETV6 gene), rs2287505 and rs1263612 (KLF7 gene) are significantly associated with CDKN1A expression level. The functional analysis revealed that rs6974232 (RPA3 gene), involved in mismatch repair (p value = 9.68e-04) as well as in RNA repair (p value = 1.4e-03) might have an important role in that process. Two missense polymorphisms with possible deleterious effect in humans were identified: rs1133833 (AKIP1 gene) and rs17362588 (CCDC141 gene). In summary, the data presented here support the validity of this novel integrative data analysis strategy to provide insights into the identification of SNPs potentially influencing radiation sensitivity. Further investigations in radiation response research at the genomic level should be therefore continued to confirm these findings.Peer reviewe

Germline DNA Retention in Murine and Human Rearranged T Cell Receptor Gene Coding Joints: Alternative Recombination Signal Sequences and V(D)J Recombinase Errors

International audienceThe genes coding for the antigenic T cell receptor (TR) subunits are assembled in thymocytes from discrete V, D, and J genes by a site-specific recombination process. A tight control of this activity is required to prevent potentially detrimental recombination events. V, D, and J genes are flanked by semi-conserved nucleotide motives called recombination signal sequences (RSSs). V(D)J recombination is initiated by the precise introduction of a DNA double-strand break exactly at the border of the genes and their RSSs by the RAG recombinase. RSSs are therefore physically separated from the coding region of the genes before assembly of a rearranged TR gene. During a high throughput profiling of TRB genes in mice, we identified rearranged TRB genes in which part or all of a flanking RSS was retained in V-D or D-J coding joints. In some instances, this retention of germline DNA resulted from the use of an upstream alternative RSS. However, we also identified TRB sequences where retention of germline DNA occurred in the absence of alternative RSS, suggesting that RAG activity was mis-targeted during recombination. Similar events were also identified in human rearranged TRB and TRG genes. The use of alternative RSSs during V(D)J recombination illustrates the complexity of RAG-RSSs interactions during V(D)J recombination. While the frequency of errors resulting from mis-targeted RAG activity is very low, we believe that these RAG errors may be at the origin of oncogenic translocations and are a threat for genetic stability in developing lymphocytes

A minimally invasive assay for individual assessment of the ATM/CHEK2/p53 pathway activity

Ionizing radiation induces DNA Double-Strand Breaks (DSBs), which activate the ATM/CHEK2/p53 pathway leading to cell cycle arrest and apoptosis through transcription of genes including CDKN1A (p21) and BBC3 (PUMA). This pathway prevents genomic instability and tumorigenesis as demonstrated in heritable syndromes [e.g., Ataxia Telangiectasia (AT); Li-Fraumeni syndrome (LFS)]. Here, a simple assay based on gene expression in peripheral blood to measure accurately ATM/CHEK2/p53 pathway activity is described. The expression of p21, Puma and Sesn2 was determined in blood from mice with different gene copy numbers of Atm, Trp53 (p53), Chek2 or Arf and in human blood and mitogen stimulated T-lymphocyte (MSTL) cultures from AT, AT carriers, LFS patients and controls, both before and after ex vivo ionizing irradiation. Mouse Atm/Chek2/p53 activity was highly dependent on the copy number of each gene except Arf. In human MSTL, an AT case, AT carriers and LFS patients showed responses distinct from healthy donors. The relationship between gene copy number and transcriptional induction upon radiation was linear for p21 and Puma and correlated well with cancer incidence in p53 variant mice. This reliable blood test provides an assay to determine ATM/CHEK2/p53 pathway activity and demonstrates the feasibility of assessing the activity of this essential cancer protection pathway in simple assays. These findings may have implications for the individualized prediction of cancer susceptibility

Investigation of transcriptional responses of juvenile mouse bone marrow to power frequency magnetic fields.

To seek alterations in gene transcription in bone marrow cells following in vivo exposure of juvenile mice to power frequency magnetic fields, young (21-24-day old) C57BL/6 mice were exposed to a 100μT 50Hz magnetic field for 2h. Transcription was analysed by three methods, High Coverage Expression Profiling (HiCEP), Illumina microarrays and quantitative real-time polymerase chain reaction (QRT-PCR). A pilot HiCEP experiment with 6 exposed (E) and 6 non-exposed (NE) mice identified four candidate responsive transcripts (two unknown transcripts (AK152075 and F10-NED), phosphatidylinositol binding clathrin assembly protein (Picalm) and exportin 7 (Xpo7)). A larger experiment compared 19 E and 15 NE mice using two independent QRT-PCR assays and repeated microarray assays. No significant field-dependent changes were seen, although Picalm showed a trend to significance in one QRT-PCR assay (E/NE=0.91; P=0.06). However, the study was underpowered to detect an effect of this magnitude (52% power at P=0.05). These data indicate the current experimental constraints in detecting small changes in transcription that may occur in response to magnetic fields. These constraints result from technical limitations in the accuracy of assays and biological variation, which together were sufficient to account statistically for the number of differentially expressed transcripts identified in the pilot experiment

Studying biomarkers reflecting the adverse outcome pathways from exposures to diseases using molecular epidemiology: example of the molecular epidemiology protocol developed as part of the Concerted Uranium Research in Europe (CURE) project

International audienceResults produced during recent years have shown that epidemiology still has a strong potential to quantify radiation-related health effects after low dose exposures, even below 100 mGy. However below certain dose levels, which may differ according to the disease considered and population characteristics (e.g.: age at exposure), direct observations of dose-risk relationships by classical epidemiological studies are not possible. Indirect evidence, including subtle changes in various physiological functions and biomarkers (sub-clinical/ pre-pathological) reflecting key events in response to low or even very low doses will need to be determined in order to draw inferences on risk after exposures to very low doses. Such inference will be possible for instance, if very large cohorts, - which can even be non-radiation cohorts - quantify the relationships between (combinations of) these biomarkers or changes in physiological function and disease risks. Such inferences are in line with the so-called “meet-in-the-middle” approach. Following this philosophy, as part of the CURE project which aimed to prepare protocols to study the health effects of chronic uranium exposures at low dose, a rational approach was employed to select biomarkers of exposures and subclinical biomarkers of effects in uranium target organs (kidney, lung, bone, brain) and in systems in which potential effects could be suspected (e.g.: vascular system). The use of non-targeted techniques (omics) is also essential to generate new hypotheses of induced signal cascades resulting finally in disease pathways and disease onset (Adverse Outcome Pathways, AOP). A full molecular epidemiology protocol was then developed, including a questionnaire and other means of data collection to measure potential confounding factors. Such an approach will be useful in order 1) to determine the changes in biomarkers which may be attributed to uranium itself and 2) to better appreciate the relative influence of uranium versus other stressors on these biomarker changes, as part of AOP leading to chronic diseases. 3) These yet hypothetical diseases and their development can subsequently be studied in classical epidemiological approaches (at higher doses) and through well-justified radiobiological experiments. Finally, indirect evidence gained from molecular epidemiology studies will prove useful to quantify risks after very low dose exposures, in addition to experimental studies and classical epidemiology

Studying biomarkers reflecting the adverse outcome pathways from exposures to diseases using molecular epidemiology: example of the molecular epidemiology protocol developed as part of the Concerted Uranium Research in Europe (CURE) project

International audienceResults produced during recent years have shown that epidemiology still has a strong potential to quantify radiation-related health effects after low dose exposures, even below 100 mGy. However below certain dose levels, which may differ according to the disease considered and population characteristics (e.g.: age at exposure), direct observations of dose-risk relationships by classical epidemiological studies are not possible. Indirect evidence, including subtle changes in various physiological functions and biomarkers (sub-clinical/ pre-pathological) reflecting key events in response to low or even very low doses will need to be determined in order to draw inferences on risk after exposures to very low doses. Such inference will be possible for instance, if very large cohorts, - which can even be non-radiation cohorts - quantify the relationships between (combinations of) these biomarkers or changes in physiological function and disease risks. Such inferences are in line with the so-called “meet-in-the-middle” approach. Following this philosophy, as part of the CURE project which aimed to prepare protocols to study the health effects of chronic uranium exposures at low dose, a rational approach was employed to select biomarkers of exposures and subclinical biomarkers of effects in uranium target organs (kidney, lung, bone, brain) and in systems in which potential effects could be suspected (e.g.: vascular system). The use of non-targeted techniques (omics) is also essential to generate new hypotheses of induced signal cascades resulting finally in disease pathways and disease onset (Adverse Outcome Pathways, AOP). A full molecular epidemiology protocol was then developed, including a questionnaire and other means of data collection to measure potential confounding factors. Such an approach will be useful in order 1) to determine the changes in biomarkers which may be attributed to uranium itself and 2) to better appreciate the relative influence of uranium versus other stressors on these biomarker changes, as part of AOP leading to chronic diseases. 3) These yet hypothetical diseases and their development can subsequently be studied in classical epidemiological approaches (at higher doses) and through well-justified radiobiological experiments. Finally, indirect evidence gained from molecular epidemiology studies will prove useful to quantify risks after very low dose exposures, in addition to experimental studies and classical epidemiology

Ionizing radiation does not impair the mechanisms controlling genetic stability during T cell receptor gene rearrangement in mice.

International audiencePURPOSE:To determine whether low dose/low dose rate radiation-induced genetic instability may result from radiation-induced inactivation of mechanisms induced by the ATM-dependent DNA damage response checkpoint. To this end, we analysed the faithfulness of T cell receptor (TR) gene rearrangement by V(D)J recombination in DNA from mice exposed to a single dose of X-ray or chronically exposed to low dose rate γ radiation.MATERIALS AND METHODS:Genomic DNA obtained from the blood or the thymus of wild type or Ogg1-deficient mice exposed to low (0.1) or intermediate/high (0.2-1 Gy) doses of radiation either by acute X-rays exposure or protracted exposure to low dose-rate γ-radiation was used to analyse by PCR the presence of illegitimate TR gene rearrangements.RESULTS:Radiation exposure does not increase the onset of TR gene trans-rearrangements in irradiated mice. In mice where it happens, trans-rearrangements remain sporadic events in developing T lymphocytes.CONCLUSION:We concluded that low dose/low dose rate ionizing radiation (IR) exposure does not lead to widespread inactivation of ATM-dependent mechanisms, and therefore that the mechanisms enforcing genetic stability are not impaired by IR in developing lymphocytes and lymphocyte progenitors, including BM-derived hematopoietic stem cells, in low dose/low dose rate exposed mice