Towards unravelling biological mechanisms behind radiation-induced oral mucositis via mass spectrometry-based proteomics.

Objective: Head and neck cancer (HNC) accounts for almost 890,000 new cases per year. Radiotherapy (RT) is used to treat the majority of these patients. A common side-effect of RT is the onset of oral mucositis, which decreases the quality of life and represents the major dose-limiting factor in RT. To understand the origin of oral mucositis, the biological mechanisms post-ionizing radiation (IR) need to be clarified. Such knowledge is valuable to develop new treatment targets for oral mucositis and markers for the early identification of "at-risk" patients. Methods: Primary keratinocytes from healthy volunteers were biopsied, irradiated in vitro (0 and 6 Gy), and subjected to mass spectrometry-based analyses 96 h after irradiation. Web-based tools were used to predict triggered biological pathways. The results were validated in the OKF6 cell culture model. Immunoblotting and mRNA validation was performed and cytokines present in cell culture media post-IR were quantified. Results: Mass spectrometry-based proteomics identified 5879 proteins in primary keratinocytes and 4597 proteins in OKF6 cells. Amongst them, 212 proteins in primary keratinocytes and 169 proteins in OKF6 cells were differentially abundant 96 h after 6 Gy irradiation compared to sham-irradiated controls. In silico pathway enrichment analysis predicted interferon (IFN) response and DNA strand elongation pathways as mostly affected pathways in both cell systems. Immunoblot validations showed a decrease in minichromosome maintenance (MCM) complex proteins 2-7 and an increase in IFN-associated proteins STAT1 and ISG15. In line with affected IFN signalling, mRNA levels of IFNß and interleukin 6 (IL-6) increased significantly following irradiation and also levels of secreted IL-1ß, IL-6, IP-10, and ISG15 were elevated. Conclusion: This study has investigated biological mechanisms in keratinocytes post-in vitro ionizing radiation. A common radiation signature in keratinocytes was identified. The role of IFN response in keratinocytes along with increased levels of pro-inflammatory cytokines and proteins could hint towards a possible mechanism for oral mucositis.

Analysis of chromosomal aberrations and γH2A.X foci to identify radiation-sensitive ataxia-telangiectasia patients.

Ataxia-telangiectasia (AT) is a rare inherited recessive disorder which is caused by a mutated Ataxia-telangiectasia mutated (ATM) gene. Hallmarks include chromosomal instability, cancer predisposition and increased sensitivity to ionizing radiation. The ATM protein plays an important role in signaling of DNA double-strand breaks (DSB), thereby phosphorylating the histone H2A.X. Non-functional ATM protein leads to defects in DNA damage response, unresolved DSBs and genomic instability. The aim of this study was to evaluate chromosomal aberrations and γH2A.X foci as potential radiation sensitivity biomarkers in AT patients. For this purpose, lymphocytes of 8 AT patients and 10 healthy controls were irradiated and induced DNA damage and DNA repair capacity were detected by the accumulation of γH2A.X foci. The results were heterogeneous among AT patients. Evaluation revealed 2 AT patients with similar γH2A.X foci numbers as controls after 1 h while 3 patients showed a lower induction. In regard to DNA repair, 3 of 5 AT patients showed poor damage repair. Therefore, DNA damage induction and DNA repair as detected by H2A.X phosphorylation revealed individual differences, seems to depend on the underlying individual mutation and thus appears not well suited as a biomarker for radiation sensitivity. In addition, chromosomal aberrations were analyzed by mFISH. An increased frequency of spontaneous chromosomal breakage was characteristic for AT cells. After irradiation, significantly increased rates for non-exchange aberrations, translocations, complex aberrations and dicentric chromosomes were observed in AT patients compared to controls. The results of this study suggested, that complex aberrations and dicentric chromosomes might be a reliable biomarker for radiation sensitivity in AT patients, while non-exchange aberrations and translocations identified both, spontaneous and radiation-induced chromosomal instability.

Risk Factors for Childhood Leukemia: Radiation and Beyond.

Childhood leukemia (CL) is undoubtedly caused by a multifactorial process with genetic as well as environmental factors playing a role. But in spite of several efforts in a variety of scientific fields, the causes of the disease and the interplay of possible risk factors are still poorly understood. To push forward the research on the causes of CL, the German Federal Office for Radiation Protection has been organizing recurring international workshops since 2008 every two to three years. In November 2019 the 6th International Workshop on the Causes of CL was held in Freising and brought together experts from diverse disciplines. The workshop was divided into two main parts focusing on genetic and environmental risk factors, respectively. Two additional special sessions addressed the influence of natural background radiation on the risk of CL and the progress in the development of mouse models used for experimental studies on acute lymphoblastic leukemia, the most common form of leukemia worldwide. The workshop presentations highlighted the role of infections as environmental risk factor for CL, specifically for acute lymphoblastic leukemia. Major support comes from two mouse models, the Pax5+/- and Sca1-ETV6-RUNX1 mouse model, one of the major achievements made in the last years. Mice of both predisposed models only develop leukemia when exposed to common infections. These results emphasize the impact of gene-environment-interactions on the development of CL and warrant further investigation of such interactions - especially because genetic predisposition is detected with increasing frequency in CL. This article summarizes the workshop presentations and discusses the results in the context of the international literature.

Low dose ionizing radiation effects on the immune system.

Ionizing radiation interacts with the immune system in many ways with a multiplicity that mirrors the complexity of the immune system itself: namely the need to maintain a delicate balance between different compartments, cells and soluble factors that work collectively to protect, maintain, and restore tissue function in the face of severe challenges including radiation damage. The cytotoxic effects of high dose radiation are less relevant after low dose exposure, where subtle quantitative and functional effects predominate that may go unnoticed until late after exposure or after a second challenge reveals or exacerbates the effects. For example, low doses may permanently alter immune fitness and therefore accelerate immune senescence and pave the way for a wide spectrum of possible pathophysiological events, including early-onset of age-related degenerative disorders and cancer. By contrast, the so called low dose radiation therapy displays beneficial, anti-inflammatory and pain relieving properties in chronic inflammatory and degenerative diseases. In this review, epidemiological, clinical and experimental data regarding the effects of low-dose radiation on the homeostasis and functional integrity of immune cells will be discussed, as will be the role of immune-mediated mechanisms in the systemic manifestation of localized exposures such as inflammatory reactions. The central conclusion is that ionizing radiation fundamentally and durably reshapes the immune system. Further, the importance of discovery of immunological pathways for modifying radiation resilience amongst other research directions in this field is implied.

Radiation Exposure of Peripheral Mononuclear Blood Cells Alters the Composition and Function of Secreted Extracellular Vesicles.

Normal tissue toxicity is a dose-limiting factor in radiation therapy. Therefore, a detailed understanding of the normal tissue response to radiation is necessary to predict the risk of normal tissue toxicity and to development strategies for tissue protection. One component of normal tissue that is continuously exposed during therapeutic irradiation is the circulating population of peripheral blood mononuclear cells (PBMC). PBMCs are highly sensitive to ionizing radiation (IR); however, little is known about how IR affects the PBMC response on a systemic level. It was the aim of this study to investigate whether IR was capable to induce changes in the composition and function of extracellular vesicles (EVs) secreted from PBMCs after radiation exposure to different doses. Therefore, whole blood samples from healthy donors were exposed to X-ray radiation in the clinically relevant doses of 0, 0.1, 2 or 6 Gy and PBMC-secreted EVs were isolated 72 h later. Proteome and miRNome analysis of EVs as well as functional studies were performed. Secreted EVs showed a dose-dependent increase in the number of significantly deregulated proteins and microRNAs. For both, proteome and microRNA data, principal component analysis showed a dose-dependent separation of control and exposed groups. Integrated pathway analysis of the radiation-regulated EV proteins and microRNAs consistently predicted an association of deregulated molecules with apoptosis, cell death and survival. Functional studies identified endothelial cells as an efficient EV recipient system, in which irradiation of recipient cells further increased the uptake. Furthermore an apoptosis suppressive effect of EVs from irradiated PBMCs in endothelial recipient cells was detected. In summary, this study demonstrates that IR modifies the communication between PBMCs and endothelial cells. EVs from irradiated PBMC donors were identified as transmitters of protective signals to irradiated endothelial cells. Thus, these data may lead to the discovery of biomarker candidates for radiation dosimetry and even more importantly, they suggest EVs as a novel systemic communication pathway between irradiated normal, non-cancer tissues.

Lifetime study in mice after acute low-dose ionizing radiation: a multifactorial study with special focus on cataract risk.

Because of the increasing application of ionizing radiation in medicine, quantitative data on effects of low-dose radiation are needed to optimize radiation protection, particularly with respect to cataract development. Using mice as mammalian animal model, we applied a single dose of 0, 0.063, 0.125 and 0.5 Gy at 10 weeks of age, determined lens opacities for up to 2 years and compared it with overall survival, cytogenetic alterations and cancer development. The highest dose was significantly associated with increased body weight and reduced survival rate. Chromosomal aberrations in bone marrow cells showed a dose-dependent increase 12 months after irradiation. Pathological screening indicated a dose-dependent risk for several types of tumors. Scheimpflug imaging of the lens revealed a significant dose-dependent effect of 1% of lens opacity. Comparison of different biological end points demonstrated long-term effects of low-dose irradiation for several biological end points.

Biomarkers for Uranium Risk Assessment for the Development of the CURE (Concerted Uranium Research in Europe) Molecular Epidemiological Protocol.

Despite substantial experimental and epidemiological research, there is limited knowledge of the uranium-induce health effects after chronic low-dose exposures in humans. Biological markers can objectively characterize pathological processes or environmental responses to uranium and confounding agents. The integration of such biological markers into a molecular epidemiological study would be a useful approach to improve and refine estimations of uranium-induced health risks. To initiate such a study, Concerted Uranium Research in Europe (CURE) was established, and involves biologists, epidemiologists and dosimetrists. The aims of the biological work package of CURE were: 1. To identify biomarkers and biological specimens relevant to uranium exposure; 2. To define standard operating procedures (SOPs); and 3. To set up a common protocol (logistic, questionnaire, ethical aspects) to perform a large-scale molecular epidemiologic study in uranium-exposed cohorts. An intensive literature review was performed and led to the identification of biomarkers related to: 1. retention organs (lungs, kidneys and bone); 2. other systems/organs with suspected effects (cardiovascular system, central nervous system and lympho-hematopoietic system); 3. target molecules (DNA damage, genomic instability); and 4. high-throughput methods for the identification of new biomarkers. To obtain high-quality biological materials, SOPs were established for the sampling and storage of different biospecimens. A questionnaire was developed to assess potential confounding factors. The proposed strategy can be adapted to other internal exposures and should improve the characterization of the biological and health effects that are relevant for risk assessment.

Natural Cubic Spline Regression Modeling Followed by Dynamic Network Reconstruction for the Identification of Radiation-Sensitivity Gene Association Networks from Time-Course Transcriptome Data.

Gene expression time-course experiments allow to study the dynamics of transcriptomic changes in cells exposed to different stimuli. However, most approaches for the reconstruction of gene association networks (GANs) do not propose prior-selection approaches tailored to time-course transcriptome data. Here, we present a workflow for the identification of GANs from time-course data using prior selection of genes differentially expressed over time identified by natural cubic spline regression modeling (NCSRM). The workflow comprises three major steps: 1) the identification of differentially expressed genes from time-course expression data by employing NCSRM, 2) the use of regularized dynamic partial correlation as implemented in GeneNet to infer GANs from differentially expressed genes and 3) the identification and functional characterization of the key nodes in the reconstructed networks. The approach was applied on a time-resolved transcriptome data set of radiation-perturbed cell culture models of non-tumor cells with normal and increased radiation sensitivity. NCSRM detected significantly more genes than another commonly used method for time-course transcriptome analysis (BETR). While most genes detected with BETR were also detected with NCSRM the false-detection rate of NCSRM was low (3%). The GANs reconstructed from genes detected with NCSRM showed a better overlap with the interactome network Reactome compared to GANs derived from BETR detected genes. After exposure to 1 Gy the normal sensitive cells showed only sparse response compared to cells with increased sensitivity, which exhibited a strong response mainly of genes related to the senescence pathway. After exposure to 10 Gy the response of the normal sensitive cells was mainly associated with senescence and that of cells with increased sensitivity with apoptosis. We discuss these results in a clinical context and underline the impact of senescence-associated pathways in acute radiation response of normal cells. The workflow of this novel approach is implemented in the open-source Bioconductor R-package splineTimeR.

Differences in DNA Repair Capacity, Cell Death and Transcriptional Response after Irradiation between a Radiosensitive and a Radioresistant Cell Line.

Normal tissue toxicity after radiotherapy shows variability between patients, indicating inter-individual differences in radiosensitivity. Genetic variation probably contributes to these differences. The aim of the present study was to determine if two cell lines, one radiosensitive (RS) and another radioresistant (RR), showed differences in DNA repair capacity, cell viability, cell cycle progression and, in turn, if this response could be characterised by a differential gene expression profile at different post-irradiation times. After irradiation, the RS cell line showed a slower rate of γ-H2AX foci disappearance, a higher frequency of incomplete chromosomal aberrations, a reduced cell viability and a longer disturbance of the cell cycle when compared to the RR cell line. Moreover, a greater and prolonged transcriptional response after irradiation was induced in the RS cell line. Functional analysis showed that 24 h after irradiation genes involved in "DNA damage response", "direct p53 effectors" and apoptosis were still differentially up-regulated in the RS cell line but not in the RR cell line. The two cell lines showed different response to IR and can be distinguished with cell-based assays and differential gene expression analysis. The results emphasise the importance to identify biomarkers of radiosensitivity for tailoring individualized radiotherapy protocols.

Concerted Uranium Research in Europe (CURE): toward a collaborative project integrating dosimetry, epidemiology and radiobiology to study the effects of occupational uranium exposure.

The potential health impacts of chronic exposures to uranium, as they occur in occupational settings, are not well characterized. Most epidemiological studies have been limited by small sample sizes, and a lack of harmonization of methods used to quantify radiation doses resulting from uranium exposure. Experimental studies have shown that uranium has biological effects, but their implications for human health are not clear. New studies that would combine the strengths of large, well-designed epidemiological datasets with those of state-of-the-art biological methods would help improve the characterization of the biological and health effects of occupational uranium exposure. The aim of the European Commission concerted action CURE (Concerted Uranium Research in Europe) was to develop protocols for such a future collaborative research project, in which dosimetry, epidemiology and biology would be integrated to better characterize the effects of occupational uranium exposure. These protocols were developed from existing European cohorts of workers exposed to uranium together with expertise in epidemiology, biology and dosimetry of CURE partner institutions. The preparatory work of CURE should allow a large scale collaborative project to be launched, in order to better characterize the effects of uranium exposure and more generally of alpha particles and low doses of ionizing radiation.

Differential Response and Priming Dose Effect on the Proteome of Human Fibroblast and Stem Cells Induced by Exposure to Low Doses of Ionizing Radiation.

It has been suggested that a mechanistic understanding of the cellular responses to low dose and dose rate may be valuable in reducing some of the uncertainties involved in current risk estimates for cancer- and non-cancer-related radiation effects that are inherited in the linear no-threshold hypothesis. In this study, the effects of low-dose radiation on the proteome in both human fibroblasts and stem cells were investigated. Particular emphasis was placed on examining: 1. the dose-response relationships for the differential expression of proteins in the low-dose range (40-140 mGy) of low-linear energy transfer (LET) radiation; and 2. the effect on differential expression of proteins of a priming dose given prior to a challenge dose (adaptive response effects). These studies were performed on cultured human fibroblasts (VH10) and human adipose-derived stem cells (ADSC). The results from the VH10 cell experiments demonstrated that low-doses of low-LET radiation induced unique patterns of differentially expressed proteins for each dose investigated. In addition, a low priming radiation dose significantly changed the protein expression induced by the subsequent challenge exposure. In the ADSC the number of differentially expressed proteins was markedly less compared to VH10 cells, indicating that ADSC differ in their intrinsic response to low doses of radiation. The proteomic results are further discussed in terms of possible pathways influenced by low-dose irradiation.

Genomics and drug profiling of fatal TCF3-HLF-positive acute lymphoblastic leukemia identifies recurrent mutation patterns and therapeutic options.

TCF3-HLF-positive acute lymphoblastic leukemia (ALL) is currently incurable. Using an integrated approach, we uncovered distinct mutation, gene expression and drug response profiles in TCF3-HLF-positive and treatment-responsive TCF3-PBX1-positive ALL. We identified recurrent intragenic deletions of PAX5 or VPREB1 in constellation with the fusion of TCF3 and HLF. Moreover somatic mutations in the non-translocated allele of TCF3 and a reduction of PAX5 gene dosage in TCF3-HLF ALL suggest cooperation within a restricted genetic context. The enrichment for stem cell and myeloid features in the TCF3-HLF signature may reflect reprogramming by TCF3-HLF of a lymphoid-committed cell of origin toward a hybrid, drug-resistant hematopoietic state. Drug response profiling of matched patient-derived xenografts revealed a distinct profile for TCF3-HLF ALL with resistance to conventional chemotherapeutics but sensitivity to glucocorticoids, anthracyclines and agents in clinical development. Striking on-target sensitivity was achieved with the BCL2-specific inhibitor venetoclax (ABT-199). This integrated approach thus provides alternative treatment options for this deadly disease.

New mutation in the mouse Xpd/Ercc2 gene leads to recessive cataracts.

Cataracts are the major eye disorder and have been associated mainly with mutations in lens-specific genes, but cataracts are also frequently associated with complex syndromes. In a large-scale high-throughput ENU mutagenesis screen we analyzed the offspring of paternally treated C3HeB/FeJ mice for obvious dysmorphologies. We identified a mutant suffering from rough coat and small eyes only in homozygotes; homozygous females turned out to be sterile. The mutation was mapped to chromosome 7 between the markers 116J6.1 and D7Mit294;4 other markers within this interval did not show any recombination among 160 F2-mutants. The critical interval (8.6 Mb) contains 3 candidate genes (Apoe, Six5, Opa3); none of them showed a mutation. Using exome sequencing, we identified a c.2209T>C mutation in the Xpd/Ercc2 gene leading to a Ser737Pro exchange. During embryonic development, the mutant eyes did not show major changes. Postnatal histological analyses demonstrated small cortical vacuoles; later, cortical cataracts developed. Since XPD/ERCC2 is involved in DNA repair, we checked also for the presence of the repair-associated histone γH2AX in the lens. During the time, when primary lens fiber cell nuclei are degraded, γH2AX was strongly expressed in the cell nuclei; later, it demarcates clearly the border of the lens cortex to the organelle-free zone. Moreover, we analyzed also whether seemingly healthy heterozygotes might be less efficient in repair of DNA damage induced by ionizing radiation than wild types. Peripheral lymphocytes irradiated by 1Gy Cs137 showed 6 hrs after irradiation significantly more γH2AX foci in heterozygotes than in wild types. These findings demonstrate the importance of XPD/ERCC2 not only for lens fiber cell differentiation, but also for the sensitivity to ionizing radiation. Based upon these data, we hypothesize that variations in the human XPD/ERCC2 gene might increase the susceptibility for several disorders besides Xeroderma pigmentosum in heterozygotes under particular environmental conditions.

Genetic factors in individual radiation sensitivity.

Cancer risk and radiation sensitivity are often associated with alterations in DNA repair, cell cycle, or apoptotic pathways. Interindividual variability in mutagen or radiation sensitivity and in cancer susceptibility may also be traced back to polymorphisms of genes affecting e.g. DNA repair capacity. We studied possible associations between 70 polymorphisms of 12 DNA repair genes with basal and initial DNA damage and with repair thereof. We investigated DNA damage induced by ionizing radiation in lymphocytes isolated from 177 young lung cancer patients and 169 cancer-free controls. We also sought replication of our findings in an independent sample of 175 families (in total 798 individuals). DNA damage was assessed by the Olive tail moment (OTM) of the comet assay. DNA repair capacity (DRC) was determined for 10, 30 and, 60min of repair. Genes involved in the single-strand-repair pathway (SSR; like XRCC1 and MSH2) as well as genes involved in the double-strand-repair pathway (DSR; like RAD50, XRCC4, MRE11 and ATM) were found to be associated with DNA damage. The most significant association was observed for marker rs3213334 (p=0.005) of XRCC1 with basal DNA damage (B), in both cases and controls. A clear additive effect on the logarithm of OTM was identified for the marker rs1001581 of the same LD-block (p=0.039): BCC=-1.06 (95%-CI: -1.16 to -0.96), BCT=-1.02 (95%-CI: -1.11 to -0.93) and BTT=-0.85 (95%-CI: -1.01 to -0.68). In both cases and controls, we observed significantly higher DNA basal damage (p=0.007) for carriers of the genotype AA of marker rs2237060 of RAD50 (involved in DSR). However, this could not be replicated in the sample of families (p=0.781). An alteration to DRC after 30min of repair with respect to cases was observed as borderline significant for marker rs611646 of ATM (involved in DSR; p=0.055), but was the most significant finding in the sample of families (p=0.009). Our data indicate that gene variation impacts measurably on DNA damage and repair, suggesting at least a partial contribution to radiation sensitivity and lung cancer susceptibility.

Radiation-induced alterations of histone post-translational modification levels in lymphoblastoid cell lines.

BACKGROUND: Radiation-induced alterations in posttranslational histone modifications (PTMs) may affect the cellular response to radiation damage in the DNA. If not reverted appropriately, altered PTM patterns may cause long-term alterations in gene expression regulation and thus lead to cancer. It is therefore important to characterize radiation-induced alterations in PTM patterns and the factors affecting them. METHODS: A lymphoblastoid cell line established from a normal donor was used to screen for alterations in methylation levels at H3K4, H3K9, H3K27, and H4K20, as well as acetylation at H3K9, H3K56, H4K5, and H4K16, by quantitative Western Blot analysis at 15 min, 1 h and 24 h after irradiation with 2 Gy and 10 Gy. The variability of alterations in acetylation marks was in addition investigated in a panel of lymphoblastoid cell lines with differing radiosensitivity established from lung cancer patients. RESULTS: The screening procedure demonstrated consistent hypomethylation at H3K4me3 and hypoacetylation at all acetylation marks tested. In the panel of lymphoblastoid cell lines, however, a high degree of inter-individual variability became apparent. Radiosensitive cell lines showed more pronounced and longer lasting H4K16 hypoacetylation than radioresistant lines, which correlates with higher levels of residual γ-H2AX foci after 24 h. CONCLUSION: So far, the factors affecting extent and duration of radiation-induced histone alterations are poorly defined. The present work hints at a high degree of inter-individual variability and a potential correlation of DNA damage repair capacity and alterations in PTM levels.

Are mouse lens epithelial cells more sensitive to γ-irradiation than lymphocytes?

In this pilot study we compared for the first time the radiation sensitivity of mouse lens epithelial cells (LECs) and mouse lymphocytes. We freshly prepared LECs and lymphocytes and irradiated them with γ-rays ((137)Cs; doses ranging from 0.25 to 2 Gy). DNA damage and repair were evaluated by alkaline comet assay and γH2AX foci assay. Using the comet assay, we observed a dose-dependent increase in DNA damage in both cell types. The faster formation of single- and double-strand breaks in LECs of C57BL/6 mice at doses below 1 Gy needs to be confirmed in other mouse strains. Immunofluorescence for γH2AX foci showed a higher degree of lesions in LECs from C57BL/6J mice compared to those of JF1 mice and to lymphocytes of both strains. Correspondingly, repair of DNA damage proceeded faster in LECs of C57BL/6J mice compared to LECs of JF1 mice and lymphocytes of both strains. It is obvious that the lymphocytes of both strains repaired DNA lesions more slowly than the corresponding LECs. In conclusion, our results demonstrate that LECs of C57Bl/6 mice show a steeper dose-response than lymphocytes in both types of experiments. It shows that both test systems are able to be used also at doses below 0.25 Gy. The observed difference in DNA repair between the LECs from C57BL/6J mice compared to the LECs from JF1 mice and to the lymphocytes of both strains warrants further experiments to identify the underlying molecular mechanisms.

Stain-Free technology as a normalization tool in Western blot analysis.

Western blots are used to specifically measure the relative quantities of proteins of interest in complex biological samples. Quantitative measurements can be subject to error due to process inconsistencies such as uneven protein transfer to the membrane. These non-sample-related variations need to be compensated for by an approach known as normalization. Two approaches to data normalization are commonly employed: housekeeping protein (HKP) normalization and total protein normalization (TPN). In this study, we evaluated the performance of Stain-Free technology as a novel TPN tool for Western blotting experiments in comparison with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a representative of the HKP normalization strategy. The target protein (TP) used for this study was MCM7, a DNA licensing replication factor, which was shown previously to be down-regulated by 20% in irradiated lymphoblastoid cell lines (LCLs). We studied the regulation of MCM7 with a multiplex Western blotting approach based on fluorescently labeled secondary antibodies and found that Stain-Free technology appears to be more reliable, more robust, and more sensitive to small effects of protein regulation when compared with HKP normalization with GAPDH. Stain-Free technology offers the additional advantages of providing checkpoints throughout the Western blotting process by allowing rapid visualization of gel separation and protein transfer.

Heritability of radiation response in lung cancer families.

Radiation sensitivity is assumed to be a cancer susceptibility factor due to impaired DNA damage signalling and repair. Relevant genetic factors may also determine the observed familial aggregation of early onset lung cancer. We investigated the heritability of radiation sensitivity in families of 177 Caucasian cases of early onset lung cancer. In total 798 individuals were characterized for their radiation-induced DNA damage response. DNA damage analysis was performed by alkaline comet assay before and after in vitro irradiation of isolated lymphocytes. The cells were exposed to a dose of 4 Gy and allowed to repair induced DNA-damage up to 60 minutes. The primary outcome parameter Olive Tail Moment was the basis for heritability estimates. Heritability was highest for basal damage (without irradiation) 70% (95%-CI: 51%-88%) and initial damage (directly after irradiation) 65% (95%-CI: 47%-83%) and decreased to 20%-48% for the residual damage after different repair times. Hence our study supports the hypothesis that genomic instability represented by the basal DNA damage as well as radiation induced and repaired damage is highly heritable. Genes influencing genome instability and DNA repair are therefore of major interest for the etiology of lung cancer in the young. The comet assay represents a proper tool to investigate heritability of the radiation sensitive phenotype. Our results are in good agreement with other mutagen sensitivity assays.

Childhood leukemia--risk factors and the need for an interdisciplinary research agenda.

The International Agency for Research on Cancer (IARC) has classified high as well as low-frequency fields as "possibly carcinogenic to humans" (Group 2B). For high frequency fields the recent assessment is based mainly on weak positive associations described in some epidemiological studies between glioma and acoustic neuroma and the use of mobile and other wireless phones. Also for lowfrequency fields the evidence is based on epidemiological findings revealing a statistic association between childhood leukemia (CL) and low-level magnetic fields. The basic findings are already 10 years old. They have since been supported by further epidemiological studies. However, the knowledge on the main/crucial question of causality has not improved. This fact and in addition the small, but statistically significant increased incidence of CL in the surrounding of German nuclear power plants have motivated the German Office for Radiation Protection (BfS) to work toward a better understanding of the main causes of CL. A long-term strategic research agenda has been developed which builds on an interdisciplinary, international network and aims at clarifying the aetiology of childhood acute lymphoblastic leukemia.

Validation of a fully automated COMET assay: 1.75 million single cells measured over a 5 year period.

The COMET assay is recognized as a rapid and sensitive method in quantifying radiation induced DNA damage. We investigated the distorting influence of endogenous, assay-inherent factors onto base (single cell level) and primary outcome measures (experimental/slide level), such as olive tail moment (OTM) and percentage DNA in the tail (%tail-DNA). From 2003 to 2008, we performed the assay on lymphocytes isolated from the blood samples of 355 lung cancer patients, 170 controls, and 610 relatives, as well as one single reference individual, repeated 170 times. In total, the data from 10,016 single experiments containing around 1,750,000 cells have been included in this study. This is the first time that the endogenous variability of the COMET assay has been validated systematically on such a huge data set over a 5 year period. Assuming that the reference sample reflects assay specific white noise, we estimated a proportion of 7-95% of the variability of the outcome measures due to assay variation (white noise) depending on parameter, exposure level, and study group. The proportion of white noise was largest for the initial radiation damage. The specific endogenous factors considered attribute to 14.8% of the total variability in the primary outcome measurements of the OTM and 6.9% of the %tail-DNA. OTM turns out to be a sensitive parameter to detect variation, but is also more susceptible to disturbance caused by endogenous factors than %tail-DNA. To reduce the experimental variability in COMET assays, we recommend a highly standardized operation protocol as well as inspecting and/or adjusting the primary outcome measures according to endogenous factors before calculating secondary outcome measures, e.g. DNA repair capacity (DRC) or testing statistical inference. A human reference (HR) sample is also useful to inspect homogeneity in the temporal progression of long lasting investigations, but not for calibrating primary outcome measurements.