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Supplementary Figures 1-5, Table 1 from DNA Damage Signaling in Hematopoietic Cells: A Role for Mre11 Complex Repair of Topoisomerase Lesions

The Mre11 complex promotes DNA double-strand break repair and regulates DNA damage signaling via activation of the ataxia-telangiectasia mutated (ATM) kinase. The hypermorphic Rad50S allele encodes a variant of Rad50, a member of the Mre11 complex. Cells expressing Rad50S experience constitutive ATM activation, which leads to precipitous apoptotic attrition in hematopoietic cells. In this study, we show that ATM activation by the Rad50S-containing Mre11 complex enhances the proliferation of LSK cells, a population consisting of hematopoietic stem cells and multipotent progenitor cells. In Rad50S/S mice, enhanced LSK proliferation triggers apoptotic attrition. This phenotype is mitigated when Rad50S/S is combined with mutations that alter either LSK cell quiescence (myeloid elf-1–like factor/ELF4–deficient mice) or hematopoietic differentiation (p21- and p27-deficient mice), indicating that the LSK population is a primary target of Rad50S pathology. We show that cells from Rad50S/S mice are hypersensitive to camptothecin, a topoisomerase I inhibitor that causes DNA damage primarily during DNA replication. On this basis, we propose that apoptotic attrition of Rad50S/S hematopoietic cells results from enhanced proliferation in the context of topoisomerase-associated DNA damage. Impairment of apoptosis in Rad50S/S mice promotes hematopoietic malignancy, suggesting that primitive hematopoietic cells serve as a reservoir of potentially oncogenic lesions in Rad50S/S mice. These data provide compelling evidence that the Mre11 complex plays a role in the metabolism of topoisomerase lesions in mammals, and further suggest that such lesions can accumulate in primitive hematopoietic cells and confer significant oncogenic potential. [Cancer Res 2008;68(7):2186–93]

We thank Jack Suess and Damian Doyle (UMBC Division of Information Technology)for their enthusiastic cooperation. Thanks to Richard Baldwin and Travis Scheponik for helpful comments. This project was supported in part by the National Science Foundation under SFS grants 1241576 and 1753681. Sherman was also supported by the National Science Foundation under SFS capacity grant 1819521 and by the U.S. Department of Defense under CAE-R grant H98230-17-1-0349 and CySP grants H98230-17-1-0387 and H98230-18-0321.

This paper does not necessarily reflect the views of the International Commission on Radiological Protection. The author passed away on 13 November 2015. Committee 1 of the International Commission on Radiological Protection (ICRP) addresses issues pertinent to tissue reactions, risks of cancer and heritable diseases, radiation dose responses, effects of dose rate, and radiation quality. In addition, it reviews data on the effects of radiation on the embryo/fetus, genetic factors in radiation response, and uncertainties in providing judgements on radiation-induced health effects. Committee 1 advises the Main Commission on the biological basis of radiation-induced health effects, and how epidemiological, experimental, and theoretical data can be combined to make quantitative judgements on health risks to humans. The emphasis is on low radiation doses, in the form of detriment-adjusted nominal risk coefficients, where there are considerable uncertainties in terms of the biology and the epidemiology. Furthermore, Committee 1 reviews data from radiation epidemiology studies and publications on the molecular and cellular effects of ionising radiation relevant to updating the basis of the 2007 Recommendations published in ICRP Publication 103. This paper will provide an overview of the activities of Committee 1, the updated work of the Task Groups and Working Parties, and the future activities being pursued.

The goal of this study was to define pathways regulated by low dose radiation to understand how biological systems respond to subtle perturbations in their environment and prioritize pathways for human health assessment. Using an in vitro 3-D human full thickness skin model, we have examined the temporal response of dermal and epidermal layers to 10 cGy X-ray using transcriptomic, proteomic, phosphoproteomic and metabolomic platforms. Bioinformatics analysis of each dataset independently revealed potential signaling mechanisms affected by low dose radiation, and integrating data shed additional insight into the mechanisms regulating low dose responses in human tissue. We examined direct interactions among datasets (top down approach) and defined several hubs as significant regulators, including transcription factors (YY1, MYC and CREB1), kinases (CDK2, PLK1) and a protease (MMP2). These data indicate a shift in response across time - with an increase in DNA repair, tissue remodeling and repression of cell proliferation acutely (24-72h). Pathway-based integration (bottom up approach) identified common molecular and pathway responses to low dose radiation, including oxidative stress, nitric oxide signaling and transcriptional regulation through the SP1 factor that would not have been identified by the individual data sets. Significant regulation of key downstream metabolites of nitrative stress was measured within these pathways. Among the features identified in our study, the regulation of MMP2 and SP1 was experimentally validated. Our results demonstrate the advantage of data integration to broadly define the pathways and networks that represent the mechanisms by which complex biological systems respond to perturbation.

Not-targeted effects represent a paradigm shift from the "DNA centric" view that ionizing radiation only elicits biological effects and subsequent health consequences as a result of an energy deposition event in the cell nucleus. While this is likely true at higher radiation doses (1 Gy), at low doses (100 mGy) non-targeted effects associated with radiation exposure might play a significant role. Here definitions of non-targeted effects are presented, the potential mechanisms for the communication of signals and signaling networks from irradiated cells/tissues are proposed, and the various effects of this intra- and intercellular signaling are described. We conclude with speculation on how these observations might lead to and impact long-term human health outcomes.

For the first time, plutonium retention in human upper airways was investigated based on the dosimetric structure of the human respiratory tract proposed by the International Commission on Radiological Protection (ICRP). This paper describes analytical work methodology, case selection criteria, and summarizes findings on soluble (ICRP 68 Type M material) plutonium distribution in the lungs of a former nuclear worker occupationally exposed to plutonium nitrate [239Pu(NO3)4]. Thirty-eight years post-intake, plutonium was found to be uniformly distributed between bronchial (BB), bronchiolar (bb) and alveolar-interstitial (AI) dosimetric compartments as well as between the left and right lungs. 239+240Pu and 238Pu total body activity was estimated to be 2333 ± 23 and 42.1 ± 0.7 Bq, respectively. The results of this work provide key information on the extent of plutonium binding in the upper airways of the human respiratory tract.

Radiation-induced genomic instability is a well-studied phenomenon that is measured as mitotically heritable genetic alterations observed in the progeny of an irradiated cell. The mechanisms that perpetuate this instability are unclear; however, a role for chronic oxidative stress has consistently been demonstrated. In the chromosomally unstable LS12 cell line, oxidative stress and genomic instability were correlated with mitochondrial dysfunction. To clarify this mitochondrial dysfunction and gain insight into the mechanisms underlying radiation-induced genomic instability we have evaluated the mitochondrial subproteome and performed quantitative mass spectrometry analysis of LS12 cells. Of 98 quantified mitochondrial proteins, 17 met criteria for fold changes and reproducibility; and 11 were statistically significant in comparison with the stable parental GM10115 cell line. Previous observations implicated defects in the electron transport chain (ETC) in the LS12 cell mitochondrial dysfunction. Proteomic analysis supports these observations, demonstrating significantly reduced levels of mitochondrial cytochrome c, the intermediary between complexes III and IV of the ETC. Results also suggest that LS12 cells compensate for ETC dysfunction and oxidative stress through increased levels of tricarboxylic acid cycle enzymes and upregulation of proteins that protect against oxidative stress and apoptosis. More than one cellular defect is likely to contribute to the genomic instability phenotype, and evaluation of gene and microRNA expression suggests that epigenetics play a role in the phenotype. These data suggest that LS12 cells have adapted mechanisms that allow survival under suboptimal conditions of oxidative stress and compromised mitochondrial function to perpetuate genomic instability.

Gas chromatography-mass spectrometry (GC-MS)-based global metabolomics requires complete chemical derivatization and matching of experimental spectra with those in various commercial or freely available databases in a high throughput manner.[1–6] Although several chemical methods have been developed for converting active functional groups in metabolites,[7, 8] the combination of methoxyamination and trimethylsilyation is one of the most widely used in global metabolomics studies.[6, 9] Recently, Agilent Technologies released the Fiehn GC-MS Metabolomics RTL (retention time locked) Library based on this derivatization approach. This library contains validated retention indices and spectra for 700 metabolites,[6] increasing the confidence of metabolite identifications in complex samples through the use of two independent parameters. However, multiple peaks could be formed from some specific molecules during the derivatization processes. Some of these peaks are partial TMS derivatives, while others are well-documented artifacts.[6, 10]

Non-targeted effects, i.e., those responses in cells or tissues that were not subject to energy deposition events after localized exposure to ionizing radiaton, are well-established. While they are not universal phenotype, when they do occur they can be associated with subsequent tissue or whole body responses. Here it is argued that non-targeted effects are a tissue level response to restore equilibrium within an organ system, and thus restores tissue homeostasis. This "adaptive homeostasis" has evolved in response to a variety of environmental and other such stresses an individual is exposed to in their lifetime. These non-targeted effects are not likely to impact significantly on estimates of potential risks associated with radiation exposure because they are presumably "built into" current risk estimates. However, they could have implications for radiation carcinogenesis, by driving processes in targeted and non-targeted cells that could eliminate transformed cells or transform cells from a normal phenotype to a phenotype associated with malignancy within a tissue.

There is a plethora of data available on the DNA damages associated with exposures to ionizing radiation and the subsequent cellular responses. Indeed, much of radiation research has focused on these initial insults and induced responses, particularly DNA repair, cell signaling pathways, cell cycle checkpoint control, mutation induction, chromosomal rearrangements, transformation and apoptosis etc. While many of these endpoints correlate with exposure dose, few, if any, provide substantive information on human health risk(s) associated with radiation exposure. Here the contribution of recent advances in high throughput ‘omics technologies are evaluated to examine what they have taught us about health risk(s) to humans associated with exposure to ionizing radiation.

This paper provides an integration and discussion of the information presented at the workshop held from 2-5 May 2010 in Richland, WA, adjacent to the Pacific Northwest National Laboratory (PNNL). Consequently, this is commentary and not necessarily a consensus document. This workshop was in honor of Dr. Victor P. Bond in celebration of his numerous contributions to the radiation sciences.

It has become increasingly evident from reports in the literature that there are many confounding factors capable of modulating radiation-induced non-targeted responses, such as the bystander effect and the adaptive response. In this paper, we examine recent data which suggest that the observation of non-targeted responses may not be universally observable for differing radiation qualities. We have conducted a study of the adaptive response following low-linear energy transfer exposures for human colon carcinoma cells and failed to observe adaption for the endpoints of clonogenic survival or micronucleus formation.

Straume, T., Amundson, S. A., Blakely, W. F., Burns, F. J., Chen, A., Dainiak, N., Franklin, S., Leary, J. A., Loftus, D. J., Morgan, W. F., Pellmar, T. C., Stolc, V., Turteltaub, K. W., Vaughan, A. T., Vijayakumar, S. and Wyrobek, A. J. NASA Radiation Biomarker Workshop. September 27–28, 2007. Radiat. Res. 170, 393–405 (2008). A summary is provided of presentations and discussions at the NASA Radiation Biomarker Workshop held September 27–28, 2007 at NASA Ames Research Center in Mountain View, CA. Invited speakers were distinguished scientists representing key sectors of the radiation research community. Speakers addressed recent developments in the biomarker and biotechnology fields that may provide new opportunities for health-related assessment of radiation-exposed individuals, including those exposed during long-duration space travel. Topics discussed included the space radiation environment, biomarkers of radiation sensitivity and individual susceptibility, molecular signatures of low-dose ...

Previous work by Morgan and coworkers on radiation-induced genome instability in Chinese hamster ovary (CHO) cell lines showed that unstable LS-12 cells had persistently elevated levels of reactive oxygen species (ROS) that were likely due to dysfunctional mitochondria. To further investigate the correlation between radiation-induced genome instability and dysfunctional mitochondria, we performed quantitative high-throughput mass spectrometry on samples enriched in mitochondrial proteins from three chromosomally unstable CHO cell lines and their stable unirradiated GM10115 parental cell line. Out of several hundred identified proteins, sufficient data were collected on 74 mitochondrial proteins to test for statistically significant differences in their abundance between unstable and stable cell lines. The LS-12 cell line, which exhibited the highest level of ROS among the three unstable cell lines, was characterized by eight significantly down-regulated mitochondrial proteins, all associated with the TCA (tricarboxylic acid). Elevated levels of ROS relative to the unirradiated parental control were also statistically significant for the CS-9 cell line. The protein profile of CS-9 revealed five significantly up-regulated mitochondrial proteins, three of which are involved in oxidative phosphorylation. Elevation of ROS in the unstable 115 cell line was nearly as large as that seen in CS-9 cells but was not statistically significant. The mitochondrial protein profile of 115 cells showed significant down-regulation of acetyl-CoA-acetyltransferase, which was also down-regulated in LS-12, and two other proteins with abundances that were significantly different from control levels but were not directly related to either the TCA or oxidative phosphorylation. These results provide further evidence that elevated ROS and mitochondrial dysfunction are associated with radiation-induced genome instability; however, additional work is required to establish a firm mechanistic relationship between these end points.

Radiation induced genomic instability can be perpetuated over time by the transmission of soluble factors. This can occur via cell-to-cell gap junction communication or the secretion/shedding of soluble factors. We have investigated whether our radiation induced chromosomally unstable GM10115 human-hamster hybrid clones secrete factors that can perpetuate the instability phenotype over time. These clones do not have functional gap junctions, but do secrete significant amounts of Interleukin 8 (IL-8) into the culture medium. We then determined whether IL-8 could initiate and or perpetuate genomic instability over time in parental GM10115 cells. Contrary to our hypothesis, IL-8 could induce DNA damage, but was not responsible for the unstable phenotype. Instead it appears that IL-8 secretion provides a pro-survival function in cells that are chromosomally unstable and generally fail to thrive.

— The relative biological importance of(5–5,6–6) cyclobutane and(6–4) pyrimidine-pyrimidone dimers in mammalian cells has been determined in a xeroderma pigmentosum (XP) revertant that repairs only the(6–4) photoproduct. Surprisingly, the majority of biological effects of UV light, including cell killing, sister chromatid exchange, mutagenesis, and inhibition and recovery of DNA and RNA synthesis, appear to be due to the(6–4) photoproduct and not to the cyclobutane dimer. Although the XP revertant repairs its own genomic DNA, it fails to repair damage in shuttle vectors, including the pZ189 mutational and chloramphenicol acetyl transferase transcriptional assay systems. The revertant is therefore a cell line for which shuttle vectors are inappropriate as models for genomic DNA repair, and may highlight a general weakness of shuttle vectors for the study of infrequent DNA lesions.

RKO36 cells, a subclone of RKO colorectal carcinoma cells that have been stably transfected with the pCMV-EGFP2Xho vector, were grown to confluence and then exposed to either the radioprotector WR-1065, i.e. the active thiol form of amifostine, for 30 min at doses of 40 microM and 4 mM or the cytokine tumor necrosis factor alpha (TNFalpha, TNFA) for 30 min at a concentration of 10 ng/ml and then washed. Total protein was isolated as a function of time up to 32 h after these treatments. Both doses of WR-1065 as well as the concentration of TNFalpha used were effective in elevating intracellular levels of the antioxidant protein SOD2 (also known as MnSOD) at least 15-fold over background levels as determined by Western blot analysis, while measured SOD2 activity was elevated between 5.5- and 6.9-fold. SOD2 reached a maximal level 24 h and 20 h after WR-1065 and TNFalpha treatments, respectively. The antioxidant proteins catalase and glutathione peroxidase (GPX) were also monitored over the 32-h period. In contrast to the robust changes observed in intracellular levels of SOD2 as a function of time after exposure of cells to WR-1065, catalase levels were elevated only 2.6-fold over background as determined by Western blot analysis, while GPX activity was unaffected by WR-1065 exposure. GPX protein levels were extremely low in cells, and analysis of GPX activity using a spectrophotometric method based on the consumption of reduced NADPH also revealed no measurable change as a function of WR-1065 or TNFalpha exposure. RKO36 cells either were irradiated with X rays in the presence of either 40 microM or 4 mM WR-1065 or 10 ng/ml TNFalpha or were irradiated 24 or 20 h later, respectively, when SOD2 protein levels were most elevated. The concentrations and exposure conditions used for WR-1065 and TNFalpha were not cytotoxic and had no effect on plating efficiencies or cell survival compared to untreated controls. No protection or sensitization was observed for cells irradiated in the presence of 40 microM WR-1065 or TNFalpha. Survival was elevated 1.90-fold for cells irradiated in the presence of 4 mM WR-1065. When RKO36 cells were irradiated with 2 Gy 24 h after 40 microM or 4 mM WR-1065 and 20 h after TNFalpha treatments when SOD2 levels were the most increased, survival was elevated 1.42-, 1.48- and 1.36-fold, respectively. This increased survival represents a SOD2-mediated delayed radioprotective effect. SOD2 appears to be an important antioxidant gene whose inducible expression is an important element in adaptive cellular responses in general, and the delayed radioprotective effect in particular. It can be induced by a range of agents including cytoprotective nonprotein thiols such as WR-1065 and pleiotropic cytokines such as TNFalpha.

Radiation-induced bystander effects refer to those responses occurring in cells that were not subject to energy deposition events following ionizing radiation. These bystander cells may have been neighbors of irradiated cells, or physically separated but subject to soluble secreted signals from irradiated cells. Bystander effects have been observed in vitro and in vivo and for various radiation qualities. In tribute to an old friend and colleague, Anthony V. Carrano, who would have said "well what are the critical questions that should be addressed, and so what?", we review the evidence for non-targeted radiation-induced bystander effects with emphasis on prevailing questions in this rapidly developing research field, and the potential significance of bystander effects in evaluating the detrimental health effects of radiation exposure.

Radiation-induced genomic instability has been demonstrated in numerous in vitro model systems. This instability can manifest as chromosomal changes, gene amplifications and mutations, micronuclei formation, transformation, and reduced plating efficiency and occurs in the progeny of an irradiated cell generations after the initial insult. However, reports of induced genomic instability in humans exposed to ionizing radiation are contradictory. We will review the evidence for and against induced chromosomal instability in radiation exposed human populations. In an attempt to reconcile these conflicting observations, we propose that analysis of potential chromosomal instability in blood samples from normal healthy individuals, irrespective of their radiation history, is unlikely to reveal evidence of induced instability because observable dynamic genomic changes would be inconsistent with a normal healthy phenotype.

All humans receive some radiation exposure and the risk for radiation-induced cancer at low doses is based on the assumption that there is a linear non-threshold relationship between dose and subsequent effect. Consequently, risk is extrapolated linearly from high radiation doses to very low doses. However, adaptive responses, bystander effects, and death-inducing effect may influence health effects associated with low-dose radiation exposure. Adaptive response is the phenomenon by which cells irradiated with a sublethal radiation dose can become less susceptible to subsequent high-dose radiation exposure. Bystander effects are nontargeted effects observed in cells that were not irradiated but were either in contact with or received soluble signals from irradiated cells. These non-hit bystander cells can exhibit damage typically associated with direct radiation exposure. Death-inducing effect is a phenomenon whereby medium from human-hamster hybrid cells displaying radiation-induced chromosomal instability is toxic to unirradiated parental cells. In this study, we show that human RKO cells do not exhibit adaptive response, bystander effect, or death-inducing effect, as measured by cell killing, or delayed genomic instability in a stably transfected plasmid–based green fluorescent protein assay measuring homologous recombination and delayed mutation/deletion events. However, growth medium conditioned by some chromosomally unstable RKO derivatives induced genomic instability, indicating that these cells can secrete factor(s) that elicit responses in nonirradiated cells. Furthermore, low radiation doses suppressed the induction of delayed genomic instability by a subsequent high dose, indicative of an adaptive response for radiation-induced genomic instability. These results highlight the inherent variability in cellular responses to low-dose radiation exposure and add to the uncertainties associated with evaluating potential hazards at these low doses. [Cancer Res 2007;67(3):1099–104]

For individuals traveling in space, exposure to space radiation is unavoidable. Since adequate shielding against radiation exposure is not practical, other strategies for protecting the astronauts must be developed. Radiation is also an important therapeutic and diagnostic tool, and evidence from the clinical and experimental settings now shows a firm connection between radiation exposure and changes in cytokine and chemokine levels. These small proteins can be pro- or anti-inflammatory in nature and the balance between those two effects can be altered easily because of exogenous stresses such as radiation. The challenge to identify a common perpetrator, however, lies in the fact that the cytokines that are produced vary based on radiation dose, type of radiation, and the cell types that are exposed. Based on current knowledge, special treatments have successfully been designed by implementing administration of proteins, antibodies, and drugs that counteract some of the harmful effects of radiation. Although these treatments show promising results in animal studies, it has been difficult to transfer those practices to the human situation. Further understanding of the mechanisms by which cytokines are triggered through radiation exposure and how those proteins interact with one another may permit the generation of novel strategies for radiation protection from the damaging effects of radiation. Here, we review evidence for the connection between cytokines and the radiation response and speculate on strategies by which modulating cytokine responses may protect astronauts against the detrimental effects of ionizing radiations.

Commission 1 of the International Commission on Radiological Protection considers the risk of induction of cancer and heritable disease (stochastic effects) together with the underlying mechanisms of radiation action. Committee 1 also considers the risks, severity, and mechanisms of induction of tissue/organ damage and developmental defects (deterministic effects). The Committee was significantly revamped in 2013 and last met in Abu Dhabi in October 2013. Committee 1 evaluated progress on two ongoing task groups: Task Group 64 ‘Cancer Risk from Alpha Emitters’ and Task Group 75 ‘Stem Cell Radiobiology’. Following approval from the Main Commission, Committee 1 established two new task groups: Task Group 91 ‘Radiation Risk Inference at Low Dose and Low Dose Rate Exposure for Radiological Protection Purposes’ and Task Group 92 ‘Terminology and Definitions’. This article presents a synopsis of the current status of Committee 1 and outlines the tasks that Committee 1 may undertake in the future.

Radiation-induced genomic instability (RIGI) manifests as a heritable increased rate of genetic alterations in the progeny of irradiated cells generations after the initial insult. The progeny can show an increased frequency of chromosomal translocations, deletions, mutations, micronuclei, and decreased plating efficiency. What perpetuates RIGI is unclear; however, persistently increased levels of reactive oxygen species (ROS) are frequently associated with genomically unstable clones. Furthermore, addition of free radical scavengers (e.g., DMSO, glycerol, and cationic thiol cysteamine) reduces the incidence of instability after irradiation, implicating a ROS-mediated role in RIGI induction. Because mitochondria are a major natural cellular source of ROS, we tested the hypothesis that mitochondrial dysfunction has a role in maintaining the elevated ROS levels in our irradiated, genetically unstable GM10115 Chinese hamster ovary cells. Amplex Red fluorometry measurements indicate that the relative contribution of uncoupler-sensitive mitochondrial hydrogen peroxide production to total cellular hydrogen peroxide generation is greater in unstable cells. Measurements of mitochondrial DNA levels and cell cytometric fluorescent measurements of Mitotracker Green FM indicate that differences in mitochondrial ROS production are not due to varying mitochondrial levels. However, mitochondrial respiration measured in digitonin-permeabilized cells is impaired in unstable clones. In addition, manganese superoxide dismutase, a major mitochondrial antioxidant enzyme, exhibits increased immunoreactivity but decreased enzyme activity in unstable clones, which along with decreased respiration rates may explain the increased levels of cellular ROS. These studies show that mitochondria from unstable cells are abnormal and likely contribute to the persistent oxidative stress in the unstable clones. (Cancer Res 2006; 66(21): 10377-83)

Ionizing radiation induces delayed genomic instability in human cells, including chromosomal abnormalities and hyperrecombination. Here, we investigate delayed genome instability of cells exposed to UV radiation. We examined homologous recombination-mediated reactivation of a green fluorescent protein (GFP) gene in p53-proficient human cells. We observed an approximately 5-fold enhancement of delayed hyperrecombination (DHR) among cells surviving a low dose of UV-C (5 J/m2), revealed as mixed GFP+/- colonies. UV-B did not induce DHR at an equitoxic (75 J/m2) dose or a higher dose (150 J/m2). UV is known to induce delayed hypermutation associated with increased oxidative stress. We found that hypoxanthine phosphoribosyltransferase (HPRT) mutation frequencies were approximately 5-fold higher in strains derived from GFP+/- (DHR) colonies than in strains in which recombination was directly induced by UV (GFP+ colonies). To determine whether hypermutation was directly caused by hyperrecombination, we analyzed hprt mutation spectra. Large-scale alterations reflecting large deletions and insertions were observed in 25% of GFP+ strains, and most mutants had a single change in HPRT. In striking contrast, all mutations arising in the hypermutable GFP+/- strains were small (1- to 2-base) changes, including substitutions, deletions, and insertions (reminiscent of mutagenesis from oxidative damage), and the majority were compound, with an average of four hprt mutations per mutant. The absence of large hprt deletions in DHR strains indicates that DHR does not cause hypermutation. We propose that UV-induced DHR and hypermutation result from a common source, namely, increased oxidative stress. These two forms of delayed genome instability may collaborate in skin cancer initiation and progression.

The relatively high frequency with which ionizing radiation induces genomic instability suggests that a gene mutation occurring after irradiation is an unlikely cause of the phenotype. To search for mechanism(s) of initiation and perpetuation of this instability phenotype, gene expression profiles of clones exhibiting delayed chromosomal instability were analyzed. Microarray analysis using two pools of isogenic radiation-induced chromosomally unstable clones compared to an irradiated but chromosomally stable clone uncovered a set of 68 differentially expressed genes using two methods of analysis. Unexpectedly, all 68 genes were under-expressed relative to the chromosomally stable reference clone. Further analysis of the candidates placed the differentially expressed genes into pathways implicating differential MAP kinase signaling, ubiquitin/proteasome function, DNA repair, cell cycle control, lipid signaling, nucleotide metabolism, and other potentially disrupted pathways. Validation studies using northern and western blotting, and functional assays concluded that although differences in some of these pathways exist, no single gene or molecular pathway was found to be differentially regulated in all of the chromosomally unstable clones tested. Inferred from these data is that there are multiple potential molecular pathways and/or events that maintain the unstable phenotype, and no single expression pattern is linked to instability in the unstable clones analyzed.

Delayed reproductive cell death or lethal mutations in the survivors of irradiated cells is a well-characterized end point associated with radiation-induced genomic instability. Although the mechanism for this delayed lethality has not been identified, it is thought to be a means of eliminating cells that have sustained extensive damage, thus preventing tissue disruption after radiation exposure. In this study we have tested the hypothesis that delayed reproductive cell death in chromosomally unstable GM10115 clones is due to persistently increased levels of apoptosis. Evidence for differences in apoptosis in two representative genomically unstable clones after irradiation is presented. In addition, one of the unstable clones was found to have abnormal levels of apoptosis after radiation exposure. An understanding of apoptosis in genomically unstable clones may provide insight into the maintenance of genomic instability and the mechanism by which genomically unstable cells evade cell death, potentially contributing to carcinogenesis.

Radiation-induced genomic instability is a delayed effect of ionizing radiation that may contribute to radiation carcinogenesis. Prior microarray studies investigating gene expression changes in genomically unstable cell lines isolated after radiation exposure uncovered the differential expression of the NF-kappaB p105 mRNA. In this study, the functionality of the NF-kappaB pathway was examined to determine its role in regulating gene expression changes after oxidative stress in chromosomally stable and unstable human-hamster hybrid clones. Basal DNA-binding activity assays showed no significant differences between the clones; however, further experiments established differences in NF-kappaB induction in three chromosomally unstable clones after acute hydrogen peroxide treatment. A second assay was used to confirm this differential activity in the chromosomally unstable clones by studying reporter gene activation after treatment with hydrogen peroxide. Yet an initial upstream analysis of the pathway revealed no significant increase in the level of IkappaBalpha inhibitor protein in the unstable clones. Downstream tests analyzing the induction of the antiapoptotic target protein Bcl-2 found variable induction among the stable and unstable clones. These differences did not translate to a reduction in clonogenic survival after acute exposure to oxidative stress, as the irradiated but chromosomally stable clone displayed the most sensitivity. Due to its role in regulating a diverse set of cellular functions, including responses to oxidative stress, alterations in the NF-kappaB pathway in chromosomally unstable clones may regulate the differential physiology of a subset of chromosomally unstable clones and could contribute to the perpetuation of the phenotype. However, a specific role for defective induction and activation of this pathway remains unidentified.

Radiation-induced genomic instability (RIGI) manifests in the progeny of cells surviving ionizing radiation (IR), and can be measured using such endpoints as delayed mutation, micronuclei formation, and chromosomal instability. The frequency of RIGI is relatively high, exceeding the gene mutation rate of IR by orders of magnitude, leading to conjecture that a gene mutation is not the cause of the phenotype. We have started to explore whether differential gene expression patterns are associated with the instability phenotype, in order to shed light on its initiation and perpetuation. Using GM10115 human-hamster hybrid-derived chromosomally stable and radiation-induced unstable clones, gene expression patterns were analyzed using microarray analysis. Two methods were used to find differentially expressed genes, and all candidate genes identified by these methods were under-expressed relative to the chromosomally stable reference sample. Among this set differentially expressed genes identified were two candidates with a relationship to the ubiquitin/proteasome pathway. While follow-up gene expression analyses have confirmed the under-expression of these two genes in some of our chromosomally unstable clones, preliminary functional studies have been unable to demonstrate a link to instability. It is anticipated that as we apply this technology to the study of radiation-induced genomic instability, clues to its onset will be revealed, ultimately contributing to a greater understanding of the mechanisms of radiation carcinogenesis.

Radiation induced genomic instability can be described as the increased rate of genomic alterations occurring in the progeny of an irradiated cell. Its manifestations are the dynamic ongoing production of chromosomal rearrangements, mutations, gene amplifications, transformation, microsatellite instability, and/or cell killing. In this prospectus, we present the hypothesis that cellular exposure to ionizing radiation can result in the secretion of soluble factors by irradiated cells and/or their progeny, and that these factors can elicit responses in other cells thereby initiating and perpetuating ongoing genomic instability.

Recent advances in low dose radiation research have raised a number of compelling issues that have compounded the understanding of low dose radiation effects. Here some of them are outlined: the linear no-threshold model for predicting effects at low radiation doses, dose rate effectiveness factor, attributability, and public perception of low dose radiation effects. The impact of changes in any of these hotly debated issues on radiation protection is considered.

We recently described a unique non-targeted effect of ionizing radiation whereby growth medium from two clones of GM10115 cells exhibiting radiation-induced chromosomal instability was cytotoxic to parental GM10115 cells. We termed this the death-inducing effect (DIE). The goal of the present study was to determine how DIE killed cells. Our hypothesis was that DIE medium contained either a secreted factor(s) from unstable clones or products from dead/dying cells that were cytotoxic to parental cells. First, we investigated the apoptotic characteristics of our unstable clones by Annexin V binding and TUNEL assays. Both the parental GM10115 cells and cells from the unstable clone LS12 had a low background (approximately 2%) level of apoptosis. The unstable Fe-10-3 clone showed a high spontaneous level of apoptosis, indicating major differences in the spontaneously occurring levels of apoptosis. We then analyzed how medium from these unstable clones killed cells by investigating the induction of DNA breaks, micronucleus formation and apoptosis induction in cells exposed to medium from unstable clones. Medium from unstable clones was capable of eliciting DNA double-strand breaks and increased apoptosis. Increased micronucleus frequencies were also observed in cells exposed to medium from either unstable clone, indicating a role of mitotis-linked cell death in DIE. These data suggest that DIE most likely results from cytotoxic factors secreted into the culture medium that can cause DNA double-strand breaks in recipient cells. These breaks can then lead to mitotis-linked cell death, as measured by micronuclei, or apoptosis, which accounts for the DIE.

In addition to the damage caused by the initial exposure to ionizing radiation, it is now known that ionizing radiation induces delayed DNA damage in cells by a process known as radiation-induced genomic instability. Characterized using many different endpoints including increased mutation, frequency, increased level of reactive oxygen species (ROS), and decreased plating efficiency, radiation-induced genomic instability is best characterized in the GM10115 system by chromosomal instability. While the cellular and molecular mechanism of induced instability is not known, we are investigating the hypothesis that clones exhibiting instability display an altered pattern of gene expression. An 1152 gene microarray chip was used to uncover potential gene expression changes in two sets of two unstable clones in comparison to an irradiated but chromosomally stable clone. One candidate gene, Cu/Zn superoxide dismutase 1 (SOD1), was under-expressed in the unstable clones by an average of 3.66-fold by array analysis. Given the high levels of ROS observed in unstable clones, we performed a detailed analysis of SOD1 in four chromosomally unstable cell lines. Northern and western blotting assays could not confirm the microarray data, showing a decrease in expression of SOD1 in only one of four unstable lines, and increases in average protein level in all four unstable clones relative to control. Average SOD1 activity was also elevated in three of four unstable clones tested. These results indicate that while increased ROS levels are characteristic of many of our clones showing instability, this is not due to under-expression of SOD1 as suggested by DNA microarray analysis.

A number of nontargeted and delayed effects associated with radiation exposure have now been described. These include radiation-induced genomic instability, death-inducing and bystander effects, clastogenic factors and transgenerational effects. It is unlikely that these nontargeted effects are directly induced by cellular irradiation. Instead, it is proposed that some as yet to be identified secreted factor can be produced by irradiated cells that can stimulate effects in nonirradiated cells (death-inducing and bystander effects, clastogenic factors) and perpetuate genomic instability in the clonally expanded progeny of an irradiated cell. The proposed factor must be soluble and capable of being transported between cells by cell-to-cell gap junction communication channels. Furthermore, it must have the potential to stimulate cellular cytokines and/or reactive oxygen species. While it is difficult to imagine a role for such a secreted factor in contributing to transgenerational effects, the other nontargeted effects of radiation may all share a common mechanism.

The goal of this review is to summarize the evidence for non-targeted and delayed effects of exposure to ionizing radiation in vivo. Currently, human health risks associated with radiation exposures are based primarily on the assumption that the detrimental effects of radiation occur in irradiated cells. Over the years a number of non-targeted effects of radiation exposure in vivo have been described that challenge this concept. These include radiation-induced genomic instability, bystander effects, clastogenic factors produced in plasma from irradiated individuals that can cause chromosomal damage when cultured with nonirradiated cells, and transgenerational effects of parental irradiation that can manifest in the progeny. These effects pose new challenges to evaluating the risk(s) associated with radiation exposure and understanding radiation-induced carcinogenesis.

Appropriate cell adhesion is necessary for numerous physiological processes and can be deranged in many diseases, including cancer. It has long been recognized that the cadherins mediated cell-cell adhesion receptor is an important determinant of tumor progression, serving as a suppressor of invasion and metastasis. Recently a role for the C-terminal region of talin (the VAD fragment) in cadherin mediated cell-cell attachment has been suggested, but its molecular mechanism remained unclear. Here, we identified a small region of the VAD fragment required for its localization to cadherin-mediated cell-cell junctions and also found that its localization to cell-cell junctions is independent of its actin binding properties. To identify the binding partner of the VAD fragment at cell-cell junctions we utilized immune-pulldown and found that it interacts with catenins, an important component of cadherin-mediated cell-cell junction. Based on our data, we proposed that the VAD-fragment of talin stabilized and/or stimulated the cell-cell junction by interacting with catenins. Citation Format: Devang M. Patel, Akhilesh Kumar, William Morgan, Michael D. Birnbaum, Chaunhua Jiang, Fangliang Zhang. Talin plays an important role in cell-cell interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1341. doi:10.1158/1538-7445.AM2017-1341

Arginylation is a post-translational modification wholly mediated by Arginyltransferase 1 (Ate1). Our studies have shown that Ate1 and arginylation are upregulated during stress conditions. In addition, it was recently shown that Ate1-deficient fibroblasts demonstrate cancer-like characteristics including genomic instability and mutagenesis. While Ate1’s role in cancer is poorly understood, our preliminary studies suggest that Ate1 is an important regulator of metastasis and cancer progression in prostate cancer models. In this study, our data suggests that Ate1 is essential for normal cell stress response in conditions including cellular oxidant, apoptosis inducing drugs, and radiation. Further, we show that a loss of Ate1 in LnCaP prostate cancer cells increases anchorage-independent growth in the soft-agar assay, and that a loss of Ate1 in PC-3 prostate cancer cells increases matrigel invasion in the Boyden chamber assay. To text how Ate1 affects metastasis in vivo, PC-3 cells with Ate1 or control shRNA were orthotopically implanted into the prostates of immunocompromised mice to observe metastasis. While very little metastasis was observed in PC-3 control cells, PC-3 cells with reduced Ate1 achieved much higher levels of metastasis. In conclusion, our data suggests that the loss of Ate1 in prostate cancer promotes cancer progression phenotypes and cell survival. In future studies, we will examine if Ate1 levels in human prostate cancers in prostate cancer prognosis. Citation Format: Michael Birnbaum, Akhilesh Kumar, William Morgan, Fangliang Zhang. Arginyltransferase1 as a novel suppressor of prostate cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3523. doi:10.1158/1538-7445.AM2017-3523

Radiation induced genomic instability is a well-studied phenomenon, the underlying mechanisms of which are poorly understood. Persistent oxidative stress, mitochondrial dysfunction, elevated cytokine levels and epigenetic changes are among the mechanisms invoked in the perpetuation of the phenotype. To determine whether epigenetic aberrations affect genomic instability we measured DNA methylation, mRNA and microRNA (miR) levels in well characterized chromosomally stable and unstable clonally expanded single cell survivors of irradiation. While no changes in DNA methylation were observed for the gene promoters evaluated, increased LINE-1 methylation was observed for two unstable clones (LS12 and CS9) and decreased Alu element methylation was observed for the other two unstable clones (115 and Fe5.0–8). These relationships also manifested for mRNA and miR expression. mRNA identified for the LS12 and CS9 clones were most similar to each other (261 mRNA), while the 115 and Fe5.0–8 clones were more similar to each other, and surprisingly also similar to the two stable clones, 114 and 118 (286 mRNA among these four clones). Pathway analysis showed enrichment for pathways involved in mitochondrial function and cellular redox, themes routinely invoked in genomic instability. The commonalities between the two subgroups of clones were also observed for miR. The number of miR for which anti-correlated mRNA were identified suggests that these miR exert functional effects in each clone. The results demonstrate significant genetic and epigenetic changes in unstable cells, but similar changes are almost as equally common in chromosomally stable cells. Possible conclusions might be that the chromosomally stable clones have some other form of instability, or that some of the observed changes represent a sort of radiation signature and that other changes are related to genomic instability. Irrespective, these findings again suggest that a spectrum of changes both drive genomic instability and permit unstable cells to persist and proliferate.

To investigate the mechanisms of radiation-induced chromosomal instability, cells were irradiated in the presence of the free radical scavengers DMSO, glycerol, or cysteamine, in the presence of DMSO while frozen, or held in confluence arrest post-irradiation to permit cells to repair potentially lethal DNA damage. Clones derived from single progenitor cells surviving each treatment were then analyzed for the subsequent development of chromosomal instability. The presence of scavengers (± freezing) during irradiation, and the recovery from potentially lethal damage after irradiation led to an increase in cell survival that was accompanied by a decrease in the initial yield of chromosomal rearrangements. Furthermore, analysis of over 400 clones and 80,000 metaphases indicates that these same treatments reduced the incidence of instability at equitoxic doses when compared to controls irradiated in the absence of scavengers at ambient temperature. Results suggest that preventing reactive species from damaging DNA, promoting chemical repair of ionized DNA intermediates, or allowing enzymatic removal of genetic lesions, represent measures that reduce the total burden of DNA damage and reduce the subsequent onset of radiation-induced genomic instability.

Chromosome instability is a common occurrence in tumour cells. We examined the hypothesis that the elevated rate of mutation formation in unstable cells can lead to the development of clones of cells that are resistant to the cancer therapy. To test this hypothesis, we compared chromosome instability to radiation sensitivity in 30 independently isolated clones of GM10115 human–hamster hybrid cells. There was a broader distribution of radiosensitivity and a higher mean SF 2 in chromosomally unstable clones. Cytogenetic and DNA double-strand break rejoining assays suggest that sensitivity was a function of DNA repair efficiency. In the unstable population, the more radioresistant clones also had significantly lower plating efficiencies. These observations suggest that chromosome instability in GM10115 cells can lead to the development of cell variants that are more resistant to radiation. In addition, these results suggest that the process of chromosome breakage and recombination that accompanies chromosome instability might provide some selective pressure for more radioresistant variants. © 2001 Cancer Research Campaign http://www.bjcancer.com

DNA polymerases catalyze the synthesis of DNA using a continuous uninterrupted template strand. However, it has been shown that a 3'-->5' exonuclease-deficient form of the Klenow fragment of Escherichia coli DNA polymerase I as well as DNA polymerase of Thermus aquaticus can synthesize DNA across two unlinked DNA templates. In this study, we used an oligonucleotide-based assay to show that discontinuous DNA synthesis was present in HeLa cell extracts. DNA synthesis inhibitor studies as well as fractionation of the extracts revealed that most of the discontinuous DNA synthesis was attributable to DNA polymerase alpha. Additionally, discontinuous DNA synthesis could be eliminated by incubation with an antibody that specifically neutralized DNA polymerase alpha activity. To test the relative efficiency of each nuclear DNA polymerase for discontinuous synthesis, equal amounts (as measured by DNA polymerase activity) of DNA polymerases alpha, beta, delta (+/- PCNA) and straightepsilon (+/- PCNA) were used in the discontinuous DNA synthesis assay. DNA polymerase alpha showed the most discontinuous DNA synthesis activity, although small but detectable levels were seen for DNA polymerases delta (+PCNA) and straightepsilon (- PCNA). Klenow fragment and DNA polymerase beta showed no discontinuous DNA synthesis, although at much higher amounts of each enzyme, discontinuous synthesis was seen for both. Discontinuous DNA synthesis by DNA polymerase alpha was seen with substrates containing 3 and 4 bp single-strand stretches of complementarity; however, little synthesis was seen with blunt substrates or with 1 bp stretches. The products formed from these experiments are structurally similar to that seen in vivo for non-homologous end joining in eukaryotic cells. These data suggest that DNA polymerase alpha may be able to rejoin double-strand breaks in vivo during replication.

Nijmegen breakage syndrome (NBS) is an autosomal recessive disorder characterized by increased cancer incidence, cell cycle checkpoint defects, and ionizing radiation sensitivity. We have isolated the gene encoding p95, a member of the hMre11/hRad50 double-strand break repair complex. The p95 gene mapped to 8q21.3, the region that contains the NBS locus, and p95 was absent from NBS cells established from NBS patients. p95 deficiency in these cells completely abrogates the formation of hMre11/hRad50 ionizing radiation-induced foci. Comparison of the p95 cDNA to the NBS1 cDNA indicated that the p95 gene and NBS1 are identical. The implication of hMre11/hRad50/p95 protein complex in NBS reveals a direct molecular link between DSB repair and cell cycle checkpoint functions.

In eukaryotic and prokaryotic organisms DNA double-strand breaks with non-complementary ends can be joined by mechanisms of illegitimate recombination. We examined the joining of 3'-protruding single strand (PSS) ends, which do not have recessed 3' hydroxyls that can allow for fill-in DNA synthesis, to blunt ends. End-joining was examined by electro-transforming Escherichia coli strains with linearized plasmid DNA, sequencing the resulting junctions, and determining the transformation frequencies. Three different E.coli strains were examined: MC1061, which has no known recombination or DNA repair defects, HB101 (rec A-) and SURE (recB- recJ-). No striking differences were found in either the spectrum of products observed or the efficiency of end-joining between these strains. As in vertebrate systems, the majority of the products were overlaps between directly repeated DNA sequences. 3'-PSS are frequently preserved in vertebrate systems, but they were not preserved in our experiments unless the transforming DNA was pretreated with a DNA polymerase.

PURPOSE: To investigate the kinetics of chromosomal instability induced in clones of Chinese hamster cells following X-irradiation. MATERIALS AND METHODS: X-irradiated clones of GM10115, human-hamster hybrid cells containing a single human chromosome 4 (HC4), have been previously established. These clones were defined as unstable if they contained > or = three subpopulations of cells with unique rearrangements of HC4 as detected by FISH. Stable and unstable clones were analysed by FISH and Giemsa staining at various times post-irradiation. RESULTS: While most of the stable clones continued to show chromosomal stability of HC4 over time, one became marginally unstable at approximately 45 population doublings post-irradiation. Clones exhibiting chromosomal instability had one of several fates. Many of the unstable clones were showed similar levels of instability over time. However, one unstable clone became stable with time in culture, while another became even more unstable over time. Cytogenetic analyses of all clones after Giemsa staining indicated that in some clones the hamster chromosomes were rearranged independent of HC4, demonstrating increased frequencies of chromatid breaks and dicentric chromosomes. The majority of the unstable clones also had higher yields of chromatid gaps. CONCLUSIONS: These data demonstrate the dynamic nature of chromosomal instability as measured by two different cytogenetic assays.