Bailey, S. Telomeres and Double-Strand Breaks – All's Well that “Ends” Well. … Radiat. Res. 169, 1–7 (2008).

Sometimes one's life (including one's science) makes a lot more sense when viewed from the perspective of time, reflected back on over a number of years. That has indeed been the case for me. Strangely enough, the story begins with chromosomes and “ends” with telomeres, both at Colorado State University. And, just as with chromosomes, a lot happened in between. Telomeres were first identified based on their function—they protected the physical ends of chromosomes from interaction with broken DNA ends created by ionizing radiation. While I was at Los Alamos National Laboratory, the sequence of human telomeres was discovered, making probes available that allowed us to re-examine and provide direct support of these early observations; thus began my fascination with telomeres. Chromosome orientation in situ hybridization (CO-FISH) also came onto the scene while I was in Los Alamos. This strand-specific modification of standard FISH, especially when combined with telomeric sequence probes, has proven to be a powerful approach that provides information not available by any other means. Applications have included pericentric inversion detection, distinction between leading- and lagging-strand telomeres, and identification of telomere-double-strand break (DSB) fusions. We also provided the first direct evidence that DSB repair proteins (DNA-PK in particular) are required for mammalian telomeric end capping, and we have been characterizing telomere dysfunction in NHEJ and HR repair-deficient backgrounds ever since. Cells must correctly distinguish between DNA ends represented by telomeres and DNA ends produced by DSBs if all is to end well. Just as these studies have provided new insight into the complex, often surprising, interactions at DNA ends, they also provoke new questions. Whereas it is now well established that DSB repair proteins associate with telomeres, most recently we've been asking whether the reverse scenario holds: Do telomere proteins interact with DSBs? We find that DSBs induced by ionizing radiations are not sufficient to recruit the essential telomere protein TRF2 as an early damage response, so perhaps this interplay is a one-way street. The rest of the story waits to unfold.

Pauwels, E., Declerck, R., Van Speybroeck, V. and Waroquier, M. Evidence for a Grotthuss-Like Mechanism in the Formation of the Rhamnose Alkoxy Radical Based on Periodic DFT Calculations. Radiat. Res. 169, 8–18 (2008).

Molecular modeling adopting a periodic approach based on density functional theory (DFT) indicates that a Grotthuss-like mechanism is active in the formation of the radiation-induced alkoxy radical in α-l-rhamnose. Starting from an oxidized crystal structure, a hydroxyl proton is transferred along an infinite hydrogen bond chain pervading the entire crystal. The result of this proton shuttling mechanism is a stable radical species dubbed RHop. Only after several reorientations of crystal waters and hydroxyl groups, the more stable radical form RO4 is obtained, which differs in structure from the former by the absence of only one hydrogen bond. Calculations of the energetics associated with the mechanism as well as simulated spectroscopic properties reveal that different variants of the rhamnose alkoxy radical can be observed depending on the temperature of irradiation and consecutive EPR measurement. Cluster calculations on both radical variants provide hyperfine coupling and g tensors that are in good agreement with two independent experimental measurements at different temperatures.

Zheng, Y., Hunting, D. J., Ayotte, P. and Sanche, L. Radiosensitization of DNA by Gold Nanoparticles Irradiated with High-Energy Electrons. Radiat. Res. 168, 19–27 (2008).

Thin films of pGEM-3Zf(−) plasmid DNA were bombarded by 60 keV electrons with and without gold nanoparticles. DNA single- and double-strand breaks (SSBs and DSBs) were measured by agarose gel electrophoresis. From transmission electron micrographs, the gold nanoparticles were found to be closely linked to DNA scaffolds, probably as a result of electrostatic binding. The probabilities for formation of SSBs and DSBs from exposure of 1:1 and 2:1 gold nanoparticle:plasmid mixtures to fast electrons increase by a factor of about 2.5 compared to neat DNA samples. For monolayer DNA adsorbed on a thick gold substrate, the damage increases by an order of magnitude. The results suggest that the enhancement of radiosensitivity is due to the production of additional low-energy secondary electrons caused by the increased absorption of ionizing radiation energy by the metal, in the form of gold nanoparticles or of a thick gold substrate. Since short-range low-energy secondary electrons are produced in large amounts by any type of ionizing radiation, and since on average only one gold nanoparticle per DNA molecule is needed to increase damage considerably, targeting the DNA of cancer cells with gold nanoparticles may offer a novel approach that is generally applicable to radiotherapy treatments.

Mazor, R., Korenstein-Ilan, A., Barbul, A., Eshet, Y., Shahadi, A., Jerby, E. and Korenstein, R. Increased Levels of Numerical Chromosome Aberrations after In Vitro Exposure of Human Peripheral Blood Lymphocytes to Radiofrequency Electromagnetic Fields for 72 Hours. Radiat. Res. 169, 28–37 (2008).

We investigated the effects of 72 h in vitro exposure of 10 human lymphocyte samples to radiofrequency electromagnetic fields (800 MHz, continuous wave) on genomic instability. The lymphyocytes were exposed in a specially designed waveguide resonator at specific absorption rates (SARs) of 2.9 and 4.1 W/kg in a temperature range of 36–37°C. The induced aneuploidy of chromosomes 1, 10, 11 and 17 was determined by interphase FISH using semi-automated image analysis. We observed increased levels of aneuploidy depending on the chromosome studied as well as on the level of exposure. In chromosomes 1 and 10, there was increased aneuploidy at the higher SAR, while for chromosomes 11 and 17, the increases were observed only for the lower SAR. Multisomy (chromosomal gains) appeared to be the primary contributor to the increased aneuploidy. The effect of temperature on the level of aneuploidy was examined over the range of 33.5–40°C for 72 h with no statistically significant difference in the level of aneuploidy compared to 37°C. These findings suggest the possible existence of an athermal effect of RF radiation that causes increased levels of aneuploidy. These results contribute to the assessment of potential health risks after continuous chronic exposure to RF radiation at SARs close to the current levels set by ICNIRP guidelines.

Joubert, V., Bourthoumieu, S., Leveque, P. and Yardin, C. Apoptosis is Induced by Radiofrequency Fields through the Caspase-Independent Mitochondrial Pathway in Cortical Neurons. Radiat. Res. 169, 38–45 (2008).

In the present study, we investigated whether continuous-wave (CW) radiofrequency (RF) fields induce neuron apoptosis in vitro. Rat primary neuronal cultures were exposed to a CW 900 MHz RF field with a specific absorption rate (SAR) of 2 W/kg for 24 h. During exposure, an increase of 2°C was measured in the medium; control experiments with neurons exposed to 39°C were then performed. Apoptosis was assessed by condensation of nuclei with 4′,6-diamino-2-phenylindole (DAPI) staining observed with an epifluorescence microscope and fragmentation of DNA with TdT-mediated dUTP nick-end labeling (TUNEL) analyzed by flow cytometry. A statistically significant difference in the rate of apoptosis was found in the RF-field-exposed neurons compared to the sham-, 37°C- and 39°C-exposed neurons either 0 or 24 h after exposure using both methods. To assess whether the observed apoptosis was caspase-dependent or -independent, assays measuring caspase 3 activity and apoptosis-inducing factor (AIF) labeling were performed. No increase in the caspase 3 activity was found, whereas the percentage of AIF-positive nuclei in RF-field-exposed neurons was increased by three- to sevenfold compared to other conditions. Our results show that, under the experimental conditions used, exposure of primary rat neurons to CW RF fields may induce a caspase-independent pathway to apoptosis that involves AIF.

Kodym, E., Kodym, R., Choy, H. and Saha, D. Sustained Metaphase Arrest in Response to Ionizing Radiation in a Non-small Cell Lung Cancer Cell Line. Radiat. Res. 169, 46–58 (2008).

In solid tumors, non-apoptotic forms of tumor cell inactivation such as mitotic catastrophe appear to be predominant in the response to DNA-damaging agents. Despite its importance, the underlying molecular mechanisms of mitotic catastrophe have been only partially elucidated. We found that a large fraction of HCC2279 non-small cell lung cancer cells underwent mitotic catastrophe after irradiation. Cells were arrested in metaphase with chromosomal damage indicated by DNA fragments displaced from the metaphase plate and considerable numbers of residual γ-H2AX foci. Although TP53 was nonfunctional, we detected a prompt radiation response on the level of checkpoint kinases. In contrast, CDC25A was the only checkpoint phosphatase that was responsive to radiation. CDC25B was not detectable, and CDC25C was constitutively phosphorylated at serine 216, leading to its cytoplasmic sequestration and functional inactivation. Therefore, radiation-induced mitotic catastrophe in HCC2279 cells appears to be induced by a combination of relative insufficiencies in the p53-mediated and checkpoint kinase-mediated pathways leading to premature entry into mitosis. Displaced chromosome fragments triggering an intra-M checkpoint in cells entering mitosis presumably result in a sustained metaphase arrest. The phenomenon found in these cells, which were derived directly from a human patient, might be responsible for therapy-induced genetic instability of tumors.

Tago, F., Tsukimoto, M., Nakatsukasa, H. and Kojima. S. Repeated 0.5 Gy Gamma Irradiation Attenuates Autoimmune Disease in MRL-lpr/lpr Mice with Suppression of CD3 CD4⁻CD8⁻B220 T-Cell Proliferation and with Up-regulation of CD4 CD25 Foxp3 Regulatory T Cells. Radiat. Res. 169, 59–66 (2008).

MRL-lpr/lpr mice are used as a model of systemic lupus erythematosus. We previously reported attenuation of autoimmune disease in MRL-lpr/lpr mice by repeated γ irradiation (0.5 Gy each time). In this study, we investigated the mechanisms of this attenuation by measuring the weight of the spleen and the population of highly activated CD3 CD4⁻CD8⁻B220 T cells, which are characteristically involved in autoimmune pathology in these mice. Splenomegaly and an increase in the percentage of CD3 CD4⁻CD8⁻B220 T cells, which occur with aging in nonirradiated mice, were suppressed in irradiated mice. The high proliferation rate of CD3 CD4⁻CD8⁻B220 T cells was suppressed in the irradiated animals. The production of autoantibodies and the level of IL6, which activates B cells, were also lowered by radiation exposure. These results indicate that progression of pathology is suppressed by repeated 0.5-Gy γ irradiation. To uncover the mechanism of the immune suppression, we measured the regulatory T cells, which suppress activated T cells and excessive autoimmune responses. We found that regulatory T cells were significantly increased in irradiated mice. We therefore conclude that repeated 0.5-Gy γ irradiation suppresses the proliferation rate of CD3 CD4⁻CD8⁻B220 T cells and the production of IL6 and autoantibodies and up-regulates regulatory T cells.

Wen, B., Urano, M., Humm, J. L., Seshan, V. E., Li, G. C. and Ling, C. C. Comparison of Helzel and OxyLite Systems in the Measurements of Tumor Partial Oxygen Pressure (pO₂). Radiat. Res. 168, 67–75 (2008).

It has been demonstrated in both experimental and human malignancies that hypoxic tumor cells are linked with aggressive disease phenotype. One of the methods to identify these cells is by direct physical measurement of tumor pO₂. This study compared pO₂ values measured with two systems, the Helzel Hypoximeter (successor of the polarographic Eppendorf electrode) and the Oxford-Optronix OxyLite (fiber-optic probe), in R3327-AT and R3327-AT/tkeGFP tumors. Partial oxygen pressure was measured in individual tumors with either system or in the same tumor with both systems. The similarities and discrepancies in pO₂ measurements between the two systems were also investigated when tumor-bearing animals were breathing pure oxygen. Our data showed a considerable heterogeneity in pO₂ values in each tumor using both the Helzel and OxyLite systems. Similar results were obtained with both systems for the mean and median pO₂ values, and the distributions of pO₂ values within the interval 0 < pO₂ < 40 mmHg (the range important for defining tumor hypoxia) were found to be statistically equivalent. However, the frequencies of high pO₂ values (>40 mmHg) and zero values measured by the two systems were statistically significantly different.

Chao, A., Wang, T. H., Lee, Y. S., Hong, J. H., Tsai, C. N., Chen, C. K., Tsai, C. S., Chao, A. S. and Lai, C. H. Analysis of Functional Groups Differentially Expressed Genes in the Peripheral Blood of Patients with Cervical Cancer Undergoing Concurrent Chemoradiation Treatment. Radiat. Res. 169, 76–86 (2008).

We prospectively investigated the gene expression profiles of cervical cancer patients undergoing concurrent chemoradiation treatment. Up-regulated genes associated with anemia were analyzed. Peripheral blood of 20 patients (bulky stage IB–IVA cervical squamous cell carcinomas) undergoing concurrent chemoradiation treatment at four times was collected. Total RNA extracted by the PAXgene Blood RNA System was analyzed with microarrays and MetaCore™ functional network analyses. Fifty-three genes were significantly differentially expressed during concurrent chemoradiation treatment. Fetal and embryonic hemoglobin genes were up-regulated when patients had been severely myelosuppressed. Twenty-eight genes correlated significantly with the hemoglobin genes are involved in responses to hypoxia and oxygenation, TGF-β signaling, cell cycle suppression, G-protein signaling, and transcriptional regulation. c-Myc has the highest rank in transcriptional co-regulation. In addition, IGKV1D-13 was significantly down-regulated in patients with severe hematological toxicity. These approaches identified biological processes in peripheral blood modulated by concurrent chemoradiation treatment and subsequent anemia.

Pawel, D. J., Preston, D. L., Pierce, D. A. and Cologne, J. B. Improved Estimates of Cancer Site-Specific Risks for A-Bomb Survivors. Radiat. Res. 169, 87–98 (2008).

Simple methods are investigated for improving summary site-specific radiogenic risk estimates. Estimates in this report are derived from cancer incidence data from the Life Span Study (LSS) cohort of A-bomb survivors that are followed up by the Radiation Effects Research Foundation (RERF). Estimates from the LSS of excess relative risk (ERR) for solid cancer sites have typically been derived separately for each site. Even though the data for this are extensive, the statistical imprecision in site-specific (organ-specific) risk estimates is substantial, and it is clear that a large portion of the site-specific variation in estimates is due to this imprecision. Empirical Bayes (EB) estimates offer a reasonable approach for moderating this variation. The simple version of EB estimates that we applied to the LSS data are weighted averages of a pooled overall estimate of ERR and separately derived site-specific estimates, with weights determined by the data. Results indicate that the EB estimates are most useful for sites such as esophageal or bladder cancer, for which the separately derived ERR estimates are less precise than for other sites.

Little, M. P., Tawn, E. J., Tzoulaki, I., Wakeford, R., Hildebrandt, G., Paris, F., Tapio, S. and Elliott, P. A Systematic Review of Epidemiological Associations Between Low and Moderate Doses of Ionizing Radiation and Late Cardiovascular Effects, and Their Possible Mechanisms. Radiat. Res. 169, 99–109 (2008).

The link between high doses of ionizing radiation and damage to the heart and coronary arteries is established. In this paper, we systematically review the epidemiological evidence for associations between low and moderate doses (<5 Gy) of ionizing radiation and late-occurring cardiovascular disease. Risks per unit dose in epidemiological studies vary over at least two orders of magnitude, possibly a result of confounding factors. An examination of possible biological mechanisms indicates that the most likely causative effect of radiation exposure is damage to endothelial cells and subsequent induction of an inflammatory response, although it seems unlikely that this would extend to low-dose and low-dose-rate exposure. However, a role for somatic mutation has been proposed that would indicate a stochastic effect. In the absence of a convincing mechanistic explanation of epidemiological evidence that is less than persuasive at present, a cause-and-effect interpretation of the reported statistical associations cannot be reliably inferred, although neither can it be reliably excluded. Further epidemiological and biological evidence will allow a firmer conclusion to be drawn.

Trzeciak, A. R., Barnes, J. and Evans, M. K. A Modified Alkaline Comet Assay for Measuring DNA Repair Capacity in Human Populations. Radiat. Res. 169, 110–121 (2008).

Use of the alkaline comet assay to assess DNA repair capacity in human populations has been limited by several factors, including lack of methodology for use of unstimulated cryopreserved peripheral blood mononuclear cells (PBMCs), insufficient control of interexperimental variability, and limited analysis of DNA repair kinetics. We show that unstimulated cryopreserved PBMCs can be used in DNA repair studies performed using the comet assay. We have applied data standardization for the analysis of DNA repair capacity using negative and positive internal standards as controls for interexperimental variability. Our standardization procedure also uses negative controls, which provides a way to minimize the interference of interindividual variation in baseline DNA damage levels on DNA repair capacity measurements in populations. DNA repair capacity was assessed in a small human cohort using the parameters described in the literature including initial DNA damage, half-time of DNA repair, and residual DNA damage after 30 and 60 min. We have also introduced new DNA repair capacity parameter, initial rate of DNA repair. There was no difference in DNA repair capacity between fresh and cryopreserved PBMCs when measured by the Olive tail moment and tail DNA. The use of DNA repair capacity parameters in assessment of fast and slow single-strand break repair components is discussed.

Puskin, J. S. What Can Epidemiology Tell Us about Risks at Low Doses? Radiat. Res. 169, 122–124 (2008).

Limitations on statistical power preclude direct detection and quantification of radiogenic cancer risks at very low (environmental) levels of low-LET radiation through epidemiological studies. Given this limitation and our incomplete understanding of cellular processes leading to radiation carcinogenesis, an “effective threshold” in the dose range of interest for radiation protection cannot yet be ruled out. Ongoing epidemiological studies of chronically exposed individuals receiving very low daily doses of radiation can be used, however, together with radiobiological data, to critically test whether such a threshold is plausible.