Port, M., Boltze, C., Wang, Y., Röper, B., Meineke, V. and Abend, M. A Radiation-Induced Gene Signature Distinguishes Post-Chernobyl from Sporadic Papillary Thyroid Cancers. Radiat. Res. 168, 639–649 (2007).

We investigated selected gene targets to differentiate radiation-induced papillary thyroid cancers (PTCs) from other etiologies. Total RNA was isolated from 11 post-Chernobyl PTCs and 41 sporadic PTCs characterized by a more aggressive tumor type and lacking a radiation exposure history. RNA from 10 tumor samples from both groups was pooled and hybridized separately on a whole genome microarray for screening. Then 92 selected gene targets were examined quantitatively on each tumor sample using an RTQ-PCR-based low-density array (LDA). Screening for more than fivefold differences in gene expression between the groups by microarray detected 646 up-regulated and 677 down-regulated genes. Categorization of these genes revealed a significant (P < 0.0006) over-representation of the number of up-regulated genes coding for oxidoreductases, G-proteins and growth factors, while the number of genes coding for immunoglobulin appeared to be significantly down-regulated. With the LDA, seven genes (SFRP1, MMP1, ESM1, KRTAP2-1, COL13A1, BAALC and PAGE1) made a complete differentiation between the groups possible. Gene expression patterns known to be associated with a more aggressive tumor type in older patients appeared to be more pronounced in post-Chernobyl PTC, thus underlining the known aggressiveness of radiation-induced PTC. Seven genes were found that completely distinguished post-Chernobyl (PTC) from sporadic PTC.

Fachin, A. L., Mello, S. S., Sandrin-Garcia, P., Junta, C. M., Donadi, E. A., Passos, G. A. S. and Sakamoto-Hojo, E. T. Gene Expression Profiles in Human Lymphocytes Irradiated In Vitro with Low Doses of Gamma Rays. Radiat. Res. 168, 650–665 (2007).

The molecular mechanisms underlying responses to low radiation doses are still unknown, especially in normal lymphocytes, despite the evidence suggesting specific changes that may characterize cellular responses. Our purpose was to analyze gene expression profiles by DNA microarrays in human lymphocytes after in vitro irradiation (10, 25 and 50 cGy) with γ rays. A cytogenetic analysis was also carried out for different radiation doses. G₀ lymphocytes were irradiated and induced to proliferate for 48 h; then RNA samples were collected for gene expression analysis. ANOVA was applied to data obtained in four experiments with four healthy donors, followed by SAM analysis and hierarchical clustering. For 10, 25 and 50 cGy, the numbers of significantly (FDR ≤ 0.05) modulated genes were 86, 130 and 142, respectively, and 25, 35 and 33 genes were exclusively modulated for each dose, respectively. We found CYP4X1, MAPK10 and ATF6 (10 cGy), DUSP16 and RAD51L1 (25 cGy), and RAD50, REV3L and DCLRE1A (50 cGy). A set of 34 significant genes was common for all doses; while SERPINB2 and C14orf104 were up-regulated, CREB3L2, DDX49, STK25 and XAB2 were down-regulated. Chromosome damage was significantly induced for doses ≥10 cGy (total aberrations) and ≥50 cGy (dicentrics/ rings). Therefore, low to moderate radiation doses induced qualitative and/or quantitative differences and similarities in transcript profiles, reflecting the type and extent of DNA lesions. The main biological processes associated with modulated genes were metabolism, stress response/DNA repair, cell growth/differentiation, and transcription regulation. The results indicate a potential risk to humans regarding the development of genetic instability and acquired diseases.

Tawn, E. J., Whitehouse, C. A., De Ruyck, K., Hodgson, L., Vandenbulcke, K. and Thierens, H. The Characterization and Transmissibility of Chromosome Aberrations Induced in Peripheral Blood Lymphocytes by In Vitro α-Particle Radiation. Radiat. Res. 168, 666–674 (2007).

Peripheral blood lymphocytes were irradiated in vitro with ²¹³Bi α particles at doses of 0, 10, 20, 50, 100, 200 and 500 mGy. Chromosome analysis was performed on 47-h cultures using single-color fluorescence in situ hybridization (FISH) to paint chromosomes 1, 3 and 5. The whole genome was analyzed for unstable aberrations to derive aberration frequencies and determine cell stability. The dose response for dicentrics was 33.60 ± 0.47 × 10⁻² per Gy. A more detailed analysis revealed that the majority of aberrations scored as dicentrics were part of complex/multiple aberrations, with the proportion of cells containing complexes increasing with dose. Cells containing aberrations involving painted chromosomes (FISH aberrations) were further classified according to cell stability and complexity. The majority of cells with FISH aberrations were unstable. The proportion of aberrant FISH cells with complex/multiple aberrations ranged from 56% at 10 mGy to 89% at 500 mGy. A linear dose response for genomic frequencies of translocations in stable cells fitted the data from 0 to 200 mGy with a dose response of 7.90 ± 0.98 × 10⁻² per Gy, thus indicating that they are likely to be observed in peripheral blood lymphocytes from individuals with past or chronic exposure to high-LET radiation. Comparisons with the dose response for low-LET radiation suggest an RBE of 13.6 for dicentrics in all cells and 3.2 for translocations in stable cells. Since stochastic effects of radiation are attributable to genetic changes in viable cells, translocations in stable cells may be a better measure when considering the comparative risks of different qualities of radiation.

Groesser, T., Chun, E. and Rydberg, B. Relative Biological Effectiveness of High-Energy Iron Ions for Micronucleus Formation at Low Doses. Radiat. Res. 168, 675–682 (2007).

Dose–response curves for micronucleus (MN) formation were measured in Chinese hamster V79 and xrs6 (Ku80⁻) cells and in human mammary epithelial MCF10A cells in the dose range of 0.05–1 Gy. The Chinese hamster cells were exposed to 1 GeV/nucleon iron ions, 600 MeV/nucleon iron ions, and 300 MeV/nucleon iron ions (LETs of 151, 176 and 235 keV/μm, respectively) as well as with 320 kVp X rays as reference. Second-order polynomials were fitted to the induction curves, and the initial slopes (the alpha values) were used to calculate RBE. For the repair-proficient V79 cells, the RBE at these low doses increased with LET. The values obtained were 3.1 ± 0.8 (LET = 151 keV/μm), 4.3 ± 0.5 (LET = 176 keV/μm), and 5.7 ± 0.6 (LET = 235 keV/μm), while the RBE was close to 1 for the repair-deficient xrs6 cells regardless of LET. For the MCF10A cells, the RBE was determined for 1 GeV/nucleon iron ions and was found to be 5.5 ± 0.9, slightly higher than for V79 cells. To test the effect of shielding, the 1 GeV/nucleon iron-ion beam was intercepted by various thicknesses of high-density polyethylene plastic absorbers, which resulted in energy loss and fragmentation. It was found that the MN yield for V79 cells placed behind the absorbers decreased in proportion to the decrease in dose both before and after the iron-ion Bragg peak, indicating that RBE did not change significantly due to shielding except in the Bragg peak region. At the Bragg peak itself with an entrance dose of 0.5 Gy, where the LET is very high from stopping low-energy iron ions, the effectiveness for MN formation per unit dose was decreased compared to non-Bragg peak areas.

Vermeulen, C., Bertocci, B., Begg, A. C. and Vens, C. Ionizing Radiation Sensitivity of DNA Polymerase Lambda-Deficient Cells. Radiat. Res. 168, 683–688 (2007).

Ionizing radiation induces a diverse spectrum of DNA lesions, including strand breaks and oxidized bases. In mammalian cells, ionizing radiation-induced lesions are targets of non-homologous end joining, homologous recombination, and base excision repair. In vitro assays show a potential involvement of DNA polymerase lambda in non-homologous end joining and base excision repair. In this study, we investigated whether DNA polymerase lambda played a significant role in determining ionizing radiation sensitivity. Despite increased sensitivity to hydrogen peroxide, lambda-deficient mouse embryonic fibroblasts displayed equal survival after exposure to ionizing radiation compared to their wild-type counterparts. In addition, we found increased sensitivity to the topoisomerase inhibitors camptothecin and etoposide in the absence of polymerase lambda. These results do not reveal a major role for DNA polymerase lambda in determining radiosensitivity in vivo.

Mendonca, M. S., Chin-Sinex, H., Gomez-Millan, J., Datzman, N., Hardacre, M., Comerford, K., Nakshatri, H., Nye, M., Benjamin, L., Mehta, S., Patino, F. and Sweeney, C. Parthenolide Sensitizes Cells to X-Ray-Induced Cell Killing through Inhibition of NF-κB and Split-Dose Repair. Radiat. Res. 168, 689–697 (2007).

Human cancers have multiple alterations in cell signaling pathways that promote resistance to cytotoxic therapy such as X rays. Parthenolide is a sesquiterpene lactone that has been shown to inhibit several pro-survival cell signaling pathways, induce apoptosis, and enhance chemotherapy-induced cell killing. We investigated whether parthenolide would enhance X-ray-induced cell killing in radiation resistant, NF-κB-activated CGL1 cells. Treatment with 5 μM parthenolide for 48 to 72 h inhibited constitutive NF-κB binding and cell growth, reduced plating efficiency, and induced apoptosis through stabilization of p53 (TP53), induction of the pro-apoptosis protein BAX, and phosphorylation of BID. Parthenolide also enhanced radiation-induced cell killing, increasing the X-ray sensitivity of CGL1 cells by a dose modification factor of 1.6. Flow cytometry revealed that parthenolide reduced the percentage of X-ray-resistant S-phase cells due to induction of p21^waf1/cip1 (CDKN1A) and the onset of G₁/S and G₂/M blocks, but depletion of radioresistant S-phase cells does not explain the observed X-ray sensitization. Further studies demonstrated that the enhancement of X-ray-induced cell killing by parthenolide is due to inhibition of split-dose repair.

Pozzi, D., Grimaldi, P., Gaudenzi, S., Di Giambattista, L., Silvestri, I., Morrone, S. and Congiu Castellano, A. UVB-Radiation-Induced Apoptosis in Jurkat Cells: A Coordinated Fourier Transform Infrared Spectroscopy-Flow Cytometry Study. Radiat. Res. 168, 698–705 (2007).

We studied the induction of apoptosis in Jurkat cells by UVB radiation (wavelength 290–320 nm) at a dose of 310 mJ/ cm². We combined Fourier transform infrared (FTIR) spectroscopy with flow cytometry to determine whether the combination of both techniques could provide new and improved information about cell modifications. To do this, we looked for correspondences and correlations between spectroscopy and flow cytometry data and found three highly probable spectroscopic markers of apoptosis. The behavior of the wave number shift of both the Amide I β-sheet component and the area of the 1083 cm⁻¹ band reproduced, with a high correlation, the behavior of the early apoptotic cell population, while the behavior of the Amide I area showed a high correlation with the early plus late apoptotic cell population.

Wang, X. Q., Stanbridge, E. J., Lao, X. Y., Cai, Q., Fan, S. T. and Redpath, J. L. p53-Dependent Chk1 Phosphorylation is Required for Maintenance of Prolonged G₂ Arrest. Radiat. Res. 168, 706–715 (2007).

Targeting checkpoint kinases has been shown to have a potential chemosensitizing effect in cancer treatment. However, inhibitors of such kinases preferentially abrogate the DNA damage-induced G₂ checkpoint in p53^−/− as opposed to p53 ^/ cells. The mechanisms by which p53 (TP53) can prevent abrogation of the G₂ checkpoint are unclear. Using normal human diploid p53 ^/ and p53^−/− fibroblasts as model systems, we have compared the effects of three checkpoint inhibitors, caffeine, staurosporine and UCN-01, on γ-radiation-induced G₂ arrest. The G₂ arrest in p53 ^/ cells was abrogated by caffeine, but not by staurosporine and UCN-01, whereas the G₂ arrest in p53^−/− cells was sensitive to all three inhibitors. Chk2 (CHEK1) phosphorylation was maintained in the presence of all three inhibitors in both p53 ^/ and p53^−/− cells. Chk1 phosphorylation was maintained only in the presence of staurosporine and UCN-01 in p53 ^/ cells. In the presence of caffeine Chk1 phosphorylation was inhibited regardless of p53 status. The pathway of Chk1 phosphorylation → Cdc25A degradation → inhibition of cyclin B1/Cdk1 activity → G₂ arrest is accordingly resistant to staurosporine and UCN-01 in p53 ^/ cells. Moreover, sustained phosphorylation of Chk1 in the presence of staurosporine and UCN-01 is strongly related to phosphorylation of p53. The present study suggests the unique role of Chk1 in preventing abrogation of the G₂ checkpoint in p53 ^/ cells.

Mitchel, R. E. J., Burchart, P. and Wyatt, H. Fractionated, Low-Dose-Rate Ionizing Radiation Exposure and Chronic Ulcerative Dermatitis in Normal and Trp53 Heterozygous C57BL/6 Mice. Radiat. Res. 168, 716–724 (2007).

The influence of low-dose-rate chronic radiation exposure and adaptive responses on non-cancer diseases is largely unknown. We examined the effect of low-dose/low-dose-rate fractionated or single exposures on spontaneous chronic ulcerative dermatitis in Trp53 normal or heterozygous female C57BL/6 mice. From 6 weeks of age, mice were exposed 5 days/week to single daily doses (0.33 mGy, 0.7 mGy/h) totaling 48, 97 or 146 mGy over 30, 60 or 90 weeks, and other Trp53 ^/− mice were exposed to a single dose of 10 mGy (0.5 mGy/min) at 20 weeks of age. The 90-week exposure produced an adaptive response, decreasing both disease frequency and severity in Trp53 ^/ mice and extending the life span of older animals euthanized due to severe disease. The 30- or 60-week exposures had no significant protective or detrimental effect. In contrast, the chronic, fractionated exposure for 30 or 60 weeks significantly increased the frequency and severity of the disease in older Trp53 ^/− mice, significantly decreasing the life span of the animals required to be euthanized for disease. Similarly, the single 10-mGy exposure also increased disease frequency in older animals. However, the chronic, fractionated exposure for 90 weeks prevented these detrimental effects, with disease frequency and severity not different from unexposed controls. We conclude that very low-dose fractionated exposures can induce a protective adaptive response in both Trp53 normal and heterozygous mice, but that a lower threshold level of exposure, similar in both cases, must first be passed. In mice with reduced Trp53 functionality, doses below the threshold can produce detrimental effects.

Lacoste-Collin, L., Jozan, S., Cances-Lauwers, V., Pipy, B., Gasset, G., Caratero, C. and Courtade-Saïdi, M. Effect of Continuous Irradiation with a Very Low Dose of Gamma Rays on Life Span and the Immune System in SJL Mice Prone to B-Cell Lymphoma. Radiat. Res. 168, 725–732 (2007).

Ionizing radiation has been shown to have dose- and dose-rate-dependent carcinogenic effects on the hematopoietic and lymphoreticular systems. We report here that continuous exposure to a low dose of γ rays influences the course of spontaneous B-cell lymphoma in SJL mice. We studied the biological effects of 10 cGy year⁻¹ γ rays on the life span of 560 4-week-old SJL/J female mice and on various parameters of the cell-mediated immune response. Life span was slightly prolonged. The mean survival was 397 days for controls and 417 days for irradiated mice that died with lymphoma (P = 0.34). In lymph nodes and spleen, lower percentages of CD4 and CD8 T cells were observed in irradiated mice before 32 weeks. Interestingly, the percentages of CD49 NK cells were increased in the spleens of irradiated mice at 28 weeks (0.61 ± 0.08% compared to 0.43 ± 0.12% in controls, P = 0.01) and at 32 weeks (0.62 ± 0.24% compared to 0.33 ± 0.09%, P = 0.02), while NK cell activity remained unchanged in exposed mice. These results provide further support for the absence of harmful effects of a continuous very low dose of radiation on life span and incidence of lymphoma in SJL mice.

Saran, A., Pazzaglia, S., Mancuso, M., Rebessi, S., Di Majo, V., Tanori, M., Lovisolo, G. A., Pinto, R. and Marino, C. Effects of Exposure of Newborn Patched1 Heterozygous Mice to GSM, 900 MHz. Radiat. Res. 168, 733–740 (2007).

Patched1 heterozygous knockout mice (Ptc1 ^/−), an animal model of multiorgan tumorigenesis in which ionizing radiation dramatically accelerates tumor development, were used to study the potential tumorigenic effects of electromagnetic fields (EMFs) on neonatal mice. Two hundred Ptc1 ^/− mice and their wild-type siblings were enrolled in this study. Newborn mice were exposed to 900 MHz radiofrequency radiation (average SAR: 0.4 W/kg for 5 days, 0.5 h twice a day) or were sham exposed. We found that RF EMFs simulating the Global System for Mobile Communications (GSM) did not affect the survival of the mice, because no statistically significant differences in survival were found between exposed and sham-exposed animals. Also, no effects attributable to radiofrequency radiation were observed on the incidence and histology of Ptc1-associated cerebellar tumors. Moreover, the skin phenotype was analyzed to look for proliferative effects of RF EMFs on the epidermal basal layer and for acceleration of preneoplastic lesions typical of the basal cell carcinoma phenotype of this model. We found no evidence of proliferative or promotional effects in the skin from neonatal exposure to radiofrequency radiation. Furthermore, no difference in Ptc1-associated rhabdomyosarcomas was detected between sham-exposed and exposed mice. Thus, under the experimental conditions tested, there was no evidence of life shortening or tumorigenic effects of neonatal exposure to GSM RF radiation in a highly tumor-susceptible mouse model.

Shuryak, I., Sachs, R. K. and Brenner, D. J. Biophysical Models of Radiation Bystander Effects: 1. Spatial Effects in Three-Dimensional Tissues. Radiat. Res. 168, 741–749 (2007).

Non-targeted (bystander) effects of ionizing radiation are caused by intercellular signaling; they include production of DNA damage and alterations in cell fate (i.e. apoptosis, differentiation, senescence or proliferation). Biophysical models capable of quantifying these effects may improve cancer risk estimation at radiation doses below the epidemiological detection threshold. Understanding the spatial patterns of bystander responses is important, because it provides estimates of how many bystander cells are affected per irradiated cell. In a first approach to modeling of bystander spatial effects in a three-dimensional artificial tissue, we assume the following: (1) The bystander phenomenon results from signaling molecules (S) that rapidly propagate from irradiated cells and decrease in concentration (exponentially in the case of planar symmetry) as distance increases. (2) These signals can convert cells to a long-lived epigenetically activated state, e.g. a state of oxidative stress; cells in this state are more prone to DNA damage and behavior alterations than normal and therefore exhibit an increased response (R) for many end points (e.g. apoptosis, differentiation, micronucleation). These assumptions are implemented by a mathematical formalism and computational algorithms. The model adequately describes data on bystander responses in the 3D system using a small number of adjustable parameters.

Heidenreich, W. F., Cullings, H. M., Funamoto, S. and Paretzke, H. G. Promoting Action of Radiation in the Atomic Bomb Survivor Carcinogenesis Data? Radiat. Res. 168, 750– 756 (2007).

The age–time patterns of risk in the atomic bomb survivor data on incidence of solid cancers suggest an action of low-LET radiation not only on the initiating event but also on promotion in a biologically motivated model that allows for both actions. The favored model indicates a decrease of radiation risks with age at exposure due to the initiating effect and with time since exposure due to the promoting effect. These result in a relative risk that depends mostly on attained age for ages at exposure above 20 years. According to the model, a dose of 100 mGy is inducing about the same number of initiating events that occur spontaneously in 1 year. Assuming that several mutations are needed to obtain intermediate cells with growth advantage does not improve the quality of fit. The estimated promoting effect could be explained if the number of intermediate cells increases by 80% at 1 Gy, e.g. due to stimulated cell repopulation.

Stayner, L., Vrijheid, M., Cardis, E., Stram, D. O., Deltour, I., Gilbert, S. J. and Howe, G. A Monte Carlo Maximum Likelihood Method for Estimating Uncertainty Arising from Shared Errors in Exposures in Epidemiological Studies of Nuclear Workers. Radiat. Res. 168, 757–763 (2007).

Errors in the estimation of exposures or doses are a major source of uncertainty in epidemiological studies of cancer among nuclear workers. This paper presents a Monte Carlo maximum likelihood method that can be used for estimating a confidence interval that reflects both statistical sampling error and uncertainty in the measurement of exposures. The method is illustrated by application to an analysis of all cancer (excluding leukemia) mortality in a study of nuclear workers at the Oak Ridge National Laboratory (ORNL). Monte Carlo methods were used to generate 10,000 data sets with a simulated corrected dose estimate for each member of the cohort based on the estimated distribution of errors in doses. A Cox proportional hazards model was applied to each of these simulated data sets. A partial likelihood, averaged over all of the simulations, was generated; the central risk estimate and confidence interval were estimated from this partial likelihood. The conventional unsimulated analysis of the ORNL study yielded an excess relative risk (ERR) of 5.38 per Sv (90% confidence interval 0.54–12.58). The Monte Carlo maximum likelihood method yielded a slightly lower ERR (4.82 per Sv) and wider confidence interval (0.41–13.31).