Radulescu, I., Elmroth, K. and Stenerlöw, B. Chromatin Organization Contributes to Non-randomly Distributed Double-Strand Breaks after Exposure to High-LET Radiation. Radiat. Res. 161, 1–8 (2004).

The influence of higher-order chromatin structure on the non-random distribution of DNA double-strand breaks induced by high-LET radiation was investigated. Five different chromatin structures (intact cells, condensed and decondensed chromatin, nucleoids and naked genomic DNA) from GM5758 cells or K562 cells were irradiated with ¹³⁷Cs γ-ray photons and 125 keV/μm nitrogen ions (16–25 MeV/nucleon). DNA was purified with a modified lysis procedure to avoid release of heat-labile sites, and fragment size distributions and double-strand break yields were analyzed by different pulsed-field gel electrophoresis protocols. Whereas double-strand breaks in photon-irradiated cells were randomly distributed, irradiation of intact K562 cells with high-LET nitrogen ions produced an excess of non-randomly distributed DNA fragments 10 kb–1 Mbp in size. Complete removal of proteins eliminated this non-random component. There was a gradual increase in the yield of double-strand breaks for each chromatin decondensation step, and compared to intact cells, the yields for naked DNA (in buffer without scavengers) increased 83 and 25 times after photon and nitrogen-ion irradiation, respectively. The corresponding relative biological effectiveness decreased from 1.6–1.8 for intact cells to 0.49 for the naked DNA. We conclude that the organization of DNA into chromatin fiber and higher-order structures is responsible for the majority of non-randomly distributed double-strand breaks induced by high-LET radiation. However, our data suggest a complex interaction between track structure and chromatin organization over several levels.

Wang, B., Ohyama, H., Shang, Y., Fujita, K., Tanaka, K., Nakajima, T., Aizawa, S., Yukawa, O. and Hayata, I. Adaptive Response in Embryogenesis: IV. Protective and Detrimental Bystander Effects Induced by X Radiation in Cultured Limb Bud Cells of Fetal Mice. Radiat. Res. 161, 9–16 (2004).

The radioadaptive response and the bystander effect represent important phenomena in radiobiology that have an impact on novel biological response mechanisms and risk estimates. Micromass cultures of limb bud cells provide an in vitro cellular maturation system in which the progression of cell proliferation and differentiation parallels that in vivo. This paper presents for the first time evidence for the correlation and interaction in a micromass culture system between the radioadaptive response and the bystander effect. A radioadaptive response was induced in limb bud cells of embryonic day 11 ICR mice. Conditioning irradiation of the embryonic day 11 cells with 0.3 Gy resulted in a significant protective effect against the occurrence of apoptosis, inhibition of cell proliferation, and differentiation induced by a challenging dose of 5 Gy given the next day. Both protective and detrimental bystander effects were observed; namely, irradiating 50% of the embryonic day 11 cells with 0.3 Gy led to a successful induction of the protective effect, and irradiating 70% of the embryonic day 12 cells with 5 Gy produced a detrimental effect comparable to that seen when all the cells were irradiated. Further, the bystander effect was markedly decreased by pretreatment of the cells with an inhibitor to block the gap junction-mediated intercellular communication. These results indicate that the bystander effect plays an important role in both the induction of a protective effect by the conditioning dose and the detrimental effect of the challenge irradiation. Gap junction-mediated intercellular communication was suggested to be involved in the induction of the bystander effect.

Limoli, C. L., Giedzinski, E., Rola, R., Otsuka, S., Palmer, T. D. and Fike, J. R. Radiation Response of Neural Precursor Cells: Linking Cellular Sensitivity to Cell Cycle Checkpoints, Apoptosis and Oxidative Stress. Radiat. Res. 161, 17–27 (2004).

Therapeutic irradiation of the brain can cause a progressive cognitive dysfunction that may involve defects in neurogenesis. In an effort to understand the mechanisms underlying radiation-induced stem cell dysfunction, neural precursor cells isolated from the adult rat hippocampus were analyzed for acute (0–24 h) and chronic (3–33 days) changes in apoptosis and reactive oxygen species (ROS) after exposure to X rays. Irradiated neural precursor cells exhibited an acute dose-dependent apoptosis accompanied by an increase in ROS that persisted over a 3–4-week period. The radiation effects included the activation of cell cycle checkpoints that were associated with increased Trp53 phosphorylation and Trp53 and p21 (Cdkn1a) protein levels. In vivo, neural precursor cells within the hippocampal dentate subgranular zone exhibited significant sensitivity to radiation. Proliferating precursor cells and their progeny (i.e. immature neurons) exhibited dose-dependent reductions in cell number. These reductions were less severe in Trp53-null mice, possibly due to the disruption of apoptosis. These data suggest that the apoptotic and ROS responses may be tied to Trp53-dependent regulation of cell cycle control and stress-activated pathways. The temporal coincidence between in vitro and in vivo measurements of apoptosis suggests that oxidative stress may provide a mechanistic explanation for radiation-induced inhibition of neurogenesis in the development of cognitive impairment.

Kruse, J. J. C. M., te Poele, J. A. M., Velds, A., Kerkhoven, R. M., Boersma, L. J., Russell, N. S. and Stewart, F. A. Identification of Differentially Expressed Genes in Mouse Kidney after Irradiation using Microarray Analysis. Radiat. Res. 161, 28–38 (2004).

Irradiation of the kidney induces dose-dependent, progressive renal functional impairment, which is partly mediated by vascular damage. The molecular mechanisms underlying the development of radiation-induced nephropathy are unclear. Given the complexity of radiation-induced responses, microarrays may offer new opportunities to identify a wider range of genes involved in the development of radiation injury. The aim of the present study was to determine whether microarrays are a useful tool for identifying time-related changes in gene expression and potential mechanisms of radiation-induced nephropathy. Microarray experiments were performed using amplified RNA from irradiated mouse kidneys (1 × 16 Gy) and from sham-irradiated control tissue at different intervals (1–30 weeks) after irradiation. After normalization procedures (using information from straight-color, color-reverse and self-self experiments), the differentially expressed genes were identified. Control and repeat experiments were done to confirm that the observations were not artifacts of the array procedure (RNA amplification, probe synthesis, hybridizations and data analysis). To provide independent confirmation of microarray data, semi-quantitative PCR was performed on a selection of genes. At 1 week after irradiation (before the onset of vascular and functional damage), 16 genes were significantly up-regulated and 9 genes were down-regulated. During the period of developing nephropathy (10 to 20 weeks), 31 and 42 genes were up-regulated and 9 and 4 genes were down-regulated. At the later time of 30 weeks, the vast majority of differentially expressed genes (191 out of 203) were down-regulated. Potential genes of interest included TSA-1 (also known as Ly6e) and Jagged 1 (Jag1). Increased expression of TSA-1, a member of the Ly-6 family, has previously been reported in response to proteinuria. Jagged 1, a ligand for the Notch receptor, is known to play a role in angiogenesis, and is particularly interesting in the context of radiation-induced vascular injury. The present study demonstrates the potential of microarrays to identify changing patterns of gene expression in irradiated kidney. Further studies will be required to evaluate functional involvement of these genes in vascular-mediated normal tissue injury.

Lu-Hesselmann, J., Abend, M. and van Beuningen, D. Comparison of Endogenous TP53 Genomic Status with Clonogenicity and Different Modes of Cell Death after X Irradiation. Radiat. Res. 161, 39–47 (2004).

Although extensive data indicate that the tumor suppressor TP53 modifies the radiation responses of human and rodent cells, the exact relationship between TP53 and radiation responsiveness remains controversial. To elucidate the relevance of endogenous TP53 genomic status to radiosensitivity in a cell-type-independent manner, different cells of 10 human tumor cell lines with different tissues of origin were examined for TP53 status. The TP53 status was compared with radiation-related cell survival parameters (D_q, D₀, SF2) and with the mode of cell death. Different modes of cell death were examined by measuring radiation-induced micronucleation, apoptosis and abnormal cells. Alterations of the TP53 gene were detected in eight cell lines. No splicing mutation was found. Five cell lines showed codon 68 polymorphism. Codon 72 alterations were found in four cell lines. “Hot spot” alterations were detected in only two of 10 cell lines. Although the cells differed widely in survival parameters (D_q, D₀, SF2) and modes of cell death (micronucleation/apoptosis/abnormal cells) after irradiation, significant cell-type-independent correlations were obtained between the multiple cell death parameter micronucleation/apoptosis/abnormal cells and SF2 (P < 0.001) and D_q (P = 0.003). Moreover, cells with a wild-type TP53 gene were more resistant to X rays than cells with a mutated TP53 gene or cells that were TP53-deficient. The alterations within exons 5–10 of the TP53 correlated with a enhanced radiosensitivity. For the first time, we demonstrated a correlation between endogenous genetic alterations within exons 5–10 of TP53 and radiation-related cell survival and cell death. This indicates a new molecular relevance of TP53 status to intrinsic cellular radiosensitivity.

Ortmann, E. K., Mayerhofer, T., Getoff, N. and Kodym, R. Effect of Antioxidant Vitamins on Radiation-Induced Apoptosis in Cells of a Human Lymphoblastic Cell Line. Radiat. Res. 161, 48–55 (2004).

Modulating the amount of radiation-induced apoptosis by administering antioxidant vitamins offers a possible way to influence radiation-induced side effects in normal tissues. Therefore, we investigated the effect of beta-carotene, vitamin C and alpha-tocopherol on radiation-induced apoptosis in cells in culture. Human T-lymphoblastic MOLT-3 cells were irradiated with a dose of 3 Gy 1 h after or immediately prior to the addition of vitamins in three concentrations (0.01 μM, 1 μM and 100 μM). Eight hours later, apoptosis was scored morphologically by staining the nuclear DNA with Hoechst 33342. When given prior to irradiation, beta-carotene and vitamin E reduced the amount of radiation-induced apoptosis significantly at concentrations of 0.01 μM and 1 μM. In contrast, vitamin C did not show any protective effect when given at these two concentrations and caused a slight but significant radiosensitization at 100 μM. At 0.01 μM, all combinations of two vitamins showed a protective effect. This was also observed for the combination of all three vitamins at concentrations of 0.01 and 1 μM. When given immediately after irradiation, each of the three vitamins showed a protective effect at 0.01 μM. In addition, the combination of alpha-tocopherol and vitamin C reduced radiation-induced apoptosis slightly when given at 1 μM. In all other cases, no statistically significant modulation of radiation-induced apoptosis was observed. In our experimental system, the protective effect of beta-carotene and vitamin E was dependent on concentration and occurred only in the micromolar and sub-micromolar concentration range, while vitamin C alone, but not in combinations, had a sensitizing effect, thus arguing for a careful consideration of vitamin concentrations in clinical settings.

Petin, V. G. and Kim, J. K. Survival and Recovery of Yeast Cells after Combined Treatment with Ionizing Radiation and Heat. Radiat. Res. 161, 56–63 (2004).

Cell survival, recovery kinetics and inactivation forms after successive and simultaneous treatments with γ rays (⁶⁰Co) and high temperatures were studied in diploid yeast cells capable of recovery. Both the extent and the rate of the recovery were shown to be greatly decreased with increase in the duration of heat treatment (60°C) followed by radiation and with increase in exposure temperature after simultaneous treatment with heat and radiation. A quantitative approach describing the recovery process was used to estimate the probability of recovery per unit time and the irreversible component of damage after the combined treatment with heat and radiation. It was shown that the probability of recovery was independent of the conditions of the treatment with heat and radiation, while the irreversible component gradually increased as a function of the duration of heat treatment (60°C) after sequential treatment with heat and radiation and as a function of the exposure temperature after simultaneous treatment with heat and radiation. The increase in the irreversible component was accompanied by an increase in cell death without postirradiation division. It is concluded on this basis that the synergistic interaction of ionizing radiation and hyperthermia in yeast cells is not related to the impairment of the recovery capacity itself and that it may be attributed to an increased yield of irreversible damage.

Guetersloh, S. B., Borak, T. B., Taddei, P. J., Zeitlin, C., Heilbronn, L., Miller, J., Murakami, T. and Iwata, Y. The Response of a Spherical Tissue-Equivalent Proportional Counter to Different Ions Having Similar LET. Radiat. Res. 161, 64–71 (2004).

The response of a tissue-equivalent proportional counter (TEPC) to different ions having a similar linear energy transfer (LET) has been studied. Three ions, ¹⁴N, ²⁰Ne and ²⁸Si, were investigated using the HIMAC accelerator at the National Institute of Radiological Sciences at Chiba, Japan. The calculated linear energy transfer (LET_∞) of all ions was 44 ± 2 keV/μm at the sensitive volume of the TEPC. A particle spectrometer was used to record the charge and position of each incident beam particle. This enabled reconstruction of the location of the track as it passed though the TEPC and ensured that the particle survived without fragmentation. The spectrum of energy deposition events in the TEPC could be evaluated as a function of trajectory through the TEPC. The data indicated that there are many events from particles that did not pass through the sensitive volume. The fraction of these events increased as the energy of the particle increased due to changes in the maximum energy of the δ rays. Even though the LET of the incident particles was nearly identical, the frequency-averaged lineal energy, ȳ_F, as well as the dose-averaged lineal energy, ȳ_D, varied with the velocity of the incident particle. However, both values were within 15% of LET in all cases.

Heidenreich, W. F., Luebeck, E. G. and Moolgavkar, S. H. Effects of Exposure Uncertainties in the TSCE Model and Application to the Colorado Miners Data. Radiat. Res. 161, 72–81 (2004).

The simulations in this paper show that exposure measurement error affects the parameter estimates of the biologically motivated two-stage clonal expansion (TSCE) model. For both Berkson and classical error models, we show that likelihood-based techniques of correction work reliably. For classical errors, the distribution of true exposures needs to be known or estimated in addition to the distribution of recorded exposures conditional on true exposures. Usually the exposure uncertainty biases the model parameters toward the null and underestimates the precision. But when several parameters are allowed to be dependent on exposure, e.g. initiation and promotion, then their relative importance is also influenced, and more complicated effects of exposure uncertainty can occur. The application part of this paper shows for two different types of Berkson errors that a recent analysis of the data for the Colorado plateau miners with the TSCE model is not changed substantially when correcting for such errors. Specifically, the conjectured promoting action of radon remains as the dominant radiation effect for explaining these data. The estimated promoting action of radon increases by a factor of up to 1.2 for the largest assumed exposure uncertainties.

Slijepcevic, P. Is There a Link between Telomere Maintenance and Radiosensitivity? Radiat. Res. 160, 82–86 (2004).

Several recent studies point to the possibility that telomere maintenance may constitute a potential genetic marker of radiosensitivity. For example, the human diseases ataxia telangiectasia and Nijmegen breakage syndrome, which are characterized by clinical radiosensitivity, show alterations in telomere maintenance. In addition, Fanconi's anemia patients, who are characterized by mild cellular radiosensitivity and in some cases marked clinical radiosensitivity, have altered telomere maintenance. Similarly, a correlation between telomere maintenance and cellular radiosensitivity was reported in a group of breast cancer patients. Another study demonstrated that radiosensitivity may be more pronounced in human fibroblasts with short telomeres than in their counterparts with long telomeres. Several mouse models including mice deficient in Ku, DNA-PKcs (Prkdc), Parp and Atm, all of which are radiosensitive in vivo, show clear telomere alterations. The link between telomere maintenance and radiosensitivity is also apparent in mice genetically engineered to have dysfunctional telomeres. Finally, studies using non-mammalian model systems such as C. elegans and yeast point to the link between radiosensitivity and telomere maintenance. These results warrant further investigation to identify the extent to which these two phenotypes, namely radiosensitivity and telomere maintenance, are linked.