Cecchini, S., Girouard, S., Huels, M. A., Sanche, L. and Hunting, D. J. Single-Strand-Specific Radiosensitization of DNA by Bromodeoxyuridine. Radiat. Res. 162, 604–615 (2004).

The effects of bromodeoxyuridine (BrdUrd) substitution for thymidine on γ-ray-induced strand breakage were determined in single- and double-stranded oligonucleotides and double-stranded oligonucleotides containing a mismatched bubble region. BrdUrd does not sensitize complementary double-stranded DNA to γ-ray-induced strand breakage, but it greatly sensitizes single-stranded DNA. However, when the BrdUrd is present in a single-stranded bubble of a double-stranded oligonucleotide, the non-base-paired nucleotides adjacent to the BrdUrd as well as several unpaired sites on the opposite unsubstituted strand are strongly sensitized. The radiosensitization properties of BrdUrd result primarily from the electrophilic nature of the bromine, making it a good leaving group and leading to the irreversible formation of the uridine-yl radical (dUrd^·) or the uridine-yl anion (dUrd⁻) upon addition of an electron. The radiolytic loss of the bromine atom is greatly suppressed in double-stranded compared to single-stranded DNA. Thus we propose that the radiosensitization effects of bromouracil in vivo will likely be limited to single-strand regions such as found in transcription bubbles, replication forks, DNA bulges and the loop region of telomeres. Our results may have profound implications for the clinical use of bromodeoxyuridine (BrdUrd) as a radiosensitizer as well as for the development of targeted radiosensitizers.

Engalytcheff, A., Debuyst, R., Vanhaelewyn, G. C. A. M., Callens, F. J. and Tilquin, B. Attempts at Correlation of the Radiolytic Species of Irradiated Solid-State Captopril Studied by Multi-frequency EPR and HPLC. Radiat. Res. 162, 616– 622 (2004).

The purpose of this study was to provide insight into the processes that occur after the irradiation of solid-state drugs. Electron paramagnetic resonance (EPR) experiments were performed at two different frequencies, X-band (about 9.5 GHz) and Q-band (about 34 GHz), to identify the radicals present in irradiated captopril. The results confirmed that an irradiated drug can trap several main radicals. Moreover, the radical composition varied as a function of the treatment. In addition, non-volatile final products were studied by liquid chromatography coupled to UV and to mass spectrometry (LC-MS). The variation of the radical composition did not influence the profile of the final products; this appears to indicate that, in the case of captopril, the trapped radicals observed by EPR are not the main precursors of the final products. Finally, high-performance liquid chromatography data appear to indicate that radiosterilization of captopril is feasible.

Mack, P. C., Jones, A. A., Gustafsson, M. H., Gandara, D. R., Gumerlock, P. H. and Goldberg, Z. Enhancement of Radiation Cytotoxicity by UCN-01 in Non-small Cell Lung Carcinoma Cells. Radiat. Res. 162, 623–634 (2004).

Thoracic ionizing radiation is a standard component of combined-modality therapy for locally advanced non-small cell lung cancer. To improve low 5-year survival rates (5– 15%), new strategies for enhancing the effectiveness of ionizing radiation are needed. The kinase inhibitor UCN-01 has multiple cell cycle effects, including abrogation of DNA damage-induced S- and G₂-phase arrest, which may limit DNA repair prior to mitosis. To test the hypothesis that therapy-induced cell cycle effects would have an impact on the efficacy of a combination of UCN-01 plus ionizing radiation, the cell cycle responses of the non-small cell lung cancer cell lines Calu1 (TP53-null) and A549 (wild-type TP53) to 2 Gy ionizing radiation were correlated with clonogenic survival after irradiation plus UCN-01. Irradiated cells were exposed to UCN-01 simultaneously and at 3-h increments after irradiation. In Calu1 cells but not A549 cells, sequence-dependent potentiation of radiation by UCN-01 was observed, with maximal interaction occurring when UCN-01 was administered 6 h after irradiation. This coincided with the postirradiation time with the greatest depletion of cells from G₁. Abrogation of G₂ arrest was observed regardless of TP53 status. The role of TP53 was investigated using siRNA to achieve gene silencing. These studies demonstrated that radiation plus UCN-01 was more effective in cells with diminished TP53 activity, associated with a reduced G₁ checkpoint arrest. These studies indicate that simultaneous elimination of multiple DNA damage-induced checkpoints in G₁, S and G₂ may enhance the effects of radiation and that drug scheduling may have an impact on clinical efficacy.

Inanami, O., Iizuka, D., Iwahara, A., Yamamori, T., Kon, Y., Asanuma, T., Matsuda, A., Kashiwakura, I., Kitazato, K. and Kuwabara, M. A Novel Anticancer Ribonucleoside, 1-(3-C-Ethynyl-β-d-ribo-pentofuranosyl)Cytosine, Enhances Radiation-Induced Cell Death in Tumor Cells. Radiat. Res. 162, 635–645 (2004).

1-(3-C-Ethynyl-β-d-ribo-pentofuranosyl)cytosine (ECyd, TAS106) is a newly developed anti-tumor agent that targets RNA synthesis. We report here that a low dose of ECyd induces radiosensitization of caspase-dependent apoptosis and reproductive cell death in cells of the gastric tumor cell lines MKN45 and MKN28 and murine rectum adenocarcinoma Colon26. Flow cytometry demonstrated that TAS106 induced the abrogation of the X-ray-induced G₂/M checkpoint. Western blot analysis showed that X rays increased the expression of cyclin B1, phospho-Cdc2 and Wee1, whereas co-treatment with X rays and TAS106 decreased the expression of these cell cycle proteins associated with the G₂/M checkpoint. Furthermore, TAS106 was shown to decrease the radiation-induced expression of survivin but not Bcl2 and BclX_L regardless of TP53 status and cell type. Overexpression of wild-type survivin in MKN45 cells inhibited the induction of apoptosis induced by co-treatment with X rays and TAS106. These results suggest that TAS106 enhances X-ray-induced cell death through down-regulation of survivin and abrogation of the cell cycle machinery.

Ko, S. J., Liao, X-Y., Molloi, S., Elmore, E. and Redpath, J. L. Neoplastic Transformation In Vitro after Exposure to Low Doses of Mammographic-Energy X Rays: Quantitative and Mechanistic Aspects. Radiat. Res. 162, 646–654 (2004).

The induction of neoplastic transformation in vitro after exposure of HeLa × skin fibroblast hybrid cells to low doses of mammography-energy (28 kVp) X rays has been studied. The data indicate no evidence of an increase in transformation frequency over the range 0.05 to 22 cGy, and doses in the range 0.05 to 1.1 cGy may result in suppression of transformation frequencies to levels below that seen spontaneously. This finding is not consistent with a linear, no-threshold dose– response curve. The dose range at which possible suppression is evident includes doses typically experienced in mammographic examination of the human breast. Experiments are described that attempt to elucidate any possible role of bystander effects in modulating this low-dose radiation response. Not unexpectedly, inhibition of gap junction intercellular communication (GJIC) with the inhibitor lindane did not result in any significant alteration of transformation frequencies seen at doses of 0.27 or 5.4 cGy in these subconfluent cultures. Furthermore, no evidence of a bystander effect associated with factors secreted into the extracellular medium was seen in medium transfer experiments. Thus, in this system and under the experimental conditions used, bystander effects would not appear to be playing a major role in modulating the shape of the dose–response curve.

Zhao, Y., Shao, G., Piao, C. Q., Berenguer, J. and Hei, T. K. Down-regulation of Betaig-h3 Gene is Involved in the Tumorigenesis in Human Bronchial Epithelial Cells Induced by Heavy-Ion Radiation. Radiat. Res. 162, 655–659 (2004).

High-energy (HZE) heavy ions, when compared to low-LET radiation, are highly effective in inducing gene mutation, chromosomal aberrations and neoplastic transformation. However, the underlying molecular mechanisms are not clearly understood. We have recently shown that the down-regulation of Betaig-h3 expression is causally linked to the tumorigenic phenotype of papillomavirus-immortalized human bronchial epithelial (BEP2D) cells treated with high-LET α-particle radiation. Using the BEP2D cell culture system, a radiation-induced transformation model has been established by a single 60-cGy dose of ⁵⁶Fe heavy-ion radiation. To determine whether the Betaig-h3 gene is involved in ⁵⁶Fe ion-induced tumorigenesis, the expression levels of the Betaig-h3 gene in tumorigenic cell lines and the ability of in vivo tumor suppression through the reintroduction of the Betaig-h3 gene in tumorigenic cells were determined. We found that the expression level of this gene is markedly decreased in three tumorigenic cell lines (⁵⁶FeT1–T3) compared with parental BEP2D cells. Ectopic expression of its cDNA in the ⁵⁶FeT2 tumorigenic cells significantly suppressed their tumorigenicity. Although biologically active TGFB1 is elevated in two of three tumorigenic cell lines, all these cell lines are resistant to the induction of Betaig-h3 expression by incubating the transformed cells with exogenous TGFB1 relative to control cells. Our data strongly suggest that down-regulation of Betaig-h3 expression results from the defect in the TGFB1 signaling pathway and plays a pivotal role in the tumorigenic process induced by ⁵⁶Fe heavy-ion radiation.

Medvedeva, N. G., Ford, J. R. and Braby, L. A. Changes in Micronucleus Frequency Resulting from Preirradiation of Cell Culture Surfaces. Radiat. Res. 162, 660–666 (2004).

We have initiated a series of experiments to quantify the impact of environmental variables on the observed frequency of micronuclei in monolayer cultures. In this paper the influence of preirradiation of cell culture vessels on micronucleus formation in Chinese hamster ovary cells was examined. Dry cell culture vessels were preirradiated with 2 Gy of either α particles or X rays and immediately plated with nonirradiated cells. About 48 h later a group of randomly chosen containers was set aside, and the rest of the containers were exposed to a range of doses of X rays or α-particle radiation. Nonirradiated cells plated on previously irradiated cell culture surfaces manifested nearly as many micronuclei as the irradiated cells. In all experiments, preirradiation of the cell substrate (the culture dish) led to a significantly increased micronucleus frequency relative to unirradiated substrate. These results suggest that methods of cell culture vessel sterilization and the composition of cell attachment surfaces could be a confounding factor, particularly in low-dose experiments.

Hill, M. A., Herdman, M. T., Stevens, D. L., Jones, N. J., Thacker, J. and Goodhead, D. T. Relative Sensitivities of Repair-Deficient Mammalian Cells for Clonogenic Survival after α-Particle Irradiation. Radiat. Res. 162, 667–676 (2004).

The clonogenic survival of cells of the radiation-sensitive hamster cell lines irs1, irs2, irs3 and xrs5, representing different DNA repair pathways, was compared to that of their parent lines after α-particle irradiation. Measurements of nuclear area were made to calculate the probability of surviving a single α-particle traversal, the average number of lethal lesions per track and per unit dose, along with the “intrinsic radiosensitivity” of these cells, allowing for the potential of multiple lethal lesions per traversal. For all cell lines studied, α particles were found to be more biologically effective per unit absorbed dose than X rays at inducing cell inactivation. The repair-deficient cells showed an enhanced sensitivity to α particles compared to their parent line, but the degree of enhancement was less than for X rays. The reduction in additional sensitivity for α-particle irradiation was shown not to be due predominantly to differences in cell geometry limiting the probability of a cell nucleus being traversed. The results suggest that both the nonhomologous end-joining pathway and to a lesser extent the homologous recombination repair pathway play a role in successful repair of α-particle-induced damage, although a large proportion of damage is not repaired by either pathway.

Balajee, A. S., Ponnaiya, B., Baskar, R. and Geard, C. R. Induction of Replication Protein A in Bystander Cells. Radiat. Res. 162, 677–686 (2004).

The bystander effect is a biological phenomenon whereby cells not directly targeted by DNA-damaging agents elicit a response similar to that of targeted cells. Understanding the mechanisms underlying the bystander effect is important not only for radiation risk assessment but also for evaluation of protocols for radiotherapy of tumors. Identification of DNA repair and signal transduction proteins that are induced specifically in bystander cells may help in deducing the molecular mechanism(s) responsible for this complex phenomenon. With this objective, we have studied the expression of replication protein A (RPA), which is involved in various DNA metabolic activities such as replication, repair and recombination. We analyzed RPA expression by immunofluorescence and Western blot techniques in both γ-irradiated primary human fibroblast cells and bystander cells that were recipients of conditioned growth medium harvested from γ-irradiated cell cultures. A two- to threefold induction of RPA was observed in bystander MRC5 cells treated with conditioned medium collected from γ-irradiated WI38 or MRC5 cells. Lack of induction of RPA in sham-manipulated MRC5 cells treated with irradiated medium alone (without cells) indicates that the signal elicited from the irradiated cells is responsible for induction of RPA in bystander cells. RPA was induced more effectively in bystander cells than in irradiated cells at the earliest time analyzed (30 min), and the RPA level declined to that of sham-treated control cells by 24 h after treatment. In addition to RPA, apurinic/apyrimidinic endonuclease (APE, a key enzyme of the base excision repair pathway) also showed enhanced expression in bystander cells. Our findings suggest that the induction of RPA and APE is due to a combination of DNA strand breaks and oxidized base lesions in the genomic DNA of bystander cells.

Borak, T. B., Doke, T., Fuse, T., Guetersloh, S., Heilbronn, L., Hara, K., Moyers, M., Suzuki, S., Taddei, P., Terasawa, K. and Zeitlin, C. J. Comparisons of LET Distributions for Protons with Energies between 50 and 200 MeV Determined Using a Spherical Tissue-Equivalent Proportional Counter (TEPC) and a Position-Sensitive Silicon Spectrometer (RRMD-III). Radiat. Res. 162, 687–692 (2004).

Experiments have been performed to measure the response of a spherical tissue-equivalent proportional counter (TEPC) and a silicon-based LET spectrometer (RRMD-III) to protons with energies ranging from 50–200 MeV. This represents a large portion of the energy distribution for trapped protons encountered by astronauts in low-Earth orbit. The beam energies were obtained using plastic polycarbonate degraders with a monoenergetic beam that was extracted from a proton synchrotron. The LET spectrometer provided excellent agreement with the expected LET distribution emerging from the energy degraders. The TEPC cannot measure the LET distribution directly. However, the frequency mean value of lineal energy, ȳ_f, provided a good approximation to LET. This is in contrast to previous results for high-energy heavy ions where ȳ_f underestimated LET, whereas the dose-averaged lineal energy, ȳ_D, provided a good approximation to LET.

Buffa, F. M. and Verhaegen, F. Backscatter and Dose Perturbations for Low- to Medium-Energy Electron Point Sources at the Interface between Materials with Different Atomic Numbers. Radiat. Res. 162, 693–701 (2004).

Electron backscatter at interfaces between dissimilar media can affect dosimetry and should be taken into consideration in radiotherapy and in radiobiology experiments. Backscatter dose perturbations depend upon factors such as electron energy, medium atomic number (Z), and distance from the interface. This study quantifies the backscatter dose factor (BSDF) for electron point sources of energy between 0.1 to 3 MeV in water at the interface with scattering materials ranging in Z from ¹³Al to ⁷⁹Au. A Monte Carlo code that performs dose calculations for monoenergetic and continuous-spectrum electron sources was developed using EGSnrc transport routines. The BSDF was quantified in a parallel layers geometry (BSDF_1D) and three-dimensional voxel geometry (BSDF_3D). The BSDF_1D near the interface increased up to 52% with decreasing energy from 3 to 0.1 MeV and increasing Z from 13 to 79. The analysis of the BSDF_3D showed a significant dependence of the scattered electron angular distribution on Z and energy, with a decrease in isotropy going from high to low Z. This effect proves the importance of considering the correct geometry when quantifying the BSDF for electron sources, especially when the dimensions of the relevant dose-collecting volume are comparable with the CSDA range of the source.

Santa Cruz, G. A. and Zamenhof, R. G. The Microdosimetry of the ¹⁰B Reaction in Boron Neutron Capture Therapy: A New Generalized Theory. Radiat. Res. 162, 702–710 (2004).

The microdosimetry of ¹⁰B thermal neutron capture reactions should be considered as an essential step to be followed before studying the radiobiological aspects of boron neutron capture therapy. The boron dose itself is insufficient as the only quantity used to describe the biological effectiveness of the ¹⁰B reaction for two important reasons: the specific microdistribution that the ¹⁰B carrier compound exhibits at the cellular level and the primarily stochastic nature of the energy deposition process, which influences the biological response to the particulate radiation. In this work, these two aspects are analyzed in detail and an innovative rigorous analytical framework is developed in the microdosimetry domain. This formalism provides the necessary microdosimetric tools for more precisely describing the ¹⁰B dose distribution deposited in sensitive microscopic structures and offers improved approaches for analyzing the biological dose–effect relationship of ¹⁰B reactions.

Stone, H. B., Moulder, J. E., Coleman, C. N., Ang, K. K., Anscher, M. S., Barcellos-Hoff, M. H., Dynan, W. S., Fike, J. R., Grdina, D. J., Greenberger, J. S., Hauer-Jensen, M., Hill, R. P., Kolesnick, R. N., MacVittie, T. J., Marks, C., McBride, W. H., Metting, N., Pellmar, T., Purucker, M., Robbins, M. E., Schiestl, R. H., Seed, T. M., Tomaszewski, J., Travis, E. L., Wallner, P. E., Wolpert, M. and Zaharevitz, D. Models for Evaluating Agents Intended for the Prophylaxis, Mitigation and Treatment of Radiation Injuries. Report of an NCI Workshop, December 3–4, 2003. Radiat. Res. 162, 711–728 (2004).

To develop approaches to prophylaxis/protection, mitigation and treatment of radiation injuries, appropriate models are needed that integrate the complex events that occur in the radiation-exposed organism. While the spectrum of agents in clinical use or preclinical development is limited, new research findings promise improvements in survival after whole-body irradiation and reductions in the risk of adverse effects of radiotherapy. Approaches include agents that act on the initial radiochemical events, agents that prevent or reduce progression of radiation damage, and agents that facilitate recovery from radiation injuries. While the mechanisms of action for most of the agents with known efficacy are yet to be fully determined, many seem to be operating at the tissue, organ or whole animal level as well as the cellular level. Thus research on prophylaxis/protection, mitigation and treatment of radiation injuries will require studies in whole animal models. Discovery, development and delivery of effective radiation modulators will also require collaboration among researchers in diverse fields such as radiation biology, inflammation, physiology, toxicology, immunology, tissue injury, drug development and radiation oncology. Additional investment in training more scientists in radiation biology and in the research portfolio addressing radiological and nuclear terrorism would benefit the general population in case of a radiological terrorism event or a large-scale accidental event as well as benefit patients treated with radiation.