Baluna, R. G., Eng, T. Y. and Thomas, C. R., Jr. Adhesion Molecules in Radiotherapy. Radiat. Res. 166, 819–831 (2006).

Recent studies have documented changes in adhesion molecule expression and function after exposure to ionizing radiation. Adhesion molecules mediate cell–cell and cell–matrix interactions and are essential for a variety of physiological and pathological processes including maintenance of normal tissue integrity as well as tumor development and progression. Consequently, modulation of adhesion molecules by radiation may have a role in radiation-induced tumor control and normal tissue damage by interfering with cell signaling, radioresistance, metastasis, angiogenesis, carcinogenesis, immune response, inflammation and fibrosis. In addition, the interactions of radiation with adhesion molecules could have a major impact in developing new strategies to increase the efficacy of radiation therapy. Remarkable progress has been made in recent years to design targeted drug delivery to radiation-up-regulated adhesion molecules. Furthermore, the inhibition of adhesion, migration, invasion and angiogenesis by blocking adhesion receptors may represent a new therapeutic approach to improve tumor control and decrease radiation toxicity. This review is focused on current data concerning the mechanistic interactions of radiation with adhesion molecules and the possible clinical-pathological implications in radiotherapy.

Elmore, E., Lao, X-Y., Kapadia, R. and Redpath, J. L. The Effect of Dose Rate on Radiation-Induced Neoplastic Transformation In Vitro by Low Doses of Low-LET Radiation. Radiat. Res. 166, 832–838 (2006).

The dependence of the incidence of radiation-induced cancer on the dose rate of the radiation exposure is a question of considerable importance to the estimation of risk of cancer induction by low-dose-rate radiation. Currently a dose and dose-rate effectiveness factor (DDREF) is used to convert high-dose-rate risk estimates to low dose rates. In this study, the end point of neoplastic transformation in vitro has been used to explore this question. It has been shown previously that for low doses of low-LET radiation delivered at high dose rates, there is a suppression of neoplastic transformation frequency at doses less than around 100 mGy. In the present study, dose–response curves up to a total dose of 1000 mGy have been generated for photons from ¹²⁵I decay (approximately 30 keV) delivered at doses rates of 0.19, 0.47, 0.91 and 1.9 mGy/min. The results indicate that at dose rates of 1.9 and 0.91 mGy/min the slope of the induction curve is about 1.5 times less than that measured at high dose rate in previous studies with a similar quality of radiation (28 kVp mammographic energy X rays). In the dose region of 0 to 100 mGy, the data were equally well fitted by a threshold or linear no-threshold model. At dose rates of 0.19 and 0.47 mGy/min there was no induction of transformation even at doses up to 1000 mGy, and there was evidence for a possible suppressive effect. These results show that for this in vitro end point the DDREF is very dependent on dose rate and at very low doses and dose rates approaches infinity. The relative risks for the in vitro data compare well with those from epidemiological studies of breast cancer induction by low- and high-dose-rate radiation.

Jobling, M. F., Mott, J. D., Finnegan, M. T., Jurukovski, V., Erickson, A. C., Walian, P. J., Taylor, S. E., Ledbetter, S., Lawrence, C. M., Rifkin, D. B. and Barcellos-Hoff, M. H. Isoform-Specific Activation of Latent Transforming Growth Factor β (LTGF-β) by Reactive Oxygen Species. Radiat. Res. 166, 839–848 (2006).

The three mammalian transforming growth factor β (TGF-β) isoforms are each secreted in a latent complex in which TGF-β homodimers are non-covalently associated with homodimers of their respective pro-peptide called the latency-associated peptide (LAP). Release of TGF-β from its LAP, called activation, is required for binding of TGF-β to cellular receptors, making extracellular activation a critical regulatory point for TGF-β bioavailability. Our previous work demonstrated that latent TGF-β1 (LTGF-β1) is efficiently activated by ionizing radiation in vivo and by reactive oxygen species (ROS) generated by Fenton chemistry in vitro. In the current study, we determined the specific ROS and protein target that render LTGF-β1 redox sensitive. First, we compared LTGF-β1, LTGF-β2 and LTGF-β3 to determine the generality of this mechanism of activation and found that redox-mediated activation is restricted to the LTGF-β1 isoform. Next, we used scavengers to determine that ROS activation was a function of OH^· availability, confirming oxidation as the primary mechanism. To identify which partner of the LTGF-β1 complex was functionally modified, each was exposed to ROS and tested for the ability to form a latent complex. Exposure of TGF-β1 did not alter its ability to associate with LAP, but exposing LAP-β1 to ROS prohibited this phenomenon, while treatment of ROS-exposed LAP-β1 with a mild reducing agent restored its ability to neutralize TGF-β1 activity. Taken together, these results suggest that ROS-induced oxidation in LAP-β1 triggers a conformational change that releases TGF-β1. Using site-specific mutation, we identified a methionine residue at amino acid position 253 unique to LAP-β1 as critical to ROS-mediated activation. We propose that LTGF-β1 contains a redox switch centered at methionine 253, which allows LTGF-β1 to act uniquely as an extracellular sensor of oxidative stress in tissues.

de Toledo, S. M., Asaad, N., Venkatachalam, P., Li, L., Howell, R. W., Spitz, D. R. and Azzam, E. I. Adaptive Responses to Low-Dose/Low-Dose-Rate γ Rays in Normal Human Fibroblasts: The Role of Growth Architecture and Oxidative Metabolism. Radiat. Res. 166, 849–857 (2006).

To investigate low-dose/low-dose-rate effects of low-linear energy transfer (LET) ionizing radiation, we used γ-irradiated cells adapted to grow in a three-dimensional architecture that mimics cell growth in vivo. We determined the cellular, molecular and biochemical changes in these cells. Quiescent normal human fibroblasts were irradiated with single acute or chronic doses (1–10 cGy) of ¹³⁷Cs γ rays. Whereas exposure to an acute dose of 10 cGy increased micronucleus formation, protraction of the dose over 48 h reduced micronucleus frequency to a level similar to or lower than what occurs spontaneously. The protracted treatment also up-regulated the cellular content of the antioxidant glutathione. These changes correlated with modulation of phospho-TP53 (serine 15), a stress marker that was regulated by doses as low as 1 cGy. The DNA damage that occurred after exposure to an acute dose of 10 cGy was protected against in two ways: (1) up-regulation of cellular antioxidant enzyme activity by ectopic overexpression of MnSOD, catalase or glutathione peroxidase, and (2) inhibition of superoxide anion generation by flavin-containing oxidases. These results support a significant role for oxidative metabolism in mediating low-dose radiation effects and demonstrate that cell culture in three dimensions is ideal to investigate radiation-induced adaptive responses. Expression of connexin 43, a constitutive protein of gap junctions, and the G₁ checkpoint were more sensitive to regulation by γ rays in cells maintained in a three-dimensional than in a two-dimensional configuration.

Hofman-Hüther, H., Peuckert, H., Ritter, S. and Virsik-Köpp, P. Chromosomal Instability and Delayed Apoptosis in Long-Term T-Lymphocyte Cultures Irradiated with Carbon Ions and X Rays. Radiat. Res. 166, 858–869 (2006).

In this study, we examined genomic instability induced by 250 kV X rays and 100 MeV/nucleon carbon ions in long-term lymphocyte cultures from two healthy donors. Two biological end points, delayed apoptosis and chromosomal instability, were studied in descendants of cells irradiated with three different doses of the particular radiation up to 22 population doublings. The delayed apoptosis showed no clear dependence on radiation dose, culture time or radiation quality. A persistent significant increase in the rate of apoptosis up to 36 days after X irradiation was observed for a dose of 4 Gy in donor 1 only. For both donors and radiations, de novo aberration yields were significantly increased in comparison to control values up to day 36. For both radiations, chromosome-type aberrations were seen more frequently than chromatid-type aberrations in both donors up to 22 days postirradiation. In both donors, carbon ions were more effective than X rays with respect to the induction of chromosome instability. A dose of 0.25 Gy of carbon ions corresponding to 1.4 ion traversals per cell nucleus was effective in the induction of instability in our cell system.

Iwamoto, K. S., Yano, S., Barber, C. L., MacPhee, D. G., and Tokuoka, S. A Dose-Dependent Decrease in the Fraction of Cases Harboring M6P/IGF2R Mutations in Hepatocellular Carcinomas from the Atomic Bomb Survivors. Radiat. Res. 166, 870–876 (2006).

The risk for hepatocellular carcinoma (HCC) development is significantly heightened in the atomic bomb survivors, but the mechanism is unclear. We have previously reported finding a radiation dose-dependent increase in HCCs with TP53 mutations from the survivors. We now show that, in the same HCC samples, the frequency of 3′-untranslated region (3′UTR) mutations in M6P/IGF2R, a candidate HCC tumor suppressor gene, decreases with dose (P = 0.0091), implying a radiation dose-dependent negative selection of cells harboring such mutations. The fact that they were in the 3′UTR implicates changes in transcript stability rather than in protein function as the mechanism. Moreover, these M6P/IGF2R 3′UTR mutations and the TP53 mutations detected previously were mutually exclusive in most of the tumors, suggesting two independent pathways to HCC development, with the TP53 pathway being more favored with increasing radiation dose than the M6P/IGF2R pathway. These results suggest that tumors attributable to radiation may be genotypically different from tumors of other etiologies and hence may provide a way of distinguishing radiation-induced cancers from “background” cancers—a shift from the current paradigm.

Narai, S., Kodama, Y., Maeda, Y., Yokoyama, M., Takagi, R. and Kominami, R. Trp53 Affects the Developmental Anomaly of Clefts of the Palate in Irradiated Mouse Embryos but not Clefts of the Lip with or without the Palate. Radiat. Res. 166, 877–882 (2006).

Trp53-deficient mice exhibit increased incidences of developmental anomalies when irradiated, probably due to lack of Trp53-dependent apoptosis. A/J strain-derived CL/Fr mice develop clefts of the lip with or without the palate (CL/P) in approximately one-fifth of the embryos. We produced Trp53-deficient CL/Fr mice and examined the susceptibility to spontaneous development of CL/P and clefts of palate only (CPO), which differ in their developmental mechanisms, CL/P resulting from clefts of the primary palate and CPO from clefts of the secondary palate. The effect of radiation on the two phenotypes was also studied. Unexpectedly, no increase in the frequency of CL/P was observed under either condition, indicating that Trp53 deficiency does not contribute to genesis of CL/P. On the other hand, radiation enhanced the incidence of CPO in Trp53 ^/ embryos but not in Trp53 ^/− and Trp53^−/−/ embryos, suggesting that the absence or presence of only one allele of Trp53 is insufficient to hinder differentiation and proliferation of cells involved in the secondary palate formation. These results indicate that Trp53 function adversely affects the development of CPO when certain damaging agents such as radiation are given.

Villasana, L., Acevedo, S., Poage, C. and Raber, J. Sex- and APOE Isoform-Dependent Effects of Radiation on Cognitive Function. Radiat. Res. 166, 883–891 (2006).

Clinical irradiation of the brain induces hippocampus-dependent cognitive impairments in some but not all individuals, suggesting the involvement of genetic risk factors. Deficiency of apolipoprotein E (APOE), which is important for the metabolism and redistribution of lipoproteins and cholesterol, increases behavioral impairments after irradiation, supporting a protective role for APOE against radiation-induced cognitive injury. Compared to APOE3, APOE4 increases while APOE2 decreases the risk of developing age-related cognitive decline and Alzheimer's disease, particularly in women. To determine the potential effects of APOE isoform and sex on radiation-induced cognitive impairments, we irradiated 2-month-old male and female APOE2, APOE3 and APOE4 mice and assessed their cognitive performance 3 months later. When hippocampus-dependent spatial learning and memory were assessed in the water maze, sham-irradiated female APOE2, APOE3 and APOE4 and irradiated female APOE2 mice showed spatial memory retention, but irradiated female APOE3 and APOE4 mice did not. Compared to sham-irradiated female APOE4 mice, irradiated female APOE4 mice also required more trials to reach criterion in the hippocampus-dependent passive avoidance test. Radiation had no effects on water maze or passive avoidance learning and memory of male APOE2, APOE3 or APOE4 mice, indicating that the effects of radiation on cognitive performance are dependent on sex- and APOE isoform.

Shi, L., Adams, M. M., Long, A., Carter, C. C., Bennett, C., Sonntag, W. E., Nicolle, M. M., Robbins, M., D'Agostino, R., Jr. and Brunso-Bechtold, J. K. Spatial Learning and Memory Deficits after Whole-Brain Irradiation are Associated with Changes in NMDA Receptor Subunits in the Hippocampus. Radiat. Res. 166, 892–899 (2006).

Whole-brain irradiation is used for the treatment of brain tumors, but can it also induce neural changes, with progressive dementia occurring in 20–50% of long-term survivors. The present study investigated whether 45 Gy of whole-brain irradiation delivered to 12-month-old Fischer 344 × Brown Norway rats as nine fractions over 4.5 weeks leads to impaired Morris water maze (MWM) performance 12 months later. Compared to sham-irradiated rats, the irradiated rats demonstrated impaired MWM performance. The relative levels of the NR1 and NR2A but not the NR2B subunits of the NMDA receptor were significantly higher in hippocampal CA1 of irradiated rats compared to control rats. No significant differences were detected for these NMDA subunits in CA3 or dentate gyrus. Further analysis of CA1 revealed that the relative levels of the GluR1 and GluR2 subunits of the AMPA receptor and synaptophysin were not altered by whole-brain irradiation. In summary, a clinically relevant regimen of fractionated whole-brain irradiation led to significant impairments in spatial learning and reference memory and alterations in the relative levels of subunits of the NMDA, but not the AMPA, receptors in hippocampal CA1. These findings suggest for the first time that radiation-induced cognitive impairments may be associated with alterations in glutamate receptor composition.

Liu, W-C., Wang, S-C., Tsai, M-L., Chen, M-C., Wang, Y-C., Hong, J-H., McBride, W. H. and Chiang, C-S. Protection against Radiation-Induced Bone Marrow and Intestinal Injuries by Cordyceps sinensis, a Chinese Herbal Medicine. Radiat. Res. 166, 900–907 (2006).

Bone marrow and intestinal damage limits the efficacy of radiotherapy for cancer and can result in death if the whole body is exposed to too high a dose, as might be the case in a nuclear accident or terrorist incident. Identification of an effective nontoxic biological radioprotector is therefore a matter of some urgency. In this study, we show that an orally administered hot-water extract from a Chinese herbal medicine, Cordyceps sinensis (CS), protects mice from bone marrow and intestinal injuries after total-body irradiation (TBI). CS increased the median time to death from 13 to 20 days after 8 Gy TBI and from 9 to 18 days after 10 Gy TBI. Although CS-treated mice receiving 10 Gy TBI survived intestinal injury, most died from bone marrow failure, as shown by severe marrow hypoplasia in mice dying between 18 and 24 days. At lower TBI doses of 5.5 and 6.5 Gy, CS protected against bone marrow death, an effect that was confirmed by the finding that white blood cell counts recovered more rapidly. In vitro, CS reduced the levels of free radical species (ROS) within cells, and this is one likely mechanism for the radioprotective effects of CS, although probably not the only one.

Ponomarev, A. L., Belli, M., Hahnfeldt, P. J., Hlatky, L., Sachs, R. K. and Cucinotta F. A. A Robust Procedure for Removing Background Damage in Assays of Radiation-Induced DNA Fragment Distributions. Radiat. Res. 166, 908–916 (2006).

The non-random distribution of DNA breakage in PFGE (pulsed-field gel electrophoresis) experiments poses a problem of proper subtraction of the background DNA damage to obtain a fragment-size distribution due to radiation only. A naive bin-to-bin subtraction of the background signal will not result in the right DNA mass distribution histogram. This problem could become more pronounced for high-LET (linear energy transfer) radiation, because the fragment-size distribution manifests a higher frequency of smaller fragments. Previous systematic subtraction methods have been based on random breakage, appropriate for low-LET radiation. Moreover, an investigation is needed to determine whether the background breakage is itself random or non-random. We consider two limiting cases: (1) the background damage is present in all cells, and (2) it is present in only a small subset of cells, while other cells are not contributing to the background DNA fragmentation. We give a generalized formalism based on stochastic processes for the subtraction of the background damage in PFGE experiments for any LET and apply it to two sets of PFGE data for iron ions.

Fakir, H., Sachs, R. K., Stenerlöw, B. and Hofmann, W. Clusters of DNA Double-Strand Breaks Induced by Different Doses of Nitrogen Ions for Various LETs: Experimental Measurements and Theoretical Analyses. Radiat. Res. 166, 917– 927 (2006).

The yields and clustering of DNA double-strand breaks (DSBs) were investigated in normal human skin fibroblasts exposed to γ rays or to a wide range of doses of nitrogen ions with various linear energy transfers (LETs). Data obtained by pulsed-field gel electrophoresis on the dose and LET dependence of DNA fragmentation were analyzed with the randomly located clusters (RLC) formalism. The formalism considers stochastic clustering of DSBs along a chromosome due to chromatin structure, particle track structure, and multitrack action. The relative biological effectiveness (RBE) for the total DSB yield did not depend strongly on LET, but particles with higher LET produced higher fractions of small DNA fragments, corresponding in the formalism to an increase in the average number of DSBs per DSB cluster. The results are consistent with the idea that DSB clustering along chromosomes is what leads to large RBEs of high-LET radiations for major biological end points. At a given dose, large fragments are less affected by the variability in LET than small fragments, suggesting that the two free ends in large fragments are often produced by two different tracks. The formalism successfully described an extra increase in small DNA fragments as dose increases and a related decrease in large fragments, mainly due to interlacing of DSB clusters produced along a chromosome by different tracks, since interlacing cuts larger DNA fragments into smaller ones.

Mokrini, R., Trouillas, P., Kaouadji, M., Champavier, Y., Houée-Lévin, C., Calliste, C. A., Fagnère, C. and Duroux, J. L. Reactivity of Chalcones with 1-Hydroxymethyl Radicals. Radiat. Res. 166, 928–941 (2006).

Reactivity of chalcones with reactive species issued from methanol radiolysis was investigated in the absence or presence of dioxygen. Chalcones are natural antioxidants that are present in fruit and vegetables. Their degradation in the radiolysed solutions was followed by HPLC, NMR, FAB-LSIMS mass spectroscopy and analytical TLC in deaerated solution. Among the 18 identified radiolytic compounds, 16 were new. The formation of the radiolytic products was not influenced by A- and B-ring substitutions. To explain the degradation process, we thus suggested that the primary step was an attack of the α,β-double bond by the 1-hydroxymethyl radical, either at C_α or at C_β. This step was followed by addition, cyclization or bond dissociations. Different chemical pathways were discussed that implicate the reactive species issued from methanol radiolysis. This paper highlights the relative importance of the different radical species, especially the carbon-centered radical, 1-hydroxymethyl (HMR) and the corresponding oxygen-centered isomer. In addition, an interesting unusual role of dioxygen should be noted; indeed, in the presence of dioxygen, degradation of chalcones was inhibited.