Epperly, M. W., Guo, H., Shen, H., Niu, Y., Zhang, X., Jefferson, M., Sikora, C. A. and Greenberger, J. S. Bone Marrow Origin of Cells with Capacity for Homing and Differentiation to Esophageal Squamous Epithelium. Radiat. Res. 162, 233–240 (2004).

Our goal was to determine whether esophageal progenitor cells could be isolated from adult mouse esophagus or bone marrow and shown to home to and proliferate in the irradiated esophagus of recipient mice. Esophageal progenitor cells were isolated from adult male C3H/HeNsd or C57BL/6J green fluorescent protein (GFP ) mice by a serial in vitro preplate technique or the technique of side population cell sorting. When injected intravenously (i.v.), these cells homed to the 30-Gy-irradiated esophagus of GFP⁻ female recipient mice and formed donor-origin esophageal foci. GFP whole murine bone marrow cells injected i.v. also formed donor-origin esophageal squamous cell foci and protected recipient GFP⁻ mice from upper-body irradiation in a cell dose-dependent manner. Marrow chimeric GFP⁻ mice reconstituted with GFP cells showed migration of GFP marrow cells to the esophagus after 30 Gy irradiation. Purified esophageal progenitor cells isolated from first-generation preplate cell recipients engrafted after i.v. injection to the esophagus of second-generation-irradiated recipient mice. These data establish that esophageal progenitor cells can home to the irradiated esophagus and show limited differentiation capacity to squamous epithelium.

Davis, S., Stepanenko, V., Rivkind, N., Kopecky, K. J., Voillequé, P., Shakhtarin, V., Parshkov, E., Kulikov, S., Lushnikov, E., Abrosimov, A., Troshin, V., Romanova, G., Doroschenko, V., Proshin A. and Tsyb, A. Risk of Thyroid Cancer in the Bryansk Oblast of the Russian Federation after the Chernobyl Power Station Accident. Radiat. Res. 162, 241–248 (2004).

This population-based case–control study investigated whether exposure to radiation from the Chernobyl Power Station accident is associated with an increased risk of thyroid cancer in children and adolescents aged 0–19 years at the time of the accident who were residing in the more highly contaminated areas of the Bryansk Oblast. Cases were diagnosed with thyroid cancer before October 1, 1997 (n = 26); two controls per case were identified from the Russian State Medical Dosimetrical Registry and were matched by gender, birth year, and raion of residence and type of settlement (urban, town, rural) on April 26, 1986 (n = 52). Individual radiation doses to the thyroid were estimated using a semi-empirical model and data were collected in interviews, primarily of the participants' mothers. Based on a loglinear dose–response model treating estimated dose as a continuous variable, the trend of increasing risk with increasing dose was statistically significant (one-sided P = 0.009). These data suggest that exposure to radiation from Chernobyl is associated with an increased risk of thyroid cancer, and that the relationship is dependent on dose. These findings are consistent with descriptive reports from contaminated areas of Ukraine and Belarus, and the quantitative estimate of thyroid cancer risk is generally consistent with estimates from other radiation-exposed populations.

Tawn, E. J., Whitehouse, C. A. and Tarone, R. E. FISH Chromosome Aberration Analysis on Retired Radiation Workers from the Sellafield Nuclear Facility. Radiat. Res. 162, 249–256 (2004).

Chromosome analysis using fluorescence in situ hybridization was undertaken on 294 retired workers from the British Nuclear Fuels plc facility at Sellafield, 95 with external occupational exposure <50 mSv, 108 with 50–499 mSv, and 91 with >500 mSv. In univariate analyses, external dose (P < 10⁻⁵) and age (P = 0.0075) were significantly associated with translocation frequency, but no effect was found for smoking status. In a multivariate analysis with age and external dose as continuous variables, the slopes were 0.017 ± 0.0075 × 10⁻² translocations per cell per year for age (P = 0.024) and 1.11 ± 0.190 × 10⁻² translocations per cell per sievert for external dose (P < 10⁻⁵). The dose response for translocation induction for occupational workers is similar to the linear component of in vitro dose–response curves, thus supporting the use of translocation frequency for retrospective biological dosimetry in situations of chronic low-dose exposure occurring over many years. The dose response obtained in this study is lower than the linear component of the dose response for stable chromosome aberrations obtained for the Japanese atomic bomb survivors. Thus, if chromosome aberration levels are indicative of cancer risk, this would suggest that low-dose risks derived from the Japanese atomic bomb survivor data will overestimate the risks associated with the occupational exposure encountered by the men in this study.

Mitchell, C. R., Azizova, T. V., Hande, M. P., Burak, L. E., Tsakok, J. M., Khokhryakov, V. F., Geard, C. R. and Brenner, D. J. Stable Intrachromosomal Biomarkers of Past Exposure to Densely Ionizing Radiation in Several Chromosomes of Exposed Individuals. Radiat. Res. 162, 257–263 (2004).

A multicolor banding (mBAND) fluorescence in situ hybridization technique was used to investigate the presence in human populations of a stable biomarker—intrachromosomal chromosome aberrations—of past exposure to high-LET radiation. Peripheral blood lymphocytes were taken from healthy Russian nuclear workers occupationally exposed from 1949 onward to either plutonium, γ rays or both. Metaphase spreads were produced and chromosomes 1 and 2 were hybridized with mBAND FISH probes and scored for intrachromosomal aberrations. A large yield of intrachromosomal aberrations was observed in both chromosomes of the individuals exposed to high doses of plutonium, whereas there was no significant increase over the (low) background control rate in the population who were exposed to high doses of γ rays. Interchromosome aberration yields were similar in both the high plutonium and the high γ-ray groups. These results for chromosome 1 and 2 confirm and extend data published previously for chromosome 5. Intrachromosomal aberrations thus represent a potential biomarker for past exposure to high-LET radiations such as α particles and neutrons and could possibly be used as a biodosimeter to estimate both the dose and type of radiation exposure in previously exposed populations.

Suzuki, M., Zhou, H., Geard, C. R. and Hei, T. K. Effect of Medium on Chromatin Damage in Bystander Mammalian Cells. Radiat. Res. 162, 264–269 (2004).

In the present study, we examined the potential contribution of irradiated medium to the bystander effect using custom-made double-Mylar stainless steel rings. Exponentially growing human–hamster hybrid (A_L) cells were plated on either one or both sides of double-Mylar dishes 2–4 days before irradiation. One side (with or without cells) was irradiated with α particles using the track segment mode of a 4 MeV Van de Graaff accelerator at the Radiological Research Accelerator Facility of Columbia University. Since α particles can traverse only a very limited distance (around 23 μm in water), cells plated on the other side of a medium-filled Mylar dish will not be irradiated by the α particles. The results of the cytogenetic assay of unirradiated target cells that were attached to the top Mylar layer indicate that the number of chromatid-type aberrations was higher when there was a bottom layer of cells in the medium-filled chambers than with just medium alone. Furthermore, when the medium was transferred from these cell-irradiated dishes to fresh A_L cell cultures, chromatid-type aberrations were produced in the unirradiated fresh cells. In contrast, medium irradiated in the absence of cells had no effect on chromatid aberrations. These results suggest that certain unidentified modulating factors secreted from the irradiated cells on the bottom Mylar layer into the medium induce chromatin damage in the unirradiated bystander cells.

Chu, K., Teele, N., Dewey, M. W., Albright, N. and Dewey, W. C. Computerized Video Time Lapse Study of Cell Cycle Delay and Arrest, Mitotic Catastrophe, Apoptosis and Clonogenic Survival in Irradiated 14-3-3σ and CDKN1A (p21) Knockout Cell Lines. Radiat. Res. 162, 270–286 (2004).

Computerized video time lapse (CVTL) microscopy was used to observe cellular events induced by ionizing radiation (10–12 Gy) in nonclonogenic cells of the wild-type HCT116 colorectal carcinoma cell line and its three isogenic derivative lines in which p21 (CDKN1A), 14-3-3σ or both checkpoint genes (double-knockout) had been knocked out. Cells that fused after mitosis or failed to complete mitosis were classified together as cells that underwent mitotic catastrophe. Seventeen percent of the wild-type cells and 34–47% of the knockout cells underwent mitotic catastrophe to enter generation 1 with a 4N content of DNA, i.e., the same DNA content as irradiated cells arrested in G₂ at the end of generation 0. Radiation caused a transient division delay in generation 0 before the cells divided or underwent mitotic catastrophe. Compared with the division delay for wild-type cells that express CDKN1A and 14-3-3σ, knocking out CDKN1A reduced the delay the most for cells irradiated in G₁ (from ∼15 h to ∼3– 5 h), while knocking out 14-3-3σ reduced the delay the most for cells irradiated in late S and G₂ (from ∼18 h to ∼3–4 h). However, 27% of wild-type cells and 17% of 14-3-3σ^−/− cells were arrested at 96 h in generation 0 compared with less than 1% for CDKN1A^−/− and double-knockout cells. Thus expression of CDKN1A is necessary for the prolonged delay or arrest in generation 0. Furthermore, CDKN1A plays a crucial role in generation 1, greatly inhibiting progression into subsequent generations of both diploid cells and polyploid cells produced by mitotic catastrophe. Thus, in CDKN1A-deficient cell lines, a series of mitotic catastrophe events occurred to produce highly polyploid progeny during generations 3 and 4. Most importantly, the polyploid progeny produced by mitotic catastrophe events did not die sooner than the progeny of dividing cells. Death was identified as loss of cell movement, i.e. metabolic activity. Thus mitotic catastrophe itself is not a direct mode of death. Instead, apoptosis during interphase of both uninucleated and polyploid cells was the primary mode of death observed in the four cell types. Knocking out either CDKN1A or 14-3-3σ increased the amount of cell death at 96 h, from 52% to ∼70%, with an even greater increase to 90% when both genes were knocked out. Thus, in addition to effects of CDKN1A and 14-3-3σ expression on transient cell cycle delay, CDKN1A has both an anti-proliferative and anti-apoptosis function, while 14-3-3σ has only an anti-apoptosis function. Finally, the large alterations in the amounts of cell death did not correlate overall with the small alterations in clonogenic survival (dose-modifying ratios of 1.05–1.13); however, knocking out CDKN1A resulted in a decrease in arrested cells and an increase in survival, while knocking out 14-3-3σ resulted in an increase in apoptosis and a decrease in survival.

Zhou, X., Suto, S., Ota, T. and Tatsuka, M. Nuclear Translocation of Cleaved LyGDI Dissociated from Rho and Rac during Trp53-Dependent Ionizing Radiation-Induced Apoptosis of Thymus Cells In Vitro. Radiat. Res. 162, 287–295 (2004).

LyGDI inhibits the dissociation of GDP from Rho family GTPases and is found in abundance in hematopoietic cells. Here we report truncation of LyGDI after irradiation in mouse 3SB thymus cells. A 21-kDa fragment of LyGDI, resulting from activated caspase 3-induced cleavage at an N-terminal consensus site following the Asp¹⁸ residue, accumulated at peak quantities between 5 and 12 h after irradiation. Cleavage of LyGDI was inhibited by the caspase inhibitor benzoyloxycarbonyl-Val-Asp-fluoromethylketone. Subcellular fractionation and immunofluorescence revealed the truncated 21-kDa fragment of LyGDI within the nuclear fraction of irradiated 3SB cells, whereas full-length LyGDI was found only in the cytoplasmic fraction. Truncated LyGDI within the nucleus had no association with the Rho family proteins RhoA and Rac1, since these proteins were observed only in the cytoplasmic fractions. These data demonstrate that regulation of Rho family GTPases by LyGDI is disrupted during apoptosis, suggesting that fragmentation of LyGDI implicates the transmission of a signal from the cytoplasm to the nucleus during Trp53-dependent apoptosis of thymus cells after irradiation.

Sugihara, T., Magae, J., Wadhwa, R., Kaul, S. C., Kawakami, Y., Matsumoto, T. and Tanaka, K. Dose and Dose-Rate Effects of Low-Dose Ionizing Radiation on Activation of Trp53 in Immortalized Murine Cells. Radiat. Res. 162, 296– 307 (2004).

A derivative of immortalized murine NIH/PG13Luc cells stably transfected with a Trp53-dependent luciferase reporter plasmid was used to study the transcriptional activity of Trp53 in response to radiation. The cell line was sensitive enough to detect the response of Trp53 to 0.2 cGy of ⁶⁰Co γ radiation. To examine the biological effects of low-dose-rate ⁶⁰Co γ radiation (from 0.1–10 cGy/h), we have analyzed the cell cycle, Trp53 transcriptional activity, and gene expression profiles of control and treated cells. Microarray analysis revealed up-regulation of six Trp53-mediated genes (Cdkn1a/ p21, Mdm2, Sip27, Ccng1/cyclin G1, Ei24/Pig8 and Dinb/ Polk) after exposure of cells to low-dose-rate radiation for 72 h. Using real-time PCR, a significant elevation in the expression of Ccng1/cyclin G1, Mdm2 and Cdkn1A/p21 was observed with low-dose-rate irradiation at dose rates over 5 cGy/ h. A dose-rate dependence was also observed for these three Trp53-mediated genes. The expression of Ccng1/cyclin G1 at high dose rates of γ rays was higher than that for low dose rate. However, the expression of Mdm2 for low-dose-rate γ rays was higher than for the high dose rate. Cells irradiated at low dose rates of 0.1 cGy/h and 1 cGy/h underwent G₁-phase arrest. Furthermore, G₂-phase growth arrest was observed in cells irradiated at the low dose rates of 5 cGy/h and 10 cGy/h, which correlated with Trp53-mediated Ccng1/cyclin G1 up-regulation. These results show that cellular response to radiation depended on the dose rate used; i.e., the responses seen at dose rates from 0.1–1 cGy/h were different from those observed at dose rates over 5 cGy/h.

Hosoi, Y., Matsumoto, Y., Enomoto, A., Morita, A., Green, J., Nakagawa, K., Naruse, K. and Suzuki, N. Suramin Sensitizes Cells to Ionizing Radiation by Inactivating DNA-Dependent Protein Kinase. Radiat. Res. 162, 308–314 (2004).

Here we report that suramin sensitizes LM217, MDA-MB-468, T98G and A431 cells to ionizing radiation. Suramin sensitized cells to X radiation in a dose-dependent fashion, and longer exposure to suramin before X irradiation resulted in more efficient sensitization. The dose-modifying factors calculated from the survival curves were 1.18 in LM217 cells and 1.37 in MDA-MB-468 cells. Suramin did not sensitize Scid cells that had no DNA-dependent protein kinase activity. Suramin inhibited DNA-dependent protein kinase activity in vitro and in vivo. The concentration of suramin resulting in 50% inhibition in vitro was 1.7 μM in LM217 cells and 2.4 μM in MDA-MB-468 cells. Exposure of LM217 and MDA-MB-468 cells to suramin did not affect the level of Ku70 (G22P1) or Ku80 (XRCC5), but it increased the level of DNA-PKcs (PRKDC). Suramin did not sensitize LM217 or MDA-MB-468 cells to UV radiation. Suramin's effects were not caused by accumulation of cells in a specific phase of the cell cycle. These results suggest that suramin sensitizes cells to ionizing radiation by inhibiting DNA-dependent protein kinase activity.

Ahn, G-O., Ware, D. C., Denny, W. A. and Wilson, W. R. Optimization of the Auxiliary Ligand Shell of Cobalt(III)(8-hydroxyquinoline) Complexes as Model Hypoxia-Selective Radiation-Activated Prodrugs. Radiat. Res. 162, 315–325 (2004).

A potential approach for activating prodrugs in hypoxic regions of tumors is to use ionizing radiation, rather than bioreductive enzymes, to effect reduction. This study investigates radiolytic release of 8-hydroxyquinoline (8-HQ), as a model for hydroxyaza-chloromethylbenzindoline DNA minor groove alkylators, from Co(III) complexes under hypoxia. 8-HQ release, measured by HPLC, showed higher efficiency (one-electron stoichiometry) when the auxiliary ligand was a tetraazamacrocycle [e.g. 1,4,7,10-tetraazacyclododecane (cyclen)] rather than a triazamacrocycle [1,4,7-triazacyclononane (TACN)]. These complexes differ from the bioreductive cobalt complex SN 24771 in that their reduction provides stable cobalt-containing products rather than free (aquated) Co² . Radiolytic release of 8-HQ from Co(cyclen)(8-HQ) and Co(TACN)(CN)(8-HQ) was also demonstrated in deoxygenated human plasma, selectively in the absence of oxygen, again with higher efficiency for the cyclen system. The cobalt complexes were >1000-fold less potent than free 8-HQ as inhibitors of cell proliferation and were metabolically stable in aerobic and hypoxic cell cultures. Investigation of cell uptake of total cobalt, by inductively coupled plasma mass spectrometry, showed that these complexes enter cells but do not accumulate to the high concentrations seen with SN 24771. The results demonstrate the feasibility of masking the cytotoxicity of hydroxyquinoline-based cytotoxins as Co(III) complexes and demonstrate the utility of cyclen-based auxiliary ligands for optimizing radiolytic activation of these novel prodrugs under hypoxia.

Mainardi, E., Donahue, R. J., Wilson, W. E. and Blakely, E. A. Comparison of Microdosimetric Simulations Using PENELOPE and PITS for a 25 keV Electron Microbeam in Water. Radiat. Res. 162, 326–331 (2004).

The calculations presented compared the performances of two Monte Carlo codes used for the estimation of microdosimetric quantities: Positive Ion Track Structure code (PITS) and a main user code based on the PENetration and Energy Loss of Positrons and Electrons code (PENELOPE-2000). Event-by-event track structure codes like PITS are believed to be superior for microdosimetric applications, and they are written for this purpose. PITS tracks electrons in water down to 10 eV. PENELOPE is one of the few general-purpose codes that can simulate random electron-photon showers in any material for energies from 100 eV to 1 GeV. The model used in the comparison is a water cylinder with an internal scoring geometry made of spheres 1 μm in diameter where the scoring quantities are calculated. The source is a 25 keV electron pencil beam impinging normally on the sphere surface. This work shows only the lineal energy y and spectra graphical presentation as a function of y since for microdosimetry and biology applications, and for discussion of radiation quality in general, these results are more appropriate. The computed PENELOPE results are in agreement with those obtained with the PITS code and published previously in this journal. This paper demonstrates PENELOPE's usefulness at low energies and for small geometries. What is still needed are experimental results to confirm these analyses.

Hinton, T. G., Bedford, J. S., Congdon, J. C. and Whicker, F. W. Effects of Radiation on the Environment: A Need to Question Old Paradigms and Enhance Collaboration among Radiation Biologists and Radiation Ecologists. Radiat. Res. 162, 332–338 (2004).

A historical perspective is given of the current paradigm that does not explicitly protect nonhuman biota from radiation but instead relies on the concept that if dose limits are set to protect humans, then the environment is automatically protected as well. We summarize recent international questioning of this paradigm and briefly present three frameworks for protecting biota that are being considered by the U.S. Department of Energy, the Canadian Nuclear Safety Commission, and the International Commission on Radiological Protection. We point out a controversial component in each of the three frameworks and suggest topics that need additional research. We emphasize that to properly address radiation protection of the environment, we need to understand how effects are integrated across different levels of biological organization. We caution that the proposed use of molecular end points to estimate ecological risks from radioactive contamination is applicable only if we understand the extent of the impact that molecular damage has on individual organisms and populations of exposed biota. To accomplish the latter, enhanced collaborations are required among the traditionally separate disciplines of radiation biology and radiation ecology.