Prieur-Carrillo, G., Chu, K., Lindqvist, J. and Dewey, W. C. Computerized Video Time-Lapse (CVTL) Analysis of the Fate of Giant Cells Produced by X-Irradiating EJ30 Human Bladder Carcinoma Cells. Radiat. Res. 159, 705–712 (2003).

This study was designed to examine the viability and proliferation of uninucleated and multinucleated giant cells formed after 6 Gy X irradiation. The pedigrees of 102 individual EJ30 giant cells present 5 days after irradiation were analyzed from time-lapse movies captured over 6.3 days from 100 fields (100×). Pedigree analysis enabled us to study the proliferation of giant cells. The average starting size (area) of the giant cells (14500 ± 9100 μm²) was ∼25 times larger than the normal-sized cells observed after irradiation (560 ± 200 μm²). From a total of 76 pedigrees of uninucleated giant cells, 42 had giant cells that either died or were arrested, while 34 divided at least once and produced progeny that divided again (five three times and three four times) before the progeny died or were arrested. Twenty-four pedigrees contained progeny that were lost from observation after dividing at least once. While most progeny continued to have giant cell morphology, two uninucleated giant cells ultimately produced progeny that contained two normal-sized cells. From a total of 26 multinucleated giant cells, only three divided. Two divided only once, but one produced progeny that divided two times. In all, 37 out of 102 giant cells divided at least once; eight of these divided four or five times with two of these pedigrees ultimately producing two normal-sized daughter cells. These results suggest that a small fraction of giant cells might be potentially clonogenic.

Widel, M., Jedrus, S., Lukaszczyk, B., Raczek-Zwierzycka, K. and Swierniak, A. Radiation-Induced Micronucleus Frequency in Peripheral Blood Lymphocytes is Correlated with Normal Tissue Damage in Patients with Cervical Carcinoma Undergoing Radiotherapy. Radiat. Res. 159, 713–721 (2003).

In an effort to find a test to predict the response of normal tissue to radiotherapy, the lymphocyte micronucleus assay was used on blood samples from patients with cervical carcinoma. Peripheral blood samples from 55 patients with advanced-stage (II B–IV B) cervical carcinoma were obtained before radiotherapy. The patients were treated with external-beam radiotherapy followed by high-dose-rate brachytherapy. Acute and late normal tissue reactions were scored and correlated with the micronucleus frequency in lymphocytes after irradiation with 4 Gy in vitro. Great interindividual variability was observed in the radiation-induced lymphocyte micronucleus frequency, especially at 4 Gy. The mean number of micronuclei per 100 binucleated cells in cells irradiated with 4 Gy in vitro was significantly higher in samples from patients who suffered from acute and/or late normal tissue reactions than in those from patients with no reactions (51.0 ± 17.7 and 29.6 ± 10.1, respectively). A significant correlation was also found between the micronucleus frequency at 4 Gy and the severity of acute reactions and late reactions. However, the overlap between the micronucleus frequencies of patients with high-grade late normal tissue reactions and low-grade reactions is too great to recommend the micronucleus assay in its present form for routine clinical application.

Kminek, G., Bada, J. L., Pogliano, K. and Ward, J. F. Radiation-Dependent Limit for the Viability of Bacterial Spores in Halite Fluid Inclusions and on Mars. Radiat. Res. 159, 722–729 (2003).

When claims for the long-term survival of viable organisms are made, either within terrestrial minerals or on Mars, considerations should be made of the limitations imposed by the naturally occurring radiation dose to which they have been exposed. We investigated the effect of ionizing radiation on different bacterial spores by measuring the inactivation constants for B. subtilis and S. marismortui spores in solution as well as for dry spores of B. subtilis and B. thuringiensis. S. marismortui is a halophilic spore that is genetically similar to the recently discovered 2-9-3 bacterium from a halite fluid inclusion, claimed to be 250 million years old (Vreeland et al., Nature 407, 897–900, 2000). B. thuringiensis is a soil bacterium that is genetically similar to the human pathogens B. anthracis and B. cereus (Helgason et al., Appl. Environ. Microbiol. 66, 2627–2630, 2000). To relate the inactivation constant to some realistic environments, we calculated the radiation regimen in a halite fluid inclusion and in the Martian subsurface over time. Our conclusion is that the ionizing dose of radiation in those environments limits the survival of viable bacterial spores over long periods. In the absence of an active repair mechanism in the dormant state, the long-term survival of spores is limited to less than 109 million years in halite fluid inclusions, to 100 to 160 million years in the Martian subsurface below 3 m, and to less than 600,000 years in the uppermost meter of Mars.

Schwartz, J. L., Jordan, R., Evans, H. H., Lenarczyk, M. and Liber, H. The TP53 Dependence of Radiation-Induced Chromosome Instability in Human Lymphoblastoid Cells. Radiat. Res. 159, 730–736 (2003).

The dose and TP53 dependence for the induction of chromosome instability were examined in cells of three human lymphoblastoid cell lines derived from WIL2 cells: TK6, a TP53-normal cell line, NH32, a TP53-knockout created from TK6, and WTK1, a WIL2-derived cell line that spontaneously developed a TP53 mutation. Cells of each cell line were exposed to ¹³⁷Cs γ rays, and then surviving clones were isolated and expanded in culture for approximately 35 generations before the frequency and characteristics of the instability were analyzed. The presence of dicentric chromosomes, formed by end-to-end fusions, served as a marker of chromosomal instability. Unexposed TK6 cells had low levels of chromosomal instability (0.002 ± 0.001 dicentrics/cell). Exposure of TK6 cells to doses as low as 5 cGy γ rays increased chromosome instability levels nearly 10-fold to 0.019 ± 0.008 dicentrics/cell. There was no further increase in instability levels beyond 5 cGy. In contrast to TK6 cells, unexposed cultures of WTK1 and NH32 cells had much higher levels of chromosome instability of 0.034 ± 0.007 and 0.041 ± 0.009, respectively, but showed little if any effect of radiation on levels of chromosome instability. The results suggest that radiation exposure alters the normal TP53-dependent cell cycle checkpoint controls that recognize alterations in telomere structure and activate apoptosis.

Evans, H. H., Horng, M-F., Ricanati, M., Diaz-Insua, M., Jordan, R. and Schwartz, J. L. Induction of Genomic Instability in TK6 Human Lymphoblasts Exposed to ¹³⁷Cs γ Radiation: Comparison to the Induction by Exposure to Accelerated ⁵⁶Fe Particles. Radiat. Res. 159, 737–747 (2003).

The induction of genomic instability in TK6 human lymphoblasts by exposure to ¹³⁷Cs γ radiation was investigated by measuring the frequency and characteristics of unstable clones isolated approximately 36 generations after exposure. Clones surviving irradiation and control clones were analyzed for 17 characteristics including chromosomal aberrations, growth defects, alterations in response to a second irradiation, and mutant frequencies at the thymidine kinase and Na /K ATPase loci. Putative unstable clones were defined as those that exhibited a significant alteration in one or more characteristics compared to the controls. The frequency and characteristics of the unstable clones were compared in clones exposed to ¹³⁷Cs γ rays or ⁵⁶Fe particles. The majority of the unstable clones isolated after exposure to either γ rays or ⁵⁶Fe particles exhibited chromosomal instability. Alterations in growth characteristics, radiation response and mutant frequencies occurred much less often than cytogenetic alterations in these unstable clones. The frequency and complexity of the unstable clones were greater after exposure to ⁵⁶Fe particles than to γ rays. Unstable clones that survived 36 generations after exposure to γ rays exhibited increases in the incidence of dicentric chromosomes but not of chromatid breaks, whereas unstable clones that survived 36 generations after exposure to ⁵⁶Fe particles exhibited increases in both chromatid and chromosome aberrations.

Despras, E., Miccoli, L., Créminon, C., Rouillard, D., Angulo, J. F. and Biard, D. S. F. Depletion of KIN17, a Human DNA Replication Protein, Increases the Radiosensitivity of RKO Cells. Radiat. Res. 159, 748–758 (2003).

The human KIN17 protein is a chromatin-associated protein involved in DNA replication. Certain tumor cell lines overproduce KIN17 protein. Among 16 cell lines, the highest KIN17 protein level was observed in H1299 non-small cell lung cancer cells, whereas the lowest was detected in MeWo melanoma cells. Cells displaying higher KIN17 protein levels exhibited elevated RPA70 protein contents. High KIN17 protein levels may be a consequence of the tumorigenic phenotype or a prerequisite for tumor progression. Twenty-four hours after exposure to ionizing radiation, after the completion of DNA repair, a co-induction of chromatin-bound KIN17 and RPA70 proteins was detected. Etoposide, an inhibitor of topoisomerase II generating double-strand breaks, triggered the concentration of KIN17 into punctuate intranuclear foci. KIN17 may be associated with unrepaired DNA sites. Flow cytometry analysis revealed that 48 h after transfection the uppermost KIN17-positive RKO cells shifted in the cell cycle toward higher DNA content, suggesting that KIN17 protein induced defects in chromatin conformation. Cells displaying reduced levels of KIN17 transcript exhibited a sixfold increased radiosensitivity at 2 Gy. The KIN17 protein may be a component of the DNA replication machinery that participates in the cellular response to unrepaired DSBs, and an impaired KIN17 pathway leads to an increased sensitivity to ionizing radiation.

MacPhail, S. H., Banáth, J. P., Yu, Y. T., Chu, E. and Olive, P. L. Cell Cycle-Dependent Expression of Phosphorylated Histone H2AX: Reduced Expression in Unirradiated but not X-Irradiated G₁-Phase Cells. Radiat. Res. 159, 759–767 (2003).

Exposure of cells to ionizing radiation causes phosphorylation of histone H2AX at sites flanking DNA double-strand breaks. Detection of phosphorylated H2AX (γH2AX) by antibody binding has been used as a method to identify double-strand breaks. Although generally performed by observing microscopic foci within cells, flow cytometry offers the advantage of measuring changes in γH2AX intensity in relation to cell cycle position. The importance of cell cycle position on the levels of endogenous and radiation-induced γH2AX was examined in cell lines that varied in DNA content, cell cycle distribution, and kinase activity. Bivariate analysis of γH2AX expression relative to DNA content and synchronization by centrifugal elutriation were used to measure cell cycle-specific expression of γH2AX. With the exception of xrs5 cells, γH2AX level was approximately 3 times lower in unirradiated G₁-phase cells than S- and G₂-phase cells, and the slope of the G₁-phase dose–response curve was 2.8 times larger than the slope for S-phase cells. Cell cycle differences were confirmed using immunoblotting, indicating that reduced antibody accessibility in intact cells was not responsible for the reduced antibody binding in G₁-phase cells. Early apoptotic cells could be easily identified on flow histograms as a population with 5–10-fold higher levels of γH2AX, although high expression was not maintained in apoptotic cells by 24 h. We conclude that expression of γH2AX is associated with DNA replication in unirradiated cells and that this reduces the sensitivity for detecting radiation-induced double-strand breaks in S- and G₂-phase cells.

Song, J-Y., Han, S-K., Bae, K-G., Lim, D-S., Son, S-J., Jung, I-S., Yi, S-Y. and Yun, Y-S. Radioprotective Effects of Ginsan, an Immunomodulator. Radiat. Res. 159, 768–774 (2003).

We previously reported that ginsan, a purified polysaccharide isolated from Panax ginseng, had a mitogenic activity, induced LAK cells, and increased levels of several cytokines. In an effort to identify other immunostimulatory effects, we evaluated the protective effects of ginsan injected in vivo against radiation by measuring its effects on the CFU-S bone marrow cells and spleen cells. Ginsan was found to significantly increase the number of bone marrow cells, spleen cells, granulocyte-macrophage colony-forming cells (GM-CFC), and circulating neutrophils, lymphocytes and platelets in irradiated mice. In addition, ginsan induced the endogenous production of cytokines such as Il1, Il6, Ifng and Il12, which are required for hematopoietic recovery, and was able to enhance Th1 function while interfering with the Th2 response in irradiated mice. We demonstrated that pretreatment with ginsan protected mice from the lethal effects of ionizing radiation more effectively than when it was given immediately after or at various times after irradiation. A significant increase in the LD_50/30 from 7.54 Gy for PBS injection to 10.93 Gy for mice pretreated with 100 mg/kg ginsan was observed. These findings indicate that ginsan may be a useful agent to reduce the time necessary for reconstituting hematopoietic cells after irradiation.

Onodera, H., Jin, Z., Chida, S., Suzuki, Y., Tago, H. and Itoyama, Y. Effects of 10-T Static Magnetic Field on Human Peripheral Blood Immune Cells. Radiat. Res. 159, 775–779 (2003).

The exposure of peripheral blood mononuclear cells (PBMCs) was performed in a 10-T static magnetic field. Without lymphocyte stimulation, there were no significant differences in the viability of the exposed and unexposed CD4 T cells, CD8 T cells, B cells, and natural killer (NK) cells. The expression of Th1 type chemokine receptor, CXCR3, and Th2 type receptor, CCR3, was unaltered after magnetic-field exposure. No differences were observed in the naive T cells and memory T-cell subclasses in either CD4 or CD8 T cells. In contrast to the unstimulated condition, the magnetic-field exposure reduced the viability of phytohemagglutinin (PHA)-activated T cells in both the CD4 and CD8 subclasses. In particular, the number of PHA-treated naive CD8 T cells (CD45RA CD4^–CD8 ) was markedly decreased after the magnetic-field exposure, while PHA-treated memory CD8 cells (CD45RA^–CD4^–CD8 ) were resistant to the exposure. The number of PHA-treated naive CD4 T cells (CD45RA CD4 CD8^–) and memory cells (CD45RA^–CD4 CD8^–) was markedly decreased to a similar degree. Thus the susceptibility of lymphocytes to the magnetic-field exposure differed among activated T-cell subtypes. The magnetic-field exposure significantly increased the death of PHA-stimulated lymphocytes by apoptosis. These results suggest that a strong static magnetic field has acute effects on immune cells during cell division, while the field exposure has a minimal effect on immune cells in a nondividing phase.

Fujiwara, S., Sharp, G. B., Cologne, J. B., Kusumi, S., Akahoshi, M., Kodama, K., Suzuki, G. and Yoshizawa, H. Prevalence of Hepatitis B Virus Infection among Atomic Bomb Survivors. Radiat. Res. 159, 780–786 (2003).

The aim of this study was to determine whether the prevalence of hepatitis B virus (HBV) carriers increased with atomic bomb radiation dose, and whether radiation decreased the ability to clear HBV among the atomic bomb survivors. The study subjects were 6,121 participants in the Adult Health Study of atomic bomb survivors in Hiroshima and Nagasaki. After adjustment for age, sex, city and potential confounders, the rates of seropositivity for hepatitis B surface antigen (HBsAg), indicating current HBV infections, and anti-hepatitis B core antibody, indicating either cured or current infections, increased with radiation dose. However, no relationship was observed between radiation and anti-hepatitis B surface antibody (indicating cured infection). The proportion of persons who were unable to clear the virus, as the proportion of HBsAg-positive persons among those ever infected by HBV (positive for HBsAg or surface or core hepatitis B antibody), increased significantly with radiation dose among those receiving blood transfusions. This proportion was not related to dose among those who reported no such transfusions. The findings may suggest a lower likelihood of clearance after HBV infection among those who were more likely to have been infected with HBV as adults after atomic bomb irradiation rather than as infants or adults prior to irradiation.

Shilnikova, N. S., Preston, D. L., Ron, E., Gilbert, E. S., Vassilenko, E. K., Romanov, S. A., Kuznetsova, I. S., Sokolnikov, M. E., Okatenko, P. V., Kreslov, V. V. and Koshurnikova, N. A. Cancer Mortality Risk among Workers at the Mayak Nuclear Complex. Radiat. Res. 159, 787–798 (2003).

At present, direct data on risk from protracted or fractionated radiation exposure at low dose rates have been limited largely to studies of populations exposed to low cumulative doses with resulting low statistical power. We evaluated the cancer risks associated with protracted exposure to external whole-body γ radiation at high cumulative doses (the average dose is 0.8 Gy and the highest doses exceed 10 Gy) in Russian nuclear workers. Cancer deaths in a cohort of about 21,500 nuclear workers who began working at the Mayak complex between 1948 and 1972 were ascertained from death certificates and autopsy reports with follow-up through December 1997. Excess relative risk models were used to estimate solid cancer and leukemia risks associated with external γ-radiation dose with adjustment for effects of plutonium exposures. Both solid cancer and leukemia death rates increased significantly with increasing γ-ray dose (P < 0.001). Under a linear dose–response model, the excess relative risk for lung, liver and skeletal cancers as a group (668 deaths) adjusted for plutonium exposure is 0.30 per gray (P < 0.001) and 0.08 per gray (P < 0.001) for all other solid cancers (1062 deaths). The solid cancer dose–response functions appear to be nonlinear, with the excess risk estimates at doses of less than 3 Gy being about twice those predicted by the linear model. Plutonium exposure was associated with increased risks both for lung, liver and skeletal cancers (the sites of primary plutonium deposition) and for other solid cancers as a group. A significant dose response, with no indication of plutonium exposure effects, was found for leukemia. Excess risks for leukemia exhibited a significant dependence on the time since the dose was received. For doses received within 3 to 5 years of death the excess relative risk per gray was estimated to be about 7 (P < 0.001), but this risk was only 0.45 (P = 0.02) for doses received 5 to 45 years prior to death. External γ-ray exposures significantly increased risks of both solid cancers and leukemia in this large cohort of men and women with occupational radiation exposures. Risks at doses of less than 1 Gy may be slightly lower than those seen for doses arising from acute exposures in the atomic bomb survivors. As dose estimates for the Mayak workers are improved, it should be possible to obtain more precise estimates of solid cancer and leukemia risks from protracted external radiation exposure in this cohort.

Suzuki, M., Nakamatsu, K., Kanamori, S., Masunaga, S. and Nishimura, Y. Additive Effects of Radiation and Docetaxel on Murine SCCVII Tumors In Vivo: Special Reference to Changes in the Cell Cycle. Radiat. Res. 159, 799–804 (2003).

The purpose of the present study was to investigate the effects of a combination of docetaxel and irradiation in vivo with special reference to docetaxel-arrested G₂/M-phase cells. At 24 and 48 h after intraperitoneal administration of docetaxel (90 mg/kg), tumor-bearing mice were irradiated with ⁶⁰Co γ rays. Cell cycle distribution was analyzed by a DNA-Ki-67 double staining method using flow cytometry. An accumulation of cells in the G₂/M phase of up to approximately 40% was observed 24 h after administration of docetaxel. Between 24 and 72 h, the percentage of cells arrested in G₂/M phase that expressed Ki-67 decreased from 37.2% to 13.8%, in accordance with the increase in the Ki-67-negative G₂/M-phase fraction. More than half of the cells arrested in G₂/M phase lost their expression of Ki-67 protein between 24 and 72 h. The G₁-phase fraction decreased from 28.4% to 8.6% at 24 h after docetaxel treatment; this remained unchanged at 72 h. These flow cytometry data suggested that docetaxel-arrested G₂/M-phase cells did not enter the next cell cycle and were killed by docetaxel alone. Our data showed that arrest of cells in G₂/M phase does not contribute to the synergism that has been reported for combinations of docetaxel and radiation in in vivo tumor models.

Tada, H., Ogushi, F., Tani, K., Nishioka, Y., Miyata, J., Sato, K., Asano, T. and Sone, S. Increased Binding and Chemotactic Capacities of PDGF-BB on Fibroblasts in Radiation Pneumonitis. Radiat. Res. 159, 805–811 (2003).

Although pulmonary fibrosis is a frequent and serious consequence of radiotherapy for thoracic malignant diseases such as lung cancer, the pathogenesis of this radiation-induced lung disorder remains unclear. To clarify the mechanisms underlying radiation pneumonitis and pulmonary fibrosis, we investigated the expression of platelet-derived growth factor receptor (PDGFR) on fibroblasts obtained from irradiated rat lungs and on control fibroblasts. Whole lungs of male Wistar rats were irradiated with a single dose of 15 Gy, and lung fibroblasts were isolated at 4 weeks after the irradiation. The chemotactic response of irradiated lung fibroblasts to PDGF-BB was significantly higher than that of control lung fibroblasts, whereas there was no significant difference between irradiated lung fibroblasts and control lung fibroblasts in the response to PDGF-AA. Receptor binding assay showed more specific binding sites for PDGF-BB on irradiated lung fibroblasts than on control lung fibroblasts, and the displacement of ¹²⁵I-labeled PDGF binding to fibroblasts by unlabeled PDGF showed that ¹²⁵I-labeled PDGF-BB was displaced by PDGF-BB but not by PDGF-AA. These results suggest that the increased binding sites for PDGF-BB on irradiated lung fibroblasts correspond mainly to PDGFRB. Scatchard analysis of the saturation data demonstrated an approximately twofold increase both in the number of PDGF-BB binding sites and in the binding affinity in irradiated lung fibroblasts compared to that in control lung fibroblasts. Those results suggest that the increased chemotactic response of irradiated lung fibroblasts to PDGF-BB is related to the overexpression of PDGFRB, which may have an important role in the pathogenesis of radiation-induced pneumonitis and pulmonary fibrosis.

Coleman, C. N., Blakely, W. F., Fike, J. R., MacVittie, T. J., Metting, N. F., Mitchell, J. B., Moulder, J. E., Preston, R. J., Seed, T. M., Stone, H. B., Tofilon, P. J. and Wong, R. S. L. Molecular and Cellular Biology of Moderate-Dose (1–10 Gy) Radiation and Potential Mechanisms of Radiation Protection: Report of a Workshop at Bethesda, Maryland, December 17–18, 2001. Radiat. Res. 159, 812–834 (2003).

Exposures to doses of radiation of 1–10 Gy, defined in this workshop as moderate-dose radiation, may occur during the course of radiation therapy or as the result of radiation accidents or nuclear/radiological terrorism alone or in conjunction with bioterrorism. The resulting radiation injuries would be due to a series of molecular, cellular, tissue and whole-animal processes. To address the status of research on these issues, a broad-based workshop was convened. The specific recommendations were: (1) Research: Identify the key molecular, cellular and tissue pathways that lead from the initial molecular lesions to immediate and delayed injury. The latter is a chronic progressive process for which postexposure treatment may be possible. (2) Technology: Develop high-throughput technology for studying gene, protein and other biochemical expression after radiation exposure, and cytogenetic markers of radiation exposure employing rapid and accurate techniques for analyzing multiple samples. (3) Treatment strategies: Identify additional biological targets and develop effective treatments for radiation injury. (4) Ensuring sufficient expertise: Recruit and train investigators from such fields as radiation biology, cancer biology, molecular biology, cellular biology and wound healing, and encourage collaboration on interdisciplinary research on the mechanisms and treatment of radiation injury. Communicate knowledge of the effects of radiation exposure to the general public and to investigators, policy makers and agencies involved in response to nuclear accidents/events and protection/treatment of the general public.