Little, M. P. and Wakeford, R. The Bystander Effect in C3H 10T½ Cells and Radon-Induced Lung Cancer. Radiat. Res. 156, 695–699 (2001).

Bystander effects, whereby cells that are not directly exposed to radiation exhibit adverse biological effects, have been observed in a number of experimental systems, including C3H 10T½ cells exposed to α-particle radiation. The bystander effect implies that risks from exposure to low doses of radiation obtained by linear extrapolation from data for high-dose exposures might be substantial underestimates. The best estimate of the ratio of the lung cancer risk among persons exposed to low (residential) doses of radon daughters to that among persons (underground miners) exposed to high doses of radon daughters is in the range of 2.4–4.0, with an upper 95% confidence limit of about 14. Assuming that the bystander effect observed in the in vitro C3H 10T½ cell system applies to human lung cells in vivo, these epidemiological data imply that the central estimate of the number of neighboring cells that can contribute to the bystander effect is between 0 and 1, with an upper 95% confidence limit of about 7. As a consequence, the bystander effect observed in the experimental C3H 10T½ cell system probably does not play a large part in the process of radon-induced lung carcinogenesis in humans.

Redpath, J. L., Liang, D., Taylor, T. H., Christie, C. and Elmore, E. The Shape of the Dose–Response Curve for Radiation-Induced Neoplastic Transformation In Vitro: Evidence for an Adaptive Response against Neoplastic Transformation at Low Doses of Low-LET Radiation. Radiat. Res. 156, 700–707 (2001).

A dose–response curve for γ-radiation-induced neoplastic transformation of HeLa × skin fibroblast human hybrid cells over the dose range 0.1 cGy to 1 Gy is presented. In the experimental protocol used, the spontaneous (background) frequency of neoplastic transformation of sham-irradiated cultures was compared to that of cultures which had been irradiated with ¹³⁷Cs γ radiation and either plated immediately or held for 24 h at 37°C prior to plating, for assay for neoplastic transformation. The pooled data from a minimum of three repeat large-scale experiments at each dose demonstrated a reduced transformation frequency for the irradiated compared to the sham-irradiated cells for doses of 0.1, 0.5, 1, 5 and 10 cGy for the delayed-plating arm. The probability of this happening by chance is given by 1/2ⁿ, where n is the number of observations (5); i.e., 1/32 ≅ 0.031. This is indicative of an adaptive response against spontaneous neoplastic transformation at least up to a dose of 10 cGy of γ radiation. The high-dose data obtained at 30 and 50 cGy and 1 Gy showed a good fit to a linear extrapolation through the sham-irradiated, zero-dose control. The delayed-plating data at 10 cGy and below showed a statistically significant divergence from this linear extrapolation.

Kellerer, A. M. and Walsh, L. Risk Estimation for Fast Neutrons with Regard to Solid Cancer. Radiat. Res. 156, 708–717 (2001).

In the absence of epidemiological information on the effects of neutrons, their cancer mortality risk coefficient is currently taken as the product of two low-dose extrapolations: the nominal risk coefficient for photons and the presumed maximum relative biological effectiveness of neutrons. This approach is unnecessary. Since linearity in dose is assumed for neutrons at low to moderate effect levels, the risk coefficient can be derived in terms of the excess risk from epidemiological observations at an intermediate dose of γ rays and an assumed value, R₁, of the neutron RBE relative to this reference dose of γ rays. Application of this procedure to the A-bomb data requires accounting for the effect of the neutron dose component, which, according to the current dosimetry system, DS86, amounts on average to 11 mGy in the two cities at a total dose of 1 Gy. With R₁ tentatively set to 20 or 50, it is concluded that the neutrons have caused 18% or 35%, respectively, of the total effect at 1 Gy. The excess relative risk (ERR) for neutrons then lies between 8 per Gy and 16 per Gy. Translating these values into risk coefficients in terms of the effective dose, E, requires accounting for the γ-ray component produced by the neutron field in the human body, which will require a separate analysis. The risk estimate for neutrons will remain essentially unaffected by the current reassessment of the neutron doses in Hiroshima, because the doses are unlikely to change much at the reference dose of 1 Gy.

Naumburg, E., Bellocco, R., Cnattingius, S., Hall, P., Boice, J. D. Jr. and Ekbom, A. Intrauterine Exposure to Diagnostic X Rays and Risk of Childhood Leukemia Subtypes. Radiat. Res. 156, 718–723 (2001).

The relationship between childhood leukemia and prenatal exposure to low-dose ionizing radiation remains debatable. This population-based case–control study investigated the association between prenatal exposure to diagnostic X-ray examinations (for different types of examinations and at different stages of pregnancy) and the risk of childhood lymphatic and myeloid leukemia. All children born and diagnosed with leukemia between 1973–1989 in Sweden (578 lymphatic and 74 myeloid) were selected as cases, and each was matched (by sex and year of birth) to a healthy control child (excluding Down's syndrome). Exposure data were abstracted blindly from all available medical records. Odds ratios (OR) and 95% confidence intervals (CI) were calculated by conditional logistic regression. It was found that prenatal X-ray examinations resulting in direct fetal exposure were not associated with a significant overall increased risk for childhood leukemia (OR = 1.11, 95% CI 0.83–1.47), for lymphatic leukemia (OR = 1.04, 95% CI 0.77–1.40), or for myeloid leukemia (OR = 1.49, 95% CI 0.48–4.72). There was little evidence of a dose response or variation in risk by trimester of exposure or age at diagnosis. Thus X-ray examinations performed during pregnancy in the 1970s and 1980s in Sweden did not affect the risk of childhood leukemia discernibly.

Mac Manus, M. P., Cavalleri, G., Ball, D. L., Beasley, M., Rotstein, H. and McKay, M. J. Exacerbation, then Clearance, of Mutation-Proven Darier's Disease of the Skin after Radiotherapy for Bronchial Carcinoma: A Case of Radiation-Induced Epidermal Differentiation? Radiat. Res. 156, 724–730 (2001).

We investigated a radiotherapy-induced flare and subsequent clearance of skin lesions of a patient with the rare, dominantly inherited genodermatosis, Darier's disease (DD). The DD gene, ATP2A2, was recently isolated and shown to be a cation pump responsible for regulating intracellular calcium homeostasis. A severe exacerbation of Darier's skin lesions developed within the radiation field when 40 Gy of palliative thoracic external-beam radiation therapy and concurrent chemotherapy (cisplatin and hydroxyurea) were delivered for non-small cell lung cancer. The DD lesions subsequently completely cleared from irradiated skin, as they did when a subsequent course of radiation alone was given for a loco-regional tumor recurrence. The two radiation therapy-treated areas of skin remained free from lesions of the skin disorder until the patient's death from progressive lung cancer 9 months later. The nucleotide sequence of the patient's ATP2A2 gene was determined by PCR-based cycle sequencing. We identified four nucleotide sequence variants in the ATP2A2 gene in this patient. Three were probable polymorphisms and the other appeared to be a novel disease-causing mutation (R751Q), situated in the transmembrane portion of the ATP2A2 protein. This finding confirmed the clinical diagnosis. Since epidermis turns over every 3–4 weeks, total and persistent clearance of the DD lesions by chemoradiotherapy suggests that this treatment induced sustained differentiation of the DD-affected skin by an unknown mechanism. Oncologists treating malignant disease in patients with DD should anticipate temporary deterioration in DD-involved irradiated skin. Radiation therapy has therapeutic potential in severe DD.

George, K., Durante, M., Wu, H., Willingham, V., Badhwar, G. and Cucinotta, F. A. Chromosome Aberrations in the Blood Lymphocytes of Astronauts after Space Flight. Radiat. Res. 156, 731–738 (2001).

Cytogenetic analysis of the lymphocytes of astronauts provides a direct measurement of space radiation damage in vivo, which takes into account individual radiosensitivity and considers the influence of microgravity and other stress conditions. Chromosome exchanges were measured in the blood lymphocytes of eight crew members after their respective space missions, using fluorescence in situ hybridization (FISH) with chromosome painting probes. Significant increases in aberrations were observed after the long-duration missions. The in vivo dose was derived from the frequencies of translocations and total exchanges using calibration curves determined before flight, and the RBE was estimated by comparison with individually measured physical absorbed doses. The values for average RBE were compared to the average quality factor (Q) from direct measurements of the lineal energy spectra using a tissue-equivalent proportional counter (TEPC) and radiation transport codes. The ratio of aberrations identified as complex was slightly higher after flight, which is thought to be an indication of exposure to high-LET radiation. To determine whether the frequency of complex aberrations measured in metaphase spreads after exposure to high-LET radiation was influenced by a cell cycle delay, chromosome damage was analyzed in prematurely condensed chromosome samples collected from two crew members before and after a short-duration mission. The frequency of complex exchanges after flight was higher in prematurely condensed chromosomes than in metaphase cells for one crew member.

Pearce, A. G., Segura, T. M., Rintala, A. C., Rintala-Maki, N. D. and Lee, H. The Generation and Characterization of a Radiation-Resistant Model System to Study Radioresistance in Human Breast Cancer Cells. Radiat. Res. 156, 739–750 (2001).

To systematically study the selection of radioresistant cells in clinically advanced breast cancer, a model system was generated by treating MDA-MB231 breast cancer cells with fractionated γ radiation. A clonogenic assay of the surviving cell populations showed that 2–6 Gy per fraction resulted in a rapid selection of radioresistant populations, within three to five fractions. Irradiation with additional fractions after this initial increase did not increase the radioresistance of the surviving population significantly. Doses of 0.5 and 8 Gy per fraction were not effective in selecting radioresistant cells. To further determine the cause of the changes in radiosensitivity, 15 clones were isolated from the cell populations treated with 40 or 60 Gy with 2 or 4 Gy per fraction, respectively, and were analyzed for radiosensitivity. The average D₁₀ for these clones was 6.75 ± 0.36 Gy, which was higher than that for the parental cell population (D₁₀ = 6.0 ± 0.2 Gy). The operation of cell cycle checkpoints and the doubling time were similar for both the nonirradiated parental population and the isolated radioresistant subclones. In contrast, a decrease in the apoptotic potential was correlated (r = 0.7, P < 0.01) with increased survival after irradiation, suggesting that apoptosis is an important factor in determining radioresistance under our experimental conditions. We also isolated several subclones from the nonirradiated parental cell population and analyzed them to determine their radiosensitivity after fractionated irradiation. Ten fractions of 4 Gy (40 Gy in total) did not result in a significant increase in the radioresistance of these subclones compared to the irradiated cell populations. The possible mechanisms of the increased radioresistance after fractionated irradiation are discussed.

Jeong, S-J., Jin, Y-H., Moon, C-W., Bae, H-R., Yoo, Y-H., Lee, H-S., Lee, S-H., Lim, Y-J., Lee, J-D. and Jeong, M-H. Protein Tyrosine Kinase Inhibitors Modulate Radiosensitivity and Radiation-Induced Apoptosis in K562 Cells. Radiat. Res. 156, 751–760 (2001).

We studied the modulating effect of protein tyrosine kinase inhibitors on the response of cells of the human chronic myelogenous leukemia cell line K562 to radiation. The radiosensitivity of the cells was increased by treatment with herbimycin A and decreased by treatment with genistein. This modulating effect of protein tyrosine kinase inhibitors on radiation sensitivity was associated with the alteration of the mode of radiation-induced cell death. After X irradiation, the cells arrested in the G₂ phase of the cell cycle, but these TP53^–/– cells were unable to sustain cell cycle arrest. This G₂-phase checkpoint deficit caused cell death. The morphological pattern of cell death was characterized by swelling of the cytoplasmic compartments, cytosolic vacuolation, disruption of the plasma membrane, less evident nuclear condensation, and faint DNA fragmentation, all of which were consistent with oncosis or cytoplasmic apoptosis. The nonreceptor protein tyrosine kinase inhibitor herbimycin A accelerated the induction of typical apoptosis by X irradiation, which was demonstrated by morphological assessments using nuclear staining and electron microscopy as well as oligonucleosomal fragmentation and caspase 3 activity. Herbimycin A is known to be a selective antagonist of the BCR/ABL kinase of Philadelphia chromosome-positive K562 cells; this kinase blocks the induction of apoptosis after X irradiation. Our results showed that the inhibition of protein tyrosine kinase by herbimycin A enhanced radiation-induced apoptosis in K562 cells. This effect was associated with the activation of caspase 3 and rapid abrogation of the G₂-phase checkpoint with progression out of G₂ into G₁ phase. In contrast, the receptor-type protein tyrosine kinase inhibitor genistein protected K562 cells from all types of radiation-induced cell death through the inhibition of caspase 3 activity and prolonged maintenance of G₂-phase arrest. Further investigations using this model may give valuable information about the mechanisms of radiation-induced apoptosis and about the radiosensitivity and radioresistance of chronic myelogenous leukemia cells having the Philadelphia chromosome.

Słonina, D., Hoinkis, C. and Dörr, W. Effect of Keratinocyte Growth Factor on Radiation Survival and Colony Size of Human Epidermal Keratinocytes In Vitro. Radiat. Res. 156, 761–766 (2001).

Keratinocyte growth factor (FGF7, also known as KGF) ameliorates the radiation response of mouse oral mucosa and other epithelial tissues. However, the precise mechanisms remain unclear. The aim of the present study was to investigate the effect of FGF7 on the survival and colony size of normal human epidermal keratinocytes in vitro. Primary neonatal keratinocytes (HEKn) were irradiated with doses of 0 and 2 Gy of 200 kV X rays and incubated in the presence or absence of 100 ng/ml FGF7. The plating efficiency (PE) and surviving fraction (SF2) were determined using a clonogenic assay. In cell cultures without FGF7, the mean PE was 4.6 ± 0.2%. Irradiation with 2 Gy resulted in an SF2 of 51 ± 2%. In cell cultures with FGF7, the mean PE was identical, and a similar SF2 of 54 ± 1% was observed (P = 0.4). However, the individual colony size was significantly increased in all cultures incubated with FGF7 compared to those incubated without FGF7. The number of extremely large colonies (≥2 mm) was clearly higher (P < 0.0001) in cultures with FGF7. This was accompanied by a significant reduction in the diameter of individual cells from 29 μm in controls to 23 μm with FGF7. In conclusion, FGF7 does not affect the survival of keratinocytes after irradiation, but it does stimulate proliferation of surviving cells.

Beck, B. D. and Dynlacht, J. R. Heat-Induced Aggregation of XRCC5 (Ku80) in Nontolerant and Thermotolerant Cells. Radiat. Res. 156, 767–774 (2001).

XRCC5 (also known as Ku80) is a component of the DNA-dependent protein kinase (DNA-PK), existing as a heterodimer with G22P1 (also known as Ku70). DNA-PK is involved in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair, and kinase activity is dependent upon interaction of the Ku subunits with the resultant DNA ends. Nuclear XRCC5 is normally extractable with non-ionic detergent; it is found in the soluble cytoplasmic fraction after nuclear isolation with Triton X-100. In this study, we found that heating at 45.5°C causes a decreased extractability of XRCC5 from the nuclei of human U-1 melanoma or HeLa cells. Such decreases in extractability are indicative of protein aggregation within nuclei. Recovery of extractability of XRCC5 to that of unheated control cells was observed after incubation at 37°C after heat shock. The decrease in extractability and the kinetics of recovery were dependent on dose, although the decrease in extractability reached a plateau after heating for 15 min or more. Thermotolerant U-1 cells also showed decreased extractability of XRCC5, but to a lesser degree compared to nontolerant cells. When a comparable initial reduction of extractability of XRCC5 was induced in both thermotolerant and nontolerant cells, the kinetics of recovery was nearly identical. The kinetics of recovery of the extractability of XRCC5 was different from that of total nuclear protein in nontolerant cells; recovery of extractability of XRCC5 occurred faster initially and returned to the level in unheated cells faster than total nuclear protein. Similar results were obtained for thermotolerant cells, with differences between the initial recovery of the extractability of XRCC5 and total protein being particularly evident after longer heating times. Heat has been shown to inactivate XRCC5. We speculate that inactivation of XRCC5 after heat shock results from protein aggregation, and that changes in XRCC5 may, in part, lead to inhibition of DSB repair through inactivation of the NHEJ pathway.

Heikkinen, P., Kosma, V-M., Hongisto, T., Huuskonen, H., Hyysalo, P., Komulainen, H., Kumlin, T., Lahtinen, T., Lang, S., Puranen, L. and Juutilainen, J. Effects of Mobile Phone Radiation on X-Ray-Induced Tumorigenesis in Mice. Radiat. Res. 156, 775–785 (2001).

The increased use of mobile phones has raised the question of possible health effects of such devices, particularly the risk of cancer. It seems unlikely that the low-level radiofrequency (RF) radiation emitted by them would damage DNA directly, but its ability to act as a tumor promoter is less well characterized. In the current study, we evaluated the effect of low-level RF radiation on the development of cancer initiated in mice by ionizing radiation. Two hundred female CBA/S mice were randomized into four equal groups at the age of 3 to 5 weeks. The mice in all groups except the cage-control group were exposed to ionizing radiation at the beginning of the study and then to RF radiation for 1.5 h per day, 5 days a week for 78 weeks. One group was exposed to continuous NMT (Nordic Mobile Telephones)-type frequency-modulated RF radiation at a frequency of 902.5 MHz and a nominal average specific absorption rate (SAR) of 1.5 W/kg. Another group was exposed to pulsed GSM (Global System for Mobile)-type RF radiation (carrier-wave frequency 902.4 MHz, pulse frequency 217 Hz) at a nominal average SAR of 0.35 W/kg. The control animals were sham-exposed. Body weight, clinical signs, and food and water consumption were recorded regularly. Hematological examinations and histopathological analyses of all lesions and major tissues were performed on all animals. The RF-radiation exposures did not increase the incidence of any neoplastic lesion significantly. We conclude that the results do not provide evidence for cancer promotion by RF radiation emitted by mobile phones.

Higashikubo, R., Ragouzis, M., Moros, E. G., Straube, W. L. and Roti Roti, J. L. Radiofrequency Electromagnetic Fields do not Alter the Cell Cycle Progression of C3H 10T½ and U87MG Cells. Radiat. Res. 156, 786–795 (2001).

The effects of exposure to radiofrequency electromagnetic fields (RF EMFs) on cell cycle progression of mouse fibroblasts C3H 10T½ and human glioma U87MG cells were determined by the flow cytometric bromodeoxyuridine pulse-chase method. Cells were exposed to a frequency-modulated continuous wave at 835.62 MHz or a code division multiple access RF EMF centered on 847.74 MHz at an average specific absorption rate of 0.6 W/kg. Five cell cycle parameters, including the transit of cells through G₁, G₂ and S phase and the probability of cell division, were examined immediately after the cells were placed in the fields or after they had been kept in the fields for up to 100 h. The only significant change observed in the study was that associated with C3H 10T½ cell cultures moving into plateau phase toward the later times in the long-exposure experiment. No changes in the cell cycle parameters were observed in cells exposed to either mode of RF EMFs when compared to sham-exposed cells in either of the cell lines studied during the entire experimental period. The results show that exposure to RF EMFs, at the frequencies and power tested, does not have any effect on cell progression in vitro.

Folkard, M., Schettino, G., Vojnovic, B., Gilchrist, S., Michette, A. G., Pfauntsch, S. J., Prise, K. M. and Michael, B. D. A Focused Ultrasoft X-Ray Microbeam for Targeting Cells Individually with Submicrometer Accuracy. Radiat. Res. 156, 796–804 (2001).

The application of microbeams is providing new insights into the actions of radiation at the cell and tissue levels. So far, this has been achieved exclusively through the use of collimated charged particles. One alternative is to use ultrasoft X rays, focused by X-ray diffractive optics. We have developed a unique facility that uses 0.2–0.8-mm-diameter zone plates to focus ultrasoft X rays to a beam of less than 1 μm diameter. The zone plate images characteristic K-shell X rays of carbon or aluminum, generated by focusing a beam of 5–10 keV electrons onto the appropriate target. By reflecting the X rays off a grazing-incidence mirror, the contaminating bremsstrahlung radiation is reduced to 2%. The focused X rays are then aimed at selected subcellular targets using rapid automated cell-finding and alignment procedures; up to 3000 cells per hour can be irradiated individually using this arrangement.

Yasuda, H. Responses of a Direct Ion Storage Dosimeter (DIS-1) to Heavy Charged Particles. Radiat. Res. 156, 805–808 (2001).

The responses of a direct ion storage dosimeter (DIS-1) to energetic heavy charged particles were examined using ⁴He, ¹²C, ⁴⁰Ar and ⁵⁶Fe ion beams at the HIMAC at the National Institute of Radiological Sciences. The efficiency of the DIS-1 on the basis of absorbed dose was almost unity for the helium and carbon ions and was slightly decreased for the argon and iron ions. The linearity in the dose response and the angular independence for these heavy ions were fairly good. Although further studies are necessary, these results suggest that the DIS-1 would be a suitable passive dosimeter for measurements of absorbed dose in a field dominated by heavy charged particles such as the space environment.