Iyer, R. and Lehnert, B. E. Alpha-Particle-Induced Increases in the Radioresistance of Normal Human Bystander Cells. Radiat. Res. 157, 3–7 (2002).

Numerous investigators have reported that direct exposure of cells to a low dose of ionizing radiation can induce a condition of enhanced radioresistance, i.e. a “radioadaptive” response. In this report, we investigated the hypothesis that a radioadaptive bystander effect may be induced in unirradiated cells by a transmissible factor(s) present in the supernatants of cells exposed to a low dose of α particles. Normal human lung fibroblasts (HFL-1) were irradiated with 1 cGy of α particles and their supernatants were transferred to unirradiated HFL-1 cells as a bystander cell model. Compared to directly irradiated cells that were not treated with supernatants from HFL-1 cells exposed to low-dose radiation, such treatment resulted in increased clonogenic survival after subsequent exposure to 10 and 19 cGy of α particles. Increases in protein levels of AP-endonuclease, a redox and DNA base excision repair protein, were found in the bystander cells, but not in directly irradiated cells. Supernatants from α-particle-irradiated cells were also found to increase the clonogenicity of unirradiated cells. These results, in conjunction with our earlier findings that supernatants from cells exposed to a low dose of α particles contain growth-promoting activity, suggest that this new bystander effect may be related to an increase in DNA repair and cell growth/cell cycle regulation.

Vallis, K. A., Chen, Z., Stanford, W. L., Yu, M., Hill, R. P. and Bernstein, A. Identification of Radiation-Responsive Genes In Vitro Using a Gene Trap Strategy Predicts for Modulation of Expression by Radiation In Vivo. Radiat. Res. 157, 8–18 (2002).

A large number of genes are known to be responsive to ionizing radiation, and there is strong evidence for the existence of inducible radiation resistance in mammalian cells. We have developed a gene trap insertional mutagenesis strategy to identify novel genes involved in responses to radiation. Using this approach, we have isolated four gene-trap integrations in embryonic stem cells. In three cases (9A, 3E and 9H) the trapped genes are radiation-inducible, and in one (7D) the gene is down-regulated. Sequence analysis of fusion transcripts from three of the integrations indicate one novel gene (3E), the mouse homologue (9A) of a known but uncharacterized human gene that encodes a protein with significant homology to several GTPase-activating proteins and a murine locus, Mym (9H). The embryonic stem cell clone with the 9A insertion was introduced into the mouse germline, and the in vivo expression pattern of 9A was studied in detail. A unique, spatially restricted pattern of expression in embryos and adult animals was observed. There is tissue-specific in vivo induction of the 9A gene in adult mice by radiation. This study demonstrates the potential of the gene trap approach for the identification and functional analysis of novel radiation-regulated genes. Similar strategies may facilitate the discovery and characterization of genes involved in other cellular stress responses.

Kim, Y. C., Koh, J. T., Shin, B. A., Ahn, K. Y., Choi, B. K., Kim, C. G. and Kim, K. K. An Antisense Construct of Full-Length Human RAD50 cDNA Confers Sensitivity to Ionizing Radiation and Alkylating Agents on Human Cell Lines. Radiat. Res. 157, 19–25 (2002).

In Saccharomyces cerevisiae, Rad50 is reported to participate in the repair of double-stranded DNA breaks, and most rad50 mutants are unable to repair γ-ray-induced DNA damage. In this study, we examined whether human RAD50 is involved in the repair of DNA damage induced by γ radiation, radiomimetic alkylating agents, or UVB radiation in cultured human cells. Because homozygous null RAD50 mutant cells could not be isolated, human 293 embryonic kidney cells and A431 epithelial tumor cells were transfected with antisense RAD50 cDNA to obtain viable cell lines which expressed reduced RAD50. Selected individual clones were subjected to PCR-Southern and Western blot analyses to confirm the integrity of the antisense RAD50 construct and the reduced RAD50 expression levels. The cells engineered to express reduced RAD50 levels showed significantly increased sensitivity to γ radiation, mitomycin C and methylmethane sulfonate compared with control cells that were transfected with the vector alone. However, there were no differences in viability of cells with reduced RAD50 levels and control cells treated with UVB radiation. These results indicate that human RAD50 is involved in the repair of DNA damage induced by γ radiation and alkylating agents in mammalian cells and suggest the possible application of antisense RAD50 cDNA transfection as a radiation sensitizer in radiation oncology.

Jha, M. N., Bamburg, J. R., Bernstein, B. W. and Bedford, J. S. Caffeine Eliminates Gamma-Ray-Induced G₂-Phase Delay in Human Tumor Cells but Not in Normal Cells. Radiat. Res. 157, 26–31 (2002).

It has been known for many years that caffeine reduces or eliminates the G₂-phase cell cycle delay normally seen in human HeLa cells or Chinese hamster ovary (CHO) cells after exposure to X or γ rays. In light of our recent demonstration of a consistent difference between human normal and tumor cells in a G₂-phase checkpoint response in the presence of microtubule-active drugs, we examined the effect of caffeine on the G₂-phase delays after exposure to γ rays for cells of three human normal cell lines (GM2149, GM4626, AG1522) and three human tumor cell lines (HeLa, MCF7, OVGI). The G₂-phase delays after a dose of 1 Gy were similar for all six cell lines. In agreement with the above-mentioned reports for HeLa and CHO cells, we also observed that the G₂-phase delays were eliminated by caffeine in the tumor cell lines. In sharp contrast, caffeine did not eliminate or even reduce the γ-ray-induced G₂-phase delays in any of the human normal cell lines. Since caffeine has several effects in cells, including the inhibition of cAMP and cGMP phosphodiesterases, as well as causing a release of Ca from intracellular stores, we evaluated the effects of other drugs affecting these processes on radiation-induced G₂-phase delays in the tumor cell lines. Drugs that inhibit cAMP or cGMP phosphodiesterases did not eliminate the radiation-induced G₂-phase delay either separately or in combination. The ability of caffeine to eliminate radiation-induced G₂-phase delay was, however, partially reduced by ryanodine and eliminated by thapsigargin, both of which can modulate intracellular calcium, but by different mechanisms. To determine if caffeine was acting through the release of calcium from intracellular stores, calcium was monitored in living cells using a fluorescent calcium indicator, furaII, before and after the addition of caffeine. No calcium release was seen after the addition of caffeine in either OVGI tumor cells or GM2149 normal cells, even though a large calcium release was measured in parallel experiments with ciliary neurons. Thus it is likely that caffeine is eliminating the radiation-induced G₂-phase delay through a Ca -independent mechanism, such as the inhibition of a cell cycle-regulating kinase.

Kobayashi, K., Frohlich, H., Usami, N., Takakura, K. and Le Sech, C. Enhancement of X-Ray-Induced Breaks in DNA Bound to Molecules Containing Platinum: A Possible Application to Hadrontherapy. Radiat. Res. 157, 32–37 (2002).

Complexes made of DNA and chloroterpyridine platinum (PtTC) bound to plasmid DNA were placed in aqueous solution and irradiated with monochromatic X rays tuned to the resonant photoabsorption energy of the L_III shell of the platinum atom. The number of single- and double-strand breaks (SSBs and DSBs) induced by irradiation on a supercoiled DNA plasmid was measured by the production of the circular-nicked and linear forms. To distinguish the contribution of the direct effects of ionization from the indirect effects due to a free radical attack, experiments were also performed in the presence of a hydroxyl free radical scavenger, dimethyl sulfoxide (DMSO). An enhancement of the number of SSBs and DSBs was observed when the plasmids contained the platinum intercalating molecules. A quantitative analysis was made to evaluate the respective contributions of the direct effects (Auger effect) and the indirect effects (free radical attack) to the number of DNA strand breaks. Even when off-resonant X rays were used, the strand break efficiency remained higher than expected based upon the absorption cross section, suggesting that the platinum bound to DNA might be increasing the yield of strand breaks. A mechanism is suggested that involves photoelectrons generated from the ionization of water which efficiently ionize platinum atoms. If this mechanism is correct, then heavy atoms, with a large cross section for ionization by electrons that are bound to the DNA, should behave as a radiosensitizer. This observation may provide insight into understanding the effects of new radiotherapy protocols, related chemotherapeutic agents such as cisplatin, and conventional radiotherapy for the treatment of tumors. A possible way to deliver the dose selectively in a well-defined volume, which uses the properties of the linear energy transfer of atomic ions interacting with matter, is suggested.

Aydogan, B., Marshall, D. T., Swarts, S. G., Turner, J. E., Boone, A. J., Richards, N. G. and Bolch, W. E. Site-Specific OH Attack to the Sugar Moiety of DNA: A Comparison of Experimental Data and Computational Simulation. Radiat. Res. 157, 38–44 (2002).

Little computational or experimental information is available on site-specific hydroxyl attack probabilities to DNA. In this study, an atomistic stochastic model of OH radical reactions with DNA was developed to compute relative OH attack probabilities at individual deoxyribose hydrogen atoms. A model of the self-complementary decamer duplex d(CCAACGTTGG) was created including Na counter ions and the water molecules of the first hydration layer. Additionally, a method for accounting for steric hindrance from nonreacting atoms was implemented. The model was then used to calculate OH attack probabilities at the various C-H sites of the sugar moiety. Results from this computational model show that OH radicals exhibit preferential attack at different deoxyribose hydrogens, as suggested by their corresponding percentage solvent-accessible surface areas. The percentage OH attack probabilities for the deoxyribose hydrogens [1H(5′) 2H(5′), H(4′), H(3′), 1H(2′) 2H(2′), H(1′)] were calculated as approximately 54.6%, 20.6%, 15.0%, 8.5% and 1.3%, respectively, averaged across the sequence. These results are in good agreement with the latest experimental site-specific DNA strand break data of Balasubramanian et al. [Proc. Natl. Acad. Sci. USA 95, 9738–9742 (1998)]. The data from this stochastic model suggest that steric hindrance from nonreacting atoms significantly influences site-specific hydroxyl radical attack probabilities in DNA. A number of previous DNA damage models have been based on the assumption that C(4′) is the preferred site, or perhaps the only site, for OH-mediated DNA damage. However, the results of the present study are in good agreement the experimental results of Balasubramanian et al. in which OH radicals exhibit preferential initial attack at sugar hydrogen atoms in the order 1H(5′) 2H(5′) > H(4′) > H(3′) > 1H(2′) 2H(2′) > H(1′).

Ning, S., Laird, D., Cherrington, J. M. and Knox, S. J. The Antiangiogenic Agents SU5416 and SU6668 Increase the Antitumor Effects of Fractionated Irradiation. Radiat. Res. 157, 45–51 (2002).

Angiogenesis is critical for tumor development, growth and metastasis. The vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF) and their tyrosine kinase receptors are major regulators of angiogenesis. Radiation induces the production of VEGF, FGF and PDGF in many tumor cells. We hypothesized that inhibition of the function of these growth factors could inhibit tumor angiogenesis and thereby enhance the efficacy of radiation therapy. To test this hypothesis, we used the small molecule inhibitors SU5416 (an inhibitor for Vegf receptor) and SU6668 (an inhibitor for Vegf, Fgf and Pdgf receptors) alone and in combination with fractionated irradiation to treat C3H mice bearing SCC VII carcinomas. The SCC VII tumors express Vegf, Fgf2 (also known as bFGF), Pdgf and their associated receptors. Animals were given either SU5416 or SU6668 daily before or after irradiation (2 Gy per fraction per day for 5 days). The results from these experiments demonstrate that administration of either SU5416 or SU6668 without radiation delayed tumor growth. Administration of SU5416 at a dose of 25 mg/kg per day (the maximum tolerated effective dose) inhibited tumor growth by 17.9% on day 7 (P < 0.05 compared to untreated control mice) and produced an average tumor growth delay time of 0.5–2.0 days. When combined with fractionated irradiation, administration of SU5416 increased the inhibition of tumor growth to 50–53% on day 7 and the tumor growth delay time to 5.7–6.5 days (P < 0.001 compared with SU5416 alone; P ≤ 0.05 compared with radiation alone). SU6668 alone inhibited tumor growth in a dose-dependent manner. Administration of SU6668 at a dose of 75 mg/kg per day (a suboptimal dose) inhibited tumor growth by 36% on day 7 and produced an average tumor growth delay time of 3.3 ± 1.4 days. The combination of SU6668 with fractionated radiation increased inhibition of tumor growth to 66–70% and the tumor growth delay time from 3.3 days to 11.9 days (P ≤ 0.001 compared with either radiation alone or SU6668 alone). Administration of these agents before or after irradiation produced similar results (P = 0.40 for SU5416; P = 0.98 for SU6668). SU5416 or SU6668 alone or in combination with radiation was very well tolerated with little or no toxicity. These results suggest that inhibition of Vegf, Fgf and Pdgf receptor function by SU5416 and SU6668 can enhance the efficacy of irradiation. The targeting of multiple tyrosine kinase receptors by SU6668 is more effective than inhibition of the Vegf receptor alone by SU5416 for the enhancement of tumor cell killing by fractionated irradiation.

Dublineau, I., Morel, E. and Griffiths, N. M. Characterization of Altered Absorptive and Secretory Functions in the Rat Colon after Abdominal Irradiation: Comparison with the Effects of Total-Body Irradiation. Radiat. Res. 157, 52–61 (2002).

The aim of this work was to determine the alterations in the absorptive and secretory functions of the rat colon after abdominal irradiation and to compare the effects of abdominal and whole-body irradiation. Rats received an abdominal irradiation with 8 to 12 Gy and were studied at 1, 4 and 7 days after exposure. Water and electrolyte absorption was measured in vivo by insertion of an agarose cylinder into the colons of anesthetized rats. In vitro measurements of potential difference, short-circuit current and tissue conductance were performed in Ussing chambers under basal and agonist-stimulated conditions. Most of the changes appeared at 4 days after abdominal irradiation. At this time, a decrease in water and electrolyte absorption in the colon was observed for radiation doses ≥9 Gy. The response to secretagogues (VIP, 5-HT and forskolin) was attenuated after 10 and 12 Gy. Epithelial integrity, estimated by potential difference and tissue conductance, was altered from 1 to 7 days after 12 Gy abdominal irradiation. These results show that the function of the colon was affected by abdominal irradiation. Comparison with earlier results for total-body irradiation demonstrated a difference of 2 Gy in the radiation dose needed to induce changes in the function of the colon.

Pateder, D. B., Sheu, T. J., O'Keefe, R. J., Puzas, J. E., Schwarz, E. M., Constine, L. S., Okunieff, P. and Rosier, R. N. Role of Pentoxifylline in Preventing Radiation Damage to Epiphyseal Growth Plate Chondrocytes. Radiat. Res. 157, 62–68 (2002).

Radiation therapy plays an important role as part of multimodality treatment for a number of childhood malignancies. The damaging effects of radiation on bone formation in children have been well documented. Recent work suggests that the postirradiation increase in cytosolic calcium is probably responsible for the deleterious effects of radiation on growth plate chondrocytes because it causes a specific suppression of the mitogen PTHrP. Using an in vitro model of avian growth plate chondrocytes, this study demonstrates that pentoxifylline is effective in increasing basal PTHrP mRNA levels and partially preventing the radiation-induced decrease in PTHrP mRNA. This effect of pentoxifylline is probably due to its ability to lower basal levels of cytosolic calcium and the radiation-induced increase in cytosolic calcium in chondrocytes. Pentoxifylline also prevented the radiation-induced decreases in [³H]thymidine uptake and BCL2 and PTHrP receptor mRNA levels in chondrocytes. The effects of pentoxifylline appear to be specific for the PTHrP signaling pathway because it did not alter basal TGFB mRNA levels or TGFB mRNA expression in irradiated chondrocytes. The results of the current study suggest that by decreasing basal cytosolic calcium levels and curtailing the radiation-induced increase in cytosolic calcium levels in chondrocytes, pentoxifylline is able to sustain PTHrP signaling in chondrocytes and maintains the proliferative signal that is necessary to prevent chondrocytes from undergoing apoptosis.

Badhwar, G. D. Shuttle Radiation Dose Measurements in the International Space Station Orbits. Radiat. Res. 157, 69–75 (2002).

The International Space Station (ISS) is now a reality with the start of a permanent human presence on board. Radiation presents a serious risk to the health and safety of the astronauts, and there is a clear requirement for estimating their exposures prior to and after flights. Predictions of the dose rate at times other than solar minimum or solar maximum have not been possible, because there has been no method to calculate the trapped-particle spectrum at intermediate times. Over the last few years, a tissue-equivalent proportional counter (TEPC) has been flown at a fixed mid-deck location on board the Space Shuttle in 51.65° inclination flights. These flights have provided data that cover the expected changes in the dose rates due to changes in altitude and changes in solar activity from the solar minimum to the solar maximum of the current 23rd solar cycle. Based on these data, a simple function of the solar deceleration potential has been derived that can be used to predict the galactic cosmic radiation (GCR) dose rates to within ±10%. For altitudes to be covered by the ISS, the dose rate due to the trapped particles is found to be a power-law function, ρ^−2/3, of the atmospheric density, ρ. This relationship can be used to predict trapped dose rates inside these spacecraft to ±10% throughout the solar cycle. Thus, given the shielding distribution for a location inside the Space Shuttle or inside an ISS module, this approach can be used to predict the combined GCR trapped dose rate to better than ±15% for quiet solar conditions.

Badhwar, G. D., Atwell, W., Badavi, F. F., Yang, T. C. and Cleghorn, T. F. Space Radiation Absorbed Dose Distribution in a Human Phantom. Radiat. Res. 157, 76–91 (2002).

The radiation risk to astronauts has always been based on measurements using passive thermoluminescent dosimeters (TLDs). The skin dose is converted to dose equivalent using an average radiation quality factor based on model calculations. The radiological risk estimates, however, are based on organ and tissue doses. This paper describes results from the first space flight (STS-91, 51.65° inclination and ∼380 km altitude) of a fully instrumented Alderson Rando™ phantom torso (with head) to relate the skin dose to organ doses. Spatial distributions of absorbed dose in 34 1-inch-thick sections measured using TLDs are described. There is about a 30% change in dose as one moves from the front to the back of the phantom body. Small active dosimeters were developed specifically to provide time-resolved measurements of absorbed dose rates and quality factors at five organ locations (brain, thyroid, heart/lung, stomach and colon) inside the phantom. Using these dosimeters, it was possible to separate the trapped-proton and the galactic cosmic radiation components of the doses. A tissue-equivalent proportional counter (TEPC) and a charged-particle directional spectrometer (CPDS) were flown next to the phantom torso to provide data on the incident internal radiation environment. Accurate models of the shielding distributions at the site of the TEPC, the CPDS and a scalable Computerized Anatomical Male (CAM) model of the phantom torso were developed. These measurements provided a comprehensive data set to map the dose distribution inside a human phantom, and to assess the accuracy and validity of radiation transport models throughout the human body. The results show that for the conditions in the International Space Station (ISS) orbit during periods near the solar minimum, the ratio of the blood-forming organ dose rate to the skin absorbed dose rate is about 80%, and the ratio of the dose equivalents is almost one. The results show that the GCR model dose-rate predictions are 20% lower than the observations. Assuming that the trapped-belt models lead to a correct orbit-averaged energy spectrum, the measurements of dose rates inside the phantom cannot be fully understood. Passive measurements using ⁶Li- and ⁷Li-based detectors on the astronauts and inside the brain and thyroid of the phantom show the presence of a significant contribution due to thermal neutrons, an area requiring additional study.

Nikezic, D. and Yu, K. N. Distributions of Specific Energy in Sensitive Layers of the Human Respiratory Tract. Radiat. Res. 157, 92–98 (2002).

The purpose of the present work was to calculate the specific energy distribution in sensitive cells of the human lung. Specific energy distributions were calculated by applying Monte Carlo methods and the ICRP 66 model of the human respiratory tract. Specific energies were calculated at various depths in the epithelium and combined according to the relative cell abundance. Distributions are given for various combinations of sources, α-particle energies, targets and regions of the lung. The chord length does not follow the triangular distribution when the particle range is comparable to the diameter of the target. The notion of “effective volume” is introduced and defined, which is needed for estimation of hit frequency in some particular targets. It has been shown that basal cells are subjected to a larger proportion of α-particle hits with small energy transfer than secretory cells. Small energy transfer events that lead to minor damage of the DNA are more efficient in cancer induction than are hits with large energy deposition that lead to cell killing.

Frankenberg, D., Kelnhofer, K., Bär, K. and Frankenberg-Schwager, M. Enhanced Neoplastic Transformation by Mammography X Rays Relative to 200 kVp X Rays: Indication for a Strong Dependence on Photon Energy of the RBE_M for Various End Points. Radiat. Res. 157, 99–105 (2002).

The fundamental assumption implicit in the use of the atomic bomb survivor data to derive risk estimates is that the γ rays of Hiroshima and Nagasaki are considered to have biological efficiencies equal to those of other low-LET radiations up to 10 keV/μm, including mammography X rays. Microdosimetric and radiobiological data contradict this assumption. It is therefore of scientific and public interest to evaluate the efficiency of mammography X rays (25–30 kVp) to induce cancer. In this study, the efficiency of mammography X rays relative to 200 kVp X rays to induce neoplastic cell transformation was evaluated using cells of a human hybrid cell line (CGL1). For both radiations, a linear-quadratic dose–effect relationship was observed for neoplastic transformation of CGL1 cells; there was a strong linear component for the 29 kVp X rays. The RBE_M of mammography X rays relative to 200 kVp X rays was determined to be about 4 for doses ≤0.5 Gy. A comparison of the electron fluences for both X rays provides strong evidence that electrons with energies of ≤15 keV can induce neoplastic transformation of CGL1 cells. Both the data available in the literature and the results of the present study strongly suggest an increase of RBE_M for carcinogenesis in animals, neoplastic cell transformation, and clastogenic effects with decreasing photon energy or increasing LET to an RBE_M approximately 8 for mammography X rays relative to ⁶⁰Co γ rays.

Radivoyevitch, T., Kozubek, S. and Sachs, R. K. The Risk of Chronic Myeloid Leukemia: Can the Dose–Response Curve be U-Shaped? Radiat. Res. 157, 106–109 (2002).

Chronic myeloid leukemia (CML) is caused by a BCR-ABL chromosome translocation in a primitive hematopoietic stem cell. The number of hematopoietic stem cells in the body is thus a major factor in CML risk. Evidence suggests that the number of hematopoietic stem cells in the body is only loosely regulated, having a broad “dead-band” of physiologically acceptable values. The existence of a dead-band is important, because it would imply that low levels of hematopoietic stem cell killing can be permanent; i.e., it would imply that low doses of ionizing radiation can cause permanent reductions in the total number of CML target cells and thus permanent reductions in the subsequent risk of spontaneous CML. Such reductions in risk could be substantial if hematopoietic stem cells are also hypersensitive to radiation killing at low dose. Our calculations indicate that, due to dead-band hematopoietic stem cell control, if hematopoietic stem cells are as hypersensitive to killing at low doses as epithelial cells, reductions in the spontaneous CML risk could exceed the low-dose risks of induced CML; i.e., the net lifetime CML risk could have a U-shaped dose–response curve.