Chang, P. Y., Bjornstad, K. A., Chang, E., McNamara, M., Barcellos-Hoff, M. H., Lin, S. P., Aragon, G., Polansky, J. R., Lui, G. M. and Blakely, E. A. Particle Irradiation Induces FGF2 Expression in Normal Human Lens Cells.

Particle radiations, including both proton and helium-ion beams, have been used to successfully treat choroidal melanoma, but with the complication of radiation-induced cataract. We have investigated a role for radiation-induced changes in the expression of basic fibroblast growth factor (FGF2) gene expression as part of the mechanism(s) underlying lens cell injury associated with cataract. Normal human lens epithelial (HLE) cells were cultured in vitro on extracellular matrix (ECM) originated from bovine corneal endothelial cells. This study reports evidence for rapid but transient induction of FGF2 transcripts, an increase of between 5- and 8-fold, within 0.5 h after exposure to particle radiation, followed by another wave of increased transcription at 2–3 h postirradiation. Immunofluorescence results confirm the enhanced levels of FGF2 protein rapidly after exposure to protons or helium ions, followed by another wave of increased activity unique to helium at 6 h postirradiation. This second wave of increased immunoreactivity was not observed in the proton-irradiated samples. Total FGF2 protein analysis after helium-ion exposures shows induced expression of three FGF2 isoforms, with an increase of up to 2-fold in the 18-kDa low-molecular-weight species. Studies of the effects of protons on individual FGF2 protein isoforms are in progress. Several mechanisms involving a role for FGF2 in radiation-induced cataract are discussed.

Furusawa, Y., Fukutsu, K., Aoki, M., Itsukaichi, H., Eguchi-Kasai, K., Ohara, H., Yatagai, F., Kanai, T. and Ando, K. Inactivation of Aerobic and Hypoxic Cells from Three Different Cell Lines by Accelerated ³He-, ¹²C- and ²⁰Ne-Ion Beams.

The LET-RBE spectra for cell killing for cultured mammalian cells exposed to accelerated heavy ions were investigated to design a spread-out Bragg peak beam for cancer therapy at HIMAC, National Institute of Radiological Sciences, Chiba, prior to clinical trials. Cells that originated from a human salivary gland tumor (HSG cells) as well as V79 and T1 cells were exposed to ³He-, ¹²C- and ²⁰Ne-ion beams with an LET ranging from approximately 20–600 keV/μm under both aerobic and hypoxic conditions. Cell survival curves were fitted by equations from the linear-quadratic model and the target model to obtain survival parameters. RBE, OER, α and D₀ were analyzed as a function of LET. The RBE increased with LET, reaching a maximum at around 200 keV/μm, then decreased with a further increase in LET. Clear splits of the LET-RBE or -OER spectra were found among ion species and/or cell lines. At a given LET, the RBE value for ³He ions was higher than that for the other ions. The position of the maximum RBE shifts to higher LET values for heavier ions. The OER value was 3 for X rays but started to decrease at an LET of around 50 keV/μm, passed below 2 at around 100 keV/μm, and then reached a minimum above 300 keV/μm, but the values remained greater than 1. The OER was significantly lower for ³He ions than the others.

Kinnaert, E., Morandini, R., Simon, S., Hill, H. Z., Ghanem, G. and Van Houtte, P. The Degree of Pigmentation Modulates the Radiosensitivity of Human Melanoma Cells.

The relationship between cell pigmentation and radiosensitivity was investigated in two selected human melanoma cell lines with different melanin content (mixed type: eumelanin and pheomelanin, and pheomelanotic phenotypes). The same study was also done after stimulation of melanogenesis (1) by addition of the melanin precursor l-tyrosine to each of the cell lines separately and (2) by irradiation alone with doses ranging from 0 to 10 Gy. We found that a decrease in cell radiosensitivity was correlated with the type of melanin, with a clear involvement of eumelanin rather than pheomelanin. Increasing the intracellular content of both melanins promoted the growth of irradiated cells. Moreover, at a dose of 10 Gy, both tyrosinase activity and melanin cell content were significantly increased in the absence of any other melanogenesis promoter. Our data suggest that the amount of intracellular melanin is inversely related to the radiosensitivity of melanoma cells and may explain at least in part the controversial responses to ionizing radiations reported for melanoma.

Al-Assar, O., Robson, T., McKeown, S. R., Gardin, I., Wilson, G. D. and Hirst, D. G. Regulation of FOS by Different Compartmental Stresses Induced by Low Levels of Ionizing Radiation.

We irradiated different cellular compartments and measured changes in expression of the FOS gene at the mRNA and protein levels. [³H]Thymidine and tritiated water were used to irradiate the nucleus and the whole cell, respectively. ¹²⁵I-Concanavalin A binding was used to irradiate the cell membrane differentially. Changes in FOS mRNA and protein levels were measured using semi-quantitative RT-PCR and SDS-PAGE Western blotting, respectively. Irradiation of the nucleus or the whole cell at a dose rate of 0.075 Gy/h caused no change in the level of FOS mRNA expression, but modestly (1.5-fold) induced FOS protein after 0.5 h. Irradiation of the nucleus at a dose rate of 0.43 Gy/h induced FOS mRNA by 1.5-fold after 0.5 h, but there was no significant effect after whole-cell irradiation. FOS protein was transiently induced 2.5-fold above control levels 0.5 h after a 0.43-Gy/h exposure of the nucleus or the whole cell. Irradiation of the cell membrane at a dose rate of 1.8 Gy/h for up to 2 h caused no change in the levels of expression of FOS mRNA or protein, but a dose rate of 6.8 Gy/h transiently increased the level of FOS mRNA 3-fold after 0.5 h. These data demonstrate the complexity of the cellular response to radiation-induced damage at low doses. The lack of quantitative agreement between the transcript and protein levels for FOS suggests a role for post-transcriptional regulation.

Dynlacht, J. R., Roberts, Z. V., Earles, M., Henthorn, J. and Seno, J. D. Different Patterns of DNA Fragmentation and Degradation of Nuclear Matrix Proteins during Apoptosis Induced by Radiation, Hyperthermia or Etoposide.

Several nuclear matrix proteins are substrates for proteolytic cleavage during apoptosis. Using Western blotting, the temporal patterns of cleavage of three nuclear matrix proteins (lamin B, NUMA and the nucleoporin TPR) were compared in HL60 cells induced to undergo apoptosis after irradiation, heat shock or treatment with etoposide. Flow cytometry was used to compare the kinetics of post-cleavage degradation of lamin B, NUMA and TPR after irradiation, and to correlate DNA fragmentation with protein degradation in cells induced to undergo apoptosis with different agents. During radiation-induced apoptosis, cleavage and subsequent degradation of lamin B, NUMA and TPR occurred with different kinetics. Low-molecular-weight DNA fragmentation occurred subsequent to the initiation of NUMA cleavage, coincided with lamin B cleavage, but occurred before more extensive degradation of lamin B and NUMA. A similar sequence was observed for cells treated with etoposide. However, during heat-induced apoptosis, cleavage of lamin B and NUMA occurred much sooner compared to other agents, with NUMA cleaved into multiple fragments within 15 min after heating. We conclude that the hierarchical sequence and kinetics of degradative events contributing to nuclear disassembly during apoptosis are highly dependent on the inducing agent. Furthermore, the nuclear pore complex, like the nuclear lamina and internal nuclear matrix, is a target for proteolytic cleavage.

Nguyen, V., Gaber, M. W., Sontag, M. R. and Kiani, M. F. Late Effects of Ionizing Radiation on the Microvascular Networks in Normal Tissue.

Damage to the microvascular networks constitutes one of the most important components of ionizing radiation damage to normal tissue. Previously, we have reported the early (3, 7 and 30 days postirradiation) effects of ionizing radiation on the structure and function of normal tissue microvascular networks. Here we report on the late effects of ionizing radiation on the structural and functional changes in microvascular networks in locally irradiated (single 10-Gy dose) hamster cremaster muscles observed 60, 120 and 180 days postirradiation; age-matched animals were used as controls. As in the previous study, intravital microscopy was used to measure structural and functional parameters in complete microvascular networks in vivo. A factorial design was used to examine the effects of radiation status, time postirradiation, and network vessel type on the structure and function of microvascular networks. Our results indicate that the progression of radiation-induced microvascular damage continues during the late times but that there is partial recovery from radiation damage within 6 months postirradiation. Red blood cell flux, red blood cell velocity, and capillary blood flow in irradiated networks at 180 days postirradiation were significantly greater than control levels. As at the early times, all vessel types were not damaged equally by radiation at every time.

Cromheecke, M., Piers, B. A., Beekhuis, H., ter Veen, H., Sluiter, W. J., Grond, J. A. K. and Hoekstra, H. J. Tissue Damage after Single High-Dose Intraoperative Irradiation of the Canine Liver: Evaluation in Time by Means of Radionuclide Imaging and Light Microscopy.

To establish the tolerance of liver tissue to single high-dose intraoperative irradiation, the histopathological changes in the canine liver after single high-dose intraoperative irradiation were investigated by means of radionuclide imaging and light microscopy. Intraoperative irradiation at doses of 0, 10, 20, 25 or 30 Gy was applied to a part of the liver of 25 beagles. Radionuclide imaging using ^99mTc-sulfur colloid was performed at several times during follow-up. Elective humane killing was done 3 months and 1, 2, 3 and 5 years after irradiation. Light microscopy was used to identify histopathological alterations. There was no morbidity or mortality during a maximal follow-up of 5 years. In 40% of the animals, a region of diminished uptake was observed at the irradiation site. The regions of diminished uptake of the radiopharmaceutical agent became smaller with time. Light microscopic examination revealed severe parenchymal fibrosis, liver cell atrophy, and bile duct proliferation at the irradiated area 1 to 2 years after irradiation. At 3 and 5 years, vascular changes with endothelial proliferation and focal arteriolar hyalinosis were observed. This study demonstrates that intraoperative irradiation of a part of the liver in the canine model can be applied safely. Light microscopy confirmed that histological damage was not always accompanied by diminished uptake of the radiopharmaceutical agent at the irradiation site.

Bussink, J., Kaanders, J. H. A. M., Strik, A. M., Vojnovic, B. and van der Kogel, A. J. Optical Sensor-Based Oxygen Tension Measurements Correspond with Hypoxia Marker Binding in Three Human Tumor Xenograft Lines.

Hypoxia has a negative effect on the outcome of radiotherapy and surgery and is also related to an increased incidence of distant metastasis. In this study, tumor pO₂ measurements using a newly developed time-resolved luminescence-based optical sensor (OxyLite™) were compared with bioreductive hypoxia marker binding (pimonidazole). Single pO₂ measurements per tumor were compared to hypoxia marker binding in tissue sections using image analysis. Both assays were performed in the same tumors of three human tumor lines grown as xenografts. Both assays demonstrated statistically significant differences in the oxygenation status of the three tumor lines. There was also a good correlation between hypoxia marker binding and the pO₂ measurements with the OxyLite™ device. A limitation of the OxyLite™ system is that it is not yet suited for sampling multiple sites in one tumor. An important strength is that continuous measurements can be taken at the same position and dynamic information on the oxygenation status of tumors can be obtained. The high spatial resolution of the hypoxia marker binding method can complement the limitations of the OxyLite™ system. In the future, a bioreductive hypoxic cell marker for global assessment of tumor hypoxia may be combined with analysis of temporal changes in pO₂ with the OxyLite™ to study the effects of oxygenation-modifying treatment on an individual basis.

Goorley, T. and Nikjoo, H. Electron and Photon Spectra for Three Gadolinium-Based Cancer Therapy Approaches.

Some recent neutron capture therapy research has focused on using compounds containing the element gadolinium, which produces internal conversion and Auger cascade electrons. The low-energy, short-range Auger electrons are absorbed locally and increase cell killing dramatically as the gadolinium compounds are introduced into the cell nucleus and bind to the DNA. Detailed electron and photon spectra are needed for biophysical modeling and Monte Carlo calculations of damage to DNA. This paper presents calculated electron and photon spectra for three cases: thermal neutron absorption by ¹⁵⁷Gd, the β-particle decay of ¹⁵⁹Gd, and the K-shell photoelectric event in gadolinium. The Monte Carlo sampling of atomic and nuclear transitions for each of the three cases was used to calculate a large number of representative decays. The sampled decays were used to determine average emissions and energy deposited in small spheres of tissue. The kinetic energy nuclear recoil from γ-ray and electron emissions was calculated and found to be more than 10 eV for 26% of all ¹⁵⁷Gd neutron capture reactions.

Mercier, J. R., Kopp, D. T., McDavid, W. D., Dove, S. B., Lancaster, J. L. and Tucker, D. M. Measurement and Validation of Benchmark-Quality Thick-Target Tungsten X-Ray Spectra below 150 kVp.

Pulse-height distributions of two constant potential X-ray tubes with fixed anode tungsten targets were measured and unfolded. The measurements employed quantitative alignment of the beam, the use of two different semiconductor detectors (high-purity germanium and cadmium-zinc-telluride), two different ion chamber systems with beam-specific calibration factors, and various filter and tube potential combinations. Monte Carlo response matrices were generated for each detector for unfolding the pulse-height distributions into spectra incident on the detectors. These response matrices were validated for the low error bars assigned to the data. A significant aspect of the validation of spectra, and a detailed characterization of the X-ray tubes, involved measuring filtered and unfiltered beams at multiple tube potentials (30–150 kVp). Full corrections to ion chamber readings were employed to convert normalized fluence spectra into absolute fluence spectra. The characterization of fixed anode pitting and its dominance over exit window plating and/or detector dead layer was determined. An Appendix of tabulated benchmark spectra with assigned error ranges was developed for future reference.

Iida, Y., Inanami, O., Inoue, H., Ohtsuka, E. and Kuwabara, M. Unique Response of Double-Stranded Oligonucleotides Containing a Single 8-Oxo-7,8-Dihydroguanine to Gamma Rays in the Frozen Aqueous State at 77 K.

Two kinds of double-stranded oligonucleotides containing a single 8-oxo-7,8-dihydroguanine were labeled with ³²P at their 5′ ends and exposed to γ rays in the frozen aqueous state at 77 K, where both direct and quasi-direct effects of ionizing radiation predominate. Analysis of the oligonucleotides with 20% denaturing polyacrylamide gel electrophoresis revealed no difference in the immediate induction of strand breaks between oligonucleotides containing 8-oxo-7,8-dihydroguanine and their corresponding oligonucleotides with normal guanine, but piperidine-sensitive damage was induced more frequently in the former than in the latter. Sequence analysis of irradiated oligonucleotides showed that not only 8-oxo-7,8-dihydroguanine but also its neighboring bases and the cytosine residue that is paired to it became piperidine-sensitive in both oligonucleotides. These results suggest that 8-oxo-7,8-dihydroguanine, its neighboring bases and the opposite cytosine are candidates for radiation damage hot spots.

Carlson, C. A. and Ethier, S. P. Lack of RB Protein Correlates with Increased Sensitivity to UV-Radiation-Induced Apoptosis in Human Breast Cancer Cells.

The underlying causes for different apoptotic responses in neoplastic cells are still not fully understood. We demonstrate here that a human breast cancer cell line, MDA-MB-468, which lacks the retinoblastoma protein (RB), is particularly sensitive to low doses of ultraviolet (UV) radiation. These cells are 15–20-fold more sensitive to UV radiation than RB-positive cell lines, as measured by both apoptosis and clonogenic assays. In addition, a prostate cancer cell line that lacks functional RB, DU-145, was found to have a similar apoptotic response to low doses of UV radiation. Based on these data, we hypothesized that the lack of RB is responsible for the extreme sensitivity of these cells to UV-radiation-induced apoptosis. To further examine the role of RB in apoptosis, cells of RB-positive human breast cancer and normal cell lines were infected with the human papilloma virus type 16 (HPV-16) E7 and assessed for UV-radiation sensitivity. The HPV-16 E7 protein is known to decrease levels of free RB in cells. Infection of RB-positive human breast cancer or normal cells with E7 resulted in a 4–5-fold increase in sensitivity to UV radiation compared to controls. The above data suggest a role for the RB protein in protecting cells from undergoing apoptosis in response to UV radiation.

Jordan, A., Schmidt, W. and Scholz, R. A New Model of Thermal Inactivation and its Application to Clonogenic Survival Data for WiDr Human Colonic Adenocarcinoma Cells.

Based on the analysis of clonogenic survival data for human colonic adenocarcinoma cells (WiDr) after a single heating, a new model is proposed to describe cell survival after hyperthermia quantitatively. The effects of heat are explained as heat-induced cell damage assuming a first-order (single-hit) and a second-order (cumulative damage) process. Thus cell survival at a specified temperature can be described by the linear-quadratic (LQ) model. The proposed model is based on an alternative definition of the (single) thermal dose, given as the (normalized) product of heating time and a specified nonlinear function of the increase in temperature (relative to a threshold temperature) to be interpreted as the thermal dose rate. In further analogy to the modeling of the effects of low-dose-rate radiation, an inherent capacity of the cells to repair sublethal damage is assumed, and these effects are quantified by the usual g factor measuring incomplete repair effects. The model defines thermal dose–response and isoeffect dose relationships, enabling a direct (i.e. single-step) analysis of the available thermal response data. Additionally, the analysis of our data based on heating times in the range from 0 to 360 min and temperatures from 41 to 46°C and covering a broad spectrum of different densities of cells seeded for colony formation did not yield any evidence of the existence of a breaking point usually derived from Arrhenius plots based on the single-hit, multitarget model and the Arrhenius equation. The model includes no specific assumptions describing the development of thermotolerance, which can be assumed to be negligible under our experimental conditions. The proposed thermal dose–response model correlates satisfactorily with the in vitro survival data for WiDr adenocarcinoma cells.