Deveaux, Y., Alonso, B., Pierrugues, O., Godon, C. and Kazmaier, M. Molecular Cloning and Developmental Expression of AtGR1, a New Growth-Related Arabidopsis Gene Strongly Induced by Ionizing Radiation.

Screening for mRNAs that accumulate after DNA damage induced by ionizing radiation, we have isolated a 2.0-kb cDNA coding for a new Arabidopsis PEST-box protein named AtGR1 (A. thaliana gamma response 1) with an expression profile similar to that observed for several plant cell cycle-related proteins. Using an anti-AtGR1 antibody, we have shown that the AtGR1 protein is expressed at basal levels in mitotically dividing cells (meristematic tissues and organ primordia) and at a strongly enhanced level in endoreduplicating cells (stipules, trichomes). Using transgenic Arabidopsis plants that express the GUS reporter gene under the control of the AtGR1 promoter, we have demonstrated that the observed AtGR1 protein distribution is due to the promoter activity. Our results suggest that basal AtGR1 levels are associated with progression through mitosis, whereas elevated intracellular levels of AtGR1 seem to induce changes between the S and M phases of the cell cycle that trigger somatic cells to enter the endoreduplication cycle. Ionizing radiation-induced rapid and dose-dependent accumulation of AtGR1 mRNA in cell cultures and plant tissues leads to tissue-specific accumulation of AtGR1 protein, best observed in ovules, which never undergo an endoreduplication cycle. It therefore appears that the radiation-induced transient AtGR1 accumulation reflects DNA damage-dependent transient cell cycle arrest before mitosis, which is necessary to accomplish DNA repair prior to chromosome segregation and cytokinesis.

Epperly, M. W., Epstein, C. J., Travis, E. L. and Greenberger, J. S. Decreased Pulmonary Radiation Resistance of Manganese Superoxide Dismutase (MnSOD)-Deficient Mice is Corrected by Human Manganese Superoxide Dismutase-Plasmid/Liposome (SOD2-PL) Intratracheal Gene Therapy.

The pulmonary ionizing radiation sensitivity of C57BL/6 Sod2 ^/– mice heterozygous for MnSOD deficiency was compared to that Sod2 ^/ control littermates. Embryo fibroblast cell lines from Sod2^–/– (neonatal lethal) or Sod2 ^/– mice produced less biochemically active MnSOD and demonstrated a significantly greater in vitro radiosensitivity. No G₂/M-phase cell cycle arrest after 5 Gy was observed in Sod2^–/– cells compared to the Sod2 ^/– or Sod2 ^/ lines. Subclonal Sod2^–/– or Sod2 ^/– embryo fibroblast lines expressing the human SOD2 transgene showed increased biochemical activity of MnSOD and radioresistance. Sod2 ^/– mice receiving 18 Gy whole-lung irradiation died sooner and had an increased percentage of lung with organizing alveolitis between 100 and 160 days compared to Sod2 ^/ wild-type littermates. Both Sod2 ^/– and Sod2 ^/ littermates injected intratracheally with human manganese superoxide dismutase-plasmid/liposome (SOD2-PL) complex 24 h prior to whole-lung irradiation showed decreased DNA strand breaks and improved survival with decreased organizing alveolitis. Thus underexpression of MnSOD in the lungs of heterozygous Sod2 ^/– knockout mice is associated with increased pulmonary radiation sensitivity and parallels increased radiation sensitivity of embryo fibroblast cell lines in vitro. The restoration of cellular radioresistance in vitro and in lungs in vivo by SOD2-PL transgene expression supports a potential role for SOD2-PL gene therapy in organ-specific radioprotection.

van Kleef, E., Verheij, M., te Poele, H., Oussoren, Y., Dewit, L. and Stewart, F. In Vitro and In Vivo Expression of Endothelial von Willebrand Factor and Leukocyte Accumulation after Fractionated Irradiation.

Previous investigations have demonstrated an increased release of von Willebrand factor (VWF; also known as vWF) in endothelial cells after high single-dose irradiation in vitro. We have also found increased levels of Vwf protein in mouse glomeruli after a high single dose of renal irradiation in vivo. In addition, increased numbers of leukocytes were observed in the renal cortex after irradiation in vivo. The aim of the present study was to investigate and quantify these biological processes after clinically relevant fractionated irradiation and to relate them to changes in renal function. A significantly greater increase in release of VWF was observed in cultured human umbilical vein endothelial cells (HUVECs) after fractionated irradiation (20 × 1.0 Gy) than after a single dose of 20 Gy (147% compared to 115% of control, respectively, P < 0.0005). In contrast with the in vitro observations, glomerular Vwf staining was lower after fractionated irradiation in vivo (20 × 2.0 Gy or 10 × 1.6 Gy ± re-irradiation) than after a single dose of 16 Gy. The number of leukocytes accumulating in the renal cortex was also lower after fractionated in vivo irradiation than after a single radiation dose. The onset of these events preceded renal functional and histopathological changes by approximately 10 weeks. These data indicate that radiation-induced changes in endothelial VWF expression after in vivo irradiation may be distinct from the in vitro observations. Increased VWF expression may reflect pivotal processes in the pathogenesis of late radiation nephropathy and provide a clue to appropriate timing of pharmacological intervention.

Schneider, U., Lomax, A. and Lombriser, N. Comparative Risk Assessment of Secondary Cancer Incidence after Treatment of Hodgkin's Disease with Photon and Proton Radiation.

Probabilities for secondary cancer incidence have been estimated for a patient with Hodgkin's disease for whom treatment has been planned with different radiation modalities using photons and protons. The ICRP calculation scheme has been used to calculate cancer incidence from dose distributions. For this purpose, target volumes as well as critical structures have been outlined in the CT set of a patient with Hodgkin's disease. Dose distributions have been calculated using conventional as well as intensity-modulated treatment techniques using photon and proton radiation. The cancer incidence has been derived from the mean doses for each organ. The results of this work are: (a) Intensity-modulated treatment of Hodgkin's disease using nine photon fields (15 MV) results in nearly the same cancer incidence as treating with two opposed photon fields (6 MV). (b) Intensity-modulated treatment using nine proton fields (maximum energy 177.25 MeV) results in nearly the same cancer incidence as treating with one proton field (160 MeV). (c) Irradiation with protons using the spot scanning technique decreases the avoidable cancer incidence compared to photon treatment by a factor of about two. This result is independent of the number of beams used. Our work suggests that there are radiotherapy indications in which intensity-modulated treatments will result in little or no reduction of cancer incidence compared to conventional treatments. However, proton treatment can result in a lower cancer incidence than photon treatment.

Costes, S., Streuli, C. H. and Barcellos-Hoff, M. H. Quantitative Image Analysis of Laminin Immunoreactivity in Skin Basement Membrane Irradiated with 1 GeV/nucleon Iron Particles.

We previously reported that laminin immunoreactivity in mouse mammary epithelium is altered shortly after whole-body irradiation with 0.8 Gy from 600 MeV/nucleon iron ions but is unaffected after exposure to sparsely ionizing radiation. This observation led us to propose that the effect could be due to protein damage from the high ionization density of the ion tracks. If so, we predicted that it would be evident soon after radiation exposure in basement membranes of other tissues and would depend on ion fluence. To test this hypothesis, we used immunofluorescence, confocal laser scanning microscopy, and image segmentation techniques to quantify changes in the basement membrane of mouse skin epidermis. At 1 h after exposure to 1 GeV/nucleon iron ions with doses from 0.03 to 1.6 Gy, neither the visual appearance nor the mean pixel intensity of laminin in the basement membrane of mouse dorsal skin epidermis was altered compared to sham-irradiated tissue. This result does not support the hypothesis that particle traversal directly affects laminin protein integrity. However, the mean pixel intensity of laminin immunoreactivity was significantly decreased in epidermal basement membrane at 48 and 96 h after exposure to 0.8 Gy 1 GeV/nucleon iron ions. We confirmed this effect with two additional antibodies raised against affinity-purified laminin 1 and the E3 fragment of the long-arm of laminin 1. In contrast, collagen type IV, another component of the basement membrane, was unaffected. Our studies demonstrate quantitatively that densely ionizing radiation elicits changes in skin microenvironments distinct from those induced by sparsely ionizing radiation. Such effects may might contribute to the carcinogenic potential of densely ionizing radiation by altering cellular signaling cascades mediated by cell–extracellular matrix interactions.

Jakob, B., Scholz, M. and Taucher-Scholz, G. Immediate Localized CDKN1A (p21) Radiation Response after Damage Produced by Heavy-Ion Tracks.

Using confocal microscopy on immunofluorescence-stained cells, we have investigated the response of CDKN1A (p21), one of the key proteins involved in the DNA damage response pathway, after irradiation with accelerated lead or chromium ions. Each traversal of an accelerated ion leads to the formation of a single, bright focus of the CDKN1A protein in the nuclei of human fibroblasts within 2 min after irradiation at 4°C. This immediate, localized CDKN1A response is specific for particle irradiation with a high linear energy transfer (LET), whereas X irradiation, after a period of induction, yields a diffusely spread pattern, in line with the differences in the microscopic dose deposition pattern of both radiation types. The particle-induced CDKN1A foci persist for several hours until they become diffuse and vanish. These findings suggest that CDKN1A accumulates at the sites of primary DNA damage, possibly mediated by the interaction with proteins involved in DNA repair. Here, for the first time, an immediate biological response confined to the radial extension of low-energy particle tracks (∼1 μm) is directly visualized and correlated to ion traversals. This indicates that particle irradiation represents an ideal tool to study the processing of biological damage induced in defined subnuclear regions.

Dionet, C., Tchirkov, A., Alard, J-P., Arnold, J., Dhermain, J., Rapp, M., Bodez, V., Tamain, J-C., Monbel, I., Malet, P., Kwiatkowski, F., Donnarieix, D., Veyre, A. and Verrelle, P. Effects of Low-Dose Neutrons Applied at Reduced Dose Rate on Human Melanoma Cells.

Human melanoma cells that are resistant to γ rays were irradiated with 14 MeV neutrons given at low doses ranging from 5 cGy to 1.12 Gy at a very low dose rate of 0.8 mGy min^–1 or a moderate dose rate of 40 mGy min^–1. The biological effects of neutrons were studied by two different methods: a cell survival assay after a 14-day incubation and an analysis of chromosomal aberrations in metaphases collected 20 h after irradiation. Unusual features of the survival curve at very low dose rate were a marked increase in cell killing at 5 cGy followed by a plateau for survival from 10 to 32.5 cGy. The levels of induced chromosomal aberrations showed a similar increase for both dose rates at 7.5 cGy and the existence of a plateau at the very low dose rate from 15 to 30 cGy. The existence of a plateau suggests that a repair process after low-dose neutrons might be induced after a threshold dose of 5–7.5 cGy which compensates for induced damage from doses as high as 32.5 cGy. These findings may be of interest for understanding the relative biological effectiveness of neutrons and the effects of environmental low-dose irradiation.

Wolf, C., Lafuma, J., Masse, R., Morin, M. and Kellerer, A. M. Neutron RBE for Induction of Tumors with High Lethality in Sprague-Dawley Rats.

The effectiveness of fission neutrons is compared to that of γ rays and X rays with regard to the induction of malignancies in male Sprague-Dawley rats. The analysis is based on autopsy results. It is focused on tumors that tend to be present in animals dying early, which is indicative of a high degree of lethality. The relative biological effectiveness (RBE) is deduced from a comparison of the cumulative hazard functions. Different nonparametric models—the constant relative risk model, a time shift model, and an acceleration model—are employed in the comparison, and the resulting values of RBE are seen to be substantially independent of the choice of model. The results are in good agreement with earlier studies of nonlethal lung tumors in the same series of experiments. At neutron doses of 20 to 60 mGy, the RBE of fission neutrons is about 50.

Park, S-H., Cho, H-N., Lee, S-J., Kim, T-H., Lee, Y., Park, Y-M., Lee, Y. J., Cho, C-K., Yoo, S-Y. and Lee, Y-S. Hsp25-Induced Radioresistance is Associated with Reduction of Death by Apoptosis: Involvement of Bcl2 and the Cell Cycle.

We previously demonstrated the protective effect of the small heat-shock protein against oxidative damage induced by tumor necrosis factor α. Here we have extended our studies of the possible role of Hsp25 in ionizing radiation-induced damage. For these studies, we transfected murine fibroblast L929 cells with the Hsp25 gene and selected three stably transfected clones. Hsp25 overexpression conferred radioresistance as detected by clonogenic survival and induction of apoptosis. Interestingly, the Hsp25-transfected cells showed an increase in the level of the anti-apoptosis molecule Bcl2. We also observed alterations of cell growth in the Hsp25-transfected cells. The cell cycle time of Hsp25-transfected cells was 3–4 h slower than that of vector-transfected control cells. Flow cytometry analysis of synchronized cells at late G₁ phase by mimosine treatment also showed the growth delay in Hsp25-overexpressing cells. In addition, reduced cyclin D1, cyclin A and Cdc2 levels and increased levels of Cdkn1a (also known as p21^Waf) were observed in Hsp25-transfected cells, which probably caused the reduction in cell growth. In addition, synchronization by mimosine treatment only partially altered radioresistance in the Hsp25-transfected cells. Taken together, these data suggest that Hsp25-induced radioresistance is associated with growth delay as well as induction of Bcl2.

Woynarowska, B. A., Roberts, K., Woynarowski, J. M., MacDonald, J. R. and Herman, T. S. Targeting Apoptosis by Hydroxymethylacylfulvene in Combination with Gamma Radiation in Prostate Tumor Cells.

Hydroxymethylacylfulvene (HMAF) is a novel agent with alkylating activity and is a potent inducer of apoptosis that is currently undergoing Phase II clinical trials for prostate cancer. This study explored the pro-apoptosis and anti-proliferative potential of HMAF in combination with γ radiation in human prostate tumor cell lines. Apoptosis was assessed based on the generation of fragmented DNA, a terminal transferase flow cytometry assay, and cell morphology. In each of the tumor cell lines examined, radiation alone induced a marginal level of apoptosis, even after a prolonged 48-h incubation after exposure. In contrast, HMAF alone was a potent inducer of apoptosis in prostate tumor cells but not in normal cells. Marked levels of apoptosis in tumor cells were also observed for the combination of HMAF with γ radiation. When drug treatment preceded irradiation, at least additive levels of apoptosis were observed in both androgen-responsive and androgen-independent cells. The combined treatment with ionizing radiation and HMAF reduced the radiation dose needed for the same level of clonogenic survival up to 2.5-fold. The potentiation of apoptosis and reduction in the clonogenic survival of tumor cells occurred at HMAF concentrations lower than that which reduced survival to 10% and at doses up to 6 Gy. No potentiation of apoptosis or clonogenic inhibition was noted in normal cells. These results suggest that the combination of HMAF with γ radiation may have clinical utility for treatments of prostate cancer.

Sauer, G., Weber, K. J., Peschke, P. and Eble, M. Measurement of Hypoxia Using the Comet Assay Correlates with Preirradiation Microelectrode pO₂ Histography in R3327-AT Rodent Tumors.

Polarographic determination of tumor oxygenation by Eppendorf histography is currently under investigation as a possible predictor of radiotherapy outcome. Alternatively, the alkaline comet assay has been proposed as a radiobiological approach for the detection of hypoxia in clinical tumor samples. Direct comparisons of these methods are scarce. One earlier study with different murine tumors could not establish a correlation, whereas a weak correlation was reported for a variety of human tumors. Considering the different end points and spatial resolution of the two methods, a direct comparison for a single tumor entity appeared desirable. Anaplastic R3327-AT Dunning prostate tumors were grown on Copenhagen rats to volumes of 1–6 cm³. Eppendorf histography (100–200 readings in 5 parallel tracks) for 8 different tumors revealed various degrees of oxygenation, with median pO₂ values ranging from 1.1 to 23 mmHg. Within 5 min after an acute exposure to 8 Gy ⁶⁰Co γ rays, tumors were excised from killed animals and rapidly cooled to limit repair, and a single cell suspension was prepared for use with the comet assay. The resulting comet moment distributions did not exhibit two subpopulations (one hypoxic and the other aerobic), and a hypoxic fraction could not be calculated. Instead, the average comet moment distribution was taken as a parameter of overall strand break induction. Corresponding experiments with tumor cells grown in vitro allowed us to derive the relationship between the oxygen enhancement ratio (OER) for the average comet moment and oxygen partial pressure (Howard-Flanders and Alper formula). The validity of this relationship was inferred for cells exposed in situ, and the convolution of a pO₂ distribution with the formula of Howard-Flanders and Alper yielded an array of expected OER values for each tumor. The average expected OER correlated well with the average comet moment (r = 0.89, P < 0.01), and the in situ comet moment distributions could be predicted from the Eppendorf data when 50% repair was taken into account, assuming a 5-min damage half-life. The findings confirm the potential of interstitial polarography to reflect radiobiologically relevant intracellular oxygenation, but also underscore the confounding influence of differences in repair that may occur when cells are prepared from irradiated tissues for use with the comet assay.

Oghiso, Y. and Yamada, Y. Strain Differences in Carcinogenic and Hematopoietic Responses of Mice after Injection of Plutonium Citrate.

The carcinogenicity of injected ²³⁹Pu citrate was compared in female mice of the C3H, C57BL/6 and BC3F₁ hybrid strains with different spectra of spontaneous or radiation-induced tumors. A significant reduction in survival due to early death caused particularly by the induction of osteosarcomas was noted in each strain after injection of 500 Bq or more. The dose response of osteosarcomas appeared to have a similar pattern in each strain except for the differences in the skeletal dose ranges for the maximum induction. While the incidence of lymphoid tumors decreased as that of osteosarcomas increased sharply to the maximum at higher doses, their histological phenotypes were predominantly non-thymic, pre-B-cell leukemic lymphomas compared to the controls in each strain. Myeloid leukemias were not highly induced in any of the control and ²³⁹Pu-injected mice, and solid tumors involving the other organs were reduced in each strain after injection of 500 Bq or more. To follow up the hematological kinetics related to α-particle irradiation of bone marrow stem cells, sequential examinations were done in mice of each strain within 1 year after injection of 5000 Bq. The numbers of peripheral white blood cells and bone marrow cells were consistently reduced in each strain from 90 days on, while spleen cells increased from 180 days on. Granulocyte-macrophage and macrophage colony-forming cells were also consistently reduced in the bone marrow, with a compensatory increase in the spleen from 90 days on. These findings indicate that the carcinogenic and hematopoietic responses were specific to α-particle irradiation and were independent of mouse strain after injection with ²³⁹Pu citrate.

Uma Devi, P., Ganasoundari, A., Vrinda, B., Srinivasan, K. K. and Unnikrishnan, M. K. Radiation Protection by the Ocimum Flavonoids Orientin and Vicenin: Mechanisms of Action.

In previous studies, flavonoids, orientin and vicenin, that were isolated from the leaf extract of Ocimum sanctum, were found to protect mice against radiation injury. Several flavonoids are known to be good antioxidants. Therefore, the effect of orientin and vicenin on radiation-induced lipid peroxidation in vivo and their antioxidant activity in vitro were studied. Adult mice were injected intraperitoneally with 50 μg/kg of orientin or vicenin and exposed whole-body to 3 Gy of γ radiation. Lipid peroxidation was measured in the liver 15 min to 8 h postirradiation. The antioxidant activity of orientin/vicenin (10–500 μM) was studied by measuring inhibition of hydroxyl radicals generated by the Fenton reaction (Fe³ -EDTA-ascorbic acid-H₂O₂) in vitro. The compounds were also tested for possible pro-oxidant and iron chelation activities at the above concentrations in the in vitro system. Orientin and vicenin provided almost equal protection against radiation-induced lipid peroxidation in mouse liver. Both compounds showed a significantly greater free radical-inhibiting activity in vitro than DMSO. Neither orientin nor vicenin showed any pro-oxidant activity at the concentrations tested. Both compounds inhibited free radical formation in the absence of EDTA. Free radical scavenging appears to be a likely mechanism of radiation protection by these flavonoids.

Gaffney, D. K., Lundquist, M., Warters, R. L. and Rowley, R. Effects of Modifying Topoisomerase II Levels on Cellular Recovery from Radiation Damage.

Experiments were performed with the budding yeast, Saccharomyces cerevisiae, to test whether DNA topoisomerase II is involved in repair of DNA damage induced by ionizing radiation. Topoisomerase II was inactivated by use of a temperature-sensitive mutation. Enzyme inactivation increased cellular radiosensitivity, blocked the restitution of broken chromosomes, assayed by pulsed-field gel electrophoresis, and prolonged the induction of a DNA damage-inducible gene (RNR3). Overexpression of the topoisomerase II gene did not alter cellular radiosensitivity. The data support a role for topoisomerase II in the repair of DNA strand breaks.

Vanhaelewyn, G. C. A. M., Morent, R. A., Callens, F. J. and Matthys, P. F. A. E. X- and Q-band Electron Paramagnetic Resonance of CO₂⁻ in Hydroxyapatite Single Crystals.

Both X- and Q-band electron paramagnetic resonance (EPR) research has been conducted using slightly carbonated hydroxyapatite (HAp) single crystals after exposure to ionizing radiation. Below a temperature of 90 K, O⁻ and CO₂⁻ radicals were detected, whereas at room temperature only CO₂⁻ spectra could be observed. The O⁻ ion has previously been investigated in high-purity HAp single crystals, whereas EPR spectra of CO₂⁻ in HAp single crystals have not been reported. Both paramagnetic defects exhibit EPR angular variations in planes containing the c axis of the crystal from which spin Hamiltonian parameters were derived. Arguments are given for the presence of two CO₂⁻ defects in the irradiated HAp single crystals.

Anderson, C. W. and Allalunis-Turner, M. J. Human TP53 from the Malignant Glioma-Derived Cell Lines M059J and M059K Has a Cancer-Associated Mutation in Exon 8.

The protein coding segment of the TP53 genes from the glioma-derived cell lines M059J and M059K was sequenced. The sequences from both cell lines were identical over 5039 bp, including the gene segment containing exons 2 through 9, exon 10, and the proximal segment of exon 11. In both cells, the first nucleotide of codon 286 (GAA, Glu) is changed to an A (AAA, Lys). Comparison with the same TP53 segment from the A549 human lung carcinoma cell line revealed several differences in intron sequence.