Chu, K., Leonhardt, E. A., Trinh, M., Prieur-Carrillo, G., Lindqvist, J., Albright, N., Ling, C. C. and Dewey, W. C. Computerized Video Time-Lapse (CVTL) Analysis of Cell Death Kinetics in Human Bladder Carcinoma Cells (EJ30) X-Irradiated in Different Phases of the Cell Cycle. Radiat. Res. 158, 667–677 (2002).

The purpose of this study was to quantify the modes and kinetics of cell death for EJ30 human bladder carcinoma cells irradiated in different phases of the cell cycle. Asynchronous human bladder carcinoma cells were observed in multiple fields by computerized video time-lapse (CVTL) microscopy for one to two cell divisions before irradiation (6 Gy) and for 6–11 days afterward. By analyzing time-lapse movies collected from these fields, pedigrees were constructed showing the behaviors of 231 cells irradiated in different phases of the cell cycle (i.e. at different times after mitosis). A total of 219 irradiated cells were determined to be non-colony-forming over the time spans of the experiments. In these nonclonogenic pedigrees, cells died primarily by necrosis either without entering mitosis or over 1 to 10 postirradiation generations. A total of 105 giant cells developed from the irradiated cells or their progeny, and 30% (31/105) divided successfully. Most nonclonogenic cells irradiated in mid-S phase (9–12 h after mitosis) died by the second generation, while those irradiated either before or after this short period in mid-S phase had cell deaths occurring over one to nine postirradiation generations. The nonclonogenic cells irradiated in mid-S phase also experienced the longest average delay before their first division. Clonogenic cells (11/12 cells) divided sooner after irradiation than the average nonclonogenic cells derived from the same phase of the cell cycle. The early death and long division delay observed for nonclonogenic cells irradiated in mid-S phase could possibly result from an increase in damage induced during the transition from the replication of euchromatin to the replication of heterochromatin.

Goto, S., Watanabe, M. and Yatagai, F. Delayed Cell Cycle Progression in Human Lymphoblastoid Cells after Exposure to High-LET Radiation Correlates with Extremely Localized DNA Damage. Radiat. Res. 158, 678–686 (2002).

To compare the genotoxic effects of high-LET ionizing radiation to those of low-LET radiation, we investigated the responses of human lymphoblastoid cells to DNA damage TK6 after treatment with either low-LET X rays or high-LET iron ions (1000 keV/μm). A highly localized distribution of γH2AX/RAD51 foci was observed in the nuclei of cells irradiated with iron ions, in sharp contrast to cells exposed to X rays, where the distribution of foci was much more uniform. This implied the occurrence of a relatively high frequency of closely spaced double-strand breaks, i.e. clustered DNA damage, after iron-ion exposure. Despite the well-established notion that clustered DNA damage is refractory to repair compared to isolated DNA lesions, there were no significant differences in the levels of clonogenic survival and apoptosis between cells treated with iron ions or X rays. Strikingly, however, cells accumulated in G₂/M phase to a much lesser extent after iron-ion exposure than after X-ray exposure. This differential accumulation could be attributed to a much slower evacuation of the S-phase compartment in the case of cells irradiated with iron ions. Taken together, our results indicate that, relative to the situation for low-LET X rays, exposure to high-LET iron ions results in a substantially greater inhibition of S-phase progression as a result of a higher frequency of DNA replication-blocking clustered DNA damage.

Evans, H. H., Horng, M-F., Ricanati, M., Diaz-Insua, M., Jordan, R. and Schwartz, J. L. Characteristics of Genomic Instability in Clones of TK6 Human Lymphoblasts Surviving Exposure to ⁵⁶Fe Ions. Radiat. Res. 158, 687–698 (2002).

Genomic instability in the human lymphoblast cell line TK6 was studied in clones surviving 36 generations after exposure to accelerated ⁵⁶Fe ions. Clones were assayed for 20 characteristics, including chromosome aberrations, plating efficiency, apoptosis, cell cycle distribution, response to a second irradiation, and mutant frequency at two loci. The primary effect of the ⁵⁶Fe-ion exposure on the surviving clones was a significant increase in the frequency of unstable chromosome aberrations compared to the very low spontaneous frequency, along with an increase in the phenotypic complexity of the unstable clones. The radiation-induced increase in the frequency of unstable chromosome aberrations was much greater than that observed previously in clones of the related cell line, WTK1, which in comparison to the TK6 cell line expresses an increased radiation resistance, a mutant TP53 protein, and an increased frequency of spontaneous unstable chromosome aberrations. The characteristics of the unstable clones of the two cell lines also differed. Most of the TK6 clones surviving exposure to ⁵⁶Fe ions showed unstable cytogenetic abnormalities, while the phenotype of the WTK1 clones was more diverse. The results underscore the importance of genotype in the characteristics of instability after radiation exposure.

Schäfer, J., Bachtler, J., Engling, A., Little, J. B., Weber, K.-J. and Wenz, F. Suppression of Apoptosis and Clonogenic Survival in Irradiated Human Lymphoblasts with Different TP53 Status. Radiat. Res. 158, 699–706 (2002).

The influence of radiation-induced apoptosis on radiosensitivity was studied in a set of closely related human lymphoblastoid cell lines differing in TP53 status. The clonogenic survival of irradiated TK6 cells (expressing wild-type TP53), WTK1 cells (overexpressing mutant TP53), and TK6E6 cells (negative for TP53 owing to transfection with HPV16 E6) was assessed in relation to the induction of apoptosis and its suppression by caspase inhibition or treatment with PMA as well as after treatment with caffeine. Measurements using the alkaline comet assay and pulsed-field electrophoresis of the induction and repair of DNA strand breaks showed similar kinetics of the processing of early DNA damage in these cell lines. The cytochalasin B micronucleus assay revealed identical levels of residual damage in the first postirradiation mitosis of these cells. Abrogation of TP53-dependent apoptosis in TK6E6 cells resulted in a distinct increase in radioresistance. Further suppression of apoptosis as observed in WTK1 cells overexpressing mutant TP53 apparently was not responsible for the high radioresistance of WTK1 cells, since other means of highly efficient suppression of apoptosis (caspase inhibition or PMA treatment) increased the clonogenic survival of irradiated TK6 cells only to levels similar to those of TK6E6 cells with abrogated TP53-dependent apoptosis. Considering the similar levels of residual chromosomal damage in TK6E6 cells and WTK1 cells, a hitherto unknown mechanism of tolerance needs to be inferred for these TP53 mutant cells. This residual damage tolerance, however, appears to require an intact G₂/M-phase checkpoint function since the relative radioresistance of the WTK1 cells was completely lost upon caffeine treatment, which also resulted in a failure of the TK6 and TK6E6 cells to execute apoptosis. In this situation, the cellular response seems to be dominated entirely by TP53-independent mitotic failure.

Franken, N. A. P., van Bree, C., ten Cate, R., van Oven, C. H. and Haveman, J. Importance of TP53 and RB in the Repair of Potentially Lethal Damage and Induction of Color Junctions after Exposure to Ionizing Radiation. Radiat. Res. 158, 707–714 (2002).

Repair of potentially lethal damage (PLD) was investigated in cells with functional G₁-phase arrest with wild-type TP53 and wild-type RB and in cells in which G₁-phase arrest was abrogated by inactivation of TP53 or RB. Confluent cultures of cells were plated for clonogenic survival assay either immediately or 24 h after irradiation. Induction of color junctions, an exchange between a painted and unpainted chromosome, was studied in chromosomes 18 and 19 after irradiation with 4 Gy γ rays. Significant repair of PLD was found in cells carrying both wild-type TP53 and wild-type RB. In cells in which TP53 or RB was inactivated, the survival curves from immediately plated and delayed-plated cells were not significantly different. The numbers of radiation-induced color junctions in chromosomes 18 and 19 were similar in all cell lines. From this study we conclude that a functional G₁-phase arrest is important for repair of PLD and that TP53 and RB do not affect the frequencies of induction of color junctions in chromosome 18 or 19.

Kusunoki, Y., Yamaoka, M., Kasagi, F., Hayashi, T., Koyama, K., Kodama, K., MacPhee, D. G. and Kyoizumi, S. T Cells of Atomic Bomb Survivors Respond Poorly to Stimulation by Staphylococcus aureus Toxins In Vitro: Does This Stem from their Peripheral Lymphocyte Populations Having a Diminished Naïve CD4 T-Cell Content? Radiat. Res. 158, 715–724 (2002).

We found previously that the peripheral CD4 T-cell populations of heavily exposed A-bomb survivors contained fewer naïve T cells than we detected in the corresponding unexposed controls. To determine whether this demonstrable impairment of the CD4 T-cell immunity of A-bomb survivors was likely to affect the responsiveness of their immune systems to infection by common pathogens, we tested the T cells of 723 survivors for their ability to proliferate in vitro after a challenge by each of the Staphylococcus aureus toxins SEB, SEC-2, SEC-3, SEE and TSST-1. The results presented here reveal that the proliferative responses of T cells of A-bomb survivors became progressively weaker as the radiation dose increased and did so in a manner that correlated well with the decreasing CD45RA-positive (naïve) [but not CD45RA-negative (memory)] CD4 T-cell percentages that we found in their peripheral blood lymphocyte (PBL) populations. We also noted that the T cells of survivors with a history of myocardial infarction tended to respond poorly to several (or even all) of the S. aureus toxins, and that these same individuals had proportionally fewer CD45RA-positive (naïve) CD4 T cells in their PBL populations than we detected in survivors with no myocardial infarction in their history. Taken together, these results clearly indicate that A-bomb irradiation led to an impairment of the ability of exposed individuals to maintain their naïve T-cell pools. This may explain why A-bomb survivors tend to respond poorly to toxins encoded by the common pathogenic bacterium S. aureus.

Denisova, N. A., Shukitt-Hale, B., Rabin, B. M. and Joseph, J. A. Brain Signaling and Behavioral Responses Induced by Exposure to ⁵⁶Fe-Particle Radiation. Radiat. Res. 158, 725–734 (2002).

Previous experiments have demonstrated that exposure to ⁵⁶Fe-particle irradiation (1.5 Gy, 1 GeV) produced aging-like accelerations in neuronal and behavioral deficits. Astronauts on long-term space flights will be exposed to similar heavy-particle radiations that might have similar deleterious effects on neuronal signaling and cognitive behavior. Therefore, the present study evaluated whether radiation-induced spatial learning and memory behavioral deficits are associated with region-specific brain signaling deficits by measuring signaling molecules previously found to be essential for behavior [pre-synaptic vesicle proteins, synaptobrevin and synaptophysin, and protein kinases, calcium-dependent PRKCs (also known as PKCs) and PRKA (PRKA RIIβ)]. The results demonstrated a significant radiation-induced increase in reference memory errors. The increases in reference memory errors were significantly negatively correlated with striatal synaptobrevin and frontal cortical synaptophysin expression. Both synaptophysin and synaptobrevin are synaptic vesicle proteins that are important in cognition. Striatal PRKA, a memory signaling molecule, was also significantly negatively correlated with reference memory errors. Overall, our findings suggest that radiation-induced pre-synaptic facilitation may contribute to some previously reported radiation-induced decrease in striatal dopamine release and for the disruption of the central dopaminergic system integrity and dopamine-mediated behavior.

Shimura, T., Toyoshima, M., Taga, M., Shiraishi, K., Uematsu, N., Inoue, M. and Niwa, O. The Novel Surveillance Mechanism of the Trp53-Dependent S-Phase Checkpoint Ensures Chromosome Damage Repair and Preimplantation-Stage Development of Mouse Embryos Fertilized with X-Irradiated Sperm. Radiat. Res. 158, 735–742 (2002).

Cell cycle checkpoints and apoptosis function as surveillance mechanisms in somatic tissues. However, some of these mechanisms are lacking or are restricted during the preimplantation stage. Previously, we reported the presence of a novel Trp53-dependent S-phase checkpoint that suppresses pronuclear DNA synthesis in mouse zygotes fertilized with X-irradiated sperm (sperm-irradiated zygotes) (Shimura et al., Mol. Cell. Biol. 22, 2220–2228, 2002). Here we studied the role of the Trp53-dependent S-phase checkpoint in the early stage of development of sperm-irradiated zygotes. In the Trp53 ^/ genetic background, all of the sperm-irradiated zygotes cleaved successfully to the two-cell stage despite the fact that half of them carried a sub-2N amount of DNA. These zygotes progressed normally to the eight-cell stage and then implanted, but the subsequent fetal development was suppressed in a dose-dependent manner. In contrast, sperm-irradiated Trp53^−/− embryos lacking an S-phase checkpoint exhibited an abnormal segregation of chromosomes at the first cleavage, even though they carried an apparently normal 2N amount of DNA. They were morphologically abnormal with numerous micronuclei, and they degenerated before reaching the eight-cell stage. As a consequence, no implants were observed for sperm-irradiated Trp53^−/− embryos. These results suggest that the Trp53-dependent S-phase checkpoint is a surveillance mechanism involved in the repair of chromosome damage and ensures the preimplantation-stage development of sperm-irradiated embryos.

Morris, G. M., Micca, P. L., Nawrocky, M. M., Weissfloch, L. E. and Coderre, J. A. Long-Term Infusions of p-Boronophenylalanine for Boron Neutron Capture Therapy: Evaluation Using Rat Brain Tumor and Spinal Cord Models. Radiat. Res. 158, 743–752 (2002).

Rat 9L gliosarcoma cells infiltrating the normal brain have been shown previously to accumulate only ∼30% as much boron as the intact tumor after administration of the boronated amino acid p-boronophenylalanine (BPA). Long-term i.v. infusions of BPA were shown previously to increase the boron content of these infiltrating tumor cells significantly. Experiments to determine whether this improved BPA distribution into infiltrating tumor cells after a long-term i.v. infusion improves tumor control after BNCT in this brain tumor model and whether it has any deleterious effects in the response of the rat spinal cord to BNCT are the subjects of the present report. BPA was administered in a fructose solution at a dose of 650 mg BPA/kg by single i.p. injection or by i.v. infusion for 2 h or 6 h, at 330 mg BPA/kg h^–1. At 1 h after the end of either the 2-h or the 6-h infusion, the CNS:blood ¹⁰B partition ratio was 0.9:1. At 3 h after the single i.p. injection, the ratio was 0.6:1. After spinal cord irradiations, the ED₅₀ for myeloparesis was 14.7 ± 0.4 Gy after i.p. administration of BPA and 12.9 ± 0.3 Gy in rats irradiated after a 6-h i.v. infusion of BPA; these values were significantly different (P < 0.001). After irradiation with 100 kVp X rays, the ED₅₀ was 18.6 ± 0.1 Gy. The boron compound biological effectiveness (CBE) factors calculated for the boron neutron capture dose component were 1.2 ± 0.1 for the i.p. BPA administration protocol and 1.5 ± 0.1 after irradiation using the 6-h i.v. BPA infusion protocol (P < 0.05). In the rat 9L gliosarcoma brain tumor model, the blood boron concentrations at 1 h after the end of the 2-h infusion (330 mg BPA/kg h^–1; n = 15) or after the 6-h infusion (190 mg BPA/kg h^–1; n = 13) were 18.9 ± 2.2 μg ¹⁰B/g and 20.7 ± 1.8 μg ¹⁰B/g, respectively. The irradiation times were adjusted individually, based on the preirradiation blood sample, to deliver a predicted 50% tumor control dose of 8.2 Gy (∼30 photon-equivalent Gy) to all tumors. In the present study, the long-term survival was approximately 50% and was not significantly different between the 2-h and the 6-h infusion groups. The mode of BPA administration and the time between administration and irradiation influence the ¹⁰B partition ratio between the CNS and the blood, which in turn influences the measured CBE factor. These findings underline the need for clinical biodistribution studies to be carried out to establish ¹⁰B partition ratios as a key component in the evaluation of modified administration protocols involving BPA.

Kriste, A. G., Tercel, M., Anderson, R. F., Ferry, D. M. and Wilson, W. R. Pathways of Reductive Fragmentation of Heterocyclic Nitroarylmethyl Quaternary Ammonium Prodrugs of Mechlorethamine. Radiat. Res. 158, 753–762 (2002).

Nitroarylmethyl quaternary (NMQ) ammonium salts have potential as prodrugs for enzymatic or radiolytic reduction to release amine effectors under hypoxia. Earlier studies demonstrated one-electron release of the cytotoxic amine mechlorethamine (HN2) from 4-nitroimidazolyl and 2-nitropyrrolyl NMQ prodrugs (but not from nitrobenzyl analogs) through intramolecular electron transfer. In this study we determined whether this is a general feature of heterocyclic NMQ prodrugs of HN2 and examined the reductive pathways in detail using pulse and steady-state radiolysis. The kinetics of radical fragmentation varied by more than four orders of magnitude, independently of the one-electron reduction potential, within the series of eight nitroheterocycles examined. In addition to the compounds identified previously, new 2-nitropyrrole and 3-nitrothiophene NMQ prodrugs were found to provide efficient HN2 release (G > 0.5 μmol/J in anoxic formate buffer). However, the nitrothiophene was sensitive to nucleophilic displacement of HN2, making it less promising. Product analysis by HPLC/mass spectrometry identified symmetrical dimers arising from benzyl-type radical intermediates but also demonstrated that these dimers are not reliable markers for the intramolecular fragmentation of the initial nitro radical anion. This study elucidated multiple competing pathways for reductive fragmentation of NMQ prodrugs and identified the preferred electron acceptors for use in the development of analogs that release more potent cytotoxins.

Corde, S., Biston, M. C., Elleaume, H., Estève, F., Charvet, A. M., Joubert, A., Ducros, V., Bohic, S., Simionovici, A., Brochard, T., Nemoz, C., Renier, Troprès, I., Fiedler, S., Bravin, A., M., Thomlinson, W., Le Bas, J. F. and Balosso, J. Lack of Cell Death Enhancement after Irradiation with Monochromatic Synchrotron X Rays at the K-Shell Edge of Platinum Incorporated in Living SQ20B Human Cells as cis-Diamminedichloroplatinum (II). Radiat. Res. 158, 763–770 (2002).

In this paper we describe the results of experiments using synchrotron radiation to trigger the Auger effect in living human cancer cells treated with a widely used chemotherapy drug: cis-diamminedichloroplatinum (II) (cisplatin). The experiments were carried out at the ID17 beamline of the European Synchrotron Radiation Facility, which produces a high-fluence monochromatic beam that is adjustable from 20 to 80 keV. Cisplatin was chosen as the carrier of platinum atoms in the cells because of its alkylating-like activity and the irradiation was done with monochromatic beams above and below the platinum K-shell edge (78.39 keV). Cell survival curves were comparable with those obtained for the same cells under conventional irradiation conditions. At a low dose of cisplatin (0.1 μM, 48 h), no difference was seen in survival when the cells were irradiated above and below the K-shell edge of platinum. Higher cisplatin concentrations were investigated to enhance the cellular platinum content. The results with 1 μM cisplatin for 12 h showed no difference when the cells were irradiated with beams above or below the platinum K-shell edge with the exception of the higher cell death resulting from drug toxicity. The intracellular content of platinum was significant, as measured macroscopically by inductively coupled plasma mass spectrometry. Its subcellular localization and particularly its presence in the cell nucleus were verified by microscopic synchrotron X-ray fluorescence. This was the first known attempt at K-shell edge photon activation of stable platinum in living cells with a platinum complex used for chemotherapy. Its evident toxicity in these cells leads us to put forth the hypothesis that cisplatin toxicity can mask the enhancement of cell death induced by the irradiation above the K-shell edge. However, K-shell edge photon activation of stable elements provides a powerful technique for the understanding of the biological effects of Auger processes. Further avenues of development are discussed.

Schmid, E., Regulla, D., Kramer, H-M. and Harder, D. The Effect of 29 kV X Rays on the Dose Response of Chromosome Aberrations in Human Lymphocytes. Radiat. Res. 158, 771–777 (2002).

The induction of chromosome aberrations in human lymphocytes irradiated in vitro with X rays generated at a tube voltage of 29 kV was examined to assess the maximum low-dose RBE (RBE_M) relative to higher-energy X rays or ⁶⁰Co γ rays. Since blood was taken from the same male donor whose blood had been used for previous irradiation experiments using widely varying photon energies, the greatest possible accuracy was available for such an estimation of the RBE_M, avoiding the interindividual variations in sensitivity or differences in methodology usually associated with interlaboratory comparisons. The magnitude of the linear coefficient α of the linear-quadratic dose–effect relationship obtained for the production of dicentric chromosomes by 29 kV X rays (α = 0.0655 ± 0.0097 Gy⁻¹) confirms earlier observations of a strong increase in α with decreasing photon energy. Relating this value to previously published values of α for the dose–effect curves for dicentrics obtained in our own laboratory, RBE_M values of 1.6 ± 0.3 in comparison with weakly filtered 220 kV X rays, 3.0 ± 0.7 compared to heavily filtered 220 kV X rays, and 6.1 ± 2.5 compared to ⁶⁰Co γ rays have been obtained. These data emphasize that the choice of the reference radiation is of fundamental importance for the RBE_M obtained. A special survey of the RBE_M values obtained by different investigators in the narrow quality range from about 30 to 350 kV X rays indicates that the present RBE is in fairly good agreement with previously published findings for the induction of chromosome aberrations or micronuclei in human lymphocytes but differs from recently published findings for neoplastic transformation in a human hybrid cell line.

Schmid, E. Is There Reliable Experimental Evidence for a Low-Dose RBE of about 4 for Mammography X Rays Relative to 200 kV X Rays? Radiat. Res. 158, 778–781 (2002).

Frankenberg et al. (Radiat. Res. 157, 99–105, 2002) recently reported, on the basis of observations of neoplastic transformation in human hybrid CGL1 cells, a low-dose relative biological effectiveness (RBE_M) of 4.3 for mammography X rays (29 kV) relative to 200 kV X rays. With reference to data in the literature, they inferred a factor of about 8 relative to ⁶⁰Co γ rays and concluded that this result is relevant to risk estimation. However, the conclusions do not appear to be valid. The data from the transformation study exhibit uncertainties in the statistical analysis that preclude any generalization of the inferred RBE_M. The data selected or inferred from the literature are likewise insufficient to support the stated RBEs. Our own uniform data set for the yields of dicentrics was obtained for widely varying photon energies with blood samples from the same donor, and it avoids interindividual variations in sensitivity as well as the differences in methodology that are associated with interlaboratory comparisons. Our data provide RBE_M values for 29 kV X rays of 1.64 ± 0.27 relative to 220 kV X rays and 4.75 ± 1.67 and 6.12 ± 2.51 relative to ⁶⁰Co γ rays.