Cornforth, M. N. Analyzing Radiation-Induced Complex Chromosome Rearrangements by Combinatorial Painting. Radiat. Res. 155, 643–659 (2001).

Prior to the advent of whole-chromosome painting, it was universally assumed that virtually all radiation-induced exchanges represented a simple rejoining between pairs of chromosome breaks. It is now known that a substantial proportion of such exchanges are actually complex, meaning that they involve the interaction of three (or more) breaks distributed among two (or more) chromosomes. The purpose of this review is to discuss some of the implications of aberration analysis using whole-chromosome painting, with emphasis given to newer combinatorial painting schemes that allow for the unambiguous identification of all homologous chromosome pairs. Such analysis requires reconsideration of how resulting information is to be handled for the purposes of tabulating and communicating raw data, quantifying aberration yields, and presenting experimental results in a cogent manner. Facilitating these objectives requires the introduction of certain concepts and terminologies that have no counterpart in conventional cytogenetic analyses.

Loucas, B. D. and Cornforth, M. N. Complex Chromosome Exchanges Induced by Gamma Rays in Human Lymphocytes: An mFISH Study. Radiat. Res. 155, 660–671 (2001).

Combinatorial multi-fluor fluorescence in situ hybridization (mFISH) allows the simultaneous painting of each pair of homologous chromosomes, thereby eliminating many of the difficulties previously associated with the analysis of complex rearrangements. We employed mFISH to visualize exchanges in human lymphocytes and found significant frequencies of these aberrations after γ-ray doses of 2 and 4 Gy. At 4 Gy, roughly half of the cells contained at least one complex exchange that required anywhere from 3 to 11 initial chromosome breaks. At this dose, more than 40% of gross cytogenetic damage, as measured by the total number of exchange breakpoints, was complex in origin. Both simple and complex exchanges were found to have nonlinear dose responses, although the latter showed significantly more upward curvature. In many cases, it could be deduced that the initial breaks leading to a particular complex exchange were proximate, meaning that the resulting broken chromosome ends all must have been capable of interacting freely during the exchange process. For other complex exchanges, the rearrangement could just as well have resulted from two or more simpler exchanges that occurred sequentially. The results demonstrate the utility of mFISH in visualizing intricacies of the exchange process, but also highlight the various sources of ambiguity concerning cytogenetic analysis that remain despite the power of this approach.

MacPhail, S. H. and Olive, P. L. RPA Foci are Associated with Cell Death after Irradiation. Radiat. Res. 155, 672–679 (2001).

Complexes containing replication protein A (RPA) were observed in human TK6 and WIL-2NS lymphoblast cells and SiHa cervical carcinoma cells exposed to 250 kV X rays. Image analysis of individual cells with fluorescence-tagged anti-RPA antibodies was used to measure numbers of discrete foci per cell. RPA foci formed in S-phase cells in response to radiation doses as low as 0.5 Gy, and the number of foci/nucleus was linearly related to dose up to 50 Gy. The maximum number of cells with foci occurred 4–8 h after exposure to 4 Gy, and subsequently declined. However, the number of RPA foci per nucleus (in those cells with foci) reached a maximum after 2–4 h. Apoptotic nuclei from irradiated TK6 and WIL-2NS cells initially contained foci, but these were lost as degradation continued. Radiation-induced micronuclei in SiHa cells were greatly enriched for RPA foci, and cells with nuclei without foci often contained micronuclei with multiple RPA foci. In SiHa cells examined up to 7 days after 4 Gy, RPA foci reappeared in one or more cells in up to 90% of the surviving colonies, and some cells contained 150 or more distinct foci. Reappearance of these complexes could be indicative of radiation-induced genomic instability. These results are consistent with the idea that RPA foci observed several hours after irradiation represent irreparable lesions and as such might be useful in identifying radiosensitive cells.

Utsumi, H., Tano, K., Takata, M., Takeda, S. and Elkind, M. M. Requirement for Repair of DNA Double-Strand Breaks by Homologous Recombination in Split-Dose Recovery. Radiat. Res. 155, 680–686 (2001).

Split-dose recovery has been observed under a variety of experimental conditions in many cell systems and is believed to be the result of the repair of sublethal damage. It is considered to be one of the most widespread and important cellular responses in clinical radiotherapy. To study the molecular mechanism(s) of this repair, we analyzed the knockout mutants KU70^–/–, RAD54^–/–, and KU70^–/–/RAD54^–/– of the chicken B-cell line, DT40. RAD54 participates in the recombinational repair of DNA double-strand breaks (DSBs), while members of the KU family of proteins are involved in nonhomologous end joining. Split-dose recovery was observed in the parent DT40 and the KU70^–/– cells. Moreover, the split-dose survival enhancement had all of the characteristics demonstrated earlier for the repair of sublethal damage, e.g., the reappearance of the shoulder on the survival curve with dose fractionation; cyclic fluctuation in cell survival at 37°C; repair and no cyclic fluctuation at 25°C. These results strongly suggest that repair of sublethal damage is due to DSB repair mediated by homologous recombination, and that these DNA DSBs constitute sublethal damage.

Debije, M. G. and Bernhard, W. A. Electron Paramagnetic Resonance Evidence for a C3′ Sugar Radical in Crystalline d(CTCTCGAGAG) X-Irradiated at 4 K. Radiat. Res. 155, 687–692 (2001).

A neutral sugar radical formed by the net loss of hydrogen from C3′ has been identified in crystalline DNA X-irradiated at 4 K. Crystals of duplex d(CTCTCGAGAG), known to be of B conformation, were studied using electron paramagnetic resonance (EPR) spectroscopy. The C3′ radical was identified by using information from dose saturation, power saturation, thermal annealing, and spectrum simulation. The yield of the C3′ radical, G(C3′), is 0.03 ± 0.01 μmol/J, and its concentration does not appear to saturate up to at least 100 kGy. In the region in which total radical concentration increases linearly with dose, the C3′ radical makes up about 4.5% of the total radical population trapped in the oligodeoxynucleotide crystal at 4 K. Based on free base release measured in other oligodeoxynucleotides, we suggest that in d(CTCTCGAGAG) the C3′ radical is responsible for about one-third of the strand breakage events.

Neuhof, D., Ruess, A., Wenz, F. and Weber, K. J. Induction of Telomerase Activity by Irradiation in Human Lymphoblasts. Radiat. Res. 155, 693–697 (2001).

Telomerase activity is a radiation-inducible function, which suggests a role of this enzyme in DNA damage processing. Since the tumor suppressor TP53 plays a central role in the regulation of the cellular response to DNA damage, our study explored the ability of ionizing radiation to change telomerase activity and telomere length in two closely related human lymphoblast cell lines with different TP53 status. TK6 cells (wild-type TP53) and WTK1 cells (mutated TP53) were exposed to different doses of X rays, and telomerase activity was measured by PCR ELISA at different times after irradiation. A dose-dependent increase in telomerase activity was observed. One hour after irradiation with 4 Gy, TK6 and WTK1 cells showed an approximately 2.5-fold increase; for lower doses (0.1 to 1 Gy), telomerase induction was seen only in TK6 cells. Telomerase induction was observed by 0.5 h after irradiation, with a further increase up to 24 h. Irradiated TK6 and WTK1 cells had longer telomeres ( 1.3 kb) than unirradiated cells 14 days after exposure. Our data demonstrate a dose-dependent induction of telomerase activity and lengthening of telomeres by ionizing radiation in human lymphoblasts. Induction of telomerase activity by radiation does not generally appear to be controlled by the TP53-dependent DNA damage response pathway. However, for low doses, induction of telomerase requires wild-type TP53.

Hawkins, R. B. A Microdosimetric-Kinetic Model for the Sensitization of V79 Cells to Radiation by Incorporation of Bromodeoxyuridine. Radiat. Res. 155, 698–702 (2001).

The sensitization of G₁-phase V79 cells to killing by ionizing radiation through incorporation of bromodeoxyuridine (BrdU) in their DNA has been reported to occur exclusively through an increase in the value of the quadratic parameter of the linear-quadratic survival relationship (β) with no change in the linear parameter (α). The consequence of this, as understood through the microdosimetric-kinetic model of cell survival, is discussed. It is shown that the invariance of α implies that sensitization is due solely to a decrease in the rate of repair of the initial (potentially lethal) lesions in DNA containing BrdU. Further, for α to be unchanged, the average size of the compartments into which the nucleus is partitioned (domains), as postulated in the microdosimetric-kinetic model, must vary in proportion to the reciprocal of the square root of the rate constant for repair of the DNA lesions. This implies that the domain is not a structural subunit of the nucleus. It is a surrogate representation of the consequence of lesions in DNA being restricted to a region in the vicinity of the location in the nucleus at which they are created. A lesion is confined because the distance it can diffuse by random flight to react with another lesion to form a lethal lesion is restricted because a lesion's lifetime is limited by the repair process.

Friedland, W., Li, W. B., Jacob, P. and Paretzke, H. G. Simulation of Exon Deletion Mutations Induced by Low-LET Radiation at the HPRT Locus. Radiat. Res. 155, 703–715 (2001).

The induction of HPRT mutants with exon deletions after irradiation with photons was simulated using the biophysical radiation track structure model PARTRAC. The exon-intron structure of the human HPRT gene was incorporated into the chromatin fiber model in PARTRAC. After γ and X irradiation, simulated double-stranded DNA fragments that overlapped with exons were assumed to result in exon deletion mutations with a probability that depended on the genomic or the geometric distance between the breakpoints. The consequences of different assumptions about this probability of deletion formation were evaluated on the basis of the resulting fractions of total, terminal and intragenic deletions. Agreement with corresponding measurements was obtained assuming a constant probability of deletion formation for fragments smaller than about 0.1 Mbp, and a probability of deletion formation decreasing with increasing geometric or genomic distance between the end points for larger fragments. For these two assumptions, yields of mutants with exon deletions, size distributions of deletions, patterns of deleted exons, and patterns of deleted STS marker sites surrounding the gene were calculated and compared with experimental data. The yields, size distributions and exon deletion patterns were grossly consistent, whereas larger deviations were found for the STS marker deletion patterns in this comparison.

Nuyts, S., Van Mellaert, L., Theys, J., Landuyt, W., Lambin, P. and Anné, J. The Use of Radiation-Induced Bacterial Promoters in Anaerobic Conditions: A Means to Control Gene Expression in Clostridium-Mediated Therapy for Cancer. Radiat. Res. 155, 716–723 (2001).

Apathogenic clostridia, which have been genetically engineered to express therapeutic genes, will specifically target hypoxic and necrotic regions in tumors. This specificity can be improved further if the expression of these genes is controlled by a radiation-induced promoter, leading to spatial and temporal control of gene expression. We isolated two radiation-inducible genes of the SOS repair system of Clostridium. Northern blot experiments confirmed radiation activation of the recA and recN genes at a dose of 2 Gy. The promoter region of these genes was isolated and used to regulate expression of the lacZ gene under anaerobic conditions. For the recA promoter, a significant increase of β-galactosidase activity of 20–30% was seen after 2 Gy irradiation. The recN promoter did not show a significant induction and had a 50–100 times lower basal expression. Treatment of the recombinant clostridial cultures with the cytostatic agent mitomycin C also resulted in a significant increase of β-galactosidase activity that was under the control of recA or recN promoter. Oxygen does not appear to be necessary in the activation of the SOS repair system by irradiation as tested with Escherichia coli since recA-deficient and recA-containing strains showed similar survival after treatment with UV and ionizing radiation in the presence or absence of oxygen.

Braun, R. D., Lanzen, J. L., Turnage, J. A., Rosner, G. and Dewhirst, M. W. Effects of the Interaction between Carbogen and Nicotinamide on R3230 Ac Tumor Blood Flow in Fischer 344 Rats. Radiat. Res. 155, 724–733 (2001).

The purpose of this study was to determine whether there are interactions between carbogen breathing and various doses of nicotinamide at the level of the tumor arteriole that might contribute to the improvement in tumor blood flow and pO₂ that is often seen with this combination treatment. R3230 adenocarcinomas were implanted and grown to 4–5 mm in dorsal skin flap window chambers in F344 rats. Saline or 65, 200 or 500 mg/kg nicotinamide was injected i.p. while the rat breathed air through a face mask. After 20 min, either the breathing gas was switched to carbogen for 60 min or the animal remained on air. Measured end points included diameter of tumor arterioles, tumor perfusion, mean arterial blood pressure, and heart rate. None of the measured parameters were affected by injection of saline or nicotinamide, except at the highest nicotinamide dose (500 mg/kg). Mean arterial blood pressure showed a median decrease of 25% when 500 mg/kg nicotinamide was given. Diameter of tumor arterioles decreased significantly from 5–15 min after 500 mg/kg nicotinamide was given but was back to baseline by 20 min. Blood flow decreased significantly 5–20 min after administration of 500 mg/kg nicotinamide compared to the baseline prior to injection. Carbogen breathing resulted in a small increase in mean arterial blood pressure in all groups. There was a transient decrease in the diameter of tumor arterioles and blood flow during the first 5 min of carbogen breathing that was statistically significant in several groups. In the group injected with 500 mg/kg nicotinamide, the diameter of tumor arterioles increased by about 10% during the first 25 min of carbogen breathing, and blood flow increased by a median of 75% over the level prior to carbogen breathing up to 40 min after carbogen breathing. The increase in flow in this group was most likely caused by the concomitant arteriolar vasodilation. Thus there was direct evidence for an interaction between carbogen breathing and nicotinamide, but only at the dose of 500 mg/kg nicotinamide. Since this dose yields plasma levels of nicotinamide that are higher than can be tolerated clinically, it is uncertain whether these changes in arteriolar diameter and blood flow would occur in human tumors.

Datta, P. K., Moulder, J. E., Fish, B. L., Cohen, E. P. and Lianos, E. A. Induction of Heme Oxygenase 1 in Radiation Nephropathy: Role of Angiotensin II. Radiat. Res. 155, 734–739 (2001).

In a rat model of radiation-induced nephropathy, we investigated changes in expression of heme oxygenase 1 (Hmox1, also known as HO-1), an enzyme that catalyzes conversion of heme into biliverdin, carbon monoxide and iron. The study explored whether radiation induces Hmox1 expression in the irradiated kidney and whether angiotensin II (AII) mediates Hmox1 expression in glomeruli isolated from irradiated kidneys. To assess the effects of radiation on Hmox1 expression, rats received 20 Gy bilateral renal irradiation and were randomized to groups receiving an AII type 1 (AT₁) receptor antagonist (L-158,809) or no treatment. Drug treatment began 9 days prior to bilateral renal irradiation and continued for the duration of the study. Estimation of Hmox1 levels in glomerular protein lysates assessed by Western blot analysis revealed a significant increase in Hmox1 protein at 50 and 65 days postirradiation. In animals treated with the AT₁ receptor antagonist, there was no induction of Hmox1, suggesting that AII may be a mediator of Hmox1 induction. To confirm that AII stimulates Hmox1 expression, animals were infused with 200, 400 or 800 ng/kg min^–1 of AII for 18–19 days, and Hmox1 protein levels in glomeruli were assessed. There was a significant induction of Hmox1 in glomeruli of animals infused with 800 ng/kg min^–1 of AII. These studies demonstrate that glomerular Hmox1 expression is elevated in the middle phase of radiation nephropathy and that AII can increase glomerular Hmox1 levels.

Norman, A., Cochran, S. T. and Sayre, J. W. Meta-analysis of Increases in Micronuclei in Peripheral Blood Lymphocytes after Angiography or Excretory Urography. Radiat. Res. 155, 740–743 (2001).

Meta-analysis of 10 studies confirms a significant increase in the frequency of micronuclei in peripheral blood lymphocytes after angiography or excretory urography; the weighted average increase is 4.2 (95% confidence interval 2.8–5.6) per 1000 binucleate lymphocytes, about the same increase in micronuclei as that produced in vitro by a diagnostic X-ray dose of 4 cGy. The analysis failed to reveal a significant effect of the specific contrast medium used in the X-ray examinations on the increased frequency of micronuclei. These results are consistent with the hypothesis that the effect of the contrast media is limited to the enhancement, by the photoelectric effect, of the X-ray dose absorbed by the lymphocytes irradiated while suspended in the contrast medium. Therefore, an estimate of increased cancer risk based on elevated frequencies of micronuclei or chromosome aberrations in peripheral blood lymphocytes may be greatly exaggerated whenever the radiation damage is largely confined to the cells circulating in the blood, as it is in people who have recently had X-ray examinations that use intravenous injections of contrast medium. Such examinations include angiography, excretory urography and CT scans, which are received annually by millions of people.

Regulla, D., Panzer, W., Schmid, E., Stephan, G. and Harder, D. Detection of Elevated RBE in Human Lymphocytes Exposed to Secondary Electrons Released from X-Irradiated Metal Surfaces. Radiat. Res. 155, 744–747 (2001).

Monolayers of human lymphocytes, attached to a 2-μm Mylar film, were irradiated with 60 kV X rays in the presence and absence of a 150-μm gold film backing the Mylar film. With the gold film present, the absorbed dose imparted to the cells was increased by a factor of 45.4 due to the release of photoelectrons from the gold film. The frequencies of dicentric chromosomes and centric rings as well as of excess acentric fragments were increased in agreement with this dose enhancement, and in addition an RBE of about 1.7 compared to the frequencies observed in the absence of the gold film was found. These radiation effects, which contribute to risk considerations in radiology, are interpreted in terms of the increased dose-mean restricted LET of the photoelectrons backscattered from the metal and slowed down in the Mylar film before they enter the cell layer.

Bourlier, V., Diserbo, M., Gourmelon, P. and Verdetti, J. Prolonged Effects of Acute Gamma Irradiation on Acetylcholine-Induced Potassium Currents in Human Umbilical Vein Endothelial Cells. Radiat. Res. 155, 748–752 (2001).

We have recently reported an acute effect of γ irradiation (15 Gy, 1 Gy/min) on acetylcholine-mediated endothelium-dependent relaxation in rat aortic rings. Given the importance of permeability to K to endothelium-dependent relaxation, we have evaluated the effect of the same radiation on K currents in human endothelial cells in culture using the patch-clamp technique in the whole-cell recording configuration. Our results indicate that, in resting cells, γ irradiation has no effect on endothelial permeability to K . However, irradiation during stimulation of endothelial cells with acetylcholine reduces the sustained increase in permeability to K observed in the acetylcholine-stimulated, nonirradiated cells. Additional experiments using K channel inhibitors (TEA, charybdotoxin, apamin) suggest that irradiation may in part decrease the prolonged activation of Ca² -activated K channels by acetylcholine. Taken together with our previous finding that irradiation inhibits the acute relaxing effects of acetylcholine, these results show that γ irradiation also affects the delayed effects of acetylcholine on permeability to K .