Hoppe, B. S., Jensen, R. B. and Kirchgessner, C.U. Complementation of the Radiosensitive M059J Cell Line.

M059J is a radiosensitive cell line established from a human glioblastoma tumor that fails to express the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs, now known as PRKDC). Another cell line, M059K, established from the same tumor is radioresistant. Neither M059J nor M059K cells have been fully characterized, beyond the lack of expression of PRKDC and low expression of ATM in M059J cells. To determine whether its radiosensitive phenotype is due to a defect in the gene that encodes PRKDC, we show here that M059J cells can be complemented with the PRKDC gene by introducing a fragment of human chromosome 8 containing a copy of the human PRKDC gene. Two hybrid cell lines that retain an extra copy of PRKDC display active kinase activity and are radioresistant, demonstrating that the primary defect in M059J cells is in PRKDC. In addition, these cell lines derived from M059J cells provide us with a closer genetic match to M059J than M059K cells in studies to elucidate the function of DNA-PK.

DeSimone, J.N., Dolezalova, H., Redpath, J.L. and Stanbridge, E.J. Prolonged Cell Cycle Arrest in Irradiated Human Diploid Skin Fibroblasts: The Role of Nutrient Deprivation.

Ionizing radiation has been reported to cause an irreversible cell cycle arrest in normal human diploid fibroblasts. However, colony survival assays show that even at high doses of γ radiation, human diploid fibroblasts do not irreversibly arrest, and that a dose-dependent fraction is capable of continued cycling. In this study, we resolve the apparent discrepancy between colony survival assays and the observed radiation-induced prolonged arrest. Using flow cytometry analysis, we have confirmed that human diploid fibroblasts do exhibit a prolonged cell cycle arrest in both G₁ and G₂/M phases of the cell cycle. However, a single replacement of fresh growth medium stimulated a fraction of the arrested population of cells to transiently re-enter the cell cycle. Daily medium changes stimulated these irradiated human diploid fibroblasts to continue cycling until they were contact-inhibited. Thus the fraction of human diploid fibroblasts which survive radiation exposure and are capable of cycling appears to permanently arrest as a result of nutrient insufficiency. Western blot analysis demonstrated a radiation-induced elevation in TP53 (formerly known as p53) protein levels within 2 h postirradiation, followed by a decrease to levels comparable to those in unirradiated controls. The TP53 and CDKN1A (formerly known as p21) protein levels were indistinguishable after 24 h and remained elevated for a 6-day period of observation in both control and irradiated cultures. Our studies indicate that human diploid fibroblasts are capable of re-entering the cell cycle after exposure to ionizing radiation and that this re-entry is dependent on a constant supply of nutrients provided by fresh medium changes. The fraction of cells capable of resuming cell cycling is consistent with the surviving fraction of cells in colony assays.

Kashiwakura, I., Kuwabara, M., Inanami, O., Murakami, M., Hayase, Y., Takahashi, T.A. and Takagi Y. Radiation Sensitivity of Megakaryocyte Colony-Forming Cells in Human Placental and Umbilical Cord Blood.

The in vitro radiation sensitivity of CFU-Meg isolated from human placental and umbilical cord blood was evaluated in plasma clot cultures stimulated by recombinant human cytokines, including thrombopoietin, the FLT3 ligand (FLT3LG), interleukin-3, interleukin-11 and stem cell factor. The CD34 cells were irradiated with X rays at a dose rate of 73 cGy/min. The megakaryocyte colonies were identified by using an FITC-conjugated antibody to glycoprotein IIbIIIa and were classified into two groups based on colony size: large colonies (immature CFU-Meg) and small colonies (mature CFU-Meg). Treatment with thrombopoietin alone or in combination with FLT3LG and/or interleukin-11 gave exponential radiation survival curves (D₀ for immature CFU-Meg = 56–77 cGy, D₀ for mature CFU-Meg = 86 cGy–1.12 Gy), while marked shoulders were observed on the survival curves for colonies supported by the combination of thrombopoietin, interleukin-3 and stem cell factor (D₀ for immature CFU-Meg = 89–98 cGy; D₀ for mature CFU-Meg = 1.25–1.31 Gy). Our results showed that the immature CFU-Meg were more radiosensitive than the mature CFU-Meg and that the combination of cytokines, including thrombopoietin, interleukin-3 and stem cell factor, affected the radiation sensitivity of CFU-Meg to the same extent as with thrombopoietin alone or in combination with FLT3LG and/or interleukin-11.

Mayberg, M.R., London, S., Rasey, J. and Gajdusek.C. Inhibition of Rat Smooth Muscle Proliferation by Radiation after Arterial Injury: Temporal Characteristics In Vivo and In Vitro.

Although several studies have suggested that inhibition of arterial narrowing by radiation after angioplasty is dependent on both time and dose, little is known regarding the temporal aspects of this effect and the mechanisms by which radiation affects the response of smooth muscle cells to injury. To determine the time course of inhibition of intimal hyperplasia by radiation, 135 rats were given single-fraction external γ irradiation (1–10 Gy) to one carotid artery at intervals from 5 days before to 5 days after bilateral carotid artery balloon catheter injury, and intimal cross-sectional area was determined from histological sections at 20 days after injury. There was a prominent time- and dose-dependent inhibition of intimal hyperplasia by radiation when it was administered before or after balloon injury, with the greatest effect noted within 24 h before or after injury. To investigate the effect of radiation on smooth muscle cell growth (by cell counting) and proliferation, cell cycle kinetics (by BrdU incorporation), and cell killing (by clonogenic assay), smooth muscle cell cultures derived from rat aortic explants were seeded in equine plasma to induce quiescence, and radiation (2.5–10 Gy) was administered at various intervals before or after synchronous growth stimulation by 10% whole blood serum. A similar time and dose dependence was noted in growth kinetics, BrdU incorporation and cell killing for smooth muscle cells irradiated in vitro; in each case, the effect was most prominent for radiation administered in temporal proximity to stimulation with whole blood serum. By Western blot analysis, cultured smooth muscle cells showed a rapid time-dependent increase in Cdkn1a (formerly known as p21) protein expression, followed by a delayed increase in Tp53 (formerly known as p53) expression after irradiation. Activation of intracellular caspases, manifest by proteolytic poly(ADP-ribose) polymerase (PARP) cleavage, was not detected in smooth muscle cell cultures after irradiation. These observations suggest that radiation limits intimal hyperplasia in vivo by a transient, reversible process. Although apparent cytotoxic injury occurs in vitro, apoptosis of smooth muscle cells is not apparent. Both inhibition of proliferation of smooth muscle cells and cell cycle delay may contribute to inhibition of intimal hyperplasia in vivo by radiation.

Kirichenko, A.V., Mason, K.A., Straume, M., Teates, C.D. and Rich, T.A. Nuclear Scintigraphic Assessment of Radiation-Induced Intestinal Dysfunction.

Radiation-induced damage to the intestine can be measured by abnormalities in the absorption of various nutrients. Changes in intestinal absorption occur after irradiation because of loss of the intestinal absorptive surface and a consequent decrease in active transport. In our study, the jejunal absorption of ^99mTc-pertechnetate, an actively transported γ-ray emitter, was assessed in C3H/Kam mice given total-body irradiation with doses of 4, 6, 8 and 12.5 Gy and correlated with morphological changes in the intestinal epithelium. The absorption of ^99mTc-pertechnetate from the intestinal lumen into the circulation was studied with a dynamic γ-ray-scintigraphy assay combined with a multichannel analyzer to record the radiometry data automatically in a time-dependent regimen. The resulting radioactivity–time curves obtained for irradiated animals were compared to those for control animals. A dose-dependent decrease in absorptive function was observed 3.5 days after irradiation. The mean absorption rate was reduced to 78.8 ± 9.3% of control levels in response to 4 Gy total-body irradiation (mean ± SEM tracer absorption lifetime was 237 ± 23 s compared to 187 ± 12 s in nonirradiated controls) and to 28.3 ± 3.7% in response to 12.5 Gy (660 ± 76 s). The decrease in absorption of ^99mTc-pertechnetate at 3.5 days after irradiation correlated strongly (P < 0.001) with TBI dose, with the number of cells per villus, and with the percentage of cells in the crypt compartment that were apoptotic or mitotic. A jejunal microcolony assay showed no loss of crypts and hence no measured dose–response effects after 4, 6 or 8 Gy TBI. These results show that dynamic enteroscintigraphy with sodium ^99mTc-pertechnetate is a sensitive functional assay for rapid evaluation of radiation-induced intestinal damage in the clinically relevant dose range and has a cellular basis.

Wittig, A., Sauerwein, W.A. and Coderre, J.A. Mechanisms of Transport of p-Borono-Phenylalanine through the Cell Membrane. Radiat. Res. 153, 173–180 (2000).

The mechanisms of transport of p-(dihydroxyboryl)-phenylalanine (BPA) through the cell membrane were investigated in vitro, evaluating especially the systems responsible for the transport of neutral amino acids as potential carriers for BPA. Rat 9L gliosarcoma cells and Chinese hamster V79 cells were exposed to BPA under controlled conditions and in a defined medium that was free of amino acids. The time course of ¹⁰B (delivered by BPA) uptake and efflux was measured under different conditions. To analyze the intracellular boron content, direct-current plasma atomic emission spectroscopy (DCP-AES) was used after separating the cells from extracellular boron in the cell medium using an oil filtration technique. The dependence of factors such as cell type, temperature, composition and concentration of amino acids and specific substrates for amino acid transport systems in the culture medium or in intracellular compartments on BPA uptake and efflux were studied. The results of this study support the hypothesis that BPA is transported by the L system and that transport can be further stimulated by amino acids preaccumulated in the cell by either the L or A amino acid transport system.

Kohli, R., Gupta, P.K. and Dube, A. Helium-Neon Laser Preirradiation Induces Protection against UVC Radiation in Wild-Type E. coli Strain K12AB1157.

We have observed that preirradiation with a helium-neon laser (632.8 nm) induces protection against UVC radiation in wild-type E. coli strain K12AB1157. The magnitude of protection was found to depend on the helium-neon laser irradiance, exposure time, and period of incubation between helium-neon laser exposure and subsequent UVC irradiation. The optimum values for dose, irradiance and interval between the two exposures were found to be 7 kJ/m², 100 W/m² and 1 h, respectively. The possible involvement of singlet oxygen in the helium-neon laser-induced protection is also discussed.

Mendez, F., Sandigursky, M., Franklin, W.A., Kenny, M.K., Kureekattil, R. and Bases, R. Heat-Shock Proteins Associated with Base Excision Repair Enzymes in HeLa Cells.

Two enzymes of base excision repair (BER), uracil DNA glycosylase (UDG) and DNA polymerase β (β pol), from HeLa cells co-eluted from Superose 12 FPLC columns. The UDG was completely displaced from 150–180-kDa fractions to 30–70-kDa fractions by brief treatment with 0.5 N NaCl, pH 3.0, as expected when protein–protein associations are disrupted, but β pol was not displaced by this treatment. UDG was not essential to the presence of β pol in the 150–180-kDa enzyme complex. β pol and UDG apparently reside in separate but co-eluting structures. Immunoaffinity chromatography showed that the association of UDG and β pol was accounted for by attachment in common to DNA and that the association was abolished by eliminating DNA. Evidence for base excision repairosomes containing UDG and β pol in protein–protein assemblies was not found. However, UDG and human AP endonuclease (HAP1) were associated with HSP70 and HSP27, which are present in 150–180-kDa and 30–70-kDa proteins of cell sonicates. The association of HSPs with BER enzymes was confirmed by hydroxyl radical protein–protein footprinting and immunoaffinity tests. The association of HSPs and BER enzymes is a novel finding. HSP binding may account for the presence of BER enzymes in the two large size class fractions and HSPs may have functional roles in BER.

LaVerne, J.A. OH Radicals and Oxidizing Products in the Gamma Radiolysis of Water.

The production of OH radicals in the γ radiolysis of water has been examined with radical scavenger techniques employing formic acid. OH radical yields were found to vary from 2.4 radicals/100 eV at the low scavenger concentration limit to 4.2 at a formic acid concentration of 3 M. An inverse Laplace transform technique was applied to the scavenger concentration dependence to obtain the temporal dependence of OH radicals in pure water. It was found that the relative decrease in OH radical yields from 200 ps to 3 ns was virtually the same for the transform of the scavenger data and the directly measured time-resolved results. The absolute yields for the time-resolved experiments are about 10% higher than expected from the present results with scavengers. The agreement can be considered to be good, and reasons for the observed difference are given. Approximately 40% of the OH radicals produced lead to the formation of hydrogen peroxide, which is the only other major oxidizing species in the γ radiolysis of water. The net water decomposition for γ rays was found to vary from an initial value of 5.6 ± 0.3 molecules/100 eV to 3.8 ± 0.2 molecules/100 eV at 1 μs.

Hindo, J., Hauville, C., Rémita, S., Thérond, P., Couturier, M., Jore, D. and Gardès-Albert, M. Evidence of the Formation of Different Hydroperoxides in Irradiated Gamma-Linolenate Solutions: Effect of Micelle Formation. Radiat. Res. 153, 201–207 (2000).

Peroxidation of unconjugated polyunsaturated fatty acids such as linolenic acid proceeds through a free radical chain mechanism and is accompanied by the formation of conjugated dienes such as hydroperoxides. In an investigation of radiation-induced oxidation of aqueous linolenate, we have measured two indexes of peroxidation: (1) conjugated dienes by means of absorption spectroscopy and (2) hydroperoxides by high-pressure liquid chromatography using detection of chemiluminescence. The experimental results indicate a strong effect of the concentration of linolenate on the yields of oxidized products. In addition, this work shows the quantitative production of two kinds of hydroperoxides. The ratio of these hydroperoxides is independent of the radiation dose but is dependent on the linolenate concentration. One hydroperoxide is formed predominantly below the critical micellar concentration (3 mM under our conditions), while the second is observed predominantly when micelles are formed in the aqueous medium. The influence of the composition of the medium on the nature of both hydroperoxides is discussed.

Bichsel, H., Hiraoka, T. and Omata, K. Aspects of Fast-Ion Dosimetry.

A first step in the dosimetry of fast-ion beams is the determination of accurate Bragg (ionization) functions. Bragg functions for several substances have been measured and calculated for 3480 MeV carbon ions. In the measurements, the ions first traverse an absorber in which the energy is reduced to either 1900 or 1200 MeV, then a “range gauge” followed by a thin ionization chamber. Functions are calculated with an analytical method using convolutions of straggling functions. This approach gives results without the stochastic variations implicit in Monte Carlo methods. The comparison of measured and calculated functions shows how reliable the calculations are. An important part of the calculations is the determination of the total range of the ions. The range can be determined from the Bragg function. The measured range is given by the sum of the thickness of the absorber and the residual range measured with the range gauge. For water, the range is about 150 mm, and the precision of the measurements is ±0.05 mm. Because the ion energy at the surface of the absorber fluctuates with time, measurements with water are used to define this energy. Thus the ranges (or average stopping powers) in absorbers are obtained relative to those in water. Measured ranges R_m are compared with ranges R₀ calculated with a current version of the Bethe theory. For light absorbers (atomic number Z < 20), differences between R_m and R₀ are less than ±0.3 mm; for Z > 20 differences are between 0 and ±0.6 mm. This agreement between calculated and measured ranges confirms the value I = 80 eV for water measured earlier for protons. The ionization by nuclear fragments is obtained from the difference between measured and calculated ionization functions, and has little influence on the ranges of the primary ions.

The extended abstracts that follow provide a summary of the Proceedings of the 4th International Workshop: Microbeam Probes of Cellular Radiation Response, held in Killiney Bay, Dublin, on July 17–18, 1999, which was jointly organized by the Columbia University Radiological Research Accelerator Facility and the MIT Laboratory for Accelerator Beam Applications.

There is increasing interest in the use of microbeam systems, which can deliver beams of different radiations with a spatial resolution of a few micrometers or less, for radiobiological research. Single-particle microbeams can be used to address such questions as the relative sensitivities of different parts of the cell (e.g. nucleus compared to cytoplasm), and the effects of irradiation of neighboring (bystander) cells. For particle (e.g. α-particle) beams, irradiation with exactly one (or more) particle per cell can be achieved, allowing questions of risks of very low doses of ionizing radiations, such as radon, to be addressed. Several microbeams are now in operation, and others are being developed. The workshop provided a forum to assess the current state of microbeam technology and current biological applications, and to discuss future directions, both technological and biological.

Roughly 75 scientists (about equal numbers of physicists and biologists) attended the workshop, the fourth in a biannual series (1). A list of attendees can be obtained from David Brenner (djb3@columbia.edu). A fifth meeting is planned for the year 2001.

Support for this workshop from the U.S. National Center for Research Resources (grant P41 RR11623-03), the U.S. National Cancer Institute, and the U.S. Department of Energy, is gratefully acknowledged.

Reference

1.  B.D. Michael, M. Folkard and K.M. Prise, Meeting report: Microbeam probes of cellular radiation response, 4th L.H. Gray Workshop, 8–10 July 1993. Int.J. Radiat. Biol. 65, 503–508 (1994).