Galloway, A. M. and Allalunis-Turner, J. cDNA Expression Array Analysis of DNA Repair Genes in Human Glioma Cells that Lack or Express DNA-PK.

M059J cells provide the only example of DNA-PKcs (now known as PRKDC) deficiency in a human cell line. M059K cells, derived from the same tumor specimen, express PRKDC protein and activity and, together with M059J, provide a useful model in which to study the role of DNA-PK in cellular responses to DNA-damaging agents. Because these cells are of tumor origin, we used Atlas™ human cancer cDNA expression arrays to investigate possible differential expression of other DNA repair genes in control and irradiated samples. cDNA array results indicated differential expression of 14 genes. Northern blotting confirmed relatively greater expression of replication factor C 37-kDa subunit mRNA in M059J cells compared to M059K cells and reduced expression of DNA ligase IV compared to ligase III in both cell lines independent of irradiation. These results suggest that other DNA repair proteins are altered in these cell lines and that repair mechanisms predicted from the study of normal tissues may be fundamentally altered in human cancer cells.

Albanese, J. and Dainiak, N. Regulation of TNFRSF6 (Fas) Expression in Ataxia Telangiectasia Cells by Ionizing Radiation.

Several studies have shown that ionizing radiation induces transcription of the TNFRSF6 (Fas) gene, leading to augmented TNFRSF6 protein levels at the surface of irradiated cells. We have examined TNFRSF6 expression in an apparently normal lymphocyte line and in a lymphocyte cell line derived from a patient with ataxia telangiectasia (AT) before and after exposure to radiation (0–10 Gy). Plasma membranes were isolated from normal lymphocytes and AT cells and subjected to Western blot analysis, using a TNFRSF6-specific monoclonal antibody to probe resolved proteins transferred onto nitrocellulose membranes. In both cell types, the presence of a 48-kDa band corresponding to the molecular mass of TNFRSF6 was revealed. Analysis of FITC-conjugated anti-TNFRSF6 antibody-stained normal lymphocytes and AT cells confirmed TNFRSF6 expression in both cell types. In MTT assays, AT cells treated with agonistic anti-TNFRSF6 Ab (CH.11) displayed a 25.9% decrease in cell viability, relative to cells treated with isotype-matched IgM Ab, suggesting the presence of a biologically active TNFRSF6 receptor at the AT cell surface. Exposure to cycloheximide (0–5 μg/ml), a metabolic inhibitor, enhanced sensitivity of AT cells to CH.11. Normal lymphocytes exhibited increased levels of apoptosis (approximately 34% cell death relative to cells treated with isotype-matched IgM Ab) when exposed to CH.11; however, the degree of cell death was not altered significantly with increasing concentrations of cycloheximide. When AT cells were exposed to 0.1, 0.5, 2 and 10 Gy, the activities of caspases 3 and 8 increased in a dose-dependent manner at 24 h postirradiation and reached a plateau by 72 h. A similar trend for activation of caspase 3 and 8 was observed in normal lymphocytes after irradiation. To assess the roles of TNFRSF6 and/or caspase 8 in radiation-induced cell death of AT and normal lymphocytes, and to determine whether hyper-radiosensitivity in AT cells is correlated with increased activity of these two components of the TNFRSF6 pathway, AT and normal lymphocytes were irradiated in the presence of ZB4, an anti-TNFRSF6 blocking antibody, and a caspase 8 inhibitor (Z-IETD-FMK). Apoptosis was determined by Annexin V staining using flow cytometry. Incubation with ZB4 anti-TNFRSF6 antibody did not alter the fraction of apoptotic cells in either AT cells or normal lymphocytes treated with doses of radiation ranging from 0–10 Gy. In contrast, apoptosis was significantly reduced in both cell lines in the presence of Z-IETD-FMK when samples were exposed to low-dose (≤2 Gy) radiation. Relative to control samples (those not incubated with Z-IETD-FMK), no difference in the level of apoptosis was observed in AT or normal lymphocytes treated with 10 Gy. These data indicate that: (a) despite radiation-induced up-regulation of TNFRSF6 at the cell surface, the death-promoting receptor does not play a role in radiation-mediated cytotoxicity; (b) apoptosis in lymphocytes irradiated with low (≤2 Gy) but not high doses (>2 Gy) proceeds at least in part through activation of caspase 8; and (3) since blocking anti-TNFRSF6 antibody (ZB4) did not reduce levels of apoptosis in irradiated AT cells to those of normal lymphocytes, TNFRSF6 is unlikely to play a significant role in the hyper-radiosensitivity exhibited by cells having the AT phenotype.

Forrester, H. B., Albright, N., Ling, C. C. and Dewey, W. C. Computerized Video Time-Lapse Analysis of Apoptosis of REC:Myc Cells X-Irradiated in Different Phases of the Cell Cycle.

Asynchronous rat embryo cells expressing Myc were followed in 50 fields by computerized video time lapse (CVTL) for three to four cycles before irradiation (4 Gy) and then for 6–7 days thereafter. Pedigrees were constructed for single cells that had been irradiated in different parts of the cycle, i.e. at different times after they were born. Over 95% of the cell death occurred by postmitotic apoptosis after the cells and their progeny had divided from one to six times. The duration of the process of apoptosis once it was initiated was independent of the phase in which the cell was irradiated. Cell death was defined as cessation of movement, typically 20–60 min after the cell rounded with membrane blebbing, but membrane rupture did not occur until 5 to 40 h later. The times to apoptosis and the number of divisions after irradiation were less for cells irradiated late in the cycle. Cells irradiated in G₁ phase divided one to six times and survived 40–120 h before undergoing apoptosis compared to only one to two times and 5–40 h for cells irradiated in G₂ phase. The only cells that died without dividing after irradiation were irradiated in mid to late S phase. Essentially the same results were observed for a dose of 9.5 Gy, although the progeny died sooner and after fewer divisions than after 4 Gy. Regardless of the phase in which they were irradiated, the cells underwent apoptosis from 2 to 150 h after their last division. Therefore, the postmitotic apoptosis did not occur in a predictable or programmed manner, although apoptosis was associated with lengthening of both the generation time and the duration of mitosis immediately prior to the death of the daughter cells. After the non-clonogenic cells divided and yielded progeny entering the first generation after irradiation with 4 Gy, 60% of the progeny either had micronuclei or were sisters of cells that had micronuclei, compared to none of the progeny of clonogenic cells having micronuclei in generation 1. However, another 20% of the non-clonogenic cells had progeny with micronuclei appearing first in generation 2 or 3. As a result, 80% of the non-clonogenic cells had progeny with micronuclei. Furthermore, cells with micronuclei were more likely to die during the generation in which the micronuclei were observed than cells not having micronuclei. Also, micronuclei were occasionally observed in the progeny from clonogenic cells in later generations at about the same time that lethal sectoring was observed. Thus cell death was associated with formation of micronuclei. Most importantly, cells irradiated in late S or G₂ phase were more radiosensitive than cells irradiated in G₁ phase for both loss of clonogenic survival and the time of death and number of divisions completed after irradiation. Finally, the cumulative percentage of apoptosis scored in whole populations of asynchronous or synchronous populations, without distinguishing between the progeny of individually irradiated cells, underestimates the true amount of apoptosis that occurs in cells that undergo postmitotic apoptosis after irradiation. Scoring cell death in whole populations of cells gives erroneous results since both clonogenic and non-clonogenic cells are dividing as non-clonogenic cells are undergoing apoptosis over a period of many days.

Binder, A., Serafin, A. and Bohm, L. Abrogation of G₂/M-Phase Block Enhances the Cytotoxicity of Daunorubicin, Melphalan and Cisplatin in TP53 Mutant Human Tumor Cells.

Irradiation of human melanoma (MeWo, Be11) and squamous cell carcinoma (4451, 4197) cells induces cell cycle blocks from which the cells recover to re-enter mitosis after 40–60 h. In the TP53 mutant cell lines, MeWo and 4451, irradiation induces a G₂-phase block, where the fraction of cells in G₂ phase reaches a maximum after 18–20 h. In the TP53 wild-type cell lines, 4197 and Be11, a G₁- and G₂-phase block is reached 12 and 16 h postirradiation, respectively. Addition of pentoxifylline after irradiation at the time when the number of cells in G₂ phase has reached a maximum shortens the normal recovery from G₂-phase block to approximately 7 h. Addition of daunorubicin, melphalan and cisplatin under these conditions markedly enhanced drug toxicity. In the TP53-mutated cell lines MeWo and 4451, the survival ratio at 7 Gy measured by colony formation was 2.3–2.8, 8.6–85 and 52–74 for daunorubicin, melphalan and cisplatin, respectively. In the TP53 wild-type cell lines, the corresponding survival ratios were found to be 1.3–1.4, 2.3–3.0 and 1.2–2.6, respectively. The survival ratios are for clonogenic survival after 7 Gy and 2 mM pentoxifylline and measure the influence of drug doses that ensure 95% survival in nonirradiated controls. The results indicate that the G₂-phase block is a crucial event in the damage response that can be manipulated to achieve a significant enhancement of drug toxicity. These effects are particularly pronounced in TP53 mutant cells and are observed at drug doses well below the clinical range.

Manti, L., Davies, H. E., Venables, S., Bowen, I. D. and Court, J. B. Correlation between the Clonogenic Initial Slope and the Response of Polykaryon-Forming Units: The Behavior of Strains Defective in XRCC5 and ATM and the Heritability of Small Variations in Radioresponse.

The polykaryon-forming unit (PFU) assay measures the survival of multiple cycles of DNA synthesis after exposure to ionizing radiation, and it is known that there is a strong correlation between the slope of the PFU dose–response curve and the clonogenic initial slope. This suggests that DNA lesions expressed in clonogens are also important in PFU. Cells having a mutation in XRCC5 (also known as Ku80; strain xrs-6) and ATM (strain AT5BIVA) were hypersensitive in the PFU assay and in clonogens, while a strain of xrs-6 cells transfected with hamster wild-type XRCC5 cDNA displayed wild-type resistance in both assays. These data suggest that the DNA double-strand break (DSB) is an important lesion in PFU, although the relative radioresistance of PFU compared to clonogens indicates differential DSB toxicity. We propose that this results from the absence of cytokinesis-related loss of DNA fragments. Small variations in the radioresponse of PFU were observed between CHO K1 cell substrains, such that the xrs parental substrain RR-CHOK1 (carrying wild-type XRCC5) was more sensitive than an independent K1 substrain (E-CHOK1). Somatic hybridization showed that this variation is heritable and that the resistant E phenotype is dominant. In RR-CHOK1 cells there was a biphasic PFU radioresponse, which suggests that there may be transient expression at a locus selectively affecting PFU sensitivity.

Roy, K., Kodama, S., Suzuki, K., Fukase, K. and Watanabe, M. Hypoxia Relieves X-Ray-Induced Delayed Effects in Normal Human Embryo Cells.

We studied the effect of hypoxia on X-ray-induced delayed effects in normal human embryo cells to elucidate the role of oxidative stress in the susceptibility of cells to induction of genetic instability by radiation. We examined X-ray-induced delayed cell death, giant cell formation, and chromosome aberrations under normally oxygenated (20%) and hypoxic (2%) conditions at 28–38 population doublings postirradiation. The results revealed that hypoxia reduced the X-ray-induced delayed effects, suggesting that radiation enhances cellular oxidative stress, which plays a significant role in determining the susceptibility of irradiated cells to genetic instability. The present study emphasizes the biological significance of epigenetic effects, such as oxygen tension, as well as direct DNA damage in the induction of genetic instability by radiation.

Hacker-Klom, U. B., Köhnlein, W. and Göhde, W. Effects of Single and Split Doses of Cobalt-60 Gamma Rays and 14 MeV Neutrons on Mouse Stem Cell Spermatogonia.

The long-term effects of ionizing radiation on male gonads may be the result of damage to spermatogonial stem cells. Doses of 10 cGy to 15 Gy ⁶⁰Co γ rays or 10 cGy to 7 Gy 14 MeV neutrons were given to NMRI mice as single or split doses separated by a 24-h interval. The ratios of haploid spermatids/2c cells and the coefficients of variation of DNA histogram peaks as measures of both the cytocidal and the clastogenic actions of radiation were analyzed by DNA flow cytometry after DAPI staining. The coefficient of variation is not only a statistical examination of the data but is also used here as a measure of residual damage to DNA (i.e. a biological dosimeter). Testicular histology was examined in parallel. At 70 days after irradiation, the relative biological effectiveness for neutrons at 50% survival of spermatogonial stem cells was 3.6 for single doses and 2.8 for split doses. The average coefficient of variation of unirradiated controls of elongated spermatids was doubled when stem cells were irradiated with single doses of approximately 14 Gy ⁶⁰Co γ rays or 3 Gy neutrons and observed 70 days later. Split doses of ⁶⁰Co γ rays were more effective than single doses, doubling DNA dispersion at 7 Gy. No fractionation effect was found with neutrons with coefficients of variation.

Wang, B., Ohyama, H., Haginoya, K., Odaka, T., Yamada, T. and Hayata, I. Prenatal Radiation-Induced Limb Defects Mediated by Trp53-Dependent Apoptosis in Mice.

We reported previously that in utero radiation-induced apoptosis in the predigital regions of embryonic limb buds was responsible for digital defects in mice. To investigate the possible involvement of the Trp53 gene, the present study was conducted using embryonic C57BL/6J mice with different Trp53 status. Susceptibility to radiation-induced apoptosis in the predigital regions and digital defects depended on both Trp53 status and the radiation dose; i.e., Trp53 wild-type (Trp53 ^/ ) mice appeared to be the most sensitive, Trp53 heterozygous (Trp53 ^/–) mice were intermediate, and Trp53 knockout (Trp53^–/–) mice were the most resistant. These results indicate that induction of apoptosis and digital defects by prenatal irradiation in the later period of organogenesis are mediated by the Trp53 gene. These findings suggest that the wild-type Trp53 gene may be an intrinsic genetic susceptibility factor that is responsible for certain congenital defects induced by prenatal irradiation.

Takahashi, M., Kojima, S., Yamaoka, K. and Niki, E. Prevention of Type I Diabetes by Low-Dose Gamma Irradiation in NOD Mice.

Pretreatment with nonlethal, low-dose irradiation has been shown to have a protective effect against oxidative injury in animal tissues. Since oxidative injury of tissues is known to be a major cause of many human diseases, we examined the effect of low-dose irradiation on the progression of type I diabetes in mice. Nonobese diabetic (NOD) mice were treated with γ irradiation and the progression of the disease was monitored. An elevated level of glucose in urine was first detected at 15 weeks of age in the control NOD mice, whereas the detection was delayed as long as 7 weeks when the mice received a single dose of 0.5 Gy total-body irradiation between 12 and 14 weeks of age. The greatest effect was observed in the mice irradiated at 13 weeks of age. The increase in blood glucose and decrease in blood insulin were effectively suppressed by irradiation at 13 weeks of age. Both suppression of cell death by apoptosis and an increase in superoxide dismutase (SOD) activity were observed in the pancreas 1 week after irradiation. The results indicate that treatment with 0.5 Gy γ rays suppresses progression of type I diabetes in NOD mice. This is the first report on the preventive effect of low-dose irradiation on disease progression.

Takahashi, S., Takeda, E., Kubota, Y. and Okayasu, R. Inhibition of Repair of Radiation-Induced DNA Double-Strand Breaks by Nickel and Arsenite.

The effect of arsenite or nickel on the repair of DNA double-strand breaks (DSBs) was studied in γ-irradiated Chinese hamster ovary cells using pulsed-field gel electrophoresis. After treatment with nickel chloride or arsenite for 2 h, cells were irradiated with γ rays at a dose of 40 Gy, and the numbers of DNA DSBs were measured immediately after irradiation as well as at 30 min postirradiation. Both arsenite and nickel(II) inhibited repair of DNA DSBs in a concentration-dependent manner; 0.08 mM arsenite significantly inhibited the rejoining of DSBs, while 76 mM nickel was necessary to observe a clear inhibition. The mean lethal concentrations for the arsenite and nickel(II) treatments were approximately 0.12 and 13 mM, respectively. This indicates that the inhibition of repair by arsenite occurred at a concentration at which appreciable cell survival occurred, but that nickel(II) inhibited repair only at cytotoxic concentrations at which the cells lost their proliferative ability. These novel observations provide insight into the mechanisms underlying the effects of combined exposure to arsenite and ionizing radiation in our environment.

Getoff, N. Pulse Radiolysis Studies of β-Carotene in Oxygenated DMSO Solution. Formation of β-Carotene Radical Cation.

The spectroscopic and kinetic characteristics of β-carotene radical cation (β-carotene^· ) were studied by pulse radiolysis in aerated DMSO solution. The buildup of β-carotene^· with k₁ = (4.8 ± 0.2) × 10⁸ dm³ mol^–1 s^–1 [λ_max = 942 nm, ϵ = (1.6 ± 0.1) × 10⁴ dm³ mol^–1 cm^–1] results from an electron transfer from β-carotene to DMSO^· . The β-carotene^· species decays exclusively by first-order reaction, k = (2.1 ± 0.1) × 10³ s^–1, probably by two processes: (1) at low substrate concentration by hydrolysis and (2) at high concentrations also by formation of dimer radical cation (β-carotene)₂^· . Under the experimental conditions, a small additional β-carotene triplet-state absorption (³β-carotene) in the range of 525 to 660 nm was observed. This triplet absorption is quenched by oxygen (k = 7 × 10⁴ s^–1), resulting in singlet oxygen (¹O₂), whose reactions can also lead to additional formation of β-carotene^· .

Badhwar, G. D., Huff, H. and Wilkins, R. Alterations in Dose and Lineal Energy Spectra under Different Shieldings in the Los Alamos High-Energy Neutron Field.

Nuclear interactions of space radiation with shielding materials result in alterations in dose and lineal energy spectra that depend on the specific elemental composition, density and thickness of the material. The shielding characteristics of materials have been studied using charged-particle beams and radiation transport models by examining the risk reduction using the conventional dose-equivalent approach. Secondary neutrons contribute a significant fraction of the total radiation exposure in space. An experiment to study the changes in dose and lineal energy spectra by shielding materials was carried out at the Los Alamos Nuclear Science Center neutron facility. In the energy range of about 2 to 200 MeV, this neutron spectrum is similar in shape within a factor of about 2 to the spectrum expected in the International Space Station habitable modules. It is shown that with a shielding thickness of about 5 g cm⁻², the conventional radiation risk increases, in some cases by as much as a factor of 2, but decreases with thicknesses of about of 20 g cm⁻². This suggests that care must be taken in evaluating the shielding effectiveness of a given material by including both the charged-particle and neutron components of space radiation.

Yasuda, H., Badhwar, G. D., Komiyama, T. and Fujitaka, K. Effective Dose Equivalent on the Ninth Shuttle–Mir Mission (STS-91).

Organ and tissue doses and effective dose equivalent were measured using a life-size human phantom on the ninth Shuttle–Mir Mission (STS-91, June 1998), a 9.8-day spaceflight at low-Earth orbit (about 400 km in altitude and 51.65° in inclination). The doses were measured at 59 positions using a combination of thermoluminescent dosimeters of Mg₂SiO₄:Tb (TDMS) and plastic nuclear track detectors (PNTD). In correcting the change in efficiency of the TDMS, it was assumed that reduction of efficiency is attributed predominantly to HZE particles with energy greater than 100 MeV nucleon^–1. A conservative calibration curve was chosen for determining LET from the PNTD track-formation sensitivities. The organ and tissue absorbed doses during the mission ranged from 1.7 to 2.7 mGy and varied by a factor of 1.6. The dose equivalent ranged from 3.4 to 5.2 mSv and varied by a factor of 1.5 on the basis of the dependence of Q on LET in the 1990 recommendations of the ICRP. The effective quality factor (Q_e) varied from 1.7 to 2.4. The dose equivalents for several radiation-sensitive organs, such as the stomach, lung, gonad and breast, were not significantly different from the skin dose equivalent (H_skin). The effective dose equivalent was evaluated as 4.1 mSv, which was about 90% of the H_skin.