A conserved Asn in TM7 of the thyrotropin receptor is a common requirement for activation by both mutations and its natural agonist

Arsenic trioxide (As2O3) inhibits cell growth and induces apoptosis in certain types of cancer cells including acute promyelocytic leukemia, prostate and ovarian carcinomas, but its effect on response of tumor cells to ionizing radiation has never been explored before. Here we demonstrate that As2O3 can sensitize human cervical cancer cells to ionizing radiation both in vitro and in vivo. As2O3 in combination with ionizing radiation have a synergistic effect in decreasing clonogenic survival and in the regression of established human cervical tumor xenografts. Pretreatment of the cells with As2O3 also synergistically enhanced radiation-induced apoptosis. Apoptosis of the cells by combined treatment of As2O3 and radiation was associated with reactive oxygen species generation and loss of mitochondrial membrane potential, resulting in the activation of caspase-9 and caspase-3. The combined treatment also resulted in an increased G2/M cell cycle distribution at the concentration of As2O3 which did not alter cell cycle when applied alone. These results indicate that As2O3 can synergistically enhance radiosensitivity of human cervix carcinoma cells in vitro and in vivo, suggesting a potential clinical applicability of combination treatment of As2O3 and ionizing radiation in cancer therapies.     

RAC2-P38 MAPK-dependent NADPH oxidase activity is associated with the resistance of quiescent cells to ionizing radiation

Our recent study showed that quiescent G0 cells are more resistant to ionizing radiation than G1 cells; however, the underlying mechanism for this increased radioresistance is unknown. Based on the relatively lower DNA damage induced in G0 cells, we hypothesize that these cells are exposed to less oxidative stress during exposure. As a catalytic subunit of NADPH oxidase, Ras-related C3 botulinum toxin substrate 2 (RAC2) may be involved in the cellular response to ionizing radiation. Here, we show that RAC2 was expressed at low levels in G0 cells but increased substantially in G1 cells. Relative to G1 cells, the total antioxidant capacity in G0 phase cells increased upon exposure to X-ray radiation, whereas the intracellular concentration of ROS and malondialdehyde increased only slightly. The induction of DNA single- and double-stranded breaks in G1 cells by X-ray radiation was inhibited by knockdown of RAC2. P38 MAPK interaction with RAC2 resulted in a decrease of functional RAC2. Increased phosphorylation of P38 MAPK in G0 cells also increased cellular radioresistance; however, excessive production of ROS caused P38 MAPK dephosphorylation. P38 MAPK, phosphorylated P38 MAPK, and RAC2 regulated in mutual feedback and negative feedback regulatory pathways, resulting in the radioresistance of G0 cells.     

Mechanisms of the synergistic action of ionizing radiation and hydrogen peroxide on the viability of bacteria

The combined effect of ionizing radiation and minor concentrations of hydrogen peroxide (0.1-0.01 M) has been studied on bacteria differing in the cell wall structure (Gy+, Gy-), radioresistance, and activity of DNA repair system. The H2O2 concentrations applied, which have a slight antibacterial action, enhance significantly the bactericidal effect of ionizing radiation producing a synergistic effect due to the decreased effectiveness of repair of DNA single-strand breaks.     

Enhancement of radiation cytotoxicity by UCN-01 in non-small cell lung carcinoma cells

Thoracic ionizing radiation is a standard component of combined-modality therapy for locally advanced non-small cell lung cancer. To improve low 5-year survival rates (5- 15%), new strategies for enhancing the effectiveness of ionizing radiation are needed. The kinase inhibitor UCN-01 has multiple cell cycle effects, including abrogation of DNA damage-induced S- and G(2)-phase arrest, which may limit DNA repair prior to mitosis. To test the hypothesis that therapy-induced cell cycle effects would have an impact on the efficacy of a combination of UCN-01 plus ionizing radiation, the cell cycle responses of the non-small cell lung cancer cell lines Calu1 (TP53-null) and A549 (wild-type TP53) to 2 Gy ionizing radiation were correlated with clonogenic survival after irradiation plus UCN-01. Irradiated cells were exposed to UCN-01 simultaneously and at 3-h increments after irradiation. In Calu1 cells but not A549 cells, sequence-dependent potentiation of radiation by UCN-01 was observed, with maximal interaction occurring when UCN-01 was administered 6 h after irradiation. This coincided with the postirradiation time with the greatest depletion of cells from G(1). Abrogation of G(2) arrest was observed regardless of TP53 status. The role of TP53 was investigated using siRNA to achieve gene silencing. These studies demonstrated that radiation plus UCN-01 was more effective in cells with diminished TP53 activity, associated with a reduced G(1) checkpoint arrest. These studies indicate that simultaneous elimination of multiple DNA damage-induced checkpoints in G(1), S and G(2) may enhance the effects of radiation and that drug scheduling may have an impact on clinical efficacy.     

Evaluation of the antioxidant effects of carotenoids from Deinococcus radiodurans through targeted mutagenesis, chemiluminescence, and DNA damage analyses

Deinococcus radiodurans is highly resistant to reactive oxygen species (ROS). The antioxidant effect of carotenoids in D. radiodurans was investigated by using a targeted mutation of the phytoene synthase gene to block the carotenoid synthesis pathway and by evaluating the survival of cells under environmental stresses. The colorless mutant R1DeltacrtB of D. radiodurans failed to synthesize carotenoids, and was more sensitive to ionizing radiation, hydrogen peroxide, and desiccation than the wild type, suggesting that carotenoids in D. radiodurans help in combating environmental stresses. Chemiluminescence analyses showed that deinoxanthin, a major product in the carotenoid synthesis pathway, had significantly stronger scavenging ability on H2O2 and singlet oxygen than two carotenes (lycopene and beta-carotene) and two xanthophylls (zeaxanthin and lutein). Deinoxanthin also exhibited protective effect on DNA. Our findings suggest that the stronger antioxidant effect of deinoxanthin contribute to the resistance of D. radiodurans. The higher antioxidant effect of deinoxanthin may be attributed to its distinct chemical structure which has an extended conjugated double bonds and the presence of a hydroxyl group at C-1' position, compared with other tested carotenoids.     

Trifluoperazine stimulates ionizing radiation induced cell killing through inhibition of DNA repair

The effect of trifluoperazine (TFP), a phenothiazine derivative antipsychotic drug, on ionizing radiation (IR) induced cell killing through inhibition of DNA repair was investigated in human cell lines. In clonogenic survival assay, TFP augmented IR induced cell killing. Also, TFP enhanced micronucleus formation in irradiated human lymphocytes. The effect of TFP and other known DNA repair inhibitors like wortmannin and caffeine, on irradiated cells, was compared by MTT assay. On the other hand, TFP failed to increase the toxicity induced by H2O2. Repair of DNA double strand breaks induced by IR was markedly inhibited by TFP, as determined by field inversion gel electrophoresis (FIGE). Further, TFP increased radiation induced apoptosis, which was accompanied by enhanced G2/M arrest. Thus, our results strongly suggest that TFP inhibits repair of DNA damage induced by IR, which significantly implicates the possibility of using TFP as an adjuvant to radiotherapy.     

Decreasing peroxiredoxin II expression decreases glutathione, alters cell cycle distribution, and sensitizes glioma cells to ionizing radiation and H(2)O(2)

Glioblastomas are notorious for their resistance to ionizing radiation and chemotherapy. We hypothesize that this resistance to ionizing radiation is due, in part, to alterations in antioxidant enzymes. Here, we show that rat and human glioma cells overexpress the antioxidant enzyme peroxiredoxin II (Prx II). Glioma cells in which Prx II is decreased using shRNA exhibit increased hyperoxidation of the remaining cellular Prxs, suggesting that the redox environment is more oxidizing. Of interest, decreasing Prx II does not alter other antioxidant enzymes (i.e., catalase, GPx, Prx I, Prx III, CuZnSOD, and MnSOD). Analysis of the redox environment revealed that decreasing Prx II increased intracellular reactive oxygen species in 36B10 cells; extracellular levels of H(2)O(2) were also increased in both C6 and 36B10 cells. Treatment with H(2)O(2) led to a further elevation in intracellular reactive oxygen species in cells where Prx II was decreased. Decreasing Prx II expression in glioma cells also reduced clonogenic cell survival following exposure to ionizing radiation and H(2)O(2). Furthermore, lowering Prx II expression decreased intracellular glutathione and resulted in a significant decline in glutathione reductase activity, suggesting a possible mechanism for the observed increased sensitivity to oxidative insults. Additionally, decreasing Prx II expression increased cell cycle doubling times, with fewer cells distributed to S phase in C6 glioma cells and more cells redistributed to the most radiosensitive phase of the cell cycle, G2/M, in 36B10 glioma cells. These findings support the hypothesis that inhibiting Prx II sensitizes glioma cells to oxidative stress, presenting Prxs as potential therapeutic targets.     

Mutagenesis and carcinogenesis caused by the oxidation of nucleic acids

Genomes and their precursor nucleotides are highly exposed to reactive oxygen species, which are generated both as byproducts of oxygen respiration or molecular executors in the host defense, and by environmental exposure to ionizing radiation and chemicals. To counteract such oxidative damage in nucleic acids, mammalian cells are equipped with three distinct enzymes. MTH1 protein hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-2'-deoxyguanosine triphosphate and 2-hydroxy-2'-deoxyadenosine triphosphate (2-OH-dATP), to the corresponding monophosphates. We observed increased susceptibility to spontaneous carcinogenesis in MTH1-null mice, which exhibit an increased occurrence of A:T-->C:G and G:C-->T:A transversion mutations. 8-Oxoguanine (8-oxoG) DNA glycosylase, encoded by the OGG1 gene, and adenine DNA glycosylase, encoded by the MUTYH gene, are responsible for the suppression of G:C to T:A transversions caused by the accumulation of 8-oxoG in the genome. Deficiency of these enzymes leads to increased tumorigenesis in the lung and intestinal tract in mice, respectively. MUTYH deficiency may also increase G:C to T:A transversions through the misincorporation of 2-OH-dATP, especially in the intestinal tract, since MUTYH can excise 2-hydroxyadenine opposite guanine in genomic DNA and the repair activity is selectively impaired by a mutation found in patients with autosomal recessive colorectal adenomatous polyposis.     

DNA lesions that signal the induction of radioresistance and DNA repair in yeast

DNA recombinational repair, and an increase in its capacity induced by DNA damage, is believed to be the major mechanism that confers resistance to killing by ionizing radiation in yeast. We have examined the nature of the DNA lesions generated by ionizing radiation that induce this mechanism, using two different end points: resistance to cell killing and ability of the error-free recombinational repair system to compete for other DNA lesions and thereby suppress chemical mutation. Under the various conditions examined in this study, the "maximum" inducible radiation resistance was increased approximately 1.5- to 3-fold and suppression of mutation about 10-fold. DNA lesions produced by low-LET gamma rays at doses greater than about 20 Gy given in oxygen were shown to be more efficient, per unit dose, at inducing radioresistance to killing than were lesions produced by neutrons (high-LET radiation). This suggests that DNA single-strand breaks are more important lesions in the induction of radioresistance than DNA double-strand breaks. Oxygen-modified lesions produced by gamma rays (low-LET radiation) were particularly efficient as induction signals. DNA damage due to hydroxyl radicals (OH.) derived from the radiolytic decomposition of H2O produced lesions that strongly induced this DNA repair mechanism. Similarly, OH. derived from aqueous electrons (e-aq) in the presence of N2O also efficiently induced the response. Cells induced to radioresistance to killing with high-LET radiation did not suppress N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-generated mutations as well as cells induced with low-LET radiation, supporting the conclusion that the type of DNA damage produced by low-LET radiation is a better inducer of recombinational repair. Surprisingly, however, cells induced with gamma radiation in the presence of N2O that became radioresistant to killing were unable to suppress MNNG mutations. This result indicates that OH. generated via e-aq (in N2O) may produce unusual DNA lesions which retard normal repair and render the system unavailable to compete for MNNG-generated lesions. We suggest that the repairability of these unique lesions is restricted by either their chemical nature or topological accessibility. Attempted repair of these lesions has lethal consequences and accounts for N2O radiosensitization of repair-competent but not incompetent cells. We conclude that induction of radioresistance in yeast by ionizing radiation responds variably to different DNA lesions, and these affect the availability of the induced recombinational repair system to deal with subsequent damage.     

Resistance of Bacillus subtilis Spore DNA to Lethal Ionizing Radiation Damage Relies Primarily on Spore Core Components and DNA Repair,with Minor Effects of Oxygen Radical Detoxification

The roles of various core components, including α/β/γ-type small acid-soluble spore proteins (SASP), dipicolinic acid (DPA), core water content, and DNA repair by apurinic/apyrimidinic (AP) endonucleases or nonhomologous end joining (NHEJ), in Bacillus subtilis spore resistance to different types of ionizing radiation including X rays, protons, and high-energy charged iron ions have been studied. Spores deficient in DNA repair by NHEJ or AP endonucleases, the oxidative stress response, or protection by major α/β-type SASP, DPA, and decreased core water content were significantly more sensitive to ionizing radiation than wild-type spores, with highest sensitivity to high-energy-charged iron ions. DNA repair via NHEJ and AP endonucleases appears to be the most important mechanism for spore resistance to ionizing radiation, whereas oxygen radical detoxification via the MrgA-mediated oxidative stress response or KatX catalase activity plays only a very minor role. Synergistic radioprotective effects of α/β-type but not γ-type SASP were also identified, indicating that α/β-type SASP''s binding to spore DNA is important in preventing DNA damage due to reactive oxygen species generated by ionizing radiation.     

The range of oxygenation in SiHa tumor xenografts

Tumor cells at very low oxygen tensions are known to be about three times more resistant to killing by ionizing radiation. Since cells at intermediate oxygen tensions (defined here as greater than 0.1% and less than 2% O(2)) show partial radioresistance, they should be a consideration in tumor treatment. In an effort to estimate the extent and range of oxygenation in SiHa human cervical carcinoma xenografts, patterns of cell killing and DNA damage by radiation and two bioreductive drugs, PD-144872 and RSU-1069, were compared to those seen in SiHa cells grown as spheroids. These drugs produce DNA interstrand crosslinks that are largely responsible for cell killing, and the degree of crosslinking increases as the oxygenation is reduced. About 60% of the cells in SiHa xenografts exhibited drug-induced crosslinks, but only about 35% showed extensive crosslinking indicative of hypoxia below 0.1% oxygen. Patterns of toxicity and DNA damage in xenografts were comparable to those of spheroids equilibrated with about 2% oxygen, indicating that most cells in the xenografts exhibit some radioresistance due to lack of oxygen. Similarly, pimonidazole binding indicated that about 60% of the cells in SiHa xenografts were either intermediate in oxygenation or hypoxic, but only about half of those were consistent with extreme oxygen depletion. The apparent size of the population of "intermediately hypoxic" cells has implications for the use of ionizing radiation, hypoxic cell cytotoxins, and other antitumor agents whose cytotoxicity is dependent on cellular oxygen content.     

Inactivation of biologically active DNA by gamma-ray-induced superoxide radicals and their dismutation products singlet molecular oxygen and hydrogen peroxide.

Since superoxide radicals are involved in many metabolically important as well as in some other, detrimental cellular processes, the reactivity of gamma-ray-induced superoxide radicals and its dismutation products singlet molecular oxygen and hydrogen peroxide with DNA have been studied. Superoxide dismutase which removes superoxide radicals and inhibits the formation of singlet oxygen in the solution protects the biologically active replicative form of DNA (from bacteriophage theta X174) against inactivation by ionizing radiation. Catalase which removes hydrogen peroxide also protects the DNA. Attempts with various chemical sources of singlet oxygen to determine whether this species inactivates DNA did not give an unequivocal answer. It is concluded from the presented experiments that a combination of the protonated form of the superoxide radical (HO-2) and H2O2 do inactivate DNA.     

Evaluation of the degree of medical radiation damage with a highly sensitive chemiluminescence method.

A highly sensitive chemiluminescence (CL) method for evaluation of medical radiation damage degree is presented. According to the principle of cell stress response to ionizing radiation, lymphocytes will produce reactive oxygen species (ROS) after irradiation. The ROS produced can react with 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha] pyrazin-3-one (MCLA), a specific CL probe for superoxide anion (O(.-) (2)) and singlet oxygen ((1)O(2)), to emit light at 465 nm. The CL intensity is positively related to the amount of generated ROS detected 30 min after irradiation. Cell viability, which is inversely related to cell mortality, was determined by MTT assay after 3 days' culture. The results show that both CL intensity and cell mortality of lymphocytes increase with the increase of the radiation dose when the dosage is no more than 3 Gy, suggesting a positive relationship between the degree of lymphocyte cell damage and the amount of ROS generated. In addition, the effects of catalase, Cu-Zn superoxide dismutase (SOD), mannitol, sodium azide (NaN(3)), and D(2)O on MCLA-dependent CL of lymphocytes are discussed. We believe that the MCLA-dependent CL method would potentially provide an easy way for evaluating the degree of lymphocyte damage induced by radiation.     

Dose-rate and oxygen effects in models of lipid membranes: linoleic acid.

Cellular membranes have been suggested as possible loci for the development of the oxygen effect in radiobiology. Unsaturated lipids from membranes are subject to very efficient radiation-induced peroxidation, and the deleterious effects generally associated with lipid autoxidation could be initiated by ionizing radiation. Oxidative damage in lipids is characterized not only by high yields but also by a profound dose-rate effect. At dose-rates of X-irradiation below 100 rad/min, a very sharp rise occurs in oxidative damage. This damage has been quantified spectrophotometrically in terms of diene conjugation (O.D. 234 mm) and chromatographically in terms of specific 9- and 13-hydroperoxide formation in linoleic acid micelles. Radical scavenging experiments indicate that hydroxyl radical attack initiates the oxidative damage. Dimethyl sulphoxide is exceptional in that it does not protect, but sensitizes, linoleic acid to radiation induced peroxidation. The yields of hydroperoxides are substantial (G=10--40) and can be related to biological changes known to be effected by autoxidizing lipids.     

A manganese porphyrin complex is a novel radiation protector

Exposure of cells to ionizing radiation leads to formation of reactive oxygen species, which are associated with radiation-induced cytotoxicity. Therefore, compounds that scavenge reactive oxygen species may confer radioprotective effects. Superoxide dismutase (SOD) mimetics have been shown to be protective against cell injury caused by reactive oxygen species. The objective of this study was to investigate the effects of manganese(III) tetrakis(N-methyl-2-pyridyl)porphyrin (MnTMPyP), a cell-permeable SOD mimetic, on radiation-dependent toxicity. We investigated the protective role of MnTMPyP against ionizing radiation in U937 cells and mice. On exposure to ionizing radiation, there was a distinct difference between control cells and cells pretreated with MnTMPyP with respect to viability, cellular redox status, and oxidative damage to cells. Lipid peroxidation, oxidative DNA damage, and protein oxidation were significantly lower in the cells treated with MnTMPyP when the cells were exposed to ionizing radiation. The [GSSG]/[GSH + GSSG] ratio and the generation of intracellular reactive oxygen species were higher and the [NADPH]/[NADP+ + NADPH] ratio was lower in control cells compared with MnTMPyP-treated cells. Ionizing radiation-induced mitochondrial damage, as reflected by the altered mitochondrial permeability transition, increase in accumulation of reactive oxygen species, reduction of ATP production, and morphological change, was significantly higher in control cells than in MnTMPyP-treated cells. MnTMPyP administration for 14 days at a daily dosage of 5 mg/kg provided substantial protection against killing and oxidative damage in mice exposed to whole-body irradiation. These data indicate that MnTMPyP may have great application potential as a new class of in vivo, non-sulfur-containing radiation protectors.     

Lack of sensitivity of primary Fanconi's anemia fibroblasts to UV and ionizing radiation

Clinical observations and theoretical considerations suggest some degree of radiosensitivity in Fanconi's anemia (FA), but experimental evidence remains controversial. We tested the sensitivity of primary skin fibroblast cultures from all known FA complementation groups to ionizing radiation and ultraviolet light using conventional cell growth and colony formation assays. In contrast to previous studies, and because FA fibroblasts grow and clone poorly at ambient oxygen, we performed our sensitivity tests under hypoxic cell culture conditions. Fibroblast strains from healthy donors served as negative controls and those from patients with ataxia telangiectasia (AT) and Cockayne syndrome (CS) as positive controls. We observed interstrain variation but no systematic difference in the response of FA and non-FA control fibroblasts to ionizing radiation. After exposure to UV radiation, only complementation group A, G and D2 strains displayed values for colony formation EC50 that were intermediate between those for the negative and positive controls. Because of considerable interstrain variation, minor alterations of the response of individual FA strains to ionizing and UV radiation should be interpreted with caution and should not be taken as evidence for genotype-specific sensitivities of primary FA fibroblasts. All together, our data indicate neither systematic nor major sensitivities of primary FA fibroblast cultures of any complementation group grown under hypoxic cell culture conditions to ionizing or UV radiation.     

Roles of vitamin C in radiation-induced DNA damage in presence and absence of copper

Exposure to either ionizing radiation or certain transition metals results in generation of reactive oxygen species that induce DNA damage, mutation, and cancer. Vitamin C (a reactive oxygen scavenger) is considered to be a dietary radioprotective agent. However, it has been reported to be genotoxic in the presence of certain transition metals, including copper. In order to explore the capacity of vitamin C to protect DNA from radiation-induced damage, and the influence of the presence of copper on this protection, we investigated vitamin C-mediated protection against radiation-induced damage to calf thymus DNA in vitro in the presence or absence of copper(II). Vitamin C (0.08-8.00 mM, pH 7.0) significantly reduced DNA damage induced by gamma-irradiation (30-150 Gy) by 30-50%, similar to the protective effect of glutathione. However, vitamin C plus copper (50 microM) significantly enhanced gamma-radiation-induced DNA damage. Low levels of added copper (5 microM), or chelation of copper with 1-N-benzyltriethylenetetraine tetrahydrochloride (BzTrien) and bathocuprinedisulfonic acid (BCSA), abolished the enhanced damage without diminishing the protective effect of vitamin C. These results indicate that vitamin C can act as: (1) an antioxidant to protect DNA damage from ionizing radiation; and (2) a reducing agent in the presence of copper to induce DNA damage. These effects are important in assessing the role of vitamin C, in the presence of mineral supplements or radioprotective therapeutic agents, particularly in patients with abnormally high tissue copper levels.