Recovery of ionizing-radiation damage after high doses of gamma ray in the hyperthermophilic archaeon Thermococcus gammatolerans

The recently discovered hyperthermophilic and radioresistant archaeon Thermococcus gammatolerans is of great interest to compare and contrast the impact of its physiology on radioresistance and its ability to repair damaged chromosomes after exposure to gamma irradiation with radioresistant bacteria. We showed that, in contrast to other organisms, cell survival was not modified by the cellular growth phase under optimal growth conditions but nutrient-limited conditions did affect the T. gammatolerans radioresistance. We determined the first kinetics of damaged DNA recovery in an archaeon after exposure to massive doses of gamma irradiation and compared the efficiency of chromosomal DNA repair according to the cellular growth phase, nutrient availability and culture conditions. Chromosomal DNA repair kinetics showed that stationary phase cells reconstitute disrupted chromosomes more rapidly than exponential phase cells. Our data also revealed that this radioresistant archaeon was proficient to reconstitute shattered chromosomes either slowly or rapidly without any loss of viability. These results suggest that rapid DNA repair is not required for the extreme radioresistance of T. gammatolerans. Angels Tapias and Christophe Leplat contributed equally to this work.     

Recovery from potentially lethal damage and recruitment time of noncycling clonogenic cells in 9L confluent monolayers and spheroids

Cells that have been grown as multicell tumor spheroids exhibit radioresistance compared to the same cells grown in monolayers. Comparison of potentially lethal damage (PLD) repair and its kinetics was made between 9L cells grown as spheroids and confluent monolayers. Survival curves of cells plated immediately after irradiation showed the typical radioresistance associated with spheroid culture compared to plateau-phase monolayers. The dose-modification factor for spheroid cell survival is 1.44. Postirradiation incubations in normal phosphate-buffered saline (PBS), conditioned media, or 0.5 M NaCl in PBS reduced the differences in radiosensitivity between the two culture conditions. Postirradiation treatment in PBS or conditioned medium promoted repair of potentially lethal damage, and 0.5 M NaCl prevented the removal of PLD and allowed the fixation of damage resulting in lower survival. Survival of spheroid and monolayer cells after hypertonic NaCl treatment was identical. NaCl treatment reduced Do more than it did the shoulder (Dq) of the survival curve. PLD repair kinetics measured after postirradiation incubation in PBS followed by hypertonic NaCl treatment was the same for spheroids and for plateau-phase monolayers. The kinetics of PLD repair indicates a biphasic phenomenon. There is an initial fast component with a repair half-time of 7.9 min and a slow component with a repair half-time of 56.6 min. Most of the damage (59%) is repaired slowly. Since the repair capacity and kinetics are the same for spheroids and monolayers, the radioresistance of spheroids cannot be explained on this basis. Evidence indicates that the time to return from a Go (noncycling G1 cells) state to a proliferative state (recruitment) for cells from confluent monolayers and from spheroids after dissociation by protease treatment may be the most important determinant of the degree of PLD repair that occurs. Growth curves and flow cytometry cell cycle analysis indicate that spheroid cells have a lag period for reentry into a proliferative state. Since plating efficiency remains high and unchanging during this period, one cannot account for the delay on the basis of the existence of a large fraction of Go cells which are not potentially clonogenic. The cell cycle progression begins in 6-8 h for monolayer cells and in 14-15 h for spheroids. It is hypothesized that the slower reentry of spheroid cells into a cycling phase allows more time for repair than for the rapidly proliferating monolayer cells.     

Inhibition of phosphatidylinositol-3-OH kinase/Akt signaling impairs DNA repair in glioblastoma cells following ionizing radiation

Radiation therapy is a mainstay in the treatment of glioblastomas, but these tumors are often associated with radioresistance. Activation of the phosphatidylinositol-3-OH kinase (PI3K)/Akt pathway, which occurs frequently in glioblastomas due to inactivation of the tumor suppressor phosphatase and tensin homologue (PTEN), correlates with radioresistance. To directly test the link between Akt activation and radioresistance, we utilized PTEN-deficient U251 glioblastoma cells engineered to inducibly restore PTEN upon exposure to doxycycline. These cells showed high basal levels of Akt activation (i.e. high levels of phospho-Akt), but induction of PTEN led to substantially decreased phospho-Akt and was associated with radiosensitization. To investigate whether the PTEN-induced radiosensitization was attributable to impaired sensing versus repair of DNA damage, we assessed levels of gamma-H2AX after ionizing radiation in U251 cells induced for PTEN. Initial post-radiation levels of gamma-H2AX foci were not decreased in PTEN-induced cells; however, the resolution of these foci was significantly delayed. In contrast to these results, induction of phosphatase-dead PTEN showed no appreciable effect. Finally, exposure of cells to the PI3K inhibitor LY294002 did not decrease the occurrence of gamma-H2AX foci after irradiation but did markedly delay their resolution. These results together support a direct link between Akt activation, repair of DNA damage, and radioresistance in glioblastoma. Targeting the PI3K/Akt pathway may modulate DNA repair to improve the efficacy of radiation therapy.     

Genetic analysis of the DNA-dependent protein kinase reveals an inhibitory role of Ku in late S-G2 phase DNA double-strand break repair

Two major complementary double-strand break (DSB) repair pathways exist in vertebrates, homologous recombination (HR), which involves Rad54, and non-homologous end-joining, which requires the DNA-dependent protein kinase (DNA-PK). DNA-PK comprises a catalytic subunit (DNA-PKcs) and a DNA-binding Ku70 and Ku80 heterodimer. To define the activities of individual DNA-PK components in DSB repair, we targeted the DNA-PKcs gene in chicken DT40 cells. DNA-PKcs deficiency caused a DSB repair defect that was, unexpectedly, suppressed by KU70 disruption. We have shown previously that genetic ablation of Ku70 confers RAD54-dependent radioresistance on S-G(2) phase cells, when sister chromatids are available for HR repair. To test whether direct interference by Ku70 with HR might explain the Ku70(-/-)/DNA-PKcs(-/-/-) radioresistance, we monitored HR activities directly in Ku- and DNA-PKcs-deficient cells. The frequency of intrachromosomal HR induced by the I-SceI restriction enzyme was increased in the absence of Ku but not of DNA-PKcs. Significantly, abrogation of HR activity by targeting RAD54 in Ku70(-/-) or DNA-PKcs(-/-/-) cells caused extreme radiosensitivity, suggesting that the relative radioresistance seen with loss of Ku70 was because of HR-dependent repair pathways. Our findings suggest that Ku can interfere with HR-mediated DSB repair, perhaps competing with HR for DSB recognition.     

Effect of a water soluble derivative of alpha-tocopherol on radiation response of Saccharomyces cerevisiae

The radioprotection conferred by a highly water soluble glucose derivative of alpha-tocopherol, namely, 2-(alpha-D-glucopyranosyl) methyl-2,5,7,8-tetramethylchroman-6-ol (TMG) in Saccharomyces cerevisiae was studied. Cells grown in standard YEPD-agar medium and irradiated in the presence of TMG showed a concentration dependent higher survival up to 10 mM of TMG in comparison to cells irradiated in distilled water. Treatment of TMG to cells given either before or immediately after irradiation but not during irradiation, had no effect on their radiation response. S. cerevisiae strain LP1383 (rad52) which is defective in recombination repair showed enhanced radioresistance only when subjected to irradiation in presence of TMG. Cells of rad52 strain grown in the medium containing TMG showed a radiation response similar to that of cells grown in the medium without TMG. The nature of TMG dependent enhanced radioresistance was studied by scoring the mutations in the strain D-7, which behaved like wild type strain in complete medium, at trp and ilv loci. Our study indicated that TMG confers radioresistance in S. cerevisiae possibly by two mechanisms viz. (i), by eliminating radiation induced reactive free radicals when the irradiation is carried out in the presence of TMG and (ii), by activating an error prone repair process involving RAD52 gene, when the cells are grown in the medium containing TMG.     

The dependence of thiol-inducible radiation resistance in Escherichia coli K12 on the medium and catalytic metal

Radiation protection by thiols in procaryotes and lower eucaryotes has been demonstrated repeatedly to require a competent DNA repair phenotype, suggesting that simple chemical radical scavenging and hydrogen donation are only a portion of the mechanism of radiation protection by thiols. In the present report, thiol-induced radiation resistance--a model in which cells are pretreated with dithiothreitol and then irradiated in the absence of thiol--is shown to be a medium-dependent process. Wild-type log-phase cells treated with dithiothreitol in minimal-glucose medium are induced to radioresistance that persists after the thiol has been removed. Although the thiol pretreatment affected the antioxidants (catalase, superoxide dismutase, and glutathione) in cells at the time of irradiation, various antioxidant levels did not predict radiation resistance. Thiol-induced radioresistance is not expressed in rich medium-treated cells or in DNA repair (recA)-deficient cells. Addition of the efficient chelator, DETAPAC, to the thiol treatment medium leads to additional radioresistance in the case of minimal medium and a moderate expression of resistance in rich medium. Experiments using the intracellular chelator, 1,10-phenanthroline, in the presence of thiol led to inhibition of thiol-induced resistance in minimal medium and radiosensitization in rich medium. These results can be explained by a "site-specific" mechanism of thiol oxidation in which the chelators control the site(s) and rate of thiol oxidation, subsequently determining the type of cellular response.     

Inhibiting UHRF1 expression enhances radiosensitivity in human esophageal squamous cell carcinoma

Radiotherapy is an effective treatment for some esophageal cancers, but the molecular mechanisms of radiosensitivity remain unknown. Ubiquitin-like with PHD and ring finger domains 1 (UHRF1) is a novel nuclear protein which is overexpressed in various cancers but not yet examined in esophageal squamous cell carcinoma (ESCC). The correlation between UHRF1 and the radioresistance in ESCC is still unclear. In the present study, the expression of UHRF1 was examined by immunohistochemistry in specimens of ESCC patients treated with radiotherapy. The results showed that UHRF1 was significantly overexpressed in ESCC specimens. Overexpression of UHRF1 correlated significantly with advanced T-stage, positive lymph node metastasis and poor differentiation. In addition, UHRF1 was associated with radiotherapy response, in which overexpression of UHRF1 was observed more frequently in the radioresistant group than in the effective group. At the molecular level, inhibition of UHRF1 by lentivirus-mediated shRNA targeting UHRF1 increased the radiosensitivity and apoptosis, while decreased radiation-induced G2/M phase arrest in TE-1 cells. Moreover, inhibition of UHRF1 resulted in higher residual γH2AX expression after irradiation, but not initial γH2AX. Further study showed that inhibition of UHRF1 down-regulated the endogenous expressions of DNA repair protein Ku70 and Ku80 in TE-1 cells, and significantly inhibited the increase of these proteins after irradiation. Above all, our data suggested that UHRF1 might play an important role in radioresistance of ESCC, and inhibition of UHRF1 can increase the radiosensitivity of TE-1 cells by altering cell cycle progression, enhancing apoptosis, and decreasing DNA damage repair capacity.     

Induction and assessment of persistent radioresistance in murine leukocytes in vivo

The aim of the present study was to investigate whether weekly exposure to gamma rays causes a persistent increase in the number of radioresistant leukocytes in mice in vivo. Using the comet assay, 1 Gy radiation exposure decreased the percentage of leukocytes with less than 5% DNA in the tail (<5% DNAT), and we propose that radioresistance induction might increase the number of cells with <5% DNAT after radiation exposure. We exposed mice to 1 Gy gamma rays weekly for four weeks or 2 Gy per week for nine weeks. We observed a significant increase in cells with <5% DNAT after the third week and up to nine weeks of exposure. We exposed animals to gradually increasing radiation doses and finally challenged the lymphocytes with 1 Gy radiation both in vivo and in vitro. We observed increased radioresistance in vitro, providing evidence that a cellular process is involved. However, more radioresistance was observed in vivo than in vitro, suggesting a physiological effect. Cells challenged in vitro were maintained on ice during and after exposure, which likely caused a reduction in DNA repair. Radioresistance induction likely arose from mutation selection in stem cells because leukocytes are unable to proliferate in peripheral blood.     

Dysregulation of IRP1-Mediated Iron Metabolism Causes Gamma Ray-specific Radioresistance in Leukemia Cells

Iron is required for nearly all organisms, playing important roles in oxygen transport and many enzymatic reactions. Excess iron, however, can be cytotoxic. Emerging evidence suggests that radioresistance can be achieved in lower organisms by the protection of proteins, but not DNA, immediately following ionizing radiation (IR) exposure, allowing for improved DNA repair. One potential mechanism for protein protection is controlling and limiting the amount of free iron in cells, as has been demonstrated in the extremophile Deinococcus Radiodurans, reducing the potential for oxidative damage to proteins during exposure to IR. We found that iron regulatory protein 1 (IRP1) expression was markedly reduced in human myeloid leukemia HL60 cells resistant to low linear energy transfer (LET) gamma rays, but not to high LET alpha particles. Stable knockdown of IRP1 by short-hairpin RNA (shRNA) interference in radiosensitive parental cells led to radioresistance to low LET IR, reduced intracellular Fenton chemistry, reduced protein oxidation, and more rapid DNA double-strand break (DSB) repair. The mechanism of radioresistance appeared to be related to attenuated free radical-mediated cell death. Control of intracellular iron by IRPs may be a novel radioresistance mechanism in mammalian cells.     

Genetic determination and mechanism of realization of temperature-induced radioresistance

A study was made of the influence of the repair genotype of E. coli cells on the realization of the effect of enhanced radioresistance during gamma-irradiation at elevated temperatures (40-45 degrees C). The effect of the thermoinduced radioresistance (TIR) was diminished significantly but not abolished completely in mutant cells selectively deficient in excision or recombination repair systems (po1A1, recB21C22sbcB15, recF143 mutants). However mutations which exclude the recA gene product (recA13, recA13B21C22 or lexA3 mutants) inhibited TIR completely. The introduction of recA+ gene into recA- or lexA- mutants almost normalized TIR. On the basis of the data obtained the authors discuss the role of recA protein in activation of the membrane-associated repair complex whose efficiency depends on the temperature of gamma-irradiation.     

Two types of X-ray-induced radioresistance in mice: Presence of 4 dose ranges with distinct biological effects

Preirradiation with 0.05 Gy of X rays 2 months before a second exposure to a mid-lethal dose significantly enhanced the survival rate in both female and male ICR strain mice. The radioresistance was observed between 2–2.5 months after exposure to 0.05 Gy. It did not appear within 1.5 months, and disappeared after 3 months. This radioresistance was induced only by whole-body preirradiation (not by partial irradiation of the head or the trunk). On the other hand, preirradiation with 0.30 Gy as well as 0.50 Gy resulted in radioresistance 2 weeks later, but not 2 months later. The radioresistance was induced by whole-body preirradiation or partial preirradiation of the trunk. No radioresistance was evident after exposure of intermediate preirradiation doses of 0.15 and 0.20 Gy administered before 2 months and 2–5 weeks, respectively. The present and previous results show that the biological effects of ionizing radiation may be distinguished with the following four radiation dose ranges; (1) below 0.025 Gy: no radioresistance after 2 months; (2) 0.05–0.10 Gy: significant radioresistance after 2–2.5 months; (3) 0.20 Gy: no radioresistance after 2–5 weeks; and (4) 0.30–0.50 Gy or more: significant radioresistance after 2 weeks. These results conflict with previous findings of the biological effects of ionizing radiation in which the radiation hazard increases in relation to increasing accumulated doses. Some stimulation, in addition to adaptation, by low dose irradiation may have occurred.     

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.     

RPA3 is a potential marker of prognosis and radioresistance for nasopharyngeal carcinoma

Radioresistance‐induced residual and recurrent tumours are the main cause of treatment failure in nasopharyngeal carcinoma (NPC). Thus, the mechanisms of NPC radioresistance and predictive markers of NPC prognosis and radioresistance need to be investigated and identified. In this study, we identified RPA3 as a candidate radioresistance marker using RNA‐seq of NPC samples. In vitro studies further confirmed that RPA3 affected the radiosensitivity of NPC cells. Specifically, the overexpression of RPA3 enhanced radioresistance and the capacity for DNA repair of NPC cells, whereas inhibiting RPA3 expression sensitized NPC cells to irradiation and decreased the DNA repair capacity. Furthermore, the overexpression of RPA3 enhanced RAD51 foci formation in NPC cells after irradiation. Immunohistochemical assays in 104 NPC specimens and 21 normal epithelium specimens indicated that RPA3 was significantly up‐regulated in NPC tissues, and a log‐rank test suggested that in patients with NPC, high RPA3 expression was associated with shorter overall survival (OS) and a higher recurrence rate compared with low expression (5‐year OS rates: 67.2% versus 86.2%; 5‐year recurrence rates: 14.8% versus 2.3%). Moreover, TCGA data also indicated that high RPA3 expression correlated with poor OS and a high recurrence rate in patients with head and neck squamous cell carcinoma (HNSC) after radiotherapy. Taken together, the results of our study demonstrated that RPA3 regulated the radiosensitivity and DNA repair capacity of NPC cells. Thus, RPA3 may serve as a new predictive biomarker for NPC prognosis and radioresistance to help guide the diagnosis and individualized treatment of patients with NPC.     

Cell-cycle dependent changes in sensitivity to γ-rays in synchronously dividing yeast culture

Changes in survival of yeast cells following γ-irradiation at different stages of the cell cycle were studied using a well synchronized culture. Maximum radioresistance occurs at the end of the S phase. Maximum radiosensitivity is observed just before entry into the S phase. The high degree of synchrony obtained allows more precise measurement of the extent of survival changes than has been achieved until now with partially synchronized cultures. Indeed, after a 60 krad irradiation we find a 100 % survival for cells which have just finished the S phase of the first cell cycle, against a 2 % survival for cells which are ready to enter the S phase of the second cell cycle. As the culture desynchronizes through successive cell cycles we have been able to follow the way in which survival curves are modified. We can extrapolate that with a perfectly synchronized culture the survival of ‘early S’ cells to a 60 krad irradiation would not be 2 % but 0.01 %. The high radioresistance observed at the end of S phase can hardly be explained simply in terms of DNA target or accumulation of radioprotectors. More likely the end of the S phase is a favourable stage for repair processes, at which time two genomes are able to recombine.     

Suppression of apoptosis and clonogenic survival in irradiated human lymphoblasts with different TP53 status

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.     

Adaptation to irradiation in vivo

The data about the increasing of radioresistance of cells and organism after the acute, chronic and prolonged irradiation in vivo were presented. The possible mechanisms of adaptation to irradiation connected with selection, stimulation of proliferative activity, forming of protecting system (protected proteins, antioxidant enzymes e.a.), activation of DNA repair and cAMP-, and Ca(2+)-dependent phosphorylation systems were observed. The conclusion about complex mechanism of radiation adaptation was drawn.     

Catalase and superoxide dismutase activity in isogenous bacterial strains with different degrees of radioresistance

Superoxide dismutase and catalase activity has been studied in isogenous strains of various radioresistance bacteria. In mutants Micrococcus radiodurans having defects in the systems of DNA repair the superoxide dismutase activity is lower than in cells of wild type. The changes of catalase and superoxide dismutase activity have not been revealed in investigated strains Escherichia coli differing in radioresistance. It has been concluded that the survival of bacteria exposed to ionizing radiation is determined by the effectiveness of DNA repair systems realiability of which depends on the catalase and superoxide dismutase activity.     

Restoration of G1 chemo/radioresistance and double-strand-break repair proficiency by wild-type but not endonuclease-deficient Artemis

Deficiency in Artemis is associated with lack of V(D)J recombination, sensitivity to radiation and radiomimetic drugs, and failure to repair a subset of DNA double-strand breaks (DSBs). Artemis harbors an endonuclease activity that trims both 5'- and 3'-ends of DSBs. To examine whether endonucleolytic trimming of terminally blocked DSBs by Artemis is a biologically relevant function, Artemis-deficient fibroblasts were stably complemented with either wild-type Artemis or an endonuclease-deficient D165N mutant. Wild-type Artemis completely restored resistance to γ-rays, bleomycin and neocarzinostatin, and also restored DSB-repair proficiency in G0/G1 phase as measured by pulsed-field gel electrophoresis and repair focus resolution. In contrast, cells expressing the D165N mutant, even at very high levels, remained as chemo/radiosensitive and repair deficient as the parental cells, as evidenced by persistent γ-H2AX, 53BP1 and Mre11 foci that slowly increased in size and ultimately became juxtaposed with promyelocytic leukemia protein nuclear bodies. In normal fibroblasts, overexpression of wild-type Artemis increased radioresistance, while D165N overexpression conferred partial repair deficiency following high-dose radiation. Restoration of chemo/radioresistance by wild-type, but not D165N Artemis suggests that the lack of endonucleolytic trimming of DNA ends is the principal cause of sensitivity to double-strand cleaving agents in Artemis-deficient cells.