Ionizing radiation induces stemness in cancer cells

The cancer stem cell (CSC) model posits the presence of a small number of CSCs in the heterogeneous cancer cell population that are ultimately responsible for tumor initiation, as well as cancer recurrence and metastasis. CSCs have been isolated from a variety of human cancers and are able to generate a hierarchical and heterogeneous cancer cell population. CSCs are also resistant to conventional chemo- and radio-therapies. Here we report that ionizing radiation can induce stem cell-like properties in heterogeneous cancer cells. Exposure of non-stem cancer cells to ionizing radiation enhanced spherogenesis, and this was accompanied by upregulation of the pluripotency genes Sox2 and Oct3/4. Knockdown of Sox2 or Oct3/4 inhibited radiation-induced spherogenesis and increased cellular sensitivity to radiation. These data demonstrate that ionizing radiation can activate stemness pathways in heterogeneous cancer cells, resulting in the enrichment of a CSC subpopulation with higher resistance to radiotherapy.     

Ionizing radiation induces delayed hyperrecombination in Mammalian cells

Exposure to ionizing radiation can result in delayed effects that can be detected in the progeny of an irradiated cell multiple generations after the initial exposure. These effects are described under the rubric of radiation-induced genomic instability and encompass multiple genotoxic endpoints. We have developed a green fluorescence protein (GFP)-based assay and demonstrated that ionizing radiation induces genomic instability in human RKO-derived cells and in human hamster hybrid GM10115 cells, manifested as increased homologous recombination (HR). Up to 10% of cells cultured after irradiation produce mixed GFP(+/-) colonies indicative of delayed HR or, in the case of RKO-derived cells, mutation and deletion. Consistent with prior studies, delayed chromosomal instability correlated with delayed reproductive cell death. In contrast, cells displaying delayed HR showed no evidence of delayed reproductive cell death, and there was no correlation between delayed chromosomal instability and delayed HR, indicating that these forms of genome instability arise by distinct mechanisms. Because delayed hyperrecombination can be induced at doses of ionizing radiation that are not associated with significantly reduced cell viability, these data may have important implications for assessment of radiation risk and understanding the mechanisms of radiation carcinogenesis.     

Endogenous opioid-mediated analgesia is dependent on adaptive T cell response in mice

Pain is an inherent component of inflammation often accompanying immune response. A large spectrum of molecules released within the inflamed tissue induces pain by stimulating primary afferent neurons in situ. Activity of primary sensitive fibers can be counteracted by local opioid release by leukocytes. In this study, we investigated the endogenous regulation of CFA-induced inflammatory pain in the context of adaptive T cell immune response. The nociceptive response to mechanical stimuli was studied using von Frey filaments in mice immunized with OVA in CFA. The nociceptive response of nude versus wild-type mice was dramatically increased, demonstrating T cell deficiency associated with increased pain sensitivity. Based on adoptive transfer experiments of OVA-specific CD4(+) T lymphocytes into nude mice, we show that Ag-specific activated, but not resting T lymphocytes are responsible for the spontaneous relief of inflammation-induced pain following Ag challenge. The analgesia was dependent on opioid release by Ag-primed CD4(+) T lymphocytes at the inflammatory site. Indeed, T cell-mediated analgesia was inhibited by local injection of an opioid receptor antagonist, unable to cross the blood-brain barrier. Notably, we found opioid precursor mRNA to be >7-fold increased in Ag-specific activated CD4(+) T lymphocytes, as compared with resting T lymphocytes in vivo. Taken together, our results show that CD4(+) T lymphocytes acquire antinociceptive effector properties when specifically primed by Ag and point out analgesia as a property linked to the effector phase of adaptive T cell response.     

Ionizing radiation at low doses induces inflammatory reactions in human blood

Irradiation of whole blood with 137Cs gamma rays intensifies the oxidative burst. Oxidant production was used as an indicator of inflammatory cell reactions and was measured by luminol-amplified chemiluminescence after treatment with inflammatory activators including bacteria, the neutrophil taxin formyl-Met-Leu-Phe, the Ca2+ ionophore A23187, the detergent saponin, and the tumor promoter phorbol ester. The irradiation response is dose-dependent up to about 100 microGy, is detectable within minutes, persists at least 1 h, and is transmitted intercellularly by a soluble mediator. The response is completely inhibited by Ca2+ sequestration in the presence of A23187 or by adenosine, indicating its Ca2+ dependency, and by the phospholipase A2 blocker p-bromphenacyl bromide. However, inhibition by the cyclooxygenase blocker aspirin is sporadic or absent. Blood taken after diagnostic examination of lungs with X rays also exhibited intensified chemiluminescence. These reactions implicate a role for specific amplifying mediator pathways, especially metabolites of the arachidonic acid cascade, in the response: "damage and repair" to cells or DNA plays little or no role. Our results provide evidence for a new mechanism of radiation action with possible consequences for the homeostasis of reactions involving inflammation and second messengers in human health and early development.     

Ionizing radiation

Everyone is exposed to radiation from natural, man-made and medical sources, and world-wide average annual exposure can be set at about 3.5 mSv. Exposure to natural sources is characterised by very large fluctuations, not excluding a range covering two orders of magnitude. Millions of inhabitants are continuously exposed to external doses as high as 10 mSv per year, delivered at low dose rates, very few workers are exposed above the legal limit of 50 mSv/year, and referring to accidental exposures, only 5% of the 116,000 people evacuated following the Chernobyl disaster encountered doses above 100 mSv. Epidemiological survey of accidentally, occupationally or medically exposed groups have revealed radio-induced cancers, mostly following high dose-rate exposure levels, only above 100 mSv. Risk coefficients were derived from these studies and projected into linear models of risk (linear non-threshold hypothesis: LNT), for the purpose of risk management following exposures at low doses and low dose-rates. The legitimacy of this approach has been questioned, by the Academy of sciences and the Academy of medicine in France, arguing: that LNT was not supported by Hiroshima and Nagasaki studies when neutron dose was revisited; that linear modelling failed to explain why so many site-related cancers were obviously non-linearly related to the dose, and especially when theory predicted they ought to be; that no evidence could be found of radio-induced cancers related to natural exposures or to low exposures at the work place; and that no evidence of genetic disease could be shown from any of the exposed groups. Arguments were provided from cellular and molecular biology helping to solve this issue, all resulting in dismissing the LNT hypothesis. These arguments included: different mechanisms of DNA repair at high and low dose rate; influence of inducible stress responses modifying mutagenesis and lethality; bystander effects allowing it to be considered that individual cellular responses reflected in fact the results of multiple cellular interactions. Following the conclusion of the French Academy of medicine, LNT modelling resulted in public anxiety by changing an hypothetical residual risk at low doses into a real one, calling on regulators, continuously, for a more and more severe control of tiny sources which may result in considerable collective doses when considered as being exposed to billions of people for hundreds of years. Examples were provided that showed that the perception of risk of radioactive sources was not related to the severity of the risk itself but to the importance attributed to the situation by the media. In some instances, such as those resulting from the loss of gammagraphy sources, it resulted in a dangerous underestimate of the necessary remedial actions.     

Ionizing radiation induces a Yap1-dependent peroxide stress response in yeast

Repair of DNA damage is fundamental for cellular tolerance to ionizing radiation (IR) and many IR-induced DNA lesions are thought to occur as a result of oxidative stress. We investigated the physiological effects of IR in Saccharomyces cerevisiae by performing protein expression profiles in cells exposed to electron pulse irradiation. Transient induction of several antioxidant enzymes in wild-type cells, but not in cells lacking the oxidative stress regulator Yap1, indicated that IR exposure causes cellular oxidative stress. Yap1 activation involved oxidation to the intramolecular disulfide bond, a signature of activation by peroxide, and was dependent on the Yap1 peroxide sensor Orp1/Gpx3. H(2)O(2) was produced in the culture medium of irradiated cells and was both necessary and sufficient for IR-induced Yap1 activation. When IR was performed in the presence of N(2)O, obviating H(2)O(2) production and increasing hydroxyl radical ((*)OH) production, the Yap1 response was lost, indicating that Yap1 was unable to respond to (*)OH or (*)OH-induced damage. However, the Yap1 response to IR did not seem to be a primary determinant of cellular IR tolerance. Altogether, these data provide a molecular demonstration that cells experience in vivo peroxide stress during IR and indicate that the H(2)O(2) produced cannot account for IR toxicity.     

Ionizing radiation induces iNOS-mediated epithelial dysfunction in the absence of an inflammatory response

Ionizing radiation induces intestinal epithelial hyporesponsiveness to secretagogues through an unknown mechanism. We investigated the role of the inducible isoform of nitric oxide (NO) synthase (iNOS)-derived NO in radiation-induced hyporesponsiveness. C57BL/6 mice were sham treated or exposed to 10-Gy gamma-radiation and were studied 3 days later. Tissues were mounted in Ussing-type diffusion chambers to assess chloride secretion in response to electrical field stimulation (EFS) and forskolin (10 microM). Transport studies were also repeated in iNOS-deficient mice. White blood cell counts were significantly lower in irradiated mice, and there was no inflammatory response as shown by myeloperoxidase activity and histological assessment. iNOS mRNA levels and nitrate/nitrite concentrations were significantly elevated in irradiated colons. iNOS immunoreactivity localized to the epithelium. Colons from irradiated wild-type, but not iNOS-deficient, mice exhibited a significant reduction in the responsiveness of the tissue to EFS and forskolin. The hyporesponsiveness was reversed by L-N(6)-(1-iminoethyl)lysine, 1400W, and dexamethasone treatments. iNOS-derived NO mediates colonic hyporesponsiveness 3 days after irradiation in the mouse in the absence of an inflammatory response.     

Ionizing radiation induces a stress response in primary cultures of rainbow trout skin

Fish skin is very vulnerable to damage from physical and chemical pollutants because it is in direct contact with the aquatic environment. In this study, the effect of gamma radiation on primary cultures of rainbow trout skin was investigated. Primary cultures containing two cell types, epidermal cells and goblet mucous cells, were exposed to doses ranging from 0.5-15 Gy 60Co gamma radiation. Expression of PCNA, c-myc and BCL2 was investigated as well as growth and levels of apoptosis and necrosis. Morphological and functional changes were also studied. The irradiated cultures showed evidence of a dose-dependent increase in necrosis and enhanced proliferation as well as morphological damage. In addition, mucous cell area was found to decrease significantly after irradiation. The study shows the value of these primary cultures as in vitro models for studying radiation effects. They provide an effective alternative to whole-animal exposures for radiation risk assessment.     

Ionizing radiation-induced mutant frequencies increase transiently in male germ cells of older mice

Spontaneous mutant frequency in the male germline increases with age, thereby increasing the risk of siring offspring with genetic disorders. In the present study we investigated the effect of age on ionizing radiation-induced male germline mutagenesis. lacI transgenic mice were treated with ionizing radiation at 4-, 15- and 26-month-old, and mutant frequencies were determined for pachytene spermatocytes and round spermatids at 15 days or 49 days after ionizing radiation treatment. Cells collected 15 days after treatment were derivatives of irradiated differentiating spermatogenic cells while cells collected 49 days later were derivatives of spermatogonial stem cells. The results showed that (1) spontaneous mutant frequency increased in spermatogenic cells recovered from nonirradiated old mice (26-months-old), particularly in the round spermatids; (2) mutant frequencies were significantly increased in round spermatids obtained from middle-aged mice (15-months-old) and old age mice (26-months-old) at 15 and 49 days after irradiation compared to the sham-treated old mice; and (3) pachytene spermatocytes obtained from 15- or 26-month-old mice displayed a significantly increased mutant frequency at 15 days post irradiation. This study indicates that age modulates the mutagenic response to ionizing radiation in the male germline.     

Ionizing radiation promotes protozoan reproduction

This experiment was performed to determine whether ionizing radiation is essential for maximum growth rate in a ciliated protozoan. When extraneous ionizing radiation was reduced to 0.15 mrad/day, the reproduction rate of Tetrahymena pyriformis was significantly less (P less than 0.01) than it was at near ambient levels, 0.5 or 1.8 mrad/day. Significantly higher growth rates (P less than 0.01) were obtained when chronic radiation was increased. The data suggest that ionizing radiation is essential for optimum reproduction rate in this organism.     

Fetal radiation exposure induces testicular cancer in genetically susceptible mice

The prevalence of testicular germ cell tumors (TGCT), a common solid tissue malignancy in young men, has been annually increasing at an alarming rate of 3%. Since the majority of testicular cancers are derived from germ cells at the stage of transformation of primordial germ cell (PGC) into gonocytes, the increase has been attributed to maternal/fetal exposures to environmental factors. We examined the effects of an estrogen (diethylstilbestrol, DES), an antiandrogen (flutamide), or radiation on the incidence of testicular germ cell tumors in genetically predisposed 129.MOLF-L1 (L1) congenic mice by exposing them to these agents on days 10.5 and 11.5 of pregnancy. Neither flutamide nor DES produced noticeable increases in testis cancer incidence at 4 weeks of age. In contrast, two doses of 0.8-Gy radiation increased the incidence of TGCT from 45% to 100% in the offspring. The percentage of mice with bilateral tumors, weights of testes with TGCT, and the percentage of tumors that were clearly teratomas were higher in the irradiated mice than in controls, indicating that irradiation induced more aggressive tumors and/or more foci of initiation sites in each testis. This radiation dose did not disrupt spermatogenesis, which was qualitatively normal in tumor-free testes although they were reduced in size. This is the first proof of induction of testicular cancer by an environmental agent and suggests that the male fetus of women exposed to radiation at about 5-6 weeks of pregnancy might have an increased risk of developing testicular cancer. Furthermore, it provides a novel tool for studying the molecular and cellular events of testicular cancer pathogenesis.     

Chemical protection against genetic effect of radiation in male mice

A study was made of the protective effect of some radioprotective agents against dominant lethal mutations (DLM) in postspermatogonial stages and reciprocal translocations (RT) in spermatogonia induced by gamma-radiation. Among the radioprotective agents used, cystaphos, a combination of cystamine and 5-MOT and a mixture of 6 components proved to be most effective against DLM, and cystaphos, gammaphos and cystamine combined with 5-MOT proved effective against RT. The degree of radioprotective efficacy was relatively low. The efficacy of cystamine in protecting against RT was higher with exposure of gonocytes of 18.5-day embryos than spermatogonia of pubertal animals. The degree of the radioprotective effect varied depending on the stage of spermatogenesis, and, in all cases, it was lower than that observed in studies of protection against lethal effects of ionizing radiation.     

Ionizing radiation biomarkers for potential use in epidemiological studies

Ionizing radiation is a known human carcinogen that can induce a variety of biological effects depending on the physical nature, duration, doses and dose-rates of exposure. However, the magnitude of health risks at low doses and dose-rates (below 100mSv and/or 0.1mSvmin(-1)) remains controversial due to a lack of direct human evidence. It is anticipated that significant insights will emerge from the integration of epidemiological and biological research, made possible by molecular epidemiology studies incorporating biomarkers and bioassays. A number of these have been used to investigate exposure, effects and susceptibility to ionizing radiation, albeit often at higher doses and dose rates, with each reflecting time-limited cellular or physiological alterations. This review summarises the multidisciplinary work undertaken in the framework of the European project DoReMi (Low Dose Research towards Multidisciplinary Integration) to identify the most appropriate biomarkers for use in population studies. In addition to logistical and ethical considerations for conducting large-scale epidemiological studies, we discuss the relevance of their use for assessing the effects of low dose ionizing radiation exposure at the cellular and physiological level. We also propose a temporal classification of biomarkers that may be relevant for molecular epidemiology studies which need to take into account the time elapsed since exposure. Finally, the integration of biology with epidemiology requires careful planning and enhanced discussions between the epidemiology, biology and dosimetry communities in order to determine the most important questions to be addressed in light of pragmatic considerations including the appropriate population to be investigated (occupationally, environmentally or medically exposed), and study design. The consideration of the logistics of biological sample collection, processing and storing and the choice of biomarker or bioassay, as well as awareness of potential confounding factors, are also essential.     

Ionizing radiation sensitivity of DNA polymerase lambda-deficient cells

Ionizing radiation induces a diverse spectrum of DNA lesions, including strand breaks and oxidized bases. In mammalian cells, ionizing radiation-induced lesions are targets of non-homologous end joining, homologous recombination, and base excision repair. In vitro assays show a potential involvement of DNA polymerase lambda in non-homologous end joining and base excision repair. In this study, we investigated whether DNA polymerase lambda played a significant role in determining ionizing radiation sensitivity. Despite increased sensitivity to hydrogen peroxide, lambda-deficient mouse embryonic fibroblasts displayed equal survival after exposure to ionizing radiation compared to their wild-type counterparts. In addition, we found increased sensitivity to the topoisomerase inhibitors camptothecin and etoposide in the absence of polymerase lambda. These results do not reveal a major role for DNA polymerase lambda in determining radiosensitivity in vivo.     

Ionizing radiation damage to DNA: molecular aspects

Radioresistant tumor cells are found in tumor specimens from patients in whom radiotherapy has failed or whose tumors have recurred after therapy. This suggests that inherent cellular radioresistance may in part underlie the failure of radiotherapy, and therefore determination of the presence of resistant cells within a tumor might be a useful predictor of response to radiation therapy. Most standard clonogenic assays of radiation response are time-consuming, and alternative assays of radiation response are being sought. In an earlier publication (J. L. Schwartz et al., Int. J. Radiat. Oncol. Biol. Phys. 15, 907-912, 1988), we reported that radioresistant human tumor cells rejoin DNA double-strand breaks, as measured by DNA neutral filter elution (pH 9.6), faster than more sensitive cell lines. To determine whether DNA elution might have potential as a rapid predictive assay, we examined the relationship between the rate of DNA double-strand break rejoining and radiosensitivity in nine first-passage-after-explant squamous cell carcinomas under conditions that minimized the influence of nontumor and nonclonogenic cells. The frequency of DNA double-strand breaks measured 1 h after irradiation with 100 Gy 60Co gamma rays was used as an estimate of relative rejoining rate. This number is a reflection of both the initial DNA double-strand break frequency and the amount of repair that occurs in 1 h. The relative break frequency was compared to radiosensitivity as measured by standard clonogenic survival assays in later passages (p3-p14) of these same cells. A significant relationship (r = 0.61, P less than 0.01) was found between break frequency measured in first-passage cells and radiosensitivity measured in later passages, suggesting that the neutral elution assay as described here has some promise as a relatively rapid assay of the radiosensitivity of human tumor cells.     

Ionizing radiation and DNA-chain elongation in ataxia telangiectasia lymphoblastoid cells

DNA-chain elongation rates, determined by sedimentation analysis, were found to be similar in control and ataxia-telangiectasia lymphoblastoid cells. A γ-radiation dose of 6 Gray, which had previously been shown to have a marked inhibitory effect on initiation of DNA replication, had no appreciable effect on elongation rates in either cell type. Elongation rates were also determined at 20 Gray of γ-rays by pulsing cells with [³H]thymidine prior to irradiation to avoid anomalous sedimentation behaviour. At this radiation dose elongation was almost completely inhibited in control cells while little or no inhibition was observed in ataxia-telangiectasia cells. Deoxyribonucleoside triphosphate pool equilibration times were not altered at either dose.     

The female pheromone 2,5-dimethylpyrazine induces sperm head abnormalities in male CBA mice

The effect of the house mouse female pheromone 2,5-dimethylpyrazine (2,5-DMP) on sperm differentiation in male CBA mice has been studied. For this purpose, mature males were treated with a 0.01% aqueous solution of the pheromone for six days. Control mice were similarly treated with physiologic saline. The mice were sacrificed 23 days after the treatment, and material for the analysis of sperm-head abnormalities was sampled from the caudal portion of the epididymis. Analysis of the frequency of abnormal sperms has demonstrated that the pheromonal treatment significantly increases the frequencies of various sperm-head abnormalities. Apparently, this results from disturbances in sex-cell differentiation germline cells caused by the induction of genetic damage at stages immediately preceding meiosis, as well as during the first and second meiotic divisions. The relationship between the effect of 2,5-DMP and the decrease in the fertility of male CBA mice that was earlier observed after a similar treatment is discussed.