Impact of the redox environment on propagation of radiation bystander effects: The modulating effect of oxidative metabolism and oxygen partial pressure.

Redox modulated pathways play important roles in out-of-field effects of ionizing radiation. We investigated how the redox environment impacts the magnitude of propagation of stressful effects from irradiated to bystander cells. Normal human fibroblasts that have incorporated [3H]-thymidine were intimately co-cultured with bystander cells in a strategy that allowed isolation of bystander cells with high purity. The antioxidant glutathione peroxidase (GPX) was maintained either at wild-type conditions or overexpressed in the bystanders. Following 24 h of coculture, levels of stress-responsive p21Waf1, p-Hdm2, and connexin43 proteins were increased in bystander cells expressing wild-type GPX relative to respective controls. These levels were significantly attenuated when GPX was ectopically overexpressed, demonstrating by direct approach the involvement of a regulator of intracellular redox homeostasis. Evidence of participation of pro-oxidant compounds was generated by exposing confluent cell cultures to low fluences of 3.7 MeV α particles in presence or absence of t-butyl hydroperoxide. By 3 h post-exposure to fluences wherein only ∼2% of cells are traversed through the nucleus by a particle track, increases in chromosomal damage were greater than expected in absence of the drug (p < 0.001) and further enhanced in its presence (p < 0.05). While maintenance and irradiation of cell cultures at low oxygen pressure (pO2 3.8 mm Hg) to mimic in vivo still supported the participation of bystander cells in responses assessed by chromosomal damage and stress-responsive protein levels (p < 0.001), the effects were attenuated compared to ambient pO2 (155 mm Hg) (p < 0.05). Together, the results show that bystander effects are attenuated at below ambient pO2 and when metabolic oxidative stress is reduced but increased when the basal redox environment tilts towards oxidizing conditions. They are consistent with bystander effects being independent of radiation dose rate.

Acquired radioresistance in cancer associated fibroblasts is concomitant with enhanced antioxidant potential and DNA repair capacity.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are a major component of the cancer stroma, and their response to therapeutic treatments likely impacts the outcome. We tested the hypothesis that CAFs develop unique characteristics that enhance their resistance to ionizing radiation. METHODS: CAFs were generated through intimate coculture of normal human fibroblasts of skin or lung origin with various human cancer cell types using permeable microporous membrane inserts. Fibroblasts and cancer cells are grown intimately, yet separately, on either side of the insert's membrane for extended times to generate activated fibroblast populations highly enriched in CAFs. RESULTS: The generated CAFs exhibited a decrease in Caveolin-1 protein expression levels, a CAF biomarker, which was further enhanced when the coculture was maintained under in-vivo-like oxygen tension conditions. The level of p21Waf1 was also attenuated, a characteristic also associated with accelerated tumor growth. Furthermore, the generated CAFs experienced perturbations in their redox environment as demonstrated by increases in protein carbonylation, mitochondrial superoxide anion levels, and modulation of the activity of the antioxidants, manganese superoxide dismutase and catalase. Propagation of the isolated CAFs for 25 population doublings was associated with enhanced genomic instability and a decrease in expression of the senescence markers ß-galactosidase and p16INK4a. With relevance to radiotherapeutic treatments, CAFs in coculture with cancer cells of diverse origins (breast, brain, lung, and prostate) were resistant to the clastogenic effects of 137Cs γ rays compared to naïve fibroblasts. Addition of repair inhibitors of single- or double-stranded DNA breaks attenuated the resistance of CAFs to the clastogenic effects of γ rays, supporting a role for increased ability to repair DNA damage in CAF radioresistance. CONCLUSIONS: This study reveals that CAFs are radioresistant and experience significant changes in indices of oxidative metabolism. The CAFs that survive radiation treatment likely modulate the fate of the associated cancer cells. Identifying them together with their mode of communication with cancer cells, and eradicating them, particularly when they may exist at the margin of the radiotherapy planning target volume, may improve the efficacy of cancer treatments. Video Abstract.

c-Jun N-Terminal Kinase Inhibition Induces Mitochondrial Oxidative Stress and Decreases Survival in Human Neural Stem Progenitors.

Neural stem cells are attracting enormous attention in regenerative medicine due to their ability to self-renew and differentiate into the cell lineages that constitute the central nervous system. However, little is known about the mechanism underlying the regulation of their redox environment, which is essential for homeostatic cellular functions. The redox-modulated c-Jun N-terminal kinases (JNK) are a molecular switch in stress signal transduction and are involved in numerous brain functions. Using a selective but broad-spectrum inhibitor of JNK 1/2/3, we investigated the role of JNK in regulating the levels of reactive oxygen species in mitochondria, mitochondrial membrane potential, viability, proliferation and lineage alterations in human H9-derived neural stem/progenitor cells (NSPs). Relative to diluent control, incubation of the NSPs for 24 h with SP600125, an anthrapyrazolone inhibitor of JNK, resulted in increased abundance of mitochondrial superoxide radicals (p < 0.05), concomitant with decreases in mitochondrial membrane potential (p < 0.001), while maintaining a consistent and stable mitochondrial mass. Whereas H9-derived NSPs collectively express Nestin, a marker for neural stem cells, a panel of cell surface markers analyzed by flow cytometry revealed that they are a heterogeneous population that sustains this diversity after JNK inhibition. In addition, the levels of nuclear forkhead homeobox type O3a (FoxO3a), a regulator of redox homeostasis, decreased, which was associated with a decrease in overall cell viability as measured by Annexin V staining (p < 0.001), and supported by an increased level of cleaved Poly-ADP-ribose polymerase and decreased survivin expression. However, staining with the proliferation marker, Ki67, revealed the presence of a significant percentage of proliferating cells in the treated population. Together, the results support a role for JNK in the redox-homeostasis and fate of NSPs. Identifying regulators of the cellular redox environment will enhance our understanding of the mechanisms that modulate neural stem cell functions and optimize therapeutic applications targeting JNK.

The Translationally Controlled Tumor Protein and the Cellular Response to Ionizing Radiation-Induced DNA Damage.

The absorption of ionizing radiation by living cells can directly disrupt atomic structures, producing chemical and biological changes. It can also act indirectly through radiolysis of water, thereby generating reactive chemical species that may damage nucleic acids, proteins, and lipids. Together, the direct and indirect effects of radiation initiate a series of biochemical and molecular signaling events that may repair the damage or culminate in permanent physiological changes or cell death. In efforts to gain insight into the mechanisms underlying these effects, we observed a prominent upregulation of the Translationally Controlled Tumor Protein (TCTP) in low dose/low dose rate 137Cs γ-irradiated cells that was associated with adaptive responses that reduced chromosomal damage to a level lower than what occurs spontaneously. Therefore, TCTP may support the survival and genomic integrity of irradiated cells through a role in the DNA damage response. Consistent with this postulate, TCTP was shown to physically interact with ATM, an early sensor of DNA damage, and to exist in a complex with γH2A.X, in agreement with its distinct localization with the foci of the DNA damage marker proteins γH2A.X, 53BP1, and P-ATM. Cells lacking TCTP failed to repair chromosomal damage induced by γ-rays. Further, TCTP was shown to interact with the DNA-binding subunits, Ku70 and Ku80, of DNA-PK, a major participant in nonhomologous end joining of DNA double strand breaks. Moreover, TCTP physically interacted with p53, and its knockdown attenuated the radiation-induced G1 delay, but prolonged the G2 delay. Here, we briefly review the biochemical events leading to DNA damage by ionizing radiation and to its sensing and repair, and highlight TCTP's critical role in maintaining genomic integrity in response to DNA-damaging agents.

Genomic instability induced in distant progeny of bystander cells depends on the connexins expressed in the irradiated cells.

PURPOSE: To examine the time window during which intercellular signaling though gap junctions mediates non-targeted (bystander) effects induced by moderate doses of ionizing radiation; and to investigate the impact of gap junction communication on genomic instability in distant progeny of bystander cells. MATERIALS AND METHODS: A layered cell culture system was developed to investigate the propagation of harmful effects from irradiated normal or tumor cells that express specific connexins to contiguous bystander normal human fibroblasts. Irradiated cells were exposed to moderate mean absorbed doses from 3.7 MeV α particle, 1000 MeV/u iron ions, 600 MeV/u silicon ions, or 137Cs γ rays. Following 5 h of co-culture, pure populations of bystander cells, unexposed to secondary radiation, were isolated and DNA damage and oxidative stress was assessed in them and in their distant progeny (20-25 population doublings). RESULTS: Increased frequency of micronucleus formation and enhanced oxidative changes were observed in bystander cells co-cultured with confluent cells exposed to either sparsely ionizing (137Cs γ rays) or densely ionizing (α particles, energetic iron or silicon ions) radiations. The irradiated cells propagated signals leading to biological changes in bystander cells within 1 h of irradiation, and the effect required cellular coupling by gap junctions. Notably, the distant progeny of isolated bystander cells also exhibited increased levels of spontaneous micronuclei. This effect was dependent on the type of junctional channels that coupled the irradiated donor cells with the bystander cells. Previous work showed that gap junctions composed of connexin26 (Cx26) or connexin43 (Cx43) mediate toxic bystander effects within 5 h of co-culture, whereas gap junctions composed of connexin32 (Cx32) mediate protective effects. In contrast, the long-term progeny of bystander cells expressing Cx26 or Cx43 did not display elevated DNA damage, whereas those coupled by Cx32 had enhanced DNA damage. CONCLUSIONS: In response to moderate doses from either sparsely or densely ionizing radiations, toxic and protective effects are rapidly communicated to bystander cells through gap junctions. We infer that bystander cells damaged by the initial co-culture (expressing Cx26 or Cx43) die or undergo proliferative arrest, but that the bystander cells that were initially protected (expressing Cx32) express DNA damage upon sequential passaging. Together, the results inform the roles that intercellular communication play under stress conditions, and aid assessment of the health risks of exposure to ionizing radiation. Identification of the communicated molecules may enhance the efficacy of radiotherapy and help attenuate its debilitating side-effects.

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication.

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events leading to stromal activation and establishment of the tumor microenvironment (TME). Several in vitro and in vivo models of the TME have been developed; however, in general these models do not readily permit isolation of individual cell populations, under non-perturbing conditions, for further study. To circumvent this difficulty, we have employed an in vitro TME model using a cell growth substrate consisting of a permeable microporous membrane insert that permits simple generation of highly enriched cell populations grown intimately, yet separately, on either side of the insert's membrane for extended co-culture times. Through use of this model, we are capable of generating greatly enriched cancer-associated fibroblast (CAF) populations from normal diploid human fibroblasts following co-culture (120 hr) with highly metastatic human breast carcinoma cells, without the use of fluorescent tagging and/or cell sorting. Additionally, by modulating the pore-size of the insert, we can control for the mode of intercellular communication (e.g., gap-junction communication, secreted factors) between the two heterotypic cell populations, which permits investigation of the mechanisms underlying the development of the TME, including the role of gap-junction permeability. This model serves as a valuable tool in enhancing our understanding of the initial events leading to cancer-stroma initiation, the early evolution of the TME, and the modulating effect of the stroma on the responses of cancer cells to therapeutic agents.

Diffusible Factors Secreted by Glioblastoma and Medulloblastoma Cells Induce Oxidative Stress in Bystander Neural Stem Progenitors.

Harmful effects that alter the homeostasis of neural stem or progenitor cells (NSPs) can affect regenerative processes in the central nervous system. We investigated the effect of soluble factors secreted by control or (137)Cs-γ-irradiated glioblastoma or medulloblastoma cells on redox-modulated endpoints in recipient human NSPs. Growth medium harvested from the nonirradiated brain tumor cells, following 24 h of growth, induced prominent oxidative stress in recipient NSPs as judged by overall increases in mitochondrial superoxide radical levels (p < .001), activation of c-jun N-terminal kinase, and decrease in the active form of FoxO3a. The induced oxidative stress was associated with phosphorylation of p53 on serine 15, a marker of DNA damage, induction of the cyclin-cyclin dependent kinase inhibitors p21(Waf1) and p27(Kip1), and perturbations in cell cycle progression (p < .001). These changes were also associated with increased apoptosis as determined by enhanced annexin V staining (p < .001) and caspase 8 activation (p < .05) and altered expression of critical regulators of self-renewal, proliferation, and differentiation. Exposure of the tumor cells to radiation only slightly altered the induced oxidative changes in the bystander NSPs, except for medium from irradiated medulloblastoma cells that was more potent at inducing apoptosis in the NSPs than medium from nonirradiated cells (p < .001). The elucidation of such stressful bystander effects provides avenues to understand the biochemical events underlying the development or exacerbation of degenerative outcomes associated with brain cancers. It is also relevant to tissue culture protocols whereby growth medium conditioned by tumor cells is often used to support the growth of stem cells.

Delayed activation of human microglial cells by high dose ionizing radiation.

Recent studies have shown that microglia affects the fate of neural stem cells in response to ionizing radiation, which suggests a role for microglia in radiation-induced degenerative outcomes. We therefore investigated the effects of γ-irradiation on cell survival, proliferation, and activation of microglia and explored associated mechanisms. Specifically, we evaluated cellular and molecular changes associated with exposure of human microglial cells (CHME5) to low and high doses of acute cesium-137 γ rays. Twenty-four hours after irradiation, cell cycle analyses revealed dose-dependent decreases in the fraction of cells in S and G2/M phase, which correlated with significant oxidative stress. By one week after irradiation, 20-30% of the cells exposed to high doses of γ rays underwent apoptosis, which correlated with significant concomitant decrease in metabolic activity as assessed by the MTT assay, and microglial activation as judged by both morphological changes and increased expression of Glut-5 and CR43. These changes were associated with increases in the mRNA levels for IL-1α, IL-10 and TNFα. Together, the results show that human CHME5 microglia are relatively resistant to low and moderate doses of γ rays, but are sensitive to acute high doses, and that CHME5 cells are a useful tool for in vitro study of human microglia.

Is Ionizing Radiation Harmful at any Exposure? An Echo That Continues to Vibrate.

The health risks to humans and non-human biota exposed to low dose ionizing radiation remain ambiguous and are the subject of intense debate. The need to establish risk assessment standards based on the mechanisms underlying low-level radiation exposure has been recognized by regulatory agencies as critical to adequately protect people and to make the most effective use of national resources. Here, the authors briefly review evidence showing that the molecular and biochemical changes induced by low doses of radiation differ from those induced by high doses. In particular, an array of redundant and inter-related mechanisms act in both prokaryotes and eukaryotes to restore DNA integrity following exposures to relatively low doses of sparsely ionizing radiation. Furthermore, the radiation-induced protective mechanisms often overcompensate and minimize the mutagenic potential of the byproducts of normal oxidative metabolism. In contrast to adaptive protection observed at low doses of sparsely ionizing radiation, there is evidence that even a single nuclear traversal by a densely ionizing particle track can trigger harmful effects that spread beyond the traversed cell and induce damaging effects in the nearby bystander cells. In vivo studies examining whether exposure to low dose radiation at younger age modulates the latency of expression of age-related diseases such as cancer, together with studies on the role of genetic susceptibility, will further illuminate the magnitude of risk of exposure to low dose radiation.

S-Nitrosylation in Organs of Mice Exposed to Low or High Doses of γ-Rays: The Modulating Effect of Iodine Contrast Agent at a Low Radiation Dose.

The covalent addition of nitric oxide (NO•) onto cysteine thiols, or S-nitrosylation, modulates the activity of key signaling proteins. The dysregulation of normal S-nitrosylation contributes to degenerative conditions and to cancer. To gain insight into the biochemical changes induced by low-dose ionizing radiation, we determined global S-nitrosylation by the "biotin switch" assay coupled with mass spectrometry analyses in organs of C57BL/6J mice exposed to acute 0.1 Gy of 137Cs γ-rays. The dose of radiation was delivered to the whole body in the presence or absence of iopamidol, an iodinated contrast agent used during radiological examinations. To investigate whether similar or distinct nitrosylation patterns are induced following high-dose irradiation, mice were exposed in parallel to acute 4 Gy of 137Cs γ rays. Analysis of modulated S-nitrosothiols (SNO-proteins) in freshly-harvested organs of animals sacrificed 13 days after irradiation revealed radiation dose- and contrast agent-dependent changes. The major results were as follows: (i) iopamidol alone had significant effects on S-nitrosylation in brain, lung and liver; (ii) relative to the control, exposure to 0.1 Gy without iopamidol resulted in statistically-significant SNO changes in proteins that differ in molecular weight in liver, lung, brain and blood plasma; (iii) iopamidol enhanced the decrease in S-nitrosylation induced by 0.1 Gy in brain; (iv) whereas a decrease in S-nitrosylation occurred at 0.1 Gy for proteins of ~50 kDa in brain and for proteins of ~37 kDa in liver, an increase was detected at 4 Gy in both organs; (v) mass spectrometry analyses of nitrosylated proteins in brain revealed differential modulation of SNO proteins (e.g., sodium/potassium-transporting ATPase subunit beta-1; beta tubulins; ADP-ribosylation factor 5) by low- and high-dose irradiation; and (vi) ingenuity pathway analysis identified major signaling networks to be modulated, in particular the neuronal nitric oxide synthase signaling pathway was differentially modulated by low- and high-dose γ-irradiation.

Ionizing Radiation Perturbs Cell Cycle Progression of Neural Precursors in the Subventricular Zone Without Affecting Their Long-Term Self-Renewal.

Damage to normal human brain cells from exposure to ionizing radiation may occur during the course of radiotherapy or from accidental exposure. Delayed effects may complicate the immediate effects resulting in neurodegeneration and cognitive decline. We examined cellular and molecular changes associated with exposure of neural stem/progenitor cells (NSPs) to (137)Cs γ-ray doses in the range of 0 to 8 Gy. Subventricular zone NSPs isolated from newborn mouse pups were analyzed for proliferation, self-renewal, and differentiation, shortly after irradiation. Strikingly, there was no apparent increase in the fraction of dying cells after irradiation, and the number of single cells that formed neurospheres showed no significant change from control. Upon differentiation, irradiated neural precursors did not differ in their ability to generate neurons, astrocytes, and oligodendrocytes. By contrast, progression of NSPs through the cell cycle decreased dramatically after exposure to 8 Gy (p < .001). Mice at postnatal day 10 were exposed to 8 Gy of γ rays delivered to the whole body and NSPs of the subventricular zone were analyzed using a four-color flow cytometry panel combined with ethynyl deoxyuridine incorporation. Similar flow cytometric analyses were performed on NSPs cultured as neurospheres. These studies revealed that neither the percentage of neural stem cells nor their proliferation was affected. By contrast, γ-irradiation decreased the proliferation of two classes of multipotent cells and increased the proliferation of a specific glial-restricted precursor. Altogether, these results support the conclusion that primitive neural precursors are radioresistant, but their proliferation is slowed down as a consequence of γ-ray exposure.

Low-dose energetic protons induce adaptive and bystander effects that protect human cells against DNA damage caused by a subsequent exposure to energetic iron ions.

During interplanetary missions, astronauts are exposed to mixed types of ionizing radiation. The low 'flux' of the high atomic number and high energy (HZE) radiations relative to the higher 'flux' of low linear energy transfer (LET) protons makes it highly probable that for any given cell in the body, proton events will precede any HZE event. Whereas progress has been made in our understanding of the biological effects of low-LET protons and high-LET HZE particles, the interplay between the biochemical processes modulated by these radiations is unclear. Here we show that exposure of normal human fibroblasts to a low mean absorbed dose of 20 cGy of 0.05 or 1-GeV protons (LET ∼ 1.25 or 0.2 keV/µm, respectively) protects the irradiated cells (P < 0.0001) against chromosomal damage induced by a subsequent exposure to a mean absorbed dose of 50 cGy from 1 GeV/u iron ions (LET ∼ 151 keV/µm). Surprisingly, unirradiated (i.e. bystander) cells with which the proton-irradiated cells were co-cultured were also significantly protected from the DNA-damaging effects of the challenge dose. The mitigating effect persisted for at least 24 h. These results highlight the interactions of biological effects due to direct cellular traversal by radiation with those due to bystander effects in cell populations exposed to mixed radiation fields. They show that protective adaptive responses can spread from cells targeted by low-LET space radiation to bystander cells in their vicinity. The findings are relevant to understanding the health hazards of space travel.

Health risks of space exploration: targeted and nontargeted oxidative injury by high-charge and high-energy particles.

SIGNIFICANCE: During deep space travel, astronauts are often exposed to high atomic number (Z) and high-energy (E) (high charge and high energy [HZE]) particles. On interaction with cells, these particles cause severe oxidative injury and result in unique biological responses. When cell populations are exposed to low fluences of HZE particles, a significant fraction of the cells are not traversed by a primary radiation track, and yet, oxidative stress induced in the targeted cells may spread to nearby bystander cells. The long-term effects are more complex because the oxidative effects persist in progeny of the targeted and affected bystander cells, which promote genomic instability and may increase the risk of age-related cancer and degenerative diseases. RECENT ADVANCES: Greater understanding of the spatial and temporal features of reactive oxygen species bursts along the tracks of HZE particles, and the availability of facilities that can simulate exposure to space radiations have supported the characterization of oxidative stress from targeted and nontargeted effects. CRITICAL ISSUES: The significance of secondary radiations generated from the interaction of the primary HZE particles with biological material and the mitigating effects of antioxidants on various cellular injuries are central to understanding nontargeted effects and alleviating tissue injury. FUTURE DIRECTIONS: Elucidation of the mechanisms underlying the cellular responses to HZE particles, particularly under reduced gravity and situations of exposure to additional radiations, such as protons, should be useful in reducing the uncertainty associated with current models for predicting long-term health risks of space radiation. These studies are also relevant to hadron therapy of cancer.

Nontargeted stressful effects in normal human fibroblast cultures exposed to low fluences of high charge, high energy (HZE) particles: kinetics of biologic responses and significance of secondary radiations.

The induction of nontargeted stressful effects in cell populations exposed to low fluences of high charge (Z) and high energy (E) particles is relevant to estimates of the health risks of space radiation. We investigated the up-regulation of stress markers in confluent normal human fibroblast cultures exposed to 1,000 MeV/u iron ions [linear energy transfer (LET) ∼151 keV/µm] or 600 MeV/u silicon ions (LET ∼50 keV/µm) at mean absorbed doses as low as 0.2 cGy, wherein 1-3% of the cells were targeted through the nucleus by a primary particle. Within 24 h postirradiation, significant increases in the levels of phospho-TP53 (serine 15), p21(Waf1) (CDKN1A), HDM2, phospho-ERK1/2, protein carbonylation and lipid peroxidation were detected, which suggested participation in the stress response of cells not targeted by primary particles. This was supported by in situ studies that indicated greater increases in 53BP1 foci formation, a marker of DNA damage. than expected from the number of primary particle traversals. The effect was expressed as early as 15 min after exposure, peaked at 1 h and decreased by 24 h. A similar tendency occurred after exposure of the cell cultures to 0.2 cGy of 3.7 MeV α particles (LET ∼109 keV/µm) that targets ∼1.6% of nuclei, but not after 0.2 cGy from 290 MeV/u carbon ions (LET ∼13 keV/µm) by which, on average, ∼13% of the nuclei were hit, which highlights the importance of radiation quality in the induced effect. Simulations with the FLUKA multi-particle transport code revealed that fragmentation products, other than electrons, in cell cultures exposed to HZE particles comprise <1% of the absorbed dose. Further, the radial spread of dose due to secondary heavy ion fragments is confined to approximately 10-20 µm. Thus, the latter are unlikely to significantly contribute to stressful effects in cells not targeted by primary HZE particles.

Human cell responses to ionizing radiation are differentially affected by the expressed connexins.

In multicellular organisms, intercellular communication is essential for homeostatic functions and has a major role in tissue responses to stress. Here, we describe the effects of expression of different connexins, which form gap junction channels with different permeabilities, on the responses of human cells to ionizing radiation. Exposure of confluent HeLa cell cultures to (137)Cs γ rays, 3.7 MeV α particles, 1000 MeV protons or 1000 MeV/u iron ions resulted in distinct effects when the cells expressed gap junction channels composed of either connexin26 (Cx26) or connexin32 (Cx32). Irradiated HeLa cells expressing Cx26 generally showed decreased clonogenic survival and reduced metabolic activity relative to parental cells lacking gap junction communication. In contrast, irradiated HeLa cells expressing Cx32 generally showed enhanced survival and greater metabolic activity relative to the control cells. The effects on clonogenic survival correlated more strongly with effects on metabolic activity than with DNA damage as assessed by micronucleus formation. The data also showed that the ability of a connexin to affect clonogenic survival following ionizing radiation can depend on the specific type of radiation. Together, these findings show that specific types of connexin channels are targets that may be exploited to enhance radiotherapeutic efficacy and to formulate countermeasures to the harmful effects of specific types of ionizing radiation.

Role of the translationally controlled tumor protein in DNA damage sensing and repair.

The translationally controlled tumor protein (TCTP) is essential for survival by mechanisms that as yet are incompletely defined. Here we describe an important role of TCTP in response to DNA damage. Upon exposure of normal human cells to low-dose γ rays, the TCTP protein level was greatly increased, with a significant enrichment in nuclei. TCTP up-regulation occurred in a manner dependent on ataxia-telangiectasia mutated (ATM) kinase and the DNA-dependent protein kinase and was associated with protective effects against DNA damage. In chromatin of irradiated cells, coimmunoprecipitation experiments showed that TCTP forms a complex with ATM and γH2A.X, in agreement with its distinct localization with the foci of the DNA damage-marker proteins γH2A.X, 53BP1, and P-ATM. In cells lacking TCTP, repair of chromosomal damage induced by γ rays was compromised significantly. TCTP also was shown to interact with p53 and the DNA-binding subunits, Ku70 and Ku80, of DNA-dependent protein kinase. TCTP knockdown led to decreased levels of Ku70 and Ku80 in nuclei of irradiated cells and attenuated their DNA-binding activity. It also attenuated the radiation-induced G(1) delay but prolonged the G(2) delay. TCTP therefore may play a critical role in maintaining genomic integrity in response to DNA-damaging agents.

Micro RNA responses to chronic or acute exposures to low dose ionizing radiation.

Human health risks of exposure to low dose ionizing radiation remain ambiguous and are the subject of intense debate. A wide variety of biological effects are induced after cellular exposure to ionizing radiation, but the underlying molecular mechanism(s) remain to be completely understood. We hypothesized that low dose γ-radiation-induced effects are controlled by the modulation of micro RNA (miRNA) that participate in the control of gene expression at the posttranscriptional level and are involved in many cellular processes. We monitored the expression of several miRNA in human cells exposed to acute or chronic low doses of 10 cGy or a moderate dose of 400 cGy of (137)Cs γ-rays. Dose, dose rate and time dependent differences in the relative expression of several miRNA were investigated. The expression patterns of many miRNA differed after exposure to either chronic or acute 10 cGy. The expression of miRNA let-7e, a negative regulator of RAS oncogene, and the c-MYC miRNA cluster were upregulated after 10 cGy chronic dose but were downregulated after 3 h of acute 10 cGy. The miR-21 was upregulated in chronic or acute low dose and moderate dose treated cells and its target genes hPDCD4, hPTEN, hSPRY2, and hTPM1 were found to be downregulated. These findings provide evidence that low dose and dose rate γ-irradiation dictate the modulation of miRNA, which can result in a differential cellular response than occurs at high doses. This information will contribute to understanding the risks to human health after exposure to low dose radiation.

Increased frequency of spontaneous neoplastic transformation in progeny of bystander cells from cultures exposed to densely ionizing radiation.

An increased risk of carcinogenesis caused by exposure to space radiation during prolonged space travel is a limiting factor for human space exploration. Typically, astronauts are exposed to low fluences of ionizing particles that target only a few cells in a tissue at any one time. The propagation of stressful effects from irradiated to neighboring bystander cells and their transmission to progeny cells would be of importance in estimates of the health risks of exposure to space radiation. With relevance to the risk of carcinogenesis, we investigated, in model C3H 10T½ mouse embryo fibroblasts (MEFs), modulation of the spontaneous frequency of neoplastic transformation in the progeny of bystander MEFs that had been in co-culture 10 population doublings earlier with MEFs exposed to moderate doses of densely ionizing iron ions (1 GeV/nucleon) or sparsely ionizing protons (1 GeV). An increase (P<0.05) in neoplastic transformation frequency, likely mediated by intercellular communication through gap junctions, was observed in the progeny of bystander cells that had been in co-culture with cells irradiated with iron ions, but not with protons.