The double-stranded RNA-activated protein kinase mediates radiation resistance in mouse embryo fibroblasts through nuclear factor kappaB and Akt activation

PURPOSE: Activation of the double-stranded RNA-activated protein kinase (PKR) leads to the induction of various pathways including the down-regulation of translation through phosphorylation of the eukaryotic translation initiation factor 2alpha (eIF-2alpha). There have been no reports to date about the role of PKR in radiation sensitivity. EXPERIMENTAL DESIGN: A clonogenic survival assay was used to investigate the sensitivity of PKR mouse embryo fibroblasts (MEF) to radiation therapy. 2-Aminopurine (2-AP), a chemical inhibitor of PKR, was used to inhibit PKR activation. Nuclear factor-kappaB (NF-kappaB) activation was assessed by electrophoretic mobility shift assay (EMSA). Expression of PKR and downstream targets was examined by Western blot analysis and immunofluorescence. RESULTS: Ionizing radiation leads to dose- and time-dependent increases in PKR expression and function that contributes to increased cellular radiation resistance as shown by clonogenic survival and terminal nucleotidyl transferase-mediated nick end labeling (TUNEL) apoptosis assays. Specific inhibition of PKR with the chemical inhibitor 2-AP restores radiation sensitivity. Plasmid transfection of the PKR wild-type (wt) gene into PKR(-/-) MEFs leads to increased radiation resistance. The protective effect of PKR to radiation may be mediated in part through NF-kappaB and Akt because both NF-kappaB and Akt are activated after ionizing radiation in PKR+/+ but not PKR-/- cells. CONCLUSIONS: We suggest a novel role for PKR as a mediator of radiation resistance modulated in part through the protective effects of NF-kappaB and Akt activation. The modification of PKR activity may be a novel strategy in the future to overcome radiation resistance.

The effect of ultraviolet radiation on the pathogenesis of {\it Candida albicans} in mice

Ultraviolet (UV) radiation produces immunological alterations in both humans and animals that include a decrease in the delayed type hypersensitivity (DTH) response to complex antigens, and to the induction of the suppressor T cell pathway. Cell-mediated immunity of the type that is altered by UV radiation has been shown to be important in host resistance against microorganisms. My dissertation addresses questions concerning the effects of UV radiation on the pathogenesis of opportunistic fungal pathogens such as Candida albicans.^ The (DTH) response of C3H mice exposed to ultraviolet (UV) radiation before (afferent arm of DTH) or after (efferent arm of DTH) infection with Candida albicans was markedly and systemically suppressed. Although suppression of both the afferent and efferent phases of DTH were caused by similar wavebands within the ultraviolet region, the dose of UV radiation that suppressed the efferent arm of DTH was 10-fold higher than the dose that suppressed the afferent arm of the DTH reaction.^ The DTH response of C57BL/6 mice was also suppressed by UV radiation; however the suppression was accomplished by exposure to significantly lower doses UV radiation compared to C3H mice. In C57BL/6 mice, the dose of UV radiation that suppressed the afferent phase of DTH was 5-fold higher than the dose that suppressed the efferent phase.^ Exposure of C3H mice to UV radiation before sensitization induced splenic suppressor T cells that upon transfer to normal recipients, impaired the induction of DTH to Candida. In contrast, the suppression caused by UV irradiation of mice after sensitization was not transferable. Spleen cells from sensitized mice exhibited altered homing patterns in animals that were exposed to UV radiation shortly before receiving cells, suggesting that UV-induced suppression of the efferent arm of DTH could result from an alteration in the distribution of effector cells.^ UV radiation decreased the survival of Candida-infected mice; however, no correlation was found between suppression of the DTH response and the course of lethal infection. This suggested that DTH was not protective against lethal disease with this organism. UV radiation also changed the persistence of the organism in the internal organs. UV-irradiated, infected animals had increased numbers of Candida in their kidneys compared to non-irradiated mice. Sensitization prior to UV irradiation aided clearance of the organism from the kidneys of UV-irradiated mice.^ These data show that UV radiation suppresses cell-mediated immunity to Candida albicans in mice and increases mortality of Candida-infected mice. Moreover, the data suggest that an increase in environmental UV radiation could increase the severity of pathogenic infections. ^

Contrast agents and ionization with respect to safety for patients and doctors

In hemodialysis patients, radiographic imaging with iodinated contrast medium (ICM) application plays a central role in the diagnosis and/or follow-up of disease-related conditions. Therefore, safety aspects concerning ICM administration and radiation exposure have a great impact on this group of patients. Current hardware and software improvements including the design and synthesis of modern contrast compounds allow the use of very small amounts of ICM in concert with low radiation exposure. Undesirable ICM side effects are divided into type A (predictable reactions such as heat feeling, headache, and contrast-induced acute kidney injury, for example) and type B (nonpredictable or hypersensitivity) reactions; this chapter deals with the latter. The first onset cannot be prevented. To prevent hypersensitivity upon reexposure of ICM, an allergological workup is recommended. If this is not possible and ICM is necessary, the patient should receive a premedication (H1 antihistamine with or without corticosteroids). Current imaging hardware and software improvements (e.g. such as additional filtration of the X-ray beam) allow the use of very small amount of ICM and small X-ray doses. Proper communication among the team involved in the treatment of a patient may allow to apply imaging protocols and efficient imaging strategies limiting radiation exposure to a minimum. Practical recommendations will guide the reader how to use radiation and ICM efficiently to improve both patient and staff safety.

TUMOR PROGRESSION: ANALYSIS OF THE INSTABILITY OF THE METASTATIC PHENOTYPE, SENSITIVITY TO RADIATION AND CHEMOTHERAPY (PHENOTYPIC DRIFT, BREAST CANCER)

The major complications for tumor therapy are (i) tumor spread (metastasis); (ii) the mixed nature of tumors (heterogeneity); and (iii) the capacity of tumors to evolve (progress). To study these tumor characteristics, the rat 13762NF mammary adenocarcinoma was cloned and studied for metastatic properties and sensitivities to therapy (chemotherapy, radiation and hyperthermia). The cell clones were heterogeneous and no correlation between metastatic potential and therapeutic sensitivities was observed. Further, these phenotypes were unstable during passage in vitro; yet, the changes were clone dependent and reproducible using different cryoprotected cell stocks. To understand the phenotypic instability, subclones were isolated from low and high passage cell clones. Each subclone possessed a unique composite phenotype. Again, no apparent correlation was seen between metastatic potential and sensitivity to therapy. The results demonstrated that (1) tumor cells are heterogeneous for multiple phenotypes; (2) tumor cells are unstable for multiple phenotypes; (3) the magnitude, direction and time of occurrence of phenotypic drift is clone dependent; (4) the sensitivity of cell clones to ionizing radiation (gamma or heat) and chemotherapy agents is independent of their metastatic potential; (5) shifts in metastatic potential and sensitivity to therapy may occur simultaneously but are not linked; and (6) tumor cells independently diverge to form several subpopulations with unique phenotypic profiles. ^

Modification of mutagen sensitivity by chemopreventive agents {\it in vitro\/}

The clinical application of chemopreventive agents is expected to prevent the appearance of cancer by arresting carcinogenesis or reversing it in the precancerous stages. The hypothesis of the present investigations was that chemopreventive agents (retinoids and antioxidant vitamins) may counteract the clastogenic effects of bleomycin in vitro in both lymphoblastoid cell lines and primary lymphocyte cultures and that a similar phenomenon can be detected in lymphocytes from individuals treated with 13-cis-retinoic acid. The efficacy of 13-cis-retinoic acid, n-(4-hydroxyphenyl)-retinamide, ascorbic acid, n-acetyl-l-cysteine, alpha-tocopherol, and alpha-tocopherol-acid succinate was tested against bleomycin-induced chromosomal breakage.^ The results provided direct evidence of the concentration-related protective effects of these agents against bleomycin-induced clastogenicity in cultures of human lymphoblastoid cell lines in vitro. Similar anticlastogenic protection was demonstrated with 13-cis-retinoic acid, ascorbic acid, n-acetyl-l-cysteine, and alpha-tocopherol-acid succinate in primary lymphocyte cultures in vitro. The in vitro anticlastogenic effect of 13-cis-retinoic acid was also demonstrated in lymphocyte cultures from peripheral blood samples from patients treated with this retinoid.^ An important consideration is that the concentrations used in the present investigations are comparable to those achieved in clinical situations.^ The in vitro anticlastogenic effect of these retinoids and antioxidants may constitute an important element of their chemopreventive properties. The results corroborate the hypothesis that these compounds may be effective in clinical chemoprevention trials. The bleomycin-assay may also be used as a short-term test to evaluate the antimutagenic effects of various agents. ^

Intrinsic oxidative stress in cancer cells: A biochemical basis for therapeutic selectivity using ROS -generating agents

A major goal of chemotherapy is to selectively kill cancer cells while minimizing toxicity to normal cells. Identifying biological differences between cancer and normal cells is essential in designing new strategies to improve therapeutic selectivity. Superoxide dismutases (SOD) are crucial antioxidant enzymes required for the elimination of superoxide (O2·− ), a free radical produced during normal cellular metabolism. Previous studies in our laboratory demonstrated that 2-methoxyestradiol (2-ME), an estradiol derivative, inhibits the function of SOD and selectively kills human leukemia cells without exhibiting significant cytotoxicity in normal lymphocytes. The present work was initiated to examine the biochemical basis for the selective anticancer activity of 2-ME. Investigations using two-parameter flow cytometric analyses and ROS scavengers established that O2·− is a primary and essential mediator of 2-ME-induced apoptosis in cancer cells. In addition, experiments using SOD overexpression vectors and SOD knockout cells found that SOD is a critical target of 2-ME. Importantly, the administration of 2-ME resulted in the selective accumulation of O 2·− and apoptosis in leukemia and ovarian cancer cells. The preferential activity of 2-ME was found to be due to increased intrinsic oxidative stress in these cancer cells versus their normal counterparts. This intrinsic oxidative stress was associated with the upregulation of the antioxidant enzymes SOD and catalase as a mechanism to cope with the increase in ROS. Furthermore, oxygen consumption experiments revealed that normal lymphocytes decrease their respiration rate in response to 2-ME-induced oxidative stress, while human leukemia cells seem to lack this regulatory mechanism. This leads to an uncontrolled production of O2·−, severe accumulation of ROS, and ultimately ROS-mediated apoptosis in leukemia cells treated with 2-ME. The biochemical differences between cancer and normal cells identified here provide a basis for the development of drug combination strategies using 2-ME with other ROS-generating agents to enhance anticancer activity. The effectiveness of such a combination strategy in killing cancer cells was demonstrated by the use of 2-ME with agents/modalities such as ionizing radiation and doxorubicin. Collectively, the data presented here strongly suggests that 2-ME may have important clinical implications for the selective killing of cancer cells. ^

Protective long-term antibody memory by antigen-driven and T help-dependent differentiation of long-lived memory B cells to short-lived plasma cells independent of secondary lymphoid organs

Memory is a hallmark of immunity. Memory carried by antibodies is largely responsible for protection against reinfection with most known acutely lethal infectious agents and is the basis for most clinically successful vaccines. However, the nature of long-term B cell and antibody memory is still unclear. B cell memory was studied here after infection of mice with the rabies-like cytopathic vesicular stomatitis virus, the noncytopathic lymphocytic choriomeningitis virus (Armstrong and WE), and after immunization with various inert viral antigens inducing naive B cells to differentiate either to plasma cells or memory B cells in germinal centers of secondary lymphoid organs. The results show that in contrast to very low background levels against internal viral antigens, no significant neutralizing antibody memory was observed in the absence of antigen and suggest that memory B cells (i) are long-lived in the absence of antigen, nondividing, and relatively resistant to irradiation, and (ii) must be stimulated by antigen to differentiate to short-lived antibody-secreting plasma cells, a process that is also efficient in the bone marrow and always depends on radiosensitive, specific T help. Therefore, for vaccines to induce long-term protective antibody titers, they need to repeatedly provide, or continuously maintain, antigen in minimal quantities over a prolonged time period in secondary lymphoid organs or the bone marrow for sufficient numbers of long-lived memory B cells to mature to short-lived plasma cells.

Hypersensitivity of Ku80-deficient cell lines and mice to DNA damage: The effects of ionizing radiation on growth, survival, and development

We recently have shown that mice deficient for the 86-kDa component (Ku80) of the DNA-dependent protein kinase exhibit growth retardation and a profound deficiency in V(D)J (variable, diversity, and joining) recombination. These defects may be related to abnormalities in DNA metabolism that arise from the inability of Ku80 mutant cells to process DNA double-strand breaks. To further characterize the role of Ku80 in DNA double-strand break repair, we have generated embryonic stem cells and pre-B cells and examined their response to ionizing radiation. Ku80−/− embryonic stem cells are more sensitive than controls to γ-irradiation, and pre-B cells derived from Ku80 mutant mice display enhanced spontaneous and γ-ray-induced apoptosis. We then determined the effects of ionizing radiation on the survival, growth, and lymphocyte development in Ku80-deficient mice. Ku80−/− mice display a hypersensitivity to γ-irradiation, characterized by loss of hair pigmentation, severe injury to the gastrointestinal tract, and enhanced mortality. Exposure of newborn Ku80−/− mice to sublethal doses of ionizing radiation enhances their growth retardation and results in the induction of T cell-specific differentiation. However, unlike severe combined immunodeficient mice, radiation-induced T cell development in Ku80−/− mice is not accompanied by extensive thymocyte proliferation. The response of Ku80-deficient cell lines and mice to DNA-damaging agents provides important insights into the role of Ku80 in growth regulation, lymphocyte development, and DNA repair.

Ambient UV-B radiation causes deformities in amphibian embryos

There has been a great deal of recent attention on the suspected increase in amphibian deformities. However, most reports of amphibian deformities have been anecdotal, and no experiments in the field under natural conditions have been performed to investigate this phenomenon. Under laboratory conditions, a variety of agents can induce deformities in amphibians. We investigated one of these agents, UV-B radiation, in field experiments, as a cause for amphibian deformities. We monitored hatching success and development in long-toed salamanders under UV-B shields and in regimes that allowed UV-B radiation. Embryos under UV-B shields had a significantly higher hatching rate and fewer deformities, and developed more quickly than those exposed to UV-B. Deformities may contribute directly to embryo mortality, and they may affect an individual’s subsequent survival after hatching.

The stress response to ionizing radiation involoves c-Abl-dependent phosphorylation of SHPTP1.

c-Abl is a nonreceptor tyrosine kinase that is activated by certain DNA-damaging agents. The present studies demonstrate that nuclear c-Abl binds constitutively to the protein tyrosine phosphatase SHPTP1. Treatment with ionizing radiation is associated with c-Abl-dependent tyrosine phosphorylation of SHPTP1. The results demonstrate that the SH3 domain of c-Abl interacts with a WPDHGVPSEP motif (residues 417-426) in the catalytic domain of SHPTP1 and that c-Abl phosphorylates C terminal Y536 and Y564 sites. The functional significance of the c-Abl-SHPTP1 interaction is supported by the demonstration that, like c-Abl, SHPTP1 regulates the induction of Jun kinase activity following DNA damage. These findings indicate that SHPTP1 is involved in the response to genotoxic stress through a c-Abl-dependent mechanism.

Synergism between UV-B radiation and a pathogen magnifies amphibian embryo mortality in nature.

Previous research has shown that amphibians have differential sensitivity to ultraviolet-B (UV-B) radiation. In some species, ambient levels of UV-B radiation cause embryonic mortality in nature. The detrimental effects of UV-B alone or with other agents may ultimately affect amphibians at the population level. Here, we experimentally demonstrate a synergistic effect between UV-B radiation and a pathogenic fungus in the field that increases the mortality of amphibian embryos compared with either factor alone. Studies investigating single factors for causes of amphibian egg mortality or population declines may not reveal the complex factors involved in declines.

Transfection of the human heme oxygenase gene into rabbit coronary microvessel endothelial cells: protective effect against heme and hemoglobin toxicity.

Heme oxygenase (HO) is a stress protein and has been suggested to participate in defense mechanisms against agents that may induce oxidative injury such as metals, endotoxin, heme/hemoglobin, and various cytokines. Overexpression of HO in cells might therefore protect against oxidative stress produced by certain of these agents, specifically heme and hemoglobin, by catalyzing their degradation to bilirubin, which itself has antioxidant properties. We report here the successful in vitro transfection of rabbit coronary microvessel endothelial cells with a functioning gene encoding the human HO enzyme. A plasmid containing the cytomegalovirus promoter and the human HO cDNA complexed to cationic liposomes (Lipofectin) was used to transfect rabbit endothelial cells. Cells transfected with human HO exhibited an approximately 3.0-fold increase in enzyme activity and expressed a severalfold induction of human HO mRNA as compared with endogenous rabbit HO mRNA. Transfected and nontransfected cells expressed factor VIII antigen and exhibited similar acetylated low-density lipoprotein uptake (two important features that characterize endothelial cells) with > 85% of cells staining positive for each marker. Moreover, cells transfected with the human HO gene acquired substantial resistance to toxicity produced by exposure to recombinant hemoglobin and heme as compared with nontransfected cells. The protective effect of HO overexpression against heme/hemoglobin toxicity in endothelial cells shown in these studies provides direct evidence that the inductive response of human HO to such injurious stimuli represents an important tissue adaptive mechanism for moderating the severity of cell damage produced by these blood components.

Genetically attenuated, P36p-deficient malarial sporozoites induce protective immunity and apoptosis of infected liver cells.

Immunization with Plasmodium sporozoites that have been attenuated by gamma-irradiation or specific genetic modification can induce protective immunity against subsequent malaria infection. The mechanism of protection is only known for radiation-attenuated sporozoites, involving cell-mediated and humoral immune responses invoked by infected hepatocytes cells that contain long-lived, partially developed parasites. Here we analyzed sporozoites of Plasmodium berghei that are deficient in P36p (p36p(-)), a member of the P48/45 family of surface proteins. P36p plays no role in the ability of sporozoites to infect and traverse hepatocytes, but p36p(-) sporozoites abort during development within the hepatocyte. Immunization with p36p(-) sporozoites results in a protective immunity against subsequent challenge with infectious wild-type sporozoites, another example of a specifically genetically attenuated sporozoite (GAS) conferring protective immunity. Comparison of biological characteristics of p36p(-) sporozoites with radiation-attenuated sporozoites demonstrates that liver cells infected with p36p(-) sporozoites disappear rapidly as a result of apoptosis of host cells that may potentiate the immune response. Such knowledge of the biological characteristics of GAS and their evoked immune responses are essential for further investigation of the utility of an optimized GAS-based malaria vaccine.

Manual of protective action guides and protective actions for nuclear incidents.

"EPA 520/1-75-001-A"--Cover.

Evaluation of occupational hazards from industrial radiation : a survey of selected states /

"NIOSH technical information."