Radiation-induced Stress Response Research Articles

Physiological and Psychological Stress of Microwave Radiation-Induced Cardiac Injury in Rats

Electromagnetic waves are widely used in both military and civilian fields, which could cause long-term and high-power exposure to certain populations and may pose a health hazard. The aim of this study was to simulate the long-term and high-power working environment of workers using special electromagnetic radiation occupations to clarify the radiation-induced stress response and cardiac damage and thus gain insights into the mechanisms of injuries caused by electromagnetic radiation. In this study, the combination of microwave and stress was an innovative point, aiming to broaden the research direction with regard to the effect and mechanism of cardiac injury caused by radiation. The myocardial structure was observed by optical and transmission electron microscope, mitochondrial function was detected by flow cytometry, oxidative-stress markers were detected by microplate reader, serum stress hormone was detected by radioimmunoassay, and heart rate variability (HRV) was analyzed by multichannel-physiological recorder. The rats were weighed and subjected to an open field experiment. Western blot (WB) and immunofluorescence (IF) were used to detect the expressions and distributions of JNK (c-Jun N-terminal kinase), p-JNK (phosphorylated c-Jun N-terminal kinase), HSF1 (heat shock factor), and NFATc4 (nuclear factor of activated T-cell 4). This study found that radiation could lead to the disorganization, fragmentation, and dissolution of myocardial fibers, severe mitochondrial cavitation, mitochondrial dysfunction, oxidative-stress injury in myocardium, increase to stress hormone in serum, significant changes in HRV, and a slow gain in weight. The open field experiment indicated that the rats experienced anxiety and depression and had decreased exercise capacity after radiation. The expressions of JNK, p-JNK, HSF1, and NFATc4 in myocardial tissue were all increased. The above results suggested that 30 mW/cm2 of S-band microwave radiation for 35 min could cause both physiological and psychological stress damage in rats; the damage was related to the activation of the JNK pathway, which provided new ideas for research on protection from radiation.

P300 promotes tumor recurrence by regulating radiation-induced conversion of glioma stem cells to vascular-like cells

Glioma stem cells (GSC) exhibit plasticity in response to environmental and therapeutic stress leading to tumor recurrence, but the underlying mechanisms remain largely unknown. Here, we employ single-cell and whole transcriptomic analyses to uncover that radiation induces a dynamic shift in functional states of glioma cells allowing for acquisition of vascular endothelial-like and pericyte-like cell phenotypes. These vascular-like cells provide trophic support to promote proliferation of tumor cells, and their selective depletion results in reduced tumor growth post-treatment in vivo. Mechanistically, the acquisition of vascular-like phenotype is driven by increased chromatin accessibility and H3K27 acetylation in specific vascular genes allowing for their increased expression post-treatment. Blocking P300 histone acetyltransferase activity reverses the epigenetic changes induced by radiation and inhibits the adaptive conversion of GSC into vascular-like cells and tumor growth. Our findings highlight a role for P300 in radiation-induced stress response, suggesting a therapeutic approach to prevent glioma recurrence.

Non-thermal near-infrared exposure photobiomodulates cellular responses to ionizing radiation in human full thickness skin models

Ionizing and near-infrared radiation are both part of the therapeutic spectrum in cancer treatment. During cancer therapy ionizing radiation is typically used for non-invasive reduction of malignant tissue, while near-infrared photobiomodulation is utilized in palliative medical approaches, e.g. for pain reduction or impairment of wound healing. Furthermore, near-infrared is part of the solar wavelength spectrum. A combined exposure of these two irradiation qualities – either intentionally during medical treatment or unintentionally due to solar exposure – is therefore presumable for cancer patients. Several studies in different model organisms and cell cultures show a strong impact of near-infrared pretreatment on ionizing radiation-induced stress response. To investigate the risks of non-thermal near-infrared (NIR) pretreatment in patients, a human in vitro full thickness skin models (FTSM) was evaluated for radiation research. FTSM were pretreated with therapy-relevant doses of NIR followed by X-radiation, and then examined for DNA-double-strand break (DSB) repair, cell proliferation and apoptosis. Double-treated FTSM revealed a clear influence of NIR on X-radiation-induced stress responses in cells in their typical tissue environment. Furthermore, over a 24h time period, double-treated FTSM presented a significant persistence of DSBs, as compared to samples exclusively irradiated by X-rays. In addition, NIR pretreatment inhibited apoptosis induction of integrated fibroblasts, and counteracted the radiation-induced proliferation inhibition of basal keratinocytes. Our work suggests that cancer patients treated with X-rays should be prevented from uncontrolled NIR irradiation. On the other hand, controlled double-treatment could provide an alternative therapy approach, exposing the patient to less radiation.

Radiotherapy-induced plasticity of prostate cancer mobilizes stem-like non-adherent, Erk signaling-dependent cells

Fractionated ionizing radiation combined with surgery or hormone therapy represents the first-choice treatment for medium to high-risk localized prostate carcinoma. One of the main reasons for the failure of radiotherapy in prostate cancer is radioresistance and further dissemination of surviving cells. In this study, exposure of four metastasis-derived human prostate cancer cell lines (DU145, PC-3, LNCaP and 22RV1) to clinically relevant daily fractions of ionizing radiation (35 doses of 2 Gy) resulted in generation of two radiation-surviving populations: adherent senescent-like cells expressing common senescence-associated markers and non-adherent anoikis-resistant stem cell-like cells with active Notch signaling and expression of stem cell markers CD133, Oct-4, Sox2 and Nanog. While a subset of the radiation-surviving adherent cells resumed proliferation shortly after completion of the irradiation regimen, the non-adherent cells started to proliferate only on their reattachment several weeks after the radiation-induced loss of adhesion. Like the parental non-irradiated cells, radiation-surviving re-adherent DU145 cells were tumorigenic in immunocompromised mice. The radiation-induced loss of adhesion was dependent on expression of Snail, as siRNA/shRNA-mediated knockdown of Snail prevented cell detachment. On the other hand, survival of the non-adherent cells required active Erk signaling, as chemical inhibition of Erk1/2 by a MEK-selective inhibitor or Erk1/2 knockdown resulted in anoikis-mediated death in the non-adherent cell fraction. Notably, whereas combined inhibition of Erk and PI3K-Akt signaling triggered cell death in the non-adherent cell fraction and blocked proliferation of the adherent population of the prostate cancer cells, such combined treatment had only marginal if any impact on growth of control normal human diploid cells. These results contribute to better understanding of radiation-induced stress response and heterogeneity of human metastatic prostate cancer cells, document treatment-induced plasticity and phenotypically distinct cell subsets, and suggest the way to exploit their differential sensitivity to radiosensitizing drugs in overcoming radioresistance.

Amelioration of radiation-induced oral cavity mucositis and distant bone marrow suppression in fanconi anemia Fancd2-/- (FVB/N) mice by intraoral GS-nitroxide JP4-039.

The altered DNA damage response pathway in patients with Fanconi anemia (FA) may increase the toxicity of clinical radiotherapy. We quantitated oral cavity mucositis in irradiated Fanconi anemia Fancd2(-/-) mice, comparing this to Fancd2(+/-) and Fancd2(+/+) mice, and we measured distant bone marrow suppression and quantitated the effect of the intraoral radioprotector GS-nitroxide, JP4-039 in F15 emulsion. We found that FA mice were more susceptible to radiation injury and that protection from radiation injury by JP4-039/F15 was observed at all radiation doses. Adult 10-12-week-old mice, of FVB/N background Fancd2(-/-), Fancd2(+/-) and Fancd2(+/+) were head and neck irradiated with 24, 26, 28 or 30 Gy (large fraction sizes typical of stereotactic radiosurgery treatments) and subgroups received intraoral JP4-039 (0.4 mg/mouse in 100 μL F15 liposome emulsion) preirradiation. On day 2 or 5 postirradiation, mice were sacrificed, tongue tissue and femur marrow were excised for quantitation of radiation-induced stress response, inflammatory and antioxidant gene transcripts, histopathology and assay for femur marrow colony-forming hematopoietic progenitor cells. Fancd2(-/-) mice had a significantly higher percentage of oral mucosal ulceration at day 5 after 26 Gy irradiation (59.4 ± 8.2%) compared to control Fancd2(+/+) mice (21.7 ± 2.9%, P = 0.0063). After 24 Gy irradiation, Fancd2(-/-) mice had a higher oral cavity percentage of tongue ulceration compared to Fancd2(+/+) mice irradiated with higher doses of 26 Gy (P = 0.0123). Baseline and postirradiation oral cavity gene transcripts were altered in Fancd2(-/-) mice compared to Fancd2(+/+) controls. Fancd2(-/-) mice had decreased baseline femur marrow CFU-GM, BFUe and CFU-GEMM, which further decreased after 24 or 26 Gy head and neck irradiation. These changes were not seen in head- and neck-irradiated Fancd2(+/+) mice. In radiosensitive Fancd2(-/-) mice, biomarkers of both local oral cavity and distant marrow radiation toxicity were ameliorated by intraoral JP4-039/F15. We propose that Fancd2(-/-) mice are a valuable radiosensitive animal model system, which can be used to evaluate potential radioprotective agents.

Radiation-induced stress response in peripheral blood of breast cancer patients differs between patients with severe acute skin reactions and patients with no side effects to radiotherapy

The aim of the study was to compare the radiation-induced oxidative stress response in blood samples from breast cancer patients that developed severe acute skin reactions during the radiotherapy, with the response in blood samples from patients with no side effects. Peripheral blood was collected from 12 breast cancer patients showing no early skin reactions after radiotherapy (RTOG grade 0) and from 14 breast cancer patients who developed acute severe skin reactions (RTOG grade 3–4). Whole blood was irradiated with 0, 5 and 2000mGy γ-radiation and serum was isolated. The biomarker for oxidative stress, 8-oxo-dG, was analyzed in the serum by a modified ELISA. While a significant radiation-induced increase of serum 8-oxo-dG levels was observed in serum of the RTOG 0 patients, no increase was seen in serum of the RTOG 3–4 patients. The radiation induced increase in serum 8-oxo-dG levels after 5mGy did not differ significantly from the increase observed for 2000mGy in the RTOG 3–4 cohort, thus no dose response relation was observed. A receiver operating characteristic (ROC) value of 0.97 was obtained from the radiation-induced increase in 8-oxo-dG indicating that the assay could be used to identify patients with severe acute adverse reactions to radiotherapy. The results show that samples of whole blood from patients, classified as highly radiosensitive (RTOG 3–4) based on their skin reactions to radiotherapy, differ significantly in their oxidative stress response to ionizing radiation compared to samples of whole blood from patients with no skin reactions (RTOG 0). Extracellular 8-oxo-dG is primarily a biomarker of nucleotide damage and the results indicate that the patients with severe acute skin reactions differ in their cellular response to ionizing radiation at the level of induction of oxidative stress or at the level of repair or both.

Functional Proteomics Analysis to Study ATM Dependent Signaling in Response to Ionizing Radiation

Ataxia telangiectasia (AT) is a human genetic disease characterized by radiation sensitivity, impaired neuronal development and predisposition to cancer. Using a genetically defined model cell system consisting of cells expressing a kinase dead or a kinase proficient ATM gene product, we previously reported systemic alterations in major metabolic pathways that translate at the gene expression, protein and small molecule metabolite levels. Here, we report ionizing radiation induced stress response signaling arising from perturbations in the ATM gene, by employing a functional proteomics approach. Functional pathway analysis shows robust translational and post-translational responses under ATM proficient conditions, which include enrichment of proteins in the Ephrin receptor and axonal guidance signaling pathways. These molecular networks offer a hypothesis generating function for further investigations of cellular stress responses.

Radiation Survivors: Understanding and Exploiting the Phenotype following Fractionated Radiation Therapy

Radiation oncology modalities such as intensity-modulated and image-guided radiation therapy can reduce the high dose to normal tissue and deliver a heterogeneous dose to tumors, focusing on areas deemed at highest risk for tumor persistence. Clinical radiation oncology produces daily doses ranging from 1 to 20 Gy, with tissues being exposed to 30 or more daily fractions. Hypothesizing the cells that survive fractionated radiation therapy have a substantially different phenotype than the untreated cells, which might be exploitable for targeting with molecular therapeutics or immunotherapy, three prostate cancer cell lines (PC3, DU145, and LNCaP) and normal endothelial cells were studied to understand the biology of differential effects of multifraction (MF) radiation of 0.5, 1, and/or 2 Gy fraction to 10 Gy total dose, and a single dose of 5 and 10 Gy. The resulting changes in mRNA, miRNA, and phosphoproteome were analyzed. Significant differences were observed in the MF radiation exposures including those from the 0.5 Gy MF that produces little cell killing. As expected, p53 function played a major role in response. Pathways modified by MF include immune response, DNA damage, cell-cycle arrest, TGF-β, survival, and apoptotic signal transduction. The radiation-induced stress response will set forth a unique platform for exploiting the effects of radiation therapy as "focused biology" for cancer treatment in conjunction with molecular targeted or immunologically directed therapy. Given that more normal tissue is treated, albeit to lower doses with these newer techniques, the response of the normal tissue may also influence long-term treatment outcome.

Cellular Stress Induced Alterations in MicroRNA let-7a and let-7b Expression Are Dependent on p53

Genotoxic stressors, such as radiation, induce cellular damage that activates pre-programmed repair pathways, some of which involve microRNAs (miRNA) that alter gene expression. The let-7 family of miRNA regulates multiple cellular processes including cell division and DNA repair pathways. However, the role and mechanism underlying regulation of let-7 genes in response to stress have yet to be elucidated. In this study we demonstrate that let-7a and let-7b expression decreases significantly following exposure to agents that induce stress including ionizing radiation. This decrease in expression is dependent on p53 and ATM in vitro and is not observed in a p53−/− colon cancer cell line (HCT116) or ATM−/− human fibroblasts. Chromatin Immunoprecipitation (ChIP) analysis showed p53 binding to a region upstream of the let-7 gene following radiation exposure. Luciferase transient transfections demonstrated that this p53 binding site is necessary for radiation-induced decreases in let-7 expression. A radiation-induced decrease in let-7a and let-7b expression is also observed in radiation-sensitive tissues in vivo and correlates with altered expression of proteins in p53-regulated pro-apoptotic signaling pathways. In contrast, this decreased expression is not observed in p53 knock-out mice suggesting that p53 directly repress let-7 expression. Exogenous expression of let-7a and let-7b increased radiation-induced cytotoxicity in HCT116 p53+/+ cells but not HCT116 p53−/− cells. These results are the first demonstration of a mechanistic connection between the radiation-induced stress response and the regulation of miRNA and radiation-induced cytotoxicity and suggest that this process may be a molecular target for anticancer agents.

Differential oxidative stress gene expression profile in mouse brain after proton exposure

Radiation is known to potentially interfere with cellular functions at all levels of cell organization. The radiation-induced stress response is very complex and involves altered expression of many genes. Identification of specific genes may allow the determination of pathways important in radiation responses. Although several radiation-related research have been studied extensively, the molecular and cellular processes affected by proton exposure remain poorly understood. Our earlier reports have shown that proton radiation induces reactive oxygen species (ROS) formation and lipid peroxidation and inhibits antioxidants, superoxide dismutase, and glutathione. Therefore, in this present study, we used quantitative real-time reverse transcription polymerase chain reaction approach and showed the modulation of several genes including oxidative stress, antioxidants defense mechanism, ROS metabolism, and oxygen transporters related genes expression in 2-Gy proton-exposed mouse brain. Literature evidences suggest that change in oxidants and antioxidants levels induce DNA damage, followed by cell death. In conclusion, changes in the gene profile of mouse brain after proton irradiation are complex and the exposed cells might undergo programmed cell death through alteration of genes responsible for oxidative stress signaling mechanism.

Transcriptional modulation of micro-RNA in human cells differing in radiation sensitivity

Purpose: The molecular basis of gene regulation in cells exposed to ionising radiation is not fully understood. Gene regulation occurs at the transcriptional and post-transcriptional levels. Recent studies have suggested that micro-RNA (miRNA) plays a significant role at the post-transcriptional gene regulation. miRNA are a recently identified class of RNA molecules 18–24 nucleotides in length that have been shown to negatively regulate the stability or translation of target messenger RNA. We hypothesised that ionising radiation induced stress response is controlled in part by miRNA and that a difference in tumour protein 53 (p53) status corresponds with altered expression in miRNA responses to ionising radiation.Materials and methods: To test this hypothesis, we investigated the relative expression of several miRNA by quantitative real-time polymerase chain reaction (QPCR) in human cell lines TK6 and WTK1 that differ in p53 status and radiosensitivity after exposure to high and low doses of X-radiation.Results: The suitability of several endogenous miRNA controls was tested for relative quantification by QPCR. The baseline expression of 21 miRNA targets in TK6 and WTK1 cells indicated a wide range of modulation between the two cell lines without exposure to ionising radiation. Differences in the relative expression of miRNA were observed among the two cell lines after radiation treatment. The expression patterns of many miRNA markedly differed within the same cell line after exposure to either 0.5 Gy or 2 Gy doses of X-rays. The expression of eight miRNA belonging to the lethal-7 (let-7) family, which are negative regulators of the rat sarcoma, RAS oncogene, was upregulated in irradiated TK6 cells but was downregulated in WTK1 cells. Alterations in the myelocytomatosis oncogene, c-MYC induced cluster of miRNA were also observed. The micro RNA, miR-15a and miR-16 were upregulated in 0.5 Gy-irradiated TK6 cells but were downregulated after a 2 Gy dose of X-rays. In contrast miR-15 and miR-16 were repressed in 0.5 Gy-exposed WTK1. The miR-21 was upregulated in 0.5 Gy-treated TK6 cells and its target genes programmed cell death factor 4 (hPDCD4) phosphatase and tensin homolog (hPTEN), and sprouty homolog 2 (hSPRY2) were found to be downregulated in these cells. The miR-21 was downregulated in 2 Gy-irradiated TK6 cells, and all three of its target genes were upregulated in 2 Gy-exposed TK6 cells.Conclusion: Taken together, these results establish the involvement of miRNA in radiation response and may potentially help explain the mechanisms of gene regulation in the cellular response to ionising radiation exposure.

Risks Associated with Low Doses and Low Dose Rates of Ionizing Radiation: Why Linearity May Be (Almost) the Best We Can Do

Deterministic and stochastic effects associated with high-dose ionizing radiation (x-ray) exposure have been known for almost as long as ionizing radiation itself (1–3). At lower doses, radiation risks are primarily stochastic effects, in particular, somatic effects (cancer) rather than the deterministic effects characteristic of higher-dose exposure (4–6). In contrast to deterministic effects, for stochastic effects, scientific committees generally assume that at sufficiently low doses there is a positive linear component to the dose response—that is, that there is no threshold (4–6). This does not preclude there being higher-order (eg, quadratic) powers of dose in the dose response that may be of importance at higher doses. It is on this basis that models linear (or linear-quadratic) in dose are often used to extrapolate the experience of the Japanese atomic bomb survivors (who were typically exposed at a high dose rate to moderate doses [average, 0.1 Sv]) to estimate risks from low doses and low dose rates (4–6). Most population-based cancer risk estimates are based primarily on the Japanese atomic bomb survivor Life Span Study (LSS) cohort data (4–6). However, evidence of excess risks comes from a large number of other studies as well. In the parallel editorial (7), evidence is presented for possible real (or at least “practical”) thresholds or “hormetic” (beneficial) effects of low doses of ionizing radiation. As we summarize here, and in contrast to the arguments of Tubiana et al (7), we judge that there is little epidemiologic or biologic evidence for these for cancer. The arguments are of three forms: (a) assessment of the degree of curvature in the cancer dose response within the Japanese atomic bomb survivors and other exposed groups (in particular, departure from linear or linear-quadratic curvature), (b) consistency of risks between the Japanese and other moderate- and low-dose cohorts, and (c) assessment of biologic data on mechanisms. Most of the information on radiation-induced cancer risk comes from (a) the Japanese atomic bomb survivors, (b) medically exposed populations, (c) occupationally exposed groups, and (d) environmentally exposed groups (6). In the higher-dose radiation therapy studies, where doses received are very much higher than in the LSS, sometimes in the range at which cell sterilization occurs, excess cancer risks per unit dose tend to be less than in comparable subsets of the LSS (8,9). However, as we show, risks in moderate- and low-dose medically and occupationally exposed groups are generally consistent with those in the LSS.

Real-Time PCR Analysis of Micro-RNA Expression in Ionizing Radiation-Treated Cells

The cellular response to ionizing radiation exposure is very complex and involves many pathways. A wide variety of biologic effects are induced after exposure to ionizing radiation, ranging from DNA damage processing, signal transduction, mutations, altered gene expression, cell-cycle arrest, genomic instability, and induction of carcinogenesis to cell death. To gain insight into this complex response, global alterations in the expression of genes in irradiated cells have been examined. Recent studies have provided evidence to associate micro-RNA (miRNA) with many cellular processes, including carcinogenesis, timing of cell-fate decision, apoptosis, and metabolic pathways controlling a range of events. The small noncoding miRNA are emerging as critical components in controlling the gene expression. Because miRNA target so many genes, we hypothesized that alterations in their expression may be associated with the overall response of cells to radiation treatment. To explore the role of miRNA in cellular response to ionizing radiation, we monitored the expression levels of several miRNA by employing the stem-loop real-time polymerase chain reaction in Jurkat and TK6 cells treated with gamma-radiation. The expression levels of several members of the let-7 family miRNA that functionally inhibit the mRNAs of Ras oncogenes were upregulated after ionizing radiation treatment in Jurkat cells but were downregulated in TK6 cells. The expressions of miRNA associated with MYC translocation were upregulated in both cell types. The modulation of miRNA involved in various cancers was also examined. These results provide a first glimpse to indicate the involvement of miRNA in radiation-induced stress response and will lead to functional studies dissecting the molecular details of these processes.

The Nucleotide Pool, a Target for Low-Dose γ-Ray-Induced Oxidative Stress

Oxidative stress occurs when the generation of reactive oxygen species (ROS) exceeds the cellular antioxidant capacity. The excess ROS react with and modify cellular components. Nucleic acid modifications are of principal interest because they may cause mutations. 8-Oxo-7,8-dihydro-2 -deoxyguanosine (8-oxo-dG) is a mutagenic lesion that can be formed by ROS in DNA as well as in the nucleotide pool. 8-Oxo-dG is removed from the DNA by base excision repair and from the nucleotide pool by the nucleotide sanitization enzyme hMTH1. hMTH1 hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, which is released to the extracellular environment and can serve as a marker of oxidative stress. The aim of this work was to establish the dose-response relationship for radiation-induced extracellular 8-oxo-dG and hMTH1 in the mGy range of gamma rays in three cellular model systems: human whole blood, human fibroblasts and stimulated lymphocytes. Extracellular 8-oxo-dG was analyzed with the use of an ELISA and hMTH1 by Western blotting. Our results demonstrate that low-dose ionizing radiation induces a stress response that leads to the formation of extracellular 8-oxo-dG and induction of hMTH1 in all three cellular model systems tested. This suggests that the nucleotide pool is an important target for radiation-induced stress response.

Characterization of a Radiation-Induced Stress Response Communicated in Vivo between Zebrafish

Radiation-induced communication of stress signals between rainbow trout (Oncorhynchus mykiss W) have recently been described by this group and linked to the bystander effect. This paper addresses the question of whether another totally unrelated fish species (Danio rerio L) can demonstrate the effect and also looks at attenuation of both the bystander signal, from irradiated fish, and the bystander effect, in naive fish. The data show that zebrafish produce bystander signals, and that, as with rainbow trout these can affect naïve (i.e., non-irradiated) fish placed in water with X-rayed fish or in water previously occupied by X-rayed fish. Skin explants from directly X-rayed fish still reduce HPV-G reporter cell growth 6 h after X-ray, but the bystander signal to naïve fish is lost. Twelve h after X-ray the signal is lost in X-rayed fish. The bystander effect is also attenuated if induction was by placing naïve fish in water which previously held the X-rayed fish. However, the effect is retained if induction was by placing X-rayed and naïve fish together. This suggests the signal is not retained by water for long periods of time. Individual fish data reveal unique responses by bystander fish which could indicate varying levels of sensitivity to signal strength among individuals.

Mutation of the Hematopoietic Cell Phosphatase (Hcph) Gene Is Associated with Resistance to γ-Irradiation-Induced Apoptosis in Src Homology Protein Tyrosine Phosphatase (SHP)-1-Deficient “Motheaten” Mutant Mice

To determine the role of Src homology protein tyrosine phosphatase (SHP-1) in the ionizing radiation-induced stress response, we analyzed the apoptotic response and cell cycle function in irradiated spleen cells of motheaten (me/me) mice. The defect in me/me mice has been attributed to mutations of the HCPH: gene, which encodes SHP-1. Homozygotes develop severe systemic autoimmune and inflammatory disease, whereas heterozygotes live longer and develop hematopoietic and lymphoid malignance. Spleen cells from C57BL/6 (B6)-me/me and B6-+/+ controls were analyzed after gamma-irradiation from a (137)Cs source. B6-me/me cells were significantly more resistant than B6-+/+ cells to gamma-irradiation-induced apoptosis exhibiting a higher LD(50). The defective apoptosis response of the B6-me/me cells was exhibited by T and B cells and macrophages. Of the Bcl-2 family members analyzed, a significant difference was observed in the transcription of Bax mRNA, which was up-regulated early after irradiation in B6-+/+ cells, but not B6-me/me cells. Analysis of 3,3'-dihexyloxacarbocyanine iodide revealed resistance to the gamma-irradiation-induced mitochondrial transmembrane permeability transition in the B6-me/me cells. The blocking of the cell cycle in the G(0)/G(1) phase characteristic of the irradiated B6-+/+ cells was not observed in the B6-me/me cells. There was decreased phosphorylation of p38 mitogen-activated protein kinase and increased phosphorylation of p53 from spleen cell lysates of irradiated B6-me/me mice compared with wild-type mice. These data suggest that SHP-1 plays an important role in regulation of apoptosis and cell cycle arrest after a gamma-irradiation-induced stress response.

Induction of interleukin 6 by ionizing radiation in a human epithelial cell line: control by corticosteroids

The cutaneous radiation syndrome after therapeutic or accidental exposure of human skin to ionizing radiation (IR) is accompanied by inflammatory processes which are controlled partly by proinflammatory cytokines. Besides tumour necrosis factor (TNF) alpha and interleukin (IL)1, the pluripotent cytokine IL-6 belongs to the key mediators of inflammation. So far, there are no reports about the regulation of IL-6 by IR in epidermal cells. As an in vitro model to study the effects of IR on IL-6 gene expression, we treated the human epithelial HeLa cell line with different single X-ray doses between 1 and 20 Gy. Twenty-four hours after irradiation the IL-6 secretion was dose-dependently enhanced as measured by ELISA. At the transcriptional level, a slight increase of IL-6 transcripts was already detectable 1 h after irradiation, with maximum levels at 2 h, and a decline to baseline levels between 8 and 24 h. Addition of the transcriptional inhibitor actinomycin D inhibited the inducibility of IL-6 mRNA by TPA and IR. As the IL-6 promoter contains multiple binding sites for activated glucocorticoid receptors within the 5 regulatory region, the potential modulation of IL-6 expression by the corticosteroids hydrocortisone, dexamethasone and mometasone furoate was included in our study to modify the radiationinduced stress response. All corticosteroids applied could efficiently downregulate TPA- or radiation-induced IL-6 expression on both gene expression and protein levels. Mometasone furoate, followed by dexamethasone, was found to be most effective at low concentrations (1 nm), whereas hydrocortisone had to be applied at about 100-fold higher concentrations to achieve comparable inhibition. This experimental model is aimed at understanding the molecular circuits following IR, and thus to provide a basis for the treatment of radiation effects in skin.