Radiation-induced NF-κB Activation Research Articles

Up-stream events in the nuclear factor κB activation cascade in response to sparsely ionizing radiation

Radiation is a potentially limiting factor for manned long-term space missions. Prolonged exposure to galactic cosmic rays may shorten the healthy life-span after return to Earth due to cancer induction. During the mission, a solar flare can be life threatening. For better risk estimation and development of appropriate countermeasures, the study of the cellular radiation response is necessary. Since apoptosis may be a mechanism the body uses to eliminate damaged cells, the induction by cosmic radiation of the nuclear anti-apoptotic transcription factor nuclear factor κB (NF-κB) could influence the cancer risk of astronauts exposed to cosmic radiation by improving the survival of radiation-damaged cells. In previous studies using a screening assay for the detection of NF-κB-dependent gene induction (HEK-pNF-κB-d2EGFP/Neo cells), the activation of this transcription factor by heavy ions was shown [Baumstark-Khan, C., Hellweg, C.E., Arenz, A., Meier, M.M. Cellular monitoring of the nuclear factor kappa B pathway for assessment of space environmental radiation. Radiat. Res. 164, 527–530, 2005]. Studies with NF-κB inhibitors can map functional details of the NF-κB pathway and the influence of radiation-induced NF-κB activation on various cellular outcomes such as survival or cell cycle arrest. In this work, the efficacy and cytotoxicity of four different NF-κB inhibitors, caffeic acid phenethyl ester (CAPE), capsaicin, the proteasome inhibitor MG-132, and the cell permeable peptide NF-κB SN50 were analyzed using HEK-pNF-κB-d2EGFP/Neo cells. In the recommended concentration range, only CAPE displayed considerable cytotoxicity. CAPE and capsaicin partially inhibited NF-κB activation by the cytokine tumor necrosis factor α. MG-132 completely abolished the activation and was therefore used for experiments with X-rays. NF-κB SN-50 could not reduce NF-κB dependent expression of the reporter destabilized Enhanced Green Fluorescent Protein (d2EGFP). MG-132 entirely suppressed the X-ray induced NF-κB activation in HEK-pNF-κB-d2EGFP/Neo cells. In conclusion, the degradation of the inhibitor of NF-κB (IκB) in the proteasome is essential for X-ray induced NF-κB activation, and MG-132 will be useful in studies of the NF-κB pathway involvement in the cellular response to heavy ion exposure and other space-relevant radiation qualities.

Ionizing radiation-induced NF-kappaB activation requires PARP-1 function to confer radioresistance.

Recent reports implicate poly(ADP-ribose) polymerase-1 (PARP-1) in the activation of NF-κB. We investigated the role of PARP-1 in the NF-κB signalling cascade induced by ionizing radiation (IR). AG14361, a potent PARP-1 inhibitor, was used in two breast cancer cell lines expressing different levels of constitutively activated NF-κB, as well as mouse embryonic fibroblasts (MEFs) proficient or deficient for PARP-1 or NF-κB p65. In the breast cancer cell lines, AG14361 had no effect on IR-induced degradation of IκBα or nuclear translocation of p50 or p65. However, AG14361 inhibited IR-induced NF-κB dependent transcription of a luciferase reporter gene. Similarly, in PARP-1-/- MEFs, IR-induced nuclear translocation of p50 and p65 was normal, but κB binding and transcriptional activation did not occur. AG14361 sensitized both breast cancer cell lines to IR-induced cell killing, inhibited IR-induced XIAP expression and increased caspase-3 activity. However, AG14361 failed to increase IR-induced caspase activity when p65 was knocked down by siRNA. Consistent with this, AG14361 sensitized p65+/+ but not p65-/- MEFs to IR. We conclude that PARP-1 activity is essential in the upstream regulation of IR-induced NF-κB activation. These data indicate that potentiation of IR-induced cytotoxicity by AG14361 is mediated soley by inhibition of NF-κB activation.

Increased cytotoxicity by the combination of radiation and diospyrin diethylether in fibrosarcoma in culture and in tumor

Objective: Radio-resistance in tumor cells and associated escape from apoptotic mechanism is a major problem in clinical cancer radiotherapy. Therefore, as a strategy to enhance the apoptosis, a combination of radiation and tumor-selective cytotoxic agents might improve the efficacy of treatment. Thus, the radiomodifying potential of diospyrin diethylether (D7), a plant-derived antitumor agent, was studied in fibrosarcoma tumor, both in vitro and in vivo.Material and methods: Mouse and human fibrosarcoma (Wehi164; HT1080) cells were treated with D7, alone, or in combination with radiation, for determination of cytotoxicity, clonogenic survival, and apoptotic death assays. Involvement of oxidative mechanism and nuclear factor kappa B (NF-κB) was studied in different treatment groups. In addition, fibrosarcoma-bearing mice were treated with D7 (intravenously, two doses, each of 1 mg/kg body weight) combined with radiation (two fractions of 2.5 Gy each) at appropriate intervals. The tumor volume was measured to assess ‘tumor growth delay’, and liver function enzymes in the serum of mice were estimated after the treatments.Results: A combination treatment with D7 and radiation showed enhancement in cytotoxicity and apoptotic induction and decrease in clonogenic survival of tumor cells, as compared to the treatments with the drug or radiation alone. Moreover, D7 in combination with radiation could significantly inhibit the radiation-induced NF-κB activation, and showed the generation of comparatively more intracellular reactive oxygen species (ROS). Similarly, a combination of D7 and radiation in vivo caused significant inhibition of tumor growth in vivo, and restoring the liver enzyme activity to the ‘normal’ level.Conclusion: The combined treatment with quinonoid D7 and radiation caused increased cytotoxicity compared to single treatment with either agent alone in fibrosarcoma tumor systems, both in vitro and in vivo.

Transcriptional response of human cells to microbeam irradiation with 2.1 MeV α-particles

Within the next decades, an increasing number of human beings will be brought into space to carry out technical and scientific tasks. There, they will be exposed simultaneously to combined stimuli, especially microgravity and radiation. In the endeavour to assess the risks for humans during long-duration space missions, it is necessary to understand already at the cellular level the complex interplay of these parameters. Cellular stress protection responses lead to an increased transcription of several genes via the modulation of transcription factors. The activation of the nuclear factor κB (NF-κB) pathway as a possible anti-apoptotic route represents such an important cellular stress response. It is hypothesized that the activation of NF-κB and the subsequent expression of NF-κB-dependent genes is involved in the cellular response to components of the cosmic radiation. Irradiation of human embryonic kidney cells (HEK/293) with α-particles (2.1 MeV, LET ∼160 keV/μm) was performed at the PTB, Braunschweig, Germany. Using the microbeam facility, cells were exposed to nuclear hits or, for the purpose of comparison, to a diffuse irradiation of the whole cell. After irradiation the following biological endpoints were determined: (i) cell survival (by means of the colony forming ability test), and (ii) quantitative RT-PCR analysis of selected NF-κB target genes (IκBα GADD45β, bcl-2, and bcl-X L). One nuclear α-particle traversal reduces the probability to survive to ∼75%. Exposure to two α-particles per nucleus resulted in an upregulation of the expression of the GADD45β gene. After exposure of HEK cells to five nuclear hits, about 43% of the irradiated cells survived, and the transcriptional response was not significant. Ten nuclear hits activated the IκBα expression, this increased IκBα production might be involved in the termination of the radiation-induced NF-κB activation. Diffuse irradiation increased the transcription of IκBα and GADD45β. Expression of the antiapoptotic genes bcl-2 and bcl-X L remained almost unchanged after α-particle irradiation. These findings suggest a role for GADD45β in the cellular radiation response to low fluences, while IκBα is enrolled at higher fluences.

Genistein inhibits radiation-induced activation of NF-κB in prostate cancer cells promoting apoptosis and G2/M cell cycle arrest

BackgroundNew cancer therapeutic strategies must be investigated that enhance prostate cancer treatment while minimizing associated toxicities. We have previously shown that genistein, the major isoflavone found in soy, enhanced prostate cancer radiotherapy in vitro and in vivo. In this study, we investigated the cellular and molecular interaction between genistein and radiation using PC-3 human prostate cancer cells.MethodsTumor cell survival and progression was determined by clonogenic analysis, flow cytometry, EMSA analysis of NF-κB, and western blot analysis of cyclin B1, p21WAF1/Cip1, and cleaved PARP protein.ResultsGenistein combined with radiation caused greater inhibition in PC-3 colony formation compared to genistein or radiation alone. Treatment sequence of genistein followed by radiation and continuous exposure to genistein showed optimal effect. Cell cycle analysis demonstrated a significant dose- and time-dependent G2/M arrest induced by genistein and radiation that correlated with increased p21WAF1/Cip1 and decreased cyclin B1 expression. NF-κB activity was significantly decreased by genistein, yet increased by radiation. Radiation-induced activation of NF-κB activity was strongly inhibited by genistein pre-treatment. A significant and striking increase in cleaved PARP protein was measured following combined genistein and radiation treatment, indicating increased apoptosis.ConclusionA mechanism of increased cell death by genistein and radiation is proposed to occur via inhibition of NF-κB, leading to altered expression of regulatory cell cycle proteins such as cyclin B and/or p21WAF1/Cip1, thus promoting G2/M arrest and increased radiosensitivity. These findings support the important and novel strategy of combining genistein with radiation for the treatment of prostate cancer.

The suppression of radiation-induced NF-κB activity by dexamethasone correlates with increased cell death in vivo

In this study, we show that dexamethasone treatment increases ionizing radiation-induced cell death by inducing the inhibitory κBα (IκBα) pathway in mice. The effect of dexamethasone on radiation-induced cell death was assessed by changes in total spleen cellularity and bone marrow colony-forming unit–granulocyte–macrophage (CFU–GM) contents after total body irradiation. While in vivo treatment of mice with dexamethasone alone (1 mg/kg/day, for 2 days) failed to elicit cell death in spleen cells, the combined treatment with dexamethasone (1 mg/kg/day, for 2 days) and γ-rays (1 or 5 Gy) caused a 50–80% reduction in total cellularity in spleen and CFU–GM contents in bone marrow. These results demonstrate that dexamethasone has a synergistic effect on radiation-induced cellular damages in vivo. Immunoblot analysis showed that dexamethasone treatment significantly increases IκBα expression in the spleens of irradiated mice. In addition, the dexamethasone treatment significantly reduced radiation-induced nuclear translocation of the nucleus factor-κB in the spleens of irradiated mice. These results indicate that dexamethasone treatment in vivo may increase radiation-induced cell damages by increasing IκBα expression in hematopoietic organs such as spleen and bone marrow.

Inhibition of constitutively activated nuclear factor-κB radiosensitizes human melanoma cells

Abstract Melanoma tumors and cultured cell lines are relatively resistant to the cytotoxic effects of ionizing radiation, thereby limiting the use of radiotherapy for the clinical treatment of melanoma. New strategies for sensitizing melanoma cells therefore deserve examination. In an attempt to identify and target signaling pathways that contribute to radioresistance, we investigated the role of nuclear factor-κB (NF-κB), a transcription factor known to inhibit apoptosis induced by a variety of stimuli and promote radioresistance. Two human metastatic melanoma cell lines, A375 and MeWo, were used to examine the radiosensitizing effects of inhibitors of the NF-κB pathway. Nuclear extracts from these cell lines were tested for active NF-κB using the electrophoretic mobility shift assay. Both melanoma cell lines had constitutively activated NF-κB as observed by electrophoretic mobility shift assay. In an attempt to reverse NF-κB activity, cells were treated either with vehicle alone (DMSO) or with a proteasome inhibitor Z-Leu-Leu-Leu-H (MG132; 10 μmol/L for 2 hours prior to irradiation) that inhibited both constitutive and radiation-induced NF-κB activity. The clonogenic cell survival assay showed that pretreatment with MG132 enhanced tumor cell radiosensitivity with the survival factor at 2 Gy being reduced from 48 ± 0.8% and 48 ± 1.6% in vehicle-treated cells to 27.7 ± 0.32% and 34.3 ± 0.7% in MG132-treated MeWo and A375 cells, respectively. To test the role of NF-κB in radioresistance more directly, MeWo cells were stably transfected with a dominant-negative mutant IκBα construct, which led to the inhibition of both constitutive and radiation-induced NF-κB activity. A modest restoration of radiosensitivity was also observed in the stably transfected MeWo cells with survival factor at 2 Gy values being reduced from 47 ± 0.8% in parental MeWo cells to 32.9 ± 0.7% in stable transfectants. Because constitutively activated mitogen-activated protein kinase kinase (MEK) pathway has been shown to lead to activated NF-κB, we wanted to determine the relative contribution of activated MEK in the human melanoma cells. To test this, MeWo and A375 melanoma cells were exposed to the MEK inhibitor PD184352. Treatment with PD184352 partially reversed NF-κB activity but did not impart radiation sensitivity to these cells. Our results indicate that activated NF-κB may be one of the pathways responsible for the radioresistance of melanoma cells and that strategies for inhibiting its influence may be useful in restoring the radioresponse of melanomas.

The p65 subunit of nuclear factor-κB is a molecular target for radiation sensitization of human squamous carcinoma cells

Abstract The transcription factor nuclear factor-κB (NF-κB) is activated in response to various stimuli including ionizing radiation. Disruption of NF-κB activation by mutant forms of the NF-κB inhibitor IκB-α or by proteasome inhibitors enhances both sensitivity to radiation and radiation-induced apoptosis. Human squamous carcinoma SCC-35 cells stably expressing a fragment (residues 1 to 84) of human p65 have been shown to exhibit down-regulation of both endogenous p65 mRNA and its protein. The mutant protein also inhibited radiation-induced NF-κB activation by preventing the proteolysis of IκB-α. This resulted in enhancement of cellular radiosensitivity and radiation-induced apoptosis. The NH2-terminal region of p65 is thus a potential molecular target for disruption of NF-κB activation and sensitization of tumors to radiotherapy.

Par-4, A Pro-Apoptotic Gene, Inhibits Radiation-Induced NFκB Activity and Bcl-2 Expression Leading to Induction of Radiosensitivity in Human Prostate Cancer Cells PC-3

Ionizing radiation caused induction NFκB activity and Bcl-2 protein expression in the radioresistant p53 null human prostate cancer cell line, PC-3. Exposure of PC-3 cells to Ad5-IkB super-repressor inhibited radiation-induced Bcl-2 expression indicating that radiation-induced NFκB activity is required for the induction of Bcl-2 protein. PAR-4, a novel pro-apoptotic protein is a potent down-modulator of NFκB activity and bcl-2 protein expression. This study was undertaken to investigate the impact of PAR-4 expression on radiation-induced NFκB activity and Bcl-2 expression and its resultant radiation response in PC-3 cells. Western blot analysis indicated that enforced expression of PAR-4 in PC-3 cells down regulated radiation-induced bcl-2 protein, whereas in vector transfected cells radiation caused an induction of bcl-2 protein. In both transfectant cell lines, the bax protein levels remained unaltered after radiation. When compared to PC-3/Vector cells, PC-3/PAR-4 cells showed significant sensitivity to radiation-induced clonogenic inhibition and apoptosis. Thus, the down-regulation of bcl-2 protein by ectopic PAR-4 expression altered bcl-2: bax ratio in PC-3/PAR-4 cells and this led enhanced radiosensitivity. PAR-4 was found to inhibit the radiation-induced NFκB activity and NFκB transcriptional activity is essential for bcl-2 upregulation. In PC-3/Vector cells, radiation caused an increase in NFκB activity leading to upregulation of bcl-2 protein. However, in PC-3/PAR-4 cells, the radiation-induced NFκB activity was inhibited resulting in the transrepression of bcl-2 promoter and down-modulation of bcl-2 protein. In addition, PAR-4 was found to directly inhibit the phosphorylation and degradation of IκBa, which led to the loss of NFkB activity causing repression of endogenous and radiation-induced Bcl-2 protein. Together, these mechanisms suggest that PAR-4 is functionally required to cause radiation-induced apoptosis by abrogating the survival and anti-apoptotic effects of NFκB activity and bcl-2 function respectively.

The E1A Gene Therapy: A Two Way Translational Research

Adenovirus type 5 E1A is associated with multiple anti-tumor activities and transcriptionally represses HER2/mu. E1A gene therapy induced regression of HER-2/neu-overexpressing cancers in nude mice. To evaluate the feasibility of E1A gene therapy for patient with HER-2/neuoverexpressing cancer, we conducted a phase 1 clinical trial. An E1A/DC-Chol complex was injected weekly into the thoracic or peritoneal cavity of 18 patients with advanced breast and/or ovarian cancers. The most common toxicities were fever, nausea, vomiting, and/or discomfort at the injection sites. After E1A/DC-Chol complex delivery, E1A gene expression was detectable in tumor cells with HER-2/neu downregulation, increased apoptosis, and reduced proliferation. Thus intracavitary E1A gene therapy is a feasible approach resulting m clear proof of preclinical concept. Phase II trials on ovarian cancer are recently initiated under the support of M. D. Anderson Ovarian Spore. In addition to downregulation of HER-2/neu overexpression, we have recently identified molecular mechanisms that provide scientific basis for E1A-mediated antitumor activities independent of HER-2/neu status. These laboratory findings are helpful to understand E1A-associated anti-tumor activity and will be incorporated to the future clinical trials: 1.E1A sensitized TNF α – and ionizing γ – irradiation-induced cell death by inhibition of NF-κB activity. Activation of the transcription factor NF-κB has been shown to play a key role in the anti-apoptotic pathway of TNF α and radiation-induced apoptosis. We have found that TNF α- or radiation-induced activation of NF κB occurred m the cancer cells but was blocked when E1A was expressed. We further showed that this inhibition was through suppression of IκB kinase (IKK) activity and IκB phosphorylation. Since IκB phosphorylation results in degradation of IκB, suppression of IκB phosphorylation leads to increased IκB level that then in turn binds to NF-κB to inbibit NF-κB anti-apoptotic activity. These results open an interesting possibility to combine E1A gene therapy with radation therapy or TNF α therapy. It should also be mentioned that TNF α expression was found to be increased in the body fluid from the treated patients in the Phase 1 trail. The increased TNF α expression may account for the increased apoptosis of cancer cells in the treated patients. In this regards, the E1A/DC-Chol-induced TNF α expression in patients may contribute to its anti-tumor activity. 2.E1A induced bystander effect by inhibiting angiogenesis and inducing apoptosis. To investigate the E1A mediated bystander effect and the mechanisms that may be associated with it. We inoculated nude mice subcutaneously with a mixture of E1A transfectants and parental cells, we found that the E1A transfectants exhibited a bystander effect on inhibition of tumor growth. We further showed that E1A mediated suppression of angiogenesis and induction of apoptosis in the tumors, likely contributing to the bystander effect. 3. E1A inhibits expression of another transforming tyrosine kinase, Axl. To study the mechanism underlying the E1A-mediated tumor suppressing function, we exploited a modified tyrosine kinase profile assay to identify potential tyrosine kinase regulated by E1A. We identified that E1A negatively regulated the expression of the transforming receptor tyrosine kinase Ax1 at the transcriptional level and downregulation of Aχ1 by E1A is required for E1A-mediated growth suppression and induction of apoptosis. These results indicated that downregulation of Ax1 receptor by E1A is involved in E1A-mediated growth suppression and E1A-induced apoptosis and thereby may contribute to E1A’s antitumor activities. Although the clinical trial of E1A gene therapy was initiated by the known mechanism of HER-2/neu downregulation, the above results together with other studies in the literature, indicate that E1A actually associate with multiple mechanisms that may contribute to anti-tumor activity. These results provide scientific basis to open the E1A gene therapy clinical trials for patients without HER-2/neu-verexpressing cancer.

N-acetyl-L-cysteine inhibits 26S proteasome function: implications for effects on NF-κB activation

Ionizing radiation shares with cytokines, such as TNF-α, an ability to generate free radicals in cells and activate downstream proinflammatory responses through NF-κB-dependent signal transduction pathways. Support for the role of free radicals in triggering such responses comes from the use of free radical scavengers like N-acetyl-L-cysteine (NAC). The nature of the link between free radical generation and NF-κB activation is, however, unclear. In this study, we explore the possibility that scavenging of free radicals by NAC might not be the mechanism by which it inhibits NF-κB activation, but rather that NAC acts through inhibition of proteasome function. The effect of NAC on the chymotryptic function of the 26s and 20s proteasome complex was measured in extracts from EVC 304 bladder carcinoma cells by assessing degradation of fluorogenic substrates. NAC inhibited 26s but not 20s proteasome activity, suggesting that it interferes with 19s regulatory subunit function. NAC blocked radiation-induced NF-κB activity in ECV 304 cells and RAW 264.7 macrophages, as measured by a gel shift assay, at doses that inhibited proteasome activity. This provides a possible mechanism whereby NAC could block NF-κB activation and affect the expression of other molecules that are dependent on the ubiquitin/proteasome system for their degradation, other than by scavenging free radicals.

Indomethacin lowers the threshold thermal exposure for hyperthermic radiosensitization and heat-shock inhibition of ionizing radiation-induced activation of NF-κB

Purpose : It is well established that salicylate and several other non-steroidal anti-inflammatory agents (NSAID), including indomethacin, can activate the heat-shock response, albeit at high concentrations. This is significant since heat shock significantly alters the cellular cytotoxic response to ionizing radiation (IR). It was previously shown that heat shock, as well as NSAIDs, inhibits IR-induced activation of NF- κB and that NF- κB protects against IR-induced cytotoxicity. Hence, it is hypothesized that pretreatment with indomethacin before heating will lower the temperature and heating times required to inhibit the activation of NF- κB and induce significant hyperthermic radiosensitization. Materials and methods : Experiments were performed in HeLa cell lines and the DNA-binding activity was determined by EMSA. Cellular radiosensitivity was determined by clonogenic assay. Results : HeLa cells pretreated with indomethacin showed a decrease in the temperature-time combination necessary to inhibit IR-induction of NF- κB DNA binding. In addition, clonogenic cell survival assays using identical conditions showed an indomethacin dose-dependent enhancement of hyperthermic radiosensitization. Thus, similar concentrations of indomethacin both lowered the threshold thermal exposure to inhibit activation of NF- κB DNA-binding and increased the sensitivity of tumour cells to hyperthermic radiosensitization-induced cytotoxicity. In HeLa cells treated with N - α -tosylphenylalanyl-chloromethyl ketone (TPCK), a serine protease inhibitor that blocks activation of NF- κB, an increase in radiosensitivity was observed. Interestingly, no additional cell killing was observed when heat shock was added to cells treated with TPCK before IR, suggesting a possible common cytotoxic pathway. Conclusions : The results demonstrate that indomethacin lowers the temperature-time conbination necessary to induce several physiological processes associated with the heat-shock response. Furthermore, NSAID may be potential adjuvants in improving the clinical effectiveness of hyperthermia in radiation therapy.

Enhancement of radiosensitivity by proteasome inhibition: Implications for a role of NF-κB

Purpose: NF-κB is activated by tumor necrosis factor, certain chemotherapeutic agents, and ionizing radiation, leading to inhibition of apoptosis. NF-κB activation is regulated by phosphorylation of IκB inhibitor molecules that are subsequently targeted for degradation by the ubiquitin-proteasome pathway. PS-341 is a specific and selective inhibitor of the proteasome that inhibits NF-κB activation and enhances cytotoxic effects of chemotherapy in vitro and in vivo. The objective of this study was to determine if proteasome inhibition leads to enhanced radiation sensitivity. Methods and Materials: Inhibition of NF-κB activation in colorectal cancer cells was performed by treatment of LOVO cells with PS-341 or infection with an adenovirus encoding IκB super-repressor, a selective NF-κB inhibitor. Cells were irradiated at 0, 2, 4, 6, 8, and 10 Gy with or without inhibition of NF-κB. NF-κB activation was determined by electrophoretic mobility gel shift assay, and apoptosis was evaluated using the TUNEL assay. Growth and clonogenic survival data were obtained to assess effects of treatment on radiosensitization. In vitro results were tested in vivo using a LOVO xenograft model. Results: NF-κB activation was induced by radiation and inhibited by pretreatment with either PS-341 or IκBα super-repressor in all cell lines. Inhibition of radiation-induced NF-κB activation resulted in increased apoptosis and decreased cell growth and clonogenic survival. A 7–41% increase in radiosensitivity was observed for cells treated with PS-341 or IκBα. An 84% reduction in initial tumor volume was obtained in LOVO xenografts receiving radiation and PS-341. Conclusions: Inhibition of NF-κB activation increases radiation-induced apoptosis and enhances radiosensitivity in colorectal cancer cells in vitro and in vivo. Results are encouraging for the use of PS-341 as a radiosensitizing agent in the treatment of colorectal cancer.

Effects of NF-κB1 (p50) targeted gene disruption on ionizing radiation-induced NF-κB activation and TNFα, IL-1α, IL-1β and IL-6 mRNA expression in vivo

Purpose : To investigate the role of the NF- κB1 (p50) gene in ionizing radiation (IR)-induced NF- κB activation and TNF α, IL-1 α, IL-1 β and IL-6 mRNA expression in vivo. Materials and methods : NF- κB activation was analysed by the gel shift/supershift assay and the levels of TNF α, IL-1 α, IL-1 β and IL-6 mRNA were measured using RNase protection assay (RPA). Various tissues from BALB/c, B6,129P-Nfkb1 (NF- κB1 or p50 gene knockout, p50 -/-) and B6,129PF2 (wild-type, p50 +/+) mice were analysed before or after exposure to a lethal dose (8.5 Gy) of total-body γ-irradiation. Results : Exposure of BALB/c mice to total-body IR selectively activated NF- κB in the spleen, mesenteric lymph nodes (LN) and bone marrow (BM). Gel supershift assay using polyclonal antibodies against NF- κB p50, p65 or c-Rel protein revealed that the NF- κB p50 subunit is a critical component of the NF- κB complexes activated by IR in vivo. Discretely augmented TNF α, IL-1 α, IL-1 β and IL-6 mRNA expression was found in the spleen, LN and BM after BALB/c mice received IR. However, mice lacking the p50 gene (p50 -/-) showed a significant reduction in IR-induced activation of NF- κB and increases in TNF α, IL-1 α, IL-1 β and IL-6 mRNA expression, as compared with that of wild-type mice (p50 +/+) . Conclusions : The NF- κB p50 subunit is a critical component of the NF- κB complexes activated by IR and it plays an important role in mediating IR-induced TNF α, IL-1 α, IL-1 β and IL-6 mRNA expression in vivo.

Heat Shock Inhibits Radiation-induced Activation of NF-κB via Inhibition of I-κB Kinase

Radiation stimulates signaling cascades that result in the activation of several transcription factors that are believed to play a central role in protective response(s) to ionizing radiation (IR). It is also well established that heat shock alters the regulation of signaling cascades and transcription factors and is a potent radiosensitizing agent. To explore the hypothesis that heat disrupts or alters the regulation of signaling factors activated by IR, the effect of heat shock on IR-induced activation of NF-kappaB was determined. Irradiated HeLa cells demonstrated transient increases in NF-kappaB DNA binding activity and NF-kappaB protein nuclear localization. In addition, irradiated cells demonstrated increased I-kappaB phosphorylation and decreased I-kappaBalpha cytoplasmic protein levels, corresponding temporally with the increase of NF-kappaB DNA binding. Heat shock prior to IR inhibited the increase in NF-kappaB DNA binding activity, nuclear localization of NF-kappaB, and the phosphorylation and subsequent degradation of I-kappaB. I-kappaB kinase (IKK) immunoprecipitation assays demonstrated an increase in IKK catalytic activity in response to IR that was inhibited by pretreatment with heat. Kinetic experiments determined that heat-induced inhibition of NF-kappaB activation in response to IR decayed within 5 h after heating. Furthermore, pretreatment with cycloheximide, to block de novo protein synthesis, did not alter heat shock inhibition of IR induction of NF-kappaB. These experiments demonstrate that heat shock transiently inhibits IR induction of NF-kappaB DNA binding activity by preventing IKK activation and suggests a mechanism independent of protein synthesis.