Relevant Doses Of Ionizing Radiation Research Articles

An Effective Protocol for Proteome Analysis of Medaka (Oryzias latipes) after Acute Exposure to Ionizing Radiation

All terrestrial organisms are subject to evolutionary pressures associated with natural sources of ionizing radiation (IR). The legacy of human-induced IR associated with energy, weapons production, medicine, and research has changed the distribution and magnitude of these evolutionary pressures. To date, no study has systematically examined the effects of environmentally relevant doses of radiation exposure across an organismal proteome. This void in knowledge has been due, in part, to technological deficiencies that have hampered quantifiable environmentally relevant IR doses and sensitive detection of proteomic responses. Here, we describe a protocol that addresses both needs, combining quantifiable IR delivery with a reliable method to yield proteomic comparisons of control and irradiated Medaka fish. Exposures were conducted at the Savannah River Ecology Laboratory (SREL, in Aiken, SC), where fish were subsequently dissected into three tissue sets (carcasses, organs and intestines) and frozen until analysis. Tissue proteins were extracted, resolved by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), and each sample lane was divided into ten equal portions. Following in-gel tryptic digestion, peptides released from each gel portion were identified and quantified by Liquid Chromatography-Mass Spectrometry (LC-MS/MS) to obtain the most complete, comparative study to date of proteomic responses to environmentally relevant doses of IR. This method provides a simple approach for use in ongoing epidemiologic studies of chronic exposure to environmentally relevant levels of IR and should also serve well in physiological, developmental, and toxicological studies.

Inhibition of Parp1 by BMN673 Effectively Sensitizes Cells to Radiotherapy by Upsetting the Balance of Repair Pathways Processing DNA Double-Strand Breaks.

Parp inhibitors (Parpi) are commonly used as single agents for the management of tumors with homologous recombination repair (HRR) deficiencies, but combination with radiotherapy (RT) is not widely considered due to the modest radiosensitization typically observed. BMN673 is one of the most recently developed Parpi and has been shown to mediate strong cell sensitization to methylating agents. Here, we explore the mechanisms of BMN673 radiosensitization to killing, aiming to combine it with RT. We demonstrate markedly stronger radiosensitization by BMN673 at concentrations substantially lower (50 nmol/L) than olaparib (3 μmol/L) or AG14361 (0.4 μmol/L) and dramatically lower as compared with second-generation inhibitors such as PJ34 (5 μmol/L). Notably, BMN673 radiosensitization peaks after surprisingly short contact times (∼1 hour) and at pharmacologically achievable concentrations in vivo BMN673 exerts a complex set of effects on DNA double-strand break (DSB) processing, including inhibition of classic nonhomologous end-joining (cNHEJ) and alternative end-joining (altEJ) pathway at high doses of ionizing radiation (IR). BMN673 enhances resection at DSB and favors HRR and altEJ at low clinically relevant IR doses. The combined outcome of these effects is an abrogation in the inherent balance of DSB processing culminating in the formation of chromosomal translocations that underpin radiosensitization. Our observations pave the way to clinical trials exploring inherent benefits in combining BMN673 with RT for the treatment of various forms of cancer. Mol Cancer Ther; 17(10); 2206-16. ©2018 AACR.

Abstract B24: Development of a radiation modulator that functions via translation inhibition

Abstract Translation is essential for cellular maintenance and growth, as well as survival in response to a variety of stresses. Translation factors are often overexpressed in cancers, and their experimental inhibition has been shown to retard cancer cell growth in a variety of pre-clinical models. Further, clinically relevant forms of cellular stress (e.g. ionizing radiation, hypoxia) result in altered translational programs. Inhibition of translation following such stress results in impaired cellular recovery. Taken together these observations suggest that modulation of translation, alone or in combination with genotoxic stress, is of potential therapeutic relevance in oncology. Bouvardin (BVD) is a plant-derived protein synthesis inhibitor that we identified in a screen for enhancers of ionizing radiation (IR) in D. melanogaster. We previously demonstrated that BVD functions as a translation elongation inhibitor by blocking dissociation of elongation factor 2 (eEF2) from the ribosome. In preclinical models of head and neck cancer (HNC) and glioma, two cancer indications for which IR is a standard of care, we demonstrated that BVD enhanced clonogenic death induced by IR. Further, BVD enhanced the growth inhibitory effects of IR in HNC tumor xenografts in mice. We show here that a fully synthetic derivative of BVD, SVC112, exhibits promising therapeutic properties in tissue culture and xenograft models of HNC. Like BVD, SVC112 inhibits general de novo protein synthesis in cell culture. In a panel of 19 HNC cell lines SVC112 exhibits antiproliferative activity with an average IC50 of ~160 nM, with no IC50 exceeding 500 nM. Combinations of SVC112 and IR dose-response curves in HNC cells yield average combination-index (CI) values generally lower than 1, especially in maximally tolerated dose ranges. CI values of 1 are indicative of additive interactions, while CI values <1 are indicative of synergy between two agents. Further, in clonogenic assays physiologically achievable levels of SVC112 consistently exhibit dose modifying factors (DMF) of >1 in HNC cells, when combined with a range of clinically relevant IR doses. DMF values of 1 are indicative of additive interactions, while DMF values >1 indicate synergistic enhancement of IR mediated clonogenic death. Additionally in clonogenic assays, SVC112 enhances cisplatin-IR mediated clonogenic death. Finally, SVC112 enhances the growth inhibitory effects of IR-cisplatin treatment (alone and in combination) in HNC tumor xenografts in mice. Taken together these data demonstrate that inhibition of translation elongation in combination with existing standards of care is a potentially promising treatment option for cancers of the head and neck. *This project has been funded in whole or in part with Federal funds from the NCI/NIH, Department of Health and Human Services, under Contract No. HHSN261201300021C Citation Format: Nathan Gomes, Barb Frederick, Ryan Hansen, Gan Zhang, Daniel Gustafson, Gijsbertus Pronk, Tin Tin Su. Development of a radiation modulator that functions via translation inhibition. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B24.

Abscopal effect of low-LET γ-radiation mediated through Rel protein signal transduction in a mouse model of nontargeted radiation response

Ascertaining the ionizing radiation (IR)-induced bystander response and its preceding molecular regulation would increase our understanding of the mechanism of acute and delayed radiobiological effects. Recent evidence clearly prompted that radiation-induced nuclear factor kappa B (NF-κB) would play a key role in bystander responses in nontargeted cells. Accordingly, we investigated the orchestration of NF-κB signaling after IR in a nontargeted distant organ. Heart tissues from C57/BL6 mice either mock irradiated or exposed (limited to lower abdomen 1 cm diameter) to single-dose IR (SDR: 2 or 10 Gy) or fractionated IR (FIR, 2 Gy per day for 5 days) were examined for onset of abscopal NF-κB signal transduction, translated activity, downstream functional signaling and associated DNA damage. Radiation significantly induced NF-κB DNA binding activity in nontargeted heart. Transcriptional profiling showed that 51, 46 and 26 of 88 genes were significantly upregulated after 2 Gy, 10 Gy and FIR. Of these genes, 22 showed dose- and fractionation-independent upregulation. Immunohistochemistry revealed a robust increase in p65 and cMyc expression in distant heart after SDR and FIR. Immunoblotting revealed increased phosphorylation of p38 after 2 Gy and extracellular signal-regulated kinases 1/2 after 10 Gy in nontargeted heart. In addition, IR exposure significantly enhanced DNA fragmentation in nontargeted heart. Together, these data clearly indicated an induced abscopal response in distant organ after clinically relevant IR doses. More importantly, the results imply that orchestration of NF-κB signal transduction in nontargeted tissues may serve as an effector and could play a key role in induced abscopal responses.

Stromal Contributions to Radiation Resistance of Breast Cancer Stem Cells

Breast cancer that is refractory to radiation therapy causes significant morbidity and mortality. Tumor-initiating cells (TICs), also called cancer stem cells (CSCs), display a radioresistant phenotype. Normal stem cells depend on stromal cells for the formation of a stem cell “niche”, but the role of the stromal cells in breast TIC function and treatment resistance is not well understood. We sought to define the effects of stromal fibroblasts on breast TIC self-renewal and resistance to IR (ionizing radiation). For investigations into breast cancer TIC and stromal fibroblast physiology we utilized MMTV-Wnt-1 transgenic mice, which spontaneously generate mammary tumors. These tumors can be dissociated into single cell suspensions and subjected to fluorescence-activated cell sorting (FACS) for in vitro colony assays or molecular analyses. The TIC population in this model has been previously identified based on cell surface markers and has been validated using in vivo limiting dilution analyses. Using this model system, we isolated normal and tumor-associated fibroblasts from primary normal and tumor murine breast tissues by FACS, and their effects on TICs were assayed in a 3D co-culture system. We show that co-culture with primary stromal fibroblasts promotes the expansion of mouse mammary TICs in vitro and maintains their ability to form new tumors in vivo. At clinically relevant doses of IR, DNA double strand breaks are mitigated and clonogenic growth of TICs is enhanced by co-culture with the stromal fibroblasts. Consistent with these observations, the TICs that are grown in co-culture maintain a more basal differentiation phenotype and harbor lower levels of reactive oxygen species (ROS) than TICs alone. The reduction in ROS found in the co-cultured TICs is a consequence of fewer mitochondria and less active oxidative phosphorylation in these cells. Working with primary tissues, we have demonstrated that stromal fibroblasts from mammary tumors play an important role in promoting TIC proliferation and resistance to IR. Our work has led to a better characterization of the unique microenvironment within the TIC-stromal niche. Future endeavors into pharmacological disruption of the supportive cues within the niche could lead to novel radiosensitizers and CSC-directed therapies.

Abstract C130: Inhibition of NF-κB activity promotes radiosensitivity of breast cancer cells in three-dimensional culture via a β1-integrin-mediated pathway.

Abstract Radioresistance markedly impairs the efficacy of tumor radiotherapy and involves prosurvival signal transduction pathways that prevent radiation-induced cell death. Nuclear factor-κB (NF-κB), a stress-sensitive heterodimeric transcription factor in the regulation of the stress-responsive genes, has been shown to initiate prosurvival signaling in response to ionizing radiation (IR). The cooperative function of NF-κB with other key stress elements in radioresistance has not been clearly defined. We have previously reported that induction of β1-integrin is associated with the enhanced cell survival of breast cancer cells after exposure to clinically relevant doses of IR. Because a typical NF-κB binding site was identified in human β1-integrin promoter region, β1-integrin-mediated resistance to radiation may be regulated by NF-κB. The aim of the present study was to reveal a connection between NF-κB and β1-integrin pathways in radioprotection of malignant T4-2 mammary epithelial cells in 3D lrECM (three-dimensional laminin-rich extracellular matrix). We show that the elevated NF-κB activity was correlated with enhanced clonogenic survival with an increase in total and phosphorylated (Thr 788/789) β1-integrins. Inhibition of NF-κB activation significantly reduced clonogenic survival with the inhibition of β1-integrin, suggesting that NF-κB-mediated induction of β1-integrin is associated with an increased radiation resistance. Treatment of malignant human breast cell colonies, T4-2 with NF-κB activation inhibitor at Day 4 in 3D lrECM before exposure to IR (4-Gy X-ray) resulted in a reduction of the size of colonies. The surviving colonies were associated with a decrease in proliferation and increase in apoptosis, indicating a decrease of resistance to IR. Using an ELISA-based DNA binding assay, we next determined that the binding of NF-κB to its target sequence on β1-integrin promoter region was significantly increased by IR. These data suggest that IR-induced increase in β1-integrin protein expression is mediated by NF-κB activation and subsequent binding of NF-κB to the β1-integrin promoter. Taken together, our results provide the first evidence that NF-κB-mediated β1-integrin expression is responsible for tumor radioresistance. The NF-κB/β1-integrin pathway could provide new insight into the development of new therapeutic modalities to re-sensitize radioresistant tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C130.

Radiation-triggered Tumor Necrosis Factor (TNF) α-NFκB Cross-signaling Favors Survival Advantage in Human Neuroblastoma Cells

Induced radioresistance in the surviving cancer cells after radiotherapy could be associated with clonal selection leading to tumor regrowth at the treatment site. Previously we reported that post-translational modification of IκBα activates NFκB in response to ionizing radiation (IR) and plays a key role in regulating apoptotic signaling. Herein, we investigated the orchestration of NFκB after IR in human neuroblastoma. Both in vitro (SH-SY5Y, SK-N-MC, and IMR-32) and in vivo (xenograft) studies showed that IR persistently induced NFκB DNA binding activity and NFκB-dependent TNFα transactivation and secretion. Approaches including silencing NFκB transcription, blocking post-translational NFκB nuclear import, muting TNF receptor, overexpression, and physiological induction of either NFκB or TNFα precisely demonstrated the initiation and occurrence of NFκB → TNFα → NFκB positive feedback cycle after IR that leads to and sustains NFκB activation. Selective TNF-dependent NFκB regulation was confirmed with futile inhibition of AP-1 and SP-1 in TNF receptor muted cells. Moreover, IR increased both transactivation and translation of Birc1, Birc2, and Birc5 and induced metabolic activity and clonal expansion. This pathway was further defined to show that IR-induced functional p65 transcription (not NFκB1, NFκB2, or c-Rel) is necessary for activation of these survival molecules and associated survival advantage. Together, these results demonstrate for the first time the functional orchestration of NFκB in response to IR and further imply that p65-dependent survival advantage and initiation of clonal expansion may correlate with an unfavorable prognosis of human neuroblastoma.

Role of Heparanase in Radiation-Enhanced Invasiveness of Pancreatic Carcinoma

Pancreatic cancer is characterized by very low survival rates because of high intrinsic resistance to conventional therapies. Ionizing radiation (IR)-enhanced tumor invasiveness is emerging as one mechanism responsible for the limited benefit of radiotherapy in pancreatic cancer. In this study, we establish the role of heparanase-the only known mammalian endoglycosidase that cleaves heparan sulfate-in modulating the response of pancreatic cancer to radiotherapy. We found that clinically relevant doses of IR augment the invasive capability of pancreatic carcinoma cells in vitro and in vivo by upregulating heparanase. Changes in the levels of the transcription factor Egr-1 occurred in pancreatic cancer cells following radiation, underlying the stimulatory effect of IR on heparanase expression. Importantly, the specific heparanase inhibitor SST0001 abolished IR-enhanced invasiveness of pancreatic carcinoma cells in vitro, whereas combined treatment with SST0001 and IR, but not IR alone, attenuated the spread of orthotopic pancreatic tumors in vivo. Taken together, our results suggest that combining radiotherapy with heparanase inhibition is an effective strategy to prevent tumor resistance and dissemination, observed in many IR-treated pancreatic cancer patients. Further, the molecular mechanism underlying heparanase upregulation in pancreatic cancer that we identified in response to IR may help identify patients in which radiotherapeutic intervention may confer increased risk of metastatic spread, where antiheparanase therapy may be particularly beneficial.

Involvement of Oct-1 in the regulation of CDKN1A in response to clinically relevant doses of ionizing radiation

CDKN1A is a cyclin-dependent kinase inhibitor that plays a critical role in cell cycle checkpoint regulation. It is transcriptionally induced by TP53 (p53) following exposure to ionizing radiation (IR). Induction of CDKN1A after irradiation is closely related to IR-sensitivity of tumor cells, but the underlying mechanisms remain obscure because conventional reporter gene systems respond poorly to IR unless hyperlethal doses are used. Here, we performed a promoter analysis of the CDKN1A gene following irradiation with clinically relevant doses of IR using the adeno-associated virus-mediated reporter system which we have recently shown to be highly responsive to IR. We demonstrate that there are regulatory elements at − 1.1 kb, − 1.4 kb, and − 1.8 kb, and deletion of these elements attenuate induction of the CDKN1A gene promoter in response to 0.2–2.0 Gy of IR. EMSA and ChIP assays showed that Oct-1 binds constitutively to the elements at − 1.1 kb and − 1.8 kb. Functional involvement of Oct-1 was confirmed by RNA interference targeting the Oct-1 gene, which suppressed both the basal and IR-inducible components of the CDKN1A expression. Thus, our results reveal that Oct-1 is crucial to the TP53-mediated regulation of the CDKN1A gene promoter following exposure to clinically relevant doses of IR.

SN52, a novel nuclear factor-κB inhibitor, blocks nuclear import of RelB:p52 dimer and sensitizes prostate cancer cells to ionizing radiation

The activation of nuclear factor-kappaB (NF-kappaB) is thought to protect cancer cells against therapy-induced cytotoxicity. RelB, a member of the NF-kappaB family in the alternative pathway, is uniquely expressed at a high level in prostate cancer with high Gleason scores. Here, we show that ionizing radiation (IR) enhances nuclear import of RelB, leading to up-regulation of its target gene, manganese superoxide dismutase (MnSOD), and renders prostate cancer cells resistant to IR. To selectively block RelB nuclear import, we designed a cell-permeable SN52 peptide, a variant of the SN50 peptide that has been shown to block nuclear import of NF-kappaB family members in the classic pathway. Inhibition of IR-induced NF-kappaB activation by SN50 and SN52 was achieved by selectively interrupting the association of p50 and p52 with nuclear import factors importin-alpha1 and importin-beta1. Importantly, SN52 seems to be more efficient for radiosensitization of prostate cancer cells at clinically relevant radiation doses and has less cytotoxicity to normal prostate epithelial cells compared with the toxicity observed with SN50. These results suggest that targeting the alternative pathway is a promising approach to selectively radiosensitize prostate cancers and that SN52 may serve as a prototype biological agent for sensitizing prostate cancers to clinically relevant doses of IR.

ATM Phosphorylation Kinetics as a Biological Reporter of Cellular Radiosensitivity

AIM: Exposure to ionizing radiation (IR) causes DNA strand break formation, resulting in the rapid activation and autophosphorylation of ATM on Ser-1981. We hypothesize that quantitative analysis of ATM autophosphorylation may serve as a reporter of DNA damage induction and repair after IR in two ATM wild-type prostate cancer cell lines PC-3 and DU-145 and two oral squamous cell carcinoma cell lines SCC25 and SQ-20B. METHODS: Immunocytochemistry was performed on glass coverslips using polyclonal anti-phospho-H2AX or monoclonal anti-phospho-ATM phospho-specific antibodies. Cells were treated with 0 to 4 Gy of IR, incubated, fixed, and stained. Stained cells were analyzed using epifluorescence microscopy and discrete foci of brightness in each nucleus were counted. In vitro clonogenic assays were performed to determine the relative radiosensitivity of PC-3, DU-145, SCC25, and SQ-20B cells. RESULTS: Within 10 min after IR, discrete foci of ATM autophosphorylation appeared at putative sites of DNA strand breaks. The number and relative brightness of γ-H2AX and ATM autophosphorylation foci increased linearly with dose at 30 min after clinically relevant IR doses in these cell lines. The relatively radiosensitive PC-3 and SCC-25 cells retained a greater percentage of ATM autophosphorylation and γ-H2AX foci, which was most evident at 30 min after IR. CONCLUSION: Our preliminary data suggest that persistence of ATM autophosphorylation is a promising reporter of in vitro radiosensitivity. Further research is necessary to validate this finding and to develop additional applications for ATM autophosphorylation in radiation research.

Radiation-activated antitumor vectors.

Radiotherapy (RT) is a primary treatment modality for the majority of solid tumors. The objective is to destroy the tumor mass by exposure to ionizing radiation (IR) from an external beam or isotopic source. IR causes DNA damage directly and indirectly via the production of reactive oxygen intermediates (ROIs). Accumulation of sufficient damage leads to tumor cell death. The efficacy of RT is usually governed by the radiation dose given, the main limitation being the need to avoid injury to the surrounding normal tissues. To address the latter problem, physical techniques such as conformal and intensity-modulated radiotherapy have been developed to improve the precision of dose delivery to the tumor volume. However, some tumors prove refractory to conventional radiotherapy treatments, as insufficient dose can be delivered to the tumor. In such cases, other therapeutic strategies, such as chemotherapy, can be used in combination, particularly if such drugs lead to increased tumor radiosensitization. Nevertheless, many tumor types, (e.g., glioblastoma) are often resistant to even these combined approaches. Consequently, there is a need for new strategies that can improve the effectiveness of current radiotherapy regimens. Gene therapy offers the exciting possibility of significantly improving the efficacy of radiotherapy without the need for IR-dose escalation or undue increases in normal tissue morbidity. Furthermore, the potential to spatially and temporally target the activation of gene therapy vectors using clinically relevant IR doses provides a particularly attractive prospect.

Transcription factors activated in mammalian cells after clinically relevant doses of ionizing radiation.

Over the past 15 years, a wealth of information has been published on transcripts and proteins 'induced' (requiring new protein synthesis) in mammalian cells after ionizing radiation (IR) exposure. Many of these studies have also attempted to elucidate the transcription factors that are 'activated' (i.e., not requiring de novo synthesis) in specific cells by IR. Unfortunately, all too often this information has been obtained using supralethal doses of IR, with investigators assuming that induction of these proteins, or activation of corresponding transcription factors, can be 'extrapolated' to low-dose IR exposures. This review focuses on what is known at the molecular level about transcription factors induced at clinically relevant (< or =2 Gy) doses of IR. A review of the literature demonstrates that extrapolation from high doses of IR to low doses of IR is inaccurate for most transcription factors and most IR-inducible transcripts/proteins, and that induction of transactivating proteins at low doses must be empirically derived. The signal transduction pathways stimulated after high versus low doses of IR, which act to transactivate certain transcription factors in the cell, will be discussed. To date, only three transcription factors appear to be responsive (i.e. activated) after physiological doses (doses wherein cells survive or recover) of IR. These are p53, nuclear factor kappa B(NF-kappaB), and the SP1-related retinoblastoma control proteins (RCPs). Clearly, more information on transcription factors and proteins induced in mammalian cells at clinically or environmentally relevant doses of IR is needed to understand the role of these stress responses in cancer susceptibility/resistance and radio-sensitivity/resistance mechanisms.