High Doses Of Ionizing Radiation Research Articles

Pre- and post-melatonin mitigates the effect of ionizing radiation-induced damage in wheat by modulating the antioxidant machinery

As an indolamine, melatonin (C13H16N2O2) performs essential roles in the regulation of plant growth and development and ameliorates the harmful effects of abiotic stresses. This study examined two types of melatonin application, pre-sowing (prMel) and application during growth (ptMel), in wheat (Triticum aestivum L.) seedlings exposed to four different doses (100, 200, 300, and 400 Gy) of radioactive cobalt (60Co) gamma rays as dry seeds to investigate their ameliorative effects on ionizing radiation (IR) stress. Peroxidase, catalase, superoxide dismutase, ascorbate peroxidase, glutathione reductase, mono- and dihydroxyperoxidase, and phenylalanine ammonia-lyase activities, and levels of lipid peroxidation, H2O2, and total glutathione (GSH), and phenolic acids (PHAs) in soluble free, ester, glycoside and ester-bound forms were examined in the seedlings. Both melatonin applications were found to increase lipid peroxidation, H2O2, and GSH contents previously reduced by gamma irradiation. The IR treatment-induced increases in enzyme activities were significantly reduced by melatonin applications. The study findings indicated that high doses of IR resulted in significant decreases in the activity and levels of the measured traits. The predominant PHAs in the tissues were vanillic, ferulic, and p-coumaric acids. In addition, ptMel application combined with IR stress lowered the total phenolic acid contents in the soluble forms while increasing those in the cell wall-bound form. In conclusion, the antioxidant system in the seedlings exposed to the different gamma ray doses was regulated by prMel and ptMel applications in such a manner as to alleviate IR stress-induced oxidatives damages in the wheat.

The impact of cyclophosphamide versus high-level of ionizing radiation on the immune response

The immune response to various high doses of Ionizing Radiation (IR) is investigated in this study compared to the non-irradiated group and other immunosuppressive conditions. Thirty rats were divided into six different groups. Group I received no radiation or medications. Groups II, III, IV, and V were subjected to the entire body to 1, 2, 3, and 5 Gray (Gy) of IR. Cyclophosphamide 50 mg/kg was administered intraperitoneally to Group VI. Serum levels of Interleukin-2/1-beta, Tumor Necrosis Factor-alpha (TNF-α), and Interferon-gamma (INF-γ) were measured one day after irradiation. The immunosuppressive effect of cyclophosphamide reduced the majority of the evaluated parameters. When high doses of IR, notably greater than 1Gy, were investigated, all measured parameters increased consistently. Finally, high doses of IR amplify essential pro-inflammatory responses and cannot be used to suppress the immune system in a single dose. More research is needed to clarify immune responses and their therapeutic potential in response to high or low IR doses.

Can Low-Level Ionizing Radiation Do Us Any Harm?

The current system of radiological protection relies on the linear no-threshold (LNT) hypothesis of cancer risk due to humans being exposed to ionizing radiation (IR). Under this tenet, effects of low doses (i.e. of those not exceeding 100 mGy or 0.1 mGy/min. of X- or γ-rays for acute and chronic exposures, respectively) are evaluated by downward linear extrapolation from regions of higher doses and dose rates where harmful effects are actually observed. However, evidence accumulated over many years clearly indicates that exposure of humans to low doses of radiation does not cause any harm and often promotes health. In this review, we discuss results of some epidemiological analyses, clinical trials and controlled experimental animal studies. Epidemiological data indicate the presence of a threshold and departure from linearity at the lowest dose ranges. Experimental studies clearly demonstrate the qualitative difference between biological mechanisms and effects at low and at higher doses of IR. We also discuss the genesis and the likely reasons for the persistence of the LNT tenet, despite its scientific implausibility and deleterious social consequences. It is high time to replace the LNT paradigm by a scientifically based dose-effect relationship where realistic quantitative hormetic or threshold models are exploited.

Possible Protective Effect of LOXL1 Variant in the Cohort of Chernobyl Catastrophe Clean-Up Workers.

Ionising radiation (IR) is an environmental factor known to alter genomes and therefore challenge organisms to adapt. Lithuanian clean-up workers of the Chernobyl nuclear disaster (LCWC) experienced high doses of IR, leading to different consequences. This study aims to characterise a unique protective genomic variation in a relatively healthy LCWC group. This variation influenced their individual reaction to IR and potentially protects against certain diseases such as exfoliation syndrome and glaucoma. Clinical and IR dosage data were collected using a questionnaire to characterise the cohort of 93 LCWC. Genome-wide genotyping using Illumina beadchip technology was performed. The control group included 466 unrelated, self-reported healthy individuals of Lithuanian descent. Genotypes were filtered out from the microarray dataset using a catalogue of SNPs. The data were used to perform association, linkage disequilibrium, and epistasis analysis. Phenotype data analysis showed the distribution of the most common disease groups among the LCWC. A genomic variant of statistical significance (Fishers’ exact test, p = 0.019), rs3825942, was identified in LOXL1 (NM_005576.4:c.458G>A). Linkage disequilibrium and epistasis analysis for this variant identified the genes LHFPL3, GALNT6, PIH1D1, ANKS1B, and METRNL as potentially involved in the etiopathogenesis of exfoliation syndrome and glaucoma, which were not previously associated with the disease. The LOXL1 variant is mostly considered a risk factor in the development of exfoliation syndrome and glaucoma. The influence of recent positive selection, the phenomenon of allele-flipping, and the fact that only individuals with the homozygous reference allele have glaucoma in the cohort of the LCWC suggest otherwise. The identification of rs3825942 and other potentially protective genomic variants may be useful for further analysis of the genetic architecture and etiopathogenetic mechanisms of other multifactorial diseases.

Abstract PO-053: Targeting focal adhesions to limit radiation-induced glioblastoma cell invasion

Abstract Introduction: Glioblastoma multiforme (GBM) is a grade IV brain tumor that is considered incurable with a median overall survival of 15 months. This low survival rate is partially attributed to the highly infiltrative nature of GBM cells. A key component of GBM cell invasion is the degradation of extracellular matrix (ECM), which is facilitated by the formation of invadosomes: focal adhesions (FAs) and invadopodia. The majority of GBM patients will receive a conventional radiation schedule of 2 Gy in 30 fractions, while a subset of patients will receive a hypofractioned radiation schedule of ~3 Gy in 15 fractions. Recent pre-clinical reports indicate the population of GBM cells that survive ionizing radiation (IR) exhibit increased invasiveness, suggesting IR may regulate FA and invadopodia function. Understanding how IR affects invadosome biology may lead to new therapeutic strategies to halt the invasion of GBM cells and increase overall survival of patients treated with radiation. Results: We found that irradiated GBM cells were significantly more invasive in vitro. GBM cells irradiated with 0-4 Gy and plated onto fluorescently-tagged gelatin to analyze ECM degradation. Cells exposed to 0-2 Gy IR produced small dot-like patterns of degradation resembling active invadopodia. However, cells exposed to 3-4 Gy IR produced oblong-shaped patterns of degradation at the periphery of the cell resembling active FAs. Using immunofluorescent markers of invadopodia (TKS5/actin) and FAs (vinculin), we found total focal adhesion and invadopodia formation remained unchanged. Rather, we found that exposing cells to 3 Gy IR increased pFAK-397 in FA structures suggesting IR increases ECM degradation by promoting FA activity. Conclusions: These data suggest higher IR doses promote FA-mediated ECM degradation and GBM cell invasion. We are currently investigating the impact of IR on FAs, ECM degradation, and GBM cell invasion in murine brain slices ex vivo. Citation Format: Lisa R. Decotret, Rocky Shi, Kevin L. Bennewith. Targeting focal adhesions to limit radiation-induced glioblastoma cell invasion [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-053.

Proficiency in homologous recombination repair is prerequisite for activation of G2-checkpoint at low radiation doses

Pathways of repair of DNA double strand breaks (DSBs) cooperate with DNA damage cell cycle checkpoints to safeguard genomic stability when cells are exposed to ionizing radiation (IR). It is widely accepted that checkpoints facilitate the function of DSB repair pathways. Whether DSB repair proficiency feeds back into checkpoint activation is less well investigated. Here, we study activation of the G2-checkpoint in cells deficient in homologous recombination repair (HRR) after exposure to low IR doses (∼1 Gy) in the G2-phase. We report that in the absence of functional HRR, activation of the G2-checkpoint is severely impaired. This response is specific for HRR, as cells deficient in classical non-homologous end joining (c-NHEJ) develop a similar or stronger G2-checkpoint than wild-type (WT) cells. Inhibition of ATM or ATR leaves largely unaffected residual G2-checkpoint in HRR-deficient cells, suggesting that the G2-checkpoint engagement of ATM/ATR is coupled to HRR. HRR-deficient cells show in G2-phase reduced DSB-end-resection, as compared to WT-cells or c-NHEJ mutants, confirming the reported link between resection and G2-checkpoint activation. Strikingly, at higher IR doses (≥4 Gy) HRR-deficient cells irradiated in G2-phase activate a weak but readily detectable ATM/ATR-dependent G2-checkpoint, whereas HRR-deficient cells irradiated in S-phase develop a stronger G2-checkpoint than WT-cells. We conclude that HRR and the ATM/ATR-dependent G2-checkpoint are closely intertwined in cells exposed to low IR-doses in G2-phase, where HRR dominates; they uncouple as HRR becomes suppressed at higher IR doses. Notably, this coupling is specific for cells irradiated in G2-phase, and cells irradiated in S-phase utilize a different mechanistic setup.

RIF1 controls replication initiation and homologous recombination repair in a radiation dose-dependent manner.

ABSTRACTRIF1 controls both DNA replication timing and the DNA double-strand break (DSB) repair pathway to maintain genome integrity. However, it remains unclear how RIF1 links these two processes following exposure to ionizing radiation (IR). Here, we show that inhibition of homologous recombination repair (HRR) by RIF1 occurs in a dose-dependent manner and is controlled via DNA replication. RIF1 inhibits both DNA end resection and RAD51 accumulation after exposure to high doses of IR. Contrastingly, HRR inhibition by RIF1 is antagonized by BRCA1 after a low-dose IR exposure. At high IR doses, RIF1 suppresses replication initiation by dephosphorylating MCM helicase. Notably, the dephosphorylation of MCM helicase inhibits both DNA end resection and HRR, even without RIF1. Thus, our data show the importance of active DNA replication for HRR and suggest a common suppression mechanism for DNA replication and HRR at high IR doses, both of which are controlled by RIF1.This article has an associated First Person interview with the first author of the paper.

P95. Radiation exposure during the course of treatment for early-onset scoliosis: longitudinal pilot study

BACKGROUND CONTEXT To estimate the amount of ionizing radiation (IR) exposure in early-onset scoliosis for early-onset scoliosis. There is abundant evidence of the health hazards attributed to IR exposure. However, no studies have estimated the amount of IR exposure in early-onset scoliosis. PURPOSE To estimate the amount of ionizing radiation (IR) exposure in early-onset scoliosis for early-onset scoliosis. There is abundant evidence of the health hazards attributed to IR exposure. However, no studies have estimated the amount of IR exposure in early-onset scoliosis. STUDY DESIGN/SETTING Retrospective consecutive case series. PATIENT SAMPLE A total of 372 total patients, 337 had a minimum 2-year follow-up. OUTCOME MEASURES All spine-related IR imaging studies are excluding interpretative fluoroscopy were tabulated and early-onset scoliosis estimated based on historical controls in millisieverts (mSv). METHODS A consecutive single-center series of early-onset scoliosis patients were retrospectively reviewed. Of 372 total patients, 337 had a minimum 2-year follow-up and complete records available for analysis. All spine-related IR imaging studies are excluding interpretative fluoroscopy were tabulated and early-onset scoliosis estimated based on historical controls in millisieverts (mSv). RESULTS Initial X-ray evaluation for scoliosis was made at a mean age of 5.7 years (range=2 to 9 years). Mean radiographic period was 11 years (range=2 to 18). There was a statistically significant inverse correlation between patient age at time of initial IR and total mean IR (p CONCLUSIONS Compared to the general public. Early-onset scoliosis patients had 3.7 times more IR than the estimated background radiation for the same duration of time. Younger patients had significantly higher IR doses. This study stresses the importance of recognizing the amount of IR used in the management of early-onset scoliosis patients and its potential long-term risks. FDA DEVICE/DRUG STATUS This abstract does not discuss or include any applicable devices or drugs.

Chromosomal radiosensitivity of triple negative breast cancer patients

Purpose: Based on clinical and molecular data, breast cancer is a heterogeneous disease. Breast cancers that have no expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) are defined as triple negative breast cancers (TNBCs); luminal cancers have different expressions of ER, PR and/or HER2. TNBCs are frequently linked with advanced disease, poor prognosis and occurrence in young African women, and about 15% of the cases are associated with germline BRCA1/2 mutations. Since radiotherapy is utilized as a principle treatment in the management of TNBC, we aimed to investigate the chromosomal instability and radiosensitivity of lymphocytes in TNBC patients compared to luminal breast cancer patients and healthy controls using the micronucleus (MN) assay. The effect of mutations in breast cancer susceptibility genes on chromosomal radiosensitivity was also evaluated.Methods: Chromosomal radiosensitivity was evaluated in the G0 (83 patients and 90 controls) and S/G2 (34 patients and 17 controls) phase of the cell cycle by exposing blood samples from all patients and controls to 2 and 4 Gy ionizing radiation (IR).Results: In the G0 MN assay, the combined cohort of all breast cancer, TNBC and luminal patients’ exhibit significantly elevated spontaneous MN values compared to controls indicating chromosomal instability. Chromosomal radiosensitivity is also significantly elevated in the combined cohort of all breast cancer patients compared to controls. The TNBC patients, however, do not exhibit enhanced chromosomal radiosensitivity. Similarly, in the S/G2 phase, 76% of TNBC patients do not show enhanced chromosomal radiosensitivity compared to the controls. In both the G0 and S/G2 phase, luminal breast cancer patients demonstrate a shift toward chromosomal radiosensitivity compared to TNBC patients and controls.Conclusions: The observations of the MN assay suggest increased chromosomal instability and chromosomal radiosensitivity in South African breast cancer patients. However, in TNBC patients, the irradiated MN values are not elevated. Our results suggest that the healthy lymphocytes in TNBC patients could handle higher doses of IR.

Alterations in the rainbow trout (Oncorhynchus mykiss) eggs exposed to ionizing radiation during induced androgenesis.

Ionizing radiation (IR) is applied to inactivate nuclear genome in the salmonid eggs to induce androgenetic development. However, it has been considered that doses of IR used to damage maternal chromosomes may also affect morphology of the eggs and decrease their developmental potential. Thus, the main goal of the present research was to assess alterations in the rainbow trout (Oncorhynchus mykiss) eggs caused by the high dose of IR administered during androgenesis. In the present research, rainbow trout eggs were irradiated with 350Gy of X-rays, inseminated and exposed to the high hydrostatic pressure (HHP) shock to develop as androgenetic doubled haploids (DHs). The distribution of lipid droplets in the irradiated and non-irradiated rainbow trout eggs, survival rates and morphology of larvae from androgenetic and control groups were compared. It has been observed that non-irradiated and irradiated eggs exhibited altered distribution of lipid droplets. Most of the eggs before IR treatment displayed rather equal distribution of the oil droplets. In turn, majority of eggs studied after irradiation had coalesced lipid droplets, a pattern found in eggs with reduced quality. Incidences of abnormally developed larvae were more frequently observed among fish that hatched from the irradiated eggs. Observed changes suggest X-rays applied for the genetic inactivation of rainbow trout eggs may lead to decrease of their developmental competence.

Modulating effects of melatonin, vitamin C and saffron alone or in combination on radiation induced cell lethality; an in vitro study with Hela cells

Introduction: Radiation countermeasures are defined as use of an agent to minimize the deleterious effects of radiation therapy by administering the compound after the radiation exposure has occurred, regardless of toxicity. In spite of extensive study in this field, yet there is no suitable radioprotector introduced. In this study two naturally occurring antioxidants were studied on radiation induced cell lethality. Materials and Methods: Various doses of melatonin, saffron and vitamin C were examined for their antioxidant capacity by using DPPH test. Hela cells were treated with optimum doses of melatonin, saffron and vitamin C; 2 hours prior to gamma irradiation alone or in combination. Treated cells were irradiated with various doses of gamma rays (2, 4, 8 Gy), then cells were plated in 96 well plates for MTT assay. Cells were fixed at 48 and 72 hours after plating and reading was done ELISA reader. Results: Results showed cell viability reduced slightly after 48 h irradiation. Pretreatment of cells with melatonin and vitamin C increased cell lethality considerably for dopses of 2 and 4 Gy, but surprisingly presence of melatonine and vitamin C led to an increase in cell viability when combined with 8 Gy gamma rays. Combination of these agents with saffron showed similar effects. Similar results were observed for 72 h sampling time. However, protective effect of these agents was more pronounced when using 8 Gy radiation dose. Conclusion: Results indicate that despite the antioxidant potential of melatonin, saffron and vitamin C, when combined with low and moderate doses of gamma rays (2 and 4 Gy) led to increased cytotoxic effects of ionizing radiation. However, at high dose of gamma rays (8 Gy) radioprotective effect was seen. The dual action of these agents with low and high doses of ionizing radiation is not known.

Metformin Protects Against Radiation-Induced Pneumonitis and Fibrosis and Attenuates Upregulation of Dual Oxidase Genes Expression.

Purpose: Lung tissue is one of the most sensitive organs to ionizing radiation (IR). Early and late side effects of exposure to IR can limit the radiation doses delivered to tumors that are within or adjacent to this organ. Pneumonitis and fibrosis are the main side effects of radiotherapy for this organ. IL-4 and IL-13 have a key role in the development of pneumonitis and fibrosis. Metformin is a potent anti-fibrosis and redox modulatory agent that has shown radioprotective effects. In this study, we aimed to evaluate possible upregulation of these cytokines and subsequent cascades such as IL4-R1, IL-13R1, Dual oxidase 1 (DUOX1) and DUOX2. In addition, we examined the potential protective effect of metformin in these cytokines and genes, as well as histopathological changes in rat’s lung tissues.Methods: 20 rats were divided into 4 groups: control; metformin treated; radiation + metformin; and radiation. Irradiation was performed with a 60Co source delivering 15 Gray (Gy) to the chest area. After 10 weeks, rats were sacrificed and their lung tissues were removed for histopathological, real-time PCR and ELISA assays.Results: Irradiation of lung was associated with an increase in IL-4 cytokine level, as well as the expression of IL-4 receptor-a1 (IL4ra1) and DUOX2 genes. However, there was no change in the level of IL-13 and its downstream gene including IL-13 receptor-a2 (IL13ra2). Moreover, histopathological evaluations showed significant infiltration of lymphocytes and macrophages, fibrosis, as well as vascular and alveolar damages. Treatment with metformin caused suppression of upregulated genes and IL-4 cytokine level, associated with amelioration of pathological changes.Conclusion: Results of this study showed remarkable pathological damages, an increase in the levels of IL-4, IL4Ra1 and Duox2, while that of IL-13 decreased. Treatment with metformin showed ability to attenuate upregulation of IL-4–DUOX2 pathway and other pathological damages to the lung after exposure to a high dose of IR.

Serum miR-375-3p increase in mice exposed to a high dose of ionizing radiation

Exposure to high-doses of ionizing radiation (IR) leads to development of a strong acute radiation syndrome (ARS) in mammals. ARS manifests after a latency period and it is important to develop fast prognostic biomarkers for its early detection and assessment. Analysis of chromosomal aberrations in peripheral blood lymphocytes is the gold standard of biological dosimetry, but it fails after high doses of IR. Therefore, it is important to establish novel biomarkers of exposure that are fast and reliable also in the high dose range. Here, we investigated the applicability of miRNA levels in mouse serum. We found significantly increased levels of miR-375-3p following whole body exposure to 7 Gy of X-rays. In addition, we analyzed their levels in various organs of control mice and found them to be especially abundant in the pancreas and the intestine. Following a dose of 7 Gy, extensive cell death occurred in these tissues and this correlated negatively with the levels of miR-375-3p in the organs. We conclude that high expressing tissues of miR-375-3p may secrete this miRNA in serum following exposure to 7 Gy. Therefore, elevated miR-375-3p in serum may be a predictor of tissue damage induced by exposure to a high radiation dose.

Abstract PR06: The effect of low-dose fractionated versus high-dose hypofractionated radiotherapy on antitumor immunity

Abstract Fractionated, low-dose (1.8-2 Gy) ionizing radiation (IR) is an integral component of head and neck cancer management. The mechanism of tumor control has traditionally been attributed to direct cytolytic effects on tumor cells. Substantial evidence supports enhanced antitumor immune responses when IR is given in large, single or hypofractionated doses. The effect of low-dose fractionated IR on antitumor immunity is less clear. We hypothesized that high-dose (8 Gy) IR would induce antigen release and priming of antigen-specific T-lymphocytes and sensitize tumor cells to killing by effector T-lymphocytes (CTLs) to a greater degree than low-dose (2 Gy) IR. To test this hypothesis, we stably transduced murine oral cancer 1 (MOC1) cells with full-length ovalbumin as a model antigen (MOC1ova). Upon exposure of MOC1ova cells to 8 Gy IR in vitro, splenocytes endocytosed and cross-presented SIINFEKL on H2-Kb and induced proliferation of CFSE-labeled, SIINFEKL-specific OT-1 T-lymphocytes to a greater degree than when exposed to MOC1ova cells irradiated to 0 or 2 Gy. Antigen cross-presentation was dependent upon type I interferon. In in vivo correlative experiments, MOC1ova tumors irradiated to 8 Gy induced expansion of adoptively transferred OT-1 T-lymphocytes located within the spleen, draining lymph node, and tumor to a greater degree than tumors exposed to 0 or 2 Gy. To assess if higher doses of IR enhance CTL killing of target cells, a real-time impedance-based cytotoxicity assay was utilized. Exposure of target cells to 8 Gy IR dramatically enhanced OT-1 CTL killing of MOC1ova cells to a greater degree than cells exposed to 0 or 2 Gy in a process independent of antigen-presentation. We next hypothesized that 10 days of 2Gy/day (low-dose fractionated) IR would be immunosuppressive in vivo compared to 2 fractions of 8 Gy (high-dose, hypofractionated) IR. We performed a time course analysis of effector and immunosuppressive cell peripheral accumulation and tumor infiltration as well as antigen-specific draining lymph node T-lymphocyte responses comparing these two IR regimens. Cumulatively, results demonstrated significantly greater correlative and functional T-lymphocyte immune responses with high-dose hypofractionated IR compared to daily low-dose IR. Analysis of myeloid derived suppressor cell and regulatory CD4+ T-lymphocyte changes displayed mixed alterations between regimens. Taken together, these results suggest that higher-dose, hypofractionated IR results in enhanced immune activation and less immunosuppression compared to daily fractionated low-dose IR. These results have significant implications in the rational design of combination therapies utilizing IR and immunotherapy. This abstract is also being presented as Poster 32. Citation Format: Megan V. Morisada, Ellen C. Moore, Jay Friedman, Clint T. Allen. The effect of low-dose fractionated versus high-dose hypofractionated radiotherapy on antitumor immunity [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr PR06.

Cytokine profile of breast cell lines after different radiation doses

Purpose: Ionizing radiation (IR) treatment activates inflammatory processes causing the release of a great amount of molecules able to affect the cell survival. The aim of this study was to analyze the cytokine signature of conditioned medium produced by non-tumorigenic mammary epithelial cell line MCF10A, as well as MCF7 and MDA-MB-231 breast cancer cell lines, after single high doses of IR in order to understand their role in high radiation response.Materials and methods: We performed a cytokine profile of irradiated conditioned media of MCF10A, MCF7 and MDA-MB-231 cell lines treated with 9 or 23 Gy, by Luminex and ELISA analyses.Results: Overall, our results show that both 9 Gy and 23 Gy of IR induce the release within the first 72 h of cytokines and growth factors potentially able to influence the tumor outcome, with a dose-independent and cell-line dependent signature. Moreover, our results show that the cell-senescence phenomenon does not correlate with the amount of ‘senescence-associated secretory phenotype’ (SASP) molecules released in media. Thus, additional mechanisms are probably involved in this process.Conclusions: These data open the possibility to evaluate cytokine profile as useful marker in modulating the personalized radiotherapy in breast cancer care.

Abstract 2335: Targeting the DNA repair as well as survival functions of MCL-1 enhances cancer cell killing

Abstract MCL-1 is a a pro-survival BCL2 protein family member which is over-expressed in drug resistant cancer cells. Our purpose was to assess whether MCL-1 increases genomic instability along with its anti-apoptotic function in cancer cells. Depletion of MCL-1 by treating human small cell lung cancer (SCLC) lines with immunotoxin sensitizes these cells to increased killing by BH3 mimetic - Navitoclax (ABT-263) both in vitro and in mice xenograft models. These SCLC cell lines are resistant to both single agent Immunotoxin or ABT-263. In addition, inhibition of NANOG or NANOGP8 by lentivrus delivered shRNA depletes MCL-1 in human colorectal cancer cells and sensitizes these cells to increased cell killing by venetaclox (ABT-199). This increased killing depended on loss of MCL-1, was caspase-dependent and reversed by re-expression of MCL-1. Depletion of MCL-1 in cancer cells , increases cell sensitivity to ionizing radiation (IR) induced death, which was reversed by expression of MCL-1. In response to IR, MCL-1 depleted cells showed reduced survival even at radiation doses less than 6 Gy, however an increase in caspases 3/7 activity was observed at 6 Gy and above, suggesting that apoptosis is activated only when MCL-1 depleted cells are irradiated with higher IR doses. In order to explain the decreased survival post- irradiation the genomic instability and DNA repair pathways were analyzed in MCL-1 depleted cells. Post-irradiation MCL-1 depleted cells exhibited increased genomic instability as measured by increase in chromosome aberrations at different phases of the cell cycle. The increase in aberrations were significantly higher in G2 and S- phases of the cell cycle suggesting defect in Homologous recombination repair pathway (HR). Moreover, the MCL-1 depleted cells show decreased gamma-H2AX foci at earlier time points (30 and 90 minutes) post-irradiation indicating defect in DNA Damage response (DDR) and higher residual foci at 360 minutes, indicating defective DNA repair. These observations were further confirmed by decreased phosphorylation of ATR suggesting defect in DNA Damage response and higher levels of residual 53BP1 and RIF1 foci in MCL-1 depleted cells , confirming DNA DSB repair by homologous recombination (HR) was compromised. Consistent with this model, MCL-1 depleted cells exhibited a reduced frequency of IR-induced MRE11, BRCA1, RPA and Rad51 foci formation, decreased DNA end resection and decreased HR repair in the DR-GFP DSB repair model. Similarly, after Hydoxyurea (HU) induction of stalled replication forks in MCL-1 depleted cells there was a decreased ability to subsequently restart DNA synthesis, which is normally dependent upon HR mediated resolution of collapsed forks. In summary, MCL-1 is an important therapeutic target in cancer cells and its depletion increases cell killing by either increase in apoptosis or suppression of HR and increased replication stress. Citation Format: Abid Mattoo, J Milburn Milburn Jessup, Tej K. Pandita. Targeting the DNA repair as well as survival functions of MCL-1 enhances cancer cell killing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2335. doi:10.1158/1538-7445.AM2017-2335

A Genome-Wide Search for Ionizing-Radiation-Responsive Elements in Deinococcus radiodurans Reveals a Regulatory Role for the DNA Gyrase Subunit A Gene's 5' Untranslated Region in the Radiation and Desiccation Response.

Tight regulation of gene expression is important for the survival of Deinococcus radiodurans, a model bacterium of extreme stress resistance. Few studies have examined the use of regulatory RNAs as a possible contributing mechanism to ionizing radiation (IR) resistance, despite their proffered efficient and dynamic gene expression regulation under IR stress. This work presents a transcriptome-based approach for the identification of stress-responsive regulatory 5' untranslated region (5'-UTR) elements in D. radiodurans R1 that can be broadly applied to other bacteria. Using this platform and an in vivo fluorescence screen, we uncovered the presence of a radiation-responsive regulatory motif in the 5' UTR of the DNA gyrase subunit A gene. Additional screens under H2O2-induced oxidative stress revealed the specificity of the response of this element to IR stress. Further examination of the sequence revealed a regulatory motif of the radiation and desiccation response (RDR) in the 5' UTR that is necessary for the recovery of D. radiodurans from high doses of IR. Furthermore, we suggest that it is the preservation of predicted RNA structure, in addition to DNA sequence consensus of the motif, that permits this important regulatory ability.IMPORTANCEDeinococcus radiodurans is an extremely stress-resistant bacterium capable of tolerating up to 3,000 times more ionizing radiation than human cells. As an integral part of the stress response mechanism of this organism, we suspect that it maintains stringent control of gene expression. However, understanding of its regulatory pathways remains incomplete to date. Untranslated RNA elements have been demonstrated to play crucial roles in gene regulation throughout bacteria. In this work, we focus on searching for and characterizing responsive RNA elements under radiation stress and propose that multiple levels of gene regulation work simultaneously to enable this organism to efficiently recover from exposure to ionizing radiation. The model we propose serves as a generic template to investigate similar mechanisms of gene regulation under stress that have likely evolved in other bacterial species.

Effects of low dose ionizing radiation on DNA damage-caused pathways by reverse-phase protein array and Bayesian networks

Ionizing radiation (IR) causing damages to Deoxyribonucleic acid (DNA) constitutes a broad range of base damage and double strand break, and thereby, it induces the operation of relevant signaling pathways such as DNA repair, cell cycle control, and cell apoptosis. The goal of this paper is to study how the exposure to low dose radiation affects the human body by observing the signaling pathway associated with Ataxia Telangiectasia mutated (ATM) using Reverse-Phase Protein Array (RPPA) and isogenic human Ataxia Telangiectasia (A-T) cells under different amounts and durations of IR exposure. In order to verify which proteins could be involved in a DNA damage-caused pathway, only proteins that highly interact with each other under IR are selected by using correlation coefficient. The pathway inference is derived from learning Bayesian networks in combination with prior knowledge such as Protein-Protein Interactions (PPIs) and signaling pathways from well-known databases. Learning Bayesian networks is based on a score and search scheme that provides the highest scored network structure given a score function, and the prior knowledge is included in the score function as a prior probability by using Dempster-Shafer theory (DST). In this way, the inferred network can be more likely to be similar to already discovered pathways and consistent with confirmed PPIs for more reliable inference. The experimental results show which proteins are involved in signaling pathways under IR, how the inferred pathways are different under low and high doses of IR, and how the selected proteins regulate each other in the inferred pathways. As our main contribution, overall results confirm that low dose IR could cause DNA damage and thereby induce and affect related signaling pathways such as apoptosis, cell cycle, and DNA repair.

The hypoxia-activated prodrug evofosfamide in combination with multiple regimens of radiotherapy.

The promising treatment combination of ionizing radiation (IR) with a hypoxia-activated prodrug (HAP) is based on biological cooperation. Here we investigated the hypoxia-activated prodrug evofosfamide in combination with different treatment regimens of IR against lung A549- and head&neck UT-SCC-14-derived tumor xenografts. DNA damage-related endpoints and clonogenic cell survival of A549 and UT-SCC-14 carcinoma cells were probed under normoxia and hypoxia.Evofosfamide (TH-302) induced DNA-damage and a dose-dependent antiproliferative response in A549 cells on cellular pretreatment under hypoxia, and supra-additively reduced clonogenic survival in combination with IR. Concomitant treatment of A549-derived tumor xenografts with evofosfamide and fractionated irradiation induced the strongest treatment response in comparison to the corresponding neoadjuvant and adjuvant regimens. Adjuvant evofosfamide was more potent than concomitant and neoadjuvant evofosfamide when combined with a single high dose of IR. Hypoxic UT-SCC-14 cells and tumor xenografts thereof were resistant to evofosfamide alone and in combination with IR, most probably due to reduced P450 oxidoreductase expression, which might act as major predictive determinant of sensitivity to HAPs.In conclusion, evofosfamide with IR is a potent combined treatment modality against hypoxic tumors. However, the efficacy and the therapeutic outcome of this combined treatment modality is, as indicated here in preclinical tumor models, dependent on scheduling parameters and tumor type, which is most probably related to the status of respective HAP-activating oxidoreductases. Further biomarker development is necessary for the launch of successful clinical trials.

Biochemical Changes Induced by Gamma Irradiation in the Ground Beetle Blaps polycresta

The exposure to low-dose gamma radiation from natural sources is a continuing and inescapable feature and it is common in certain occupations. The present study was designed to investigate the effects of low-dose gamma radiation on some detoxification enzymes such as; AST, ALT, ACP, ALP, GSH, GPX, SOD and CAT, as well as, its effect on total protein and DNA content in the soft tissues homogenate of male’s ground beetle Blaps polycresta. Insects were divided into three groups (group A, B and C) of 10 beetles each. Two groups (group B and C) were irradiated with doses at a rate of 32 Gy and 64 Gy; group A was considered as the control group. The study revealed a dose-dependent effect on the tested parameters. Significant increase in the enzyme activity of AST, ALT, ACP and ALP were observed in group B and C respectively compared to the control group (group A). The findings also showed a significant inhibitions in the enzymatic activity of GSH, GST, GPX, SOD and CAT in the irradiated groups compared to the control group. Furthermore, their radiated groups reported a decrease in protein and DNA content compared to the control group. Our observations suggested that the genotoxic effects of gamma radiation induced damaging effects on enzyme molecules, protein and nucleic acid (DNA) and may explain the increase in health risk from high doses of ionizing radiation.