Adeola Y. Makinde

Low-Dose Photons Modify Liver Response to Simulated Solar Particle Event Protons

Abstract Gridley, D. S., Coutrakon, G. B., Rizvi, A., Bayeta, E. J. M., Luo-Owen, X., Makinde, A. Y., Baqai, F., Koss, P., Slater, J. M. and Pecaut, M. J. Low-Dose Photons Modify Liver Response to Simulated Solar Particle Event Protons. Radiat. Res. 169, 280–287 (2008). The health consequences of exposure to low-dose radiation combined with a solar particle event during space travel remain unresolved. The goal of this study was to determine whether protracted radiation exposure alters gene expression and oxidative burst capacity in the liver, an organ vital in many biological processes. C57BL/6 mice were whole-body irradiated with 2 Gy simulated solar particle event (SPE) protons over 36 h, both with and without pre-exposure to low-dose/low-dose-rate photons (57Co, 0.049 Gy total at 0.024 cGy/h). Livers were excised immediately after irradiation (day 0) or on day 21 thereafter for analysis of 84 oxidative stress-related genes using RT-PCR; genes up or down-regulated by more than twofold were noted. On day 0, genes with increased expression were: photons, none; simulated SPE, Id1; photons + simulated SPE, Bax, Id1, Snrp70. Down-regulated genes at this same time were: photons, Igfbp1; simulated SPE, Arnt2, Igfbp1, Il6, Lct, Mybl2, Ptx3. By day 21, a much greater effect was noted than on day 0. Exposure to photons + simulated SPE up-regulated completely different genes than those up-regulated after either photons or the simulated SPE alone (photons, Cstb; simulated SPE, Dctn2, Khsrp, Man2b1, Snrp70; photons + simulated SPE, Casp1, Col1a1, Hspcb, Il6st, Rpl28, Spnb2). There were many down-regulated genes in all irradiated groups on day 21 (photons, 13; simulated SPE, 16; photons + simulated SPE, 16), with very little overlap among groups. Oxygen radical production by liver phagocytes was significantly enhanced by photons on day 21. The results demonstrate that whole-body irradiation with low-dose-rate photons, as well as time after exposure, had a great impact on liver response to a simulated solar particle event.

Low-dose Photon and Simulated Solar Particle Event Proton Effects on Foxp3+ T Regulatory Cells and other Leukocytes

Radiation is a major factor in the spaceflight environment that has carcinogenic potential. Astronauts on missions are continuously exposed to low-dose/low-dose-rate (LDR) radiation and may receive relatively high doses during a solar particle event (SPE) that consists primarily of protons. However, there are very few reports in which LDR photons were combined with protons. In this study, C57BL/6 mice were exposed to 1.7 Gy simulated SPE (sSPE) protons over 36 h, both with and without pre-exposure to 0.01 Gray (Gy) LDR γ-rays at 0.018 cGy/h. Apoptosis in skin samples was determined by immunohistochemistry immediately post-irradiation (day 0). Spleen mass relative to body mass, white blood cells (WBC), major leukocyte populations, lymphocyte subsets (T, Th, Tc, B, NK), and CD4+ CD25+ Foxp3+ T regulatory (Treg) cells were analyzed on days 4 and 21. Apoptosis in skin samples was evident in all irradiated groups; the LDR+sSPE mice had the greatest expression of activated caspase-3. On day 4 post-irradiation, the sSPE and LDR+sSPE groups had significantly lower WBC counts in blood and spleen compared to non-irradiated controls (p < 0.05 vs. 0 Gy). CD4+ CD25+ Foxp3+ Treg cell numbers in spleen were decreased at day 4, but proportions were increased in the sSPE and LDR+sSPE groups (p < 0.05 vs. 0 Gy). By day 21, lymphocyte counts were still low in blood from the LDR+sSPE mice, especially due to reductions in B, NK, and CD8+ T cytotoxic cells. The data demonstrate, for the first time, that pre-exposure to LDR photons did not protect against the adverse effects of radiation mimicking a large solar storm. The increased proportion of immunosuppressive CD4+ CD25+ Foxp3+ Treg and persistent reduction in circulating lymphocytes may adversely impact immune defenses that include removal of sub-lethally damaged cells with carcinogenic potential, at least for a period of time post-irradiation.

Differential Expression of Stress and Immune Response Pathway Transcripts and miRNAs in Normal Human Endothelial Cells Subjected to Fractionated or Single-Dose Radiation

Although modern radiotherapy technologies can precisely deliver higher doses of radiation to tumors, thus, reducing overall radiation exposure to normal tissues, moderate dose, and normal tissue toxicity still remains a significant limitation. The present study profiled the global effects on transcript and miR expression in human coronary artery endothelial cells using single-dose irradiation (SD, 10 Gy) or multifractionated irradiation (MF, 2 Gy × 5) regimens. Longitudinal time points were collected after an SD or final dose of MF irradiation for analysis using Agilent Human Gene Expression and miRNA microarray platforms. Compared with SD, the exposure to MF resulted in robust transcript and miR expression changes in terms of the number and magnitude. For data analysis, statistically significant mRNAs (2-fold) and miRs (1.5-fold) were processed by Ingenuity Pathway Analysis to uncover miRs associated with target transcripts from several cellular pathways after irradiation. Interestingly, MF radiation induced a cohort of mRNAs and miRs that coordinate the induction of immune response pathway under tight regulation. In addition, mRNAs and miRs associated with DNA replication, recombination and repair, apoptosis, cardiovascular events, and angiogenesis were revealed. Implications: Radiation-induced alterations in stress and immune response genes in endothelial cells contribute to changes in normal tissue and tumor microenvironment, and affect the outcome of radiotherapy. Mol Cancer Res; 12(7); 1002–15. ©2014 AACR.

Comparison of proton and electron radiation effects on biological responses in liver, spleen and blood.

Purpose: To determine whether differences exist between proton and electron radiations on biological responses after total-body exposure. Materials and methods: ICR mice (n = 45) were irradiated to 2 Gray (Gy) using fully modulated 70 MeV protons (0.5 Gy/min) and 21 MeV electrons (3 Gy/min). At 36 h post-irradiation liver gene expression, white blood cell (WBC), natural killer (NK) cell and other analyses were performed. Results: Oxidative stress-related gene expression patterns were strikingly different for irradiated groups compared to 0 Gy (P < 0.05). Proton radiation up-regulated 15 genes (Ctsb, Dnm2, Gpx5, Il19, Il22, Kif9, Lpo, Nox4, Park7, Prdx4, Prdx6, Rag2, Sod3, Srxn1, Xpa) and down-regulated 2 genes (Apoe, Prdx1). After electron irradiation, 20 genes were up-regulated (Aass, Ctsb, Dnm2, Gpx1, Gpx4, Gpx5, Gpx6, Gstk1, Il22, Kif9, Lpo, Nox4, Park7, Prdx3, Prdx4, Prdx5, Rag2, Sod1, Txnrd3, Xpa) and 1 was down-regulated (Mpp4). Of the modified genes, only 11 were common to both forms of radiation. Comparison between the two irradiated groups showed that electrons significantly up-regulated three genes (Gstk1, Prdx3, Scd1). Numbers of WBC and major leukocyte types were low in the irradiated groups (P < 0.001 vs. 0 Gy). Hemoglobin and platelet counts were low in the electron-irradiated group (P < 0.05 vs. 0 Gy). However, spleens from electron-irradiated mice had higher WBC and lymphocyte counts, as well as enhanced NK cell cytotoxicity, compared to animals exposed to protons (P < 0.05). There were no differences between the two irradiated groups in body mass, organ masses, and other assessed parameters, although some differences were noted compared to 0 Gy. Conclusion: Collectively, the data demonstrate that at least some biological effects induced by electrons may not be directly extrapolated to protons.

A Metalloporphyrin Antioxidant Alters Cytokine Responses after Irradiation in a Prostate Tumor Model

Abstract The goal of this study was to evaluate cytokine secretion capacity in a mouse model of prostate cancer, both with and without metalloporphyrin antioxidant and radiation treatment. C57BL/6 mice with subcutaneous RM-9 tumors were treated daily for 12 days with MnTE-2-PyP5+ [Mn (III) tetrakis (N-ethylpyridinium-2-yl) porphyrin], beginning 1 day after injection of RM-9 cells; a 10-Gy tumor-localized dose of 60Co γ rays was administered in a single fraction on day 7. Spleen, tumors and plasma were collected on day 12. T cells in the spleen were activated with anti-CD3 antibody and supernatants were collected. Twenty-two cytokines were quantified in spleen supernatants, five in tumor homogenates, and three in plasma using multiplex bead array technology and ELISA. The presence of a tumor had significant effects on many of the cytokines quantified (P < 0.05). Tumor-induced depression was evident for eight spleen cytokines (TNF-α, G-CSF, GM-CSF, IFN-γ, IL10, IP-10, MIP-1α and mKC), whereas only three were enhanced (IL1β, IL6 and MCP-1). Radiotherapy resulted in enhanced splenocyte capacity to produce IL4 and IL13 and increased IL4, MCP-1 and VEGF in tumors (P < 0.05). Addition of MnTE-2-PyP5+ to radiation decreased the concentrations of IL4, IL13 and TGF-β1 in spleen supernatants and IL4 and VEGF in tumors (P < 0.05 compared to radiation alone). Some differences were also noted in plasma cytokines. Overall, the findings suggest that administration of MnTE-2-PyP5+ together with radiotherapy may enhance anti-tumor immune responsiveness and decrease the risk for radiation-induced normal tissue toxicities.

Space-relevant radiation modifies cytokine profiles, signaling proteins and Foxp3+ T cells

Abstract Purpose: The major goal was to evaluate effects of various radiation regimens on leukocyte populations relatively long-term after whole-body irradiation. Materials and methods: C57BL/6 mice were exposed to-low-dose/low-dose rate (LDR) 57Co γ-rays (0.01 Gy, 0.03 cGy/h), with and without acute 2 Gy proton (1 Gy/min) or γ-ray (0.9 Gy/min) irradiation; analyses were done on days 21 and 56 post-exposure. Results: Numerous radiation-induced changes were noted at one or both time points. Among the most striking differences (P < 0.05) were: (i) High percentage of CD4+CD25+Foxp3+ T cells in spleens from the Proton vs. LDR, Gamma and LDR + Proton groups (day 56); (ii) high interleukin-2 (IL-2) in spleen supernatants from the LDR and LDR + Proton groups vs. 0 Gy (day 56), whereas IL-10 was high in the LDR + Gamma group vs. 0 Gy (day 56); (iii) difference in transforming growth factor-β1 (TGF-β1) in spleen supernatants from Proton and LDR + Proton groups vs. Gamma and LDR + Gamma groups (both days); (iv) low TGF-β1 in blood from LDR + Proton vs. LDR + Gamma group (day 21); and (v) high level of activated cJun N-terminal kinase (JNK) in CD4+ T cells from LDR + Proton vs. LDR + Gamma group (day 21). Conclusions: The findings demonstrate that at least some immune responses to acute 2 Gy radiation were dependent on radiation quality time of assessment, and pre-exposure to LDR γ-rays.

Radiation-Induced Long Noncoding RNAs in a Mouse Model after Whole-Body Irradiation

Long noncoding RNAs (lncRNAs) are emerging as key molecules in regulating many biological processes and have been implicated in development and disease pathogenesis. Biomarkers of cancer and normal tissue response to treatment are of great interest in precision medicine, as well as in public health and medical management, such as for assessment of radiation injury after an accidental or intentional exposure. Circulating and functional RNAs, including microRNAs (miRNAs) and lncRNAs, in whole blood and other body fluids are potential valuable candidates as biomarkers. Early prediction of possible acute, intermediate and delayed effects of radiation exposure enables timely therapeutic interventions. To address whether long noncoding RNAs (lncRNAs) could serve as biomarkers for radiation biodosimetry we performed whole genome transcriptome analysis in a mouse model after whole-body irradiation. Differential lncRNA expression patterns were evaluated at 16, 24 and 48 h postirradiation in total RNA isolated from whole blood of mice exposed to 1, 2, 4, 8 and 12 Gy of X rays. Sham-irradiated animals served as controls. Significant alterations in the expression patterns of lncRNAs were observed after different radiation doses at the various time points. We identified several radiation-induced lncRNAs known for DNA damage response as well as immune response. Long noncoding RNA targets of tumor protein 53 (P53), Trp53cor1, Dino, Pvt1 and Tug1 and an upstream regulator of p53, Meg3, were altered in response to radiation. Gm14005 (Morrbid) and Tmevpg1 were regulated by radiation across all time points and doses. These two lncRNAs have important potential as blood-based radiation biomarkers; Gm14005 (Morrbid) has recently been shown to play a key role in inflammatory response, while Tmevpg1 has been implicated in the regulation of interferon gamma. Precise molecular biomarkers, likely involving a diverse group of inducible molecules, will not only enable the development and effective use of medical countermeasures but may also be used to detect and circumvent or mitigate normal tissue injury in cancer radiotherapy.

Microarray analysis of miRNA expression profiles following whole body irradiation in a mouse model

Abstract Context: Accidental exposure to life-threatening radiation in a nuclear event is a major concern; there is an enormous need for identifying biomarkers for radiation biodosimetry to triage populations and treat critically exposed individuals. Objective: To identify dose-differentiating miRNA signatures from whole blood samples of whole body irradiated mice. Methods: Mice were whole body irradiated with X-rays (2 Gy–15 Gy); blood was collected at various time-points post-exposure; total RNA was isolated; miRNA microarrays were performed; miRNAs differentially expressed in irradiated vs. unirradiated controls were identified; feature extraction and classification models were applied to predict dose-differentiating miRNA signature. Results: We observed a time and dose responsive alteration in the expression levels of miRNAs. Maximum number of miRNAs were altered at 24-h and 48-h time-points post-irradiation. A 23-miRNA signature was identified using feature selection algorithms and classifier models. An inverse correlation in the expression level changes of miR-17 members, and their targets were observed in whole body irradiated mice and non-human primates. Conclusion: Whole blood-based miRNA expression signatures might be used for predicting radiation exposures in a mass casualty nuclear incident.

Abstract C146: Radiation-inducible molecular targets in a human prostate cancer mouse model

Background: In order to understand the changes induced in tumor cells following multi-fraction (MF) radiation therapy, we have previously studied molecular changes using prostate cancer cells and endothelial cells treated in vitro with MF doses of 0.5 Gy/1 Gy x 10 and 2 Gy x 5 and single-dose (SD) of 5 Gy and 10 Gy. The hypothesis being tested is that the response and adaptation to radiation-induced stress will produce a druggable phenotype. This might increase the utility of molecularly targeted therapeutics and also help address tumor cell heterogeneity. The data indicate more genes and pathways are induced by MF compared to SD and that the change in phenotype is more stable following MF. In this report, the focus is on new data from PC-3 cells irradiated in vivo and comparing it to MF and SD in vitro using MF 1 Gy x 10 and SD 10 Gy. Methods: PC-3 prostate cancer cells were implanted subcutaneously into the lateral aspect of rear leg of nude mice. Mice were divided into three groups (n = 3), based on radiation dose/schedule- control, SD, and MF. SD and MF employed similar dose/schedule as used for the in vitro studies, 10 Gy x 1 and 1 Gy x 10 respectively. RNA was isolated 24 h after radiation treatment. mRNA microarray analysis was performed using Agilent Technologies Human Gene Expression 4 × 44 K V2 microarrays. The data was generated and analyzed with GeneSpring® software (Agilent Technologies, Santa Clara, CA) and IPA software (IPA, QIAGEN, Redwood City, CA). Results: 6,374 genes were significantly altered by MF, with a cohort of genes, based on the > 250 gene ontology categories, involved in DNA response to stimulus, DNA repair, mitosis, cell cycle, and metabolism. In contrast, only 453 genes were significantly altered by SD, with ontological categories associated with cell morphology, assembly and organization such as actin filament-based process, extracellular matrix organization and biogenesis, fibril organization and biogenesis and collagen catabolism. Further bioinformatics analysis of the gene expression data with IPA, identified multiple pathways with functions correlated with the ontological categories. Significantly altered MF-induced genes are members of pathways which play a central role in DNA replication, recombination, and repair, cell proliferation and metabolism such as HIPPO Signaling, Protein Ubiquitination Pathway, JNK/SAPK Signaling, ERK/MAPK Signaling, G2/M DNA Damage Checkpoint Regulation, ATM Signaling, PI3K/AKT Signaling and Oxidative Phosphorylation. These pathways were uniquely up-regulated by MF treatment, as none of these changes were identified with SD radiation exposure. Conclusion: Our result show the differential expression pattern between SD and MF, with MF inducing changes in “targetable” molecular pathways. Ongoing studies: Currently we are in the process of evaluating radiation-induced targets in ATM signaling, DNA damage and repair, and multiple metabolic targets, and their potential for using radiation to prime cells for molecular-targeted drug therapy. Citation Format: Adeola Y. Makinde, Iris Eke, Molykutty J. Aryankalayil, Mansoor Ahmed, C. Norman Coleman. Radiation-inducible molecular targets in a human prostate cancer mouse model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C146.

Abstract 425: Fractionated radiation-induced tumor suppressor microRNAs in human prostate carcinoma cells.

To understand the role of microRNAs (miRNA) in regulation of radiation-induced gene expression and to help define potential radiation-inducible targets, miRNA expression was studied in wild type p53 LNCaP and p53-mutated PC3 and DU145 cells. We have previously investigated the changes in the molecular profiles of tumor cells that were exposed to single dose (SD) versus fractionated radiation (MF) in vitro, and identified immune and stress response pathways that were induced by fractionated but not single dose radiation. Methods: Cells were exposed to 5Gy and 10Gy either as SD or MF radiation radiation. Microarray analyses were done using human Agilent miRNA Microarray Kit (V2). Data were analyzed using Gene Spring software. Validation of the miRNA expression and gene expression of miRNA targets was evaluated by real-time RT-PCR analysis. Target filter analysis of differentially expressed miRNAs (>1.5 fold change and p value 1.5fold change, p This work was supported by the Intramural Research Program of the NIH, NCI, CCR. Citation Format: C. Norman Coleman, Molykutty John-Aryankalayil, Adeola Y. Makinde, Sanjeewani T. Palayoor. Fractionated radiation-induced tumor suppressor microRNAs in human prostate carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 425. doi:10.1158/1538-7445.AM2013-425