Sublethal Irradiation Research Articles

Impaired hematopoiesis and delayed thrombopoietic recovery following sublethal irradiation in SRC-3 knockout mice

The objective of the present study was to investigate the role of the steroid receptor coactivator-3 (SRC-3) in hematopoiesis of mouse bone marrow (BM) following total body irradiation (TBI). SRC-3−/− mice and wild-type (WT) mice were exposed to 4.5 Gy γ rays. Immunoblotting analysis revealed that the SRC-3 protein (p160) levels in normal BM-nucleated cells in WT were higher than in SRC-3−/− mice. Furthermore, peripheral blood cell counts, BM cellularity and colony-forming unit (CFU) assays were performed following irradiation. The results showed that peripheral blood cells were significantly lower in number and recovered less rapidly in irradiated SRC-3−/− mice as compared with control animals. BM-nucleated cell and CFU counts were significantly decreased in SRC-3−/− mice on the 7th and 14th day. Of note, the recovery of platelet (PLT) and megakaryocytic lineage were more depressed than the granulocytic and erythroid lineage in SRC-3−/− mice. In conclusion, the present study demonstrated that the hematopoietic ability in SRC-3 knockout mice is severely impaired following a sublethal dose of irradiation.

Complete remission of cancer in late-stage disease by radiation and transfer of allogeneic MHC-matched immune T cells: lessons from GvL studies in animals.

Most immunotherapy studies in animal tumor models are performed in early stages of the disease. Reports on the studies of treatment in late stages of tumor growth and metastasis are much rarer. To guide future efforts for treatment in late-stage disease, a model of effective immune rejection of advanced metastasized cancer is reviewed and lessons therefrom are summarized. Already cachectic DBA/2 mice with a subcutaneously transplanted syngeneic tumor (ESb-MP lymphoma) of 1.5 cm diameter and with macroscopic liver and kidney metastases at 4 weeks could be successfully treated by a combination of sublethal (5 Gy) irradiation followed by a single transfer of 20 million anti-tumor immune spleen cells from tumor-resistant allogeneic MHC-B10.D2 mice. Following intravenous cell transfer, the primary tumors became encapsulated and were eventually rejected from the skin while visceral metastases gradually disappeared leaving behind only scar tissue. There was wound-healing at the site of the rejected primary tumor, and the animals survived long term without any tumor recurrence. The complete eradication of late-stage disease by adoptive cellular immunotherapy could be corroborated noninvasively by (31)P-NMR spectroscopy of primary tumors and by (1)H-NMR microimaging of liver metastases. Conclusions from functional mechanistic studies in this model are summarized and clinical implications discussed.

Effect of irradiation of renal cell carcinoma (RCC) cell lines on RCC tumor-associated antigen (TAA) specific CD8+ T-cell stimulation.

542 Background: Radiation increases the immunogenicity of many cancers. We have previously shown increased immunogenicity of RCC with cell line irradiation. Herein, we describe the effect of irradiation of RCC cell lines on the expression of TAAs as well as several molecules involved in CD8+ T Cell (CTL) mediated tumor cytotoxicty and the in vitro effect of tumor irradiation on NY-ESO-1 specific CTL clones. Methods: Six RCC cell lines (ACHN, 786-0, 769-P, Caki-1, A-498, A-704) were cultured and exposed to 0, 12 or 16 Gy of ionizing radiation. Cells were isolated at 2, 24 and 48 hours after irradiation. Expression of cell surface molecules (CD95, CD54, Calreticulin, NYESO-1, CA-9, 5T4, MUC-1 and HSP-70) was assessed using multi-color flow cytometry. Results are reported as mean relative increase from control (dose 0 Gy, time 2 hours post irradiation). In a second experiment, NY-ESO-1 specific CTL clones were cultured in the presence of A-498 cells that were subjected to radiation under the above noted conditions and assessed using an ELISPOT assay. Negative control consisted of only CD8+ T Cells. Results: The ELISPOT experiment demonstrated stimulation of NY-ESO-1-specific CTL by irradiated A-498 cells. Negative control showed no stimulation, with only 4 spots. An increase in spots was observed from 79 and 82 spots at 2 hours post-irradiation to 166 and 205 spots at 48 hours post-irradiation for 0 Gy and 16 Gy, respectively. Conclusions: Sublethal irradiation of human RCC cell lines increases their immunogenicity, provoking TAA-specific T cell stimulation. [Table: see text]

Mathematical Modeling Predicts Enhanced Growth of X-Ray Irradiated Pigmented Fungi

Ionizing radiation is known for its cytotoxic and mutagenic properties. However, recent evidence suggests that chronic sub-lethal irradiation stimulates the growth of melanin-pigmented (melanized) fungi, supporting the hypothesis that interactions between melanin and ionizing photons generate energy useful for fungal growth, and/or regulate growth-promoting genes. There are no quantitative models of how fungal proliferation is affected by ionizing photon energy, dose rate, and presence versus absence of melanin on the same genetic background. Here we present such a model, which we test using experimental data on melanin-modulated radiation-induced proliferation enhancement in the fungus Cryptococcus neoformans, exposed to two different peak energies (150 and 320 kVp) over a wide range of X-ray dose rates. Our analysis demonstrates that radiation-induced proliferation enhancement in C. neoformans behaves as a binary “on/off” phenomenon, which is triggered by dose rates <0.002 mGy/h, and stays in the “on” position. A competing dose rate-dependent growth inhibition becomes apparent at dose rates >5000 mGy/h. Proliferation enhancement of irradiated cells compared with unirradiated controls occurs at both X-ray peak energies, but its magnitude is modulated by X-ray peak energy and cell melanization. At dose rates <5000 mGy/h, both melanized and non-melanized cells exposed to 150 kVp X-rays, and non-melanized cells exposed to 320 kVp X-rays, all exhibit the same proliferation enhancement: on average, chronic irradiation stimulates each founder cell to produce 100 (95% CI: 83, 116) extra descendants over 48 hours. Interactions between melanin and 320 kVp X-rays result in a significant (2-tailed p-value = 4.8×10−5) additional increase in the number of radiation-induced descendants per founder cell: by 55 (95% CI: 29, 81). These results show that both melanin-dependent and melanin-independent mechanisms are involved in radiation-induced fungal growth enhancement, and implicate direct and/or indirect interactions of melanin with high energy ionizing photons as an important pro-proliferative factor.

Radiation-induced immunogenic modulation of tumor enhances antigen processing and calreticulin exposure, resulting in enhanced T-cell killing

Radiation therapy (RT) is used for local tumor control through direct killing of tumor cells. Radiation-induced cell death can trigger tumor antigen-specific immune responses, but these are often noncurative. Radiation has been demonstrated to induce immunogenic modulation (IM) in various tumor types by altering the biology of surviving cells to render them more susceptible to T cell-mediated killing. Little is known about the mechanism(s) underlying IM elicited by sub-lethal radiation dosing. We have examined the molecular and immunogenic consequences of radiation exposure in breast, lung, and prostate human carcinoma cells. Radiation induced secretion of ATP and HMGB1 in both dying and surviving tumor cells. In vitro and in vivo tumor irradiation induced significant upregulation of multiple components of the antigen-processing machinery and calreticulin cell-surface expression. Augmented CTL lysis specific for several tumor-associated antigens was largely dictated by the presence of calreticulin on the surface of tumor cells and constituted an adaptive response to endoplasmic reticulum stress, mediated by activation of the unfolded protein response. This study provides evidence that radiation induces a continuum of immunogenic alterations in tumor biology, from immunogenic modulation to immunogenic cell death. We also expand the concept of immunogenic modulation, where surviving tumor cells recovering from radiation-induced endoplasmic reticulum stress become more sensitive to CTL killing. These observations offer a rationale for the combined use of radiation with immunotherapy, including for patients failing RT alone.

SRSF2 Is Essential For Hematopoiesis and Its Mutations Dysregulate Alternative RNA Splicing In MDS

Myelodysplastic syndromes (MDS) are a group of neoplasms that are ineffective in generating multiple lineages of myeloid cells and have various risks to progress to acute myeloid leukemia. Recent genome-wide sequencing studies reveal that mutations in genes of splicing factors are commonly associated with MDS. However, the importance of these splicing factors in hematopoiesis has been unclear and the causal effect of their mutations on MDS development remains to be determined. One of these newly identified genes is SRSF2, and its mutations have been linked to poor survival among MDS patients. Interestingly, most of SRSF2 mutations occur at proline 95 and the majority of these mutations change this proline to histidine (P95H). Given that SRSF2 is a well-characterized splicing factor involved in both constitutive and regulated splicing, we hypothesize that SRSF2 plays an important role in normal hematopoiesis and the SRSF2 mutations induce specific changes in alternative splicing that favor disease progression. We first examined the role of SRSF2 in hematopoiesis by generating Srsf2 null mutation in mouse blood cells via crossing conditional Srsf2 knockout mice (Srsf2f/f) with blood cell-specific Cre transgenic mice (Vav-Cre). The mutant mice produced significantly fewer definitive blood cells (10% of wild type controls), exhibited increased apoptosis in the remaining blood cells, and died during embryonic development. Importantly, we detected no hematopoietic stem/progenitor cells (lineage-/cKit+) in E14 fetal livers of Vav-Cre/Srsf2f/f mice. These results indicate that SRSF2 is essential for hematopoiesis during embryonic development. We next examined the role of SRSF2 in adult hematopoiesis by injecting polyIC into mice that carry a polyIC inducible Cre expression unit. Unexpectedly, after multiple polyIC treatments, the Srsf2f/f mice stayed alive during several months of observation. Time course genotyping analyses of polyIC treated mice revealed an increased rate of incomplete Srsf2 deletion in peripheral blood cells. These observations suggest that Srsf2 ablation did not cause immediate cell lethality in differentiated blood cells, but the gene is indispensable for the function of blood stem/progenitor cells. Since mutations of splicing factors are generally heterozygous in MDS patients, we also examined mice with Srsf2+/- blood cells. No obvious defect of hematopoiesis was observed under normal conditions or in response to stress with 5-FU treatment and sublethal irradiation. To gain molecular insight into the splicing activity of MDS-associated mutant forms of SRSF2, we performed large-scale alternative splicing surveys by using RNA-mediated oligonucleotide annealing, selection, and ligation coupled with next-generation sequencing (RASL-seq) previously developed in our lab, which offers a robust and cost-effective platform for splicing profiling. Compared to vector transduction controls, we found that overexpression of both wild type and P95H SRSF2 induced many, but distinct changes in alternative splicing in lineage-negative bone marrow cells, and importantly, we noted several changes in genes with known roles in hematopoietic malignancies that were uniquely induced by the mutant SRSF2. To further link the mutations to altered splicing in MDS patients, we also applied RASL-seq to a large number of MDS patient samples with or without mutations in SRSF2 or other splicing regulators. The data revealed a specific set of alternative splicing events that are commonly linked to MDS with splicing factor mutations. These findings strongly suggest that many of these mutations in splicing regulators are gain-of-function mutations that are causal to MDS. In conclusion, we report that SRSF2 plays an essential role in hematopoietic stem/progenitor cells and that the MDS-associated mutations in SRSF2 have a dominant effect on RNA alternative splicing. These findings provide functional information and molecular basis of SRSF2 and its MDS-related mutations in hematopoiesis and related clinical disorders. Disclosures:No relevant conflicts of interest to declare.

Hhex Is a Critical Gene In The Development Of Normal and Malignant Lymphoid Cells

Hematopoietically expressed homeobox (Hhex) is a T-cell oncogene. It is frequently deregulated in murine retroviral insertional mutagenesis screens and its enforced expression induces T-cell leukemia in bone marrow transduction and transplantation experiments. We discovered that HHEX is a direct transcriptional target of an LIM domain Only-2 (LMO2)-associated protein complex. HHEX clusters with LMO2-overexpressing T-ALLs and is especially overexpressed in Early T-cell Precursor (ETP) – ALL where it is a direct transcriptional target of LMO2. To further understand Hhex's function, we induced a conditional knockout in floxed Hhex mice with the Vav-iCre transgene. Mice were viable and showed normal blood cell counts with highly efficient deletion of Hhex in all hematopoietic tissues. Thymocytes from conditional knockouts showed a normal pattern of development. Most impressively, Hhex conditional knockout markedly prolonged the latency of T-ALL onset in CD2-Lmo2 transgenic mice (figure 1). Hhex conditional knockouts (Hhex cKOs) also had a significant decrease in mature B cells in the spleen and bone marrow. Interestingly, hematopoietic stem and progenitor cells plated on OP9-GFP or OP9-DL1 stromal cells showed proliferative defects and incomplete differentiation towards both B and T lineage. Also under stress conditions such as sublethal irradiation and competitive bone marrow transplants, Hhex conditional knockouts show a marked defect in both B and T lineages but an increase in early progenitor populations. Our experiments show that Hhex is a critical transcription factor in lymphoid development and in LMO2-induced T-ALL. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Lentiviral Transduction Of Notch Ligand Expanded Cord Blood HSC's To Express a CD19-Specific CAR Generates NK and Myeloid Progeny With Antitumor Activity

Over the past decade, significant progress has been made using chimeric antigen receptors (CAR) to redirect the specificity of T cells against an array of cell surface tumor antigens. Using this same technology, we hypothesized that CD34+ hematopoietic stem and progenitor cells (HSPC) derived from cord blood (CB) could be transduced with lentivirus to efficiently express a CAR specific for CD19 that would enable targeted lysis through its continued expression in differentiated progeny effector cells. As previously reported, the Notch-mediated ex vivo expansion of CB HSPC is a clinically validated cell therapy product that is well tolerated, can be given off the shelf without HLA matching, and provides transient myeloid engraftment in both the HCT and intensive chemotherapy setting. CB-derived CD34+ cells are selected using the Miltenyi CliniMACS or AutoMACS platform and plated in StemSpan serum free expansion media supplemented with IL-6, IL-3, TPO, Flt3L, and SCF and in the presence of immobilized Notch ligand and retronectin. The cells are cultured for 14-16 days resulting in >150 fold expansion of CD34+ cells on average and a final heterogeneous cell product consisting of 14.5% (range 6.2-26) CD34+, 20.5% (6-36) CD15+, 11.3 (1.8-23) CD14+, 5.4%(2.2-13.6) CD56+CD16+, and 0.1(0-0.2)CD20+ which is cryopreserved for future clinical use. Importantly, no T cells are derived from this culture system. Off the shelf expanded units have been infused into 45 patients and no serious adverse events have been noted except for one allergic reaction attributed to DMSO. Additionally, there has been no persistent engraftment beyond day 180 in the HCT setting and 14 days post infusion in the chemotherapy setting.To engineer anti-CD19 activity into this product, cells are transduced at an MOI of 3 on day 3 of culture using a self inactivating lentivirus that encodes for a second generation CD19 CAR with 41BB costimulation as well as an EGFR tag which lacks an intracellular signaling domain and serves as a marker of transduction and a selection marker. Transduction efficiency ranges from 20-40% and equally transduces the CD34+ and CD34- populations. Copy number analysis demonstrates between 1-4 copies/cell. Expression of the transgene does not affect the final culture phenotype at 14 days and transgene expression is seen in all cell subsets and is stable over the culture period. Due to concerns of expression of the CAR on HSC with potential signaling capacity, irradiated LCL was added on day 7 of culture at a 1:1 ratio to provide antigen stimulation and did not negatively affect the growth of the cultures, nor the expression of the transgene. Functional NK cells were derived in vitro from the end culture population by an additional week of culture with RPMI supplemented with L-glutamine, human serum, IL-2 and IL-15. The CD19-CAR expressing NK cells had enhanced cytotoxic activity in a CRA against LCL compared with non-transduced NK cells (50 v 30%) whereas both killed K562 targets equally (75 v 80%). Ongoing experiments are being conducted to further evaluate the expression, signaling capacity and function of the CAR on each of the cell compartments.The NOG mouse model was used for in vivo functional assays to look at effects of CD19 CAR expression on human repopulating ability and to assess anti-CD19 activity. NOG mice underwent sub-lethal irradiation followed by tail vein injection of the expanded progeny of 20,000 selected CD34+ CB cells. There was no effect on early engraftment of transduced cells, however there was no detectable CD19 engraftment in the mice transplanted with CD19 CAR expressing cells compared with untransduced cells, in which the CD19 population was >20% of the engrafted cells, indicating anti-CD19 activity. NK cell populations were increased using NS0-IL15 secreting cells, irradiated and injected subcutaneously three times per week starting at week 3 to provide enhanced effector function. Current studies are ongoing to evaluate the anti-CD19 activity using the Raji CD19+ tumor model in the NOG mice.These initial results are promising as a way to engineer a graft versus leukemia effect into cord blood transplant. Furthermore, transduction of a CD19 CAR into our existing universal donor expanded CB cell therapy product allows for infusion of an anti-CD19 cell product to be given immediately following identification of a patient with clinical need for therapy, for example one in relapse or with persistent MRD. Disclosures:Jensen:ZetaRx: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Engraftment Of Human Polycythemia Vera CD34+ Cells In hSIRPα-Transgenic-Human-TPO-Expressing RAG2-/-, IL2Rγ-/- Immunodeficient Mice

IntroductionXenotransplantation of human hematopoietic malignancies into immunodeficient mice represents the most appropriate in vivo model system for human malignant hematopoiesis. While a diversity of immunodeficient mouse strains on the NOD/SCID and RAG2-/-/IL2Rγ-/--BALB/C background are described for the most aggressive human malignancies like acute leukemias, xenotransplantation models of less aggressive human hematologic disorders like myeloproliferative neoplasms and myelodysplastic syndromes show only limited engraftment levels. We recently developed next generation mouse strains expressing human cytokines and key factors of xenogeneic cell acceptance (e.g. hSIRPα to inactivate mouse macrophage activation by human cells) and hypothesized that these would represent suitable models for the assessment of human less aggressive hematologic disorders in vivo by providing an optimized “humanized” microenvironment. MethodsPeripheral blood (PB) and bone marrow (BM) samples of polycythemia vera (PV) patients were collected after informed consent at the Division of Hematology, Zurich University Hospital. Human CD34+ cells were isolated by density gradient centrifugation followed by immunomagnetical selection using anti-CD34 coupled beads. Purity of magnetical selection process was confirmed by FACS analysis. Newborn (24h-48h old) hSIRPα-tg-hTPO-knockin mice on the RAG2-/-/IL2Rγ-/--BALB/C background received sublethal irradiation (split dose of 2x1.5 Gy) and were transplanted intra-hepatically 24 hours later. Transplanted cell dose was dependent on availability of CD34+ stem and progenitor cells isolated from one phlebotomy sample (∼400ml of PB) of the respective patient. Mice were bled 4 weeks after transplantation and chimerism in peripheral blood was analyzed by flow cytometry using a panel of antigens (mCD45.2, hCD45, hCD33, hCD34, hCD3, hCD19). Mice showing positive chimerism in PB (i.e.>0.1% hCD45+ of total MNCs) at week 4 were sacrificed between week 8-16 and engraftment in BM, spleen and PB was analyzed by flow cytometry. To verify engraftment of human malignant hematopoiesis we quantified allele-burden of JAK2V617F point mutation in mouse BM using allele specific (AS)-PCR for the pathognomonic point mutation of the JAK2 gene. ResultsBy transplantation of 4-10x105 CD34+ cells into newborn hSIRPα-tg-hTPO-RAG2-/-/IL2Rγ-/- mice we could detect engraftment of hCD45+ cells in PB at week 4 (median 0.68%, range 0.12-23.8%). At week 8, BM engraftment of hCD45+ cells ranged 0.88-54.1% (median 4.43%) with a high proportion of human myeloid cells detected by hCD45/hCD33 co-staining (median 3.07 %, range 0.6-17.6% of total MNCs). We could detect engraftment until week 16, the latest timepoint assessed. Since all transplanted PV patient samples were positive for the common point mutation JAK2V617F, AS-PCR was used to quantify human malignant hematopoiesis. In tested BM samples of engrafted mice we found JAK2V617F positive alleles with a frequency of 2-12% (median 8%). To further assess the clonal composition of the engrafted population we established single cell sorting of primary and engrafted human PV-CD34+ cells in a 96 well format followed by liquid culture expansion and AS-PCR. In pilot studies we could show the clonal composition of a BM engrafted CD34+ population that split into 80% JAK2 WT expressing, 10% JAK2V617F heterozygous and 10 % JAK2V617F homozygous clones. We are currently extending these findings by side by side comparison of the clonal composition of primary vs. xenografted human PV-CD34+ cells of the same patient to test for the influence of a xenogeneic humanized microenvironment on maintenance of malignant cells in vivo. ConclusionsBy using hSIRPα-tg-hTPO-RAG2-/-/IL2Rγ-/- mice we could show engraftment of PV-CD34+ cells that extends previous reported engraftment levels in other model organisms. To our knowledge this is the first study assessing the clonal composition of human PV engrafted cells in the xenogeneic environment aiming at identifying components that are critical for the maintenance of human malignant hematopoiesis in vivo. This model will thus be a useful tool to test targeted therapies in vivo. Disclosures:No relevant conflicts of interest to declare.

A method to generate enhanced GFP+ chimeric mice to study the role of bone marrow-derived cells in the eye

GFP-chimeric mice are important tools to study the role of bone marrow-derived cells in eye physiology. A method is described to generate GFP-chimeric mice using whole-body, sub-lethal radiation (600 rad) of wild-type C57BL/6 recipients followed by tail vein injection of bone marrow cells derived from GFP+ (GFP-transgenic C57/BL/6-Tg(UBC-GFP)30 Scha/J) mice. This method yields stable GFP+ chimeras with greater than 95% chimerism (range 95–99%), achieved within one month of bone marrow transfer confirmed by microscopy and fluorescence-assisted cell sorting (FACS) analysis, with lower mortality after irradiation than prior methods. To demonstrate the efficacy of GFP+ bone marrow chimeric mice, the role of circulating GFP+ bone marrow–derived cells in myofibroblast generation after irregular photo-therapeutic keratectomy (PTK) was analyzed. Many SMA+ myofibroblasts that were generated at one month after PTK were derived from GFP+ bone marrow-derived cells. The GFP+ bone marrow chimeric mouse provides an excellent model for studying the role of bone marrow-derived cells in corneal wound healing, glaucoma surgery, optic nerve head pathology and retinal pathophysiology and wound healing.

Cold Storage Enhances the Efficacy and Margin of Security in Postharvest Irradiation Treatments Against Fruit Flies (Diptera: Tephritidae)

Cold storage is used to preserve fruit quality after harvest during transportation in marketing channels. Low temperature can be a stressor for insects that reduces survivorship, and cold storage may contribute to the efficacy of postharvest quarantine treatments such as irradiation against quarantine insect pests. The combined effect of irradiation and cold storage was examined in a radiation-tolerant fruit fly, Bactrocera cucurbitae Coquillet (melon fly), and a radiation-intolerant fruit fly, Ceratitis capitata (Wiedemann) (Mediterranean fruit fly) (Diptera: Tephritidae). Third instars on diet or in papaya were treated with a sublethal radiation dose of 30 Gy and stored at 4 or 11 degrees C for 3-13 d and held for adult emergence. For both fruit fly species, survival of third instars to the adult stage generally decreased with increasing cold storage duration at 4 or 11 degrees C in diet or papaya. Survivorship differences were highly significant for the effects of substrate (diet > papaya), temperature (11 > 4 degrees C),and irradiation (0 > 30 Gy). Few Mediterranean fruit flies survived in any cold storage treatment after receiving a radiation dose of 30 Gy. No melon fly larvae survived to the adult stage after irradiation and 11 d cold storage at 4 or 11 degrees C in papayas. Cold storage enhances the efficacy and widens the margin of security in postharvest irradiation treatments. Potentially irradiation and cold storage can be used in combination to reduce the irradiation exposure requirements of quarantine treatments.

Influence of Modified Atmosphere Packaging on Radiation Tolerance in the Phytosanitary Pest Melon Fly (Diptera: Tephritidae)

Modified atmosphere packaging (MAP) produces a low-oxygen (O2) environment that can increase produce shelf life by decreasing product respiration and growth of pathogens. However, low O2 is known to increase insect tolerance to irradiation, and the use of MAP with products treated by irradiation before export to control quarantine pests may inadvertently compromise treatment efficacy. Melon fly, Bactrocera cucurbitae Coquillet (Diptera: Tephritidae), is an important economic and quarantine pest of tropical fruits and vegetables, and one of the most radiation-tolerant tephritid fruit flies known. The effect of low O2 generated by MAP on the radiation tolerance of B. cucurbitae was examined. Third-instar larval B. cucurbitae were inoculated into ripe papayas and treated by 1) MAP + irradiation, 2) irradiation alone, 3) MAP alone, or (4) no MAP and no irradiation, and held for adult emergence. Three types of commercially available MAP products were tested that produced O2 concentrations between 1 and 15%, and a sublethal radiation dose (50 Gy) was used to allow comparisons between treatments. Ziploc storage bags (1-4% O2) increased survivorship to adult from 14 to 25%, whereas Xtend PP61 bags (3-8% O2) and Xtend PP53 bags (11-15% O2) did not enhance survivorship to the adult stage in B. cucurbitae irradiated at 50 Gy. Radiation doses approved by the United States Department of Agriculture and the International Plant Protection Commission for B. cucurbitae and Ceratitis capitata (Wiedemann) (Mediterranean fruit fly) are 150 and 100 Gy, respectively. In large-scale tests, 9,000 B. cucurbitae and 3,800 C. capitata larvae infesting papayas in Ziploc bags were irradiated at 150 and 100 Gy, respectively, with no survivors to the adult stage. MAP can increase insect survivorship during irradiation treatment at certain doses and O2 concentrations, but should not compromise the efficacy of the 150-Gy generic radiation treatment for tephritid fruit flies or the 100-Gy radiation treatment for C. capitata.

Effect of gamma radiation on Phenoloxidase pathway, antioxidant defense mechanism in Helicoverpa armigera (Lepidoptera: Noctuidae) and its implication in inherited sterility towards pest suppression

Purpose: To investigate age-correlated radiosensitivity in highly radioresistant lepidopteran pest, Helicoverpa armigera, upon exposure to ionizing radiation and to examine the irradiation impact on stress-molecular responses in F1 (first-filial) progeny of irradiated (100 Gy) male moths in relation to its reproductive behavior.Materials and methods: Efficacy of sub-lethal gamma radiation was evaluated on two markedly apart ontogenic stages, neonates and adult moths. Differential growth, reproductive behavior and stress-indicating molecular responses were examined upto F1 progeny of sub-sterilized moths. Free-radical scavenging enzymes, superoxide dismutase (SOD), catalase (CAT) and Phenoloxidase cascade enzymes, pro-phenoloxidase (PPO), its activating enzyme (PPAE) were studied in irradiated and irradiated plus microbial challenge regimen (dual-stress) by Real-time RT-PCR (reverse-transcription-polymerase-chain-reaction).Results: An inverse correlation of radiosensitivity with developmental age of insect was observed. F1 sterility was higher than parent sterility. F1 progeny exhibited protraction in development and decreased survival upon irradiation. Sex ratio in F1 progeny was skewed towards males. PPO, PPAE, SOD and CAT transcripts were downregulated upon neonate irradiation resulting in enhanced vulnerability of larvae to incidental microbial challenge. These transcripts were upregulated in F1 progeny of sub-sterilized male moths (100 Gy) upon dual-stress.Conclusions: Irradiation impact on stress-indicating molecular responses in F1 progeny is correlated with its reproductive performance. These observations will permit defining regimen having pragmatic viability of ‘F1 sterility technique’ for pest suppression. Gamma dose of 100 Gy would ensure balance between induced sterility of males and their field competitiveness. These parameters would facilitate integration of biocontrol strategy with parabiological ‘Sterile Insect Release Technique’.

Evaluation of potential ionizing irradiation protectors and mitigators using clonogenic survival of human umbilical cord blood hematopoietic progenitor cells

We evaluated the use of colony formation (colony-forming unit-granulocyte macrophage [CFU-GM], burst-forming unit erythroid [BFU-E], and colony-forming unit-granulocyte-erythroid-megakaryocyte-monocytes [CFU-GEMM]) by human umbilical cord blood (CB) hematopoietic progenitor cells for testing novel small molecule ionizing irradiation protectors and mitigators. The following compounds were added before (protection) or after (mitigation) ionizing irradiation: GS-nitroxides (JP4-039 and XJB-5-131), the bifunctional sulfoxide MMS-350, the phosphoinositol-3-kinase inhibitor LY29400, triphenylphosphonium-imidazole fatty acid, the nitric oxide synthase inhibitor (MCF-201-89), the p53/mdm2/mdm4 inhibitor (BEB55), methoxamine, isoproterenol, propranolol, and the adenosine triphosphate-sensitive potassium channel blocker (glyburide). The drugs XJB-5-131, JP4-039, and MMS-350 were radiation protectors for CFU-GM. JP4-039 was also a radiation protector for CFU-GEMM. The drugs XJB-5-131, JP4-039, and MMS-350 were radiation mitigators for BFU-E, MMS-350 and JP4-039 were mitigators for CFU-GM, and MMS350 was a mitigator for CFU-GEMM. In contrast, other drugs were effective in murine assays; TTP-IOA, LY294002, MCF201-89, BEB55, propranolol, isoproterenol, methoxamine, and glyburide but showed no significant protection or mitigation in human CB assays. These data support the testing of new candidate clinical radiation protectors and mitigators using human CB clonogenic assays early in the drug discovery process, thus reducing the need for animal experiments.

Improving the Therapeutic Ratio in Stereotactic Radiosurgery: Optimizing Treatment Protocols Based on Kinetics of Repair of Sublethal Radiation Damage

Sublethal damage after radiation exposure may become lethal or be repaired according to repair kinetics. This is a well-established concept in conventional radiotherapy. It also plays an important role in single-dose stereotactic radiotherapy treatments, often called stereotactic radiosurgery, when duration of treatment is extended due to source decay or treatment planning protocol. The purpose of this study is to look into the radiobiological characteristics of normal brain tissue and treatment protocols and find a way to optimize the time course of these protocols. The general problem is nonlinear and can be solved numerically. For numerical optimization of the time course of radiation protocol, a biexponential repair model with slow and fast components was considered. With the clinically imposed constraints of a fixed total dose and total treatment time, three parameters for each fraction (dose-rate, fraction duration, time of each fraction) were simultaneously optimized. A biological optimization can be performed by maximizing the therapeutic difference between tumor control probability and normal tissue complication probability. Specifically, for gamma knife radiosurgery, this approach can be implemented for normal brain tissue or tumor voxels separately in a treatment plan. Differences in repair kinetics of normal tissue and tumors can be used to find clinically optimized protocols. Thus, in addition to considering the physical dose in tumor and normal tissue, we also account for repair of sublethal damage in both these tissues.

The effects of CX3CR1 deficiency and irradiation on the homing of monocyte-derived cell populations in the mouse eye.

This study examined whether CX3CR1 deficiency altered monocytic cell replenishment dynamics in ocular tissues in the context of radiation chimeras. Long-term effects of irradiation and effects of sublethal irradiation on ocular macrophages were also assessed. Bone marrow from BALB/c Cx 3 cr1 +/gfp or Cx 3 cr1 gfp/gfp mice was used to reconstitute full body irradiated WT mice and donor cell densities in the uveal tract were compared at 4 and 8 weeks post-transplantation. BALB/c and C57BL/6J chimeric mice were examined at 6 months of age to determine strain-related differences in microglial replenishment and radiation sensitivity. A separate cohort of mice were sublethally irradiated (5.5 Gy) and retinal tissue assessed 8 and 12 weeks later. CX3CR1 deficiency altered the early replenishment of monocytes in the posterior iris but not in the iris stroma, choroid or retina. In six month old chimeric mice, there were significantly higher GFP+ cell densities in the uveal tract when compared to non-irradiated 8-12 week old Cx 3 cr1 +/gfp mice. Additionally, MHC Class II expression was upregulated on hyalocytes and GFP+ cells in the peripheral retina and the repopulation of microglia appeared to be more rapid in C57BL/6J mice compared to BALB/c mice. Transient expression of MHC Class II was observed on retinal vasculature in sublethally irradiated mice. These data indicate CX3CR1-deficiency only slightly alters monocyte-derived cell replenishment in the murine uveal tract. Lethal irradiation leads to long-term increase in monocytic cell density in the uveal tract and retinal microglial activation, possibly as a sequelae to local irradiation induced injury. Microglial replenishment in this model appears to be strain dependent.

Turning T cells on: epigenetically enhanced effector T cell activity following tumor cellirradiation (P2091)

Abstract While sub-lethal tumor irradiation enhances killing of colorectal carcinoma cells by tumor-specific CD8+ CTLs, the mechanisms responsible for increased activity of CTLs to irradiated tumor cells remain unknown. Two members of the tumor necrosis family super family of receptors, OX40L/TNFSF4 and 41BBL/TNFSF9, enhance CTL activation and survival. Our results indicate sub-lethal doses of radiation increase tumor cell expression of 41BBL and OX40L via epigenetic mechanisms as inhibition of HDACs (with TSA) and of DNMTs (with 5-AZA) results in significantly increased OX40L and 41BBL mRNA and protein. The combination of radiation and epigenetic modifiers results in greater expression of both receptors, suggesting overlap in the mechanisms by which radiation, 5-AZA, and TSA regulate OX40L and 41BBL. Finally, chromatin immunoprecipitation experiments reveal significantly increased H3 acetylation of 41BBL promoters following irradiation. Our results show non-lethal doses of radiation make human tumors amenable to immune system attack through epigenetic modulation of genes stimulatory to CTLs. The use of ionizing radiation to specifically enhance cancer immunotherapy strategies, through epigenetic modulation of genes stimulatory to CTLs, will have a broad impact on cancer therapy strategies and will extend the use of radiation in new directions.

Promoting regulation via the inhibition of DNAM-1 after transplantation

AbstractDonor T cells play pivotal roles in graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effects following bone marrow transplantation (BMT). DNAX accessory molecule 1 (DNAM-1) is a costimulatory and adhesion molecule, expressed mainly by natural killer cells and CD8+ T cells at steady state to promote adhesion to ligand-expressing targets and enhance cytolysis. We have analyzed the role of this pathway in GVHD and GVL. The absence of DNAM-1 on the donor graft attenuated GVHD in major histocompatibility complex (MHC)-mismatched and MHC-matched BMT following conditioning with lethal and sublethal irradiation. In contrast, DNAM-1 was not critical for GVL effects against ligand (CD155) expressing and nonexpressing leukemia. The effects on GVHD following myeloablative conditioning were independent of CD8+ T cells and dependent on CD4+ T cells, and specifically donor FoxP3+ regulatory T cells (Treg). The absence of DNAM-1 promoted the expansion and suppressive function of Treg after BMT. These findings provide support for therapeutic DNAM-1 inhibition to promote tolerance in relevant inflammatory-based diseases characterized by T-cell activation.

Metabolomics Reveals Aging-associated Attenuation of Noninvasive Radiation Biomarkers in Mice: Potential Role of Polyamine Catabolism and Incoherent DNA Damage-repair

Development of methods for rapid screening and stratification of subjects after exposure is an integral part of countermeasures against radiation. The potential demographic and exposure history-related heterogeneity of exposed populations warrants robust biomarkers that withstand and reflect such differences. In this study, the effect of aging and repeated exposure on the metabolic response to sublethal irradiation was examined in mice using UPLC-ESI-QTOF mass spectrometry. Aging attenuated postexposure elevation in excretions of DNA damage biomarkers as well as N(1)-acetylspermidine. Although N(1)-acetylspermidine and 2'-deoxyuridine elevation was highly correlated in all age groups, xanthine and N(1)-acetylspermidine elevation was poorly correlated in older mice. These results may reflect the established decline in DNA damage-repair efficiency associated with aging and indicate a novel role for polyamine metabolism in the process. Although repeated irradiation at long intervals did not affect the elevation of N(1)-acetylspermidine, 2'-deoxyuridine, and xanthine, it did significantly attenuate the elevation of 2'-deoxycytidine and thymidine compared to a single exposure. However, these biomarkers were found to identify exposed subjects with accuracy ranging from 82% (xanthosine) to 98% (2'-deoxyuridine), irrespective of their age and exposure history. This indicates that metabolic biomarkers can act as robust noninvasive signatures of sublethal radiation exposure.

Abstract 440: The sublethal dose radiation induces cellular senescence and potent bystander effects through c-Myc oncogene.

Abstract The sublethal dose radiation may induce cellular senescence and promote secondary maligancy during radiotherapy. However, the molecular mechanisms are unclear. It has been reported that a variety of early response oncogenes can be up-regulated by the low dose radiation. Because oncogene-induced senescence (OIS) has been considered important for tumor development, we are interested in investigating whether the sublethal dose radiation also raises OIS to affect the malignancy. To this end, human diploid fibroblasts WI-38 cells and human non-small lung cancer H1299 cells were exposed to less than 2Gy of X-rays delivered by the RS2000 X-rays machine. Both cell types exhibited senescent phenotypes within a dose-dependent manner using the senescence-associated β-galactosidase (SA-β-gal) staining. Subsequently, we investigated the expression of c-Myc and c-Ras oncogenes, and the results showed that sublethal dose radiation could induce c-Myc, but not c-Ras, in both transcriptional and translational levels. Additionally, up-regulated c-Myc exhibited transcriptional activity as demonstrated by the promoter-based reporter gene assys. Although the sublethal dose radiation caused cellular senescence and growth delay in exposed cells, the collected culture medium could increase the motility of non-irradiated H1299 cancer cells using the wound healing assay. Interestingly, the culture medium obtained from irradiated H1299 cancer cells caused higher motility in non-irradiated cells than that from irradiated WI-38 fibroblasts. Furthermore, the sublethal dose radiation induced senescent phenotype was suppressed in cells treated with siRNA against c-Myc. Meanwhile, the motility of non-irradiated cells was not increased by the cultured medium obtained from irradiated c-Myc silenced cells. Taken together, the current data suggest that the sublethal dose radiation causes a c-Myc oncogene induced senescence, and this event may promote the bystandard effects that influence the motility of non-irradiated cancer cells. Citation Format: Chia-Chien Lo, Yen-Ting Chou, Su-Jun Chiu, Jeng-Jung Hwang, Yi-Jang Lee. The sublethal dose radiation induces cellular senescence and potent bystander effects through c-Myc oncogene. [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 440. doi:10.1158/1538-7445.AM2013-440 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.