32D Cl Research Articles

Combination Mitigators, GS-Nitroxide JP4-039 and water Soluble Oxetanyl Sulfoxide MMS350 Improve Survival of Lethally Irradiated Mice

New small molecule radiation mitigator drugs are needed to administer 24 hr or later after a mass casualty radiation event. We have developed two small molecule mitigators with applicability as clinical radiation protectors. The mitochondrial targeted GS-nitroxide, JP4-039, acts as a superoxide dismutase mimic. A novel oxetanyl sulfoxide, MMS350, acts as a hydroxyl radical scavenger and mitigates both acute total body irradiation damage and prevents late radiation pulmonary fibrosis (Kalash, Epperly, Cao et. al., Radiation Research 180:474-490, 2013). We tested the effect of both compounds together in the C57BL/6NTac mouse TBI irradiation model. Each drug was a radiation protector and mitigator with murine hematopoietic progenitor cell line 32D cl3 in vitro. JP4-039 (10 μM) or MMS350 (100 μM) were incubated with the cells one hour before irradiation or added 30 min following irradiation. The irradiated cells were plated in methylcellulose, incubated for 7 days at 37oC, and colonies of greater than 50 cells counted. The data was analyzed using linear quadratic or single hit, multi-target models. In vivo total body irradiated C57BL/6NTac mice received 9.25 Gy and were injected intravenously 24 hr later with JP4-039 (20 mg/kg) in F14 emulsion, MMS350 (20 mg/kg) or both JP4-039 (20 mg/kg) + MMS350 (20 mg/kg). Mice were followed for development of the hematopoietic syndrome (irradiation induced bone marrow failure). In vitro radiation survival curves demonstrated that both JP4-039 and MMS350 protected and mitigated 32D cl 3 cells by increasing the shoulder on the clonagenic survival curves. The shoulder on survival curve for 32D cl 3 cells was 2.0 ± 0.3 compared to 3.8 ± 0.5 when JP4-039 was administered pre irradiation (p = 0.0092), 5.4 ± 0.5 when JP4-039 was administered post irradiation (p = 0.0002), 3.5 ± 0.3 for cells incubated with MMS350 pre irradiation (p = 0.0104) or 5.3 ± 1.5 when MMS350 is administered post irradiation (p = 0.0096). Mice treated with JP4-039 had increased survival at 60 days following 9.25 Gy from 20% to 65% (p = 0.0092). Mice treated with MMS350 showed an increased survival of 55% (p = 0.0097). The combination of JP4-039 and MMS350 was additive and increased survival to 95% (p < 0.0001). The combination of JP4-039 and MMS350 increased mitigation of irradiation damage better than one drug alone. DisclosuresNo relevant conflicts of interest to declare.

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.

Identification of Druggable Targets for Radiation Mitigation Using a Small Interfering RNA Screening Assay

Currently, there is a serious absence of pharmaceutically attractive small molecules that mitigate the lethal effects of an accidental or intentional public exposure to toxic doses of ionizing radiation. Moreover, cellular systems that emulate the radiobiologically relevant cell populations and that are suitable for high-throughput screening have not been established. Therefore, we examined two human pluripotent embryonal carcinoma cell lines for use in an unbiased phenotypic small interfering RNA (siRNA) assay to identify proteins with the potential of being drug targets for the protection of human cell populations against clinically relevant ionizing radiation doses that cause acute radiation syndrome. Of the two human cell lines tested, NCCIT cells had optimal growth characteristics in a 384 well format, exhibited radiation sensitivity (D(0) = 1.3 ± 0.1 Gy and ñ = 2.0 ± 0.6) comparable to the radiosensitivity of stem cell populations associated with human death within 30 days after total-body irradiation. Moreover, they internalized siRNA after 4 Gy irradiation enabling siRNA library screening. Therefore, we used the human NCCIT cell line for the radiation mitigation study with a siRNA library that silenced 5,520 genes known or hypothesized to be potential therapeutic targets. Exploiting computational methodologies, we identified 113 siRNAs with potential radiomitigative properties, which were further refined to 29 siRNAs with phosphoinositide-3-kinase regulatory subunit 1 (p85α) being among the highest confidence candidate gene products. Colony formation assays revealed radiation mitigation when the phosphoinositide-3-kinase inhibitor LY294002 was given after irradiation of 32D cl 3 cells (D(0) = 1.3 ± 0.1 Gy and ñ = 2.3 ± 0.3 for the vehicle control treated cells compared to D(0) = 1.2 ± 0.1 Gy and ñ = 6.0 ± 0.8 for the LY294002 treated cells, P = 0.0004). LY294002 and two other PI3K inhibitors, PI 828 and GSK 1059615, also mitigated radiation-induced apoptosis in NCCIT cells. Treatment of mice with a single intraperitoneal LY294002 dose of 30 mg/kg at 10 min, 4, or 24 h after LD(50/30) whole-body dose of irradiation (9.25 Gy) enhanced survival. This study documents that an unbiased siRNA assay can identify new genes, signaling pathways, and chemotypes as radiation mitigators and implicate the PI3K pathway in the human radiation response.

L-Arginine is a Radioprotector for Hematopoietic Progenitor Cells

L-arginine is shown to protect hematopoietic progenitor (32D cl 3) cells from death due to exposure to γ radiation ((137)Cs). Some of the other intermediates in the urea cycle, namely ornithine and citrulline, plus urea itself, were not found to have any significant impact on cell survival after irradiation. Intriguingly, supplementation of irradiated cells with L-arginine results in decreased production of peroxynitrite, suggesting that suppression of superoxide generation by nitric oxide synthase in one or more microenvironments is an important factor in the observed radioprotection. The absence of any radioprotective effect of L-arginine in cells at 3% oxygen also confirms the involvement of one or more oxygen-derived species. Knockdown experiments with nitric oxide synthase (NOS) siRNAs in cells and NOS knockout animals confirm that the observed radioprotection is associated with nNOS (NOS-1). L-arginine also ameliorates the transient inhibition of the electron-transport chain complex I that occurs within 30 min of completing the dose (10 Gy) and that appears to be a functional marker for postirradiation mitochondrial oxidant production.

Ionizing Irradiation Protection and Mitigation by Carbamazepine Is p53 and Autophagy Independent,

Abstract 3400 Introduction:Carbamazepine (CBZ), an established sodium channel blocker, used in treatment of epilepsy and trigeminal neuralgia, induces autophagy. CBZ has recently been demonstrated to be an ionizing radiation mitigator and protector (Kim,H.et. al IJRB-in press). CBZ protected C57BL/6NHsd female mice from irradiation if given 24 hr before total body irradiation (TBI) and mitigated if given 12 hours after irradiation.Materials and Methods: To elucidate the mechanism of CBZ action, autophagy incompetent (ATG5−/−) and competent (ATG5+/+) mouse embryo fibroblasts (MEF), p53−/− and p53+/+ bone marrow stromal cells and 32D cl 3 murine IL-3 dependent hematopoietic progenitor cells were tested for CBZ mediated radiation protection and mitigation in clonagenic irradiation survival curves. We also measured CBZ effect on irradiation-induced apoptosis, and depletion of antioxidant stores in vitro and after (TBI) in vivo in control mice and in hind limb irradiated mice with orthotopic tumors. Results:CBZ was a significant radiation protector and mitigator for both ATG5−/− and ATG5 +/+ cell lines by an increased ñ (a measurement of the shoulder on the clonogenic survival curve). CBZ treated ATG5 +/+ cells has an increased ñ of 11.1 +/− 0.2 or 8.8 +/− 0.2 for CBZ added before or after irradiation respectively compared to 5.4 +/− 0.9 for irradiation control cells (p = 0.0287 or 0.0119,respectively). ATG −/− cells were also protected and mitigated by CBZ (ñ of 16.1 +/− 2.6 as a radioprotector or 9.8 +/− 1.5 as a mitigator compared to 4.6 +/− 0.7 for irradiated control cells (p = 0.0002 or 0.0037, respectively). Thus, CBZ functions independent of autophagy. Incubation of p53 +/+ and p53−/− cell lines in 10 μM CBZ for one hour before irradiation protected (ñ of 5.6 +/− 0.9 compared to 1.9 +/− 0.5 for control irradiated p53 +/+ cells (p = 0.0126) and 3.7 +/− 1.9 compared to 1.8 +/− 0.4 for irradiated control p53 −/− cells (p = 0.0018). Thus, CBZ functions in a p53 independent manner. Other pro-autophagy drugs, Valproic Acid and Lithium Carbonate, were ineffective radiation protectors or mitigators. CBZ treatment of 32D cl 3 cells for one hour before or immediately following irradiation had no effect on mitochondrial membrane depolarization, apoptosis or viability but was protective and mitigative in clonagenic survival curve assays. Irradiation initially decreased antioxidant stores in both ATG5+/+ and ATG5−/− cells 10 to 30 min following irradiation. However, cells treated with CBZ had a faster recovery of antioxidant stores to pre-irradiation levels by 90 min compared to 120 min for control irradiated cells. To determine if CBZ protected both normal tissues and tumors in vivo mice with 3LL Lewis Lung carcinoma hind limb xenografts were treated with 10 mg/kg CBZ before or after 20 Gy limb irradiation. There was no significant difference in tumor growth inhibition by 20 Gy in mice treated with CBZ before or after irradiation (p = 0.2431 or 0.5439, respectively). Conclusion:Thus, CBZ is an effective radiation protector and mitigator of normal but not tumor tissues by a novel mechanism independent of autophagy, p53 and apoptosis. Acknowledgments:This project was supported by U191A168021–06. Disclosures:No relevant conflicts of interest to declare.

The Autophagy Inducing Drug Carbamazepine is a Radiation Protector and Mitigator

AbstractPurpose: To determine whether Carbamazepine (CBZ) is a radiation protector and/or mitigator.Materials and methods: Murine hematopoietic progenitor 32D cl 3 cells were incubated in 1, 10, or 100 μM CBZ 1 h before or immediately after 0–8 Gy irradiation and assayed for clonogenic survival. Autophagy was assayed by immunoblot for microtubule-associated protein light chain 3 (LC3). In vivo radioprotection and mitigation were determined with C57BL/6NTac mice.Results: CBZ treatment at 1, 10 or 100 μM for 1 h prior to irradiation increased radioresistance (the dose for 37% survival or D0) from control 1.5 ± 0.1 Gy to 2.1 ± 0.2 Gy (P = 0.012), 2.3 ± 0.1 Gy (P = 0.010), and 3.6 ± 0.7 Gy (P = 0.003), respectively; after irradiation increased the extrapolation number (n) from 1.5 ± 0.3 to 10.1 ± 4.2 (P = 0.011), 5.5 ± 1.7 (P = 0.019), and 3.6 ± 0.8 (P = 0.014), respectively, and increased autophagy. CBZ treated mice 10 min or 24 h before or 10 min or 12 h after 9.25 Gy total body irradiation (TBI) showed inc...

The autophagy-inducing drug carbamazepine is a radiation protector and mitigator

Purpose: To determine whether Carbamazepine (CBZ) is a radiation protector and/or mitigator.Materials and methods: Murine hematopoietic progenitor 32D cl 3 cells were incubated in 1, 10, or 100 μM CBZ 1 h before or immediately after 0–8 Gy irradiation and assayed for clonogenic survival. Autophagy was assayed by immunoblot for microtubule-associated protein light chain 3 (LC3). In vivo radioprotection and mitigation were determined with C57BL/6NTac mice.Results: CBZ treatment at 1, 10 or 100 μM for 1 h prior to irradiation increased radioresistance (the dose for 37% survival or D0) from control 1.5 ± 0.1 Gy to 2.1 ± 0.2 Gy (P = 0.012), 2.3 ± 0.1 Gy (P = 0.010), and 3.6 ± 0.7 Gy (P = 0.003), respectively; after irradiation increased the extrapolation number (ñ) from 1.5 ± 0.3 to 10.1 ± 4.2 (P = 0.011), 5.5 ± 1.7 (P = 0.019), and 3.6 ± 0.8 (P = 0.014), respectively, and increased autophagy. CBZ treated mice 10 min or 24 h before or 10 min or 12 h after 9.25 Gy total body irradiation (TBI) showed increased survival (P = 0.012, 0.011, 0.0002, and 0.017, respectively).Conclusion: CBZ may be a useful radiation protector and mitigator.

Two Strategies for the Development of Mitochondrion-Targeted Small Molecule Radiation Damage Mitigators

To evaluate the effectiveness of mitigation of acute ionizing radiation damage by mitochondrion-targeted small molecules. We evaluated the ability of nitroxide-linked alkene peptide isostere JP4-039, the nitric oxide synthase inhibitor-linked alkene peptide esostere MCF201-89, and the p53/mdm2/mdm4 protein complex inhibitor BEB55 to mitigate radiation effects by clonogenic survival curves with the murine hematopoietic progenitor cell line 32D cl 3 and the human bone marrow stromal (KM101) and pulmonary epithelial (IB3) cell lines. The p53-dependent mechanism of action was tested with p53(+/+) and p53(-/-) murine bone marrow stromal cell lines. C57BL/6 NHsd female mice were injected i.p. with JP4-039, MCF201-89, or BEB55 individually or in combination, after receiving 9.5 Gy total body irradiation (TBI). Each drug, JP4-039, MCF201-89, or BEB55, individually or as a mixture of all three compounds increased the survival of 32D cl 3 (p = 0.0021, p = 0.0011, p = 0.0038, and p = 0.0073, respectively) and IB3 cells (p = 0.0193, p = 0.0452, p = 0.0017, and p = 0.0019, respectively) significantly relative to that of control irradiated cells. KM101 cells were protected by individual drugs (p = 0.0007, p = 0.0235, p = 0.0044, respectively). JP4-039 and MCF201-89 increased irradiation survival of both p53(+/+) (p = 0.0396 and p = 0.0071, respectively) and p53(-/-) cells (p = 0.0007 and p = 0.0188, respectively), while BEB55 was ineffective with p53(-/-) cells. Drugs administered individually or as a mixtures of all three after TBI significantly increased mouse survival (p = 0.0234, 0.0009, 0.0052, and 0.0167, respectively). Mitochondrial targeting of small molecule radiation mitigators decreases irradiation-induced cell death in vitro and prolongs survival of lethally irradiated mice.

Carbamazepine Is a Radioprotector and Radiation Damage Mitigator for Murine Hematopoietic Cell Line 32D Cl 3

AbstractAbstract 4772The search for new radiation mitigators includes targeted new- indication testing of known drugs. Carbamazepine (cbz) has been used for decades in the treatment of trigeminal neuralgia, epilepsy, mania and bipolar disorders, and has recently been demonstrated to reduce liver damage in a mouse model of alpha-1-antitrypsin deficiency by a mechanism that stimulates the proteasomal and autophagic pathway allowing degradation and clearing of abnormally folded antitrypsin. Because of its potential mitochondrial membrane ion channel effects, we tested cbz action on the irradiation survival of murine hematopoietic progenitor 32D cl 3 cells that were incubated for 1 hour in 1, 10 or 100 μ M cbz either before or after irradiation. Cells were irradiated to doses ranging from 0 to 8 Gy, plated in 4% methylcellulose, incubated for seven days at 37°C, and colonies of greater than 50 cells counted. Data was analyzed using linear quadratic or single-hit, multi-target models. Cbz added before irradiation resulted in radioprotection by an increased Do of 2.1 + 0.1 Gy (p = 0.0117), 2.3 + 0.1 Gy (p = 0.010), or 3.6 + 0.7 Gy (p = 0.0026) for 1, 10 or 100 μ M, respectively, compared to 1.5 + 0.1 Gy for control irradiated 32D cl 3 cells, with no significant change in the ñ (shoulder on the survival curve). Cbz added after irradiation mitigated against damage by an increase in the shoulder (ñ) on the survival curve with no significant change in the Do. Control irradiated 32D cl 3 cells had a ñ of 1.4 + 0.3 compared to 10.1 + 4.2 (p = 0.0113), 5.5 + 1.7 (p = 0.0185) or 3.6 + 0.8 (p = 0.0144) for cells treated after irradiation with 1, 10 or 100 μ M cbz, respectively. Cbz is known to stabilize ion channels in both cellular and mitochondrial membranes. Since irradiation induced apoptosis involves influx of calcium across the mitochondrial membrane followed by induction of nitric oxide synthase, nitric oxide and peroxynitrite formation, destabilization of the mitochondrial membrane and cytoplasmic release of cytochrome C; cbz-stabilization of mitochondrial ion channels may be the mechanism of this novel radiation mitigative effect of a commonly used drug.Supported by NIAID grant U19AI068021. Disclosures:No relevant conflicts of interest to declare.

5030 High resolution array Comparative Genomic Hybridization (aCGH) of breast carcinoma identifies Mouse double minutes 4 (Mdm4) as one of the early genetic changes in breast cancer development – Mdm4 is a new independent prognostic and predictive marker

To evaluate the effectiveness of mitigation of acute ionizing radiation damage by mitochondrion-targeted small molecules.We evaluated the ability of nitroxide-linked alkene peptide isostere JP4-039, the nitric oxide synthase inhibitor-linked alkene peptide esostere MCF201-89, and the p53/mdm2/mdm4 protein complex inhibitor BEB55 to mitigate radiation effects by clonogenic survival curves with the murine hematopoietic progenitor cell line 32D cl 3 and the human bone marrow stromal (KM101) and pulmonary epithelial (IB3) cell lines. The p53-dependent mechanism of action was tested with p53+/+ and p53−/− murine bone marrow stromal cell lines. C57BL/6 NHsd female mice were injected i.p. with JP4-039, MCF201-89, or BEB55 individually or in combination, after receiving 9.5 Gy total body irradiation (TBI).Each drug, JP4-039, MCF201-89, or BEB55, individually or as a mixture of all three compounds increased the survival of 32D cl 3 (p = 0.0021, p = 0.0011, p = 0.0038, and p = 0.0073, respectively) and IB3 cells (p = 0.0193, p = 0.0452, p = 0.0017, and p = 0.0019, respectively) significantly relative to that of control irradiated cells. KM101 cells were protected by individual drugs (p = 0.0007, p = 0.0235, p = 0.0044, respectively). JP4-039 and MCF201-89 increased irradiation survival of both p53+/+ (p = 0.0396 and p = 0.0071, respectively) and p53−/− cells (p = 0.0007 and p = 0.0188, respectively), while BEB55 was ineffective with p53−/− cells. Drugs administered individually or as a mixtures of all three after TBI significantly increased mouse survival (p = 0.0234, 0.0009, 0.0052, and 0.0167, respectively).Mitochondrial targeting of small molecule radiation mitigators decreases irradiation-induced cell death in vitro and prolongs survival of lethally irradiated mice.

Mitochondrial Targeting of a Catalase Transgene Product by Plasmid Liposomes Increases RadioresistanceIn VitroandIn Vivo

To determine whether increased mitochondrially localized catalase was radioprotective, a human catalase transgene was cloned into a small pSVZeo plasmid and localized to the mitochondria of 32D cl 3 cells by adding the mitochondrial localization sequence of MnSOD (mt-catalase). The cell lines 32D-Cat and 32D-mt-Cat had increased catalase biochemical activity as confirmed by Western blot analysis compared to the 32D cl 3 parent cells. The MnSOD-overexpressing 32D cl 3 cell line, 2C6, had decreased baseline catalase activity that was increased in 2C6-Cat and 2C6-mt-Cat subclonal cell lines. 32D-mt-Cat cells were more radioresistant than 32D-Cat cells, but both were radioresistant relative to 32D cl 3 cells. 2C6-mt-Cat cells but not 2C6-Cat cells were radioresistant compared to 2C6 cells. Intratracheal injection of the mt-catalase-plasmid liposome complex (mt-Cat-PL) but not the catalase-plasmid liposome complex (Cat-PL) increased the resistance of C57BL/6NHsd female mice to 20 Gy thoracic irradiation compared to MnSOD-plasmid liposomes. Thus mitochondrially targeted overexpression of the catalase transgene is radioprotective in vitro and in vivo.

A Mitochondrially Targeted Nitroxide JP4-039 Protects and Mitigates against Total Body Irradiation Induced Hematopoietic Syndrome

Nitroxides are efficient scavengers of free radicals and have been proposed for use as radioprotective agents. To circumvent the need for high concentrations for in vivo effects, and to target drugs to the mitochondria to prevent ionizing irradiation-induced apoptosis, we tested a family of compounds based on combining a hemi-gramicidin linker to a nitroxide. One small molecule (JP4-039) produced a 32-fold increased mitochondrial nitroxide localization in 32D cl 3 cells in vitro after one hour incubation at 37°C compared to the unlinked nitroxide tempo at 10uM. The cells were lyzed by rapidly freezing then thawing, followed by centrifugation to yield the nuclear, cytoplasmic, and mitochondrial fractions. EPR was used to quantitate nitroxide in each JP4-039 or tempo treated cell fraction. There was a 32-fold increase in the nitroxide signal for JP4-039 accumulation in the mitochondria compared to the nitroxide tempo. Irradiation survival curves were performed by incubating 32D cl 3 murine hematopoietic progenitor cells in JP4-039 or tempo (1 μM) for one hour before irradiation or by addition immediately following irradiation. The cells were irradiated to doses ranging from 0 to 8 Gy, suspended in methylcellulose-containing media, incubated for seven days at 37°C and colonies of greater than 50 cells were scored. The data was analyzed using linear quadratic and single-hit, multi-target models. Incubation of 32D cl 3 cells in JP4-039 for one hour prior to irradiation resulted in a significant increase in Do to 2.25 ± 0.11 Gy compared to 1.19 ± 0.13 Gy for 32D cl 3 cells alone or 1.32 ± 0.09 Gy for 32D cl 3 cells incubated in tempo (p=0.0042 or 0.0073, respectively). Similar results were achieved giving JP4-039 after irradiation with a Do of 1.97 ± 0.01 Gy (p < 0.0001). To test effectiveness in vivo, C57BL/6NHsd female mice were injected intraperitoneally with JP4-039 or tempo (10 mg/kg) 10 minutes before (or four hours after) total body irradiation to the LD 75/30 dose of 9.75 Gy. Mice were monitored and those moribund from the hematopoietic syndrome were sacrificed. Mice injected with 10 mg/kg JP4-039 before irradiation had a significant increase in survival of 60% compared to 28% for control irradiated mice (p = 0.0005) or 44% for tempo treated mice (p = 0.2252). JP4-039 given after irradiation had a survival of 53% at 30 days after irradiation (p = 0.0474). Thus, JP4-039, a mitochondrial targeted nitroxide, is an effective radioprotector, and mitigator.

Mitigation of Irradiation Induced Potentially Lethal Damage (PLD) in Hematopoietic Cells by Mitochondrial Localized GS-Nitroxide, JP4-039

PLD is an ionizing irradiation pathway believed to apply to quiescent hematopoietic cells in the marrow microenvironment. To determine if the mitochondrial targeted JP4-039 which has been shown to mitigate sublethal irradiation damage (SLD) in non-quiescent clonagenic cells protected cells from PLD, 32D cl 3 hematopoietic cells were irradiated in three sets of cultures to doses ranging from 0 to 8 Gy. To one group of cells, to measure SLD mitigation effects, JP4-039 (10 μM) was added immediately, then cells plated in methycellulose, and incubated at 37°C for seven days at which time colonies of greater than 50 cells counted. The second set of irradiated cells was grown in flasks in 10 ml of liquid media. The third set was tested for PLD effects and cells were centrifuged and maintained in pellets in 1 ml of media (PLD conditions preventing cell growth or division) and these latter two sets were kept at 37°C. After 24, 48 or 72 hrs irradiation, subsets of each were removed, viability and cell counts were determined and JP4-039 was then added to subgroups of cultured cells at each time point, cells re-suspended in methycellulose, and incubated at 37°C for seven days at which time colonies of greater than 50 cells counted. Viability of cells in non-irradiated pellets decreased from 24 to 72 hours from 60.3 ± 2.7% to 37.3 ± 2.3% (p = 0.0030), while after 8 Gy the cell viability was 57. 3 ± 0.3% at 24 hr and 25.7 ± 6.1% at 72 hr (p = 0.0065). There was no significant decrease in percent viability at 24 hrs of non-irradiated or 8 Gy irradiated cells (60.3 ± 2.7% and 57.3 ± 0.3%, respectively) or at 72 hr (37.3 ± 2.3% and 25.7 ± 6.1%, respectively). In contrast, non-irradiated cells re-suspended in flasks grew and thus viability increased from 77.0 ± 4.0% at 24 hr to 96.7 ± 0.3% at 72 hr (p = 0.0080). Cell viability after 8 Gy of surviving irradiated cells grown in flasks was not significantly changed from 64.0 ± 10.6% at 24 hr to 49.3 + 3.2% at 72 hr. There was no significant change in cell viability in flasks at 24 hrs of non-irradiated or 8 Gy irradiated cells (77 ± 4.0% and 64.0 ± 10.6%, respectively). Cell viability was significantly decreased at 72 hr in non-irradiated cell flasks of 96.7 ± 0.3% compared to 8 Gy irradiated flasks with a viability of 49.3 ± 3.2% (p = 0.0001). Cells maintained in pellets showed there was no cell growth between 24 to 72 hours while those maintained in flasks did significantly grow for non-irradiated cells from 6.3 ± 0.2 × 105 cells at 24 hr to 85.3 ± 6.8 × 105 cells at 72 hr (p = 0.0003). For 8 Gy irradiated cells grown in flasks, there was no significant change in the number of cells between 24 and 72 hr (4.9 ± 0.9 × 105 and 4.9 ± 1.2 × 105, respectively). Thus, PLD conditions were established in pellets. Irradiation survival curves showed a JP4-039 mediated PLD radiation resistance when cells were incubated in JP4-039 at 24 hr or 72 hr after irradiation by increase in the Do. At 24 hr, the Do for pelleted cells incubated in JP4-039 was 1.92 ± 0.01 Gy compared to pelleted irradiated control 32D cl 3 cells, 1.36 ± 0.02 Gy (p = 0.0014). At 24 hrs, cells grown in flasks and incubated in JP4-039 had a Do of 1.70 ± 0.02 Gy compared to 1.40 ± 0.05 Gy for irradiated control 32D cl 3 cells (p = 0.0261). By 72 hrs after irradiation, the Do for pelleted cells was increased to 1.97 ± 0.06 Gy by adding JP4-039 compared to the pelleted irradiated control 32D cl 3 cells with a Do of 1.51 ± 0.04 Gy (p = 0.0218). In cells grown in flasks for 72 hr before the addition of JP4-039 the Do was 2.01 ± 0.02 Gy compared to irradiated controls which had a Do of 1.62 ± 0.07 Gy (p = 0.0346). Therefore, JP4-039 displays a clear potential to mitigate irradiation induced PLD damage. These data correlate with significant mitigation of the hematopoietic syndrome by JP4-039 in total body irradiated C57BL/6NHsd mice.

Druggable Genome siRNA-Screening Identifies Glybenclamide as a Radioprotector against Total Body Irradiation

New drugs to protect or mitigate total body irradiation (TBI) damage are urgently required. To determine whether existing pharmacologic agents have this second function, we used siRNA to screen 5,520 druggable targets in the Silencer Druggable Genome siRNA library (Version 1.1, Ambion, Austin, TX). We sought to identify genes which if silenced protected against or mitigated radiation damage. T98G human glioblastoma cells were transfected with each target in the siRNA library in triplicate at a final concentration of 20nM/target in a one-gene, one-well format using DharmaFECT2 transfection reagent and 48 hours later were irradiated to 25 Gy. Viability was measured 72 hours later using the CellTiter-Blue cell viability assay. The siRNA screen was performed three times and a hit was scored as a gene, which when silenced, protected T98G cells by at least 15% in each of the triplicate screens. We identified 117 candidate protective genes. These were further analyzed using Ingenuity Pathways Analysis to identify potential radiation inhibitor signaling pathways and drug candidates. One pharmacologically attractive candidate was glybenclamide. Glybenclamide is a sulfonylurea currently used to treat diabetes that inhibits the ATP-binding cassette, sub-family A (ABC1) and selectively blocks ATP sensitive potassium channels. Glybenclamide (10 μM) was added to 32D cl 3 murine hematopoietic progenitor cells one hour before irradiation, or immediately following irradiation. Cells were then irradiated to doses ranging from 0 to 8 Gy, plated in methylcellulose, and incubated for seven days at 37° C in a humidified CO2 incubator at which time colonies of greater than 50 cells were counted. The data were analyzed using linear quadratic or single-hit, multi-target models. Glybenclamide pre-irradiation treated cells were protected from irradiation as demonstrated by an increased shoulder on the survival curve with a n of 34.9 ± 0.5 compared to untreated 32D cl 3 cells or cells treated with glybenclamide after irradiation with a n of 3.1 ± 1.3 or 2.5 ± 0.9 (p = 0.0018 or < 0.0001, respectively). To determine if glybenclamide was radioprotective in vivo, we dissolved the drug in 50% Cremophor EL and 50% ethanol and then diluted 1:4 in water. C57BL/6NHsd female mice were injected intraperitoneally with 5 mg/kg glybenclamide either 10 minutes before or 10 minutes after TBI to the LD50/30 dose of 9.5 Gy. Duplicate groups received sucrose supplement to counteract potential hypoglycemia. We found that 60% of mice injected with glybenclamide before irradiation survived for 35 days compared to 33% for the TBI alone group (p = 0.0361), while 44% of mice injected with glybenclamide following irradiation survived. There was no significant effect of sucrose supplement or difference between irradiated controls, mice injected with vehicle only or glybenclamide following irradiation. These results demonstrate the power of unbiased siRNA screening with a druggable genome and suggest that glybenclamide might be a clinically useful radioprotector.

Acetylated Resveratrol: A New Small Molecule Radioprotector

New small molecules with radioprotective capacity are required for treatment of radiation spills or as countermeasures against radiological terrorism. Small molecules which can be easily stored, easily transported, inexpensive to produce and easily administered are optimal. We have tested the dietary component resveratrol and an acetylated moiety for protection of cells against radiation damage. Irradiation survival curves were carried out with resveratrol or acetylated resveratrol. Cells from the murine hematopoietic progenitor cell line 32D cl 3 were incubated with resveratrol or acetylated resveratrol (10 μM) for 1 hr and then were irradiated to doses ranging from 0 to 8 Gy. The cells were suspended in methylcellulose, incubated for 7 days at 37oC, colonies of greater than 50 cells counted and data analyzed by linear quadratic and single-hit, multi-target models. In vivo studies were performed by injecting intraperitoneally female C57BL/6NHsd mice either 10 minutes before or 10 minutes after an LD50/30 dose of 9.5 Gy total body irradiation. We compared resveratrol or acetylated resveratrol (1, 10, or 25 mg/kg). The mice were followed for irradiation induced hematopoietic syndrome. Incubation of 32D cl 3 cells in resveratrol or acetylated resveratrol before irradiation resulted in an increase in radiation resistance by an increased shoulder on the survival curve of 33.2 ± 5.7 or 37.5 ± 9.9, respectively, compared to 6.9 + 1.8 for 32D cl 3 cells (p = 0.0122 or 0.0072, respectively). The mice injected with acetylated-resveratrol had increased survival at 10 and 25 mg/kg compared to control irradiated mice (p < 0.0001 or = 0.0004, respectively). In contrast, resveratrol did not increase mouse survival. Neither resveratrol nor acetylated resveratrol were effective mitigators when given after irradiation. Acetylated resveratrol administered before irradiation in a mouse model is radioprotective. Further studies are in progress to determine if acetylated resveratrol can be translated to clinical use as a radioprotective agent.

Radioprotection In Vitro and In Vivo by Minicircle Plasmid Carrying the Human Manganese Superoxide Dismutase Transgene

Manganese superoxide dismutase plasmid liposomes (MnSOD-PL) confer organ-specific in vivo ionizing irradiation protection. To prepare for potential intravenous clinical trials of systemic MnSOD-PL for radioprotection in humans, plasmid and bacterial sequences were removed and a new minicircle construct was tested. Minicircle MnSOD was purified and then cotransfected into 32D cl 3 murine interleukin-3-dependent hematopoietic progenitor cells along with another plasmid carrying the neo gene. Cells were selected in G418 (50 microg/ml) and cloned by limiting dilution. Biochemical analysis of minicircle MnSOD-transfected cells showed an MnSOD biochemical activity level of 5.8 +/- 0.5 U/mg compared with 2.7 +/- 0.1 U/mg for control 32D cl 3 cells (p = 0.0039). 32D-mc-MnSOD cells were as radioresistant as full-length MnSOD-PL transgene-expressing 2C6 cells, relative to 32D cl 3 parent cells, with an increased shoulder on the radiation survival curve (n = 4.8 +/- 0.2 and n = 4.6 +/- 0.2, respectively, compared with 1.5 +/- 0.5 for 32D cl 3 cells; p = 0.007). C57BL/6NHsd mice received intraoral mc-MnSOD-PL, mc-DsRed-PL control, full-length MnSOD-PL, or blank-PL and then were irradiated 24 hr later with 31 Gy to the esophagus. Mice receiving mc-MnSOD-PL showed increased survival compared with control mice or mice treated with mc-DsRed-PL (p = 0.0003 and 0.039, respectively), and comparable to full-length MnSOD-PL. Intravenous, systemic administration of mc-MnSOD-PL protected mice from total body irradiation (9.75 Gy). Therefore, minicircle DNA containing the human MnSOD transgene confers undiminished radioprotection in vitro and in vivo.

Radioprotection In Vitro and In Vivo by Mini Circle Plasmid Containing the Human Manganese Superoxide Dismutase (MnSOD) Transgene

Manganese superoxide dismutase-plasmid liposomes (MnSOD-PL) confer organ specific in vivo ionizing irradiation protection. To prepare for potential intravenous clinical trials of systemic MnSOD-PL for radioprotection in humans, plasmid and bacterial sequences were removed and a new mini circle construct was tested. Mini circle-MnSOD was purified and then co-transfected into 32D cl 3 murine IL-3 dependent hematopoietic progenitor cells along with another plasmid containing the neo gene. Cells were selected in G418 (50 μg/ml) and cloned by limiting dilution. Biochemical analysis of mini circle MnSOD transfected cells showed 5.8 ± 0.5 U/mg MnSOD biochemical activity compared to 2.7 ± 0.1 U/mg for control 32D cl 3 cells (p = 0.0039). 32D -MC-MnSOD cells were comparably radioresistant to full length MnSOD-PL transgene 2C6 cells, relative to 32D cl 3 parent cells, with an increased shoulder on the radiation survival curve (n = 4.8 ± 0.2 and n = 4.6 ± 0.2 respectively compared to 1.5 ± 0.5 for 32D cl 3, p = 0.007). C57BL/6NHsd mice received intra-oral MC-MnSOD-PL, MC-DS-red-PL control, full length MnSOD-PL, or blank-PL then were irradiated 24 hours later to 31 Gy to the esophagus. Mice receiving MC-MnSOD-PL showed increased survival compared to control mice or mice treated with MC-DS-red-PL (p = 0.0099, or 0.039, respectively), and comparable to full length MnSOD-PL. Intravenous, systemic administration of MC-MnSOD-PL protected mice from 9.75 Gy total body irradiation. Therefore, mini circle DNA containing the human MnSOD transgene confers undiminished radioprotection in vitro and in vivo.

561. Overexpression of Mitochondrially Targeted Catalase and Manganese Superoxide Dismutase (MnSOD) in 32D cl 3 Mouse Hematopoietic Progenitor Cells Increases Antioxidant Levels and Radioresistance

561. Overexpression of Mitochondrially Targeted Catalase and Manganese Superoxide Dismutase (MnSOD) in 32D cl 3 Mouse Hematopoietic Progenitor Cells Increases Antioxidant Levels and Radioresistance

Overexpression of the MnSOD Transgene Product Protects Cryopreserved Bone Marrow Hematopoietic Progenitor Cells from Ionizing Radiation

We determined whether manganese superoxide dismutase (MnSOD)-plasmid liposome (PL) transfection of C57BL/ 6NHsd mouse bone marrow protected cells irradiated at room temperature (24 degrees C) or in the cryopreserved state. MnSOD-overexpressing hematopoietic progenitor 2C6 cells were radioresistant compared to the parent 32D cl 3 cells when irradiated frozen or at 24 degrees C. Fresh whole marrow from mice injected intravenously with MnSOD-PL prior to explant as well as explanted marrow single cell suspensions transfected in vitro were irradiated at 24 degrees C or -80 degrees C. In vivo or in vitro transfection of marrow with MnSOD-PL produced significant radiation protection of irradiated marrow progenitor cells compared to controls at 24 degrees C or -80 degrees C. (in vivo transfection D(0) 2.19 +/- 0.21 at 24 degrees C, D(0) 2.10 +/- 0.07 at -80 degrees C compared to control D(0) 1.56 +/- 0.06 or 1.66 +/- 0.04, P = 0.047 and 0.017 respectively; in vitro transfection D(0) 2.35 +/- 0.11 at 24 degrees C, D(0) 3.42 +/- 0.13 at -80 degrees C compared to D(0) 1.81 +/- 0.01 or 2.53 +/- 0.05, P = 0.0087 and 0.0026, respectively). Thus the MnSOD transgene product protects frozen marrow cells as well as marrow cells irradiated at 24 degrees C.

199. Overexpression of Mitochondrial Localized (mt)-Catalase Further Increases Radioresistance Induced by Manganese Superoxide Dismutase (MnSOD) Overexpression in 32D cl 3 Murine Hematopoietic Progenitor Cells

199. Overexpression of Mitochondrial Localized (mt)-Catalase Further Increases Radioresistance Induced by Manganese Superoxide Dismutase (MnSOD) Overexpression in 32D cl 3 Murine Hematopoietic Progenitor Cells