Amelioration of Mucositis in Proton Therapy of Fanconi Anemia Fanca Mice by JP4-039.

Background/aim: We tested JP4-039, a GS-nitroxide radiation damage mitigator in proton therapy of Fanconi anemia (FA) mice.

Materials And Methods: Fanca and Fanca bone marrow stromal cells were pre-treated with JP4-039 and irradiated with either protons or photons (0-10 GyRBE) followed by clonogenic survival and β-Galactosidase senescence analysis. Fanca and Fanca mice were pretreated with JP4-039 for 10 min prior to oropharyngeal irradiation with either protons or photons (0 or 30 GyRBE) followed by sacrifice and measurement of oral cavity ulceration, distant hematopoietic suppression, and real-time polymerase chain reaction analysis.

Malignant Transformation of Fanconi Anemia Complementation Group D2-deficient ( ) Hematopoietic Progenitor Cells by a Single HPV16 Oncogene.

Aim: To demonstrate that Fanconi anemia complementation group D2-deficient (Fancd2) hematopoietic progenitor cell lines can be transformed by transfection with a plasmid containing either the E6 or E7 oncogene of human papillomavirus (HPV) to generate malignant plasmacytoma-inducing cell lines.

Materials And Methods: In order to determine whether a single HPV type 16 (HPV16) oncogene induced malignant transformation, Fancd2 and Fancd2 interleukin 3 (IL3)-dependent hematopoietic progenitor cell lines were transfected with plasmids containing E6 or E7 oncogene, or control empty plasmid.

Results: Fancd2 but not Fancd2 cells were transformed into malignant IL3-independent cells by both E6, and E7 oncogenes, but not by empty plasmid.

Continuous One Year Oral Administration of the Radiation Mitigator, MMS350, after Total-Body Irradiation, Restores Bone Marrow Stromal Cell Proliferative Capacity and Reduces Senescence in Fanconi Anemia (Fanca) Mice.

We quantitated age-related accumulation of senescent cells in irradiated Fanconi anemia (FA) (Fanca mouse cell lines in vitro, and monitored the effect of continuous administration (via drinking water) of the water-soluble radiation mitigator, MMS350, on tissues in vivo over one year after 7.5 Gy total-body irradiation (TBI). Irradiated Fanca mouse bone marrow stromal cell lines showed increased numbers of beta-galactosidase- and p21-positive senescent cells compared to Fanca cell lines, which was reduced by MMS350.

Evaluation of Different Formulations and Routes for the Delivery of the Ionizing Radiation Mitigator GS-Nitroxide (JP4-039).

Background/aim: The mitochondrial targeted GS-nitroxide, JP4-039, is an effective total body irradiation (TBI) mitigator when delivered intravenously (IV) up to 72 h after exposure. Effective systemic and localized administration to oral cavity/oropharynx and esophagus has been demonstrated. The objective of the study was to establish alternatives to IV administration suitable for JP4-039 delivery to mass casualties.

Reduced Competitive Repopulation Capacity of Multipotential Hematopoietic Stem Cells in the Bone Marrow of Friend Virus-infected Fv2-resistant Mice.

Background/aim: The polycythemia form of Friend leukemia virus (FVP) causes splenomegaly and lethal erythroleukemia in Fv-2-susceptible mouse strains. We sought to determine whether the hematopoietic stem cell (HSC) pool was expanded in Fv-2-resistant mice.

Materials And Methods: The 120-day bone marrow transplantation competitive repopulation assay was used to determine whether FVP-infected Fv-2 C57BL/6 mice demonstrated expansion of the HSC pool compared to the pool of committed hematopoietic progenitor cells in the same marrow assayed in vitro.

Induction of TGF-β by Irradiation or Chemotherapy in Fanconi Anemia (FA) Mouse Bone Marrow Is Modulated by Small Molecule Radiation Mitigators JP4-039 and MMS350.

Background/aim: Total-body irradiation and/or administration of chemotherapy drugs in bone marrow transplantation induce cytokines that can suppress engraftment. Fanconi Anemia (FA) patients have a hyperactive responsiveness to the inhibitory cytokine, transforming growth factor-beta (TGF-β). Small molecule radiation mitigator drugs, JP4-039 and MMS350, were evaluated for suppression of irradiation or drug-induced TGF-β.

Evolution of malignant plasmacytoma cell lines from K14E7 Fancd2-/- mouse long-term bone marrow cultures.

We tested the effect of expression of the Human Papilloma Virus (HPV E7) oncogene on hematopoiesis in long-term bone marrow cultures (LTBMCs) derived from K14E7 (FVB) Fancd2-/- (129/Sv), K14E7 Fancd2+/+, Fancd2-/-, and control (FVB X 129/Sv) Fl mice. K14E7 Fancd2-/- and Fancd2-/- LTBMCs showed decreased duration of production of total nonadherent hematopoietic cells and progenitors forming day 7 and day 14 multilineage CFU-GEMM colonies in secondary cultures (7 wks and 8 wks respectively) compared to cultures from K14E7 Fancd2+/+ (17 wks) or control mice (18 wks) p < 0.0001.

Intraoral Mitochondrial-Targeted GS-Nitroxide, JP4-039, Radioprotects Normal Tissue in Tumor-Bearing Radiosensitive Fancd2(-/-) (C57BL/6) Mice.

We evaluated normal tissue specific radioprotection of the oral cavity in radiosensitive Fanconi Anemia (FA) Fancd2(-/-) mice with orally established tumors using mitochondrial-targeted GS-nitroxide (JP4-039). Adult (10-12 weeks old) Fancd2(+/+), Fancd2(+/-) and Fancd2(-/-) mice (C57BL/6 background) and subgroups with orally established TC-1 epithelial cell tumors received a single fraction of 28 Gy or four daily fractions of 8 Gy to the head and neck. Subgroups received JP4-039 in F15 emulsion (F15/JP4-039; 0.

Improved hematopoiesis in GS-nitroxide (JP4-039)-treated mouse long-term bone marrow cultures and radioresistance of derived bone marrow stromal cell lines.

Aim: To determine if the small-molecule radioprotector GS-nitroxide, JP4-039, improved hematopoiesis in long-term bone marrow cultures (LTBMCs), explanted marrow from in vivo drug-treated C57BL/6NTac mice was maintained in JP4-039 for 25 weeks. Hematopoietic cell production and radiobiology of derived stromal cell lines was measured.

Materials And Methods: Groups of LTBMCs were established from mouse groups.

Effects of the bifunctional sulfoxide MMS350, a radiation mitigator, on hematopoiesis in long-term bone marrow cultures and on radioresistance of marrow stromal cell lines.

The ionizing irradiation mitigator MMS350 prolongs survival of mice treated with total-body irradiation and prevents radiation-induced pulmonary fibrosis when added to drinking water at day 100 after thoracic irradiation. The effects of MMS350 on hematopoiesis in long-term bone marrow culture and on the radiobiology of derived bone marrow stromal cell lines were tested. Long-term bone marrow cultures were established from C57BL/6NTac mice and maintained in a high-humidity incubator, with 7% CO2 and the addition of 100 μM MMS350 at the weekly media change.

Increased hematopoiesis in long-term bone marrow cultures and reduced irradiation-induced pulmonary fibrosis in Von Willebrand factor homologous deletion recombinant mice.

Aim: We investigated whether homologous recombinant deletion of the endothelial cell-specific protein Von Willebrand factor (vWF) affected hematopoiesis in long-term bone marrow cultures, and irradiation induction of pulmonary fibrosis/organizing alveolitis.

Materials And Methods: We established long-term bone marrow cultures from vWF(-/-) (C57BL/6 background) and vWF(+/+) littermate mice. Non-adherent cells removed weekly were tested for formation of multi-lineage hematopoietic stem cells forming colonies at 7 and 14 days in secondary semi-solid medium cultures.

Improved longevity of hematopoiesis in long-term bone marrow cultures and reduced irradiation-induced pulmonary fibrosis in Toll-like receptor-4 deletion recombinant-negative mice.

Aim: We measured long-term hematopoiesis in continuous bone marrow cultures derived from Toll-like receptor-4 (Tlr4(-/-))(C57BL/6J) mice.

Materials And Methods: We measured hematopoiesis in vitro over 27 weeks in long-term bone marrow cultures from Tlr4(-/-) and control mice, and irradiation-induced pulmonary fibrosis in mice irradiated to 20 Gy to the thorax.

Results: There was a significant increase in the duration of hematopoiesis in long-term bone marrow cultures from Tlr4(-/-) mice in production of total non-adherent cells and day 7 and day 14 multi-lineage colony-forming cells.

Esophageal radioprotection by swallowed JP4-039/F15 in thoracic-irradiated mice with transgenic lung tumors.

Background/aim: To determine whether Gramicidin S (GS)-nitroxide, JP4-039, esophageal radiation protection protected lung tumors in a transgenic model, LoxP-Stoop-LoxP Kristen Rat Sarcoma Viral oncogene (LSL-K-RAS) mice were administered intra-tracheal- Carbapenem-resistant Enterobacteriaceae (CRE) recombinase, bilateral lung tumors were confirmed at 11 weeks, then thoracic irradiation was delivered.

Materials And Methods: Mice received single-fraction 15 Gy or 24 Gy to both lungs, in subgroups receiving intraesophageal administration 10 min before irradiation of JP4-039 (in F15 emulsion) tumor size reduction and survival were investigated. Mice were followed for survival, and reduction in tumor size.

Amelioration of radiation-induced oral cavity mucositis and distant bone marrow suppression in fanconi anemia Fancd2-/- (FVB/N) mice by intraoral GS-nitroxide JP4-039.

The altered DNA damage response pathway in patients with Fanconi anemia (FA) may increase the toxicity of clinical radiotherapy. We quantitated oral cavity mucositis in irradiated Fanconi anemia Fancd2(-/-) mice, comparing this to Fancd2(+/-) and Fancd2(+/+) mice, and we measured distant bone marrow suppression and quantitated the effect of the intraoral radioprotector GS-nitroxide, JP4-039 in F15 emulsion. We found that FA mice were more susceptible to radiation injury and that protection from radiation injury by JP4-039/F15 was observed at all radiation doses.

Differences in irradiated lung gene transcription between fibrosis-prone C57BL/6NHsd and fibrosis-resistant C3H/HeNHsd mice.

Background/aim: We compared pulmonary irradiation-induced whole-lung, gene transcripts over 200 days after 20 Gy thoracic irradiation in female fibrosis-prone C57BL/6NHsd mice with fibrosis-resistant C3H/HeNHsd mice.

Materials And Methods: Lung specimens were analyzed by real time polymerase chain reaction (rt-PCR) and changes over time in representative gene transcript levels were correlated with protein levels using western blot.

Results: C3H/HeNHsd mice showed a significantly longer duration of elevation of gene transcripts for stress-response genes nuclear factor kappa-light-chain-enhancer of activated B cells (Nfkb), nuclear factor (erythroid-derived 2)-like 2 (Nrf2), transcription factor SP1 (SP1), activator protein 1 (AP1), radioprotection gene manganese superoxide dismutase (Sod2), and endothelial cell-associated genes von Willebrand factor (Vwf) and vascular endothelial growth factor (Vegf).

Radiologic differences between bone marrow stromal and hematopoietic progenitor cell lines from Fanconi Anemia (Fancd2(-/-)) mice.

FancD2 plays a central role in the human Fanconi anemia DNA damage response (DDR) pathway. Fancd2(-/-) mice exhibit many features of human Fanconi anemia including cellular DNA repair defects. Whether the DNA repair defect in Fancd2(-/-) mice results in radiologic changes in all cell lineages is unknown.

Amelioration of radiation-induced pulmonary fibrosis by a water-soluble bifunctional sulfoxide radiation mitigator (MMS350).

A water-soluble ionizing radiation mitigator would have considerable advantages for the management of acute and chronic effects of ionizing radiation. We report that a novel oxetanyl sulfoxide (MMS350) is effective both as a protector and a mitigator of clonal mouse bone marrow stromal cell lines in vitro, and is an effective in vivo mitigator when administered 24 h after 9.5 Gy (LD100/30) total-body irradiation of C57BL/6NHsd mice, significantly improving survival (P = 0.

Radioresistance of bone marrow stromal and hematopoietic progenitor cell lines derived from Nrf2-/- homozygous deletion recombinant-negative mice.

Aim: We determined whether bone marrow from Nrf2(-/-) compared with Nrf2(+/+) mice differed in response to the oxidative stress of continuous marrow culture, and in radiosensitivity of derived stromal and interleukin-3 (IL-3)-dependent hematopoietic progenitor cells.

Materials And Methods: Hematopoiesis longevity in Nrf2(-/-) was compared with Nrf2(+/+) mice in long-term bone marrow cultures. Clonogenic irradiation survival curves were performed on derived cell lines.

Conditional radioresistance of Tet-inducible manganese superoxide dismutase bone marrow stromal cell lines.

Mitochondrial targeted manganese superoxide dismutase is a major antioxidant enzyme, the levels of which modulate the response of cells, tissues and organs to ionizing irradiation. We developed a Tet-regulated MnSOD mouse (MnSOD(tet)) to examine the detailed relationship between cellular MnSOD concentration and radioresistance and carried out in vitro studies using bone marrow culture derived stromal cell lines (mesenchymal stem cells). Homozygous MnSOD(tet/tet) cells had low levels of MnSOD, reduced viability and proliferation, increased radiosensitivity, elevated overall antioxidant stores, and defects in cell proliferation and DNA strand-break repair.

Increased longevity of hematopoiesis in continuous marrow cultures and radiation resistance of marrow stromal and hematopoietic progenitor cells from caspase-1 homozygous recombinant-negative (knockout) mice.

Aim: We determined whether absence of caspase-1 altered the stress response of hematopoietic and bone marrow stromal cells in vitro.

Materials And Methods: Long-term bone marrow cultures from caspase-1 -/- and control caspase-1 +/+ mice were established and the derived bone marrow stromal and interleukin-3 (Il-3)-dependent hematopoietic progenitor cell lines were evaluated for radiosensitivity.

Results: Long-term bone marrow cultures from caspase-1 -/- mice generated hematopoietic cells for over 30 weeks in vitro, significantly longer than controls did (p=0.

Effects of thoracic irradiation on pulmonary endothelial compared to alveolar type-II cells in fibrosis-prone C57BL/6NTac mice.

Background/aim: Thoracic irradiation results in an acute inflammatory response, latent period, and late fibrosis. Little is known about the mechanisms involved in triggering late radiation fibrosis.

Materials And Methods: Thoracic irradiated fibrosis prone C57BL/6NTac mice were followed for detectable mRNA transcripts in isolated lung cells and micro-RNA in whole-tissues, and the effect of administration of water-soluble oxetanyl sulfoxide MMS350 was studied.

Ionizing irradiation protection and mitigation of murine cells by carbamazepine is p53 and autophagy independent.

Background: Carbamazepine, a sodium channel blocker and pro-autophagy agent used in the treatment of epilepsy and trigeminal neuralgia, is also an ionizing radiation mitigator and protector.

Materials And Methods: We measured the effect of carbamazepine, compared to other pro-autophagy drugs (i.e.

Repopulation of the irradiation damaged lung with bone marrow-derived cells.

Aim: The effect of lung irradiation on reduction of lung stem cells and repopulation with bone marrow-derived cells was measured.

Materials And Methods: Expression of green fluorescent protein positive cells (GFP(+)) in the lungs of thoracic irradiated FVB/NHsd mice (Harlan Sprague Dawley, Indianapolis, IN, USA) was determined. This was compared to the repopulation of bone marrow-derived cells found in the lungs from naphthalene treated male FVB/NHsd mice and gangciclovir (GCV) treated FeVBN GFP(+) male marrow chimeric HSV-TK-CCSP.

GS-nitroxide (JP4-039)-mediated radioprotection of human Fanconi anemia cell lines.

Fanconi anemia (FA) is an inherited disorder characterized by defective DNA repair and cellular sensitivity to DNA crosslinking agents. Clinically, FA is associated with high risk for marrow failure, leukemia and head and neck squamous cell carcinoma (HNSCC). Radiosensitivity in FA patients compromises the use of total-body irradiation for hematopoietic stem cell transplantation and radiation therapy for HNSCC.