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. We measured stress of hematopoiesis in long-term marrow cultures and radiosensitivity in clonogenic survival curves, as well as comet tail intensity, total antioxidant stores and radiation-induced gene expression in hematopoietic progenitor compared to bone marrow stromal cell lines. We further evaluated radioprotection by a mitochondrial-targeted antioxidant GS-nitroxide, JP4-039. Hematopoiesis longevity in Fancd2−/− mouse long-term marrow cultures was diminished and bone marrow stromal cell lines were radiosensitive compared to Fancd2+/+ stromal cells (Fancd2−/− D0 = 1.4 ± 0.1 Gy, ñ = 5.0 ± 0.6 vs. Fancd2+/+ D0 = 1.6 ± 0.1 Gy, ñ = 6.7 ± 1.6), P = 0.0124 for D0 and P = 0.0023 for ñ, respectively). In contrast, Fancd2−/− IL-3-dependent hematopoietic progenitor cells were radioresistant (D0 = 1.71 ± 0.04 Gy and ñ = 5.07 ± 0.52) compared to Fancd2+/+ (D0 = 1.39 ± 0.09 Gy and ñ = 2.31 ± 0.85, P = 0.001 for D0). CFU-GM from freshly explanted Fancd2−/− marrow was also radioresistant. Consistent with radiosensitivity, irradiated Fancd2−/− stromal cells had higher DNA damage by comet tail intensity assay compared to Fancd2+/+ cells (P < 0.0001), slower DNA damage recovery, lower baseline total antioxidant capacity, enhanced radiation-induced depletion of antioxidants, and increased CDKN1A-p21 gene transcripts and protein. Consistent with radioresistance, Fancd2−/− IL-3-dependent hematopoietic cells had higher baseline and post irradiation total antioxidant capacity. While, there was no detectable alteration of radiation-induced cell cycle arrest with Fancd2−/− stromal cells, hematopoietic progenitor cells showed reduced G2/M cell cycle arrest. The absence of the mouse Fancd2 gene product confers radiosensitivity to bone marrow stromal but not hematopoietic progenitor cells.

Purpose-To evaluate the effectiveness of mitigation of acute ionizing radiation damage by mitochondria-targeted small molecules.Materials and Methods-We evaluated the nitroxide-linked alkene peptide isostere JP4-039, the nitric oxide synthase inhibitor-linked alkene peptide esostere MCF201-89, and the p53/mdm2/ mdm4 inhibitor BEB55 in radiation mitigation by clonogenic survival curves with the murine hematopoietic progenitor cell line 32D cl 3, human bone marrow stromal (KM101) and pulmonary epithelial (IB3) cell line. 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. after 9.5 Gy total body irradiation (TBI) with JP4-039, MCF201-89, or BEB55 individually or in combination.Results-Each drug, JP4-039, MCF201-89, or BEB55, individually or as a mixture of all 3 compounds, increased the survival of 32D cl 3 cells and IB3 cells significantly over control irradiated cells (p=0.0021, p=0.0011, p=0.0038, and p=0.0073, respectively), and (p=0.0193, p=0.0452, p=0.0017, and p=0.0019 respectively). 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 3 after TBI significantly increased mouse survival (p=0.0234, 0.0009, 0.0052 and 0.0167 respectively).Corresponding Author: Joel S. Greenberger M.D., Department of Radiation Oncology, University of Pittsburgh Cancer Institute, 5150 Centre Avenue, Rm. 533, Pittsburgh, PA 15232, Tel: 412-647-3602, Fax: 412-647-1161, greenbergerjs@upmc

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. Adult 10–12-week-old mice, of FVB/N background Fancd2−/−, Fancd2+/− and Fancd2+/+ were head and neck irradiated with 24, 26, 28 or 30 Gy (large fraction sizes typical of stereotactic radiosurgery treatments) and subgroups received intraoral JP4-039 (0.4 mg/mouse in 100 μL F15 liposome emulsion) preirradiation. On day 2 or 5 postirradiation, mice were sacrificed, tongue tissue and femur marrow were excised for quantitation of radiation-induced stress response, inflammatory and antioxidant gene transcripts, histopathology and assay for femur marrow colony-forming hematopoietic progenitor cells. Fancd2−/− mice had a significantly higher percentage of oral mucosal ulceration at day 5 after 26 Gy irradiation (59.4 ± 8.2%) compared to control Fancd2+/+ mice (21.7 ± 2.9%, P = 0.0063). After 24 Gy irradiation, Fancd2−/− mice had a higher oral cavity percentage of tongue ulceration compared to Fancd2+/+ mice irradiated with higher doses of 26 Gy (P = 0.0123). Baseline and postirradiation oral cavity gene transcripts were altered in Fancd2−/− mice compared to Fancd2+/+ controls. Fancd2−/− mice had decreased baseline femur marrow CFU-GM, BFUe and CFU-GEMM, which further decreased after 24 or 26 Gy head and neck irradiation. These changes were not seen in head- and neck-irradiated Fancd2+/+ mice. In radiosensitive Fancd2−/− mice, biomarkers of both local oral cavity and distant marrow radiation toxicity were ameliorated by intraoral JP4-039/F15. We propose that Fancd2−/− mice are a valuable radiosensitive animal model system, which can be used to evaluate potential radioprotective agents.

Mammalian POLQ (pol theta) is a specialized DNA polymerase with an unknown function in vivo. Roles have been proposed in chromosome stability, as a backup enzyme in DNA base excision repair, and in somatic hypermutation of immunoglobulin genes. The purified enzyme can bypass AP sites and thymine glycol. Mice defective in POLQ are viable and have been reported to have elevated spontaneous and radiation-induced frequencies of micronuclei in circulating red blood cells. To examine the potential roles of POLQ in hematopoiesis and in responses to oxidative stress responses, including ionizing radiation, bone marrow cultures and marrow stromal cell lines were established from Polq(+/+) and Polq(-/-) mice. Aging of bone marrow cultures was not altered, but Polq(-/-) cells were more sensitive to gamma radiation than were Polq(+/+) cells. The D(0) was 1.38 +/- 0.06 Gy for Polq(+/+) cells compared to 1.27 +/- 0.16 and 0.98 +/- 0.10 Gy (P = 0.032) for two Polq(-/-) clones. Polq(-/-) cells were moderately more sensitive to bleomycin than Polq(+/+) cells and were not hypersensitive to paraquat or hydrogen peroxide. ATM kinase activation appeared to be normal in gamma-irradiated Polq(-/-) cells. Inhibition of ATM kinase activity increased the radiosensitivity of Polq(+/+) cells slightly but did not affect Polq(-/-) cells. Polq(-/-) mice had more spontaneous and radiation-induced micronucleated reticulocytes than Polq+/+ and (+/-) mice. The sensitivity of POLQ-defective bone marrow stromal cells to ionizing radiation and bleomycin and the increase in micronuclei in red blood cells support a role for this DNA polymerase in cellular tolerance of DNA damage that can lead to double-strand DNA breaks.

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; 0.4 mg/mouse), 4-amino-Tempo in F15 emulsion (F15/4-amino-Tempo; 0.2 mg/ mouse) or F15 emulsion alone prior to each irradiation. Oral mucosa of Fancd2−/− mice showed baseline elevated RNA transcripts for Sod2, p53, p21 and Rad51 (all P < 0.0012) and suppressed levels of Nfkb and Tgfb, (all P < 0.0020) compared with Fancd2+/+ mice. The oral mucosa in tumor-bearing mice of all genotypes showed decreased levels of p53 and elevated Tgfb and Gadd45a (P ≤ 0.0001 for all three genotypes). Intraoral F15/JP4-039, but not F15/4-amino-Tempo, modulated radiation-induced normal tissue transcript elevation, ameliorated mucosal ulceration and reduced the depletion of antioxidant stores in oral cavity tissue of all genotypes, but did not radioprotect tumors. Mitochondrial targeting makes F15/JP4-039 an effective normal tissue radioprotector for Fancd2−/− mice, as well as wild-type mice.