Transgenic expression of human C19MC miRNAs impacts placental morphogenesis.

INTRODUCTION: The chromosome 19 miRNA cluster (C19MC) encodes a large family of microRNAs (miRNAs) that are abundantly expressed in the placenta of higher primates and also in certain cancers. In the placenta, miRNAs from this cluster account for nearly 40% of all miRNAs present in trophoblasts. However, the function of these miRNAs in the placenta remains poorly understood. Recent observations reveal a role for these miRNAs in cell migration, and suggest that they are involved in the development and function of the human placenta. Here, we examine the placenta in transgenic mice expressing the human C19MC miRNAs. METHODS: We produced transgenic mice using pronuclear microinjection of a bacterial artificial chromosome plasmid carrying the entire human C19MC locus and derived a homozygous line using crossbreeding. We performed morphological characterization and profiled gene expression changes in the placentas of the transgenic mice. RESULTS: C19MC transgenic mice delivered on time with no gross malformations. The placentas of transgenic mice expressed C19MC miRNAs and were larger than wild type placentas. Histologically, we found that the transgenic placenta exhibited projections of spongiotrophoblasts that penetrated deep into the labyrinth. Gene expression analysis revealed alterations in the expression of several genes involved in cell migration, with evidence of enhanced cell proliferation. DISCUSSION: Mice that were humanized for transgenically overexpressed C19MC miRNAs exhibit enlarged placentas with aberrant delineation of cell layers. The observed phenotype and the related gene expression changes suggest disrupted migration of placental cell subpopulations.

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; 0.4 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.

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. Stromal cell lines were established from the adherent layer of JP4-039-treated and untreated control groups. RESULTS: LTBMCs maintained in JP4-039 exhibited increased production of total non-adherent and 7-day and 14-day hematopoietic colony-forming cells. Stromal cell lines derived from JP4-039-treated cultures were radioresistant in vitro, demonstrated a distinct squamous/epithelial morphology and overexpressed Nrf2, Ctgf, Lox, Tlr1, collagen 1a, Brd3, and Brd4. CONCLUSION: Chronic treatment of bone marrow cultures and derived stromal cell lines with JP4-039 was non-toxic, and conferred resistance to oxidative stress.

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. The histology of bone marrow hematopoietic and stromal cell lines was indistinguishable between different mouse strains. There was no detectable late irradiation pulmonary fibrosis in Tlr4(-/-) mice. CONCLUSION: Homozygous deletion of both alleles of Tlr4, encoding for an inflammatory mediator receptor, improves the duration of hematopoiesis in vitro and reduces irradiation-induced lung fibrosis.

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. Irradiation fibrosis in the lungs of 20-Gy thoracic irradiated mice was quantitated and scored. RESULTS: Hematopoiesis was increased over 20 weeks in vWF(-/-) compared to vWF(+/+) cultures in production of non-adherent cells, and cells forming colonies at 7 or 14 days in secondary semi-solid medium culture. The irradiated lungs showed no increased fibrosis. CONCLUSION: Absence of vWF increases hematopoiesis in long-term bone marrow cultures and has a protective effect in irradiated lungs.

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. RESULTS: There was no evidence of tumor radioprotection in mice receiving JP4-039/F15. CONCLUSION: Intraesophageal radioprotective small-molecule antioxidant therapy protects normal tissue but not tumor tissue in mice with transgenic lung tumors.

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. 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.

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). C57BL/6NHsd mice showed acute elevation then down-regulation and a second elevation in gene transcripts for Nfkb, connective tissue growth factor (Ctgf), insulin-like growth factor-binding protein 7 (Igfbp7), tumor necrosis factor-alpha (Tnfa) Ctgf, Igfbp7, Tnfa, collagen 1a, and toll like receptor 4 (Tlr4). There were reciprocal patterns of elevation and decrease in levels of transcripts for epigenetic reader proteins bromodomain coding protein 1 (Brd1)Brd2,-3, and -4 between mouse strains. CONCLUSION: Regulatory pathways linked to radiation pulmonary fibrosis may identify new targets for mitigators of radiation-induced fibrosis.

Effects of mouse genotype on bone wound healing and irradiation-induced delay of healing.

We tested the effects of mouse genotype (C57BL/6NHsd, NOD/SCID, SAMR1, and SAMP6) and ionizing irradiation on bone wound healing. Unicortical wounds were made in the proximal tibiae, and the time course of spontaneous healing and effects of irradiation were monitored radiographically and histologically. There was reproducible healing beginning with intramedullary osteogenesis, subsequent bone resorption by osteoclasts, gradual bridging of the cortical wound, and re-population of medullary hematopoietic cells. The most rapid wound closure was noted in SAMR1 mice, followed by SAMP6, C57BL/6NHsd, and NOD/SCID. Ionizing irradiation (20 Gy) to the leg significantly delayed bone wound healing in mice of all four genotypes. Mice with genetically-determined predisposition to early osteopenia (SAMP6) or with immune deficiency (NOD/SCID) had impairments in bone wound healing. These mouse models should be valuable for determining the effects of irradiation on bone healing and also for the design and testing of novel bone growth-enhancing drugs and mitigators of ionizing irradiation.

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. 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.

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 = 0.0097). Furthermore, MMS350 (400 µM) added weekly to drinking water after 20 Gy thoracic irradiation significantly decreased: expression of pulmonary inflammatory and profibrotic gene transcripts and proteins; migration into the lungs of bone marrow origin luciferase+/GFP+ (luc+/GFP+) fibroblast progenitors (in both luc+ marrow chimeric and luc+ stromal cell line injected mouse models) and decreased radiation-induced pulmonary fibrosis (P < 0.0001). This nontoxic and orally administered small molecule may be an effective therapeutic in clinical radiotherapy and as a counter measure against the acute and chronic effects of ionizing radiation.

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.

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. Total antioxidant capacity at baseline in nonirradiated cells and at 24 hours after 5 Gy and 10 Gy irradiation was quantitated using an antioxidant reductive capacity assay. RESULTS: Long-term cultures of bone marrow from Nrf2(-/-) compared to Nrf2(+/+) mice demonstrated equivalent longevity of production of total cells and hematopoietic progenitor cells forming multi-lineage hematopoietic colonies over 26 weeks in culture. Both bone marrow stromal cell lines and Il-3-dependent hematopoietic progenitor cell lines derived from Nrf2(-/-) mouse marrow cultures were radioresistant compared to Nrf2(+/+)-derived cell lines. Both DNA repair assay and total antioxidant capacity assay showed no defect in Nrf2(-/-) compared to Nrf2(+/+) stromal cells and IL-3-dependent cells. CONCLUSION: The absence of a functional Nrf2 gene product does not alter cellular interactions in continuous marrow culture, nor response to dsDNA damage repair and antioxidant response. However, lack of the Nrf2 gene does confer radioresistance on marrow stromal and hematopoietic cells.

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=0.0018). Bone marrow stromal (mesenchymal stem cell) and Il-3-dependent hematopoietic progenitor cell lines from caspase-1-/- marrow cultures compared to caspase-1 +/+ were radioresistant (p=0.0486 and p=0.0235 respectively). Total-body irradiated caspase-1 -/- mice were not significantly radioresistant compared to controls (p=0.6542). CONCLUSION: Caspase-1 deletion increases hematopoiesis and radioresistance of bone marrow cells in vitro.

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. Doxycycline (doxy) treatment of MnSOD(tet/tet) cells increased MnSOD levels and radioresistance from ñ of 2.79 ± 1.04 to 8.69 ± 1.09 (P = 0.0060) and normalized other biologic parameters. In contrast, MnSOD(tet/tet) cells showed minimal difference in baseline and radiation induced mRNA and protein levels of TGF-ß, Nrf2 and NF-κB and radiation induced cell cycle arrest was not dependent upon MnSOD level. These novel MnSOD(tet/tet) mouse derived cells should be valuable for elucidating several parameters of the oxidative stress response to ionizing radiation.

Synthesis of analogs of the radiation mitigator JP4-039 and visualization of BODIPY derivatives in mitochondria.

JP4-039 is a lead structure in a series of nitroxide conjugates that are capable of accumulating in mitochondria and scavenging reactive oxygen species (ROS). To explore structure-activity relationships (SAR), new analogs with variable nitroxide moieties were prepared. Furthermore, fluorophore-tagged analogs were synthesized and provided the opportunity for visualization in mitochondria. All analogs were tested for radioprotective and radiomitigative effects in 32Dcl3 cells.

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. Marrow stromal cell motility in medium from fibrotic-phase explanted pulmonary endothelial and alveolar type-II cells was measured. RESULTS: RNA and micro-RNA expression in lung correlated with fibrosis. MMS350 reduced pro-fibrotic gene expression in both endothelial and alveolar type-II cells in irradiated mice. Conditioned medium from irradiated cells did not alter cell motility in vitro. CONCLUSION: These studies should facilitate identification of potential new drug targets for ameliorating irradiation-induced pulmonary fibrosis.

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. lithium and valproic acid), on irradiation of autophagy incompetent (Atg5(-/-)) and competent (Atg5(+/+)) mouse embryonic fibroblasts, p53(-/-) and p53(+/+) bone marrow stromal cells, and human IB3, KM101, HeLa, and umbilical cord blood cell and in total body-irradiated or orthotopic tumor-bearing mice. RESULTS: Carbamazepine, but not other pro-autophagy drugs, was a radiation protector and mitigator for mouse cell lines, independent of apoptosis, autophagy, p53, antioxidant store depletion, and class I phosphatidylinositol 3-kinase, but was ineffective with human cells. Carbamazepine was effective when delivered 24 hours before or 12 hours after total body irradiation of C57BL/6HNsd mice and did not protect orthotopic Lewis lung tumors. CONCLUSION: Carbamazepine is a murine radiation protector and mitigator.

Dysregulated in vitro hematopoiesis, radiosensitivity, proliferation, and osteoblastogenesis with marrow from SAMP6 mice.

The senescence accelerated-prone mouse variant 6 (SAMP6) shows normal growth followed by rapid aging, development of osteopenia, and shortened lifespan, compared with control R1 mice. Because oxidative stress is a fundamental mechanism of tissue aging, we tested whether cellular parameters that are associated with oxidative stress are impaired with marrow from SAMP6 mice. We compared in vitro hematopoiesis, irradiation sensitivity, proliferative potential, and osteoblastogenesis with marrow cells from SAMP6 and R1 mice. Marrow cells from SAMP6 mice showed shortened in vitro hematopoiesis; their stromal cells showed greater radiation sensitivity and decreased proliferation. Consistent with those properties, there was constitutive upregulation of transforming growth factor-ß(1), an inhibitor of hematopoiesis, and of cell cycle inhibitory genes, p16(INK4A) and p19(ARF). Paradoxically, there was constitutive expression of osteoblast genes in stromal cells from SAMP6 mice, but in vitro matrix mineralization was impaired. These studies and data included in other reports indicate that impaired proliferation of osteoblast progenitors in SAMP6 marrow may be a major factor contributing to accelerated loss of bone mass. In sum, marrow from SAMP6 mice had diminished capacity for long-term hematopoiesis, increased radiosensitivity, and reduced proliferative capacity.

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. The level of mRNA for lung stem cell markers clara cell (CCSP), epithelium 1 (FOXJ1) and surfactant protein C (SP-C), and sorted single cells positive for marrow origin epithelial cells (GFP(+)CD45(-)) was measured. RESULTS: The expression of pulmonary stem cells as determined by PCR was reduced most by GCV, then naphthalene, and least by thoracic irradiation. Irradiation, like GCV, reduced mRNA expression of CCSP, CYP2F2, and FOXJ1, while naphthalene reduced that of CCSP and CYP2F2. Ultrastructural analysis showed GFP(+) pulmonary cells of bone marrow origin, with the highest frequency being found in GCV-treated groups. CONCLUSION: Bone marrow progenitor cells may not participate in the repopulation of the lung following irradiation.