Ferroptosis induces membrane blebbing in placental trophoblasts.

Ferroptosis is a regulated, non-apoptotic form of cell death, characterized by hydroxy-peroxidation of discrete phospholipid hydroperoxides, particularly hydroperoxyl (Hp) forms of arachidonoyl- and adrenoyl-phosphatidylethanolamine, with a downstream cascade of oxidative damage to membrane lipids, proteins and DNA, culminating in cell death. We recently showed that human trophoblasts are particularly sensitive to ferroptosis caused by depletion or inhibition of glutathione peroxidase 4 (GPX4) or the lipase PLA2G6. Here, we show that trophoblastic ferroptosis is accompanied by a dramatic change in the trophoblast plasma membrane, with macro-blebbing and vesiculation. Immunofluorescence revealed that ferroptotic cell-derived blebs stained positive for F-actin, but negative for cytoplasmic organelle markers. Transfer of conditioned medium that contained detached macrovesicles or co-culture of wild-type target cells with blebbing cells did not stimulate ferroptosis in target cells. Molecular modeling showed that the presence of Hp-phosphatidylethanolamine in the cell membrane promoted its cell ability to be stretched. Together, our data establish that membrane macro-blebbing is characteristic of trophoblast ferroptosis and can serve as a useful marker of this process. Whether or not these blebs are physiologically functional remains to be established.

PLA2G6 guards placental trophoblasts against ferroptotic injury.

The recently identified ferroptotic cell death is characterized by excessive accumulation of hydroperoxy-arachidonoyl (C20:4)- or adrenoyl (C22:4)- phosphatidylethanolamine (Hp-PE). The selenium-dependent glutathione peroxidase 4 (GPX4) inhibits ferroptosis, converting unstable ferroptotic lipid hydroperoxides to nontoxic lipid alcohols in a tissue-specific manner. While placental oxidative stress and lipotoxicity are hallmarks of placental dysfunction, the possible role of ferroptosis in placental dysfunction is largely unknown. We found that spontaneous preterm birth is associated with ferroptosis and that inhibition of GPX4 causes ferroptotic injury in primary human trophoblasts and during mouse pregnancy. Importantly, we uncovered a role for the phospholipase PLA2G6 (PNPLA9, iPLA2beta), known to metabolize Hp-PE to lyso-PE and oxidized fatty acid, in mitigating ferroptosis induced by GPX4 inhibition in vitro or by hypoxia/reoxygenation injury in vivo. Together, we identified ferroptosis signaling in the human and mouse placenta, established a role for PLA2G6 in attenuating trophoblastic ferroptosis, and provided mechanistic insights into the ill-defined placental lipotoxicity that may inspire PLA2G6-targeted therapeutic strategies.

Diet influences community dynamics following vaginal group B streptococcus colonization.

The vaginal microbiota plays a pivotal role in reproductive, sexual, and perinatal health and disease. Unlike the well-established connections between diet, metabolism, and the intestinal microbiota, parallel mechanisms influencing the vaginal microbiota and pathogen colonization remain overlooked. In this study, we combine a mouse model of Streptococcus agalactiae strain COH1 [group B Streptococcus (GBS)] vaginal colonization with a mouse model of pubertal-onset obesity to assess diet as a determinant of vaginal microbiota composition and its role in colonization resistance. We leveraged culture-dependent assessment of GBS clearance and culture-independent, sequencing-based reconstruction of the vaginal microbiota in relation to diet, obesity, glucose tolerance, and microbial dynamics across time scales. Our findings demonstrate that excessive body weight gain and glucose intolerance are not associated with vaginal GBS density or timing of clearance. Diets high in fat and low in soluble fiber are associated with vaginal GBS persistence, and changes in vaginal microbiota structure and composition due to diet contribute to GBS clearance patterns in nonpregnant mice. These findings underscore a critical need for studies on diet as a key determinant of vaginal microbiota composition and its relevance to reproductive and perinatal outcomes.IMPORTANCEThis work sheds light on diet as a key determinant influencing the composition of vaginal microbiota and its involvement in group B Streptococcus (GBS) colonization in a mouse model. This study shows that mice fed diets with different nutritional composition display differences in GBS density and timing of clearance in the female reproductive tract. These findings are particularly significant given clear links between GBS and adverse reproductive and neonatal outcomes, advancing our understanding by identifying critical connections between dietary components, factors originating from the intestinal tract, vaginal microbiota, and reproductive outcomes.

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.

The lipase cofactor CGI58 controls placental lipolysis.

In eutherians, the placenta plays a critical role in the uptake, storage, and metabolism of lipids. These processes govern the availability of fatty acids to the developing fetus, where inadequate supply has been associated with substandard fetal growth. Whereas lipid droplets are essential for the storage of neutral lipids in the placenta and many other tissues, the processes that regulate placental lipid droplet lipolysis remain largely unknown. To assess the role of triglyceride lipases and their cofactors in determining placental lipid droplet and lipid accumulation, we assessed the role of patatin like phospholipase domain containing 2 (PNPLA2) and comparative gene identification-58 (CGI58) in lipid droplet dynamics in the human and mouse placenta. While both proteins are expressed in the placenta, the absence of CGI58, not PNPLA2, markedly increased placental lipid and lipid droplet accumulation. These changes were reversed upon restoration of CGI58 levels selectively in the CGI58-deficient mouse placenta. Using co-immunoprecipitation, we found that, in addition to PNPLA2, PNPLA9 interacts with CGI58. PNPLA9 was dispensable for lipolysis in the mouse placenta yet contributed to lipolysis in human placental trophoblasts. Our findings establish a crucial role for CGI58 in placental lipid droplet dynamics and, by extension, in nutrient supply to the developing fetus.

Intestinal microbial circadian rhythms drive sex differences in host immunity and metabolism.

Circadian rhythms dynamically regulate sex differences in metabolism and immunity, and circadian disruption increases the risk of metabolic disorders. We investigated the role of sex-specific intestinal microbial circadian rhythms in host metabolism using germ-free and conventionalized mice and manipulation of dietary-derived fat, fiber, and microbiota-accessible carbohydrates. Our findings demonstrate that sex differences in circadian rhythms of genes involved in immunity and metabolism depend on oscillations in microbiota, microbial metabolic functions, and microbial metabolites. Further, we show that consuming an obesogenic, high-fat, low-fiber diet produced sex-specific changes in circadian rhythms in microbiota, metabolites, and host gene expression, which were linked to sex differences in the severity of metabolic dysfunction. Our results reveal that microbial circadian rhythms contribute to sex differences in immunity and metabolism and that dietary factors can entrain new circadian rhythms and modify the magnitude of sex differences in host-microbe circadian dynamics.

Myeloperoxidase-dependent oxidation of etoposide in human myeloid progenitor CD34+ cells.

Etoposide is a widely used anticancer drug successfully used for the treatment of many types of cancer in children and adults. Its use, however, is associated with an increased risk of development of secondary acute myelogenous leukemia involving the mixed-lineage leukemia (MLL) gene (11q23) translocations. Previous studies demonstrated that the phenoxyl radical of etoposide can be produced by action of myeloperoxidase (MPO), an enzyme found in developing myeloid progenitor cells, the likely origin for myeloid leukemias. We hypothesized, therefore, that one-electron oxidation of etoposide by MPO to its phenoxyl radical is important for converting this anticancer drug to genotoxic and carcinogenic species in human CD34(+) myeloid progenitor cells. In the present study, using electron paramagnetic resonance spectroscopy, we provide conclusive evidence for MPO-dependent formation of etoposide phenoxyl radicals in growth factor-mobilized CD34(+) cells isolated from human umbilical cord blood and demonstrate that MPO-induced oxidation of etoposide is amplified in the presence of phenol. Formation of etoposide radicals resulted in the oxidation of endogenous thiols, thus providing evidence for etoposide-mediated MPO-catalyzed redox cycling that may play a role in enhanced etoposide genotoxicity. In separate studies, etoposide-induced DNA damage and MLL gene rearrangements were demonstrated to be dependent in part on MPO activity in CD34(+) cells. Together, our results are consistent with the idea that MPO-dependent oxidation of etoposide in human hematopoietic CD34(+) cells makes these cells especially prone to the induction of etoposide-related acute myeloid leukemia.

Small molecule GS-nitroxide ameliorates ionizing irradiation-induced delay in bone wound healing in a novel murine model.

We studied radioprotection and mitigation by mitochondrial-targeted Tempol (GS-nitroxide, JP4-039), in a mouse injury/irradiation model of combined injury (fracture/irradiation). Right hind legs of control C57BL/6NHsd female mice, mice pretreated with MnSOD-PL, JP4-039, or with amifostine were irradiated with single and fractionated doses of 0 to 20 Gy. Twenty-four hours later, unicortical holes were drilled into the tibiae of both hind legs; at intervals, tibias were excised, radiographed, and processed for histology. Bone wounds irradiated to 20 or 30 Gy showed delayed healing at 21 to 28 days. Treatment with JP4-039 MnSOD-PL or amifostine, before or after single fraction 20 Gy or during fractionated irradiation followed by drilling accelerated wound healing at days 21 and 28. Orthotopic 3LL tumors were not protected by JP4-039 or amifostine. In nonirradiated mice, pretreatment with JP4-039 accelerated bone wound healing. This test system should be useful for the development of new small molecule radioprotectors.

Lack of DNA polymerase theta (POLQ) radiosensitizes bone marrow stromal cells in vitro and increases reticulocyte micronuclei after total-body irradiation.

Abstract 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.

Investigation of the effects of aging on homologous recombination in long-term bone marrow cultures.

Fluorescent yellow direct repeat (FYDR) mice carry a transgenic reporter for homologous recombination (HR) and have been used to reveal an age-dependent increase in HR in the pancreas. An established in vitro model system for accelerated aging of the marrow is the mouse long-term bone marrow culture (LTBMC) system. To determine whether the FYDR system, in which an HR event can lead to a fluorescent cell, can be used to study the effects of aging in LTBMCs, clonally expanded hematopoietic and marrow stromal cells in FYDR, positive control FYDR-Recombined (FYDR-Rec), and negative control wild-type C57BL/6NHsd (WT) LTBMCs were analysed. All groups of cultures demonstrated equivalent parameters of continuous hematopoiesis including generation of multilineage colony forming CFU-GM progenitor cells for over 22 weeks and age associated senescence of hematopoiesis. Results indicate that low expression of the FYDR transgene in bone marrow cells in vivo and in vitro prevents the use of the FYDR mice to study rare combination events in bone marrow. Using an alternative approach for detecting HR, namely the sister chromatid exchange (SCE) assay, a statistically significant increase in the number of SCEs per chromosome was observed in adherent cells subcultured from 20-week-compared to 4-week-old LTBMCs. These data suggest that adherent marrow stromal cells from LTBMCs become increasingly susceptible to HR events during aging.

Increased radioresistance, g(2)/m checkpoint inhibition, and impaired migration of bone marrow stromal cell lines derived from Smad3(-/-) mice.

Smad3 protein is a prominent member of the Tgfb receptor signaling pathway. Smad3(-/-) mice display decreased radiation-induced skin fibrosis, suggesting a defect in both Tgfb-mediated fibroblast proliferation and migration. We established bone marrow stromal cell lines from Smad3(-/-) mice and homozygous littermate(+/+) mice. Smad3(-/-) cells displayed a significant increase in radiation resistance with a D(0)=2.25+/- 0.14 Gy compared to Smad3(+/+) cells with a D(0)=1.75+/- 0.03 (P=0.023). Radioresistance was abrogated by reinsertion of the human SMAD3 transgene, resulting in a D(0)=1.49 0.10 (P=0.028) for Smad3(-/-)(3) cells. More Smad3(-/-) cells than Smad3(+/+) cells were in the G(2)/M phase; Smad3(-/-)(3) cells were similar to Smad3(+/+) cells. Smad3(+/+) cells exhibited increased apoptosis 24 h after 5 Gy (15%) or 8 Gy (43%) compared to less than 1% in Smad3(-/-) cells exposed to either dose. The movement of Smad3(-/-) cells, measured in an automated cell tracking system, was slower than that of Smad3(+/+) cells. Smad3(-/-)(3) cells resembled Smad3(+/+) cells. These studies establish concordance of a defective Tgfb signal transduction pathway, an increased proportion of G(2)/M cells, and radioresistance. The decreased migratory capacity of Smad3(-/-) cells in vitro correlates with decreased radiation fibrosis in vivo in mice deficient in Tgfb signaling.

Reduced irradiation pulmonary fibrosis and stromal cell migration in Smad3-/- marrow chimeric mice.

Pulmonary irradiation fibrosis involves migration to the lungs of bone marrow origin myofibroblast progenitor cells (marrow stromal cells (MSCs)). Smad3-/- mice display decreased ionizing irradiation-induced skin fibrosis, defective osteochondrogenesis and other abnormalities thought to be associated with a defective stromal cell response(s) to transforming growth factor-beta (TGFFbeta). Clonal bone marrow stromal cell lines were derived from the adherent layer of continuous bone marrow cultures of homozygous deletion recombinant negative Smad3-/- mice and Smad3+/+ littermates. Quantitation in an Automated Cell Tracking System of the in vitro single cell migratory capacity over five days demonstrated a significant decrease in locomotion in microns per 24 h of Smad3-/- compared to Smad3+/+ clonal MSC lines. Reexpression by retroviral vector transfection of the Smad3 but not control ds-red transgene restored in vitro migratory capacity. Intravenously injected GFP transgene product labeled Smad3-/- (MSCs) seeded 10-fold less effectively than ds-red transgene product labeled Smad3+/+ cells to the 80 days post 20 Gy irradiated lungs of C57BL/6J mice and proliferated less significantly for 60 days after cell injection. Female mice chimeric for male Smad3-/- compared to Smad3+/+ marrow showed decreased irradiation pulmonary fibrosis, Y+ stromal cell migration to the lungs, and improved survival. The data show that the reduced in vitro and in vivo migratory capacity of Smad3-/- bone marrow stromal cells correlates with decreased radiation pulmonary fibrosis observed in mice chimeric for Smad3-/- marrow.

Thrombopoietin alone or in the presence of stem cell factor supports the growth of KIT(CD117)low/ MPL(CD110)+ human mast cells from hematopoietic progenitor cells.

OBJECTIVES: Thrombopoietin (TPO) is known to promote platelet number, have growth-promoting potential for human megakaryocytes (HuMKs), and increase erythrocyte, monocyte, mast cell, and granulocyte numbers in the presence of additional growth factors. We explored the ability of TPO alone or in the presence of stem cell factor (SCF) to support human mast cells (HuMCs). METHODS: CD34+ pluripotent and CD34+/CD117+/CD13+ HuMC progenitor cells were cultured in rhTPO and examined for HuMCs. Similarly, we added rhTPO to CD34(+) cells cultured in stem cell factor (SCF), which promotes HuMC development. RESULTS: When CD34+ cells were cultured in 10 ng/mL rhTPO and 10 ng/mL rhSCF, TPO enhanced HuMC numbers compared to rhSCF alone. Higher concentrations of rhTPO (50 ng/mL) in the presence of 100 ng/mL rhSCF inhibited the rhSCF-dependent subpopulation of CD117high HuMCs, while promoting CD117low HuMCs. Human CD34+/CD117+/CD13+ cells cultured in rhTPO alone for 1 to 2 weeks differentiated into CD41+/CD110+ HuMKs (85-90%) and FcepsilonRI+/CD117low/CD13+ HuMCs (5-10%). RhTPO-induced HuMCs expressed the TPO (CD110) receptor, tryptase, and chymase and survived when recultured in rhSCF. CONCLUSION: The effect of TPO on HuMCs in the presence of rhSCF varies, depending on the relative concentration of each growth factor, while TPO alone or in combination with rhSCF supports a unique population of CD117low/CD110+ HuMCs.

Characterization of novel stem cell factor responsive human mast cell lines LAD 1 and 2 established from a patient with mast cell sarcoma/leukemia; activation following aggregation of FcepsilonRI or FcgammaRI.

Two novel stem cell factor (SCF) dependent human mast cell lines, designated LAD 1 and 2, were established from bone marrow aspirates from a patient with mast cell sarcoma/leukemia. LAD 1 and 2 cells have the ultrastructural features of human mast cells, and express FcepsilonRI, CD4, 9, 13, 14, 22, 31, 32, 45, 64, 71, 103, 117, 132, CXCR4 (CD184), CCR5 (CD195); and intracytoplasmic histamine, tryptase and chymase. LAD 1 and 2 do not exhibit activating mutations at codon 816 of c-kit. Both LAD 1 and 2 release beta-hexosaminidase following FcepsilonRI or FcgammaRI aggregation. The availability of these cell lines offers an unparalleled circumstance to examine the biology of human mast cells.

Delivery of herpes simplex virus-based vectors to stem cells.

In contrast to traditional drugs that generally act by altering existing gene product function, gene therapy aims to target the root cause of the disease by altering the genetic makeup of the cell to treat the disease. Researchers have adapted several classes of viruses as gene-transfer vectors, taking advantage of natural viral mechanisms designed to efficiently and effectively deliver DNA to the host-cell nucleus. Among these, the human herpesviruses are excellent candidate vectors for a variety of applications. Herpes simplex virus type 1 (HSV-1) is a particularly attractive gene-transfer vehicle because natural infection in humans includes a latent state in which the viral genome persists in a nonintegrated form without causing disease in an immune-competent host. HSV-1 is a large DNA virus with a broad host range that can be engineered to accommodate multiple or large therapeutic transgenes (4). HSV vectors may be generally useful for gene transfer to a variety of tissues in which short-term or extended transgene expression of therapeutic transgenes achieve a therapeutic effect. We have used therapeutic vectors to successfully treat human disease models in animals, including cancer, Parkinson's disease, and nerve damage (5-10).

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.

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.

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.

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.

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.