RESUMO
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.
Assuntos
Ferroptose , Feminino , Humanos , Peroxidação de Lipídeos , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Placenta , Gravidez , TrofoblastosRESUMO
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.
Assuntos
Ferroptose/fisiologia , Fosfolipases A2 do Grupo VI/metabolismo , Trofoblastos/metabolismo , Animais , Feminino , Glutationa Peroxidase/metabolismo , Fosfolipases A2 do Grupo VI/genética , Fosfolipases A2 do Grupo VI/fisiologia , Humanos , Ferro/metabolismo , Peróxidos Lipídicos/metabolismo , Camundongos , Camundongos Knockout , Fosfatidiletanolaminas/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Placenta/metabolismo , Gravidez , Nascimento Prematuro/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Dieta , Infecções Estreptocócicas , Streptococcus agalactiae , Vagina , Vagina/microbiologia , Feminino , Animais , Streptococcus agalactiae/crescimento & desenvolvimento , Camundongos , Infecções Estreptocócicas/microbiologia , Microbiota/fisiologia , Obesidade/microbiologia , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , HumanosRESUMO
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.
Assuntos
Compostos de Boro/análise , Corantes Fluorescentes/análise , Sequestradores de Radicais Livres/análise , Mitocôndrias/ultraestrutura , Óxidos de Nitrogênio/análise , Protetores contra Radiação/análise , Radiossensibilizantes/análise , Linhagem Celular , Sequestradores de Radicais Livres/síntese química , Sequestradores de Radicais Livres/farmacologia , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/efeitos da radiação , Modelos Moleculares , Óxidos de Nitrogênio/síntese química , Óxidos de Nitrogênio/farmacologia , Protetores contra Radiação/síntese química , Protetores contra Radiação/farmacologia , Radiossensibilizantes/síntese química , Radiossensibilizantes/farmacologiaRESUMO
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.
RESUMO
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.
Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase , Aciltransferases , Lipase , Lipólise , Placenta , Lipase/metabolismo , Humanos , Animais , Camundongos , Placenta/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Aciltransferases/metabolismo , Trofoblastos , Feminino , Gotículas LipídicasRESUMO
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.
Assuntos
Antígenos CD34/metabolismo , Antineoplásicos Fitogênicos/metabolismo , Etoposídeo/metabolismo , Células Progenitoras Mieloides/metabolismo , Peroxidase/metabolismo , Ensaio Cometa , Espectroscopia de Ressonância de Spin Eletrônica , Citometria de Fluxo , Rearranjo Gênico , Guaiacol/metabolismo , Humanos , Immunoblotting , OxirreduçãoRESUMO
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.
Assuntos
Óxidos N-Cíclicos/uso terapêutico , Lesões por Radiação/tratamento farmacológico , Protetores contra Radiação/uso terapêutico , Tíbia/lesões , Cicatrização/efeitos da radiação , Animais , Modelos Animais de Doenças , Relação Dose-Resposta à Radiação , Feminino , Membro Posterior , Camundongos , Camundongos Endogâmicos C57BL , Lesões por Radiação/prevenção & controle , Radiação Ionizante , Radiografia , Marcadores de Spin , Tíbia/diagnóstico por imagem , Tíbia/efeitos dos fármacosRESUMO
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.
Assuntos
Cromossomos Humanos Par 19 , MicroRNAs/metabolismo , Placentação , Animais , Feminino , Perfilação da Expressão Gênica , Humanos , Antígeno Ki-67/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Placenta/metabolismo , GravidezRESUMO
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.
Assuntos
Células da Medula Óssea/efeitos da radiação , DNA Polimerase Dirigida por DNA/metabolismo , Micronúcleos com Defeito Cromossômico/efeitos da radiação , Tolerância a Radiação/fisiologia , Reticulócitos/efeitos da radiação , Irradiação Corporal Total , Animais , Células da Medula Óssea/citologia , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , DNA Polimerase Dirigida por DNA/genética , Relação Dose-Resposta à Radiação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doses de Radiação , Reticulócitos/citologia , DNA Polimerase tetaRESUMO
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.
Assuntos
Células da Medula Óssea/citologia , Senescência Celular/genética , Hematopoese/genética , Recombinação Genética , Animais , Células da Medula Óssea/fisiologia , Células Cultivadas , Células Clonais , Feminino , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Camundongos , Troca de Cromátide Irmã , Células Estromais/citologia , Células Estromais/fisiologiaRESUMO
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.
Assuntos
Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteína Smad3/metabolismo , Animais , Apoptose/efeitos da radiação , Células da Medula Óssea/efeitos da radiação , Linhagem Celular , Movimento Celular/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Genes cdc/fisiologia , Genes cdc/efeitos da radiação , Camundongos , Tolerância a Radiação/fisiologia , Células Estromais/citologia , Células Estromais/fisiologia , Células Estromais/efeitos da radiaçãoRESUMO
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.
Assuntos
Células da Medula Óssea/fisiologia , Movimento Celular , Fibrose Pulmonar/induzido quimicamente , Proteína Smad3/genética , Células Estromais/fisiologia , Animais , Medula Óssea/metabolismo , Medula Óssea/fisiologia , Transplante de Medula Óssea/fisiologia , Proliferação de Células , Células Clonais/fisiologia , Feminino , Proteínas Luminescentes/genética , Pulmão/fisiologia , Pulmão/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibrose Pulmonar/etiologia , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Proliferação de Células/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Mastócitos/citologia , Fator de Células-Tronco/farmacologia , Trombopoetina/farmacologia , Técnicas de Cultura de Células , Células Cultivadas , Interações Medicamentosas , Citometria de Fluxo , Humanos , Imunofenotipagem , Proteínas Proto-Oncogênicas , Proteínas Proto-Oncogênicas c-kit , Receptores de Citocinas , Receptores de TrombopoetinaRESUMO
OBJECTIVE: To determine the role of Smad3 in modulating hematopoiesis, continuous bone marrow cultures were established from Smad-/- mice, and the longevity of hematopoiesis and extent of adipogenesis in the supportive hematopoietic microenvironment were compared to those from cultures of control, Smad3+/+ or heterozygous Smad3+/- mice. MATERIALS AND METHODS: Long-term bone marrow cultures (LTBMCs) were established from Smad3+/+, Smad3+/-, or Smad3-/- mice. On a weekly basis, the number of cobblestone islands, number of nonadherent cells, confluence of the adherent cells, or CFU-GEMM colonies was determined. Bone marrow stromal cell lines were established and cobblestone island production on these cell lines determined in the presence of nonadherent cells from week-42 Smad3-/- or week-4 C57BL/6J LTBMCs. RESULTS: Initial proliferative capacity of the LTBMCs was similar in all groups through week 20, at which time there was an increase in cobblestone islands and production of nonadherent cells and CFU-GEMM colonies in the Smad3-/- group. By week 28, only the Smad3-/- LTBMCs had significantly maintained increased production of these parameters. Maintenance of cobblestone islands indicative of the most primitive hematopoietic progenitor cells persisted past 45 weeks in Smad3-/- cultures. The Smad3-/- stromal cell line also demonstrated increased support of cobblestone island production when incubated with nonadherent cells from week-42 Smad3-/- or week-4 C57BL/6J LTBMCs. Evaluation of adipocytogenesis in stromal cells showed significantly greater accumulation of adipocytes in lines from Smad3-/- than from Smad3+/+ mice. CONCLUSIONS: These data provide evidence for a significant effect of deletion of the Smad3 signaling pathway in increased hematopoiesis in LTBMCs and support the negative regulatory influence of TGFbeta signaling on adipocytogenesis and long-term hematopoiesis in vitro.
Assuntos
Adipócitos/fisiologia , Medula Óssea/fisiologia , Proteínas de Ligação a DNA/metabolismo , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Animais , Células Cultivadas , Proteínas de Ligação a DNA/genética , Hematopoese/genética , Masculino , Camundongos , Camundongos Knockout , Transdução de Sinais/genética , Proteína Smad3 , Células Estromais/fisiologia , Fatores de Tempo , Transativadores/genética , Fator de Crescimento Transformador beta/fisiologiaRESUMO
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.
Assuntos
Mitocôndrias/efeitos dos fármacos , Mitocôndrias/efeitos da radiação , Mucosa Bucal/efeitos dos fármacos , Mucosa Bucal/efeitos da radiação , Neoplasias Bucais/radioterapia , Óxidos de Nitrogênio/administração & dosagem , Administração Oral , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta à Radiação , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mucosa Bucal/patologia , Neoplasias Bucais/patologia , Órgãos em Risco , Tolerância a Radiação/efeitos dos fármacos , Protetores contra Radiação/administração & dosagem , Resultado do TratamentoRESUMO
BACKGROUND: Time-lapse microscopic imaging provides a powerful approach for following changes in cell phenotype over time. Visible responses of whole cells can yield insight into functional changes that underlie physiological processes in health and disease. For example, features of cell motility accompany molecular changes that are central to the immune response, to carcinogenesis and metastasis, to wound healing and tissue regeneration, and to the myriad developmental processes that generate an organism. Previously reported image processing methods for motility analysis required custom viewing devices and manual interactions that may introduce bias, that slow throughput, and that constrain the scope of experiments in terms of the number of treatment variables, time period of observation, replication and statistical options. Here we describe a fully automated system in which images are acquired 24/7 from 384 well plates and are automatically processed to yield high-content motility and morphological data. RESULTS: We have applied this technology to study the effects of different extracellular matrix compounds on human osteoblast-like cell lines to explore functional changes that may underlie processes involved in bone formation and maintenance. We show dose-response and kinetic data for induction of increased motility by laminin and collagen type I without significant effects on growth rate. Differential motility response was evident within 4 hours of plating cells; long-term responses differed depending upon cell type and surface coating. Average velocities were increased approximately 0.1 microm/min by ten-fold increases in laminin coating concentration in some cases. Comparison with manual tracking demonstrated the accuracy of the automated method and highlighted the comparative imprecision of human tracking for analysis of cell motility data. Quality statistics are reported that associate with stage noise, interference by non-cell objects, and uncertainty in the outlining and positioning of cells by automated image analysis. Exponential growth, as monitored by total cell area, did not linearly correlate with absolute cell number, but proved valuable for selection of reliable tracking data and for disclosing between-experiment variations in cell growth. CONCLUSION: These results demonstrate the applicability of a system that uses fully automated image acquisition and analysis to study cell motility and growth. Cellular motility response is determined in an unbiased and comparatively high throughput manner. Abundant ancillary data provide opportunities for uniform filtering according to criteria that select for biological relevance and for providing insight into features of system performance. Data quality measures have been developed that can serve as a basis for the design and quality control of experiments that are facilitated by automation and the 384 well plate format. This system is applicable to large-scale studies such as drug screening and research into effects of complex combinations of factors and matrices on cell phenotype.
Assuntos
Movimento Celular , Proliferação de Células , Automação , Linhagem Celular , Colágeno Tipo I/farmacologia , Técnicas Citológicas/métodos , Diagnóstico por Imagem , Relação Dose-Resposta a Droga , Humanos , Cinética , Laminina/farmacologia , Análise em Microsséries , Osteoblastos/citologia , Projetos de PesquisaRESUMO
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.
Assuntos
Linhagem Celular , Leucemia de Mastócitos/patologia , Mastócitos/patologia , Sarcoma de Mastócitos/patologia , Adulto , Células da Medula Óssea/patologia , Divisão Celular , Dimerização , Humanos , Imunofenotipagem , Cariotipagem , Leucemia de Mastócitos/enzimologia , Leucemia de Mastócitos/genética , Masculino , Mastócitos/enzimologia , Sarcoma de Mastócitos/enzimologia , Sarcoma de Mastócitos/genética , Mutação , Receptores de IgE/metabolismo , Receptores de IgG/metabolismo , Fator de Células-Tronco/farmacologiaRESUMO
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).
Assuntos
Vetores Genéticos , Simplexvirus/genética , Células-Tronco/virologia , Animais , Antígenos CD34/imunologia , Ensaio de Imunoadsorção Enzimática , Técnicas de Transferência de Genes , Humanos , Camundongos , Simplexvirus/fisiologia , Células-Tronco/imunologia , Transdução Genética , Replicação ViralRESUMO
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.