RESUMEN
APOBEC3G (A3G) restricts HIV-1 replication primarily by reducing viral cDNA and inducing G-to-A hypermutations in viral cDNA. HIV-1 encodes virion infectivity factor (Vif) to counteract A3G primarily by excluding A3G viral encapsidation. Even though the Vif-induced exclusion is robust, studies suggest that A3G is still detectable in the virion. The impact of encapsidated A3G in the HIV-1 replication is unclear. Using a highly sensitive next-generation sequencing (NGS)-based G-to-A hypermutation detecting assay, we found that wild-type HIV-1 produced from A3G-expressing T-cells induced higher G-to-A hypermutation frequency in viral cDNA than HIV-1 from non-A3G-expressing T-cells. Interestingly, although the virus produced from A3G-expressing T-cells induced higher hypermutation frequency, there was no significant difference in viral infectivity, revealing a disassociation of cDNA G-to-A hypermutation to viral infectivity. We also measured G-to-A hypermutation in the viral RNA genome. Surprisingly, our data showed that hypermutation frequency in the viral RNA genome was significantly lower than in the integrated DNA, suggesting a mechanism exists to preferentially select intact genomic RNA for viral packing. This study revealed a new insight into the mechanism of HIV-1 counteracting A3G antiviral function and might lay a foundation for new antiviral strategies.
Asunto(s)
Desaminasa APOBEC-3G , ADN Complementario , VIH-1 , Mutación , Humanos , Desaminasa APOBEC-3G/genética , Desaminasa APOBEC-3G/metabolismo , ADN Complementario/genética , ADN Viral/genética , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Infecciones por VIH/virología , VIH-1/genética , VIH-1/patogenicidad , Linfocitos T/virología , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Replicación Viral/genéticaRESUMEN
Medroxyprogesterone acetate (MPA) is one of the most widely used contraceptives in the world. Epidemiologic studies have uncovered a possible link between the use of MPA and an increased risk of HIV-1 transmission. However, the understanding of the mechanism is still limited. Our previous publication demonstrated that the lysosomal activity in human vaginal epithelial cells attenuated the trafficking of viral particles during HIV-1 transcytosis. In this study, we show that treating human primary cervical epithelial cells with MPA led to a reduction in lysosomal activity. This reduction caused an increase in the intracellular HIV-1 accumulation and, consequently, an increase in viral release. Our study uncovers a novel mechanism by which MPA enhances HIV-1 release in primary cervical epithelial cells, thus providing vital information for HIV intervention and prevention.
RESUMEN
The activation of the blastocyst, a process by which it gains competency to attach with the receptive uterus, is a prerequisite for successful implantation. However, the molecular basis of blastocyst activation remains largely unexplored. Combining molecular, pharmacological and physiological approaches, we show here that silencing of Wnt-beta-catenin signaling in mice does not adversely affect the development of preimplantation embryos to blastocysts and uterine preparation for receptivity, but, remarkably, blocks blastocyst competency to implantation. Using the physiologically relevant delayed implantation model and trophoblast stem cells in culture, we further demonstrate that a coordinated activation of canonical Wnt-beta-catenin signaling with attenuation of the non-canonical Wnt-RhoA signaling pathway ensures blastocyst competency to implantation. These findings constitute novel evidence that Wnt signaling is at least one pathway that determines blastocyst competency for implantation.
Asunto(s)
Blastocisto/citología , Blastocisto/metabolismo , Núcleo Celular/metabolismo , Implantación del Embrión , Transducción de Señal , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animales , Células Cultivadas , Transferencia de Embrión , Femenino , Silenciador del Gen , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , PPAR delta/metabolismo , Células Madre/citología , Células Madre/metabolismo , Trofoblastos/citología , Trofoblastos/metabolismo , Proteína Wnt3RESUMEN
Successful pregnancy depends on well coordinated developmental events involving both maternal and embryonic components. Although a host of signaling pathways participate in implantation, decidualization, and placentation, whether there is a common molecular link that coordinates these processes remains unknown. By exploiting genetic, molecular, pharmacological, and physiological approaches, we show here that the nuclear transcription factor peroxisome proliferator-activated receptor (PPAR) delta plays a central role at various stages of pregnancy, whereas maternal PPARdelta is critical to implantation and decidualization, and embryonic PPARdelta is vital for placentation. Using trophoblast stem cells, we further elucidate that a reciprocal relationship between PPARdelta-AKT and leukemia inhibitory factor-STAT3 signaling pathways serves as a cell lineage sensor to direct trophoblast cell fates during placentation. This novel finding of stage-specific integration of maternal and embryonic PPARdelta signaling provides evidence that PPARdelta is a molecular link that coordinates implantation, decidualization, and placentation crucial to pregnancy success. This study is clinically relevant because deferral of on time implantation leads to spontaneous pregnancy loss, and defective trophoblast invasion is one cause of preeclampsia in humans.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , PPAR delta/metabolismo , Preñez/fisiología , Animales , Decidua/metabolismo , Implantación del Embrión , Femenino , Fertilización , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Ovulación , Placenta/metabolismo , Embarazo , Factor de Transcripción STAT3/fisiología , Transducción de Señal , Células Madre/citología , Factores de Tiempo , Trofoblastos/metabolismoRESUMEN
Ex vivo gene therapy approaches can improve the outcome of islet transplantation for treating type I diabetes. We have recently shown improvement in islet survival and function following ex vivo infection of islets with a mixture of adenoviral vectors encoding human vascular endothelial growth factor (Adv-hVEGF) and human interleukin-1 receptor antagonist (Adv-hIL-1Ra). In this study, we constructed a bicistronic vector encoding these two genes (phVEGF-hIL-1Ra) by cloning hIL-1Ra under the cytomegalovirus (CMV) promoter and hVEGF under the elongation factor-1alpha (EF-1 alpha) promoter in pBudCE4.1 vector. There was dose and time dependent expression of hVEGF and hIL-1Ra at both mRNA and protein levels after transfection with human islets. Transfected islets were viable, as evidenced by insulin release upon glucose challenge. Coexpression of hVEGF and hIL-1Ra suppressed nitric oxide production, total caspases, apoptosis, and necrosis in the presence of inflammatory cytokine cocktail consisting of IL-1beta, TNFalpha, and IFNgamma. In conclusion, our results indicated that coexpression of growth factor and antiapoptic genes can improve islet survival and function.
Asunto(s)
Regulación de la Expresión Génica , Vectores Genéticos/genética , Proteína Antagonista del Receptor de Interleucina 1/metabolismo , Islotes Pancreáticos/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Caspasas/metabolismo , Muerte Celular , Células Cultivadas , Citocinas/farmacología , Humanos , Óxido Nítrico/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
This study was aimed to evaluate the in vivo antitumor effect of genetically modified myeloma cell vaccine on human myeloma xenografts implanted into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Human immune system was established in NOD/SCID mice by intraperitoneal injection of human peripheral blood lymphocytes (PBLs). After being inoculated subcutaneously with irradiated myeloma cell line sko-007, adenovirally transferred with GFP or p53, granulocyte-macrophage colony-stimulating factor (GM-CSF) and B7-1 genes, huPBL-NOD/SCID mice were challenged by subcutaneous injection of non-transferred sko-007 cells. The results indicated that Ad-p53/GM-CSF/B7-1-infected sko-007 cell vaccination significantly reduced local tumor growth compared with controls. Histopathological and immunohistochemical analysis showed that tumor tissues increasingly displayed diffuse necrosis, mainly caused by apoptosis, accompanied with significant fibroplasias and blood vessel hyperplasia, and human T cells infiltrated into the tumor tissues. It is concluded that transgenic p53, GM-CSF and B7-1 expression produces an immune response against myeloma cells and may be of therapeutic value for multiple myeloma in human being.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Mieloma Múltiple/inmunología , Adenoviridae/genética , Animales , Antígeno B7-1/genética , Antígeno B7-1/inmunología , Genes p53/inmunología , Vectores Genéticos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Inmunoterapia , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trasplante de NeoplasiasRESUMEN
Multiple myeloma (MM) remains incurable despite the use of high-dose chemotherapy and stem cell transplantation. However, immunotherapy is expected to offer long-term disease control, or even possibly a cure. We have previously demonstrated the suppressive effect of a recombinant adenovirus carrying human wild-type p53, granulocyte-macrophage colony-stimulating factor, and B7-1 genes (Ad-p53/GM-CSF/B7-1) on the growth of laryngeal cancer cells. In the present study, we evaluated the effects of an Ad-p53/GM-CSF/B7-1-modified myeloma cell vaccine strategy aimed to induce apoptosis and to augment the immunogenicity of MM cells. Both MM cell lines and purified primary myeloma cells were infected with Ad-p53/GM-CSF/B7-1. High expression levels of these three genes were confirmed separately by Western blot, enzyme-linked immunosorbent assay (ELISA), and flow cytometry. When wild-type p53, GM-CSF and B7-1 genes were introduced, the growth of MM cells was inhibited via enhanced apoptosis and the immunogenicity of tumor cells was augmented. The combinatorial effect of these three genes on inducing cytotoxic T lymphocytes (CTLs) was more evident than that of p53 individually or any combinations of two (p53 plus GM-CSF or p53 plus B7-1). Furthermore, significant proliferation of autologous peripheral blood lymphocytes (PBLs) and specific cytotoxicity against autologous primary MM cells were induced in vitro. These results suggest that myeloma cell vaccination co-transferred with p53, GM-CSF and B7-1 genes may be a promising immunotherapeutic approach against MM.
Asunto(s)
Antígeno B7-1/genética , Vacunas contra el Cáncer/inmunología , Genes p53 , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Mieloma Múltiple/inmunología , Adenoviridae/genética , Apoptosis , Antígeno B7-1/inmunología , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Genes p53/inmunología , Terapia Genética , Vectores Genéticos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Humanos , Inmunoterapia/métodos , Linfocitos T Citotóxicos/inmunología , Células Tumorales CultivadasRESUMEN
AIM: To assess whether hepatocyte growth factor recruits bone marrow-derived endothelial progenitor cells into blood circulation to participate in postnatal angiogenesis and endothelium repair. METHODS: The adenovirus vector encoding HGF gene (Ad-HGF) were intravenous administrated into BALB/c mice, and then serum HGF was determined by enzyme-linked immunosorbent assay, the number of CD34+ cells in peripheral blood was assayed by flow cytometry, and the nucleated cells in peripheral blood were isolated, cultured and the endothelial cell colonies were characterized by staining with antibodies against tie-2, vWF. The carbon tetrachloride-induced liver damage model of female mice was established. The peripheral blood nucleated cells of Ad-HGF treated male mice were intravenous administrated into these mice, and 4 weeks later, in situ hybridization for the sry gene was used to identify the implanted cells in the damaged tissues. RESULTS: Intravenous administration of Ad-HGF resulted in significant elevation of serum hepatocyte growth factor level and induced profoundly increase of endothelial progenitor cells in the peripheral blood, which were characterized by their ability to form endothelial cell colonies in culture and expression of CD34, tie-2, and vW factor. HGF-mobilized endothelial progenitors could incorporate into sites of neovascularization in a liver regeneration model. CONCLUSION: Hepatocyte growth factor could markedly recruit bone marrow-derived endothelial progenitor cells into blood circulation.
Asunto(s)
Células de la Médula Ósea/citología , Factor de Crecimiento de Hepatocito/farmacología , Células Madre/citología , Animales , Células Endoteliales/citología , Femenino , Movilización de Célula Madre Hematopoyética , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
AIM: To establish the methods for determining the activity of sphingosine kinase(SPK) and the content of sphingosine 1-phosphate (S1P) in biological samples. METHODS: The ECV304 cells were transfected with pcDNA3 vector encoding Flag-labeled SPK gene. The expression of SPK was measured by Western blot assay and the activity of SPK was determined by enzymatic reaction, isotope incorporation and thin-layer chromatography methods. The S1P in biological samples was extracted, digested by alkaline phosphatase and then catalyzed by SPK. The S1P contents were determined according to the amounts of products. RESULTS: SPK gene transfection could enhance the expression and activity of SPK in cells markedly, and the cellular S1P was also increased obviously. HGF stimulation could increase the activity of SPK and cellular S1P in ECV304 cells. CONCLUSION: Methods for determining the activity of SPK and the content of SPK in biological samples were established.
Asunto(s)
Citofotometría , Marcaje Isotópico , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Línea Celular , Humanos , Lisofosfolípidos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMEN
The proliferation and apoptosis of multiple myeloma (MM) cells were regulated by bone marrow microenvironments in which Notch signal plays important role in mediating cell-cell communication. However, the regulatory effect of Notch signal on the proliferation and apoptosis of multiple myeloma cells remains unclear. In this study, regulatory effect of Notch signal on the apoptosis of MM cells induced by DMS (N, N-dimethylsphingosine) was investigated. RT-PCR was used to identify the expression of Notch receptor and related molecules such as Dll-1, Jagged-1, Deltex-1 in MM cell lines. The intracellular domain of Notch (ICN), active form of Notch, was transferred into MM cells by retrovirus. The apoptosis of MM cells was determined by trypan blue exclusion tests and TdT-mediated dUTP nick end labeling (TUNEL) assay. The results showed that multiple myeloma cells expressed the Notch-1 and its related molecules. Notch activated multiple myeloma cell lines were obtained. Activation of Notch protected the multiple myeloma cells from the apoptosis induced by DMS,which was determined by cell viability and TUNEL assay. In conclusion, Notch signal suppressed the apoptosis of multiple myeloma cells and would possibly be a novel therapeutic target.
Asunto(s)
Apoptosis , Mieloma Múltiple/patología , Receptores de Superficie Celular/fisiología , Factores de Transcripción/fisiología , División Celular , Humanos , Mieloma Múltiple/tratamiento farmacológico , Receptor Notch1 , Transducción de SeñalRESUMEN
Hepatocyte growth factor (HGF)-induced migration of endothelial cells is critical for angiogenesis. Sphingosine kinase (SPK) is a key enzyme catalyzing the formation of sphingosine-1-phosphate (S1P), a lipid messenger that is implicated in the regulation of a wide variety of important cellular events through both intracellular and extracellular mechanisms. The aim of this study was to investigate whether activation of SPK is involved in the migration of endothelial cells induced by HGF. The biological functions of HGF are mediated through the activation of its high-affinity tyrosine kinase receptor, c-met protooncogene. In the present study, Treatment of ECV304 endothelial cells with HGF resulted in tyrosine phosphorylation of c-Met and activation of SPK in a concentration-dependent manner. Either Ly294002 or PD98059, specific inhibitor of the PI3K and ERK/MAPK pathways, respectively, blocked the HGF-induced activation of SPK. HGF stimulation significantly increased intracellular S1P level, but no detectable secretion of S1P into the cell culture medium was observed. Treatment of ECV304 cells with pertussis toxin (PTX) has no effect on the HGF-induced migration, indicating extracellular S1P is dispensable for this process. Overexpression of wild-type SPK gene in ECV 304 cells increased the intracellular S1P and enhanced the HGF-induced migration, whereas inhibition of cellular SPK activity by N,N-dimethylsphingosine (DMS), a potent inhibitor of SPK, or by expression of a dominant-negative SPK (DN-SK) blocked the HGF-induced migration of ECV 304 cells. It is suggested that PI3K and ERK/MAPK mediated the activation of SPK and would be involved in the HGF-induced migration of endothelial cells. These results elucidate a novel mechanism by which intracellularly generated S1P mediates signaling from HGF/c-Met to the endothelial cell migration.
Asunto(s)
Movimiento Celular/fisiología , Células Endoteliales/enzimología , Factor de Crecimiento de Hepatocito/metabolismo , Lisofosfolípidos/metabolismo , Neovascularización Fisiológica/fisiología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Línea Celular , Relación Dosis-Respuesta a Droga , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Factor de Crecimiento de Hepatocito/farmacología , Humanos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Toxina del Pertussis/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Proto-Oncogénicas c-met/efectos de los fármacos , Proteínas Proto-Oncogénicas c-met/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Esfingosina/farmacología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Mesenchymal stem cells could differentiate into cardiomyocytes in vitro and have been shown to reconstitute the impaired myocardium in vivo. Hepatocyte growth factor, a recognized angiogenic factor and endothelial cell chemoattractant, has been applied in the treatment of myocardial ischemia. In this study, we used a ligation model of proximal left anterior descending coronary artery of rats to evaluate the effect of mesenchymal stem cells overexpressing hepatocyte growth factor in the treatment of myocardial ischemia. Bone marrow-derived mesenchymal stem cells were isolated, expanded, characterized, and infected with adenovirus carrying human hepatocyte growth factor cDNA (Ad-HGF). Mesenchymal stem cells infected by Ad-HGF released soluble HGF protein at a high level, which was maintained at least for 2 weeks. Implantation of mesenchymal stem cells overexpressing hepatocyte growth factor into left anterior descending risk areas improved the functions of impaired myocardium, including diminishing the area of ischemia, increasing the number of capillaries, and reducing collagen content. By using the sry gene as a marker, we also demonstrated that the engrafted cells or their progeny incorporated into ischemic cardiac muscle. These results showed that treatment of myocardial ischemia with bone marrow-derived mesenchymal stem cells overexpressing hepatocyte growth factor could be a novel strategy that can both restore local blood flow and regenerate lost cardiomyocytes.
Asunto(s)
Terapia Genética , Factor de Crecimiento de Hepatocito/genética , Isquemia Miocárdica/terapia , Trasplante de Células Madre , Células Madre/metabolismo , Adenoviridae , Animales , Células de la Médula Ósea/metabolismo , Vectores Genéticos , Factor de Crecimiento de Hepatocito/metabolismo , Neovascularización Fisiológica/fisiología , Ratas , Ratas WistarRESUMEN
Notch signal path plays important roles in the regulation of proliferation and differentiation of hematopoietic stem cells. An extracellular domain of human Delta-like-1 (hDll-1(ext)), one of Notch ligands, was cloned and expressed in CHO cells, and the effect of hDll-1(ext) on expansion of hematopoietic stem/progenitor cells was investigated in this study. Total RNA was isolated from human marrow mononuclear cells. hDll-1(ext) was amplified by RT-PCR and cloned to T vector, then the gene was sequenced and subcloned to pcDNA3.1/Myc-His(+)A expression vector. The constructed plasmid was transfected into CHO cells with lipofectin and the expression of secreted hDll-1(ext) in G418-resistant clones was assayed by Western blot. hDll-1(ext) high-expressed clone was cultured to collect supernatant. Fusion protein hDll-1(ext) was purified from the supernatant by immobilized metal affinity chromatography (IMAC). The results showed that expression of Notch-1 receptor was detected in cord blood-derived CD34(+) cells by RT-PCR. Human umbilical blood CD34(+) cells were cultured in serum-free medium containing SCF, IL-3, VEGF, and with or without purified hDll-1(ext) for 4 or 8 days. Effect of hDll-1(ext) on the expansion of progenitor cells was analyzed then by clonogenic assays. The number of CFU-Mix and HPP-CFC generated from the culture system containing hDll-1(ext) was 1.5 times of that from the control. In conclusion, the recombinant hDll-1(ext) promotes the expansion of primitive hematopoietic progenitors.
Asunto(s)
Glicoproteínas/genética , Células Madre Hematopoyéticas/citología , Receptores de Superficie Celular , Factores de Transcripción , Animales , Antígenos CD34/inmunología , Sitios de Unión/genética , Células CHO , División Celular/efectos de los fármacos , División Celular/fisiología , Ensayo de Unidades Formadoras de Colonias , Cricetinae , Factores de Crecimiento Endotelial/farmacología , Sangre Fetal/citología , Sangre Fetal/inmunología , Sangre Fetal/metabolismo , Expresión Génica , Vectores Genéticos/genética , Glicoproteínas/farmacología , Glicoproteínas/fisiología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Interleucina-3/farmacología , Linfocinas/farmacología , Proteínas de la Membrana/genética , ARN/genética , ARN/metabolismo , Receptor Notch1 , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Células Madre/farmacología , Transfección , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Adhesion to extracellular matrix plays important roles in the regulation of survival, proliferation, differentiation and homing of hematopoietic cells and is regulated by a wide variety of growth factors, adhesion receptors and other ligands that mediate the cell to matrix and cell to cell interaction. Stem cell factor (SCF) plays important roles in the regulating growth and self-renewal of hematopoietic stem/progenitor cells. In the report, the effects of stem cell factor on the adhesion of hematopoietic cells to fibronectin were observed by using a hematopoietic growth factor dependent cell line-Mo7e. Results showed that Mo7e cells express the very late antigen VLA-4 (beta1 alpha4) and VLA-5 (beta1 alpha5) integrins. The expression of the SCF receptor (c-kit) was also detected in the Mo7e cells. SCF enhances the adhesion of Mo7e cells to fibronectin in a concentration dependent manner. SCF enhanced adhesion of Mo7e cells to fibronectin was blocked by anti-beta1, alpha4 and alpha5 antibodies. Addition of PI-3 kinase inhibitors also blocked the adhesion of Mo7e cells to fibronectin induced by SCF. It was concluded that SCF enhances the adhesion of Mo7e cells to fibronectin, and this process is mediated by integrins and PI-3 kinase pathway.