RESUMEN
Oncostatin M (OSM) and leukemia inhibitory factor (LIF) belong to the interleukin-6 family of cytokines. The authors' previous in vitro work demonstrated that in mouse cells mouse OSM (mOSM) signals through a heterodimeric receptor complex incorporating the mOSM-specific receptor mOSMRbeta while human OSM (hOSM) and bovine OSM (bOSM) use the mouse LIF receptor mLIFRbeta rather than mOSMRbeta. These in vitro data suggest that prior studies in mouse systems with hOSM or bOSM (the usual molecules used in early studies) reflect LIF rather than OSM biology. The current work assessed whether or not this divergence in actions among these three OSMs also occurs in vivo in mouse models. Adult female (C57BL/6J x DBA/2J) F(1) mice were engineered to stably overexpress mOSM, hOSM, or bOSM by retrovirus-mediated gene transfer (n = 10 or more per group). After 4 weeks, molecular and hematologic profiles and anatomic phenotypes in multiple organs were assessed by standard techniques. Animals overexpressing either hOSM or bOSM had an identical phenotype resembling that associated with LIF activation, including significant hematologic abnormalities (anemia, neutrophilia, lymphopenia, eosinopenia, and thrombocytosis); weight loss; profound enlargement (lymph node, spleen) and/or structural reorganization (lymph node, spleen, thymus) of lymphoid organs; and severe osteosclerosis. In contrast, mice overexpressing mOSM did not develop hematologic changes, weight loss, or osteosclerosis and exhibited more modest and anatomically distinct restructuring of lymphoid organs. These data indicate that activities imputed to OSM and the mOSMRbeta signaling pathway using in vitro and in vivo mouse experimental systems are unique to mOSM.
Asunto(s)
Expresión Génica , Subunidad beta del Receptor de Oncostatina M/metabolismo , Oncostatina M/metabolismo , Fenotipo , Receptores OSM-LIF/metabolismo , Transducción de Señal/fisiología , Animales , Northern Blotting , Trasplante de Médula Ósea , Bovinos , Femenino , Vectores Genéticos/genética , Humanos , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Ratones , Células 3T3 NIH , Oligonucleótidos/genética , Oncostatina M/genética , Bazo/metabolismo , Bazo/patología , Timo/metabolismo , Timo/patologíaRESUMEN
Notch receptors mediate cell-fate decisions through interaction with specific ligands during development. The biological role of a novel Notch ligand, Dll4, in mice was explored by reconstituting lethally irradiated mice with bone marrow (BM) cells transduced with Dll4 retroviral vector. White blood cell and lymphocyte counts in Dll4-overexpressing mice were reduced at the early stage of reconstitution but increased significantly at approximately 10 weeks after BM transplantation. BM, spleen, lymph nodes, and peripheral blood of Dll4-overexpressing mice contained predominantly CD4(+)CD8(+) T cells and virtually lacked B cells. The Dll4-overexpressing mice eventually developed a lethal phenotype that was characterized by the progression of a T-cell lymphoproliferative disease (restricted to BM and lymphoid tissues) to transplantable monoclonal T-cell leukemia/lymphoma scattered to multiple organs. Results suggest that the interaction of Dll4 with Notch1 may provide key signals for T-cell development.
Asunto(s)
Expresión Génica , Leucemia-Linfoma de Células T del Adulto/etiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Retroviridae/genética , Transfección , Animales , Células de la Médula Ósea/metabolismo , Trasplante de Médula Ósea , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/patología , Femenino , Rayos gamma , Vectores Genéticos , Péptidos y Proteínas de Señalización Intracelular , Leucemia-Linfoma de Células T del Adulto/patología , Ganglios Linfáticos/patología , Recuento de Linfocitos , Linfoma de Células T/etiología , Linfoma de Células T/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Trasplante de Neoplasias , Bazo/patologíaRESUMEN
In this report, we tested whether ectopic overexpression of a cell surface receptor cDNA could be used to explore the physiological roles of that receptor. We generated c-mpl overexpressing animals by reconstituting mice with retroviral vector-transduced bone marrow (BM) cells. We observed that platelet counts in the c-mpl overexpressing mice failed to recover to normal levels and remained at <200 x 10(6)/mL post-transplantation, while platelet numbers in the control mice returned to > 800 x 10(6)/mL by 4 weeks post-transplantation. However, platelet counts in the c-mpl overexpressing mice could be stimulated to normal levels after administration of rhMGDF. No significant changes in peripheral leukocyte counts were observed, although the number of CFU-E, GM-CFC, and CFC-multi were reduced two- to threefold in the BM of the c-mpl overexpressing mice. In addition, enhanced erythropoiesis was observed in the c-mpl overexpressing mice. The mpl receptors on erythroid cells were functional as demonstrated by tyrosine-phosphorylation of mpl receptor on RBC and by in vitro erythroid colony-formation in response to MGDF stimulation, respectively. These results suggested that ectopically expressed mpl receptors competed for ligand in vivo leading to an insufficient amount of circulating thrombopoietin (Tpo) for the development of megakaryocytic lineage. These results further suggest that, in addition to sequestering circulating Tpo, overexpression of the mpl receptor on erythroid progenitors may directly contribute to enhanced erythropoiesis in vivo. Our studies demonstrate that ectopic overexpression of a receptor by retroviral-mediated gene transfer provides an approach to explore the biological roles of novel receptors.
Asunto(s)
Eritropoyesis , Hematopoyesis , Megacariocitos/citología , Proteínas de Neoplasias , Proteínas Proto-Oncogénicas/biosíntesis , Receptores de Citocinas , Animales , Ensayo de Unidades Formadoras de Colonias , ADN Complementario/genética , Vectores Genéticos/genética , Factores de Crecimiento de Célula Hematopoyética/farmacología , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/virología , Recuento de Leucocitos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Proteínas Proto-Oncogénicas/genética , Provirus/genética , Receptores de Trombopoyetina , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/fisiología , Proteínas Recombinantes/farmacología , Retroviridae/genética , Trombopoyetina/farmacología , Trombopoyetina/fisiología , TransfecciónRESUMEN
Granulocyte colony-stimulating factor (G-CSF) has proven effective in the prophylaxis of chemotherapy-induced neutropenia and as a mobilizer of peripheral blood progenitor cells. The longevity of G-CSF action is limited by its removal from the body by two mechanisms. The first is thought to be mediated via receptors (receptor mediated clearance [RMC]) predominantly on neutrophils, the second process is likely the result of renal clearance. With the intention of developing a novel form of Filgrastim (r-met HuG-CSF) with a sustained duration of action in vivo, a new derivative named SD/01 has been made by association of Filgrastim with poly(ethylene glycol). The desired properties of this new agent would include a prolonged duration of action sufficient to cover a complete single course of chemotherapy. SD/01 is shown here to sustain significantly elevated neutrophil counts in hematopoietically normal mice for 5 days. In neutropenic mice effects were noted for at least 9 days, accompanying a significant reduction in the duration of chemotherapy induced neutropenia. Normal human volunteers showed higher than baseline ANC for around 9 to 10 days after a single injection of SD/01. Data from these normal volunteers also indicate that mobilization of CD34+ cells and progenitors may occur in a more timely manner and to around the same absolute numbers as with repeated daily injections of unmodified Filgrastim. These data indicate that SD/01 represents an efficacious novel form of Filgrastim with actions sustained for between one and two weeks from a single injection.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos/análogos & derivados , Factor Estimulante de Colonias de Granulocitos/farmacología , Movilización de Célula Madre Hematopoyética , Animales , Filgrastim , Humanos , Ratones , Proteínas RecombinantesRESUMEN
Oncostatin M (OSM) is a member of a family of cytokines that includes ciliary neurotrophic factor, interleukin-6, interleukin-11, cardiotrophin-1, and leukemia inhibitory factor (LIF). The receptors for these cytokines consist of a common signaling subunit, gp130, to which other subunits are added to modify ligand specificity. We report here the isolation and characterization of a cDNA encoding a subunit of the mouse OSM receptor. In NIH 3T3 cells (which endogenously express gp130, LIF receptor beta [LIFRbeta], and the protein product, c12, of the cDNA described here), mouse LIF, human LIF, and human OSM signaled through receptors containing the LIFRbeta and gp130 but not through the mouse OSM receptor. Mouse OSM, however, signaled only through a c12-gp130 complex; it did not use the LIF receptor. Binding studies demonstrated that mouse OSM associated directly with either the c12 protein or gp130. These data highlight the species-specific differences in receptor utilization and signal transduction between mouse and human OSM. In mouse cells, only mouse OSM is capable of activating the mouse OSM receptor; human OSM instead activates the LIF receptor. Therefore, these data suggest that all previous studies with human OSM in mouse systems did not elucidate the biology of OSM but, rather, reflected the biological actions of LIF.
Asunto(s)
Interleucina-6 , Linfocinas , Péptidos/metabolismo , Precursores de Proteínas/genética , Receptores de Citocinas/genética , Células 3T3 , Secuencia de Aminoácidos , Animales , Antígenos CD/metabolismo , Secuencia de Bases , Células COS , Clonación Molecular , Receptor gp130 de Citocinas , Inhibidores de Crecimiento/metabolismo , Humanos , Factor Inhibidor de Leucemia , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia , Glicoproteínas de Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Peso Molecular , Oncostatina M , Precursores de Proteínas/metabolismo , Receptores de Citocinas/metabolismo , Receptores OSM-LIF , Receptores de Oncostatina M , Alineación de Secuencia , Transducción de Señal , Especificidad de la EspecieRESUMEN
The effect of chronic expression of flt3 ligand (FL) on in vivo hematopoiesis was studied. Retroviral vector-mediated gene transfer was used in a mouse model of bone marrow transplantation to enforce expression of mouse FL cDNA in hematopoietic tissues. As early as 2 weeks posttransplantation, peripheral blood white blood cell counts in FL-overexpressing recipients were significantly elevated compared with controls. With the exception of eosinophils, all nucleated cell lineages studied were similarly affected in these animals. Experimental animals also exhibited severe anemia and progressive loss of marrow-derived erythropoiesis. All of the FL-overexpressing animals, but none of the controls, died between 10 and 13 weeks posttransplantation. Upon histological examination, severe splenomegaly was noted, with progressive fibrosis and infiltration by abnormal lymphoreticular cells. Abnormal cell infiltration also occurred in other organ systems, including bone marrow and liver. In situ immunocytochemistry on liver sections showed that the cellular infiltrate was CD3+/NLDC145+/CD11c+, but B220- and F4/80-, suggestive of a mixed infiltrate of dendritic cells and activated T lymphocytes. Infiltration of splenic blood vessel perivascular spaces resulted in vascular compression and eventual occlusion, leading to splenic necrosis consistent with infarction. These results show that FL can affect both myeloid and lymphoid cell lineages in vivo and further demonstrate the potential toxicity of in vivo treatment with FL.
Asunto(s)
Regulación de la Expresión Génica , Leucocitos/patología , Proteínas de la Membrana/genética , Bazo/patología , Animales , Recuento de Células Sanguíneas , Diferenciación Celular/genética , Linaje de la Célula , Movimiento Celular/genética , Fibrosis/genética , Fibrosis/patología , Expresión Génica , Masculino , Proteínas de la Membrana/biosíntesis , Ratones , Ratones EndogámicosAsunto(s)
ADN Complementario/genética , Fibras Musculares de Contracción Rápida/metabolismo , Músculo Esquelético/metabolismo , Troponina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Secuencia Conservada , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Muridae , Ratas , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie , Troponina TRESUMEN
Cloning of interleukin-1 beta converting enzyme (ICE) and Caenorhabditis elegans death protein CED-3 revealed the structural and functional homology between these two proteases. It also suggested the involvement of ICE-like cysteine proteases in apoptosis. Several CED-3- and ICE-like cysteine proteases have been described, including Nedd2/Ich-1, CPP32 beta, Tx, ICErel3, and Mch2. We have previously described a mouse ortholog of cysteine protease CPP32 beta that shares strong homology with ICE and CED-3. Here, we describe the cloning of mouse and human Casp7, another member of this family of cysteine proteases. Mouse Casp7 encodes a putative 340-amino-acid polypeptide that contains all the known conserved residues required for protease function, including the QACRG sequence, aspartic acid residues for internal cleavage sites, and the residues required for substrate binding. Three RNA variants of human Casp7 were also cloned. Amino acid sequence analysis indicated that Casp7 shared high homology with CPP32 beta/Casp3 and Mch2/Casp6. Northern blot analysis demonstrated that a 2.6-kb Casp7 mRNA was expressed in various tissues except brain. Mouse interspecific backcross mapping allowed localization of Casp7 to the distal region of mouse chromosome 19, linked to Mxi1, Adra2a, and Aop1.
Asunto(s)
Caspasas , Mapeo Cromosómico , Cisteína Endopeptidasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Proteínas de Caenorhabditis elegans , Caspasa 1 , Caspasa 3 , Caspasa 7 , Clonación Molecular , Femenino , Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , ARN Mensajero , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Interleukin-1 beta converting enzyme (ICE) defines a new class of mammalian cysteine protease that shares strong homology with the Caenorhabditis elegans death gene ced-3. Both ICE and CED-3, when introduced into cultured cells, induce apoptosis, indicating that this type of cysteine protease may play an important role in the process of programmed cell death. Here, we report the cloning of a mouse and rat gene encoding a novel cysteine protease. The putative proteins encoded by these cDNAs contain the conserved sequence (QACRG) necessary for covalent linkage to the substrate as well as the three amino acids responsible for substrate binding and catalysis in ICE. Amino acid sequence analysis indicates that this rodent cysteine protease is the homolog of human CPP32 beta. Mouse CPP32 beta mRNA is highly expressed in spleen, and to a lesser degree in brain, lung, liver, and kidney. The mouse CPP32 beta genomic locus spans a region of approximately 20 kb, including seven exons and six introns. Mouse interspecific backcross mapping allowed localization of CPP32 beta to the central region of mouse chromosome 8, linked to Scvr, Lpl, Jund1 and Mlr.
Asunto(s)
Caspasas , Cisteína Endopeptidasas/genética , Proteínas del Helminto/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Proteínas de Caenorhabditis elegans , Caspasa 1 , Caspasa 3 , Mapeo Cromosómico , ADN Complementario , Exones , Femenino , Humanos , Intrones , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Ratas , Homología de Secuencia de AminoácidoRESUMEN
Elk is a member of the eph family of receptor-like tyrosine kinases. Although its function is unknown, elk is postulated to play a role in nervous system development. Using Northern analysis, we examined the developmental regulation of RNAs encoding elk, and several ligands for the eph family of RTKs, the LERKs. Expression of elk, LERK-1, and LERK-2 RNAs is high in all regions examined in the embryonic and postnatal rat brain and decreases to low levels with age. One exception is the adult olfactory bulb which continues to express a moderate level of LERK-2. In contrast, moderate LERK-4 expression was limited to the developing hippocampus and cerebral cortex. These data indicate that elk and some of the LERKs may play a role in nervous system development, maintenance, and/or regeneration.
Asunto(s)
Encéfalo/enzimología , Proteínas de Unión al ADN , Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana/biosíntesis , Proteínas del Tejido Nervioso/metabolismo , Biosíntesis de Proteínas , Proteínas Tirosina Quinasas Receptoras/metabolismo , Factores de Transcripción , Animales , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Encéfalo/ultraestructura , Inducción Enzimática , Efrina-A1 , Efrina-A2 , Efrina-A3 , Efrina-A4 , Efrina-B1 , Efrina-B2 , Glicosilfosfatidilinositoles/metabolismo , Ligandos , Proteínas de la Membrana/genética , Familia de Multigenes , Proteínas/genética , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Ratas , Ratas Sprague-Dawley , Proteínas Tirosina Quinasas Receptoras/clasificación , Organismos Libres de Patógenos Específicos , Proteína Elk-1 con Dominio etsRESUMEN
Severe combined immunodeficient (SCID) mice injected with co-isogenic CD4+/CD45RBhigh lymph node T cells from normal donors develop a wasting disease that is caused by hyperplasia of the intestinal epithelium. SCID mice injected with purified lymph node CD4+ T cells or CD4+/CD45RBlow T cells do not develop the disease. The IEL compartment from SCID mice injected with highly purified CD4+/CD45RBhigh T cells or CD4+ T cells contained significant numbers of T cells that expressed both CD4 and CD8 alpha, but not CD8 beta. The CDr+/CD8 alpha + T cells were unique to the IEL compartment of the small intestine and were not observed in significant numbers in the lamina propria, mesenteric lymph node, nor IEL compartment of the large intestine. By using Ly-5 mismatched donors and recipients, we determined that the CD4+/CD8 alpha + T cells were derived from the donor T cells. The expression of CD8 alpha was stable in vitro, and CD8 alpha mRNA was detected in sorted CD4+/CD8 alpha + T cells by reverse transcriptase-PCR (RT-PCR). Recombinase-activating gene (RAG)-1 and -2 mRNA was not detected in the intra-epithelial lymphocyte CD4+/CD8 alpha + T cell population. Thus, it appears that under conditions unique to the epithelial layer of the small intestine, mature post-thymic CD4+ T cells can be induced to express CD8 alpha.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Antígenos CD8/biosíntesis , Mucosa Intestinal/inmunología , Animales , Secuencia de Bases , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Enfermedad Injerto contra Huésped/inmunología , Antígenos Comunes de Leucocito/inmunología , Tejido Linfoide/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
We attempted to isolate novel receptor tyrosine kinase, which may play a role in hematopoietic development by screening for expressed sequences with conserved tyrosine kinase catalytic domains. Among the known tyrosine kinases identified in this screen, we found a gene with characteristics of a receptor tyrosine kinase but unusual motifs in the catalytic domain. This gene is identical to ryk described independently by other investigators. Chromosomal fluorescence in situ hybridization localization of human ryk was clarified by using monochromosomal hybrids and placing it as a single locus in 3q22. Although Northern analysis reveals widespread expression in adult mouse tissues, we have found that ryk expression is not ubiquitous. Expression increased in bone marrow cells from mice treated with 5-fluorouracil. Northern analysis on cell lines indicates expression in CD3-, CD4-, CD8- T cells (at a low level), pre-T cells, thymic epithelial cells, and mature myeloid cells, but not myeloid precursors or B cell precursors. Expression analysis with the use of RT-PCR on mouse bone marrow cells separated on the basis of cell surface markers (B220, CD4, CD8, Gr-1, Mac-1) reveals that this receptor is expressed in differentiated cells (Lin+) but is not expressed in the precursor cells (Lin-). Flow cytometric analysis with a monospecific anti-Ryk Ab demonstrates that Ryk+ cells constitute 36.7% and Lin+/Ryk+ cells constitute 33.7% of low density bone marrow cells whereas Ryk+ cells represent only 0.3% of the Lin- population. We conclude that ryk expression is regulated during hematopoietic development by lineage commitment and stage of maturation.
Asunto(s)
Células Madre Hematopoyéticas/fisiología , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Receptores de Superficie Celular/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células de la Médula Ósea , Línea Celular , Mapeo Cromosómico , Femenino , Citometría de Flujo , Hibridación Fluorescente in Situ , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
Hek is a member of the eph subfamily of receptor tyrosine kinases whose members include elk, hek2, sek, eph and eck among others. Using a soluble form of hek consisting of the extracellular region of the receptor fused to the Fc domain of human IgG1 and an expression cloning strategy, we have isolated two different but related cDNAs from the human T-lymphoma line HSB-2 that encode ligands for hek. The cDNAs encode proteins of 238 and 201 amino acids (44% amino acid identity) that are anchored to the membrane by glycosylphosphatidylinositol (GPI)-linkage. The proteins encoded by these cDNAs are bound by hek with affinity constants of 2 x 10(8) M-1. These proteins also bind the elk tyrosine kinase receptor. These cDNAs are related to other cDNAs that we have recently isolated from a human placental library that encode ligands for both hek and elk and define an emerging family of ligands for eph-related kinases (LERKs).
Asunto(s)
Proteínas Tirosina Quinasas Receptoras/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario , Efrina-A3 , Efrina-A4 , Efrina-B1 , Glicosilfosfatidilinositoles/metabolismo , Ligandos , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Proteínas/química , Receptor EphB3 , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoAsunto(s)
Proteínas Portadoras/genética , Proteínas de Unión al ADN/metabolismo , Hominidae/genética , Proteínas/genética , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción , Cromosoma X , Animales , Proteínas Portadoras/metabolismo , Mapeo Cromosómico , Cromosomas Artificiales de Levadura , Efrina-B1 , Marcadores Genéticos , Humanos , Ratones , Familia de Multigenes , Proteínas/metabolismo , Mapeo Restrictivo , Proteína Elk-1 con Dominio etsRESUMEN
We have isolated and characterized cDNA clones that encode the rat homologue of a binding protein, LERK-2, for the receptor tyrosine kinase, elk. The cDNAs contain an open reading frame of 1527 nucleotides capable of encoding a protein 345 amino acid residues in length. The nucleotide sequence of the present clones is > 90% identical to the previously identified human LERK-2 cDNA, and the predicted proteins encoded by the rat and human clones are identical at 95% of amino acid residues. Recombinant proteins expressed from the rat cDNAs bind to elk with high affinity, similar to recombinant human LERK-2 and an endogenously-expressed rat elk-binding protein. Expression of the rat LERK-2 mRNA was detected in embryonic brain, kidney, lung, skeletal muscle, thymus, liver, and heart, and diminished in the early post-natal period. Significant LERK-2 mRNA expression in the young adult rat was restricted to the lung, kidney, heart and testes.
Asunto(s)
Secuencia Conservada , Proteínas de Unión al ADN , Regulación de la Expresión Génica , Proteínas/genética , Proteínas Proto-Oncogénicas , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Oncogénicas de Retroviridae/metabolismo , Factores de Transcripción , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Evolución Biológica , Clonación Molecular , ADN Complementario , Efrina-B1 , Humanos , Masculino , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Unión Proteica , Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Homología de Secuencia de Aminoácido , Distribución Tisular , Células Tumorales Cultivadas , Proteína Elk-1 con Dominio etsRESUMEN
The human gene EPLG2 (Eph ligand-2) encodes a potential ligand for the receptor tyrosine kinase elk. High sequence conservation between the human and the rat cDNAs and developmentally regulated expression of the rat gene suggest that the protein encoded by EPLG2 plays an important role in mammalian development. To facilitate analysis of the physiological role of the protein, we have cloned and characterized a 24-kb region of mouse genomic DNA containing the mouse homologue of EPLG2 (Eplg2), including 5'- and 3'-flanking sequences. Restriction mapping, coupled with Southern blot hybridization and sequencing, was used to determine the structural organization of the gene. The Eplg2 genomic locus spans a region of approximately 12 kb, encoding five exons and four introns. The first intron comprises approximately 8.5 kb of the entire 12-kb genomic sequence. Eplg2 was mapped to the mouse X chromosome by interspecific backcross analysis and is tightly linked to the androgen receptor (Ar) locus.
Asunto(s)
Proteínas Portadoras/genética , Proteínas de Unión al ADN , Efrina-B1 , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Factores de Transcripción , Cromosoma X , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/química , Mapeo Cromosómico , Clonación Molecular , ADN Complementario , Humanos , Ratones , Datos de Secuencia Molecular , Ratas , Homología de Secuencia de Aminoácido , Proteína Elk-1 con Dominio etsAsunto(s)
Formación de Anticuerpos/fisiología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/química , Hematopoyesis/fisiología , Factores de Crecimiento de Célula Hematopoyética/fisiología , Inmunidad Celular/fisiología , Interleucina-3/química , Proteínas Recombinantes de Fusión/química , Animales , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Factores de Crecimiento de Célula Hematopoyética/aislamiento & purificación , Células Madre Hematopoyéticas/citología , Humanos , Interleucina-3/farmacología , Interleucinas/aislamiento & purificación , Interleucinas/fisiología , Macaca mulatta , Ratones/genética , Ratones/inmunología , Proteínas Recombinantes de Fusión/farmacología , Factor de Células Madre , Linfocitos T Colaboradores-Inductores/metabolismoRESUMEN
Regulation of hematopoietic stem cell proliferation and differentiation is likely to be controlled by the local concentration of both inhibitory and stimulatory cytokines. A newly described regulator of early haematopoietic cells called mast cell growth factor, stem cell factor or kit ligand (referred to here as the Sl factor) has been described. This factor is the gene product of the murine Steel locus and is the ligand for the c-kit proto-oncogene, the product of the murine W locus. The effects of Sl factor on primitive hematopoietic cells suggest that this growth factor is a major stimulator of basal hemopoiesis. Further, data indicates that Sl factor acts in synergy with virtually all of the later acting growth factors to enhance the proliferative and differentiative potential of these cells.
Asunto(s)
Citocinas/fisiología , Células Madre Hematopoyéticas/citología , Animales , Diferenciación Celular , División Celular , Hematopoyesis/genética , Factores de Crecimiento de Célula Hematopoyética/fisiología , RatonesRESUMEN
Low recovery and poor retroviral vector infection efficiency of hematopoietic stem cells has hindered application of gene therapy for disease affecting blood-forming tissues. Developmental restriction (or death) of stem cells during ex vivo infection has contributed to these difficulties. In these studies we report that the cytokine leukemia inhibitory factor (LIF) directly or indirectly supported the survival of hematopoietic stem cells during culture of bone marrow with vector-producing fibroblasts, resulting in efficient recovery of stem cells able to compete for engraftment in irradiated recipient animals. The infection efficiency of hematopoietic stem cells recovered from these cultures was approximately 80%; and all recipients (20/20) of the LIF-treated marrow were stably engrafted with the progeny of provirus-bearing stem cells. Expression of vector-encoded human adenosine deaminase (hADA) was detected in all recipients at levels averaging 15-50% of endogenous murine ADA in all their hematolymphoid tissues. Survival of stem cells in untreated cultures was approximately 10% of that observed from LIF-treated cultures, resulting in poor engraftment of recipient animals with transplanted cells. The infection efficiency of the few stem cells recovered from untreated cultures, however, was high (approximately 80%), suggesting that LIF did not have an effect on infection efficiency per se, but acted at the level of stem cell survival. Consistent with the poor engraftment observed in the control animals, expression of vector-encoded ADA was only approximately 4-20% of the endogenous levels. These results support the postulated role of LIF as a regulator of hematopoiesis and suggest that cytokine stimulation can positively affect inefficient retroviral vector transduction in hematopoietic stem cells.
Asunto(s)
Adenosina Desaminasa/genética , Trasplante de Médula Ósea , Inhibidores de Crecimiento/farmacología , Células Madre Hematopoyéticas/citología , Interleucina-6 , Linfocinas/farmacología , Animales , Antígenos Transformadores de Poliomavirus/genética , Médula Ósea/efectos de los fármacos , Células de la Médula Ósea , Línea Celular , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Femenino , Vectores Genéticos , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/enzimología , Humanos , Factor Inhibidor de Leucemia , Masculino , Ratones , Ratones Transgénicos , Virus 40 de los Simios/genética , TransfecciónRESUMEN
Conditioned media (CM) from a cloned murine marrow-derived stromal cell line, AC6.21 (ALC), was shown to stimulate retroviral vector infection of hematopoietic progenitors in culture. Inclusion of ALC CM during cocultivation of normal murine bone marrow (BM) with vector-producing fibroblasts improved infection efficiency of day 13 spleen colony-forming cells (CFU-s) from 63% (15 provirus-positive spleen colonies/24 total), without added growth factor, to 90% (36 provirus-positive colonies/40 total). In addition, stimulation of BM cells with ALC CM during cocultivation improved retroviral infection of stem cells capable of repopulating the hematopoietic system of irradiated recipient animals. Because ALC CM was found to have 50 to 100 U/ml of IL-6 activity, purified recombinant human IL-6 was tested for an effect in this system. Stimulation with IL-6 alone increased retroviral infection efficiency of CFU-s from 15% (17 colonies provirus-positive/111 total analyzed) without added growth factor to 66% (97 provirus-positive colonies/148 total analyzed). These experiments support and extend previous studies which have demonstrated the necessity for growth factor stimulation in optimizing retroviral vector transduction of hematopoietic precursors.