RESUMEN
Human macrophage colony-stimulating factor (M-CSF or CSF-1), either in purified or in recombinant form, is able to generate macrophagic colonies in a murine bone marrow colony assay, but only stimulates small macrophagic colonies of 40-50 cells in a human bone marrow colony assay. We report here that recombinant human granulocytic/macrophage colony stimulating factor (rhGM-CSF) at concentrations in the range of picograms enhances the responsiveness of bone marrow progenitors to M-CSF activity, resulting in an increased number of macrophagic colonies of up to 300 cells. Polyclonal antiserum against M-CSF did not alter colony formation of bone marrow progenitors incubated with GM-CSF at optimal concentration (1-10 ng/ml) for these in vitro assays. Thus, GM-CSF at higher concentrations (nanogram range) can by itself, elicit macrophagic colonies, and at lower concentrations (picogram range) acts to enhance the responsiveness of these progenitors to M-CSF.
Asunto(s)
Factores Estimulantes de Colonias/administración & dosificación , Sustancias de Crecimiento/administración & dosificación , Macrófagos/fisiología , Animales , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Factores Estimulantes de Colonias/farmacología , Sinergismo Farmacológico , Factor Estimulante de Colonias de Granulocitos , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Humanos , Interleucina-1/farmacología , Interleucina-3/farmacología , Ratones , Proteínas Recombinantes/farmacologíaRESUMEN
IL-1 is released by activated monocytes and is thought to be a key mediator of the host immune response. The availability of the purified and, more recently, recombinant IL-1 has allowed the characterization of other biological properties of this molecule. Thus, IL-1 is thought to have the same properties as hemopoietic 1, a growth factor that has been shown to act on primitive murine hemopoietic cells. Here we report that rIL-1 acts synergistically with granulocyte/macrophage CSF (GM-CSF) or granulocyte CSF in the stimulation of clonogenic cells from many patients with acute myeloblastic leukemia (AML). Although IL-1 by itself has no effect on AML blasts, it can support colony formation under conditions where there is detectable production of endogenous GM-CSF. IL-1 also promotes the growth of multipotential progenitors from normal human bone marrow cells in the presence of GM-CSF. These observations support the hypothesis that in the hemopoietic system, IL-1 has a selective effect on primitive precursors.
Asunto(s)
Ensayo de Unidades Formadoras de Colonias , Factores Estimulantes de Colonias/farmacología , Células Madre Hematopoyéticas/citología , Interleucina-1/fisiología , Leucemia Mieloide Aguda/patología , Proteínas Recombinantes/farmacología , Células Tumorales Cultivadas/citología , Ensayo de Tumor de Célula Madre , Células de la Médula Ósea , División Celular/efectos de los fármacos , Células Cultivadas , Factores Estimulantes de Colonias/inmunología , Sinergismo Farmacológico , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Sueros Inmunes , Interleucina-1/genética , Cinética , Hibridación de Ácido Nucleico , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Differentiation factor (D factor), also called leukemia inhibitory factor (LIF), is a glycoprotein that has been increasingly recognized to possess a wide range of physiological activities. We examined the possibility that the administration of D factor may confer beneficial effects and enhance host resistance against lethal endotoxemia. A single intravenous dose of recombinant human D factor completely protected C57/Bl6 mice from the lethal effect of Escherichia coli endotoxin (lipopolysaccharide [LPS]). The protective effects were dose dependent and observed when administered 2-24 h before LPS. Previous work has shown that interleukin 1 (IL-1) and tumor necrosis factor (TNF) also protect against a subsequent LPS challenge in a dose-dependent manner. When human D factor was combined with sub-protective doses of IL-1 beta or TNF-alpha, there was dramatic synergistic protection against a subsequent lethal LPS challenge.
Asunto(s)
Inhibidores de Crecimiento/administración & dosificación , Interleucina-1/administración & dosificación , Interleucina-6 , Lipopolisacáridos/toxicidad , Linfocinas/administración & dosificación , Factor de Necrosis Tumoral alfa/administración & dosificación , Animales , Sinergismo Farmacológico , Femenino , Factor Inhibidor de Leucemia , Ratones , Ratones Endogámicos C57BL , Proteínas RecombinantesRESUMEN
Empiric antibiotic therapy is often prescribed prior to the availability of bacterial culture results. In some cases, the organism isolated may not be susceptible to initial empiric therapy (inadequate empiric therapy or IET). In solid-organ transplant recipients, the overall incidence and clinical importance of IET is unknown. We performed a retrospective cohort study of patients admitted from 2002 to 2004. Multiple logistic regression analyses were conducted to determine associations between potential determinants and mortality. IET was administered in 169/312 (54%) patients, with a hospital mortality rate that was significantly greater than those receiving adequate therapy (24.9% vs. 7.0%; relative risk [RR] 3.55; 95% confidence interval [CI], 1.85-6.83; p < 0.001). Regression analysis demonstrated that an increasing duration of IET (adjusted odds ratio [OR] at 24 h: 1.33; 95% CI: 1.15-1.53; p < 0.001), ICU-associated infections (adjusted OR: 6.27; 95% CI: 2.79-14.09; p < 0.001), prior antibiotic use (adjusted OR: 3.56; 95% CI: 1.51-8.41; p = 0.004) and increasing APACHE-II scores (adjusted OR: 1.26; 95% CI: 1.16-1.34; p < 0.001) were independently correlated with hospital mortality. IET is common and appears to be associated with an increased hospital mortality rate in the solid-organ transplant population.
Asunto(s)
Antibacterianos/administración & dosificación , Trasplante de Órganos/mortalidad , Adulto , Bacteriemia/tratamiento farmacológico , Bacteriemia/etiología , Infecciones Bacterianas/tratamiento farmacológico , Infecciones Bacterianas/etiología , Estudios de Cohortes , Femenino , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Trasplante de Órganos/efectos adversos , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
Soluble peptide factors have been implicated as the agents responsible for embryonic inductions in vertebrates. Here, a protein (PIF) secreted by a mouse macrophage cell line is shown to change the developmental fate of Xenopus embryonic cells. Exposure to PIF causes presumptive ectodermal explants to form anterior neural and mesodermal tissues, including brain and eye, instead of ciliated epidermis. In addition, the induced tissues are organized into a rudimentary embryonic axis. These results suggest that PIF or a closely related molecule is involved in inducing anterior structures and organizing the frog body plan.
Asunto(s)
Blastocisto/fisiología , Embrión no Mamífero/fisiología , Macrófagos/fisiología , Proteínas/farmacología , Animales , Blastocisto/citología , Blastocisto/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Embrión no Mamífero/citología , Embrión no Mamífero/efectos de los fármacos , Ratones , Peso Molecular , Técnicas de Cultivo de Órganos , Proteínas/aislamiento & purificación , Proteínas/metabolismo , XenopusRESUMEN
Human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) was tested for its ability to induce colony formation in human bone marrow that had been enriched for progenitor cells. In addition to its expected granulocyte-monocyte colony-stimulating activity, the recombinant GM-CSF had burst-promoting activity for erythroid burst-forming units and also stimulated colonies derived from multipotent (mixed) progenitors. In contrast, recombinant erythroid-potentiating activity did not stimulate erythroid progenitors. The experiments prove that human GM-CSF has multilineage colony-stimulating activity.
Asunto(s)
Médula Ósea/efectos de los fármacos , Factores Estimulantes de Colonias/farmacología , Granulocitos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Proteínas Recombinantes/farmacología , Animales , Factores Estimulantes de Colonias/biosíntesis , ADN/genética , Relación Dosis-Respuesta a Droga , Eritroblastos/efectos de los fármacos , Humanos , Ratones , Células Madre/efectos de los fármacos , Linfocitos T/efectos de los fármacosRESUMEN
Clones of complementary DNA encoding the human lymphokine known as granulocyte-macrophage colony-stimulating factor (GM-CSF) were isolated by means of a mammalian cell (monkey COS cell) expression screening system. One of these clones was used to produce recombinant GM-CSF in mammalian cells. The recombinant hematopoietin was similar to the natural product that was purified to apparent homogeneity from medium conditioned by a human T-cell line. The human T-cell GM-CSF was found to be 60 percent homologous with the GM-CSF recently cloned from murine lung messenger RNA.
Asunto(s)
Clonación Molecular , Factores Estimulantes de Colonias/genética , ADN , Granulocitos , Macrófagos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Factores Estimulantes de Colonias/biosíntesis , Factores Estimulantes de Colonias/aislamiento & purificación , ADN Recombinante , Haplorrinos , Humanos , ARN Mensajero/genética , Linfocitos T , TransfecciónRESUMEN
A 4-kilobase complementary DNA (cDNA) encoding human macrophage-specific colony-stimulating factor (CSF-1) was isolated. When introduced into mammalian cells, this cDNA directs the expression of CSF-1 that is structurally and functionally indistinguishable from the natural human urinary CSF-1. Direct structural analysis of both the recombinant CSF-1 and the purified human urinary protein revealed that these species contain a sequence of at least 40 amino acids at their carboxyl termini which are not found in the coding region of a 1.6-kilobase CSF-1 cDNA that was previously described. These results demonstrate that the human CSF-1 gene can be expressed to yield at least two different messenger RNA species that encode distinct but related forms of CSF-1.
Asunto(s)
Factores Estimulantes de Colonias/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Factores Estimulantes de Colonias/orina , ADN/genética , Regulación de la Expresión Génica , Humanos , Macrófagos/fisiología , Peso Molecular , Fragmentos de Péptidos , Procesamiento Proteico-Postraduccional , ARN Mensajero/genéticaRESUMEN
Neutrophil migration inhibition factor from T lymphocytes (NIF-T) is a lymphokine that acts to localize granulocytes. Medium conditioned by the Mo human T-lymphoblast cell line was used to purify NIF-T, a glycoprotein with a molecular weight of 22,000. The NIF-T was found to potently stimulate the growth of granulocyte and macrophage colonies from human bone marrow and colony formation by the KG-1 myeloid leukemia cell line. Thus a human lymphokine (NIF-T) that modulates the activities of mature neutrophilic granulocytes is also a colony-stimulating factor acting on precursors to induce growth and differentiation of new effector cells.
Asunto(s)
Factores Estimulantes de Colonias/aislamiento & purificación , Granulocitos/citología , Factores Inhibidores de la Migración de Leucocitos/farmacología , Linfocinas/farmacología , Macrófagos/citología , Células de la Médula Ósea , División Celular , Línea Celular , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Humanos , Peso MolecularRESUMEN
To facilitate the direct study of progenitor cell biology, we have developed a simple and efficient procedure based upon negative selection by panning to purify large numbers of committed erythroid and myeloid progenitors from human fetal liver. The nonadherent, panned cells constitute a highly enriched population of progenitor cells, containing 30.4 +/- 13.1% erythrocyte burst forming units (BFU-E), 5.5 +/- 1.9% granulocyte-macrophage colony forming units (CFU-GM), and 1.4 +/- 0.7% granulocyte-erythroid-macrophage-megakaryocyte colony forming units (CFU-GEMM) as assayed in methylcellulose cultures. These cells are morphologically immature blasts with prominent Golgi. This preparative method recovers 60-100% of the committed progenitors detectable in unfractionated fetal liver and yields 2-30 X 10(6) progenitors from each fetal liver sample, and thus provides sufficient numbers of enriched progenitors to allow direct biochemical and immunologic manipulation. Using this technique, a purified recombinant protein previously thought to have only granulocyte-macrophage colony stimulating activity (GM-CSA) is shown to have both burst promoting activity and multipotential colony stimulating activity. Progenitor purification by panning thus appears to be a simple, efficient method that should facilitate the direct study of committed hematopoietic progenitors and their differentiation.
Asunto(s)
Separación Celular/métodos , Factores Estimulantes de Colonias , Hematopoyesis , Células Madre Hematopoyéticas/citología , Hígado/citología , Recuento de Células , Ensayo de Unidades Formadoras de Colonias , Factores Estimulantes de Colonias/fisiología , Eritrocitos/citología , Eritropoyesis , Feto , Humanos , Proteínas Recombinantes/fisiologíaRESUMEN
The mouse fibroblast gene, JE, was one of the first platelet-derived growth factor-inducible genes to be described as such. The protein encoded by JE (mJE) is the prototype of a large family of secreted, cytokinelike glycoproteins, all of whose members are induced by a mitogenic or activation signal in monocytes macrophages, and T lymphocytes; JE is the only member to have been identified in fibroblasts. We report the identification of a human homolog for murine JE, cloned from human fibroblasts. The protein predicted by the coding sequence of human JE (hJE) is 55 amino acids shorter than mJE, and its sequence is identical to that of a recently purified monocyte chemoattractant. When expressed in COS cells, the human JE cDNA directed the secretion of N-glycosylated proteins of Mr 16,000 to 18,000 as well as proteins of Mr 15,500, 15,000, and 13,000. Antibodies raised against mJE recognized these hJE species, all of which were secreted by human fibroblasts. hJE expression was stimulated in HL60 cells during phorbol myristate acetate-induced monocytoid differentiation. However, resting human monocytes constitutively secreted hJE; treatment with gamma interferon did not enhance hJE expression in monocytes, and treatment with phorbol myristate acetate or lipopolysaccharide inhibited its expression. Thus, human JE encodes yet another member of the large family of JE-related cytokinelike proteins, in this case a novel human monocyte and fibroblast secretory protein.
Asunto(s)
Glicoproteínas/genética , Monocitos/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Diferenciación Celular , Línea Celular , Quimiocina CCL2 , ADN/genética , Fibroblastos , Expresión Génica/efectos de los fármacos , Genes , Glicoproteínas/análisis , Glicoproteínas/biosíntesis , Humanos , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Ratones , Datos de Secuencia Molecular , Homología de Secuencia de Ácido Nucleico , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Proteins in the E2A family of basic helix-loop-helix transcription factors are important in a wide spectrum of physiologic processes as diverse as neurogenesis, myogenesis, lymphopoeisis, and sex determination. In the pancreatic beta cell, E2A proteins, in combination with tissue-specific transcription factors, regulate expression of the insulin gene and other genes critical for beta-cell function. By yeast two-hybrid screening of a cDNA library prepared from rat insulinoma (INS-1) cells, we identified a novel protein, Bridge-1, that interacts with E2A proteins and functions as a coactivator of gene transcription mediated by E12 and E47. Bridge-1 contains a PDZ-like domain, a domain known to be involved in protein-protein interactions. Bridge-1 is highly expressed in pancreatic islets and islet cell lines and the expression pattern is primarily nuclear. The interaction of Bridge-1 with E2A proteins is further demonstrated by coimmunoprecipitation of in vitro-translated Bridge-1 with E12 or E47 and by mammalian two-hybrid studies. The PDZ-like domain of Bridge-1 is required for interaction with the carboxy terminus of E12. In both yeast and mammalian two-hybrid interaction studies, Bridge-1 mutants lacking an intact PDZ-like domain interact poorly with E12. An E12 mutant (E12DeltaC) lacking the carboxy-terminal nine amino acids shows impaired interaction with Bridge-1. Bridge-1 has direct transactivational activity, since a Gal4 DNA-binding domain-Bridge-1 fusion protein transactivates a Gal4CAT reporter. Bridge-1 also functions as a coactivator by enhancing E12- or E47-mediated activation of a rat insulin I gene minienhancer promoter-reporter construct in transient-transfection experiments. Substitution of the mutant E12DeltaC for E12 reduces the coactivation of the rat insulin I minienhancer by Bridge-1. Inactivation of endogenous Bridge-1 in insulinoma (INS-1) cells by expression of a Bridge-1 antisense RNA diminishes rat insulin I promoter activity. Bridge-1, by utilizing its PDZ-like domain to interact with E12, may provide a new mechanism for the coactivation and regulation of transcription of the insulin gene.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Secuencias Hélice-Asa-Hélice , Insulina/genética , Regiones Promotoras Genéticas , Transactivadores/metabolismo , Factores de Transcripción , Activación Transcripcional , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Sitios de Unión , Línea Celular , Clonación Molecular , Cricetinae , ADN Complementario , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Estructura Terciaria de Proteína , Ratas , Homología de Secuencia de Aminoácido , Factores de Transcripción TCF , Distribución Tisular , Transactivadores/genética , Proteína 1 Similar al Factor de Transcripción 7 , Células Tumorales CultivadasRESUMEN
Granulocyte/macrophage-colony-stimulating factor (GM-CSF) is an effective growth factor for the blasts of acute myeloblastic leukemia (AML). Radioiodinated Chinese hamster ovary (CHO)-cell derived GM-CSF was prepared using Bolton-Hunter reagent to label free amino groups on the protein. Normal human neutrophils and the blast cells from AML patients were examined for binding. We found that there were fewer receptors of higher affinity on blast cells compared with neutrophils. After brief culture in suspension, receptor number increased and affinity decreased. Experiments provided evidence that GM-CSF from Escherichia coli had a higher affinity for neutrophils (kd = 20 pM) than the CHO-cell derived protein (kd = 500 pM-1 nM). This difference was reflected in the increased effectiveness of the E. coli protein over the CHO protein to stimulate colony formation in both normal bone marrow cells and AML blasts.
Asunto(s)
Factores Estimulantes de Colonias/metabolismo , Sustancias de Crecimiento/metabolismo , Leucemia Mieloide Aguda/metabolismo , Células Madre Neoplásicas/metabolismo , Receptores de Superficie Celular/análisis , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Humanos , Radioisótopos de Yodo , Neutrófilos/metabolismo , Receptores del Factor Estimulante de Colonias , Proteínas Recombinantes/metabolismoRESUMEN
The blast cells of acute myeloblastic leukemia may be considered as a renewal population maintained by stem cells that are capable of both self-renewal and differentiation. Blast stem cells grow in culture usually when stimulated by growth factors normally active on myelopoietic cells. Two culture methods permit an evaluation of the balance between self-renewal and differentiation; previous studies have shown that this balance can be affected by recombinant growth factors. These include interleukin 3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF), active on early cells in normal myelopoiesis, and G-CSF and CSF-1, restricted in normal hemopoiesis to the granulopoietic and macrophage/monocytic lineages, respectively. In this paper we report the results of evaluating the effects on these recombinant growth factors alone or in mixtures of two at optimal concentrations. The results were obtained either using titrations of colony formation in methylcellulose or growth in suspension. Star diagrams, a technique from exploratory data analysis, were used to provide quantitative and graphic displays of the results of the recombinant factors on the balance between blast self-renewal and differentiation. Blasts from 4 acute myeloblastic leukemia patients and one patient with the blast crisis of chronic myeloblastic leukemia were examined in detail. The great patient-to-patient variation usually observed was seen in both plating efficiency in methylcellulose and growth pattern in suspension. In spite of this variation, a common pattern of response to growth factors emerged. When the early acting factors, IL-3 and GM-CSF, were combined, the effect was quantitatively and qualitatively similar to the largest stimulation seen with either of the factors alone. In contrast, late-acting factors, G-CSF and CSF-1, influenced each other's effects when present together and each affected the activities of GM-CSF and IL-3. Notably, CSF-1, which often led to the accumulation of adherent, terminal cells in suspension, usually maintained or increased this differentiation-like activity in combination. G-CSF also favored differentiation in combination, although the effect was usually to increase the number of colonies in methylcellulose, most of which consist of blast cells incapable of further divisions. The results are discussed as they relate to the postulated structure of the blast population and the normal targets of the recombinant growth factors.
Asunto(s)
Factores Estimulantes de Colonias/farmacología , Sustancias de Crecimiento/farmacología , Interleucina-3/farmacología , Leucemia Mieloide Aguda/patología , Proteínas Recombinantes/farmacología , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Clonales/efectos de los fármacos , Combinación de Medicamentos , Factor Estimulante de Colonias de Granulocitos , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Humanos , Ensayo de Tumor de Célula MadreRESUMEN
Recombinant human granulocyte-macrophage colony-stimulating factor (rGM-CSF) has been previously demonstrated to stimulate colony formation from human myeloid, erythroid, and multipotential stem cells. In this investigation, we evaluated the effects of rGM-CSF on colony growth by human megakaryocyte progenitors (CFU-Meg). rGM-CSF was tested at concentrations of 0.1-100 U/ml in plasma clot cultures of adherent-depleted normal peripheral blood mononuclear cells. Control cultures were concurrently prepared containing either no stimulator or megakaryocyte colony-stimulating factor (Meg-CSF) partially purified from aplastic canine serum. rGM-CSF increased megakaryocyte colony numbers from a baseline of 4.3 +/- 1.4 (+/- SEM) in the unstimulated cultures to a maximum of 21.0 +/- 5.3 colonies at an rGM-CSF concentration of 1.0 U/ml. Corresponding megakaryocytic colony size increased from 4.4 to 8.3 cells/colony. Further increasing the rGM-CSF concentration resulted in decreasing megakaryocyte colony growth, reaching 6.8 +/- 2.9 colonies at 100 U/ml. The maximum number of megakaryocyte colonies stimulated by rGM-CSF was only 23.3% of that achieved in the control cultures containing optimal concentrations of serum-derived Meg-CSF protein (2.0 mg/ml). Megakaryocyte colonies stimulated by rGM-CSF consisted of predominantly low ploidy cells approximately equally distributed in 2N, 4N, and 8N ploidy classes. There was no increase in ploidy with any rGM-CSF concentration. These data indicate that rGM-CSF has modest activity in stimulating human megakaryocyte colony growth that is substantially less than that present in serum-derived Meg-CSF. rGM-CSF appears to primarily affect the early mitotic phase of megakaryocyte colony development with little influence on megakaryocyte endoreduplication.
Asunto(s)
Factores Estimulantes de Colonias/fisiología , Sustancias de Crecimiento/fisiología , Células Madre Hematopoyéticas/citología , Megacariocitos/citología , Proteínas Recombinantes/farmacología , División Celular/efectos de los fármacos , Células Cultivadas , ADN/sangre , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Cinética , Megacariocitos/efectos de los fármacosRESUMEN
Colony-stimulating factor from human urine (CSF-HU) has been purified to a homogeneous protein, and its complementary DNA (cDNA) has been cloned. Recombinant CSF-HU was prepared from a serum-free medium conditioned by Chinese hamster ovary cells transfected with the cDNA and purified by the same method as that for the native protein. Purified CSF-HU stimulated human bone marrow cells to form macrophage colonies. It also stimulated human mature monocytes prepared from peripheral blood of healthy volunteers to produce human active colony-stimulating activity that stimulates human bone marrow cells to form granulocyte and macrophage colonies. This activity was partially neutralized by the addition of both polyclonal antibodies against human granulocyte colony-stimulating factor and against human granulocyte-macrophage CSF, respectively. The stimulation of monocytes by CSF-HU was not inhibited by the addition of polymyxin-B, which is known as a potent inhibitor of endotoxin. On the other hand, CSF-HU did not stimulate monocyte production of interleukin-1 and interferon. These results indicate that recombinant and native CSF-HU stimulates immature cells as well as mature cells in the human monocyte lineage.
Asunto(s)
Factores Estimulantes de Colonias/biosíntesis , Factores Estimulantes de Colonias/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Orina/análisis , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Factores Estimulantes de Colonias/aislamiento & purificación , ADN/genética , Granulocitos , Humanos , Interferón Tipo I/biosíntesis , Interleucina-1/biosíntesis , Leucocitos Mononucleares/metabolismo , Macrófagos , Polimixina B/farmacología , Proteínas Recombinantes/farmacologíaRESUMEN
FoxM1 (previously named WIN, HFH-11 or Trident) is a Forkhead box (Fox) transcription factor widely expressed in proliferating cells. Various findings, including a recent analysis of FoxM1 knockout mice, suggest that FoxM1 is required for normal S-M coupling during cell cycle progression. To study the regulatory role of FoxM1 and its downstream regulatory targets, three stably transfected HeLa lines that display doxycycline (dox)-inducible FoxM1 expression were established. Over-expression of FoxM1 by dox induction facilitates growth recovery from serum starvation. Quantitation of cyclin B1 and D1 levels using flow cytometric, Western and Northern analyses reveals that elevated FoxM1 levels lead to stimulation of cyclin B1 but not cyclin D1 expression. Transient reporter assays in the dox-inducible lines and upon co-transfection with a constitutive FoxM1 expression plasmid suggest that FoxM1 can activate the cyclin B1 promoter.
Asunto(s)
Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Ciclo Celular , División Celular , Medio de Cultivo Libre de Suero , Ciclina B/genética , Ciclina B/metabolismo , Ciclina B1 , Ciclina D1/genética , Ciclina D1/metabolismo , ADN/genética , Doxiciclina/farmacología , Proteína Forkhead Box M1 , Factores de Transcripción Forkhead , Expresión Génica/efectos de los fármacos , Genes Reporteros , Células HeLa , Humanos , Luciferasas/genética , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , TransfecciónRESUMEN
Functional cDNA genes for cell surface proteins, receptors, growth factors and nuclear and cytoplasmic proteins can be isolated by a heterologous expression-function gene isolation strategy. The method of this strategy is dependent on highly efficient mammalian expression vectors, the properties of COS cells as hosts for SV40-origin-containing mammalian expression vectors, the well-established technology of constructing highly complex plasmid cDNA libraries and on the sensitivity of biological assays. These technologies are combined to identify a specific gene sequence by its biological phenotype. In theory this can be accomplished by individually testing each single gene of the cDNA library by expression in COS cells. In practice complex cDNA libraries are formed so that multiple cDNA genes can be tested and analyzed simultaneously. When the biological assay can discriminate phenotypic changes at the single cell level as typified by cellular morphology, cell membrane proteins or receptors or intracellular enzymes, then a reiterative expression screening method can be used. This was demonstrated by the isolation of cDNA genes encoding cell membrane proteins (7-10, 12). For the isolation of cDNA genes for growth factors and cytoplasmic enzymes by the expression-function cDNA gene isolation strategy, an alternative method is followed. The cDNA library is partitioned into sets of cDNA clones for transfection and analysis. The number of clones in a set is dependent on the efficiency of the expression vector used and on the sensitivity of the biological assay. For secreted cytokines and growth factors which can be simply detected by factor dependent cell lines, about 1,000 cDNA clones can be tested as a single set. By this strategy the genes for numerous hematopoietic growth factors, cytokines and interleukins have been isolated. With such numerous successes we can expect that the heterologous expression-function cDNA gene isolation strategy will become an important molecular biology method.
Asunto(s)
ADN/genética , Regulación de la Expresión Génica , Genes , Animales , Línea Celular , ADN/aislamiento & purificación , Biblioteca de Genes , TransfecciónRESUMEN
The coordinate increase in the hepatic production of the acute phase plasma proteins appears to be mediated by several cytokines produced by different cell types. One factor, hepatocyte-stimulating factor III (HSF-III), constitutively produced by human squamous carcinoma (COLO-16) cells, stimulates the synthesis of the same set of acute phase plasma proteins as the structurally distinct IL-6. The physicochemical properties of HSF-III coincide with those of the T cell-derived leukemia-inhibitory factor (LIF). Human rLIF, tested on hepatoma cells, indicated a liver-regulating activity identical to HSF-III. The LIF activity is specifically neutralized by HSF-III antibodies. COLO-16 cells contain an LIF mRNA which is characteristic for lectin-stimulated T cells, suggesting that HSF-III is an epidermal cell-derived form of LIF. This result provides additional evidence for the close relationship between acute phase regulation of the liver and control of proliferation and differentiation of hemopoietic cells by identical cytokines.
Asunto(s)
Proteínas de Fase Aguda/biosíntesis , Carcinoma Hepatocelular/metabolismo , Inhibidores de Crecimiento/fisiología , Interleucina-6 , Neoplasias Hepáticas/metabolismo , Linfocinas , Proteínas de Fase Aguda/genética , Regulación de la Expresión Génica , Inhibidores de Crecimiento/metabolismo , Humanos , Factor Inhibidor de Leucemia , ARN Mensajero/aislamiento & purificación , Transcripción Genética , Células Tumorales Cultivadas/metabolismoRESUMEN
An improved method for detecting and measuring the enzyme glucosephosphate isomerase after starch gel electrophoresis is described. Nitrocellulose filters are used in a gel overlay procedure which increases the sensitivity of the staining reaction and provides a simple means for accurate quantitation of the isozyme pattern. This staining technique may have wider application with other gel media and also with other enzymes.