RESUMEN
BACKGROUND: Arachidonic acid metabolites are implicated in the pathogenesis of asthma although only limited information is available on the impact of current smoking history on these metabolites. The aim of the study was to examine the effect of smoking status on urinary, sputum, and plasma eicosanoid concentrations and relevant enzyme transcripts in asthma. METHODS: In 108 smokers and never smokers with asthma and 45 healthy controls [smokers and never smokers], we measured urinary tetranor prostaglandin (PG)D2 (PGDM) and leukotriene (LT)E4 , induced sputum fluid LTB4 , LTE4 , PGD2 , and PGE2 , plasma secretory phospholipase A2 (sPLA2 ), and 11ß prostaglandin F2α (11ßPGF2α ), and, in a subgroup with severe asthma, airway leukocyte and epithelial cell mRNA expression levels of arachidonic acid metabolic enzymes. RESULTS: Smokers with asthma had higher urinary LTE4 ; 83 (59, 130) vs 59 (40, 90) pg/mg creatinine, P = 0.008, and PGDM; 60 (35, 100) vs 41 (28, 59) ng/mg creatinine, P = 0.012 concentrations, respectively, and lower sputum PGE2 concentrations 80 (46, 157) vs 192 (91, 301) pg/ml, P = 0.001 than never smokers with asthma. Sputum LTB4 (P = 0.013), and plasma 11ßPGF2α (P = 0.032), concentrations, respectively, were increased in smokers with asthma compared with healthy smokers. Asthma-specific and smoking-related increases (>1.5-fold expression) in arachidonate 15-lipoxygenase and gamma-glutamyltransferase transcripts were demonstrated. CONCLUSIONS: Several arachidonic acid metabolites and enzyme transcripts involving both lipoxygenase and cyclooxygenase pathways are increased in smokers with asthma and differ from never smokers with asthma. Possibly targeting specific lipoxygenase and cyclooxygenase pathways that are activated by asthma and cigarette smoking may optimize therapeutic responses.
Asunto(s)
Ácido Araquidónico/metabolismo , Asma/genética , Asma/metabolismo , Fumar , Transcripción Genética , Adulto , Antiasmáticos/farmacología , Antiasmáticos/uso terapéutico , Asma/diagnóstico , Asma/tratamiento farmacológico , Estudios Transversales , Femenino , Expresión Génica , Humanos , Leucocitos/metabolismo , Leucotrieno E4/sangre , Leucotrieno E4/metabolismo , Leucotrieno E4/orina , Masculino , Persona de Mediana Edad , Prostaglandinas/sangre , Prostaglandinas/orina , ARN Mensajero/genética , Pruebas de Función Respiratoria , Mucosa Respiratoria/metabolismo , Factores de Riesgo , Esputo/metabolismo , Encuestas y CuestionariosRESUMEN
Selective muscarinic agonists could be useful in the treatment of neurological disorders such as Alzheimer's disease, schizophrenia, and chronic pain. Many muscarinic agonists have been developed, yet most exhibit at best limited functional selectivity for a given receptor subtype perhaps because of the high degree of sequence homology within the putative binding site, which appears to be buried within the transmembrane domains. Bivalent compounds containing essentially two agonist pharmacophores within the same molecule were synthesized and tested for receptor binding affinity and muscarinic agonist activity. A series of bis-1,2,5-thiadiazole derivatives of 1,2,5,6-tetrahydropyridine linked by an alkyloxy moiety exhibited very high affinity (K(i) < 1 nM) and strong agonist activity. The degree of activity depended on the length of the linking alkyl group, which could be replaced by a poly(ethylene glycol) moiety, resulting in improved water solubility, binding affinity, and agonist potency.
Asunto(s)
Agonistas Muscarínicos/síntesis química , Piridinas/síntesis química , Tiadiazoles/síntesis química , Unión Competitiva , Línea Celular , Diseño de Fármacos , Humanos , Ligandos , Modelos Moleculares , Agonistas Muscarínicos/química , Agonistas Muscarínicos/farmacología , Fosfatidilinositoles/metabolismo , Estructura Terciaria de Proteína , Piridinas/química , Piridinas/farmacología , Ensayo de Unión Radioligante , Receptor Muscarínico M1 , Receptor Muscarínico M3 , Receptor Muscarínico M5 , Receptores Muscarínicos/metabolismo , Solubilidad , Relación Estructura-Actividad , Tiadiazoles/química , Tiadiazoles/farmacología , TransfecciónRESUMEN
The native form of soluble c-kit ligand (KL) is a noncovalent dimer. We have isolated a soluble, disulfide-linked dimer of murine KL (KL-CD) by expressing KL in Escherichia coli and refolding the denatured protein under conditions that promote the formation of both noncovalent dimers (KL-NC) and KL-CD. KL-CD exhibits a 10- to 15-fold increase in the ability to stimulate the growth of both the human megakaryocytic cell line MO7e and murine bone marrow-derived mast cells relative to KL-NC. Colony-forming assays of murine bone marrow progenitor cells also reflected this increased potency. However, KL-CD and KL-NC are equally able to prime mast cells for enhanced IgE-dependent degranulation in vitro and activate mast cells in vivo. Improving the growth-promoting activity of KL without changing its mast cell activation potential suggests that KL-CD or a related molecule could be administered in the clinic at doses that stimulate hematopoietic recovery while avoiding significant mast cell activation.
Asunto(s)
Mastocitos/efectos de los fármacos , Megacariocitos/efectos de los fármacos , Factor de Células Madre/química , Factor de Células Madre/farmacología , Animales , Degranulación de la Célula/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Dimerización , Escherichia coli , Humanos , Mastocitos/metabolismo , Mastocitos/patología , Megacariocitos/patología , Ratones , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologíaRESUMEN
The Rho subfamily of small guanosine triphosphate (GTP)-binding proteins, through their role in cytoskeletal organization, is involved in diverse cellular functions, including cell motility and morphologic changes during differentiation. Rac also has a special role in the production of superoxide, a key component in phagocytic antimicrobial function. Guanosine diphosphate (GDP)-dissociation inhibitors (GDIs) belong to one of three classes of proteins that regulate the critical cycling of GTP-binding proteins between the inactive and active states. Two homologous GDIs for the Rho subfamily have been identified. GDID4 is preferentially expressed in hematopoietic cells, while RhoGDI is ubiquitously expressed. Whether different physiologic functions are subserved by the two GDIs is unknown. We have derived embryonal stem (ES) cells with targeted disruption of both alleles of the GDID4 gene and examined hematopoiesis and phagocytic functions of macrophages derived from in vitro ES-cell differentiation. GDID4-/- ES cells develop like wild-type cells into colonies that contain heterogeneous populations of progenitor cells and differentiated erythromyeloid cells. GDID4-/- cells express no GDID4 protein, but have normal levels of RhoGDI. GDID4-/- macrophages phagocytose yeasts and antibody-opsonized erythrocytes as effectively as wild-type macrophages. However, a slight but consistent reduction in their capacity to generate superoxide was observed, which suggests new insight into the cellular role of GDID4. The minimal phenotypic effect of a loss of function of GDID4 also indicates a significant redundancy of function between GDID4 and RhoGDI. Their functional repertoire may be better revealed by a disruption of both genes. The use of hematopoietic cells derived in vitro from genotypically altered ES cells avoids the difficulties inherent in generating knockout animals and is a useful complementary approach for evaluating the gene function.
Asunto(s)
Marcación de Gen , Inhibidores de Disociación de Guanina Nucleótido , Células Madre Hematopoyéticas/citología , Macrófagos/metabolismo , Proteínas/genética , Estallido Respiratorio , Células Madre/metabolismo , Superóxidos/metabolismo , Animales , Calcimicina/farmacología , Calcio/metabolismo , Diferenciación Celular , Células Cultivadas , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/fisiología , Hematopoyesis , Liberación de Histamina/efectos de los fármacos , Inmunoglobulina E/farmacología , Ionóforos/farmacología , Mastocitos/citología , Ratones , Antígenos de Histocompatibilidad Menor , Fagocitosis , Proteínas/fisiología , Factor de Células Madre/farmacología , Células Madre/citología , Inhibidor beta de Disociación del Nucleótido Guanina rho , Inhibidores de la Disociación del Nucleótido Guanina rho-EspecíficoRESUMEN
The phenotypes of mice that harbor a defect in the genes encoding either stem cell factor (SCF) or its receptor, c-kit, indicate that this ligand/receptor pair is necessary for maintenance of normal hematopoiesis in the adult. Our objective was to determine whether SCF, like erythropoietin, is necessary for acute erythroid expansion during recovery from hemolytic anemia. Monoclonal antibody ACK2, which recognizes the murine c-kit receptor, was used to selectively block the hematopoietic growth-promoting effects of SCF. Mice were treated with phenylhydrazine on day 0 and day 1 to induce hemolytic anemia and also received no antibody, control IgG, or ACK2 on day 0. The mice were killed on day 3 and the hematocrit (Hct), reticulocyte count, and numbers of erythroid and myeloid hematopoietic progenitor cells (colony-forming unit-erythroid [CFU-E], burst-forming unit [BFU]-E, and CFU-granulocyte-macrophage [GM]) were quantitated in the femoral marrow and spleen using hematopoietic colony-forming assays. Induction of hemolytic anemia with phenylhydrazine resulted in a drop in the Hct from approximately 50% to 30%, and an approximate 8- to 10-fold increase in the reticulocyte count. The numbers of CFU-E increased modestly in the femur, and approximately 25- to 50-fold in the spleen, in comparison with normal mice. BFU-E and CFU-GM values did not increase in the femur but expanded 6- to 10-fold in the spleen, in comparison with normal mice. This confirms that much of the erythroid expansion in response to hemolytic anemia occurs in the murine spleen. Neutralizing quantities of the ACK2 antibody reduced femoral CFU-E, BFU-E, and CFU-GM content to less than half that found in phenylhydrazine-treated control mice and nearly totally ablated splenic hematopoiesis. These results suggest that c-kit receptor function may be required for optimal response to acute erythropoietic demand and that erythropoiesis in the splenic microenvironment is more dependent on SCF/c-kit receptor interaction than is erythropoiesis in the marrow microenvironment. Because expansion of late erythropoiesis in the spleen was preferentially blocked, we tested the hypothesis that homing of more primitive hematopoietic cells to the spleen was dependent on c-kit receptor function. Lethally irradiated mice were injected with marrow cells obtained from mice that had received phenylhydrazine plus control IgG or with marrow cells obtained from mice that had received phenylhydrazine plus ACK2. In parallel experiments, normal murine marrow cells were treated in vitro with control IgG or with ACK2 and were injected into lethally irradiated mice. The fraction of BFU-E and CFU-GM retrieved from the marrow and spleen of the recipient mice 4 hours later was reduced by approximately 75% when progenitor cells had been exposed to ACK2, in comparison with control IgG. These data suggest that interaction of SCF with the c-kit receptor affects the homing behavior of hematopoietic progenitor cells in the adult animal.
Asunto(s)
Células Madre Hematopoyéticas/patología , Proteínas Proto-Oncogénicas c-kit/fisiología , Bazo/patología , Factor de Células Madre/fisiología , Anemia Hemolítica/inducido químicamente , Anemia Hemolítica/patología , Animales , Anticuerpos Monoclonales/farmacología , Médula Ósea/patología , Movimiento Celular/fisiología , Ensayo de Unidades Formadoras de Colonias , Eritropoyesis/fisiología , Femenino , Células Madre Hematopoyéticas/metabolismo , Inmunoglobulina G/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Fenotipo , Fenilhidrazinas/toxicidad , Proteínas Proto-Oncogénicas c-kit/efectos de los fármacos , Quimera por Radiación , Ratas , Factor de Células Madre/antagonistas & inhibidoresRESUMEN
The c-kit ligand, KL, and its receptor, the proto-oncogene c-kit are encoded, respectively, at the steel (Sl) and white spotting (W) loci of the mouse. Both Sl and W mutations affect cellular targets in melanogenesis, gametogenesis, and hematopoiesis during development and in adult life. Although identified as a soluble protein, the predicted amino acid sequence of KL indicates that it is an integral transmembrane protein. We have investigated the relationship between the soluble and the cell associated forms of KL and the regulation of their expression. We show that the soluble form of KL is generated by efficient proteolytic cleavage from a transmembrane precursor, KL-1. An alternatively spliced version of KL-1, KL-2, in which the major proteolytic cleavage site is removed by splicing, is shown to produce a soluble biologically active form of KL as well, although with somewhat diminished efficiency. The protein kinase C inducer phorbol 12-myristate 13-acetate and the calcium ionophore A23187 were shown to induce the cleavage of both KL-1 and KL-2 at similar rates, suggesting that this process can be regulated differentially. Furthermore, proteolytic processing of both the KL-1 and KL-2 transmembrane protein products was shown to occur on the cell surface. The relative abundance of KL-1 and KL-2 is controlled in a tissue-specific manner. Sld, a viable steel allele, is shown to encode a biologically active secreted mutant KL protein. These results indicate an important function for both the soluble and the cell associate form of KL. The respective roles of the soluble and cell associated forms of KL in the proliferative and migratory functions of c-kit are discussed.
Asunto(s)
Factores de Crecimiento de Célula Hematopoyética/fisiología , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Calcimicina/farmacología , Técnicas de Cultivo , Regulación de la Expresión Génica , Factores de Crecimiento de Célula Hematopoyética/biosíntesis , Factores de Crecimiento de Célula Hematopoyética/genética , Ratones , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Empalme del ARN/genética , ARN Mensajero/aislamiento & purificación , Factor de Células Madre , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The c-kit proto-oncogene encodes a 145- to 160-Kd transmembrane tyrosine kinase, which is a member of the platelet-derived growth factor receptor family and is allelic with the murine white spotting locus (W). W mutations affect several aspects of hematopoiesis, most notably erythroid progenitors and mast cells. A monoclonal antibody, YB5.B8, had been raised against the leukemic blasts of a patient with M1-type acute myelocytic leukemia (AML) and it precipitates a 150-Kd cell surface glycoprotein from leukemic cells. The YB5.B8 epitope is expressed on mast cells, on up to 3% of normal mononuclear bone marrow cells, and it identifies a sub-group of AML patients with a poor prognosis. In view of similarities noted between the cell surface antigen identified by YB5.B8 and the c-kit protein product, we performed experiments to determine whether they are identical. c-kit RNA expression in the cell lines HEL (human erythroleukemia) and A172 (glioblastoma) was shown to parallel the expression of the YB5.B8 epitope in these lines as measured by flow cytometry. Immunoprecipitation analysis with anti-kit serum and YB5.B8 antibody indicated that the two antibodies identified proteins of identical size in HEL (155 Kd) and A172 (145 Kd) cells, and sequential immunoprecipitations with the kit and the YB5.B8 antibodies demonstrated that the two antibodies recognize the same molecule. The proteins identified by both the anti-kit and YB5.B8 antibodies displayed in vitro autophosphorylation activity in immune complex kinase assays. In addition, YB5.B8 was able to inhibit the binding of the kit ligand to HEL cells. These studies provide evidence that the YB5.B8 antigen and the c-kit protein product are identical and raise certain hypotheses regarding the role of c-kit in AML.
Asunto(s)
Anticuerpos Monoclonales , Proteínas Proto-Oncogénicas/análisis , Anticuerpos Monoclonales/inmunología , Antígenos de Neoplasias/análisis , Epítopos/inmunología , Citometría de Flujo , Expresión Génica , Glioma/inmunología , Glioma/metabolismo , Humanos , Técnicas de Inmunoadsorción , Leucemia Eritroblástica Aguda/inmunología , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/metabolismo , Mastocitos/inmunología , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/inmunología , Proteínas Proto-Oncogénicas c-kit , ARN/genética , Células Tumorales CultivadasRESUMEN
Recently, it has been shown that the c-kit proto-oncogene is encoded at the white spotting (W) locus in mice. Mutations of this gene cause depletion of germ cells, some hematopoietic cells and melanocytes. In order to define further the role of c-kit in gametogenesis, we have examined its expression in late fetal and postnatal ovaries and in postnatal testis. By RNA blot analysis, c-kit transcripts were not detected in late fetal ovaries but appeared at birth. The relative amount reached a maximum in ovaries of juvenile mice, and decreased in adult ovaries. c-kit transcripts were present in increasing amounts in isolated primordial, growing and full-grown oocytes, as well as in ovulated eggs. Little was detected in early 2-cell embryos and none in blastocysts. In situ hybridization revealed c-kit transcripts in a few oocytes of late fetal ovaries and in all oocytes (from primordial to full-grown) in ovaries from juvenile and adult mice. Expression was also observed in ovarian interstitial tissue from 14 days of age onward. Using indirect immunofluorescence, the c-kit protein was detected on the surface of primordial, growing and full-grown oocytes, as well as on embryos at the 1- and 2-cell stages; little remained in blastocysts. In situ hybridization analysis of testes from mice of different ages demonstrated expression in spermatogonia from 6 days of age onward. Using information provided by determining the stage of the cycle of the seminiferous epithelium for a given tubule and by following the age dependence of labeling, it was concluded that the period of expression of c-kit extends from at least as early as type A2 spermatogonia through type B spermatogonia and into preleptotene spermatocytes. Leydig cells were labelled at all ages examined. The expression pattern in oocytes correlates most strongly with oocyte growth and in male germ cells with gonial proliferation.
Asunto(s)
Expresión Génica/genética , Genitales/fisiología , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Animales , Northern Blotting , Femenino , Masculino , Ratones , Ratones Endogámicos , Microscopía Inmunoelectrónica , Oocitos/fisiología , Ovario/fisiología , Proteínas Proto-Oncogénicas c-kit , Espermatozoides/fisiología , Testículo/fisiologíaRESUMEN
The c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor for an unidentified ligand and is allelic with the murine white-spotting locus (W). W mutations affect melanogenesis, gametogenesis and hematopoiesis during development and in adult life. Cellular targets of W mutations in hematopoiesis include distinct cell populations in the erythroid and mast cell lineages as well as stem cells. In the absence of interleukin-3 (IL-3) mast cells derived from normal mice but not from W mutant mice can be maintained by co-culture with 3T3 fibroblasts. Based on the defective proliferative response of W mast cells in the 3T3 fibroblast co-culture system it had been proposed that fibroblasts produce the c-kit ligand. We have used a mast cell proliferation assay to purify a 30 kd protein, designated KL, from conditioned medium of Balb/3T3 fibroblasts to apparent homogeneity. KL stimulates the proliferation of normal bone marrow derived mast cells but not mast cells from W mice, although both normal and mutant mast cells respond similarly to IL-3. Connective tissue-type mast cells derived from the peritoneal cavity of normal mice were found to express a high level of c-kit protein on their surface and to proliferate in response to KL. The effect of KL on erythroid progenitor cells was investigated as well. In combination with erythropoietin, KL was found to stimulate early erythroid progenitors (BFU-E) from fetal liver and spleen cells but not from bone marrow cells of adult mice and from fetal liver cells of W/W mice.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Factores de Crecimiento de Fibroblastos/farmacología , Células Madre Hematopoyéticas/citología , Mastocitos/citología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Alelos , Animales , División Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Factores de Crecimiento de Fibroblastos/aislamiento & purificación , Factores de Crecimiento de Fibroblastos/metabolismo , Células Madre Hematopoyéticas/efectos de los fármacos , Focalización Isoeléctrica , Ligandos , Mastocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos , Proteínas Proto-Oncogénicas c-kitRESUMEN
Mutations at the steel locus (Sl) of the mouse affect the same cellular targets as mutations at the white spotting locus (W), which is allelic with the c-kit proto-oncogene. We show that KL, a hematopoietic growth factor obtained from conditioned medium of BALB/c 3T3 fibroblasts that stimulates the proliferation of mast cells and early erythroid progenitors, specifically binds to the c-kit receptor. The predicted amino acid sequence of isolated KL-specific cDNA clones suggests that KL is synthesized as an integral transmembrane protein. Linkage analysis maps the KL gene to the Sl locus on mouse chromosome 10, and KL sequences are deleted in the genome of the Sl mouse. These results indicate that the Sl locus encodes the ligand of the c-kit receptor, KL.
Asunto(s)
Mapeo Cromosómico , Factores de Crecimiento de Célula Hematopoyética/genética , Mutación , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN/genética , ADN/aislamiento & purificación , Genes , Factores de Crecimiento de Célula Hematopoyética/aislamiento & purificación , Factores de Crecimiento de Célula Hematopoyética/metabolismo , Ligandos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos , Datos de Secuencia Molecular , Peso Molecular , Proteínas Proto-Oncogénicas c-kit , Transcripción GenéticaRESUMEN
The proto-oncogene c-kit encodes a transmembrane tyrosine protein kinase receptor for an unknown ligand and is allelic with the murine white-spotting locus (W). Mutations at the W locus affect various aspects of hematopoiesis, the proliferation and migration of primordial germ cells and melanoblasts during development. The original W mutation and W37 are severe lethal mutations when homozygous. In the heterozygous state the W mutation has a weak phenotype while W37 has dominant characteristics. Wv and W41 are weak W mutations with dominant characteristics. We have characterized the molecular basis of these four W mutations and determined their effects on mast cell differentiation by using a fibroblast/mast cell co-culture assay. We show that W37, Wv and W41 are the result of missense mutations in the kinase domain of the c-kit coding sequence (W37 E----K at position 582; Wv T----M position 660 and W41 V----M position 831), which affect the c-kit associated tyrosine kinase to varying degrees. The c-kit protein products in homozygous mutant mast cells are expressed normally, although the 160 kd cell membrane form of the c-kitW37 protein displays accelerated turnover characteristics. The W mutation is the result of a 78 amino acid deletion which includes the transmembrane domain of the c-kit protein. A 125 kd c-kit protein was detected in homozygous W/W mast cells which lacks kinase activity and is not expressed on the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Mutación , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Mapeo Cromosómico , Fibroblastos/fisiología , Mastocitos/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Fenotipo , Pigmentación/genética , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-kit , ARN Mensajero/biosíntesisRESUMEN
The murine white spotting locus (W) is allelic with the proto-oncogene c-kit, which encodes a transmembrane tyrosine protein kinase receptor for an unknown ligand. Mutations at the W locus affect various aspects of hematopoiesis and the proliferation and migration of primordial germ cells and melanoblasts during development to varying degrees of severity. The W42 mutation has a particularly severe effect in both the homozygous and the heterozygous states. The molecular basis of the W42 mutation was determined. The c-kit protein products in homozygous mutant mast cells were expressed normally but displayed a defective tyrosine kinase activity in vitro. Nucleotide sequence analysis of mutant complementary DNAs revealed a missense mutation that replaces aspartic acid with asparagine at position 790 in the c-kit protein product. Aspartic acid-790 is a conserved residue in all protein kinases. These results provide an explanation for the dominant nature of the W42 mutation and provide insight into the mechanism of c-kit-mediated signal transduction.
Asunto(s)
Mutación , Fenotipo , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , ADN/genética , Expresión Génica , Homocigoto , Hígado/análisis , Hígado/citología , Hígado/embriología , Mastocitos/metabolismo , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-kit , ARN/análisis , Receptores de Superficie Celular/genética , Transducción de SeñalRESUMEN
The mature cells in the haemopoietic system arise as the result of the extensive developmental and proliferative capacity of pluripotential stem cells. In order to understand the molecular basis for these developmental processes, it will be necessary to identify and characterize the cellular genes that control early steps in haemopoiesis. Mutations at the mouse W locus on chromosome 5 lead to pleiotropic developmental defects, including sterility, coat colour abnormalities, severe macrocytic anaemia and mast cell deficiency. The defects in all these lineages are cell autonomous and intrinsic, suggesting that the W locus encodes a gene product required directly for cellular differentiation. In an attempt to understand this classical mouse developmental mutation, we have demonstrated that the c-kit proto-oncogene, which encodes a transmembrane receptor tyrosine kinase, is very closely linked to W. Several further observations are consistent with the idea that W and c-kit are allelic: first, c-kit is expressed in those cell populations affected by W mutations; second, the expression of c-kit transcripts can be affected by mutations at the W locus; third, the tyrosine kinase activity associated with the protein encoded by c-kit is functionally impaired in mast cells derived from mutant W/Wv mice; and fourth, rearrangements within the c-kit gene have been reported in two W mutant alleles. These observations suggest that the dominant phenotype associated with W mutations results from loss-of-function alterations that affect the receptor tyrosine kinase encoded by c-kit. The demonstration that the W locus encodes a transmembrane growth factor receptor provides a molecular basis for understanding the intrinsic haemopoietic defect in W mutant mice and the role that this cellular proto-oncogene plays in haemopoiesis and other developmental processes.
Asunto(s)
Cromosomas/ultraestructura , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Animales , Mapeo Cromosómico , Receptores ErbB/genética , Sistema Hematopoyético/citología , Sistema Hematopoyético/metabolismo , Sistema Hematopoyético/ultraestructura , Ratones , Ratones Mutantes , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-kitRESUMEN
The proto-oncogene c-kit, a transmembrane tyrosine protein kinase receptor for an unknown ligand, was shown recently to map to the dominant white spotting locus (W) of the mouse. Mutations at the W locus affect various aspects of hematopoiesis, as well as the proliferation and/or migration of primordial germ cells and melanoblasts during development. Here, we show that c-kit is expressed in tissues known to be affected by W mutations in fetal and adult erythropoietic tissues, mast cells, and neural-crest-derived melanocytes. We demonstrate that the c-kit associated tyrosine-specific protein kinase is functionally impaired in W/WV mast cells, thus providing a molecular basis for understanding the developmental defects that result from these mutations.
Asunto(s)
Anemia Macrocítica/genética , Ratones Mutantes/genética , Trastornos de la Pigmentación/genética , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes , Alelos , Anemia Macrocítica/enzimología , Animales , Movimiento Celular , Desarrollo Embrionario y Fetal , Genes Letales , Hematopoyesis , Células Madre Hematopoyéticas/enzimología , Heterocigoto , Hígado/embriología , Hígado/enzimología , Mastocitos/enzimología , Melanocitos/enzimología , Melanoma Experimental , Ratones , Ratones Mutantes/embriología , Ratones Mutantes/metabolismo , Cresta Neural/patología , Especificidad de Órganos , Trastornos de la Pigmentación/embriología , Trastornos de la Pigmentación/enzimología , Proteínas Tirosina Quinasas/deficiencia , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-kit , ARN Mensajero/análisis , Células Tumorales Cultivadas/enzimologíaRESUMEN
The requirement for CD8+ T lymphocytes in the stimulation of erythroid progenitor cells by prostaglandin E (PGE) was examined. When low density bone marrow (LD-BM) or non-adherent bone marrow (NA-BM) cells were depleted of CD8+ cells the enhancing effect of PGE on BFU-E proliferation was abrogated. However, further enrichment of marrow progenitor cells by depletion of accessory cells using a cocktail of specific monoclonal antibodies, immunoadherence and fluorescence activated cell sorting with the MY10 monoclonal antibody resulted in a population of erythroid progenitor cells which were responsive to the enhancing effect of PGE despite the absence of CD8+ cells. Stepwise individual cell lineage depletion of marrow cell populations indicated that prostaglandin E enhanced erythroid burst formation in the absence of CD8+ cells provided that glycophorin-A+ cells were removed from LD-BM or NA-BM cells. These results suggest that nucleated erythroid cell populations may modulate the enhancement of BFU-E by PGE. The ability of GP-A+ cells to block the enhancement of erythroid burst formation by PGE following removal of CD8+ T cells was confirmed by readdition of conditioned medium prepared from positively selected GP-A+ marrow cells. These results expand the role of CD8+ T cells in the PGE enhancement of BFU-E proliferation and suggest another mechanism by which accessory cells regulate the proliferation of BFU-E in bone marrow.
Asunto(s)
Células Presentadoras de Antígenos/fisiología , Médula Ósea/fisiología , Eritroblastos/fisiología , Eritropoyesis/efectos de los fármacos , Sustancias de Crecimiento/farmacología , Células Madre Hematopoyéticas/fisiología , Prostaglandinas E/farmacología , Adhesión Celular , Recuento de Células , Medios de Cultivo/farmacología , Humanos , Depleción Linfocítica , Linfocitos TRESUMEN
Expression of major histocompatibility complex class II Ags HLA-DR, HLA-DP, and HLA-DQ on human BM granulocyte-erythroid-macrophage-megakaryocyte CFU (CFU-GEMM), BFU-E, and CFU-GM was examined by indirect immunofluorescence, cell sorting, and complement-mediated cytotoxicity. BM, highly enriched for progenitor cells by depletion of mature hematopoietic elements, was further separated by sterile sorting into HLA-DR (-), low, intermediate, and high intensity HLA-DR (+), as well as HLA-DP (+) and HLA-DP (-) cell fractions and assayed for progenitor cell content. In addition, in the case of HLA-DR, CFU-GM response to inhibition by prostaglandin E was determined. Cell sorting and cytotoxicity data confirm that approximately 95% of assayable erythroid, myeloid, and multipotential progenitor cells expressed HLA-DR, whereas HLA-DQ Ags were undetectable. HLA-DR and HLA-DP Ags were co-expressed on 61% of these progenitor cells, predominantly those expressing HLA-DR at high intensity. Day 7 and 14 CFU-GM showed a trend toward segregation to the high HLA-DR (+) cell fractions, especially when recombinant human G-CSF was used to stimulate clone formation. Both day 7 and day 14 CFU-GMs were found predominantly in the HLA-DP (+) cell fraction. In contrast, BFU-E and CFU-GEMM were found in the low intensity HLA-DR cell fraction and predominantly in the HLA-DP (-) fraction. Both eosinophil CFU and cells giving rise to basophil/mast cells in suspension culture were found in the low and intermediate intensity HLA-DR fractions, but could be segregated into HLA-DP (+) and HLA-DP (-) cell fractions, respectively. Functional analysis of day 7 CFU-GM segregated, based upon HLA-DR intensity, indicated a positive correlation between increasing HLA-DR intensity and responsiveness to inhibition by prostaglandin E. Furthermore, only those CFU-GM expressing HLA-DR at high intensity could be removed by cytolytic treatment using a mAb anti-HLA-DR previously shown to be selective for CFU-GM responsive to PGE and in S phase of the cell cycle.
Asunto(s)
Médula Ósea/inmunología , Células Madre Hematopoyéticas/inmunología , Antígenos de Histocompatibilidad Clase II/fisiología , Adulto , Anticuerpos Monoclonales , Células Presentadoras de Antígenos/citología , Células de la Médula Ósea , Separación Celular , Ensayo de Unidades Formadoras de Colonias , Citotoxicidad Inmunológica , Hematopoyesis , HumanosRESUMEN
The effect of the iron chelator deferoxamine (DSF) on the proliferation of normal erythroid and granulocyte-macrophage progenitor cells from human and murine bone marrow was examined. The addition of DSF at a concentration equivalent to the concentration of iron present in the culture system resulted in dose dependent inhibition of colony formation by human and murine granulocyte-macrophage progenitor cells and human normal erythroid progenitor cells. The addition of FeCl3 at culture initiation completely reversed the effects of DSF. Furthermore, significant numbers of progenitor cells could be rescued from the effects of DSF by iron added back as late as 24-48 h after exposure to DSF. The cell cycle specificity of DSF was also examined using bone marrow cells treated with high specific activity tritiated thymidine. Kinetic experiments demonstrated that in the presence of DSF the number of erythroid or granulocyte-macrophage colonies that could be rescued was dependent on the length of exposure to DSF. Comparisons between control and tritiated thymidine treated cells indicated that the proliferation of progenitor cells in S phase of the cell cycle was inhibited if iron was withheld until 6 and 24 h after exposure to DSF for murine and human cells, respectively, with little to no effect observed on progenitor cells not in S phase during this time period. These results confirm the importance of iron for hematopoietic progenitor cell proliferation and represent a new method by which the proliferation of cycling cells may be investigated in situ in semisolid culture systems.
Asunto(s)
Ciclo Celular/efectos de los fármacos , Deferoxamina/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Animales , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Eritropoyesis/efectos de los fármacos , Compuestos Férricos/farmacología , Hematopoyesis/efectos de los fármacos , Humanos , Técnicas In Vitro , Ratones , Factores de TiempoRESUMEN
An interleukin-3 (IL-3) dependent mast cell line (MC) was infected with a recombinant retrovirus expressing the proto-oncogene c-myc and the drug selectable marker neo. Cells containing the transcriptionally activated c-myc gene displayed an increased growth rate in liquid culture and a higher cloning efficiency in soft agar when compared to control virus infected cells. All infected cells remained absolutely dependent on IL-3 for growth and were not tumorigenic in nude mice. Similar results were obtained with two additional IL-3 dependent cell lines, the mast cell 32D and the pre-B-cell Ea3. Thus, while constitutive expression of c-myc potentiates the response of mast cells to IL-3, it is not sufficient to eliminate their requirement for growth factors.
Asunto(s)
Sustancias de Crecimiento/farmacología , Interleucina-3/farmacología , Mastocitos/citología , Proteínas Proto-Oncogénicas/genética , Animales , División Celular , Regulación de la Expresión Génica , Mastocitos/efectos de los fármacos , Ratones , ARN Mensajero/genéticaRESUMEN
The effects of prostaglandin E (PGE) and recombinant human interferon-alpha, -beta, and -gamma alone and in combination were tested for their effects on the proliferation of human bone marrow granulocyte-macrophage colony-forming units (GM-CFU). When tested alone, both classes of cytokines inhibited GM-CFU proliferation. In combination, PGE and all three types of recombinant interferons synergized in their ability to inhibit GM-CFU proliferation. Progressive enrichment for marrow GM-CFU indicated that the synergistic effects of PGE and interferon were dependent upon the presence of marrow-adherent cells. Studies using conditioned media from marrow-adherent cells prepared in the presence of interferon-alpha, -beta, and -gamma indicated that adherent cells produced a soluble factor in the presence of interferons that subsequently synergized with PGE in inhibiting GM-CFU proliferation. Neutralization of this conditioned media with a monoclonal antibody to tumor necrosis factor abrogated the synergistic inhibition of GM-CFU observed in the presence of PGE. The addition of recombinant tumor necrosis factor and PGE to accessory cell-depleted bone marrow resulted in synergystic inhibition of GM-CFU proliferation.