RESUMEN
Syndecan-4 is a widely expressed transmembrane heparan sulfate proteoglycan which localizes to focal adhesions. Previous studies showed that the syndecan-4 cytoplasmic domain can associate with and potentiate the activity of protein kinase C, which is required for focal adhesion formation. To examine further the role of syndecan-4 in cell adhesion, we expressed syndecan-4 cDNA constructs in CHO-K1 cells. Syndecan-2 transfection was used to confirm effects seen were specific for syndecan-4. Cells overexpressing full length syndecan-4 core protein exhibited a more flattened, fibroblastic morphology, with increased focal adhesion formation and decreased cell motility. Expression of a syndecan-4 core protein with either a partial or complete deletion of the cytoplasmic domain or of an antisense construct led to markedly decreased spreading and focal adhesion formation, a more epithelioid morphology, and decreased motility. Overexpression of syndecan-2 changed the adhesive phenotype, but did not markedly alter focal adhesion and microfilament bundle formation. The data suggest that syndecan-4 is a regulator of focal adhesion and stress fiber formation, and influences both morphology and migration.
Asunto(s)
Glicoproteínas de Membrana/fisiología , Proteoglicanos/fisiología , Secuencia de Aminoácidos , Animales , Células CHO , Adhesión Celular/fisiología , División Celular , Tamaño de la Célula , Quimiotaxis , Cricetinae , Citoesqueleto/fisiología , Citoesqueleto/ultraestructura , Células Epiteliales/citología , Células Epiteliales/fisiología , Citometría de Flujo , Hígado/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Mutagénesis , Proteína Quinasa C/metabolismo , Proteoglicanos/química , Proteoglicanos/genética , Ratas , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Sindecano-2 , Sindecano-4 , TransfecciónRESUMEN
Many cytotoxic agents kill cells by invoking a specific death pathway termed physiological cell death, or apoptosis. Treatment of a murine hemopoietic stem cell line, FDCP-mix, with methylmethanesulfonate (MMS) or N'-methyl-N'-nitrosourea (MNU) leads to death by apoptosis. Retroviral gene transfer was used to overexpress the bcl-2 oncogene in FDCP-mix cells, and this was associated with a delay in apoptosis in these cells after treatment with MNU and MMS and decreased sensitivity of colony formation to the cytotoxic effects of MMS. These data suggest an explanation for the refractory nature of bcl-2-expressing follicular lymphoma to cytotoxic chemotherapy and furthermore suggest that DNA-damaging antitumor therapy may contribute to the progression of disease.
Asunto(s)
Apoptosis , Células Madre Hematopoyéticas/patología , Proteínas Proto-Oncogénicas/fisiología , Animales , Reparación del ADN , Hematopoyesis/efectos de los fármacos , Técnicas In Vitro , Metilmetanosulfonato/farmacología , Metilnitrosourea/farmacología , Ratones , Proteínas Proto-Oncogénicas c-bcl-2RESUMEN
Our previous studies have shown that haemopoietic stem cells undergo apoptotic death as a consequence of growth factor withdrawal. In this paper we review the new data that has accumulated since this observation and compare it with older data from the 'pre-apoptotic' age. Models of erythropoiesis and granulopoiesis that incorporate apoptosis as a normal physiological process controlling homeostasis are examined. The converse to cell death is cell survival, and we describe experiments which suggest that haemopoietic growth factors can not only act as mitogenic or differentiation stimuli but also act as survival signals. We, and others, have proposed that these growth factor-induced survival signals act through the membrane bound polypeptide receptors and share common features of signal transduction with proliferative responses. Enforced expression of bcl-2 in haemopoietic stem cells is able to overcome apoptosis following the withdrawal of growth factor, and the cells commit into different lineage differentiation programmes. Such cells spontaneously differentiate without cell division, suggesting a stochastic model of haemopoiesis in which the major role of haemopoietic growth factors is to suppress apoptosis and act as mitogens. We review the evidence that the underlying causes of some haematological diseases may be associated with change in the balance between cell survival and death.
Asunto(s)
Apoptosis , Hematopoyesis , Células Madre Hematopoyéticas/fisiología , Animales , División Celular , Supervivencia Celular , Eritropoyesis , Granulocitos/fisiología , HumanosRESUMEN
A temperature sensitive abl protein tyrosine kinase gene was transferred into a multipotent haemopoietic stem cell line, and the primary biological effects of expression of the gene were examined at the permissive and non-permissive temperatures. Unlike previous studies in factor-dependent cell lines, we found that expression of the functional abl protein tyrosine kinase did not lead to growth autonomy. Furthermore, the cells were still able to undergo terminal myeloid differentiation. However, expression of the functional gene did lead to a delay in maturation with a concomitant increase in cell production, had a modest effect in terms of delayed apoptosis particularly when the cells were maintained at a high cell density, and slightly increased the response to sub-optimal concentrations of IL-3. In many respects, therefore, the effects of abl protein tyrosine kinase in these cells mimics the effect of bcr/abl in primary haemopoietic cells where growth factor independence and an aberrant differentiation profile are relatively late events in clonal evolution and are not intermediate consequences of activation of the abl gene.
Asunto(s)
Células Madre Hematopoyéticas/citología , Proteínas Oncogénicas v-abl/fisiología , Apoptosis , Recuento de Células , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular , Diglicéridos/metabolismo , Resistencia a Medicamentos/genética , Genes abl , Células Madre Hematopoyéticas/metabolismo , Interleucina-3/administración & dosificación , Interleucina-3/farmacología , Neomicina , Proteínas Oncogénicas v-abl/metabolismo , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Retroviridae , TemperaturaRESUMEN
In the absence of growth factors, hemopoietic cells die rapidly by the process of apoptosis. Transfection of the human bcl-2 gene into an interleukin-3 (IL-3)-dependent, multipotent hemopoietic cell line allowed these cells to survive in the absence of IL-3, both in serum-containing and serum-deprived conditions, and this survival was accompanied by multilineage differentiation. Moreover, single cell experiments showed that differentiation could occur in the absence of cell division. While these data do not rule out the possibility that growth factors can influence the lineage choice of multipotent cells, they suggest that exposure to growth factors may not be obligatory for the differentiation of stem cells. The data also support the hypothesis that differentiation is intrinsically determined and that the role of the hemopoietic growth factors is enabling rather than inductive.
Asunto(s)
Apoptosis/genética , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes , Animales , Northern Blotting , Southern Blotting , Línea Celular , Supervivencia Celular/efectos de los fármacos , Medios de Cultivo , Medio de Cultivo Libre de Suero , ADN/análisis , Citometría de Flujo , Proteínas de Unión al GTP/genética , Humanos , Interleucina-3/farmacología , Ratones , Proteínas Proto-Oncogénicas c-bcl-2 , ARN/análisis , Células Madre , TransfecciónRESUMEN
Using the technique of differential cDNA library screening, we have molecularly cloned a gene that is highly expressed in an undifferentiated myeloid multipotent and growth factor-dependent stem cell line (FDCP-Mix) and that downregulates as these cells are induced to differentiate along monocytic, granulocytic, and erythroid cell lineages. Sequence analysis of this gene has shown homology with a previously cloned gene, cytotoxic cell protease 1 (CCP1 or Granzyme 'B'), that has been shown to be expressed only in thymocytes, activated T cells, a mast cell line, and peritoneal exudate leukocytes. In situ hybridization, Northern blot analysis, and nuclear run-off assay has confirmed that expression of CCP1 is restricted to the phenotypically primitive multipotent undifferentiated. FDCP-Mix cells that are undergoing self-renewal in the presence of growth factors such as interleukin-3.
Asunto(s)
Diferenciación Celular , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Sustancias de Crecimiento/farmacología , Interleucina-3/farmacología , Serina Endopeptidasas/genética , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Diferenciación Celular/efectos de los fármacos , Línea Celular , Clonación Molecular , Biblioteca de Genes , Factor Estimulante de Colonias de Granulocitos/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Granzimas , Interleucina-6/farmacología , Factor Estimulante de Colonias de Macrófagos/farmacología , Ratones , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , ARN/genética , ARN/aislamiento & purificación , Proteínas Recombinantes/farmacología , Células Madre , Transcripción Genética/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
The production of blood cells in the body is controlled by at least 20 polypeptide growth factors. Most of these factors have been cloned and many expressed in bacterial and eukaryotic systems to give biologically active proteins. Currently, these recombinant human proteins are undergoing intensive evaluation for their use in treating primary haemopoietic diseases, or stimulating normal haemopoiesis following drug-, radiation- or virus-induced trauma of the bone marrow. Erythropoietin (EPO) and the myeloid colony stimulating factors (IL-3, G-CSF, GM-CSF and M-CSF) were among the first to be cloned and expressed.
Asunto(s)
Factores Estimulantes de Colonias/uso terapéutico , Eritropoyetina/uso terapéutico , Animales , Clonación Molecular , Factores Estimulantes de Colonias/biosíntesis , Factores Estimulantes de Colonias/genética , Eritropoyetina/biosíntesis , Eritropoyetina/genética , Glicosilación , Humanos , Conformación Proteica , Relación Estructura-ActividadRESUMEN
We have used an antiserum raised against a purified heparan sulfate proteoglycan (HSPG) preparation isolated from rat liver to screen a lambda gt11 expression library and have obtained overlapping cDNA clones that contain the full-length coding sequence of an HSPG core protein capable of spanning the plasma membrane. The open reading frame of the rat cDNA encodes a protein of 211 amino acids. The predicted protein sequence (23 kDa) has a high degree of homology with the published partial sequence of the human lung fibroblast HSPG, fibroglycan. The deduced protein sequence contains a 24-amino acid transmembrane domain and a 33-amino acid cytoplasmic domain, both of which are identical with the corresponding regions of human fibroglycan and are highly homologous to the human, hamster, and mouse epithelial HSPG, syndecan. The putative ectodomain, which has 85% homology to fibroglycan, contains three possible glycosaminoglycan attachment sites that may be occupied by heparan sulfate chains. The major 49-kDa core protein in the liver HSPG preparation was found to be reactive to an antibody that specifically recognizes the cytoplasmic domain of fibroglycan. We have used the full-length cDNA clone to analyze the expression of this transmembrane core protein gene in whole tissues and several epithelial and fibroblastoid cell lines. It hybridizes to three mRNA species in all cell and tissue types examined, but in liver, isolated hepatocytes, and kidney, an additional 0.8-kilobase mRNA was detected. The three common messages arise from differential use of alternative polyadenylation sites, whereas the fourth tissue-restricted RNA species represents a related gene transcript. The rat equivalent of human fibroglycan therefore appears to be the major transmembrane proteoglycan in liver, and its widespread expression in many diverse tissues and cells suggests that it plays an important role in cellular interactions.
Asunto(s)
Heparitina Sulfato/genética , Hígado/metabolismo , Proteoglicanos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Western Blotting , Membrana Celular/metabolismo , Clonación Molecular , ADN/genética , Electroforesis en Gel de Poliacrilamida , Fibroblastos/metabolismo , Proteoglicanos de Heparán Sulfato , Hígado/citología , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Pruebas de Precipitina , ARN Mensajero/genética , RatasAsunto(s)
Sustancias de Crecimiento/genética , ARN Mensajero/genética , Animales , Secuencia de Bases , Femenino , Factores de Crecimiento de Célula Hematopoyética , Células Madre Hematopoyéticas/metabolismo , Ratones , Oocitos/metabolismo , Biosíntesis de Proteínas , Transcripción Genética , XenopusRESUMEN
Chicken erythrocyte chromatins containing a single species of linker histone, H1 or H5, have been prepared, using reassembly techniques developed previously. The reconstituted complexes possess the conformation of native chicken erythrocyte chromatin, as judged by chemical and structural criteria; saturation is reached when two molecules of linker histone are bound per nucleosome, as in native erythrocyte chromatin, which the resulting material resembles in its appearance in the electron microscope and quantitatively in its linear condensation factor relative to free DNA. The periodicity of micrococcal nuclease-sensitive sites in the linker regions associated with histone H1 or H5 is 10.4 base pairs, suggesting that the spatial organization of the linker region in the higher-order structure of chromatin is similar to that in isolated nucleosomes. The susceptible sites are cut at differing frequencies, as previously found for the nucleosome cores, leading to a characteristic distribution of intensities in the digests. The scission frequency of sites in the linker DNA depends additionally on the identity of the linker histone, suggesting that the higher-order structure is subject to secondary modulation by the associated histones.
Asunto(s)
Cromatina , Histonas/fisiología , Animales , Centrifugación por Gradiente de Densidad , Pollos , ADN/metabolismo , Eritrocitos , Calor , Nucleasa Microcócica/metabolismo , Microscopía Electrónica , Desnaturalización de Ácido Nucleico , Nucleosomas , Concentración Osmolar , Conformación ProteicaRESUMEN
Structural studies have revealed that chromatin is composed of repeating units or nucleosomes having two distinct domains, the nucleosome core and the linker region. The nucleosome core comprises 146 base pairs of DNA wound in one and three quarter turns around an octamer of histones made up of two symmetrical tetramers (1). It may be inferred on topological grounds that this structure must be perturbed during chromatin transcription and replication since the histone core bridges the supercoil which blocks the passage of polymerase along the template and prevents the unwinding of DNA required for enzymatic copying. A number of mechanisms for freeing the DNA template may be envisaged, and one detailed model, based on symmetrical dissociation of the histone tetramers, has been proposed (2). Here we present evidence against such unpairing or indeed any detachment of histones from the octamer during chromatin transcription, and we give reasons for favouring a transcriptional mechanism based upon the separation of the octamer from at least one of the DNA.
Asunto(s)
Cromatina/metabolismo , ADN/metabolismo , Transcripción Genética , Animales , Composición de Base , Núcleo Celular/metabolismo , Cromatina/ultraestructura , Eritrocitos/metabolismo , Histonas/metabolismo , Peso Molecular , Nucleosomas/metabolismo , Moldes GenéticosRESUMEN
Chicken reticulocyte chromatin can be reassembled from its separated constituents, viz. DNA, H1 plus H5, core histones, and non-histone proteins, to yield a product resembling the native starting material by a series of structural criteria. In particular, it possesses nucleosomes separated by spacer regions; the particles contain DNA with a unit length of approximately 200 base pairs. The recovery of the correctly reassembled product depends critically on the annealing conditions: the components are initially mixed in 2 M NaCl and 5 M urea, and it seems to be important to remove urea at a relatively high salt concentration. The results suggest that the characteristic chromatin structure is formed only when core histones bind to DNA in their native conformation and are followed by the addition of H1 and H5 to the spacer regions.
Asunto(s)
Cromatina/ultraestructura , ADN , Histonas , Reticulocitos/ultraestructura , Animales , Pollos , ADN/sangre , Histonas/sangre , Nucleasa Microcócica , Conformación de Ácido Nucleico , Conformación ProteicaRESUMEN
The nucleotide sequence of Drosophila melanogaster methionine tRNAi was determined to be: pA-G-C-A-G-A-G-U-m1G-m2G-C-G-C-A-G-U-G-G-A-A-G-C-G-U-m2G-C-U-G-G-G-C-C-C-A-U-t6A-A-C-C-C-A-G-A-G-m7G-D-m5C-C-C-G-A-G-G-A-U-C-G-m1A-A-A-C-C-U-U-G-C-U-C-U-G-C-U-A-C-C-A(OH). It differs from vertebrate initiator tRNAs in only 6 out of 75 positions.
Asunto(s)
Drosophila melanogaster/análisis , ARN de Transferencia , Animales , Secuencia de Bases , Conformación de Ácido Nucleico , Oligorribonucleótidos/análisis , Ribonucleasa T1RESUMEN
A method for the isolation and labeling to high specific radioactivity of individual isoaccepting tRNAs is described. After blocking reactive minor bases by acetylation and iodination of the crude tRNA, a single family of isoacceptors was aminoacylated. Individual isoacceptors were separated by chromatography on RPC-5 and then acylated with the 3-(4-hydroxyphenyl)propionyl ester of N-hydroxysuccinimide. The product was purified by chromatography on BD-cellulose and RPC-5. This derivatized tRNA was then iodinated with 125I- and Chloramine-T to give a product containing between 5 X 10(7) and 3 X 10(8) dpm/microgram. The suitability of such labeled tRNAs for hybridization to homologous DNA in solution and cytological preparations of chromosomes is discussed with particular reference to Drosophila melanogaster.