RESUMEN
To examine both possible correlations between anti-Ro/SS-A and anti-La/SS-B levels and their correlation with clinical disease activity in patients with Sjögren's syndrome (SS) or systemic lupus erythematosus (SLE), an ELISA was developed using purified recombinant Ro 60 kDa, Ro 52 kDa and La antigens. The ELISA was used for testing sequential serum samples from 16 patients with either SS or SLE. The patients were followed for periods between 15 and 128 months, and 3-15 serum samples per patient were analysed and compared with clinically apparent disease activity at the time of sampling in 14 patients. A temporal correlation of antibody levels to Ro and La antigens was found, and antibodies to different epitopes of the Ro 60 kDa protein showed parallel variation in seven of eight patients tested. Co-variation of autoantibody levels and disease activity was found in 11 of 14 patients. In seven of these 11 patients the anti-Ro and anti-La levels were stable and changes in disease activity were minimal during the observation period. In the other four of these 11, changes in disease activity were noted, with an associated change in autoantibody levels. The results suggest that the serological response to Ro and La antigens, as well as to different epitopes of the Ro 60 kDa protein, is antigen driven and regulated by common mechanisms, and indicate a correlation of Ro and La antibodies with pathogenic events.
Asunto(s)
Autoanticuerpos/metabolismo , Autoantígenos/inmunología , Lupus Eritematoso Sistémico/inmunología , ARN Citoplasmático Pequeño , Ribonucleoproteínas/inmunología , Síndrome de Sjögren/inmunología , Adulto , Anciano , Análisis de Varianza , Especificidad de Anticuerpos , Autoanticuerpos/efectos de los fármacos , Femenino , Estudios de Seguimiento , Humanos , Epítopos Inmunodominantes/inmunología , Inmunosupresores/uso terapéutico , Lupus Eritematoso Sistémico/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Peso Molecular , Síndrome de Sjögren/tratamiento farmacológico , Antígeno SS-BRESUMEN
The Ro 60 kDa protein is an RNA binding molecule present both in the cytoplasm and in the nucleus. Cytoplasmic Ro 60 kDa is complexed to other proteins and to certain RNAs denoted hYRNAs. This RNA-protein complex is also known as the Ro/SSA antigen recognized by sera from patients with certain autoimmune disorders. Components interacting with the nuclear Ro 60 kDa protein fraction in mammalian cells have not been identified. To look for an association with previously known nuclear structures, rabbit antisera to the amino- and carboxy-terminal parts of the Ro 60 kDa protein were used in immunomorphological studies on HeLa cells. A strong speckled nuclear pattern and a weak cytoplasmic staining were detected. Double immunofluorescence staining with affinity purified anti-Ro 60 kDa antibodies and monoclonal antibodies recognizing the Sm and RNP antigens of the U snRNPs, displayed colocalization. Another U snRNP containing nuclear compartment, the coiled bodies, did not contain any Ro 60 kDa protein. Cells infected with a toga virus demonstrated redistribution of both U snRNP antigens and the Ro 60 kDa protein with retained colocalization. These results indicate a role for the nuclear fraction of the Ro 60 kDa protein in RNA processing.
Asunto(s)
Autoantígenos/análisis , Núcleo Celular/química , ARN Citoplasmático Pequeño , Ribonucleoproteínas Nucleares Pequeñas/química , Ribonucleoproteínas/análisis , Animales , Especificidad de Anticuerpos , Autoantígenos/inmunología , Chlorocebus aethiops , Córnea/citología , Cricetinae , Perros , Técnica del Anticuerpo Fluorescente , Células HeLa/química , Células HeLa/ultraestructura , Células HeLa/virología , Humanos , Immunoblotting , Túbulos Renales Distales/citología , Fragmentos de Péptidos/inmunología , Estructura Terciaria de Proteína , Virus ARN/fisiología , Conejos , Proteínas Recombinantes/análisis , Proteínas Recombinantes/inmunología , Ribonucleoproteínas/inmunología , Ribonucleoproteínas Nucleares Pequeñas/inmunología , Células Vero/química , Células Vero/ultraestructura , Células Vero/virología , Antígeno SS-BRESUMEN
During skeletal muscle development three intermediate filament proteins are expressed: nestin, vimentin, and desmin. Vimentin and desmin belong to the class III intermediate filaments and are closely related to each other, whereas nestin is a more distantly related, class VI, intermediate filament. It was previously observed by conventional immunocytochemistry that the intracellular patterns of nestin, desmin, and vimentin appeared indistinguishable, despite nestin's more distant evolutionary relationship. We here extend this analysis by applying three-dimensional fluorescence digital imaging microscopy to compare the intracellular distribution of nestin with that of desmin, vimentin, actin, and tubulin in G6 human fetal skeletal muscle cells. We show that in vitro differentiation of G6 cells can produce an intermediate filament expression pattern similar to that observed during myogenesis in vivo, i.e., downregulation of vimentin but not of nestin and desmin during myotube maturation. The image analysis demonstrated that the degree of overlap between nestin and desmin/vimentin was very extensive in myoblasts and in multinucleate myotubes in all regions of the cells. In contrast, nestin did not colocalize with tubulin or actin in G6 myoblasts. In particular, nestin immunoreactivity was not detected at the microtubule-organizing center, and it was only sparsely observed at the cell periphery where actin stress fibers were seen. Our data lend further support to the notion that nestin interacts very closely with the two more distantly related class III intermediate filament proteins desmin and vimentin in the entire muscle cell, before and after myotube formation. A comparison of conserved amino acid residues in the different IFs suggest that charged amino acid residues in the alpha-helical rod domain may play a role in the interaction.
Asunto(s)
Desmina/aislamiento & purificación , Proteínas de Filamentos Intermediarios/aislamiento & purificación , Músculo Esquelético/metabolismo , Proteínas del Tejido Nervioso , Vimentina/aislamiento & purificación , Secuencia de Aminoácidos , Compartimento Celular , Línea Celular , Desmina/metabolismo , Embrión de Mamíferos/citología , Técnica del Anticuerpo Fluorescente , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Proteínas de Filamentos Intermediarios/metabolismo , Filamentos Intermedios/fisiología , Microscopía Fluorescente/métodos , Microtúbulos/fisiología , Datos de Secuencia Molecular , Nestina , Unión Proteica , Homología de Secuencia de Aminoácido , Vimentina/metabolismoRESUMEN
Patients with several different connective tissue diseases including Sjögren's syndrome and systemic lupus erythematosus produce autoantibodies reacting with a 52kD protein component of the Ro/SS-A antigen. Antibody recognition of recombinant Ro 52kD proteins encoded by both full-length and deletion clones was analysed by immunoblotting with patient sera. An antigenic region recognized by all anti-Ro 52kD positive sera was found in the middle part of the protein. By further mapping of residues 136-292 with overlapping clones, at lest two independent epitopes within the domain were detected. This part of the protein contains a leucine zipper motif and shows structural similarities with a predicted coiled-coil region involved in protein dimer formation. In addition, one fifth of the sera reacted weakly with another antigenic region located in the amino-terminal part of the protein containing two putative zinc fingers. These results demonstrate the presence of an immunodominant region but also heterogeneity in the human autoimmune response to the 52kD protein moiety of the Ro/SS-A antigen.
Asunto(s)
Autoantígenos/inmunología , Epítopos/inmunología , ARN Citoplasmático Pequeño , Ribonucleoproteínas/inmunología , Secuencia de Bases , Biblioteca de Genes , Humanos , Epítopos Inmunodominantes/inmunología , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
The Ro/SSA and La/SSB antigens are common targets for autoantibodies found in the sera of patients with Sjögren's syndrome and SLE. The anti-Ro/SSA and anti-La/SSB antibodies often appear together but are not cross-reactive. This paper describes the humoral autoimmune response to the Ro/SSA 60 kDa protein moiety with respect to the presence of IgM and IgG1-4 antibodies. IgM antibodies to the Ro 60 kDa protein coexisted with IgG anti-Ro 60 kDa antibodies in nearly half of the sera. A similar fraction also contained IgM anti-La/SSB antibodies. The frequency of sera with IgM antibodies of both specificities was that expected from random overlap. A predominating IgG1 anti-Ro 60 kDa response was found in all patients, but anti-Ro 60 kDa antibodies of the other IgG subclasses were present also in a high number of sera. This is in contrast to the reported IgG subclass distribution of anti-La/SSB antibodies. Mapping of IgM and IgG1-4 antibody recognition of different parts of the Ro 60 kDa protein was also performed. IgM and IgG1-4 antibodies of all sera reacted with the central part of the Ro 60 kDa protein, encompassing amino acid residues 181-320.
Asunto(s)
Autoanticuerpos/sangre , Autoantígenos/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , ARN Citoplasmático Pequeño , Enfermedades Reumáticas/inmunología , Ribonucleoproteínas/inmunología , Humanos , Immunoblotting , Antígeno SS-BRESUMEN
A myoblast clone, G6, was obtained from thigh muscle of an 11 week old human fetus, and used to examine the effect of platelet-derived growth factor (PDGF) on cell multiplication and differentiation. G6 myoblasts showed extensive fusion, and expressed creatine phosphokinase activity and muscle specific gene mRNA (myosin heavy chain, alpha-actin) when switched to a differentiation medium. The cells expressed PDGF beta-receptor mRNA, and bound 125I-PDGF-BB specifically. Expression of PDGF beta-receptors declined during in vitro differentiation. Relative levels of transcripts for the myogenic regulatory factors Myf4 (myogenin), Myf5, and Myf6 (MRF4) increased during the differentiation process, whereas Myf3 (MyoD1) was preferentially expressed in undifferentiated myoblasts. Treatment of the myoblasts with PDGF-BB increased DNA synthesis and cell density. Myogenic differentiation, analyzed as number of nuclei present in myotubes and expression of creatine phosphokinase and myosin heavy chain, was partly inhibited by the presence of PDGF-BB in the differentiation medium. PDGF-BB may, therefore, have the potential of regulating human muscle development and muscle regeneration.
Asunto(s)
Músculo Liso/citología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Células Madre/efectos de los fármacos , Becaplermina , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Células Clonales , ADN/biosíntesis , Desarrollo Embrionario y Fetal/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Genes Reguladores , Humanos , Músculo Liso/efectos de los fármacos , Músculo Liso/embriología , Factor de Crecimiento Derivado de Plaquetas/genética , Proteínas Proto-Oncogénicas c-sis , ARN Mensajero/biosíntesis , Ensayo de Unión Radioligante , Receptores del Factor de Crecimiento Derivado de Plaquetas/genética , Proteínas Recombinantes/farmacología , Células Madre/citologíaRESUMEN
Earlier studies have shown that smooth muscle cells (SMC) from arteries of neonatal and adult rats differ markedly in their in vitro growth characteristics. Since some of these differences may be relevant to the proliferation of SMC in atherosclerotic plaques we examined the expression of three proto-oncogenes (c-fos, c-jun, and c-myc) and an SMC-specific differentiation marker (alpha-actin) in cultured SMC. In presence of serum cultured adult SMC contained lower levels of alpha-actin mRNA than neonatal cells. In neonatal cells serum-starvation resulted in a distinct increase in both c-myc and alpha-actin mRNA levels, whereas the expression of these genes appeared to be unaffected in adult cells. Transfer of adult SMC proliferating in the presence of fetal calf serum to serum-free medium for 48 h almost completely inhibited DNA synthesis, whereas transfer of neonatal SMC to serum-free medium reduced DNA synthesis only to about 50%. Serum-starved adult and neonatal SMC did not contain c-fos or c-jun transcripts, but in both cell types serum-stimulation resulted in a comparable increase in the expression of both genes. The present results demonstrate clear differences in the mechanisms regulating gene expression in adult and neonatal SMC.
Asunto(s)
Animales Recién Nacidos/genética , ADN/biosíntesis , Genes myc , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Actinas/genética , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Células Cultivadas , Senescencia Celular , Medio de Cultivo Libre de Suero/farmacología , Masculino , Microscopía Fluorescente , Músculo Liso Vascular/fisiología , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-DawleyRESUMEN
Uptake of cadmium into cultured cells and its effects on cell growth and DNA synthesis are measured over a range of Cd concentrations of seven orders of magnitude. Cd uptake is found to be proportional to the external Cd concentration and to incubation time over a very broad range of concentrations. At least 200 mmol cadmium per kg dry weight of cells can be accumulated in this way, leading to exhaustion of the major intracellular Cd binding sites before cell death. On the other hand, very low cadmium concentrations down to 100 pM stimulate cell growth and DNA synthesis significantly. Stimulation is found in all three mammalian cell types examined: namely L6J1, a rat permanent myoblast cell line, LLC-PK1 porcine renal epithelial cells, and a primary rat chondrocyte culture. Cd acts as a cofactor with serum in L6J1 cultures, but is stimulatory only in serum-free cultures of chondrocytes. Stimulation occurs at Cd concentrations too low to result in a measurable induction of metallothionein. This might implicate the action of response amplifiers in the chain of events leading to Cd-stimulated DNA replication and cell growth.
Asunto(s)
Cadmio/farmacología , División Celular/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Animales , Secuencia de Bases , Cartílago/citología , Cartílago/efectos de los fármacos , Células Cultivadas , Medio de Cultivo Libre de Suero/farmacología , Relación Dosis-Respuesta a Droga , Células Epiteliales , Epitelio/efectos de los fármacos , Riñón , Metalotioneína/genética , Metalotioneína/metabolismo , Datos de Secuencia Molecular , Músculos/citología , Músculos/efectos de los fármacos , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Estimulación Química , PorcinosRESUMEN
The effects of two adenosine analogs on cyclic AMP (cAMP) accumulation and DNA synthesis were studied in cultured smooth muscle cells (SMCs) isolated from adult and neonatal rat arteries. N-ethylcarboxamido adenosine (NECA) dose-dependently increased intracellular cAMP levels and appeared to be more potent in adult than in neonatal SMCs. R-phenylisopropyl adenosine (R-PIA), in nanomolar concentrations, counteracted the increase in cAMP evoked by 10 microM forskolin in adult but not in neonatal SMCs, indicating that the enhanced "A2" response seen in adult SMCs was not due to a lack of "A1" receptors in these cultures. Binding experiments performed using the adenosine antagonist XAC did not reveal any differences in the number or affinity of the adenosine receptors between neonatal and adult SMCs. This indicates effects presumably on the G-protein level. A high capacity to spontaneously synthesize DNA and a weak response to platelet-derived growth factor (PDGF) were seen in the neonatal SMCs. Furthermore, NECA had no effect on PDGF-induced DNA synthesis in these cells. In contrast, adult SMCs presented a low rate of spontaneous DNA synthesis and a marked proliferative response to PDGF, which was inhibited by NECA. This inhibition paralleled the increase in cAMP elicited by NECA. Our findings suggest that neonatal and adult SMCs differ both in their response to growth factors and growth inhibitors.
Asunto(s)
AMP Cíclico/metabolismo , ADN/biosíntesis , Músculo Liso Vascular/metabolismo , Receptores Purinérgicos/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacología , Adenosina-5'-(N-etilcarboxamida) , Envejecimiento/metabolismo , Animales , Aorta , Células Cultivadas , Colforsina/farmacología , Músculo Liso Vascular/embriología , Fenilisopropiladenosina/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Autoantibodies reacting with the human Ro 60 kDa protein are present in anti-Ro/SS-A positive sera from patients with several different connective tissue diseases including Sjögren's syndrome and systemic lupus erythematosus. To investigate the humoral immune response to this protein, the pattern of antibody recognition of recombinant Ro 60 kDa proteins encoded by both full-length and deletion clones was analysed by immunoblotting. An antigenic region recognized by nearly all anti-Ro 60 kDa positive sera was found to reside in the middle part of the protein. In addition, some sera reacted with two other antigenic regions located in the amino-terminal and carboxyl-terminal part of the protein. For further mapping, overlapping peptides covering the most frequently recognized region of the protein were synthesized by solid-phase methods and used as antigens in ELISA. Three major patterns of reactivity to Ro 60 kDa peptides were found. These results not only indicate the presence of an immunodominant region but also heterogeneity in the autoimmune human response to the Ro 60 kDa antigen.
Asunto(s)
Autoantígenos/inmunología , Epítopos/inmunología , Fragmentos de Péptidos/inmunología , ARN Citoplasmático Pequeño , Proteínas Recombinantes de Fusión/inmunología , Ribonucleoproteínas/inmunología , Secuencia de Aminoácidos , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Enfermedades del Tejido Conjuntivo/sangre , Enfermedades del Tejido Conjuntivo/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis químicaRESUMEN
The development of atherosclerosis includes an abnormal proliferation of smooth muscle cells (SMCs) in the arterial intima. The factors responsible for this process remain to be identified, but earlier studies have suggested that age-related changes in growth-regulatory mechanisms may be involved. In the present study growth-regulatory mechanisms of neonatal and adult rat SMCs have been compared both in early passage and after subcultivation. Neonatal SMCs in early passage were found to have a high rate of spontaneous DNA synthesis and showed little response to stimulation with growth factors. Early passage adult SMCs showed a lower rate of spontaneous DNA synthesis but responded well to exogenous growth factors. There was no difference in the gene or surface expression of receptors for platelet-derived growth factor (PDGF) between neonatal and adult cells, and there was no significant difference in the amount of inositol phosphate formed in the cells after stimulation with PDGF BB. However, there was increased expression of PDGF A chain mRNA in serum-starved neonatal cells as compared to adult serum-starved SMCs. After subcultivation (seven to nine passages) neonatal SMCs started to become senescent, had a low rate of spontaneous DNA synthesis and were more sensitive to growth factor stimulation than in early passage. Adult SMCs did not demonstrate signs of senescence after subcultivation. The results demonstrate marked differences in the mechanisms regulating growth of neonatal and adult rat SMCs and suggest that the increased sensitivity of adult cells to exogenous growth factors and the inability of these cells to become senescent may be important factors in atherogenesis.
Asunto(s)
Envejecimiento/fisiología , Arteriosclerosis/fisiopatología , Músculo Liso Vascular/fisiología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Arterias , Arteriosclerosis/etiología , Northern Blotting , División Celular/fisiología , Células Cultivadas , ADN/biosíntesis , Fosfatos de Inositol/biosíntesis , Radioisótopos de Yodo , Músculo Liso Vascular/citología , Ratas , Ratas Endogámicas , Factores de RiesgoRESUMEN
We previously found that L6 myoblasts and skeletal muscle isolated from developing rats express the platelet-derived growth factor (PDGF) beta-receptor gene (Jin, P., Rahm, M., Claesson-Welsh, L., Heldin, C.-H., and Sejersen, T. (1990) J. Cell Biol. 110, 1665-1672). We now report that recombinant human PDGF-BB is a mitogen for L6 myoblasts and also a potent inhibitor of myogenic differentiation. Treatment of L6J1 myoblasts with PDGF-BB increased the rate of DNA synthesis and stimulated cell proliferation. In differentiation medium (Dulbecco's modified Eagle's medium/0.5% fetal calf serum or Dulbecco's modified Eagle's medium/insulin), PDGF-BB prevented fusion of confluent myoblasts and suppressed biochemical differentiation in L6J1 cells. Inhibition of myoblast differentiation was, however, reversible. Withdrawal of PDGF-BB from the medium allowed myoblast fusion to occur. Northern blot hybridization showed that the PDGF beta-receptor mRNA was down-regulated to an undetectable level when confluent cultures of L6J1 myoblasts in growth medium (Dulbecco's modified Eagle's medium/5% fetal calf serum) were shifted to differentiation medium. Receptor binding assays further indicated that binding of PDGF-BB to its receptors on L6J1 myoblasts declined rapidly before creatine kinase activity rose. Our results provide the first demonstration that PDGF-BB is a potent regulator of myogenesis of L6 rat myoblasts and suggest that it may regulate muscle differentiation in vivo.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Músculos/fisiología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Animales , Línea Celular , Creatina Quinasa/análisis , Creatina Quinasa/antagonistas & inhibidores , ADN/biosíntesis , Sondas de ADN , Mitógenos , Desarrollo de Músculos , Músculos/enzimología , ARN/análisis , Ratas , Receptores de Superficie Celular/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas , Proteínas Recombinantes/farmacologíaRESUMEN
Neurokinin A, a member of the tachykinin family of neuropeptides, has been identified as a mitogen for cultured smooth muscle cells. Tachykinin-induced DNA synthesis has previously been shown to be mediated by a receptor-specific mechanism and to correlate with accumulation of phosphatidylinositol 4,5-bisphosphate breakdown products. In the present experiments, we have studied intracellular pH and expression of the proto-oncogenes c-myc, c-jun and c-fos in smooth muscle cells exposed to mitogenic concentrations of neurokinin A. Growth-arrested smooth muscle cells stimulated with neurokinin A responded with an amiloride-sensitive intracellular alkalinization, indicating Na+/H+ antiport activation. c-myc and c-jun mRNA expression was only slightly elevated by neurokinin A, while c-fos expression underwent a more pronounced increase. Maximal levels of c-fos transcripts were found after 15 min and 30 min following neurokinin A stimulation. The results demonstrate that neuropeptides may influence proto-oncogene expression in smooth muscle cells and suggest a mechanism by which peripheral neurons may modulate differentiation and growth of these cells.
Asunto(s)
Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Neuroquinina A/farmacología , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes/efectos de los fármacos , Factores de Transcripción/genética , Amilorida/farmacología , Animales , Northern Blotting , ADN/biosíntesis , ADN/efectos de los fármacos , Concentración de Iones de Hidrógeno , Músculo Liso/citología , Nisoldipino/farmacología , Proteínas Proto-Oncogénicas c-fos , Proteínas Proto-Oncogénicas c-jun , Ratas , Ratas Endogámicas , Sulfonamidas/farmacologíaRESUMEN
The effect of cadmium on the expression of oncogenes c-jun, c-fos, and c-myc was studied by exposing L6J1 myoblast cultures to different doses of cadmium chloride and then analyzing the abundance of oncogene transcripts. Cadmium induced a transient accumulation of c-jun and c-myc mRNA with maximum expression at 2-4 h. At the same time, the level of c-fos transcripts remained below the detection level. Both the c-fos and c-jun genes could. However, be induced by treating the myoblasts with insulin. Cadmium induction of c-jun and c-myc mRNA occurred in a concentration-dependent manner with maximum stimulation at 5-10 microM. In the presence of cycloheximide, c-jun and c-myc genes were superinduced by the addition of cadmium. Under these conditions there was also a marked increase in c-fos transcripts. Induction of c-myc and c-jun by cadmium and c-fos by a combination of cadmium and cycloheximide could be abolished by blocking transcription with actinomycin D. The cadmium-induced increase in c-jun and c-myc mRNA was enhanced in myoblasts stably transfected with a mouse c-fos gene under a metallothionein promoter. Our present data suggest that cadmium has the potential to deregulate the expression of several important oncogenes.
Asunto(s)
Cadmio/farmacología , Proteínas de Unión al ADN/genética , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes , ARN Mensajero/genética , Factores de Transcripción/genética , Transcripción Genética/efectos de los fármacos , Animales , Cloruro de Cadmio , División Celular/efectos de los fármacos , Línea Celular , Músculos , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas c-jun , Proteínas Proto-Oncogénicas c-myc , ARN/genética , ARN/aislamiento & purificación , ARN Mensajero/aislamiento & purificación , Ratas , Mapeo Restrictivo , TransfecciónRESUMEN
The subcellular localization of the c-fos proto-oncogene product was studied in the G1, S, G2, and mitotic phases of the cell cycle by indirect immunofluorescence. For these analyses c-fos transfected L6J1 rat skeletal myoblasts and adult rat aortic smooth muscle cells in secondary culture, and c-fos- and c-myc co-transfected mouse Swiss 3T3 fibroblasts were used. During G1, S, and G2, the c-fos protein was evenly distributed in the nucleus, with exclusion of the nucleoli. In mitotic prophase the c-fos antigen was dissociated from the condensed chromosomes and became diffusely distributed in the cell cytoplasm, where it remained until telophase, when, again, it appeared to be associated with chromatin in the re-assembling nucleus. When comparing the subnuclear distribution of the c-fos product with that of densely packed DNA, stained with the fluorochrome Hoechst, an inverse relationship was found. Dispersed chromatin regions with weak Hoechst DNA fluorescence showed a stronger fos immunofluorescence than regions that contained a higher concentration of DNA. The localization of c-fos antigen partially overlapped with that of antigens typical of small nuclear ribonucleoprotein complexes participating in transcription and splicing. To examine if the c-fos protein would bind preferentially to specific interphase chromosomes the nucleus was fragmented into micronuclei containing single, or groups of, chromosomes. Immunofluorescence analysis showed that the majority of micronuclei were fos-positive. Possible roles of the c-fos proto-oncogene product are discussed in relation to other nuclear antigens.
Asunto(s)
Antígenos de Neoplasias/inmunología , Fibroblastos/citología , Músculo Liso Vascular/citología , Músculos/citología , Proteínas Proto-Oncogénicas/inmunología , Animales , Anticuerpos/inmunología , Antígenos de Neoplasias/metabolismo , Ciclo Celular/fisiología , Línea Celular , Núcleo Celular/inmunología , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Cromatina/inmunología , Cromatina/metabolismo , Cromatina/ultraestructura , ADN/genética , ADN/metabolismo , Replicación del ADN/fisiología , Fibroblastos/inmunología , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Expresión Génica/fisiología , Immunoblotting , Ratones , Mitosis/fisiología , Músculo Liso Vascular/inmunología , Músculo Liso Vascular/metabolismo , Músculos/inmunología , Músculos/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-fos , ARN/genética , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Ratas , Transcripción GenéticaRESUMEN
In order to study the antigenic properties of the La protein we have isolated a 1650 base pair (bp)-long human cDNA encoding an anti-La reactive protein. Restriction enzyme analysis and DNA sequencing was used to compare this clone with two published but inconsistent partial sequences. Our clone extends about 220 bp further towards the 5' end than the two clones previously studied and includes a putative initiation codon. When introduced into an expression vector, stable fusion proteins were made both from the initial clone and from two deletion clones. The recombinant proteins were tested by immunoblotting against a panel of anti-La sera. All reacted with the fusion protein produced by the 1650-bp clone. About half of the anti-La sera showed reactivity against the recombinant protein from the shortest deletion clone. This indicates the presence of an epitope in the amino terminal part of the La protein, encoded by sequences not present in previously published clones.
Asunto(s)
Autoantígenos/inmunología , Ribonucleoproteínas , Secuencia de Aminoácidos , Anticuerpos/inmunología , Secuencia de Bases , Sitios de Unión , Sondas de ADN , ADN Circular/análisis , ADN Circular/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Epítopos/inmunología , Humanos , Immunoblotting , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/biosíntesis , Mapeo Restrictivo , Antígeno SS-BRESUMEN
Protooncogenes expressed in murine embryonal carcinoma (EC) cells or their differentiated daughter cells include more or less ubiquitously expressed protooncogenes such as c-myc, c-K-ras, and c-abl, as well as c-onc genes with a very restricted expression pattern. Examples of the latter are N-myc, c-mos, and int-2. These c-onc genes are transcriptionally active in EC cells, as well as in germ cells and/or early embryonic cells. When EC cells are induced to differentiate some protooncogenes or oncogene-related products undergo changes in expression. Thus, EC cell differentiation has been associated with increased expression of c-src, c-fos, int-1, int-2, and the epidermal growth factor (EGF) receptor, whereas decreased expression has been observed for c-mos, c-K-ras, c-myc, N-myc, and platelet-derived growth factor. The relationships between these changes in expression and EC cell differentiation are not understood. They may be important for the differentiation process or for expression of a differentiated phenotype. They may, however, also be secondary events with no functional significance to EC cell differentiation.
Asunto(s)
Diferenciación Celular , Regulación de la Expresión Génica , Células Madre Neoplásicas/citología , Proto-Oncogenes , Animales , Células Madre de Carcinoma Embrionario , GTP Fosfohidrolasas/genética , Sustancias de Crecimiento/genética , Técnicas In Vitro , Ratones , Proteínas Nucleares/genética , Proteínas Quinasas/genéticaRESUMEN
Indirect immunofluorescence staining with human anti-centromere autoantibodies from a patient (LU 851) suffering from the CREST form of scleroderma was used to analyse chromosome topology in interphase nuclei of rat-kangaroo (PTO) and Indian muntjac (IM) cells. In some cells, centromeres were arranged in pairs suggesting association of homologous chromosomes. Clustering of centromeres at one pole of the nucleus (Rabl configuration) and other patterns suggesting higher order organization were also observed. In one fifth of the IM cells it was possible to identify the intranuclear location of each single chromosome on the basis of the morphology of the immunostained centromeres. In 30% of the IM cells in which centromeres could be identified, homologous chromosomes occupied adjacent territories within the interphase chromatin.
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Núcleo Celular/ultraestructura , Cromosomas/ultraestructura , Interfase , Animales , Línea Celular , Centrómero/inmunología , Centrómero/ultraestructura , Ciervos , Técnica del Anticuerpo Fluorescente , Macropodidae , MetafaseRESUMEN
We examined the expression of N-myc, c-myc, and c-src in four embryonic carcinoma (EC) cell lines during different states of cell growth and following induction of in vitro differentiation. N-myc mRNA was detected in undifferentiated cells of four EC cell lines (PCC7, PCC3, PCC4, F9) neither of which showed N-myc gene amplification. No N-myc transcripts could be detected in mRNA prepared from a murine neuroblastoma cell line and from a murine fibroblast line. The level of N-myc mRNA decreased by 85% when PCC7 EC cells were induced by retinoic acid and cAMP treatment to form nerve-like cells. Six days after induction, the PCC7 cells changed into aggregates of neurofilament positive cells with massive neurite outgrowths. At this stage DNA replication had been reduced by more than 95%. The decreased N-myc expression in induced PCC7 cells was parallelled by 300-500% increase in c-src expression. Slowing of cell multiplication by serum starvation, on the other hand, did not affect the level of N-myc or c-src mRNA levels in PCC7 cells. C-myc was expressed in all EC lines except PCC7, which surprisingly did not express c-myc even at an exponential rate of proliferation. Chemical induction of F9 EC cells to form visceral endoderm or parietal endoderm resulted in markedly reduced (85%) levels of N-myc transcripts. A similar decline in c-myc expression was found in differentiated F9 cells. No c-src transcripts were detected in proliferating or differentiated F9 cells. These results suggest that N-myc may be expressed not only in neural development, but also in very early, undetermined embryonic cells. The activation of c-src expression when PCC7 EC cells differentiate into nerve-like cells shows that the pattern of proto-oncogene expression may change during a differentiation process, some proto-oncogenes increasing, others decreasing their representation in the mRNA pool.
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Regulación de la Expresión Génica , Células Madre Neoplásicas/fisiología , Neuronas/patología , Proto-Oncogenes , Sangre , Diferenciación Celular , Línea Celular , Medios de Cultivo , Células Madre de Carcinoma Embrionario , Endodermo/patologíaRESUMEN
The ability of crocidolite fibers to induce point mutations and mitotic abnormalities in Chinese hamster ovary (CHO) cells was examined in cell cultures. The purpose has been to study the possibilities for establishing in vitro test methods to quantify genetic damage induced by asbestos and other mineral fibers. Results obtained with the CHO/hypoxanthine guanine phosphoribosyl transferase system indicated that crocidolite fibers per se do not significantly increase the number of thioguanine-resistant mutants. Crocidolite fibers also failed to potentiate the mutagenicity of benzo[a]pyrene. Time-lapse cinematography and microscopy showed that asbestos (crocidolite) fibers were markedly cytotoxic. Among surviving cells some underwent abnormal cell divisions which resulted in multi- and micronucleate cells. Many cells that contained a few asbestos fibers, however, underwent mitosis and successfully formed two mononucleate daughter cells capable of further divisions. Individual, fiber-containing cells were examined by time-lapse television recordings for 4-5 days. During this time period some cells underwent six divisions and generated an almost normal number of daughter cells. Cells which contained fibers that were longer or equivalent to the diameter of the mitotic cell (20 microns), showed different forms of mitotic abnormalities. The frequency of multinucleate cells was drastically increased following exposure to asbestos fibers. Only rarely, however, did these cells divide to produce viable daughter cells capable of continued cell multiplication. The frequency of multinucleate cells was dependent on the dose of exposure to asbestos fibers and could possibly be used as an index of the degree of mitotic disturbances induced by mineral fibers.