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1.
Nat Genet ; 20(4): 389-93, 1998 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-9843215

RESUMEN

Continuous regulation is required to maintain a given cell state or to allow it to change in response to the environment. Studies of the mechanisms underlying such regulation have often been hindered by the inability to control gene expression at will. Among the inducible systems available for regulating gene expression in eukaryotes, the tetracycline (tet) regulatable system has distinct advantages. It is highly specific, non-toxic and non-eukaryotic, and consequently does not have pleiotropic effects on host cell genes. Previously this system also had drawbacks, as it did not extinguish gene expression completely, precluding the study of toxic or growth-inhibitory gene products. We report here the development of a facile reversible tetracycline-inducible retroviral system (designated RetroTet-ART) in which activators and repressors together are expressed in cells. Gene expression can now be actively repressed in the absence of tet and induced in the presence of tet, as we have engineered distinct dimerization domains that allow co-expression of homodimeric tet-regulated transactivators and transrepressors in the same cells, without the formation of non-functional heterodimers. Using this system, we show that growth arrest by the cell cycle inhibitor p16 is reversible and dependent on its continuous expression.


Asunto(s)
División Celular/fisiología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/efectos de los fármacos , Tetraciclina/farmacología , Células 3T3 , Animales , Células Cultivadas , Inhibidor p16 de la Quinasa Dependiente de Ciclina/fisiología , Dimerización , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Ratones Endogámicos C3H , Retroviridae/genética
2.
Nat Med ; 3(9): 970-7, 1997 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-9288722

RESUMEN

Muscle biopsies from six patients with Duchenne muscular dystrophy (DMD) participating in a myoblast transplantation clinical trial were reexamined using a fluorescence in situ hybridization (FISH)-based method. Donor nuclei were detected in all biopsies analyzed, including nine where no donor myoblasts were previously thought to be present. In three patients, more than 10% of the original number of donor cells were calculated as present 6 months after implantation. Half of the detected donor nuclei were fused into host myofibers, and of these, nearly 50% produced dystrophin. These findings demonstrate that although donor myoblasts have persisted after injection, their microenvironment influences whether they fuse and express dystrophin. Our methodology could be used for developing new approaches to improve myoblast transfer efficacy and for the analysis of future gene- and/or cell-based therapies of numerous genetic disorders.


Asunto(s)
Músculos/trasplante , Distrofias Musculares/terapia , Recuento de Células , Fusión Celular , Núcleo Celular/patología , Trasplante de Células/efectos adversos , Distrofina/genética , Distrofina/metabolismo , Expresión Génica , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Masculino , Músculos/metabolismo , Músculos/patología , Distrofias Musculares/metabolismo , Distrofias Musculares/patología , Linfocitos T/inmunología , Linfocitos T/patología , Factores de Tiempo , Donantes de Tejidos
3.
Science ; 371(6528)2021 01 29.
Artículo en Inglés | MEDLINE | ID: mdl-33303683

RESUMEN

Treatments are lacking for sarcopenia, a debilitating age-related skeletal muscle wasting syndrome. We identifed increased amounts of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), the prostaglandin E2 (PGE2)-degrading enzyme, as a hallmark of aged tissues, including skeletal muscle. The consequent reduction in PGE2 signaling contributed to muscle atrophy in aged mice and results from 15-PGDH-expressing myofibers and interstitial cells, such as macrophages, within muscle. Overexpression of 15-PGDH in young muscles induced atrophy. Inhibition of 15-PGDH, by targeted genetic depletion or a small-molecule inhibitor, increased aged muscle mass, strength, and exercise performance. These benefits arise from a physiological increase in PGE2 concentrations, which augmented mitochondrial function and autophagy and decreased transforming growth factor-ß signaling and activity of ubiquitin-proteasome pathways. Thus, PGE2 signaling ameliorates muscle atrophy and rejuvenates muscle function, and 15-PGDH may be a suitable therapeutic target for countering sarcopenia.


Asunto(s)
Envejecimiento/metabolismo , Dinoprostona/metabolismo , Hidroxiprostaglandina Deshidrogenasas/fisiología , Músculo Esquelético/patología , Rejuvenecimiento , Sarcopenia/enzimología , Animales , Muerte Celular Autofágica/genética , Muerte Celular Autofágica/fisiología , Hidroxiprostaglandina Deshidrogenasas/antagonistas & inhibidores , Hidroxiprostaglandina Deshidrogenasas/genética , Macrófagos/enzimología , Ratones , Ratones Endogámicos C57BL , Mitocondrias Musculares/ultraestructura , Fuerza Muscular/genética , Fuerza Muscular/fisiología , Músculo Esquelético/enzimología , Miofibrillas/enzimología , Sarcopenia/genética
4.
J Cell Biol ; 102(1): 124-30, 1986 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-3941151

RESUMEN

We report that gene dosage, or the ratio of nuclei from two cell types fused to form a heterokaryon, affects the time course of differentiation-specific gene expression. The rate of appearance of the human muscle antigen, 5.1H11, is significantly faster in heterokaryons with equal or near-equal numbers of mouse muscle and human fibroblast nuclei than in heterokaryons with increased numbers of nuclei from either cell type. By 4 d after fusion, a high frequency of gene expression is evident at all ratios and greater than 75% of heterokaryons express the antigen even when the nonmuscle nuclei greatly outnumber the muscle nuclei. The kinetic differences observed with different nuclear ratios suggest that the concentration of putative trans-acting factors significantly influences the rate of muscle gene expression: a threshold concentration is necessary, but an excess may be inhibitory.


Asunto(s)
Regulación de la Expresión Génica , Células Híbridas/fisiología , Proteínas Musculares/genética , Músculos/fisiología , Animales , Anticuerpos Monoclonales/inmunología , Membrana Celular/inmunología , Humanos , Proteínas de la Membrana/inmunología , Ratones , Factores de Tiempo
5.
J Cell Biol ; 78(1): 131-51, 1978 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-566758

RESUMEN

Procedures for quantitative autoradiography were used for studying the process of secretion of eggshell (chorion) proteins in the follicular epithelium of silkmoths. The method was based on photometric measurements of the reflectance of vertically illuminated autoradiographic silver grains. Results were analyzed and plotted by computer. Secretory kinetics were also determined by analysis of labeled proteins in physically separated epithelium and chorion. Rapid accumulation of radioactivity into "clumps" visualized by light microscope autoradiography and evidence from preliminary electron microscope autoradiography indicate that, within 2 min from the time of synthesis, labeled chorion proteins move to Golgi regions scattered throughout the cytoplasm. The proteins begin to accumulate in the apical area 10-20 min later and to be discharged from the cell. The time for half-secretion is 20-25 min, and discharge is essentially complete 30-50 min after labeling. At the developmental stages examined, the kinetics of secretion appear to be similar for all proteins. Within the chorion the proteins rapidly assume a characteristic distribution, which varies for different developmental stages. Two relatively slow steps have been identified in secretion, associated with residence in Golgi regions and in the cell apex, respectively. By contrast, translocation of proteins across the cell and deposition of discharged proteins in the chorion are rapid steps.


Asunto(s)
Bombyx/fisiología , Proteínas del Huevo/metabolismo , Oogénesis , Animales , Autorradiografía , Corion/metabolismo , Computadores , Citoplasma/metabolismo , Retículo Endoplásmico/metabolismo , Femenino , Aparato de Golgi/metabolismo , Cinética , Ovario/metabolismo
6.
J Cell Biol ; 125(6): 1275-87, 1994 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-8207057

RESUMEN

The transplantation of cultured myoblasts into mature skeletal muscle is the basis for a new therapeutic approach to muscle and non-muscle diseases: myoblast-mediated gene therapy. The success of myoblast transplantation for correction of intrinsic muscle defects depends on the fusion of implanted cells with host myofibers. Previous studies in mice have been problematic because they have involved transplantation of established myogenic cell lines or primary muscle cultures. Both of these cell populations have disadvantages: myogenic cell lines are tumorigenic, and primary cultures contain a substantial percentage of non-myogenic cells which will not fuse to host fibers. Furthermore, for both cell populations, immune suppression of the host has been necessary for long-term retention of transplanted cells. To overcome these difficulties, we developed novel culture conditions that permit the purification of mouse myoblasts from primary cultures. Both enriched and clonal populations of primary myoblasts were characterized in assays of cell proliferation and differentiation. Primary myoblasts were dependent on added bFGF for growth and retained the ability to differentiate even after 30 population doublings. The fate of the pure myoblast populations after transplantation was monitored by labeling the cells with the marker enzyme beta-galactosidase (beta-gal) using retroviral mediated gene transfer. Within five days of transplantation into muscle of mature mice, primary myoblasts had fused with host muscle cells to form hybrid myofibers. To examine the immunobiology of primary myoblasts, we compared transplanted cells in syngeneic and allogeneic hosts. Even without immune suppression, the hybrid fibers persisted with continued beta-gal expression up to six months after myoblast transplantation in syngeneic hosts. In allogeneic hosts, the implanted cells were completely eliminated within three weeks. To assess tumorigenicity, primary myoblasts and myoblasts from the C2 myogenic cell line were transplanted into immunodeficient mice. Only C2 myoblasts formed tumors. The ease of isolation, growth, and transfection of primary mouse myoblasts under the conditions described here expand the opportunities to study muscle cell growth and differentiation using myoblasts from normal as well as mutant strains of mice. The properties of these cells after transplantation--the stability of resulting hybrid myofibers without immune suppression, the persistence of transgene expression, and the lack of tumorigenicity--suggest that studies of cell-mediated gene therapy using primary myoblasts can now be broadly applied to mouse models of human muscle and non-muscle diseases.


Asunto(s)
Técnicas de Cultivo/métodos , Terapia Genética/métodos , Músculos/citología , Células Madre/citología , Trasplante de Tejidos/métodos , Animales , Diferenciación Celular , División Celular , Fusión Celular , Células Clonales , Terapia de Inmunosupresión , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Desnudos , Músculos/inmunología , Neoplasias de Tejido Muscular/etiología , Fenotipo , Células Madre/inmunología , Trasplante Homólogo , Trasplante Isogénico
7.
J Cell Biol ; 121(4): 795-810, 1993 May.
Artículo en Inglés | MEDLINE | ID: mdl-8491773

RESUMEN

Vertebrate muscles are composed of an array of diverse fast and slow fiber types with different contractile properties. Differences among fibers in fast and slow MyHC expression could be due to extrinsic factors that act on the differentiated myofibers. Alternatively, the mononucleate myoblasts that fuse to form multinucleated muscle fibers could differ intrinsically due to lineage. To distinguish between these possibilities, we determined whether the changes in proportion of slow fibers were attributable to inherent differences in myoblasts. The proportion of fibers expressing slow myosin heavy chain (MyHC) was found to change markedly with time during embryonic and fetal human limb development. During the first trimester, a maximum of 75% of fibers expressed slow MyHC. Thereafter, new fibers formed which did not express this MyHC, so that the proportion of fibers expressing slow MyHC dropped to approximately 3% of the total by midgestation. Several weeks later, a subset of the new fibers began to express slow MyHC and from week 30 of gestation through adulthood, approximately 50% of fibers were slow. However, each myoblast clone (n = 2,119) derived from muscle tissues at six stages of human development (weeks 7, 9, 16, and 22 of gestation, 2 mo after birth and adult) expressed slow MyHC upon differentiation. We conclude from these results that the control of slow MyHC expression in vivo during muscle fiber formation in embryonic development is largely extrinsic to the myoblast. By contrast, human myoblast clones from the same samples differed in their expression of embryonic and neonatal MyHCs, in agreement with studies in other species, and this difference was shown to be stably heritable. Even after 25 population doublings in tissue culture, embryonic stage myoblasts did not give rise to myoblasts capable of expressing MyHCs typical of neonatal stages, indicating that stage-specific differences are not under the control of a division dependent mechanism, or intrinsic "clock." Taken together, these results suggest that, unlike embryonic and neonatal MyHCs, the expression of slow MyHC in vivo at different developmental stages during gestation is not the result of commitment to a distinct myoblast lineage, but is largely determined by the environment.


Asunto(s)
Músculos/metabolismo , Miosinas/biosíntesis , Diferenciación Celular , Células Cultivadas , Preescolar , Feto , Regulación de la Expresión Génica , Humanos , Lactante , Cinética , Músculos/citología , Músculos/embriología , Miosinas/genética
8.
J Cell Biol ; 127(6 Pt 2): 1923-32, 1994 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-7806570

RESUMEN

Normal and genetically engineered skeletal muscle cells (myoblasts) show promise as drug delivery vehicles and as therapeutic agents for treating muscle degeneration in muscular dystrophies. A limitation is the immune response of the host to the transplanted cells. Allogeneic myoblasts are rapidly rejected unless immunosuppressants are administered. However, continuous immunosuppression is associated with significant toxic side effects. Here we test whether immunosuppressive treatment, administered only transiently after allogeneic myoblast transplantation, allows the long-term survival of the transplanted cells in mice. Two immunosuppressive treatments with different modes of action were used: (a) cyclosporine A (CSA); and (b) monoclonal antibodies to intracellular adhesion molecule-1 and leukocyte function-associated molecule-1. The use of myoblasts genetically engineered to express beta-galactosidase allowed quantitation of the survival of allogeneic myoblasts at different times after cessation of the immunosuppressive treatments. Without host immunosuppression, allogeneic myoblasts were rejected from all host strains tested, although the relative time course differed as expected for low and high responder strains. The allogeneic myoblasts initially fused with host myofibers, but these hybrid cells were later destroyed by the massive immunological response of the host. However, transient immunosuppressive treatment prevented the hybrid myofiber destruction and led to their long-term retention. Even four months after the cessation of treatment, the hybrid myofibers persisted and no inflammatory infiltrate was present in the tissue. Such long-term survival indicates that transient immunosuppression may greatly increase the utility of myoblast transplantation as a therapeutic approach to the treatment of muscle and nonmuscle disease.


Asunto(s)
Refuerzo Inmunológico de Injertos , Terapia de Inmunosupresión , Músculo Esquelético/trasplante , Animales , Anticuerpos Monoclonales/farmacología , Trasplante de Células , Ciclosporina/farmacología , Rechazo de Injerto , Molécula 1 de Adhesión Intercelular/inmunología , Antígeno-1 Asociado a Función de Linfocito/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Especificidad de la Especie , Factores de Tiempo , Trasplante Homólogo
9.
J Cell Biol ; 106(4): 1027-34, 1988 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-3162914

RESUMEN

The inducibility of stably transfected alpha-cardiac actin genes differs among L cell clones. We examined the ability of muscle-specific factors to induce the expression of the human muscle alpha-cardiac actin gene promoter when stably transfected into mouse fibroblast L cells. This promoter is transcriptionally active in L cells at a low level, 2-5% of that in transfected muscle cells. Upon fusion with muscle cells to form heterokaryons, expression of the transfected alpha-cardiac actin gene promoter can be induced. However, induction is observed with only 10% of transfected L cell clones and the magnitude of this induction varies between 5- and 50-fold. These properties of the transfected L cell appear to be stably inherited. Our results are consistent with the hypothesis that muscle cells contain factors capable of increasing the transcription of the transfected gene, but that differences among L cell clones, possibly in the site of integration in the genome, determine the extent to which the gene can respond. By fusion into heterokaryons, transfectants with responsive genes can be identified. Such clones should prove useful in determining the basis for clonal variation. In addition, they provide an in vivo system for isolating functionally active tissue-specific transcription factors and the genes that encode them.


Asunto(s)
Actinas/genética , Células Clonales/fisiología , Regulación de la Expresión Génica , Acetiltransferasas/genética , Animales , Cloranfenicol O-Acetiltransferasa , Genes , Células Híbridas , Células L , Ratones , Miocardio/citología , Regiones Promotoras Genéticas , Ratas , Conteo por Cintilación , Factores de Transcripción/genética , Transcripción Genética , Transfección
10.
J Cell Biol ; 135(3): 829-35, 1996 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-8909554

RESUMEN

It has been suggested, on the basis of immunolocalization studies in vivo and antibody blocking experiments in vitro, that alpha 4 integrins interacting with vascular cell adhesion molecule 1 (VCAM-1) are involved in myogenesis and skeletal muscle development. To test this proposal, we generated embryonic stem (ES) cells homozygous null for the gene encoding the alpha 4 subunit and used them to generate chimeric mice. These chimeric mice showed high contributions of alpha 4-null cells in many tissues, including skeletal muscle, and muscles lacking any detectable (< 2%) alpha 4-positive cells did not reveal any gross morphological abnormalities. Furthermore, assays for in vitro myogenesis using either pure cultures of alpha 4-null myoblasts derived from the chimeras or alpha 4-null ES cells showed conclusively that alpha 4 integrins are not essential for muscle cell fusion and differentiation. Taking these results together, we conclude that alpha 4 integrins appear not to play essential roles in normal skeletal muscle development.


Asunto(s)
Antígenos CD/fisiología , Desarrollo de Músculos , Músculo Esquelético/crecimiento & desarrollo , Animales , Antígenos CD/análisis , Antígenos CD/genética , Diferenciación Celular , Fusión Celular , Células Cultivadas , Quimera , Integrina alfa4 , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/química , Músculo Esquelético/citología , Miocardio/química , Células Madre
11.
J Cell Biol ; 138(2): 331-6, 1997 Jul 28.
Artículo en Inglés | MEDLINE | ID: mdl-9230075

RESUMEN

Myoblast fusion is essential to muscle tissue development yet remains poorly understood. N-cadherin, like other cell surface adhesion molecules, has been implicated by others in muscle formation based on its pattern of expression and on inhibition of myoblast aggregation and fusion by antibodies or peptide mimics. Mice rendered homozygous null for N-cadherin revealed the general importance of the molecule in early development, but did not test a role in skeletal myogenesis, since the embryos died before muscle formation. To test genetically the proposed role of N-cadherin in myoblast fusion, we successfully obtained N-cadherin null primary myoblasts in culture. Fusion of myoblasts expressing or lacking N-cadherin was found to be equivalent, both in vitro by intracistronic complementation of lacZ and in vivo by injection into the muscles of adult mice. An essential role for N-cadherin in mediating the effects of basic fibroblast growth factor was also excluded. These methods for obtaining genetically homozygous null somatic cells from adult tissues should have broad applications. Here, they demonstrate clearly that the putative fusion molecule, N-cadherin, is not essential for myoblast fusion.


Asunto(s)
Cadherinas/fisiología , Fusión Celular/fisiología , Músculo Esquelético/citología , Animales , Cadherinas/genética , Células Cultivadas , Células Clonales , Factor 2 de Crecimiento de Fibroblastos/fisiología , Homocigoto , Ratones , Ratones Desnudos , Fibras Musculares Esqueléticas/citología , Mutación
12.
Science ; 290(5497): 1775-9, 2000 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-11099418

RESUMEN

After intravascular delivery of genetically marked adult mouse bone marrow into lethally irradiated normal adult hosts, donor-derived cells expressing neuronal proteins (neuronal phenotypes) developed in the central nervous system. Flow cytometry revealed a population of donor-derived cells in the brain with characteristics distinct from bone marrow. Confocal microscopy of individual cells showed that hundreds of marrow-derived cells in brain sections expressed gene products typical of neurons (NeuN, 200-kilodalton neurofilament, and class III beta-tubulin) and were able to activate the transcription factor cAMP response element-binding protein (CREB). The generation of neuronal phenotypes in the adult brain 1 to 6 months after an adult bone marrow transplant demonstrates a remarkable plasticity of adult tissues with potential clinical applications.


Asunto(s)
Células de la Médula Ósea/citología , Trasplante de Médula Ósea , Encéfalo/citología , Neuronas/citología , Animales , Biomarcadores/análisis , Diferenciación Celular , Tamaño de la Célula , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Citometría de Flujo , Expresión Génica , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/análisis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/genética , Neuronas/química , Neuronas/metabolismo , Bulbo Olfatorio/citología , Fenotipo , Fosforilación
13.
Science ; 254(5037): 1509-12, 1991 Dec 06.
Artículo en Inglés | MEDLINE | ID: mdl-1962213

RESUMEN

A recombinant gene encoding human growth hormone (hGH) was stably introduced into cultured myoblasts with a retroviral vector. After injection of genetically engineered myoblasts into mouse muscle, hGH could be detected in serum for 3 months. The fate of injected myoblasts was assessed by coinfecting the cells with two retroviral vectors, one encoding hGH and the other encoding beta-galactosidase from Escherichia coli. These results provide evidence that myoblasts, which can fuse into preexisting multinucleated myofibers that are vascularized and innervated, may be advantageous as vehicles for systemic delivery of recombinant proteins.


Asunto(s)
Terapia Genética/métodos , Hormona del Crecimiento/administración & dosificación , Músculos/citología , Proteínas Recombinantes/administración & dosificación , Transfección , Animales , Diferenciación Celular , Células Clonales , Expresión Génica , Humanos , Ratones
14.
Science ; 230(4727): 758-66, 1985 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-2414846

RESUMEN

Heterokaryons provide a model system in which to examine how tissue-specific phenotypes arise and are maintained. When muscle cells are fused with nonmuscle cells, muscle gene expression is activated in the nonmuscle cell type. Gene expression was studied either at a single cell level with monoclonal antibodies or in mass cultures at a biochemical and molecular level. In all of the nonmuscle cell types tested, including representatives of different embryonic lineages, phenotypes, and developmental stages, muscle gene expression was induced. Differences among cell types in the kinetics, frequency, and gene dosage requirements for gene expression provide clues to the underlying regulatory mechanisms. These results show that the expression of genes in the nuclei of differentiated cells is remarkably plastic and susceptible to modulation by the cytoplasm. The isolation of the genes encoding the tissue-specific trans-acting regulators responsible for muscle gene activation should now be possible.


Asunto(s)
Diferenciación Celular , Anciano , Animales , Anticuerpos Monoclonales , Fusión Celular , Núcleo Celular/ultraestructura , Células Epidérmicas , Feto/metabolismo , Fibroblastos/citología , Regulación de la Expresión Génica , Genes , Células HeLa/metabolismo , Humanos , Células Híbridas/metabolismo , Queratinas/fisiología , Cinética , Hígado/citología , Ratones , Desarrollo de Músculos , Músculos/citología , Miosinas/genética , Fenotipo , Transcripción Genética , Activación Transcripcional
15.
Trends Genet ; 5(8): 268-72, 1989 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-2686116

RESUMEN

The differentiated state is highly stable in vivo. Yet, in response to nuclear transplantation, tissue regeneration or cell fusion, the nuclei of differentiated cells exhibit a remarkable capacity to change. I review here the utility of heterokaryons, multinucleated cell hybrids, in elucidating the mechanisms that establish and maintain the differentiated state and yet allow such plasticity.


Asunto(s)
Diferenciación Celular , Replicación del ADN , Expresión Génica , Animales , Fusión Celular , Genes Reguladores , Humanos , Células Híbridas
16.
Trends Genet ; 9(8): 269-74, 1993 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-8379006

RESUMEN

The tissues of a multicellular animal are composed of diverse cell types arranged in a precisely organized pattern. Features unique to muscle allow an analysis of pattern formation and maintenance in mammals. The progeny of single cells can be taken full cycle from the animal to the culture dish and back to the animal where they fuse into mature myofibers of the host. These features not only facilitate the use of genetically engineered myoblasts in studies of pattern formation, but also in cell-mediated gene therapy: a novel mode of drug delivery for the treatment of muscle and nonmuscle diseases such as hemophilia, cardiac disease and cancer.


Asunto(s)
Terapia Genética , Músculos/citología , Animales , Vectores Genéticos , Humanos , Músculos/metabolismo , Proteínas Recombinantes/administración & dosificación
17.
Mol Cell Biol ; 8(6): 2295-301, 1988 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-3405207

RESUMEN

We examined the effects of human recombinant tumor necrosis factor-alpha (TNF) on human primary myoblasts. When added to proliferating myoblasts, TNF inhibited the expression of alpha-cardiac actin, a muscle-specific gene whose expression is observed at low levels in human myoblasts. TNF also inhibited muscle differentiation as measured by several parameters, including cell fusion and the expression of other muscle-specific genes, such as alpha-skeletal actin and myosin heavy chain. Muscle cells were sensitive to TNF in a narrow temporal window of differentiation. Northern (RNA) blot and immunofluorescence analyses revealed that human muscle gene expression became unresponsive to TNF coincident with myoblast differentiation. When TNF was added to differentiated myotubes, there was no effect on muscle gene expression. In contrast, TNF-inducible mRNAs such as interferon beta-2 still responded, suggesting that the signal mediated by TNF binding to its receptor had no effect on muscle-specific genes after differentiation.


Asunto(s)
Regulación de la Expresión Génica , Músculos/fisiología , ARN Mensajero/biosíntesis , Factor de Necrosis Tumoral alfa/farmacología , Actinas/genética , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Agar , Músculos/citología , Músculos/efectos de los fármacos , Hibridación de Ácido Nucleico
18.
Mol Cell Biol ; 7(11): 4100-14, 1987 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-3431550

RESUMEN

We evaluated the extent to which muscle-specific genes display identical patterns of mRNA accumulation during human myogenesis. Cloned satellite cells isolated from adult human skeletal muscle were expanded in culture, and RNA was isolated from low- and high-confluence cells and from fusing cultures over a 15-day time course. The accumulation of over 20 different transcripts was compared in these samples with that in fetal and adult human skeletal muscle. The expression of carbonic anhydrase 3, myoglobin, HSP83, and mRNAs encoding eight unknown proteins were examined in human myogenic cultures. In general, the expression of most of the mRNAs was induced after fusion to form myotubes. However, several exceptions, including carbonic anhydrase and myoglobin, showed no detectable expression in early myotubes. Comparison of all transcripts demonstrated little, if any, identity of mRNA accumulation patterns. Similar variability was also seen for mRNAs which were also expressed in nonmuscle cells. Accumulation of mRNAs encoding alpha-skeletal, alpha-cardiac, beta- and gamma-actin, total myosin heavy chain, and alpha- and beta-tubulin also displayed discordant regulation, which has important implications for sarcomere assembly. Cardiac actin was the only muscle-specific transcript that was detected in low-confluency cells and was the major alpha-actin mRNA at all times in fusing cultures. Skeletal actin was transiently induced in fusing cultures and then reduced by an order of magnitude. Total myosin heavy-chain mRNA accumulation lagged behind that of alpha-actin. Whereas beta- and gamma-actin displayed a sharp decrease after initiation of fusion and thereafter did not change, alpha- and beta-tubulin were transiently induced to a high level during the time course in culture. We conclude that each gene may have its own unique determinants of transcript accumulation and that the phenotype of a muscle may not be determined so much by which genes are active or silent but rather by the extent to which their transcript levels are modulated. Finally, we observed that patterns of transcript accumulation established within the myotube cultures were consistent with the hypothesis that myoblasts isolated from adult tissue recapitulate a myogenic developmental program. However, we also detected a transient appearance of adult skeletal muscle-specific transcripts in high-confluence myoblast cultures. This indicates that the initial differentiation of these myoblasts may reflect a more complex process than simple recapitulation of development.


Asunto(s)
Desarrollo de Músculos , ARN Mensajero/genética , Transcripción Genética , Tubulina (Proteína)/genética , Adulto , Diferenciación Celular , Clonación Molecular , ADN/aislamiento & purificación , Feto , Humanos , Cinética , Masculino , Persona de Mediana Edad , Músculos/citología , Músculos/embriología , Distrofias Musculares/patología , ARN Mensajero/metabolismo
19.
Mol Cell Biol ; 18(12): 7371-82, 1998 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-9819424

RESUMEN

The putative function of highly conserved regions (HCRs) within 3' untranslated regions (3'UTRs) as regulatory RNA sequences was efficiently and quantitatively assessed by using modular retroviral vectors. This strategy led to the identification of HCRs that alter gene expression in response to oxidative or mitogenic stress. Databases were screened for UTR sequences of >100 nucleotides that had retained 70% identity over more than 300 million years of evolution. The effects of 10 such HCRs on a standard reporter mRNA or protein were studied. To this end, we developed a modular retroviral vector that can allow for a direct comparison of the effects of different HCRs on gene expression independent of their gene-intrinsic 5'UTR, promoter, protein coding region, or poly(A) sequence. Five of the HCRs tested decreased mRNA steady-state levels 2- to 10-fold relative to controls, presumably by altering mRNA stability. One HCR increased translation, and one decreased translation. Elevated mitogen levels caused four HCRs to increase protein levels twofold. One HCR increased protein levels fourfold in response to hypoxia. Although nonconserved UTR sequences may also have a role, these results provide evidence that sequences that are highly conserved during evolution are good candidates for RNA motifs with posttranscriptional regulatory functions in gene expression.


Asunto(s)
Regiones no Traducidas 3'/genética , Secuencia Conservada/genética , ARN/genética , Estrés Fisiológico , Animales , Evolución Biológica , Línea Celular , Citometría de Flujo , Regulación de la Expresión Génica/genética , Genes Reguladores/genética , Genes Reporteros/genética , Hipoxia/genética , Ratones , Mitógenos/farmacología , Biosíntesis de Proteínas , ARN Mensajero/genética , Retroviridae/genética
20.
Nat Biotechnol ; 14(8): 1012-6, 1996 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-9631042

RESUMEN

To monitor the presence of introduced genes and the distribution of the encoded proteins in host tissues after gene transfer, we combined fluorescence in situ hybridization (FISH) and immunohistochemistry in two separate gene therapy paradigms. In brain tissue sections from animals injected with pHSVlac vector, we localized nuclei containing vector DNA both in cells expressing and not expressing beta-galactosidase (beta-gal). This suggests that the efficiency of gene transfer is affected not only by gene delivery, but also by cellular controls on gene expression. In a second paradigm, following myoblast transplantation, we detected donor nuclei in the muscle of a patient with Duchenne's muscular dystrophy. The donor nuclei were either surrounded by host nuclei or apparently fused in the patient's muscle fiber producing dystrophin. The combined FISH and immunohistochemistry assay offers greater sensitivity and more information than currently used polymerase chain reaction and protein detection methods.


Asunto(s)
Encéfalo/metabolismo , Protocolos Clínicos , Terapia Genética , Hibridación Fluorescente in Situ/métodos , Músculo Esquelético/metabolismo , Distrofias Musculares/terapia , Adulto , Animales , Distrofina/genética , Femenino , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/aislamiento & purificación , Humanos , Masculino , Distrofias Musculares/genética , Ratas , Ratas Sprague-Dawley
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