RESUMEN
Gene therapy using viral vectors that stably integrate into ex vivo cultured cells holds great promises for the treatment of monogenic diseases as well as cancer. However, carry-over of infectious vector particles has been described to occur upon ex vivo transduction of target cells. This, in turn, may lead to inadvertent spreading of viral particles to off-target cells in vivo, raising concerns for potential adverse effects, such as toxicity of ectopic transgene expression, immunogenicity from in vivo transduced antigen-presenting cells and, possibly, gene transfer to germline cells. Here, we have investigated factors influencing the extent of lentiviral vector (LV) shedding upon ex vivo transduction of human hematopoietic stem and progenitor cells. Our results indicate that, although vector carry-over is detectable when using laboratory-grade vector stocks, the use of clinical-grade vector stocks strongly decreases the extent of inadvertent transduction of secondary targets, likely because of the higher degree of purification. These data provide supportive evidence for the safe use of the LV platform in clinical settings.
Asunto(s)
Terapia Genética , Vectores Genéticos , Lentivirus/genética , Esparcimiento de Virus , Antígenos CD34/metabolismo , Humanos , Lentivirus/fisiología , Células Madre/metabolismoRESUMEN
Gene-transfer vectors based on lentiviruses are distinguished by their ability to transduce non-dividing cells. The HIV-1 proteins Matrix, Vpr and Integrase have been implicated in the nuclear import of the viral genome in non-dividing cells. Here we show that a sequence within pol is also required in cis. It contains structural elements previously associated with the progress of reverse transcription in target cells. We restored these elements in cis within late-generation lentiviral vectors. The new vector transduced to a much higher efficiency several types of human primary cells, when both growing and growth-arrested, including haematopoietic stem cells assayed by long-term repopulation of NOD/SCID mice. On in vivo administration into SCID mice, the vector induced higher plasma levels of human clotting factor IX (F.IX) than non-modified vector. Our results indicate that nuclear translocation of the genome is a rate-limiting step in lentiviral infection of both dividing and non-dividing cells, and that it depends on protein and nucleic acid sequence determinants. Full rescue of this step in lentivirus-based vectors improves performance for gene-therapy applications.
Asunto(s)
Núcleo Celular/genética , Productos del Gen pol/genética , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , VIH-1/genética , Transducción Genética/genética , Animales , Secuencia de Bases , División Celular , Núcleo Celular/metabolismo , Núcleo Celular/virología , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Factor IX/análisis , Factor IX/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Productos del Gen pol/fisiología , Genes Virales/genética , Genes Virales/fisiología , VIH-1/fisiología , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Linfocitos/citología , Linfocitos/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Ratones SCID , Datos de Secuencia Molecular , ARN Viral/genética , ARN Viral/metabolismo , Integración ViralRESUMEN
Human-immunodeficiency-virus (HIV)-based lentiviral vectors are a promising tool for in vivo gene therapy. Unlike Moloney-murine-leukaemia-based retroviruses (MLV), lentiviruses are believed to stably transduce quiescent (non-cycling) cells in various organs. No previous studies, however, have directly established the cell-cycle status of any transduced cell type at the time of vector administration in vivo. In vitro studies using wild-type HIV or HIV-based vectors have shown that, in some cases, cell-cycle activation is required for infection, even though cellular mitosis is not an absolute requirement for integration. Even if the block in reverse transcription is overcome in quiescent T cells, productive infection by HIV cannot be rescued in the absence of cell-cycle activation. The potential use of these vectors for gene therapy prompted our study, which establishes a cell-cycle requirement for efficient transduction of hepatocytes in vivo.
Asunto(s)
Ciclo Celular/genética , Lentivirus/genética , Hígado/metabolismo , Transducción Genética , Animales , Línea Celular , Femenino , Vectores Genéticos , Humanos , Hígado/citología , Hígado/enzimología , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Ratas , Ratas Endogámicas F344RESUMEN
RNA interference (RNAi) has been a landmark discovery in science. A typical application is to knock down the expression of endogenous genes by delivering small interfering RNA (siRNA) into cells triggering the degradation of complementary mRNA. However, RNAi can also be exploited the other way round: making use of the huge diversity of endogenous microRNAs (miRNA), the expression of exogenously introduced genes tagged with artificial miRNA target sequences can be negatively regulated according to the activity of a given miRNA which can be tissue-, lineage-, activation- or differentiation stage specific. This has significantly expanded the regulatory potential of gene transfer vectors and will benefit both basic science and therapeutic applications. This review briefly introduces the reader to the technical basis for exploiting miRNA regulation, followed by a discussion of specific applications for miRNA-regulated vectors/viruses in basic research, gene- and virotherapy.
Asunto(s)
Ingeniería Genética , MicroARNs/genética , ARN Interferente Pequeño/genética , Animales , Regulación de la Expresión Génica , Silenciador del Gen , Vectores Genéticos , Humanos , Ratones , MicroARNs/química , MicroARNs/metabolismo , Viroterapia Oncolítica , ARN Interferente Pequeño/química , ARN Interferente Pequeño/metabolismo , Transfección , Transgenes , Tropismo Viral , Virus/genéticaRESUMEN
Considered by some to be among the simpler forms of life, viruses represent highly evolved natural vectors for the transfer of foreign genetic information into cells. This attribute has led to extensive attempts to engineer recombinant viral vectors for the delivery of therapeutic genes into diseased tissues. While substantial progress has been made, and some clinical successes are over the horizon, further vector refinement and/or development is required before gene therapy will become standard care for any individual disorder.
Asunto(s)
Terapia Genética , Vectores Genéticos , Virus/genéticaRESUMEN
Metachromatic leukodystrophy (MLD) is a lipidosis caused by deficiency of arylsulfatase A (ARSA). Although the genetics of MLD are known, its pathophysiology is not understood. The disease leads to progressive demyelination and early death and no effective treatment is available. We used lentiviral vectors to deliver a functional ARSA gene (human ARSA) into the brain of adult mice with germ-line inactivation of the mouse gene encoding ARSA, As2. We report sustained expression of active enzyme throughout a large portion of the brain, with long-term protection from development of neuropathology and hippocampal-related learning impairments. We show that selective degeneration of hippocampal neurons is a central step in disease pathogenesis, and provide evidence that in vivo transfer of ARSA by lentiviral vectors reverts the disease phenotype in all investigated areas. Therefore, in vivo gene therapy offers a unique option for MLD and other storage diseases affecting the central nervous system.
Asunto(s)
Terapia Genética , Vectores Genéticos , Discapacidades para el Aprendizaje/prevención & control , Lentivirus/genética , Leucodistrofia Metacromática/terapia , Animales , Encéfalo/enzimología , Encéfalo/metabolismo , Encéfalo/patología , Cerebrósido Sulfatasa/genética , Cerebrósido Sulfatasa/metabolismo , Humanos , Discapacidades para el Aprendizaje/etiología , Leucodistrofia Metacromática/complicaciones , Leucodistrofia Metacromática/patología , Metabolismo de los Lípidos , RatonesRESUMEN
The cell-substratum interaction was studied in cultures of osteoclasts isolated from the medullary bone of laying hens kept on low calcium diet. In fully spread osteoclasts, cell-substratum adhesion mostly occurred within a continuous paramarginal area that corresponded also to the location of a thick network of intermediate filaments of the vimentin type. In this area, regular rows of short protrusions contacting the substratum and often forming a cup-shaped adhesion area were observed in the electron microscope. These short protrusions showed a core of F-actin-containing material presumably organized as a network of microfilaments and surrounded by a rosette-like structure in which vinculin and alpha-actinin were found by immunofluorescence microscopy. Rosettes were superposable to dark circles in interference-reflection microscopy and thus represented circular forms of close cell-substratum contact. The core of ventral protrusions also contained, beside F-actin, fimbrin and alpha-actinin. Villin was absent. This form of cell-substratum contact occurring at the tip of a short ventral protrusion differed from other forms of cell-substratum contact and represented an osteoclast-specific adhesion device that might also be present in in vivo osteoclasts as well as in other normal and transformed cell types.
Asunto(s)
Adhesión Celular , Glicoproteínas de Membrana , Proteínas de Microfilamentos , Osteoclastos/ultraestructura , Actinina/metabolismo , Actinas/metabolismo , Animales , Células Cultivadas , Pollos , Femenino , Técnica del Anticuerpo Fluorescente , Histocitoquímica , Proteínas de la Membrana/metabolismo , Microscopía Electrónica , Proteínas Musculares/metabolismo , Osteoclastos/metabolismo , VinculinaRESUMEN
Hepatocyte Growth Factor (HGF, also known as Scatter Factor) is a powerful mitogen or motility factor in different cells, acting through the tyrosine kinase receptor encoded by the MET protooncogene. Endothelial cells express the MET gene and expose at the cell surface the mature protein (p190MET) made of a 50 kD (alpha) subunit disulfide linked to a 145-kD (beta) subunit. HGF binding to endothelial cells identifies two sites with different affinities. The higher affinity binding site (Kd = 0.35 nM) corresponds to the p190MET receptor. Sub-nanomolar concentrations of HGF, but not of a recombinant inactive precursor, stimulate the receptor kinase activity, cell proliferation and motility. HGF induces repairs of a wound in endothelial cell monolayer. HGF stimulates the scatter of endothelial cells grown on three-dimensional collagen gels, inducing an elongated phenotype. In the rabbit cornea, highly purified HGF promotes neovascularization at sub-nanomolar concentrations. HGF lacks activities related to hemostasis-thrombosis, inflammation and endothelial cells accessory functions. These data show that HGF is an in vivo potent angiogenic factor and in vitro induces endothelial cells to proliferate and migrate.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Endotelio Vascular/fisiología , Factor de Crecimiento de Hepatocito/metabolismo , Factor de Crecimiento de Hepatocito/farmacología , Neovascularización Patológica , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Cicatrización de Heridas/efectos de los fármacos , Animales , Línea Celular , Células Cultivadas , Córnea/citología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Factor de Crecimiento de Hepatocito/genética , Humanos , Cinética , Sustancias Macromoleculares , Ratones , Mutagénesis Sitio-Dirigida , Proteínas Proto-Oncogénicas c-met , Proto-Oncogenes , Conejos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Venas UmbilicalesRESUMEN
A retroviral vector system based on the human immunodeficiency virus (HIV) was developed that, in contrast to a murine leukemia virus-based counterpart, transduced heterologous sequences into HeLa cells and rat fibroblasts blocked in the cell cycle, as well as into human primary macrophages. Additionally, the HIV vector could mediate stable in vivo gene transfer into terminally differentiated neurons. The ability of HIV-based viral vectors to deliver genes in vivo into nondividing cells could increase the applicability of retroviral vectors in human gene therapy.
Asunto(s)
Técnicas de Transferencia de Gen , Vectores Genéticos , VIH/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/citología , Encéfalo/virología , División Celular , Células Cultivadas , Femenino , Terapia Genética , VIH/fisiología , Células HeLa , Humanos , Macrófagos/citología , Macrófagos/virología , Datos de Secuencia Molecular , Neuronas/citología , Neuronas/virología , Plásmidos , Ratas , Transfección , Integración ViralRESUMEN
Successful gene therapy approaches for metachromatic leukodystrophy (MLD), based either on hematopoietic stem/progenitor cells (HSPCs) or direct central nervous system (CNS) gene transfer, highlighted a requirement for high levels of arylsulfatase A (ARSA) expression to achieve correction of disease manifestations in the mouse model. Full assessment of the safety of ARSA expression above physiological levels thus represents a prerequisite for clinical translation of these approaches. Here, using lentiviral vectors (LVs), we generated two relevant models for the stringent evaluation of the consequences of ARSA overexpression in transduced cells. We first demonstrated that ARSA overexpression in human HSPCs does not affect their clonogenic and multilineage differentiation capacities in clonogenic assays and in a neonatal hematochimeric mouse model. Further, we studied ARSA overexpression in all body tissues by generating transgenic mice overexpressing the ARSA enzyme by LV up to 15-fold above the normal range and carrying multiple copies of LV in their genome. Characterization of these mice demonstrated the safety of ARSA overexpression in two main gene therapy targets, HSPCs and neurons, with maintenance of the complex functions of the hematopoietic and nervous system in the presence of supraphysiological enzyme levels. The activity of other sulfatases dependent on the same common activator, sulfatase-modifying factor-1 (SUMF1), was tested in ARSA-overexpressing HSPCs and in transgenic mice, excluding the occurrence of saturation phenomena. Overall, these data indicate that from the perspective of clinical translation, therapeutic levels of ARSA overexpression can be safely achieved. Further, they demonstrate an experimental platform for the preclinical assessment of the safety of new gene therapy approaches.
Asunto(s)
Cerebrósido Sulfatasa/metabolismo , Terapia Genética , Leucodistrofia Metacromática/genética , Leucodistrofia Metacromática/terapia , Animales , Animales Recién Nacidos , Antígenos CD34/inmunología , Antígenos CD34/metabolismo , Southern Blotting , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Cerebrósido Sulfatasa/efectos adversos , Cerebrósido Sulfatasa/análisis , Ensayo de Unidades Formadoras de Colonias , Estudios de Factibilidad , Vectores Genéticos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Lentivirus/genética , Leucodistrofia Metacromática/metabolismo , Leucodistrofia Metacromática/patología , Ratones , Ratones Transgénicos , Modelos Animales , Neuronas/citología , Neuronas/metabolismo , Reacción en Cadena de la Polimerasa , Bazo/citología , Transducción GenéticaRESUMEN
The neuropeptide bombesin is known for its potent mitogenic activity on murine 3T3 fibroblasts and other cells. Recently it has been implicated in the pathogenesis of small cell lung carcinoma, in which it acts through an autocrine loop of growth stimulation. Phosphotyrosine (P-Tyr) antibodies have been successfully used to recognize the autophosphorylated receptors for known growth factors. In Swiss 3T3 fibroblasts, phosphotyrosine antibodies identified a 115,000-Mr cell surface protein (p115) that became phosphorylated on tyrosine as a specific response to bombesin stimulation of quiescent cells. The extent of phosphorylation was dose dependent and correlated with the mitogenic effect induced by bombesin, measured by [3H]thymidine incorporation. Tyrosine phosphorylation of p115 was detectable minutes after the addition of bombesin, and its time course paralleled that described for the binding of bombesin to its receptor. Immunocomplexes of phosphorylated p115 and phosphotyrosine antibodies bound 125I-labeled [Tyr4]bombesin in a specific and saturable manner and displayed an associated tyrosine kinase activity enhanced by bombesin. Furthermore, the 125I-labeled bombesin analog gastrin-releasing peptide, bound to intact live cells, was coprecipitated with p115. These data strongly suggest that p115 participates in the structure and function of the surface receptor for bombesin, a new member of the family of growth factor receptors with associated tyrosine kinase activity.
Asunto(s)
Bombesina/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores de Neurotransmisores/metabolismo , Aminoácidos/análisis , Animales , Complejo Antígeno-Anticuerpo , Células Cultivadas , Sueros Inmunes , Cinética , Ratones , Fosforilación , Fosfotirosina , Receptores de Bombesina , Tirosina/análogos & derivados , Tirosina/análisisRESUMEN
Antibodies against phosphotyrosine are a powerful tool with which to identify proteins phosphorylated on tyrosine residues, such as viral oncogene-encoded transforming proteins and their cellular protein substrates. Probed on human leukemia cell lines, phosphotyrosine antibodies recognized a 210,000-molecular-weight protein (p210) in K562 cells, a cell line derived from a Philadelphia (Ph)'-positive chronic myelogenous leukemia (CML), but recognized no protein in control Ph'-negative non-CML leukemia cells. The p210 protein was also recognized by antisera against v-abl-encoded polypeptides and displayed kinase activity, phosphorylating itself on tyrosine, in an immunocomplex kinase assay. These data are consistent with reported findings of the expression of a recombined bcr-abl gene in Ph'-positive CML cells, leading to the synthesis of an altered p210c-abl protein endowed with tyrosine kinase activity. Phosphotyrosine antibodies also detected the expression of the p210c-abl protein in fresh bone marrow cells harvested from CML patients in blast crisis. Besides the p210c-abl protein kinase, phosphotyrosine antibodies recognized other proteins with molecular weights of 110,000, 68,000, and 36,000 (p110, p68, and p36) in K562 cells. When [gamma-32P]ATP was added to nonionic detergent-extracted cells, these proteins became phosphorylated on tyrosine, as confirmed by phosphoamino acid analysis. A comparison with fibroblasts transformed by the v-abl, v-src, and v-fps oncogenes suggested the identity of the p36 protein with the common 36-kilodalton protein substrate of viral oncogene-encoded tyrosine kinases. Enhanced tyrosine phosphorylation of cellular proteins is thus a feature shared by cells transformed by v-abl and cells expressing a rearranged bcr-abl gene.
Asunto(s)
Leucemia Mieloide/enzimología , Proteínas de Neoplasias/análisis , Proteínas Tirosina Quinasas/análisis , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes/análisis , Tirosina/análogos & derivados , Aminoácidos/análisis , Anticuerpos , Complejo Antígeno-Anticuerpo , Médula Ósea/química , Médula Ósea/enzimología , Línea Celular , Proteínas de Fusión bcr-abl , Humanos , Radioisótopos de Fósforo , Fosfotirosina , Valores de Referencia , Tirosina/análisisRESUMEN
Protein tyrosine kinases are crucially involved in the control of cell proliferation. Therefore, the regulation of their activity in both normal and neoplastic cells has been under intense scrutiny. The product of the MET oncogene is a transmembrane receptorlike tyrosine kinase with a unique disulfide-linked heterodimeric structure. Here we show that the tyrosine kinase activity of the MET-encoded protein is powerfully activated by tyrosine autophosphorylation. The enhancement of activity was quantitated with a phosphorylation assay of exogenous substrates. It involved an increase in the Vmax of the enzyme-catalyzed phosphotransfer reaction. No change was observed in the Km (substrate). A causal relationship between tyrosine autophosphorylation and activation of the kinase activity was proved by (i) the kinetic agreement between autophosphorylation and kinase activation, (ii) the overlapping dose-response relationship for ATP, (iii) the specificity for ATP of the activation process, (iv) the phosphorylation of tyrosine residues only, in the Met protein, in the activation step, (v) the linear dependence of the activation from the input of enzyme assayed, and (vi) the reversal of the active state by phosphatase treatment. Autophosphorylation occurred predominantly on a single tryptic peptide, most likely via an intermolecular reaction. The structural features responsible for this positive modulation of kinase activity were all contained in the 45-kDa intracellular moiety of the Met protein.
Asunto(s)
Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes , Adenosina Trifosfato/metabolismo , Cromatografía Líquida de Alta Presión , Activación Enzimática , Cinética , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-met , Especificidad por SustratoRESUMEN
Retroviral vectors derived from lentiviruses such as HIV-1 are promising tools for human gene therapy because they mediate the in vivo delivery and long-term expression of transgenes in nondividing tissues. We describe an HIV vector system in which the virulence genes env, vif, vpr, vpu, and nef have been deleted. This multiply attenuated vector conserved the ability to transduce growth-arrested cells and monocyte-derived macrophages in culture, and could efficiently deliver genes in vivo into adult neurons. These data demonstrate the potential of lentiviral vectors in human gene therapy.
Asunto(s)
Regulación Viral de la Expresión Génica/genética , Terapia Genética , Vectores Genéticos/genética , VIH-1/genética , Animales , Células Cultivadas , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , ADN Viral/genética , Eliminación de Gen , Regulación Viral de la Expresión Génica/efectos de los fármacos , Genes Reporteros/genética , Vectores Genéticos/farmacología , Humanos , Macrófagos , Ratas , Transducción Genética , Transfección , Proteínas del Envoltorio Viral/genéticaRESUMEN
FOXP3 is the transcription factor ruling regulatory T cell function and maintenance of peripheral immune tolerance, and mutations in its coding gene causes IPEX autoimmune syndrome. FOXP3 is also a cell-cycle inhibitor and onco-suppressor in different cell types. In this work, we investigate the effect of ectopic FOXP3 expression on HSC differentiation and we challenged this approach as a possible HSC-based gene therapy for IPEX. FOXP3-expressing HSC showed reduced proliferation ability and increased maintenance of primitive markers in vitro in both liquid and OP9-ΔL1 co-cultures. When transplanted into immunodeficient mice, FOXP3-expressing HSC showed significantly enhanced engraftment ability. This was due to a pronounced increase in the frequency of repopulating cells, as assessed by extreme limiting dilution assay. Likely underlying the increased repopulating ability, FOXP3 expressing HSC showed significantly enhanced expression of genes controlling stemness features. However, peripheral T cells developed in the FOXP3-humanized mice were quantitatively reduced and hyporesponsive to cytokine and polyclonal stimulation. Our findings reveal unpredicted effects of FOXP3 in the biology of HSC and may provide new tools to manipulate primitive features in HSC for clinical applications. Moreover, they formally prove the need of preserving endogenous FOXP3 regulation for an HSC-based gene therapy approach for IPEX syndrome.
Asunto(s)
Diferenciación Celular , Factores de Transcripción Forkhead/metabolismo , Células Madre Hematopoyéticas/metabolismo , Linfocitos T/citología , Linfocitos T/metabolismo , Animales , Células Cultivadas , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Humanos , RatonesRESUMEN
Mucopolysaccharidosis type I (MPS I) due to deficient alpha-L-iduronidase (IDUA) activity results in the accumulation of glycosaminoglycans (GAGs) in many of the cells of affected patients. Stable gene replacement by in vivo administration of lentiviral vectors (LVs) has therapeutic potential for metabolic disorders and other systemic diseases. We have previously shown in a murine model the therapeutic potential of lentiviral IDUA vector-mediated gene therapy, in which human IDUA cDNA was driven by the cytomegalovirus promoter. However, the major limitation of this approach was the induction of an immune response against the therapeutic protein, which limited the efficacy and long-term duration of treatment. In this study, we evaluate the potential of liver-directed gene therapy, that is, programming of murine hepatocytes to secrete the enzyme with mannose 6-phosphate (M6P), which can be taken up by distant cells. Eight- to 10-week-old mice were injected via the tail vein with a lentiviral vector expressing human IDUA cDNA driven by the albumin gene promoter selectively expressed in hepatocytes. One month after treatment, IDUA activity was present in the liver and spleen of treated mice; an expression level of 1% normal IDUA activity was sufficient to reduce the GAG level in liver, spleen, kidney, heart, and lung. Interestingly, 6 months after a single injection of this vector, IDUA activity was detectable in several murine tissues; the level of enzyme activity was low but sufficient to maintain the decrease in GAG levels in liver, spleen, kidney, heart, and lung. Also, the level of enzyme-specific antibodies reached at 6 months postinjection was nearly null, and real-time polymerase chain reaction analysis showed high levels of vector DNA content in liver and spleen. Thus, these results show that the use of LV with the albumin gene promoter selectively expressed in hepatocytes limited the immune response to the transgene and allowed stable and prolonged expression of the IDUA enzyme and a partial correction of the pathology.
Asunto(s)
Terapia Genética , Vectores Genéticos/uso terapéutico , Iduronidasa/genética , Hígado/enzimología , Mucopolisacaridosis I/terapia , Transgenes/genética , Animales , Formación de Anticuerpos , Autoanticuerpos/sangre , Humanos , Iduronidasa/biosíntesis , Iduronidasa/inmunología , Lentivirus/genética , Ratones , Mucopolisacaridosis I/enzimología , Mucopolisacaridosis I/genética , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapéutico , Bazo/enzimología , Factores de TiempoRESUMEN
The tyrosine kinase encoded by the MET proto-oncogene (p190MET) is the receptor for Hepatocyte Growth Factor/Scatter Factor (HGF/SF). Previous work has shown that autophosphorylation of p190MET enhances its enzymatic activity and that the major phosphorylation site is Tyr1235, located in the catalytic domain. This residue is part of a 'three tyrosine' motif, including Tyr1230, Tyr1234, and Tyr1235, conserved in several other receptor kinases. We studied the role of these tyrosines in the positive regulation of the p190MET kinase by site-directed mutagenesis. Substitution of either Tyr1235 or Tyr1234 with phenylalanine severely reduced the in vitro kinase activity toward exogenous substrates. Kinetic experiments showed that the residual activity of these mutants could still be enhanced by autophosphorylation. Phosphopeptide mapping indicated that, in the absence of Tyr1235, Tyr1234 is phosphorylated. Only the replacement of both Tyr1234 and Tyr1235 yielded a mutant which completely lost the ability to be activated by autophosphorylation. In stable transfectants expressing the HGF/SF receptor with single substitution of either Tyr1234 or Tyr1235 the response to HGF/SF was impaired. The ligand did not induce tyrosine phosphorylation of the receptor nor stimulated chemotaxis. These data show that Tyr1234 and Tyr1235 are critical for the activation of the HGF/SF receptor kinase both in vitro and in response to the ligand in intact cells.
Asunto(s)
Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Activación Enzimática , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Proteínas Proto-Oncogénicas c-met , Relación Estructura-Actividad , Transfección , Tirosina/metabolismoRESUMEN
The human proto-oncogene c-MET encodes a heterodimeric 190 kDa transmembrane protein (p190c-met) with structural features of a tyrosine kinase receptor. The ligand for this putative receptor has not yet been identified. By Northern blot hybridization we found that, among a restricted number of human tissues, c-MET is highly expressed in the liver. This prompted us to test the hypothesis of a functional interaction between the c-MET receptor and Hepatocyte Growth Factor (HGF), a heparin-binding polypeptide consisting of heavy and light chains of 65 and 35 kDa. Nanomolar concentrations of highly purified HGF added to GTL-16 cells, which overexpress the c-MET receptor, enhanced the phosphorylation on tyrosine of the p190c-met kinase. Addition of other known growth factors or serum was ineffective. The kinase activity of the c-MET receptor was also stimulated by HGF in an in vitro assay, after detergent solubilization and partial purification of p190c-met. Moreover, elution of immunoprecipitates obtained with anti-MET antibodies from GTL-16 cell lysates yielded an HGF-responsive kinase activity. These results suggest that HGF, or a growth factor structurally related to HGF, is a candidate ligand for the receptor encoded by c-MET.
Asunto(s)
Receptores ErbB/metabolismo , Sustancias de Crecimiento/fisiología , Proteínas Tirosina Quinasas/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , Glándulas Suprarrenales/metabolismo , Femenino , Mucosa Gástrica/metabolismo , Regulación de la Expresión Génica , Factor de Crecimiento de Hepatocito , Humanos , Técnicas In Vitro , Riñón/metabolismo , Hígado/metabolismo , Pulmón/metabolismo , Ovario/metabolismo , Fosforilación/efectos de los fármacos , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-met , ARN Mensajero/biosíntesis , Bazo/metabolismo , Glándula Tiroides/metabolismo , Útero/metabolismoRESUMEN
Antibodies against the synthetic hapten azobenzyl phosphonate which specifically crossreact with phosphotyrosine have been produced and used to detect the proteins phosphorylated in tyrosine following exposure of intact quiescent Swiss 3T3 fibroblasts to the platelet-derived growth factor (PDGF). Western blotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-fractionated proteins followed by decoration with phosphotyrosine antibodies and 125I-labeled protein A have been used. The major tyrosine-phosphorylated component was a 170 kDa protein. The following lines of evidence suggest that this protein is the PDGF receptor in its tyrosine-phosphorylated form: (a) both proteins have the same (170 kDa) molecular weight; (b) the phosphorylated 170 kDa protein was detectable only in cell lines bearing the PDGF receptor; (c) the phosphorylation of the 170 kDa protein required PDGF and was dose-dependent. Kinetic studies showed that the phosphorylation of the receptor was maximal after 5-10 min at 37 degrees C and was followed by a rapid decrement of the band. The loss of the 170 kDa component was not prevented by inhibitors of membrane internalization and of lysosomal proteinases, while it was inhibited by lowering the temperature to 5 degrees C. In PDGF-stimulated cells, phosphotyrosine antibodies detected also a minor 36 kDa component phosphorylated at tyrosine.
Asunto(s)
Anticuerpos , Receptores de Superficie Celular/metabolismo , Tirosina/análogos & derivados , Adenosina Trifosfato/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Animales , Anticuerpos/inmunología , Cadaverina/análogos & derivados , Cadaverina/farmacología , Cloroquina/farmacología , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Fibroblastos/metabolismo , Técnicas Inmunológicas , Metilaminas/farmacología , Ratones , Peso Molecular , Fenilglioxal/farmacología , Fosforilación , Fosfotirosina , Factor de Crecimiento Derivado de Plaquetas/farmacología , Receptores del Factor de Crecimiento Derivado de Plaquetas , Tirosina/inmunología , Tirosina/metabolismoRESUMEN
Lentiviral vectors are proving to be effective agents for the direct delivery and sustained expression of a transgene in several tissues, including brain, retina, muscle and liver. Significant progress was achieved in the biosafety of HIV-derived vectors by eliminating all the viral sequences non-essential for transduction. Other vectors have also been developed from non-primate lentiviruses.