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1.
Cancer Invest ; 30(5): 422-32, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22571343

RESUMO

The gene encoding PTPδ is mutated or downregulated in human cancers including neuroblastoma. Here, we functionally tested the tumor-suppressive potential of PTPδ in neuroblastoma cell lines by reconstitution of both short and long PTPδ isoforms. We did not observe any significant difference in colony forming ability between cells expressing wild-type or catalytically inactive PTPδ. Although endogenous PTPδ expression was very low in neuroblastoma cells, it was also low in mouse embryo adrenal glands, suggesting that PTPδ may have little developmental function in early adrenal neuroblasts. This study, therefore, questions the significance of PTPδ as a tumor suppressor protein in neuroblastoma.


Assuntos
Neuroblastoma/patologia , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Glândulas Suprarrenais/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Camundongos
2.
J Neurobiol ; 49(2): 99-117, 2001 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-11598918

RESUMO

Receptor protein tyrosine phosphatases (RPTPs) are regulators of axon outgrowth and guidance in a variety of different vertebrate and invertebrate systems. Three RPTPs, CRYP-alpha, PTP-delta, and LAR, are expressed in overlapping but distinct patterns in the developing Xenopus retina, including expression in retinal ganglion cells (RGCs) as they send axons to the tectum (Johnson KG, Holt CE. 2000. Expression of CRYP-alpha, LAR, PTP-delta, and PTP-rho in the developing Xenopus visual system. Mech Dev 92:291-294). In order to examine the role of these RPTPs in visual system development, putative dominant negative RPTP mutants (CS-CRYP-alpha, CS-PTP-delta, and CS-LAR) were expressed either singly or in combination in retinal cells. No effect was found on either retinal cell fate determination or on gross RGC axon guidance to the tectum. However, expression of these CS-RPTP constructs differentially affected the rate of RGC axon outgrowth. In vivo, expression of all three CS-RPTPs or CS-PTP-delta alone inhibited RGC axon outgrowth, while CS-LAR and CS-CRYP-alpha had no significant effect. In vitro, expression of CS-CRYP-alpha enhanced neurite outgrowth, while CS-PTP-delta inhibited neurite outgrowth in a substrate-dependent manner. This study provides the first in vivo evidence that RPTPs regulate retinal axon outgrowth.


Assuntos
Proteínas Aviárias , Axônios/fisiologia , Moléculas de Adesão Celular/fisiologia , Proteínas do Olho/fisiologia , Proteínas do Tecido Nervoso , Nervo Óptico/embriologia , Proteínas Tirosina Fosfatases/fisiologia , Proteínas Tirosina Quinases/fisiologia , Receptores de Superfície Celular/fisiologia , Células Ganglionares da Retina/citologia , Colículos Superiores/embriologia , Vias Visuais/embriologia , Proteínas de Xenopus , Xenopus laevis/embriologia , Animais , Blastômeros , Moléculas de Adesão Celular/genética , Embrião de Galinha , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Proteínas do Olho/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes Dominantes , Microinjeções , Modelos Biológicos , Família Multigênica , Mutagênese Sítio-Dirigida , Neuritos/fisiologia , Nervo Óptico/enzimologia , Técnicas de Cultura de Órgãos , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Fosfatases Semelhantes a Receptores , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores , Receptores de Superfície Celular/genética , Proteínas Recombinantes de Fusão/fisiologia , Retina/transplante , Células Ganglionares da Retina/enzimologia , Colículos Superiores/enzimologia , Vias Visuais/citologia , Vias Visuais/enzimologia , Xenopus laevis/metabolismo
3.
Curr Opin Neurobiol ; 11(1): 95-102, 2001 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11179878

RESUMO

Receptor-like protein tyrosine phosphatases (RPTPs) continue to emerge as important signalling molecules in axons and their growth cones. Recent findings show that Drosophila RPTPs play key roles in guiding retinal axons and in preventing midline crossing of longitudinal axons. Vertebrate RPTPs are now implicated in controlling axon outgrowth, and preliminary evidence suggests that they too may influence axon guidance.


Assuntos
Axônios/fisiologia , Sistema Nervoso/crescimento & desenvolvimento , Proteínas Tirosina Fosfatases/metabolismo , Animais , Movimento Celular , Drosophila , Sanguessugas , Sistema Nervoso/citologia , Transdução de Sinais
4.
Mol Cell Neurosci ; 16(4): 470-80, 2000 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11085882

RESUMO

Receptor-like protein tyrosine phosphatases potentially play a crucial role in axon growth and targeting. We focus here on their role within the embryonic avian spinal cord, in particular the development and outgrowth of motorneurons. We have used in situ mRNA hybridization to examine the spatiotemporal expression of eight receptor-like protein tyrosine phosphatases and find that it is both dynamic and highly varied, including novel, isoform-specific expression patterns. CRYP alpha 1 is expressed in all of the ventral motorneuron pools, whereas CRYP2, RPTP gamma, and RPTP alpha are only expressed in specific subsets of these neurons. CRYP alpha 2, RPTP psi, and RPTP delta are neuronally expressed elsewhere in the cord, but not in ventral motorneurons, whereas RPTP mu is unique in being restricted to capillaries. The developmentally regulated expression of these genes strongly suggests that the encoded phosphatases play numerous roles during neurogenesis and axonogenesis in the vertebrate spinal cord.


Assuntos
Proteínas Aviárias , Proteínas Tirosina Fosfatases/genética , Receptores de Superfície Celular , Medula Espinal/embriologia , Medula Espinal/enzimologia , Animais , Embrião de Galinha , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Hibridização In Situ , Neurônios Motores/enzimologia , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/análise , Proteínas Tirosina Fosfatases Semelhantes a Receptores , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores , Medula Espinal/citologia
5.
J Cell Biol ; 147(2): 375-88, 1999 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-10525542

RESUMO

Retinal ganglion cell axons grow towards the optic fissure in close contact with the basal membrane, an excellent growth substratum. One of the ligands of receptor tyrosine phosphatase CRYPalpha is located on the retinal and tectal basal membranes. To analyze the role of this RPTP and its ligand in intraretinal growth and guidance of ganglion cell axons, we disrupted ligand- receptor interactions on the retinal basal membrane in culture. Antibodies against CRYPalpha strongly reduced retinal axon growth on the basal membrane, and induced a dramatic change in morphology of retinal growth cones, reducing the size of growth cone lamellipodia. A similar effect was observed by blocking the ligand with a CRYPalpha ectodomain fusion protein. These effects did not occur, or were much reduced, when axons were grown either on laminin-1, on matrigel or on basal membranes with glial endfeet removed. This indicates that a ligand for CRYPalpha is located on glial endfeet. These results show for the first time in vertebrates that the interaction of a receptor tyrosine phosphatase with its ligand is crucial not only for promotion of retinal axon growth but also for maintenance of retinal growth cone lamellipodia on basal membranes.


Assuntos
Proteínas Aviárias , Axônios/ultraestrutura , Proteínas Tirosina Fosfatases/fisiologia , Células Ganglionares da Retina/fisiologia , Células Ganglionares da Retina/ultraestrutura , Animais , Axônios/fisiologia , Moléculas de Adesão Celular/fisiologia , Comunicação Celular , Células Cultivadas , Laminina/fisiologia , Ligantes , Proteínas Tirosina Fosfatases Semelhantes a Receptores , Transdução de Sinais/fisiologia
6.
J Neurobiol ; 39(1): 81-96, 1999 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10213455

RESUMO

Receptor tyrosine kinases and receptor protein tyrosine phosphatases (RPTPs) appear to coordinate many aspects of neural development, including axon growth and guidance. Here, we focus on the possible roles of RPTPs in the developing avian retinotectal system. Using both in situ hybridization analysis and immunohistochemistry, we show for the first time that five RPTP genes--CRYPalpha, CRYP-2, PTPmu, PTPgamma, and PTPalpha--have different but overlapping expression patterns throughout the retina and the tectum. PTPalpha is restricted to Muller glia cells and radial glia of the tectum, indicating a possible function in controlling neuronal migration. PTPgamma expression is restricted to amacrine neurons. CRYPalpha and CRYP-2 mRNAs in contrast are expressed throughout the retinal ganglion cell layer from where axons grow out to their tectal targets. PTPmu is expressed in a subset of these ganglion cells. CRYPalpha, CRYP-2, and PTPmu proteins are also localized in growth cones of retinal ganglion cell axons and are present in defined laminae of the tectum. Thus, the spatial and temporal expression of three distinct RPTP subtypes--CRYPalpha, CRYP-2, and PTPmu--are consistent with the possibility of their involvement in axon growth and guidance of the retinotectal projection.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas Tirosina Fosfatases/genética , Retina/embriologia , Colículos Superiores/embriologia , Vias Visuais/embriologia , Animais , Axônios/fisiologia , Embrião de Galinha , Regulação Enzimológica da Expressão Gênica , Imuno-Histoquímica , Reação em Cadeia da Polimerase , Proteínas Tirosina Fosfatases/análise , Proteínas Tirosina Fosfatases/biossíntese , Retina/enzimologia , Células Ganglionares da Retina/fisiologia , Colículos Superiores/enzimologia , Vias Visuais/enzimologia
7.
Curr Biol ; 6(7): 794-7, 1996 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-8835859

RESUMO

Recent results have revealed for the first time that receptor-like protein tyrosine phosphatases help to control the navigation of motor axons in the Drosophila nervous system.


Assuntos
Axônios/metabolismo , Modelos Moleculares , Neurônios Motores/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais/fisiologia , Animais , Drosophila
8.
Brain Res Dev Brain Res ; 90(1-2): 129-40, 1995 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-8719336

RESUMO

The avian CRYP alpha gene is expressed in the embryonic nervous system and encodes a receptor-like protein tyrosine phosphatase with structural similarity to neural cell adhesion molecules. To gain further insight into the role of the CRYP alpha phosphatase in neural development, this study addresses the protein's cellular distribution in the well characterised embryonic visual system. High levels of CRYP alpha protein localise in retinal axons extending from the eye to the tectum throughout the major growth periods of these nerve processes. In addition, primitive inner plexiform layer processes in the retina, tectobulbar axons, and non-retinal fibres of the tectal stratum opticum, contain large amounts of CRYP alpha. Its presence in non-fasciculated processes suggests that CRYP alpha has a role other than in fasciculation in short range fibres. In contrast to CRYP alpha, NgCAM is confined largely to axon fascicles in the retina and tectum, consistent with its demonstrated role in fasciculation of cultured neurites. In cultured retinal neurons CRYP alpha proteins reside both in neurite processes and in growth cone membranes, implicating both of these as potential functional locations for the protein. Although CRYP alpha continues to be expressed in the later embryo, the strong, early expression suggests a significant developmental role in the initial growth or guidance of nerve processes. This applies both over the longer range in the retinotectal and tectobulbar projections and over the shorter range within plexiform layers.


Assuntos
Proteínas Aviárias , Moléculas de Adesão de Célula Nervosa/análise , Neurônios/enzimologia , Proteínas Tirosina Fosfatases/análise , Retina/química , Colículos Superiores/química , Vias Visuais/química , Animais , Axônios/química , Células Cultivadas , Embrião de Galinha , Proteínas Tirosina Fosfatases Semelhantes a Receptores , Retina/citologia , Retina/embriologia , Frações Subcelulares/química , Colículos Superiores/embriologia , Vias Visuais/embriologia
9.
Development ; 121(6): 1833-44, 1995 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-7600997

RESUMO

Migrating embryonic growth cones require multiple, membrane-associated signalling molecules to monitor and respond to guidance cues. Here we present the first evidence that vertebrate cell adhesion molecule-like protein tyrosine phosphatases are likely to be components of this signalling system. CRYP alpha, the gene for an avian cell adhesion molecule-like phosphatase, is strongly expressed in the embryonic nervous system. In this study we have immunolocalised the protein in the early chick embryo and demonstrated its predominant localisation in axons of the central and peripheral nervous systems. This location suggests that the major, early role of the enzyme is in axonal development. In a study of sensory neurites in culture, we furthermore show that this phosphatase localises in migrating growth cones, within both the lamellipodia and filopodia. The dependence of growth cone migration on both cell adhesion and signalling through phosphotyrosine turnover, places the cell adhesion molecule-like CRYP alpha phosphatase in a position to be a regulator of these processes.


Assuntos
Proteínas Aviárias , Axônios/química , Indução Embrionária , Sistema Nervoso/embriologia , Proteínas Tirosina Fosfatases/análise , Proteínas Tirosina Fosfatases/fisiologia , Transdução de Sinais , Animais , Axônios/fisiologia , Western Blotting , Adesão Celular/fisiologia , Moléculas de Adesão Celular Neuronais/fisiologia , Movimento Celular/fisiologia , Células Cultivadas , Sistema Nervoso Central/química , Sistema Nervoso Central/embriologia , Embrião de Galinha , Imuno-Histoquímica , Isoenzimas , Sistema Nervoso/química , Sistema Nervoso Periférico/química , Sistema Nervoso Periférico/embriologia , Proteínas Tirosina Fosfatases Semelhantes a Receptores
10.
Mech Dev ; 46(3): 201-17, 1994 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-7918104

RESUMO

The controlled development of embryo cells depends on their ability to monitor and respond to dynamic microenvironmental signals. This is frequently effected through membrane-associated receptor proteins which signal directly or indirectly through protein tyrosine phosphorylation. A search for such proteins in the developing nervous system of the chick has identified a new receptor-like protein tyrosine phosphatase (R-PTP) gene which may be responsible in part for this signalling. This gene, named CRYP alpha, is related to the LAR subfamily of R-PTPs and has extracellular homology to the neural cell adhesion molecules (CAMs). The gene is widely expressed in both the central and peripheral nervous systems, with particularly strong expression in motor neurons and in brain subregions such as the optic tectum and hypothalamus. Expression is seen both in early proliferating neuroepithelia and in subsets of post-mitotic nerve cells. Moreover, tissue-specific and developmentally-regulated exon use has been found in the brain, suggesting that isoforms of the R-PTP protein have stage-specific neural roles. This alternative RNA splicing event affects the encoded structure of the CAM-like domain, which may in turn influence its ligand binding properties. The novel, regulated expression of this R-PTP gene suggests that it plays a role in early neural development, and that the signalling properties of the encoded phosphatase can be modified according to the differentiated state of the cells.


Assuntos
Moléculas de Adesão Celular Neuronais/fisiologia , Embrião não Mamífero/fisiologia , Isoenzimas/fisiologia , Sistema Nervoso/enzimologia , Proteínas Tirosina Fosfatases/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Embrião de Galinha , Clonagem Molecular , Éxons , Hibridização In Situ , Isoenzimas/análise , Dados de Sequência Molecular , Sistema Nervoso/embriologia , Proteínas Tirosina Fosfatases/análise , Splicing de RNA , RNA Mensageiro/biossíntese
11.
Proc Natl Acad Sci U S A ; 88(23): 10505-9, 1991 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-1660139

RESUMO

Retroviruses are valuable tools in studies of embryonic development, both as gene expression vectors and as cell lineage markers. In this study early chicken blastoderm cells are shown to be permissive for infection by Rous sarcoma virus and derivative replication-defective vectors, and, in contrast to previously published data, these cells will readily express viral genes. In cultured blastoderm cells, Rous sarcoma virus stably integrates and is transcribed efficiently, producing infectious virus particles. Using replication-defective vectors encoding the bacterial lacZ gene, we further show that blastoderms can be infected in culture and in ovo. In ovo, lacZ expression is seen within 24 hr of virus inoculation, and by 96 hr stably expressing clones of cells are observed in diverse tissues throughout the embryo, including epidermis, somites, and heart, as well as in extraembryonic membranes. Given the rapid onset of vector expression and the broad range of permissive cell types, it should be feasible to use Rous sarcoma virus-derived retroviruses as early lineage markers and expression vectors beginning at the blastoderm stage of avian embryogenesis.


Assuntos
Vírus do Sarcoma Aviário/genética , Blastoderma/fisiologia , Vetores Genéticos , Animais , Vírus do Sarcoma Aviário/fisiologia , Blastoderma/microbiologia , Células Cultivadas , Embrião de Galinha , DNA Viral/genética , DNA Viral/isolamento & purificação , Vírus Defeituosos/genética , Vírus Defeituosos/fisiologia , Fibroblastos/fisiologia , Cinética , Microinjeções , RNA Viral/genética , RNA Viral/isolamento & purificação , Replicação Viral
12.
J Virol ; 65(12): 7020-4, 1991 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-1658394

RESUMO

Previous studies have indicated that the regression versus progression of v-src-DNA-induced sarcomas is dependent on chicken line. As a first step in analyzing the role of tumor immunity as a determinant of this line dependence, experiments were undertaken to ascertain whether an antisarcoma immune response is generated in the course of sarcoma growth in TK chickens, a regressor line. To assay for this response, test TK chickens in which v-src-induced wing web sarcomas had regressed, as well as control TK chickens that had not been exposed to v-src, were challenged in protocols known to yield v-src-dependent sarcoma formation and monitored for challenge sarcoma growth. Compared with the control chickens, the test chickens showed a significant resistance to the sarcomagenic challenge. These results raise the possibility that the antisarcoma response that is inducible in regressor lines, as demonstrated here in terms of a protective effect against a subsequent sarcomagenic challenge, may also underlie the regression of v-src-induced primary sarcomas.


Assuntos
Vírus do Sarcoma Aviário/genética , Genes src , Regressão Neoplásica Espontânea/genética , Sarcoma Experimental/microbiologia , Animais , Vírus do Sarcoma Aviário/fisiologia , Galinhas , DNA de Neoplasias/isolamento & purificação , DNA Viral/genética , DNA Viral/isolamento & purificação , Vírus Defeituosos/genética , Vírus Defeituosos/fisiologia , Regressão Neoplásica Espontânea/imunologia , Regressão Neoplásica Espontânea/patologia , Plasmídeos , Mapeamento por Restrição , Sarcoma Experimental/imunologia , Sarcoma Experimental/patologia , Especificidade da Espécie , Replicação Viral/genética
15.
J Cell Biol ; 111(1): 217-28, 1990 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-2164029

RESUMO

We demonstrate that the behavior of cells expressing v-src, a tyrosine kinase oncogene, differs profoundly between the embryonic and culture environments. V-src was introduced into avian embryo cells both in culture and in stage-24 embryo limbs, using replication-defective retroviral vectors. These vectors were used as single-hit, cellular markers to determine the environmental influences imposed by normal cells and tissues on clonal cell growth. The marker gene lacZ was coexpressed with v-src in order to locate the descendent cells. In culture, v-src induced rapid morphological transformation and anchorage-independent growth of embryo fibroblasts; the vectors were also tumorigenic in hatchling chickens. In contrast, most of the cell clones expressing v-src in the embryo grew normally without neoplasia. Expression of v-src vectors could be found in a wide range of cell types, demonstrating not only that neoplastic transformation is attenuated in ovo, but also that differentiation commitment in many lineages can be maintained concurrently with oncogene expression. Significantly, the embryonic control of cell growth could be perturbed by v-src under certain conditions. Rare, marked clones showed hyperplasia or dysplasia, and the primitive endothelium could succumb to rapid neoplasia; thus, these embryonic tissues are not inherently deficient in transformation factors. We propose that the environmental conditions imposed on cells in ovo are critical for the attenuation of neoplasia, while cultured cells lose this requisite environment.


Assuntos
Desenvolvimento Embrionário e Fetal , Proteína Oncogênica pp60(v-src)/genética , Oncogenes , Proteínas Tirosina Quinases/genética , Animais , Vírus do Sarcoma Aviário/genética , Divisão Celular , Linhagem Celular , Transformação Celular Neoplásica , Células Cultivadas , Embrião de Galinha , Células Clonais , Endotélio/citologia , Endotélio/enzimologia , Células Epiteliais , Epitélio/enzimologia , Expressão Gênica , Vetores Genéticos
16.
Proc Natl Acad Sci U S A ; 86(24): 10123-7, 1989 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-2557619

RESUMO

v-src is an effective carcinogen when expressed from Rous sarcoma virus (RSV) in vivo. Whereas RSV tumors require sustained oncogene expression, their growth is largely a balance between viral recruitment of tissues and host immune destruction of infected cells. We have therefore examined the tumorigenic potential of v-src in the absence of viral recruitment and viral antigen expression. v-src was introduced with high efficiency into chicken wing web tissues using replication-defective (rd) retroviral vectors. Clonal sarcomas were induced rapidly, and, furthermore, v-src potentiated metastatic progression in approximately 0.1%-1% of tumor clones with unexpectedly short latency. rd vectors proved effective not only in transducing v-src into tissues but also as insertional markers of tumor clonality. The rd vector present in most primary and metastatic tumors was a highly truncated form of RSV derived by viral transmission of spliced v-src mRNA; this vector should thus avoid viral recruitment and host anti-viral immune reaction through its complete lack of viral structural genes. Under such conditions v-src maintains strong carcinogenicity in vivo when restricted to clonal tumor growth and can confer rapid metastatic potential on a discrete subset of tumor clones.


Assuntos
Vírus do Sarcoma Aviário/genética , Vírus Defeituosos/genética , Expressão Gênica , Oncogenes , Sarcoma Aviário/genética , Transdução Genética , Animais , Sequência de Bases , Galinhas , DNA de Neoplasias/análise , DNA de Neoplasias/genética , Vírus Defeituosos/fisiologia , Vetores Genéticos , Metástase Neoplásica , Técnicas de Amplificação de Ácido Nucleico , Proteína Oncogênica pp60(v-src)/análise , Splicing de RNA/genética , RNA Mensageiro/genética , Mapeamento por Restrição , Sarcoma Aviário/patologia , Replicação Viral
17.
Cancer Res ; 49(22): 6419-24, 1989 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-2553256

RESUMO

Chickens given injections of Rous sarcoma virus form sarcomas at the site of inoculation (primary tumor) and at the site of experimentally introduced wounds (wound tumor). This latter finding provides a model system to study systematically the mechanisms underlying the cocarcinogenic effects of wounding. Our experiments show the following. (a) Chickens inoculated with a Rous sarcoma virus-derived, replication-defective virus construct fail to elaborate wound tumors in spite of aggressively growing primary tumors. We thus rule out metastasis as a mechanism and conclude that infectious virus is required for wound tumor formation; (b) using bromodeoxyuridine incorporation and immunofluorescence on frozen sections we demonstrate proliferation in the unwounded wing in cell types which are normally targets for Rous sarcoma virus infection and transformation and conclude that proliferation per se is not sufficient to induce wound tumors; (c) using immunohistochemistry for the viral protein p19gag we show that wounding induces virus expression in fibroblasts of newly forming granulation tissue 2 days after injury. We also demonstrate expression of viral mRNA in wound tumors by in situ hybridization with a v-src probe. We discuss the possibility of activation of integrated, silent virus or the preferential infection of a special target cell population as a result of wounding as well as the potential role of wound factors in transformation.


Assuntos
Vírus do Sarcoma Aviário/patogenicidade , Sarcoma Aviário/complicações , Ferimentos e Lesões/complicações , Animais , Vírus do Sarcoma Aviário/genética , Vírus do Sarcoma Aviário/isolamento & purificação , Bromodesoxiuridina/análise , Bromodesoxiuridina/metabolismo , Divisão Celular , Transformação Celular Neoplásica , Galinhas , Replicação do DNA , Imunofluorescência , Produtos do Gene gag/análise , Técnicas Imunoenzimáticas , RNA Mensageiro/genética , Sarcoma Aviário/patologia , Ferimentos e Lesões/patologia
18.
J Virol ; 62(3): 1008-15, 1988 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-2828649

RESUMO

We have constructed an avian leukosis virus derivative with a 5' deletion extending from within the tRNA primer binding site to a SacI site in the leader region. Our aim was to remove cis-acting replicative and/or encapsidation sequences and to use this derivative, RAV-1 psi-, to develop vector-packaging cell lines. We show that RAV-1 psi- can be stably expressed in the quail cell line QT6 and chicken embryo fibroblasts and that it is completely replication deficient in both cell types. Moreover, we have demonstrated that QT6-derived lines expressing RAV-1 psi- can efficiently package four structurally different replication-defective v-src expression vectors into infectious virus, with very low or undetectable helper virus release. These RAV-1 psi--expressing cell lines comprise the first prototype avian sarcoma and leukosis virus-based vector-packaging system. The construction of our vectors has also shown us that a sequence present within gag, thought to facilitate virus packaging, is not necessary for efficient vector expression and high virus production. We show that quantitation and characterization of replication-defective viruses can be achieved with a sensitive immunocytochemical procedure, presenting an alternative to internal selectable vector markers.


Assuntos
Vírus da Leucose Aviária/genética , Vírus Defeituosos/genética , Vetores Genéticos , Animais , Linhagem Celular , Embrião de Galinha , Coturnix , Fibroblastos , Produtos do Gene gag , Genes Virais , Vírus Auxiliares/genética , Proteína Oncogênica pp60(v-src) , Proteínas dos Retroviridae/genética , Replicação Viral
19.
J Gen Virol ; 68 ( Pt 9): 2481-5, 1987 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-2821185

RESUMO

A simple and accurate immunocytochemical focus assay is described, whereby both transforming and non-transforming avian retroviruses can be enumerated. After virus infection of chick embryo fibroblasts, an agar overlay is applied; foci of infected cells (expression foci) are detected immunocytochemically after 5 to 7 days. The primary antibodies are monoclonal sera directed against either viral p19gag or pp60v-src. Detection of expression foci after transfection of cells with cloned viral DNA is also demonstrated.


Assuntos
Transformação Celular Neoplásica , Retroviridae/crescimento & desenvolvimento , Animais , Vírus do Sarcoma Aviário/crescimento & desenvolvimento , Embrião de Galinha , Imuno-Histoquímica , Vírus da Leucemia Murina/crescimento & desenvolvimento , Retroviridae/genética , Retroviridae/imunologia , Cultura de Vírus/métodos
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