RESUMO
Four temperature-sensitive (ts) Rous sarcoma virus src gene mutants with lesions in different parts of the gene represent three classes of alteration in pp60src. These classes are composed of mutants with (i) heat-labile protein kinase activities both in vitro and in vivo (tsLA27 and tsLA29), (ii) heat-labile kinases in vivo but not in vitro (tsLA33), and (iii) neither in vivo nor in vitro heat-labile kinases (tsLA32). The latter class indicates the existence of structural or functional pp60src domains that are required for transformation but do not grossly affect tyrosine kinase activity.
Assuntos
Vírus do Sarcoma Aviário/genética , Mutação , Proteínas Quinases/genética , Proteínas Virais/genética , Aminoácidos/análise , Animais , Vírus do Sarcoma Aviário/enzimologia , Embrião de Galinha , Proteína Oncogênica pp60(v-src) , Fosforilação , Temperatura , Proteínas Virais/metabolismoRESUMO
Avian leukosis virus (ALV)-induced neoplasias are commonly found associated with integrations of proviral DNA in proximity to the myc gene. However, studies suggest that other genetic events are necessary for the complete neoplastic phenotype. A cell line (HP46) derived from an ALV-induced tumor has been analyzed and found to contain, in addition to an alteration in the myc gene, a promoter insertion in the c-rel locus. Both loci expressed large amounts of mRNA coding for their respective proteins. Several rel-related transcripts were expressed in the HP46 line, and four rel-related proteins of lower molecular weight than the wild-type p68c-rel product were detected. At least two of these transcripts contained U5 long terminal repeat sequences on the 5' end of the RNA. Structural data suggest that the messages may have evolved by an alternative splicing mechanism. This is the first example of a promoter insertion in the c-rel locus, a gene whose viral counterpart v-rel is responsible for the induction of lymphoid tumors.
Assuntos
Vírus da Leucose Aviária/genética , Elementos de DNA Transponíveis , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Animais , Vírus do Sarcoma Aviário/genética , Sequência de Bases , Southern Blotting , Linhagem Celular , Embrião de Galinha , DNA/genética , DNA/isolamento & purificação , Biblioteca Gênica , Dados de Sequência Molecular , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-rel , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência do Ácido NucleicoRESUMO
The oncogene, v-rel is a member of the rel/NF-kappaB family of transcription factors. It causes a rapidly fatal lymphoma in young chicks and is capable of transforming both fibroblasts and primitive hematopoietic cells in culture. To understand the role of v-rel in transformation we constructed an inducible form of v-rel and used it to identify potential cellular target genes for v-rel regulation. In this paper we show that High Mobility Group Protein 14 (HMG 14) is expressed in a wide variety of v-rel transformed cell types. In addition we show that v-rel participates in the transcriptional regulation of HMG 14 and that extracts from v-relER cells interact with the HMG 14 promoter. These experiments suggest a role for v-rel in the regulation of a unique gene whose protein product may influence gene transcription in a global fashion.
Assuntos
Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas Oncogênicas de Retroviridae/genética , Proteínas Oncogênicas de Retroviridae/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Diferenciação Celular/genética , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Extratos Celulares/farmacologia , Linhagem Celular Transformada , Transformação Celular Neoplásica , Embrião de Galinha , Sequência Conservada , Estradiol/análogos & derivados , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Estrogênios/farmacologia , Fibroblastos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Dados de Sequência Molecular , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas Oncogênicas v-rel , Alcamidas Poli-Insaturadas , Regiões Promotoras Genéticas , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transcrição GênicaRESUMO
Sequential deletions of approximately 100 base pairs were made in the rel-homology domain of the viral rel protein. Each deletion mutant was cloned into a replication-competent viral vector and assayed in chicken embryo fibroblasts (CEFs). The deleted v-rel proteins were analysed for localization, complex formation and ability to induce transformation. In vitro-translated mutant proteins were assayed for binding to a NF-kappa B consensus sequence. All the deletion mutants between nucleotides 37 and 798 in v-rel were transformation defective. Each of the mutants localized predominantly in the cytoplasm, whereas wild-type v-rel localizes predominantly in the nucleus of CEFs. Any disruption of the rel-homology domain reduced binding of the mutant v-rel proteins to the cellular protein, p36, while the requirements for binding to p68c-rel, p115 and p124 appeared to be more complicated. The binding of these three proteins to v-rel appeared to be linked and mediated through c-rel, suggesting that v-rel disrupts normal c-rel function. None of the deletion mutants in this region were able to bind to the NF-kappa B site. However, mutants which lie outside the rel-homology domain retained the ability to transform CEFs, localize to the nucleus, complex with p36, p115, p124 and p68c-rel and bind to the NK-kappa B site. These results suggest that transformation by v-rel requires an intact rel-homology domain and that the biochemical properties of v-rel are linked and dependent upon higher order protein structure for full function.
Assuntos
Transformação Celular Neoplásica , Mutagênese Sítio-Dirigida , Oncogenes , Proteínas Oncogênicas de Retroviridae/genética , Animais , Sequência de Bases , Sítios de Ligação , Medula Óssea , Linhagem Celular , Células Cultivadas , Embrião de Galinha , Deleção Cromossômica , Fibroblastos , Dados de Sequência Molecular , NF-kappa B/metabolismo , Sondas de Oligonucleotídeos , Proteínas Oncogênicas v-rel , Biossíntese de Proteínas , Proteínas Tirosina Quinases/genética , Proteínas Oncogênicas de Retroviridae/metabolismo , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
In this study, alterations in gene expression patterns have been examined in v-Rel-transformed avian bone marrow cells. Using a conditional v-Rel estrogen receptor chimera (v-RelER) which transforms cells in an estrogen-dependent manner, we constructed subtraction cDNA libraries from v-RelER-transformed bone marrow cells. Several different sequences were identified whose expression was altered upon hormone activation of v-RelER. These include two genes related to the MIP-1 chemokine family (mip-1beta and a tca3 homologue), a cell surface antigen sca-2 and the transcription factor nfkb1. The expression of each gene was assayed in a number of wild-type and mutant v-Rel-expressing fibroblast and hematopoietic cells. All v-Rel-transformed hematopoietic cells tested express high levels of nfkb1 and sca-2. In fibroblasts, wild-type v-Rel induced expression of mip-1beta and nfkb1, while nontransforming mutants of v-Rel failed to do so, suggesting a role for these two genes in v-Rel mediated transformation. Finally, these genes are expressed at high levels in cells overexpressing wild-type and truncated forms of c-Rel, implying that v-Rel transforms, in part, by induction of c-Rel target genes.
Assuntos
Transformação Celular Neoplásica , Regulação Neoplásica da Expressão Gênica , Proteínas Oncogênicas de Retroviridae , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Células da Medula Óssea/patologia , Embrião de Galinha , Fibroblastos , Dados de Sequência Molecular , NF-kappa B/fisiologia , Proteínas Oncogênicas v-rel , RNA Mensageiro/genética , RNA Neoplásico/genética , Relação Estrutura-Atividade , Transcrição GênicaRESUMO
The avian reticuloendotheliosis virus strain T (REV-T) transforms bone marrow cells and may cause phenotypic changes in fibroblasts. Both events are thought to result from expression of the v-rel oncoprotein, a member of the NF-kappa B family of transcription factors. Most REV stocks contain a cytopathic and immunosuppressive helper virus (REV-A) unrelated to standard avian retroviruses, and thus the degree to which v-rel expression alone contributes to the transformed phenotype in bone marrow cells and fibroblasts is complicated by helper virus expression. To gain a more accurate picture of how v-rel contributes to transformation, we have cloned the v-rel gene into a replication-competent avian retrovirus vector (RCAS) and have expressed it in both chick embryo fibroblasts (CEF) and bone marrow cells. Transfection of RCAS-rel into CEF readily produced a partially transformed phenotype, demonstrating that expression of the v-rel protein is sufficient for fibroblast transformation. The RCAS-rel virus also transformed bone marrow cells in vitro, but required culture conditions different from those normally required for transformation by REV-T. The v-rel protein expressed in transformed CEF was biochemically indistinguishable from that expressed in transformed bone marrow cells, being localized to the cytoplasm and the nucleus, and forming a complex with cellular proteins. We also demonstrate that the RCAS-rel-transformed hematopoietic cells exhibited a distinct differentiation phenotype.
Assuntos
Transformação Celular Neoplásica , Oncogenes , Proteínas Tirosina Quinases/genética , Vírus da Reticuloendoteliose/genética , Proteínas Oncogênicas de Retroviridae/genética , Transfecção , Animais , Células Cultivadas , Embrião de Galinha , Galinhas , Células Clonais , Fibroblastos/fisiologia , Células-Tronco Hematopoéticas/citologia , NF-kappa B/genética , Proteínas Oncogênicas v-rel , Vírus da Reticuloendoteliose/fisiologia , Replicação ViralRESUMO
In this report, we characterize the biological and biochemical properties of a conditional protein containing chicken c-Rel fused to the hormone-binding domain of the human estrogen receptor. This chimeric c-RelER protein causes estrogen-dependent, but otherwise c-Rel-specific, transformation of avian fibroblasts in vitro. Our results demonstrate that c-RelER heterodimerizes with wild-type c-Rel and forms specific complexes with IkappaB-alpha. Estrogen causes translocation of c-RelER to the nucleus and stabilizes its binding to DNA. Hormone-activated c-RelER induces transcription of at least four cellular genes that are constitutively active in wild-type c-Rel-transformed fibroblasts. Two distinct cell populations were examined that differed with respect to their growth phenotypes. The growth of fibroblasts with moderate expression levels of c-RelER was stimulated by estrogen. In contrast, the addition of estrogen to cells with high cRelER expression levels resulted in inhibition of cytokinesis and the arrest of growth. The carboxy terminal transactivation domain of c-Rel was required for the induction of these effects since neither v-Rel nor c-Rel deletion mutants were able to induce similar changes. Taken together, our results demonstrate that high levels of c-Rel expression can affect cell cycle control and/or cytokinesis. Furthermore, they also indicate that the biological properties of c-Rel in cell growth and differentiation will potentially differ depending on the level of expression.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Estrogênio/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Sequência de Bases , Transformação Celular Neoplásica , Embrião de Galinha , Células Clonais , DNA/metabolismo , Fibroblastos/metabolismo , Expressão Gênica , Células HeLa , Humanos , NF-kappa B/metabolismo , Fenótipo , Ligação Proteica , Proteínas Proto-Oncogênicas c-rel , Codorniz , Frações Subcelulares/metabolismo , Ativação TranscricionalRESUMO
Proteins within the Rel/NF-kappa B transcription factor family can be divided into two functional domains, a homologous amino terminal region, the Rel Homology Domain, and a divergent carboxy terminal domain. The amino terminal sequences specify DNA binding, nuclear localization, and interaction with the I kappa B family of inhibitory proteins. The carboxy terminus of each protein functions as a transcriptional activation domain, however, precise definition of sequence requirements has been difficult. To further define these sequences, small 100 bp deletions were constructed throughout the carboxy terminus of v-Rel. Each resulting mutant was assayed for DNA binding, localization, protein complex formation, activation of endogenous gene expression and ability to transform bone marrow cells and fibroblasts. Surprisingly, deletion within the carboxy terminus had marginal effects on transforming potential. However, three separate regions were required for full activation of gene expression. Taken together, these results suggest that the carboxy terminus of v-Rel contains multiple sequences that participate in the activation of gene expression.
Assuntos
Regulação da Expressão Gênica , Proteínas Oncogênicas de Retroviridae/fisiologia , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Medula Óssea/patologia , Divisão Celular , Embrião de Galinha , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Oncogênicas v-rel , Proteínas Oncogênicas de Retroviridae/análise , Proteínas Oncogênicas de Retroviridae/química , Proteínas Oncogênicas de Retroviridae/genética , Deleção de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
The rel oncogene from the avian reticuloendotheliosis virus strain T is a 59 kd phosphoprotein localized primarily to the cytoplasm of transformed cells. Recently, the v-rel protein was shown to associate with several cellular proteins with molecular weights of 124 kd, 115 kd, and 36 kd. We have analysed the subcellular distribution of v-rel protein complexes after biochemical fractionation of [35S]methionine and [32P]orthophosphate labeled cells. Our results demonstrate that the v-rel protein coprecipitates with a characteristic set of proteins, some of which are distinct to nuclear or cytoplasmic fractions. We also demonstrate that the normal cellular homolog of the viral rel protein, c-rel, coprecipitates with several cellular proteins from normal chick hematopoietic tissue. These cellular proteins have apparent molecular weights similar to those which are coprecipitated with v-rel from cytoplasmic fractions. Our results demonstrate that both v-rel and c-rel interact with a variety of cellular proteins and suggest that this association is important for the function or regulation of the rel protein.
Assuntos
Proteínas Proto-Oncogênicas/metabolismo , Vírus da Reticuloendoteliose/genética , Proteínas dos Retroviridae/metabolismo , Retroviridae/genética , Animais , Fracionamento Celular , Linhagem Celular , Linhagem Celular Transformada , Núcleo Celular/metabolismo , Centrifugação com Gradiente de Concentração , Citoplasma/metabolismo , Peso Molecular , Proteínas Oncogênicas v-rel , Fosforilação , Testes de Precipitina , Ligação Proteica , Proteínas Proto-Oncogênicas c-rel , Vírus da Reticuloendoteliose/metabolismoRESUMO
A cDNA encoding a novel chicken chemokine homologous to mammalian chemokine macrophage inflammatory protein 1 beta (MIP-1 beta) was isolated and characterised. The cDNA encodes a protein which is 75-80% homologous to human and mouse MIP-1 beta. All conserved amino acids characteristic of the mammalian chemokine family have been evolutionarily preserved in chicken MIP-1 beta, suggesting similar protein folding patterns and functional properties.
Assuntos
Galinhas/genética , Citocinas/genética , Genes , Monocinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular Transformada , Quimiocina CCL4 , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Humanos , Proteínas Inflamatórias de Macrófagos , Camundongos , Dados de Sequência Molecular , Proteínas Oncogênicas v-rel , Proteínas Oncogênicas de Retroviridae/genética , Proteínas Oncogênicas de Retroviridae/fisiologia , Homologia de Sequência de AminoácidosAssuntos
Alpharetrovirus/fisiologia , Leucose Aviária/etiologia , Sarcoma Aviário/etiologia , Alpharetrovirus/genética , Animais , Leucose Aviária/genética , Transformação Celular Viral , Regulação da Expressão Gênica , Genes Virais , Mutação , Oncogenes , Sequências Repetitivas de Ácido Nucleico , Vírus da Reticuloendoteliose/genética , Vírus da Reticuloendoteliose/fisiologia , Retroviridae/fisiologia , Sarcoma Aviário/genéticaAssuntos
Regulação da Expressão Gênica , Proteínas Oncogênicas/biossíntese , Oncogenes , Proteínas Proto-Oncogênicas/biossíntese , Receptores de Esteroides/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Animais , Linhagem Celular , Galinhas , Meios de Cultura , Técnicas de Cultura/métodos , Dexametasona/farmacologia , Dietilestilbestrol/farmacologia , Drosophila , Estradiol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônios/farmacologia , Humanos , Camundongos , Ratos , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologiaRESUMO
In order to characterize the role of the carboxy terminus of the viral myc protein in the transformation of avian fibroblasts and macrophages, several Bal31 deletion mutants were created which removed varying portions of the carboxy terminus of the myc protein. Only one such mutant, S90.9, which had lost nine amino acids of myc retained biological activity when tested in macrophages and fibroblasts. This mutant transformed avian macrophages in a manner similar to that of wild-type, but appeared to be partially transformation defective in fibroblasts. Chicken embryo fibroblast cultures infected with S90.9 exhibited an intermediate phenotype morphologically when compared to wild-type-infected cells. When tested for growth in soft agar, the presence or absence of actin cables and fibronectin on the cell surface, and growth rate, S90.9-infected cells showed intermediate behavior when compared to wild-type or helper virus-infected fibroblasts. These experiments suggest that the carboxy terminus of the myc protein, which is highly basic in nature, is involved in the transformation of avian fibroblasts.
Assuntos
Vírus da Leucose Aviária/genética , Transformação Celular Viral , Proteínas Oncogênicas Virais/fisiologia , Oncogenes , Animais , Medula Óssea/microbiologia , Divisão Celular , Células Cultivadas , Embrião de Galinha , Galinhas , Fibroblastos/microbiologia , Genes Virais , Macrófagos/microbiologia , Mutação , Proteínas Oncogênicas Virais/genética , Codorniz , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , TransfecçãoRESUMO
The rel oncogene induces rapidly fatal lymphomas in birds. The mechanism by which this occurs is not clear, but investigations into the properties of rel have shown it to be an unusual protein. Unlike other 'nuclear' oncogenes described in this issue, v-rel can be found in both the cytoplasm and nucleus. In addition, it appears to transform avian lymphoid cells regardless of its localization. Interestingly, the rel gene is highly homologous to the Drosophila gene dorsal, which is involved in determination of dorsal-ventral polarity in the developing embryo. In this review, the biology of the virus containing the rel oncogene, REV-T, will be described. The structure of the gene and the properties of the rel protein will be examined. The rel protein will be compared to the Drosophila homolog, dorsal, both structurally and functionally.
Assuntos
Neoplasias/genética , Oncogenes/fisiologia , Animais , Neoplasias/etiologiaRESUMO
The myc oncogene has been the subject of intense research of late because of its involvement in viral and non-viral carcinogenesis in the avian, murine, feline and human systems. From data obtained in each system it appears that myc, which has been highly conserved through evolution, has a key role in the control of cell proliferation. However, the precise function of this gene remains obscure. This review attempts to summarize data obtained in the various systems in order to compare sometimes conflicting results.
Assuntos
Neoplasias Experimentais/genética , Oncogenes , Animais , Vírus da Leucose Aviária , Aves , MamíferosRESUMO
The Rel family consists of a large set of related proteins containing a conserved protein region, termed the Rel Homology Domain. Although the prototype member of the Rel family, v-Rel, was originally discovered on the basis of its transforming ability, recent studies have indicated that other members are also important mediators of embryonic development and differentiation. In particular, the dorsal protein is an essential embryonic polarity gene required for the proper dorsal/ventral development of the Drosophila embryo, while the avian c-rel gene has been implicated in the control of cell death and tissue molding. Other members may play a part in the differentiation of dendritic antigen presenting cells and the control of general and lymphoid specific gene transcription.
Assuntos
Família Multigênica , Oncogenes , Proteínas/genética , Animais , Diferenciação Celular/genética , Humanos , NF-kappa B/fisiologiaRESUMO
We describe the generation and characterization of a series of deletion mutants of the avian acute leukaemia virus MC29 which allow the study of the function of the myc in transformation of quail embryo fibroblasts in vitro and tumour induction in vivo. These mutants, which are deleted in the 3' portion of the myc gene, fail to transform macrophages in vitro or induce tumours in vivo but are still able to transform morphologically fibroblasts. From one of these mutants a 'recovered' MC29 virus was generated which, like wild type MC29, transformed fibroblasts and macrophages in vitro. When tested in vivo this virus induced lymphomas of T and B cells rather that the endotheliomas induced by wild type MC29. This system allows us to investigate another question which is the mechanism by which the virus (or oncogene it contains) preferentially transforms one cell type.
Assuntos
Vírus da Leucose Aviária/genética , Genes Virais , Mutação , Oncogenes , Animais , Sequência de Bases , Transformação Celular Neoplásica , Células Cultivadas , Deleção Cromossômica , Enzimas de Restrição do DNA , Embrião não Mamífero , Fibroblastos/citologia , CodornizRESUMO
The cellular location of viral structural proteins was carried out by immunohistochemistry and by cell fractionation. Antibody against the structural protein p27 was used in immunohistochemical reactions to demonstrate the presence of viral proteins in the cytoplasm and nucleus of Rous-associated virus 2-infected chicken cells. Localization in the nucleus was found over heterochromatic regions; in the cytoplasm it was found in discrete particulate structures. These observations were extended in cell fractionation studies in which cytoplasmic and nuclear fractions were immunoprecipitated with antibody against the viral structural proteins.
Assuntos
Vírus da Leucose Aviária/análise , Núcleo Celular/análise , Citoplasma/análise , Proteínas Virais/análise , Animais , Vírus da Leucose Aviária/crescimento & desenvolvimento , Fracionamento Celular , Células Cultivadas , Embrião de Galinha , Fibroblastos , Heterocromatina/análise , Técnicas Imunoenzimáticas , Microscopia EletrônicaRESUMO
Restriction enzyme mapping and limited sequence analysis have been used to study the generation and genome structure of three partial-transformation mutants of avian acute leukemia virus MC29. The three mutants, td10A, td10C, and td10H, could be shown to have sustained overlapping deletions of 200, 400, and 600 base pairs, respectively, in their genomes. The precise location of the deletions was mapped within the v-myc gene of the mutants by limited sequence analysis of cloned MC29 DNA. The data obtained are discussed in terms of the effect of these deletions on the mechanism of transformation by MC29.