RESUMO
The gene encoding the transcriptional co-activator MN1 is the target of the reciprocal chromosome translocation (12;22)(p13;q12) in some patients with acute myeloid leukemia (AML). In addition, expression array analysis showed that MN1 was overexpressed in AML specified by inv(16), in some AML overexpressing ecotropic viral integration 1 site (EVI1) and in some AML without karyotypic abnormalities. Here we describe that mice receiving transplants of bone marrow (BM) overexpressing MN1 rapidly developed myeloproliferative disease (MPD). This BM also generated myeloid cell lines in culture. By mimicking the situation in human inv(16) AML, forced coexpression of MN1 and Cbfbeta-SMMHC rapidly caused AML in mice. These findings identify MN1 as a highly effective hematopoietic oncogene and suggest that MN1 overexpression is an important cooperative event in human inv(16) AML.
Assuntos
Inversão Cromossômica , Cromossomos Humanos Par 16/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Leucemia Mieloide/etiologia , Proteínas Oncogênicas/genética , Translocação Genética/genética , Doença Aguda , Animais , Transplante de Medula Óssea , Células Cultivadas , Feminino , Citometria de Fluxo , Humanos , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Camundongos , Camundongos Transgênicos , Transtornos Mieloproliferativos/etiologia , Transtornos Mieloproliferativos/metabolismo , Transtornos Mieloproliferativos/patologia , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Taxa de Sobrevida , Transativadores , Proteínas Supressoras de TumorRESUMO
The translocation t(12;22)(p13;q11) creates an MN1-TEL fusion gene leading to acute myeloid leukemia. MN1 is a transcription coactivator of the retinoic acid and vitamin D receptors, and TEL (ETV6) is a member of the E26-transformation-specific family of transcription factors. In MN1-TEL, the transactivating domains of MN1 are combined with the DNA-binding domain of TEL. We show that MN1-TEL inhibits retinoic acid receptor (RAR)-mediated transcription, counteracts coactivators such as p160 and p300, and acts as a dominant-negative mutant of MN1. Compared to MN1, the same transactivation domains in MN1-TEL are poorly stimulated by p160, p300 or histone deacetylase inhibitors, indicating that the block of RAR-mediated transcription by MN1-TEL is caused by dysfunctional transactivation domains rather than by recruitment of corepressors. The mechanism leading to myeloid leukemia in t(12;22) thus differs from the translocations that involve RAR itself.
Assuntos
Carcinoma Hepatocelular/patologia , Proteínas de Fusão Oncogênica/fisiologia , Receptores do Ácido Retinoico/genética , Receptores X de Retinoides/genética , Fatores de Transcrição/fisiologia , Transcrição Gênica , Carcinoma Hepatocelular/genética , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 22/genética , Proteínas de Ligação a DNA , Proteína p300 Associada a E1A/fisiologia , Inibidores Enzimáticos , Genes Dominantes , Inibidores de Histona Desacetilases , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Mutação , Proteínas Nucleares/fisiologia , Proteínas de Transporte Nucleocitoplasmático/fisiologia , Proteínas de Ligação a RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação Transcricional , Transfecção , Translocação Genética , Células Tumorais CultivadasRESUMO
MN1-TEL is the product of the recurrent t(12;22)(p12;q11) associated with human myeloid malignancies. MN1-TEL functions as an activated transcription factor, exhibiting weak transforming activity in NIH3T3 fibroblasts that depends on the presence of a functional TEL DNA-binding domain, the N-terminal transactivating sequences of MN1 and C-terminal sequences of MN1. We determined the transforming activity of MN1-TEL in mouse bone marrow (BM) by using retroviral transfer. MN1-TEL-transduced BM showed increased self-renewal capacity of primitive progenitors in vitro, and prolonged in vitro culture of MN1-TEL-expressing BM produced immortalized myeloid, interleukin (IL)-3/stem cell factor-dependent cell lines with a primitive morphology. Transplantation of such cell lines into lethally irradiated mice rescued them from irradiation-induced death and resulted in the contribution of MN1-TEL-expressing cells to all hematopoietic lineages, underscoring the primitive nature of these cells and their capacity to differentiate in vivo. Three months after transplantation, all mice succumbed to promonocytic leukemia. Transplantation of freshly MN1-TEL-transduced BM into lethally irradiated mice also caused acute myeloid leukemia within 3 months of transplantation. We infer that MN1-TEL is a hematopoietic oncogene that stimulates the growth of hematopoietic cells, but depends on secondary mutations to cause leukemia in mice.
Assuntos
Transformação Celular Neoplásica , Cromossomos Humanos Par 12 , Cromossomos Humanos Par 22 , Células-Tronco Hematopoéticas/patologia , Leucemia Mieloide/genética , Proteínas de Fusão Oncogênica/genética , Fatores de Transcrição/genética , Translocação Genética , Animais , Proliferação de Células , Transplante de Células , Humanos , Leucemia Mieloide/patologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Human SET, a target of chromosomal translocation in human leukemia encodes a highly conserved, ubiquitously expressed, nuclear phosphoprotein. SET mediates many functions including chromatin remodeling, transcription, apoptosis and cell cycle control. We report that overexpression of SET directs differentiation of the human promonocytic cell line U937 along the dendritic cell (DC) pathway, as cells display typical morphologic changes associated with DC fate and express the DC surface markers CD11b and CD86. Differentiation occurs via a calcium-dependent mechanism involving the CaMKII and MAPK/ERK pathways. Similar responses are elicited by interferon-gamma (IFN-gamma) treatment with the distinction that IFN-gamma signaling activates the DNA-binding activity of STAT1 whereas SET overexpression does not. In addition, unlike IFN-gamma signaling, SET generated stress-induced p38/MAPK activity. Interestingly, IFN-gamma treatment transiently upregulated endogenous SET in a dose-dependent manner. These results suggest that SET is part of both IFN-gamma-mediated and stress-mediated cellular responses and that SET induces cell differentiation via calcium and MAPK/ERK pathways.
Assuntos
Sinalização do Cálcio , Proteínas Cromossômicas não Histona/fisiologia , Células Dendríticas/citologia , Sistema de Sinalização das MAP Quinases , Monócitos/citologia , Fatores de Transcrição/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Diferenciação Celular , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/metabolismo , Chaperonas de Histonas , Humanos , Interferon gama/farmacologia , Fator de Transcrição STAT1 , Transativadores/metabolismo , Fatores de Transcrição/genética , Células U937 , Regulação para Cima/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Human SET encodes a nuclear phosphoprotein with a highly acidic carboxyl-terminus, forming a SET-CAN fusion gene in a patient with acute undifferentiated leukemia. SET is highly conserved between species and is ubiquitously expressed, suggesting a widespread biological role. Even though SET is involved in chromatin remodeling and transcriptional activation, its precise role in hematopoietic cells and the contribution of SET-CAN to leukemogenesis remains unknown. We determined the effect of tetracycline-regulatable expression of SET, a deletion mutant of SET, and SET-CAN on the human promonocytic cell line U937T. The expression of SET and SET-CAN inhibited proliferation of these cells. SET accomplishes this through the induction of the differentiation program, an effect that depends on the presence of its acidic domain. SET-CAN most likely inhibits growth by interfering with hCRM1, but it also partially blocks differentiation. Our results are the first demonstration of a potential role of SET in hematopoietic differentiation.
Assuntos
Diferenciação Celular , Proteínas Cromossômicas não Histona , Monócitos/citologia , Proteínas de Fusão Oncogênica/fisiologia , Fosfoproteínas/fisiologia , Proteínas/fisiologia , Receptores Citoplasmáticos e Nucleares , Fatores de Transcrição , Divisão Celular , Proteínas de Ligação a DNA , Chaperonas de Histonas , Humanos , Carioferinas , Proteínas de Fusão Oncogênica/genética , Fosfoproteínas/genética , Estrutura Terciária de Proteína , Proteínas/genética , Deleção de Sequência , Transfecção , Células U937 , Proteína Exportina 1RESUMO
TEL/ETV6 is the frequent target of translocations associated with lymphoid and myeloid leukemias and solid tumors. We show that TEL induces aggregation of immortalized and transformed fibroblasts, endothelial cells and astrocytes. These aggregates form cellular cords in NIH3T3-UCLA by a cell autonomous process, which occurs when the monolayer is made up of over 75% of cells expressing exogenous TEL. Cords with a diameter of 15-25 microm contain a lumen and occur as tube structures. The possible relevance for vasculogenic mimicry is discussed. By contrast TEL did not induce aggregation of regular NIH3T3 cells, an effect that could only be induced by co-expression of oncogenic RAS/Lys12. Also transduction of TEL and RAS retroviral vectors into the endothelial MS1 cell line and TEL alone in the highly transformed glioblastoma cell lines EH-A and EH-B resulted in extensive aggregation. Thus, the induction of cellular aggregation by TEL correlates with transformation.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Neoplasias/patologia , Proteínas Repressoras/fisiologia , Células 3T3 , Animais , Agregação Celular , Linhagem Celular , Linhagem Celular Transformada , Tamanho Celular , Proteínas de Ligação a DNA/genética , Endotélio/citologia , Vetores Genéticos , Camundongos , Microscopia de Fluorescência , Proteína Oncogênica p21(ras)/genética , Proteína Oncogênica p21(ras)/metabolismo , Proteínas Proto-Oncogênicas c-ets , Proteínas Repressoras/genética , Retroviridae/genética , Transdução Genética , Células Tumorais Cultivadas , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
An approach is described to modify yeast artificial chromosomes (YACs) with cassettes that can be easily excised for embryonic stem (ES) cell gene targeting experiments. YAC targeting technology (YTT) uses the WIBR/MIT-820 C57BL/6-mapped YAC library derived from the C57BL/6 mouse as the starting point for Internet- or PCR-based clone isolation, although in principle any YAC system can be used. Homologous recombination is initially performed in yeast using cassettes that function in Saccharomyces cerevisiae, Escherichia coli, and ES cells, followed by cloning or conversion of the targeted locus into a plasmid. The completed targeting vector can be transfected into C57BL/6 ES cells and clones selected with G418 followed by injection into Balb/c blastocysts. YTT increases the speed of targeting vector construction and obviates the need for extensive backcrossing to the C57BL/6 background.
Assuntos
Cromossomos Artificiais de Levedura , Marcação de Genes/métodos , Camundongos Endogâmicos C57BL/genética , Animais , Escherichia coli , Deleção de Genes , Camundongos , Dados de Sequência Molecular , Saccharomyces cerevisiae/genética , Células-Tronco/fisiologia , TransfecçãoRESUMO
Stem cells from bone marrow, skeletal muscle and possibly other tissues can be identified by the 'side-population' (SP) phenotype. Although it has been assumed that expression of ABC transporters is responsible for this phenotype, the specific molecules involved have not been defined. Here we show that expression of the Bcrp1 (also known as Abcg2 murine/ABCG2 human) gene is a conserved feature of stem cells from a wide variety of sources. Bcrp1 mRNA was expressed at high levels in primitive murine hematopoietic stem cells, and was sharply downregulated with differentiation. Enforced expression of the ABCG2 cDNA directly conferred the SP phenotype to bone-marrow cells and caused a reduction in maturing progeny both in vitro and in transplantation-based assays. These results show that expression of the Bcrp1/ABCG2 gene is an important determinant of the SP phenotype, and that it might serve as a marker for stem cells from various sources.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas Inflamatórias de Macrófagos , Glicoproteínas de Membrana , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Proteínas de Neoplasias , Células-Tronco/fisiologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Antígenos CD/metabolismo , Antígenos CD34/metabolismo , Biomarcadores , Células da Medula Óssea/fisiologia , Células Cultivadas , Quimiocinas CC , Citocinas/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Mutantes , Proteínas Ribossômicas/metabolismo , Células-Tronco/citologia , Tetraspanina 29 , TransplantesRESUMO
The Tel gene (or ETV6) is the target of the translocation (12;22)(p13;q11) in myeloid leukemia. TEL is a member of the ETS family of transcription factors and contains the pointed protein interaction (PNT) domain and an ETS DNA binding domain (DBD). By contrast to other chimeric proteins that contain TEL's PNT domain, such as TEL-platelet-derived growth factor beta receptor in t(5;12)(q33;p13), MN1-TEL contains the DBD of TEL. The N-terminal MN1 moiety is rich in proline residues and contains two polyglutamine stretches, suggesting that MN1-TEL may act as a deregulated transcription factor. We now show that MN1-TEL type I, unlike TEL and MN1, transforms NIH 3T3 cells. The transforming potential depends on both N-terminal MN1 sequences and a functional TEL DBD. Furthermore, we demonstrate that MN1 has transcription activity and that MN1-TEL acts as a chimeric transcription factor on the Moloney sarcoma virus long terminal repeat and a synthetic promoter containing TEL binding sites. The transactivating capacity of MN1-TEL depended on both the DBD of TEL and sequences in MN1. MN1-TEL contributes to leukemogenesis by a mechanism distinct from that of other chimeric proteins containing TEL.
Assuntos
Transformação Celular Neoplásica , Proteínas de Ligação a DNA/genética , Leucemia Mieloide/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Repressoras , Fatores de Transcrição/genética , Ativação Transcricional , Translocação Genética , Animais , Clonagem Molecular , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Genes Reguladores , Humanos , Immunoblotting , Camundongos , Microscopia Confocal , Proteínas de Fusão Oncogênica/imunologia , Proteínas de Fusão Oncogênica/metabolismo , Testes de Precipitina , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-ets , Retroviridae/genética , Retroviridae/metabolismo , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transfecção , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
The Tel gene is a major target of translocations in leukemia and loss of heterozygosity is regularly observed for the non-translocated allele, thus supporting the notion that Tel is a tumor suppressor. Most tumor suppressors influence cellular proliferation, differentiation and cell death and thereby prevent oncogenic transformation and genetic instability. We found that overexpression of Tel retards proliferation of many cell types, primary cells and immortalized cells, by inducing a G1 arrest. Tel's block of cellular proliferation is rescued by high seeding densities. Furthermore, Tel suppressed Ras-mediated colony growth in soft agar and tumor formation in nude mice. The Pointed and DNA binding (DB) domains of Tel were required for all Tel-induced phenotypes.
Assuntos
Transformação Celular Neoplásica/patologia , Proteínas de Ligação a DNA/metabolismo , Fase G1 , Proteína Oncogênica p21(ras)/antagonistas & inibidores , Proteínas Repressoras , Fatores de Transcrição/metabolismo , Células 3T3 , Ágar , Animais , Western Blotting , Contagem de Células , Divisão Celular , Linhagem Celular Transformada , Células Cultivadas , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Fibroblastos , Citometria de Fluxo , Imunofluorescência , Genes ras/genética , Genes ras/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Proteína Oncogênica p21(ras)/genética , Proteína Oncogênica p21(ras)/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-ets , Fatores de Transcrição/química , Fatores de Transcrição/genética , Ensaio Tumoral de Célula-Tronco , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
DEK is an approximately 45-kD phosphoprotein that is fused to the nucleoporin CAN as a result of a (6;9) chromosomal translocation in a subset of acute myeloid leukemias (AMLs). It has also been identified as an autoimmune antigen in juvenile rheumatoid arthritis and other rheumatic diseases. Despite the association of DEK with several human diseases, its function is not known. In this study, we demonstrate that DEK, together with SR proteins, associates with the SRm160 splicing coactivator in vitro. DEK is recruited to splicing factor-containing nuclear speckles upon concentration of SRm160 in these structures, indicating that DEK and SRm160 associate in vivo. We further demonstrate that DEK associates with splicing complexes through interactions mediated by SR proteins. Significantly, DEK remains bound to the exon-product RNA after splicing, and this association requires the prior formation of a spliceosome. Thus, DEK is a candidate factor for controlling postsplicing steps in gene expression that are influenced by the prior removal of an intron from pre-mRNA.
Assuntos
Antígenos Nucleares , Proteínas Cromossômicas não Histona , Éxons/fisiologia , Leucemia Mieloide Aguda/metabolismo , Proteínas Associadas à Matriz Nuclear , Proteínas Oncogênicas/metabolismo , Splicing de RNA/fisiologia , Proteínas de Ligação a RNA/metabolismo , Células HeLa , Humanos , Leucemia Mieloide Aguda/fisiopatologia , Proteínas Nucleares/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , RNA/metabolismoRESUMO
The ETS family of proteins is a large group of transcription factors implicated in many aspects of normal hematopoietic development, as well as oncogenesis. For example, the TEL1/ETV6 (TEL1) gene is required for normal yolk sac angiogenesis, adult bone marrow hematopoiesis, and is rearranged or deleted in numerous leukemias. This report describes the cloning and characterization of a novel ETS gene that is highly related to TEL1 and is therefore called TEL2. The TEL2 gene consists of 8 exons spanning approximately 21 kilobases (kb) in human chromosome 6p21. Unlike the ubiquitously expressed TEL1 gene, however, TEL2 appears to be expressed predominantly in hematopoietic tissues. Antibodies raised against the C-terminus of the TEL2 protein were used to show that TEL2 localizes to the nucleus. All ETS proteins can bind DNA via the highly conserved ETS domain, which recognizes a purine-rich DNA sequence with a GGAA core motif. DNA binding assays show that TEL2 can bind the same consensus DNA binding sequence recognized by TEL1/ETV6. Additionally, the TEL2 protein is capable of associating with itself and with TEL1 in doubly transfected Hela cells, and this interaction is mediated through the pointed (PNT) domain of TEL1. The striking similarities of TEL2 to the oncogenic TEL1, its expression in hematopoietic tissues, and its ability to associate with TEL1 suggest that TEL2 may be an important hematopoietic regulatory protein.
Assuntos
Cromossomos Humanos Par 8 , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Hematopoese , Proteínas Repressoras , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Adulto , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Mapeamento Cromossômico , Clonagem Molecular , Proteínas de Ligação a DNA/química , Éxons , Humanos , Hibridização in Situ Fluorescente , Fígado/embriologia , Fígado/metabolismo , Ativação Linfocitária , Linfócitos/imunologia , Linfócitos/fisiologia , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-ets , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
The INK4 family of cyclin-dependent kinase (CDK) inhibitors includes four 15- to 19-kDa polypeptides (p16(INK4a), p15(INK4b), p18(INK4c), and p19(INK4d)) that bind to CDK4 and CDK6. By disrupting cyclin D-dependent holoenzymes, INK4 proteins prevent phosphorylation of the retinoblastoma protein and block entry into the DNA-synthetic phase of the cell division cycle. The founding family member, p16(INK4a), is a potent tumor suppressor in humans, whereas involvement, if any, of other INK4 proteins in tumor surveillance is less well documented. INK4c and INK4d are expressed during mouse embryogenesis in stereotypic tissue-specific patterns and are also detected, together with INK4b, in tissues of young mice. INK4a is expressed neither before birth nor at readily appreciable levels in young animals, but its increased expression later in life suggests that it plays some checkpoint function in response to cell stress, genotoxic damage, or aging per se. We used targeted gene disruption to generate mice lacking INK4d. These animals developed into adulthood, had a normal life span, and did not spontaneously develop tumors. Tumors did not arise at increased frequency in animals neonatally exposed to ionizing radiation or the carcinogen dimethylbenzanthrene. Mouse embryo fibroblasts, bone marrow-derived macrophages, and lymphoid T and B cells isolated from these animals proliferated normally and displayed typical lineage-specific differentiation markers. Males exhibited marked testicular atrophy associated with increased apoptosis of germ cells, although they remained fertile. The absence of tumors in INK4d-deficient animals demonstrates that, unlike INK4a, INK4d is not a tumor suppressor but is instead involved in spermatogenesis.
Assuntos
Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Inibidor p16 de Quinase Dependente de Ciclina , Fertilidade/genética , Regulação da Expressão Gênica/fisiologia , Testículo/patologia , Animais , Inibidor de Quinase Dependente de Ciclina p19 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Masculino , Camundongos , Camundongos Knockout , Testículo/fisiologiaRESUMO
The Pax3-FKHR fusion protein is present in alveolar rhabdomyosarcoma and results from the t(2;13) (q35;q14) chromosomal translocation. Its oncogenic activity is dependent on a combination of protein-DNA and protein-protein interactions mediated by the Pax3 homeodomain recognition helix. In this report we demonstrate that human Daxx (hDaxx) interacts with Pax3 in vivo and with DNA-bound Pax3 in vitro. This interaction is mediated primarily through the homeodomain recognition helix with the additional involvement of the octapeptide domain and its N-terminal flanking amino acids. Through this interaction hDaxx represses the transcriptional activity of Pax3 by approximately 80%. The Pax3-FKHR fusion is unresponsive to this repressive effect despite an observed endogenous interaction with hDaxx in a rhabdomyosarcoma tumor cell line. Therefore, these data support the model that fusion of FKHR to Pax3 not only adds a strong transactivation domain, but also deregulates transcriptional control of Pax3 by overriding the natural repressive effect of hDaxx.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Nucleares , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Linhagem Celular , Clonagem Molecular , Proteínas Correpressoras , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead , Regulação Neoplásica da Expressão Gênica , Humanos , Chaperonas Moleculares , Dados de Sequência Molecular , Peso Molecular , Proteínas de Fusão Oncogênica/química , Fator de Transcrição PAX3 , Fatores de Transcrição Box Pareados , Fosforilação , Testes de Precipitina , Ligação Proteica , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/isolamento & purificação , Rabdomiossarcoma/genética , Deleção de Sequência , Transfecção , Translocação Genética/genética , Leveduras/genéticaRESUMO
Many cytokines activate two highly homologous Stat proteins, 5a and 5b. Mice deficient in both genes lack all growth hormone and prolactin functions but retain functions associated with cytokines such as erythropoietin. Here, we demonstrate that, while lymphoid development is normal, Stat5a/b mutant peripheral T cells are profoundly deficient in proliferation and fail to undergo cell cycle progression or to express genes controlling cell cycle progression. In addition, the mice lack NK cells, develop splenomegaly, and have T cells with an activated phenotype, phenotypes seen in IL-2 receptor beta chain-deficient mice. These phenotypes are not seen in mice lacking Stat5a or Stat5b alone. The results demonstrate that the Stat5 proteins, redundantly, are essential mediators of IL-2 signaling in T cells.
Assuntos
Ciclo Celular/fisiologia , Proteínas de Ligação a DNA/fisiologia , Interleucina-2/fisiologia , Proteínas do Leite , Linfócitos T/citologia , Transativadores/fisiologia , Animais , Divisão Celular , Células Cultivadas , Citometria de Fluxo , Camundongos , Camundongos Mutantes , Fator de Transcrição STAT5 , Timo/citologia , Timo/metabolismoRESUMO
Genetic evidence suggests that the POU transcription factor Oct-6 plays a pivotal role as an intracellular regulator of Schwann cell differentiation. In the absence of Oct-6 function Schwann cells are generated in appropriate numbers and these cells differentiate normally up to the promyelin stage at which they transiently arrest. During peripheral nerve development Oct-6 expression is initiated in Schwann cell precursors and is strongly upregulated in promyelin cells. Oct-6 expression is subsequently extinguished in terminally differentiating Schwann cells. Thus, identification and characterisation of the DNA elements involved in this stage specific regulation may lead us to the signaling cascade and the axon-derived signals that drive Schwann cell differentiation and initiate myelination. Here we present experiments that aim at identifying such regulatory sequences.
Assuntos
Regulação da Expressão Gênica , Células de Schwann/fisiologia , Fatores de Transcrição/genética , Transcrição Gênica , Animais , Células Cultivadas , Camundongos , Proteínas do Tecido Nervoso/genética , Fator 6 de Transcrição de Octâmero , Sequências Reguladoras de Ácido Nucleico , Células de Schwann/citologia , Transfecção , beta-Galactosidase/genéticaRESUMO
Tel is an Ets transcription factor that is the target of chromosome translocations in lymphoid and myeloid leukemias and in solid tumors. It contains two functional domains, a pointed oligomerization domain and a DNA-binding domain. Retroviral transduction of a wild-type Tel cDNA into a clonal subline of NIH3T3 fibroblasts resulted in a striking morphologic change: at confluency, the cells reorganized into a specific "bridge-like" pattern over the entire surface of the culture dish, and started migrating, thereby leaving circular holes in the monolayer. Thereafter, formation of cellular cords became apparent. This sequence of events was inhibited by coating the culture dishes with fibronectin and collagen IV. Retroviral transduction of Tel into MS1 endothelial cells reproduced the aggregation phenotype, but not the cellular cord formation. Tel-mutagenesis showed that both the pointed domain and the DNA-binding domain of Tel are required for the morphologic change. Other Ets family genes, Fli-1 and Ets-1 that are both endogenously expressed in endothelial cells, could not induce this morphologic change. Exogenous Tel expression is associated with transcriptional upregulation of entactin/nidogen, Smad5, Col3a1, CD44 and fibronectin, and downregulation of Col1a1 and secretory leukocyte protease inhibitor. Interestingly, Tel, Smad5, fibronectin, Col1a1 and Col3a1 all have essential roles during vascular development.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas da Matriz Extracelular/biossíntese , Proteínas Repressoras , Fatores de Transcrição/fisiologia , Células 3T3 , Animais , Agregação Celular , Regulação da Expressão Gênica , Camundongos , Fenótipo , Proteínas Proto-Oncogênicas c-ets , Transfecção , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
A variety of cytokines mediate the activation of Janus protein tyrosine kinases (Jaks). The Jaks then phosphorylate cellular substrates, including members of the signal transducers and activators of transcription (Stat) family of transcription factors. Among the Stats, the two highly related proteins, Stat5a and Stat5b, are activated by a variety of cytokines. To assess the role of the Stat5 proteins, mutant mice were derived that have the genes deleted individually or together. The phenotypes of the mice demonstrate an essential, and often redundant, role for the two Stat5 proteins in a spectrum of physiological responses associated with growth hormone and prolactin. Conversely, the responses to a variety of cytokines that activate the Stat5 proteins, including erythropoietin, are largely unaffected.
Assuntos
Citocinas/farmacologia , Proteínas de Ligação a DNA/metabolismo , Proteínas do Leite , Prolactina/farmacologia , Transativadores/metabolismo , Animais , Constituição Corporal , Proteínas de Ligação a DNA/genética , Eritropoetina/farmacologia , Feminino , Deleção de Genes , Hormônio do Crescimento/metabolismo , Hematopoese/genética , Infertilidade Feminina/genética , Glândulas Mamárias Animais/crescimento & desenvolvimento , Camundongos , Camundongos Mutantes , Ovário/crescimento & desenvolvimento , Fator de Transcrição STAT5 , Transativadores/genéticaRESUMO
A variety of cytokines activate receptor-associated members of the Janus family of protein tyrosine kinases (Jaks). To assess the role of Jak2, we have derived Jak2-deficient mice. The mutation causes an embryonic lethality due to the absence of definitive erythropoiesis. Fetal liver myeloid progenitors, although present based on the expression of lineage specific markers, fail to respond to erythropoietin, thrombopoietin, interleukin-3 (IL-3), or granulocyte/macrophage colony-stimulating factor. In contrast, the response to granulocyte specific colony-stimulating factor is unaffected. Jak2-deficient fibroblasts failed to respond to interferon gamma (IFNgamma), although the responses to IFNalpha/beta and IL-6 were unaffected. Lastly, reconstitution experiments demonstrate that Jak2 is not required for the generation of lymphoid progenitors, their amplification, or functional differentiation. Therefore, Jak2 plays a critical, nonredundant role in the function of a specific group of cytokines receptors.
Assuntos
Citocinas/farmacologia , Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas , Receptores de Citocinas/fisiologia , Transdução de Sinais/fisiologia , Animais , Linfócitos B/citologia , Diferenciação Celular , Fatores Estimuladores de Colônias/farmacologia , Proteínas de Ligação a DNA/genética , Eritropoese/genética , Fibroblastos/citologia , Células-Tronco Hematopoéticas/citologia , Fator Regulador 1 de Interferon , Janus Quinase 2 , Fígado/citologia , Fígado/embriologia , Camundongos , Camundongos Knockout , Fenótipo , Fosfoproteínas/genética , Fosforilação , Proteínas Tirosina Quinases/genética , RNA Mensageiro/análise , Linfócitos T/citologia , Trombopoetina/farmacologia , Timo/imunologia , Tirosina/metabolismoRESUMO
Several partner genes on different chromosomes have been reported to be fused with the ETV6 gene (located in chromosome band 12p13), with different breakpoints and different frequencies, in various hematologic malignancies, particularly acute myeloid and lymphoid leukemias and myelodysplastic syndromes. By using FISH and molecular analyses, we have analyzed five different pediatric and adult patients carrying cytogenetic abnormalities involving 12p13. Our findings demonstrate that ETV6 was rearranged in all the cases analyzed. In particular, ETV6 was disrupted by translocations with chromosomal bands 7q22, 7q36, 9q11, and 13q12, not previously described as partners of ETV6 in translocations, thus extending its promiscuity in rearranging with different partner genes.