RESUMO
Notch receptors are involved in cell-fate determination in organisms as diverse as flies, frogs and humans. In Drosophila melanogaster , loss-of-function mutations of Notch produce a 'neurogenic' phenotype in which cells destined to become epidermis switch fate and differentiate to neural cells. Upon ligand activation, the intracellular domain of Notch (ICN) translocates to the nucleus, and interacts directly with the DNA-binding protein Suppressor of hairless (Su(H)) in flies, or recombination signal binding protein Jkappa (RBP-Jkappa) in mammals, to activate gene transcription. But the precise mechanisms of Notch-induced gene expression are not completely understood. The gene mastermind has been identified in multiple genetic screens for modifiers of Notch mutations in Drosophila. Here we clone MAML1, a human homologue of the Drosophila gene Mastermind, and show that it encodes a protein of 130 kD localizing to nuclear bodies. MAML1 binds to the ankyrin repeat domain of all four mammalian NOTCH receptors, forms a DNA-binding complex with ICN and RBP-Jkappa, and amplifies NOTCH-induced transcription of HES1. These studies provide a molecular mechanism to explain the genetic links between mastermind and Notch in Drosophila and indicate that MAML1 functions as a transcriptional co-activator for NOTCH signalling.
Assuntos
Proteínas de Ligação a DNA , Proteínas de Drosophila , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Transativadores/genética , Transativadores/metabolismo , Células 3T3 , Animais , Células COS , Drosophila/genética , Genes de Insetos , Humanos , Camundongos , Dados de Sequência Molecular , Mutação , Receptores Notch , Especificidade da Espécie , Fatores de Transcrição , TransfecçãoRESUMO
Various histological subtypes of leukaemia and lymphoma are associated with diagnostic chromosome translocations, and substantial strides have been made in determining the specific oncogenes targetted by those translocations. We report the cloning of a novel fusion oncogene associated with a unique leukaemia/lymphoma syndrome. Patients afflicted with this syndrome present with lymphoblastic lymphoma and a myeloproliferative disorder, often accompanied by pronounced peripheral eosinophilia and/or prominent eosinophilic infiltrates in the affected bone marrow, which generally progress to full-blown acute myelogenous leukaemia within a year of diagnosis. A specific chromosome translocation, t(8;13)(p11;q11-12), is found in both lymphoma and myeloid leukaemia cells from these patients, supporting bi-lineage differentiation from a transformed stem cell. We find that the 8p11 translocation breakpoints, in each of four patients, interrupt intron 8 of the fibroblast growth factor receptor 1 gene (FGFR1). These translocations are associated with aberrant transcripts in which four predicted zinc-finger domains, contributed by a novel and widely expressed chromosome-13 gene (ZNF198), are fused to the FGFR1 tyrosine-kinase domain. Transient expression studies show that the ZNF198-FGFR1 fusion transcript directs the synthesis of an approximately 87-kD polypeptide, localizing predominantly to the cytoplasm. Our studies demonstrate an FGFR1 oncogenic role and suggest a tumorigenic mechanism in which ZNF198-FGFR1 activation results from ZNF198 zinc-finger-mediated homodimerization.
Assuntos
Proteínas de Transporte , Cromossomos Humanos Par 13 , Cromossomos Humanos Par 8 , Proteínas de Ligação a DNA/genética , Transtornos Mieloproliferativos/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptores Proteína Tirosina Quinases , Receptores de Fatores de Crescimento de Fibroblastos/genética , Translocação Genética , Dedos de Zinco/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Transformação Celular Neoplásica , Humanos , Camundongos , Dados de Sequência Molecular , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Síndrome , Fatores de TranscriçãoRESUMO
Previous work has demonstrated that intergenic V(D)J rearrangement, a process referred to as trans-rearrangement, occurs at an unexpectedly high frequency. These rearrangements generate novel V(D)J combinations which could conceivably have some role in the normal immune system, and since they probably arise through chromosomal rearrangements akin to those associated with lymphoid neoplasia, they may also serve as a model for investigating recombinational events which underlie oncogenesis. In view of the existence of a mechanism that permits relatively frequent intergenic trans-rearrangements, it seems reasonable that interallelic trans-rearrangements involving segments belonging to each of the two alleles of a single antigen receptor gene might also occur. To determine the frequency of such rearrangements, we examined thymocytes of F1 progeny of a cross between SWR mice, which have a deletion spanning 10 of the known V beta segments, and NZW mice, which have a deletion involving all J beta 2 segments. Rearranged TCR-beta genes containing V beta segments from the NZW chromosome and J beta segments from the SWR chromosome were amplified from the DNA of F1 thymocytes with the polymerase chain reaction. Using this approach, we found that such rearrangements are relatively uncommon, being present in about 1 in 10(5) thymocytes, a frequency lower than that of V gamma/J beta intergenic trans-rearrangements. The ratio of conventional cis-rearrangement to interallelic trans-rearrangement for any particular V beta segment appears to be about 10(4):1. The structure of the junctions in all trans-rearrangements analyzed closely resembles conventional cis-rearrangements, indicating involvement of V(D)J recombinase in the ultimate joining event. However, in contrast to cis-rearrangements, a strong bias for inclusion of D beta 1 segments over D beta 2 segments was noted, suggesting that interallelic trans-rearrangement may occur preferentially during attempted D-J joining. J beta 2 segment usage in trans-rearrangements also appeared to differ from that expected from previously studied cis-rearrangements. The results have implications with respect to the events and timing of conventional cis-rearrangement during thymocyte differentiation, and the prevalence of various types of trans-rearrangements.
Assuntos
Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Região de Junção de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologia , Alelos , Animais , Sequência de Bases , Southern Blotting , Deleção Cromossômica , DNA/genética , DNA/isolamento & purificação , Frequência do Gene , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase/métodos , Especificidade da Espécie , Timo/imunologiaRESUMO
Notch is a highly conserved transmembrane protein that is involved in cell fate decisions and is found in organisms ranging from Drosophila to humans. A human homologue of Notch, TAN1, was initially identified at the chromosomal breakpoint of a subset of T-cell lymphoblastic leukemias/lymphomas containing a t(7;9) chromosomal translocation; however, its role in oncogenesis has been unclear. Using a bone marrow reconstitution assay with cells containing retrovirally transduced TAN1 alleles, we analyzed the oncogenic potential of both nuclear and extranuclear forms of truncated TAN1 in hematopoietic cells. Although the Moloney leukemia virus long terminal repeat drives expression in most hematopoietic cell types, retroviruses encoding either form of the TAN1 protein induced clonal leukemias of exclusively immature T cell phenotypes in approximately 50% of transplanted animals. All tumors overexpressed truncated TAN1 of the size and subcellular localization predicted from the structure of the gene. These results show that TAN1 is an oncoprotein and suggest that truncation and overexpression are important determinants of transforming activity. Moreover, the murine tumors caused by TAN1 in the bone marrow transplant model are very similar to the TAN1-associated human tumors and suggest that TAN1 may be specifically oncotropic for T cells.
Assuntos
Transplante de Medula Óssea , Medula Óssea/patologia , Leucemia de Células T/patologia , Linfoma de Células T/patologia , Proteínas de Membrana/biossíntese , Receptores de Superfície Celular , Fatores de Transcrição , Animais , Células da Medula Óssea , Cromossomos Humanos Par 7 , Cromossomos Humanos Par 9 , Drosophila , Proteínas de Drosophila , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia de Células T/genética , Linfoma de Células T/genética , Camundongos , Vírus da Leucemia Murina de Moloney/genética , Receptor Notch1 , Receptores Notch , Sequências Repetitivas de Ácido Nucleico , Deleção de Sequência , Translocação Genética , Transplante Isogênico , Integração ViralRESUMO
Notch1 signaling is required for T cell development. We have previously demonstrated that expression of a dominant active Notch1 (ICN1) transgene in hematopoietic stem cells (HSCs) leads to thymic-independent development of CD4(+)CD8(+) double-positive (DP) T cells in the bone marrow (BM). To understand the function of Notch1 in early stages of T cell development, we assessed the ability of ICN1 to induce extrathymic T lineage commitment in BM progenitors from mice that varied in their capacity to form a functional pre-T cell receptor (TCR). Whereas mice repopulated with ICN1 transduced HSCs from either recombinase deficient (Rag-2(-/)-) or Src homology 2 domain--containing leukocyte protein of 76 kD (SLP-76)(-/)- mice failed to develop DP BM cells, recipients of ICN1-transduced Rag-2(-/)- progenitors contained two novel BM cell populations indicative of pre-DP T cell development. These novel BM populations are characterized by their expression of CD3 epsilon and pre-T alpha mRNA and the surface proteins CD44 and CD25. In contrast, complementation of Rag-2(-/)- mice with a TCR beta transgene restored ICN1-induced DP development in the BM within 3 wk after BM transfer (BMT). At later time points, this population selectively and consistently gave rise to T cell leukemia. These findings demonstrate that Notch signaling directs T lineage commitment from multipotent progenitor cells; however, both expansion and leukemic transformation of this population are dependent on T cell-specific signals associated with development of DP thymocytes.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Receptores de Superfície Celular , Linfócitos T/fisiologia , Fatores de Transcrição , Animais , Medula Óssea/fisiologia , Linhagem da Célula , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Leucemia de Células T/genética , Camundongos , Camundongos Transgênicos , Receptor Notch1 , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/metabolismo , Transdução de Sinais , Timo/citologiaRESUMO
The superficial cortical fiber cells of the bovine lens contain membrane-associated proteins of 150,000, 80,000, and 78,000 D that cross-react with antisera prepared against red blood cell (RBC) protein 4.1 (Aster, J. C., G. J. Brewer, S. M. Hanash, and H. Maisel, 1984, Biochem. J., 224:609-616). To further study their relationship to protein 4.1, these proteins were immunoprecipitated from detergent extracts of crude lens membranes with purified polyclonal and monoclonal anti-4.1 antibodies and resolved by SDS PAGE. The electrophoretic mobilities of the lens proteins of 80,000 and 78,000 D were found to be identical to bovine RBC protein 4.1a and protein 4.1b, respectively. One- and two-dimensional peptide mapping revealed that a high degree of structural homology exists among all three of the lens 4.1-like proteins and RBC protein 4.1a and protein 4.1b. Despite the large difference in apparent molecular mass, the 150,000-D lens protein showed only minor peptide map differences. A nitrocellulose filter overlay assay showed that all three of the lens 4.1-like proteins bind to RBC and lens spectrins. We conclude that the bovine lens contains proteins of 80,000 and 78,000 D that are highly similar to protein 4.1 in structure and functional capacity. Additionally, the lens also contains a 4.1 isomorph of 150 kD. Analogous to RBC protein 4.1, these proteins may function in the lens by promoting association of spectrin with actin and by playing a role in the coupling of lens cytoskeleton to plasma membrane.
Assuntos
Proteínas Sanguíneas/metabolismo , Proteínas do Citoesqueleto , Cristalino/metabolismo , Neuropeptídeos , Espectrina/metabolismo , Animais , Anticorpos Monoclonais , Proteínas Sanguíneas/imunologia , Proteínas de Transporte/metabolismo , Bovinos , Diferenciação Celular , Reações Cruzadas , Cristalino/citologia , Proteínas de Membrana/metabolismo , Peso Molecular , Fragmentos de Peptídeos/análise , Proteínas Quinases/metabolismoRESUMO
A biochemically active conjugate of calmodulin and tetramethylrhodamine isothiocyanate (CaM-RITC) was synthesized. When incubated with sections of chick lens, this conjugate bound to the surface membranes of lens fiber cells in the presence of absence of calcium. Incubation of lens sections with antibodies to gap junction protein of lens completely blocked the binding of the conjugate to cell membranes, whereas serum from nonimmunized animals or antibodies to others lens proteins reduced the binding only slightly. By means of a gel overlay procedure, 125I-labeled calmodulin was found to bind to the gap junction protein of lens, also in a calcium-independent manner. These results support the concept that calmodulin may interact with and regulate gap junctions in living cells.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Calmodulina/metabolismo , Proteínas do Olho/metabolismo , Junções Intercelulares/metabolismo , Cristalino/metabolismo , Proteínas de Membrana/metabolismo , Animais , Galinhas , Conexinas , Cristalino/ultraestruturaRESUMO
Activating mutations in the NOTCH1 gene have been found in about 60% of patients with T-cell acute lymphoblastic leukemia (T-ALL). In order to study the molecular mechanisms by which altered Notch signaling induces leukemia, a zebrafish model of human NOTCH1-induced T-cell leukemia was generated. Seven of sixteen mosaic fish developed a T-cell lymphoproliferative disease at about 5 months. These neoplastic cells extensively invaded tissues throughout the fish and caused an aggressive and lethal leukemia when transplanted into irradiated recipient fish. However, stable transgenic fish exhibited a longer latency for leukemia onset. When the stable transgenic line was crossed with another line overexpressing the zebrafish bcl2 gene, the leukemia onset was dramatically accelerated, indicating synergy between the Notch pathway and the bcl2-mediated antiapoptotic pathway. Reverse transcription-polymerase chain reaction analysis showed that Notch target genes such as her6 and her9 were highly expressed in NOTCH1-induced leukemias. The ability of this model to detect a strong interaction between NOTCH1 and bcl2 suggests that genetic modifier screens have a high likelihood of revealing other genes that can cooperate with NOTCH1 to induce T-ALL.
Assuntos
Transformação Celular Neoplásica/genética , Leucemia-Linfoma de Células T do Adulto/etiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Receptor Notch1/fisiologia , Animais , Animais Geneticamente Modificados , Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Feminino , Raios gama , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Rearranjo Gênico da Cadeia alfa dos Receptores de Antígenos dos Linfócitos T , Genes bcl-2 , Humanos , Leucemia-Linfoma de Células T do Adulto/genética , Masculino , Mosaicismo , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Transplante de Neoplasias , Oncogenes , Quimera por Radiação , Tolerância a Radiação , Receptor Notch1/genética , Proteínas Recombinantes de Fusão/fisiologia , Transdução de Sinais , Fatores de Tempo , Peixe-Zebra , Proteínas de Peixe-Zebra/fisiologiaRESUMO
The t(5;12)(q33;p13) translocation associated with chronic myelomonocytic leukemia (CMML) generates a TEL/PDGFbetaR fusion gene. Here, we used a murine bone marrow transplant (BMT) assay to test the transforming properties of TEL/PDGFbetaR in vivo. TEL/PDGFbetaR, introduced into whole bone marrow by retroviral transduction, caused a rapidly fatal myeloproliferative disease that closely recapitulated human CMML. TEL/PDGFbetaR transplanted mice developed leukocytosis with Gr-1(+) granulocytes, splenomegaly, evidence of extramedullary hematopoiesis, and bone marrow fibrosis, but no lymphoproliferative disease. We assayed mutant forms of the TEL/PDGFbetaR fusion protein - including 8 tyrosine to phenylalanine substitutions at phosphorylated PDGFbetaR sites to which various SH2 domain-containing signaling intermediates bind - for ability to transform hematopoietic cells. All of the phenylalanine (F-) mutants tested conferred IL-3-independence to a cultured murine hematopoietic cell line, but, in the BMT assay, different F-mutants displayed distinct transforming properties. In transplanted animals, tyrosines 579/581 proved critical for the development of myeloproliferative phenotype. F-mutants with these residues mutated showed no sign of myeloproliferation but instead developed T-cell lymphomas. In summary, TEL/PDGFbetaR is necessary and sufficient to induce a myeloproliferative disease in a murine BMT model, and PDGFbetaR residues Y579/581 are required for this phenotype.
Assuntos
Transformação Celular Neoplásica , Proteínas de Ligação a DNA/metabolismo , Leucemia Mielomonocítica Aguda/etiologia , Proteínas de Fusão Oncogênica/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Repressoras , Fatores de Transcrição/metabolismo , Tirosina/metabolismo , Animais , Células Clonais , Proteínas de Ligação a DNA/genética , Rearranjo Gênico do Linfócito T , Técnicas de Transferência de Genes , Vetores Genéticos , Linfoma de Células T , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Proto-Oncogênicas c-ets , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Retroviridae/genética , Transdução de Sinais , Síndrome , Transplante de Tecidos , Fatores de Transcrição/genética , Integração Viral , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
Signaling through Notch receptors has been implicated in the control of cellular differentiation in animals ranging from nematodes to humans. Starting from a human expressed sequence tag-containing sequence resembling that of Serrate, the gene for a ligand of Drosophila melanogaster Notch, we assembled a full-length cDNA, now called human Jagged2, from overlapping cDNA clones. The full-length cDNA encodes a polypeptide having extensive sequence homology to Serrate (40.6% identity and 58.7% similarity) and even greater homology to several putative mammalian Notch ligands that have subsequently been described. When in situ hybridization was performed, expression of the murine Jagged2 homolog was found to be highest in fetal thymus, epidermis, foregut, dorsal root ganglia, and inner ear. In Northern blot analysis of RNA from tissues of 2-week-old mice, the 5.0-kb Jagged2 transcript was most abundant in heart, lung, thymus, skeletal muscle, brain, and testis. Immunohistochemistry revealed coexpression of Jagged2 and Notch1 within thymus and other fetal murine tissues, consistent with interaction of the two proteins in vivo. Coculture of fibroblasts expressing human Jagged2 with murine C2C12 myoblasts inhibited myogenic differentiation, accompanied by increased Notch1 and the appearance of a novel 115-kDa Notch1 fragment. Exposure of C2C12 cells to Jagged2 led to increased amounts of Notch mRNA as well as mRNAs for a second Notch receptor, Notch3, and a second Notch ligand, Jagged1. Constitutively active forms of Notchl in C2C12 cells also induced increased levels of the same set of mRNAs, suggesting positive feedback control of these genes initiated by binding of Jagged2 to Notch1. This feedback control may function in vivo to coordinate differentiation across certain groups of progenitor cells adopting identical cell fates.
Assuntos
Proteínas de Transporte/genética , DNA Complementar/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Membrana/metabolismo , Receptores de Superfície Celular , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Proteínas de Transporte/análise , Proteínas de Transporte/metabolismo , Diferenciação Celular , Técnicas de Cocultura , Fibroblastos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Proteína Jagged-2 , Ligantes , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Músculos/química , Músculos/citologia , Especificidade de Órgãos , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/análise , Receptor Notch1 , Receptor Notch3 , Receptor Notch4 , Receptores Notch , Homologia de Sequência de AminoácidosRESUMO
Notch receptors participate in a conserved signaling pathway that controls the development of diverse tissues and cell types, including lymphoid cells. Signaling is normally initiated through one or more ligand-mediated proteolytic cleavages that permit nuclear translocation of the intracellular portion of the Notch receptor (ICN), which then binds and activates transcription factors of the Su(H)/CBF1 family. Several mammalian Notch receptors are oncogenic when constitutively active, including Notch1, a gene initially identified based on its involvement in a (7;9) chromosomal translocation found in sporadic T-cell lymphoblastic leukemias and lymphomas (T-ALL). To investigate which portions of ICN1 contribute to transformation, we performed a structure-transformation analysis using a robust murine bone marrow reconstitution assay. Both the ankyrin repeat and C-terminal transactivation domains were required for T-cell leukemogenesis, whereas the N-terminal RAM domain and a C-terminal domain that includes a PEST sequence were nonessential. Induction of T-ALL correlated with the transactivation activity of each Notch1 polypeptide when fused to the DNA-binding domain of GAL4, with the exception of polypeptides deleted of the ankyrin repeats, which lacked transforming activity while retaining strong transactivation activity. Transforming polypeptides also demonstrated moderate to strong activation of the Su(H)/CBF1-sensitive HES-1 promoter, while polypeptides with weak or absent activity on this promoter failed to cause leukemia. These experiments define a minimal transforming region for Notch1 in T-cell progenitors and suggest that leukemogenic signaling involves recruitment of transcriptional coactivators to ICN1 nuclear complexes.
Assuntos
Repetição de Anquirina , Transformação Celular Neoplásica/patologia , Leucemia de Células T/patologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Receptores de Superfície Celular , Fatores de Transcrição , Ativação Transcricional , Animais , Transplante de Medula Óssea , Transformação Celular Neoplásica/metabolismo , Células Clonais , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imunofenotipagem , Leucemia de Células T/genética , Leucemia de Células T/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/genética , Estrutura Terciária de Proteína , Receptor Notch1 , Proteínas Recombinantes de Fusão , Retroviridae/genética , Deleção de Sequência , TransfecçãoRESUMO
Notch receptors participate in a highly conserved signaling pathway that regulates morphogenesis in multicellular animals. Maturation of Notch receptors requires the proteolytic cleavage of a single precursor polypeptide to produce a heterodimer composed of a ligand-binding extracellular domain (N(EC)) and a single-pass transmembrane signaling domain (N(TM)). Notch signaling has been correlated with additional ligand-induced proteolytic cleavages, as well as with nuclear translocation of the intracellular portion of N(TM) (N(ICD)). In the current work, we show that the N(EC) and N(TM) subunits of Drosophila Notch and human Notch1 (hN1) interact noncovalently. N(EC)-N(TM) interaction was disrupted by 0.1% sodium dodecyl sulfate or divalent cation chelators such as EDTA, and stabilized by millimolar Ca(2+). Deletion of the Ca(2+)-binding Lin12-Notch (LN) repeats from the N(EC) subunit resulted in spontaneous shedding of N(EC) into conditioned medium, implying that the LN repeats are important in maintaining the interaction of N(EC) and N(TM). The functional consequences of EDTA-induced N(EC) dissociation were studied by using hN1-expressing NIH 3T3 cells. Treatment of these cells for 10 to 15 min with 0.5 to 10 mM EDTA resulted in the rapid shedding of N(EC), the transient appearance of a polypeptide of the expected size of N(ICD), increased intranuclear anti-Notch1 staining, and the transient activation of an Notch-sensitive reporter gene. EDTA treatment of HeLa cells expressing endogenous Notch1 also stimulated reporter gene activity to a degree equivalent to that resulting from exposure of the cells to the ligand Delta1. These findings indicate that receptor activation can occur as a consequence of N(EC) dissociation, which relieves inhibition of the intrinsically active N(TM) subunit.
Assuntos
Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais , Células 3T3 , Animais , Dimerização , Drosophila , Proteínas de Drosophila , Humanos , Transporte de Íons , Proteínas de Membrana/genética , Camundongos , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores NotchRESUMO
Over the last decade, genetic characterization of T-cell acute lymphoblastic leukemia (T-ALL) has led to the identification of a variety of chromosomal abnormalities. In this study, we used array-comparative genome hybridization (array-CGH) and identified a novel recurrent 9q34 amplification in 33% (12/36) of pediatric T-ALL samples, which is therefore one of the most frequent cytogenetic abnormalities observed in T-ALL thus far. The exact size of the amplified region differed among patients, but the critical region encloses approximately 4 Mb and includes NOTCH1. The 9q34 amplification may lead to elevated expression of various genes, and MRLP41, SSNA1 and PHPT1 were found significantly expressed at higher levels. Fluorescence in situ hybridization (FISH) analysis revealed that this 9q34 amplification was in fact a 9q34 duplication on one chromosome and could be identified in 17-39 percent of leukemic cells at diagnosis. Although this leukemic subclone did not predict for poor outcome, leukemic cells carrying this duplication were still present at relapse, indicating that these cells survived chemotherapeutic treatment. Episomal NUP214-ABL1 amplification and activating mutations in NOTCH1, two other recently identified 9q34 abnormalities in T-ALL, were also detected in our patient cohort. We showed that both of these genetic abnormalities occur independently from this newly identified 9q34 duplication.
Assuntos
Cromossomos Humanos Par 9 , Duplicação Gênica , Leucemia-Linfoma de Células T do Adulto/genética , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Hibridização in Situ Fluorescente , Lactente , Masculino , Proteínas de Fusão Oncogênica/genética , Valor Preditivo dos Testes , Receptor Notch1/genética , Recidiva , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Activating mutations in NOTCH1 are found in over 50% of human T-cell lymphoblastic leukemias (T-ALLs). Here, we report the analysis for activating NOTCH1 mutations in a large number of acute myeloid leukemia (AML) primary samples and cell lines. We found activating mutations in NOTCH1 in a single M0 primary AML sample, in three (ML1, ML2 and CTV-1) out of 23 AML cell lines and in the diagnostic (myeloid) and relapsed (T-lymphoid) clones in a patient with lineage switch leukemia. Importantly, the ML1 and ML2 AML cell lines are derived from an AML relapse in a patient initially diagnosed with T-ALL. Overall, these results demonstrate that activating mutations in NOTCH1 are mostly restricted to T-ALL and are rare in AMLs. The presence of NOTCH1 mutations in myeloid and T-lymphoid clones in lineage switch leukemias establishes the common clonal origin of the diagnostic and relapse blast populations and suggests a stem cell origin of NOTCH1 mutations during the molecular pathogenesis of these tumors.
Assuntos
Linhagem da Célula/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide/genética , Leucemia Mieloide/patologia , Receptor Notch1/genética , Doença Aguda , Sequência de Bases , Linhagem Celular Tumoral , Deleção de Genes , Células-Tronco Hematopoéticas/patologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Mutação Puntual , Recidiva , Linfócitos T/patologiaRESUMO
Notch receptors have been implicated as oncogenic drivers in several cancers, the most notable example being NOTCH1 in T-cell acute lymphoblastic leukemia (T-ALL). To characterize the role of activated NOTCH3 in cancer, we generated an antibody that detects the neo-epitope created upon gamma-secretase cleavage of NOTCH3 to release its intracellular domain (ICD3), and sequenced the negative regulatory region (NRR) and PEST (proline, glutamate, serine, threonine) domain coding regions of NOTCH3 in a panel of cell lines. We also characterize NOTCH3 tumor-associated mutations that result in activation of signaling and report new inhibitory antibodies. We determined the structural basis for receptor inhibition by obtaining the first co-crystal structure of a NOTCH3 antibody with the NRR protein and defined two distinct epitopes for NRR antibodies. The antibodies exhibit potent anti-leukemic activity in cell lines and tumor xenografts harboring NOTCH3 activating mutations. Screening of primary T-ALL samples reveals that 2 of 40 tumors examined show active NOTCH3 signaling. We also identified evidence of NOTCH3 activation in 12 of 24 patient-derived orthotopic xenograft models, 2 of which exhibit activation of NOTCH3 without activation of NOTCH1. Our studies provide additional insights into NOTCH3 activation and offer a path forward for identification of cancers that are likely to respond to therapy with NOTCH3 selective inhibitory antibodies.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Mutação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Receptor Notch3/antagonistas & inibidores , Receptor Notch3/genética , Substituição de Aminoácidos , Animais , Linhagem Celular Tumoral , Códon , Modelos Animais de Doenças , Epitopos/química , Epitopos/imunologia , Feminino , Humanos , Camundongos , Modelos Moleculares , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Conformação Proteica , Receptor Notch3/química , Receptor Notch3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The diagnostic category of Burkitt's lymphoma encompasses a closely related group of aggressive B-cell tumors that includes sporadic, endemic, and human immunodeficiency virus-associated subtypes. All subtypes are characterized by chromosomal rearrangements involving the c-myc proto-oncogene that lead to its inappropriate expression. This review focuses on the roles of c-myc dysregulation and Epstein-Barr virus infection in Burkitt's lymphoma. Although the normal function of c-Myc remains enigmatic, recent data indicate that it has a central role in several fundamental aspects of cellular biology, including proliferation, differentiation, metabolism, apoptosis, and telomere maintenance. We discuss new insights into the molecular mechanisms of these c-Myc activities and their potential relevance to the pathogenesis of Burkitt's lymphoma and speculate on the role of Epstein-Barr virus.
Assuntos
Linfoma de Burkitt/genética , Animais , Linfoma de Burkitt/patologia , Regulação Neoplásica da Expressão Gênica , Genes myc/genética , Humanos , Proto-Oncogene Mas , Translocação GenéticaRESUMO
PURPOSE: To determine the incidence, natural history, and risk factors associated with myelodysplastic syndrome (MDS) occurring as a late complication following autologous bone marrow transplantation for patients with non-Hodgkin's lymphoma. METHODS: We retrospectively reviewed the charts of all 262 patients who underwent autologous bone marrow transplantation for non-Hodgkin's lymphoma at the Dana-Farber Cancer Institute from 1982 through 1991. Although patients received a variety of treatments before they were eligible for transplant, identical myeloablative therapy (cyclophosphamide 60 mg/kg/d for 2 days plus total-body irradiation twice daily for 3 days) was administered in each case. By collecting data on pretransplant and early posttransplant variables, we attempted to identify risk factors for the development of MDS. RESULTS: The crude overall incidence of posttransplant MDS or acute myeloid leukemia (AML) was 7.6%. The actuarial risk at 6 years was 18% +/- 9%. The median time of onset was 31 months (range, 10 to 101) after transplant or 69 months (range, 27 to 141) after initial treatment for lymphoma. Pretreatment variables predictive for the development of MDS (univariate analysis) included prolonged interval between initial treatment and the transplant procedure (P = .003), increased duration of exposure to chemotherapy (P = .019) or to alkylating agents (P = .045), and use of radiation therapy (P = .032) or pelvic radiation (P = .003) before transplant. CONCLUSION: MDS is a potential complication of autologous bone marrow transplantation for non-Hodgkin's lymphoma; bone marrow stem-cell damage sustained before the transplant may be the most important risk factor.
Assuntos
Transplante de Medula Óssea/efeitos adversos , Linfoma não Hodgkin/terapia , Síndromes Mielodisplásicas/etiologia , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Terapia Combinada , Feminino , Humanos , Incidência , Leucemia Mieloide Aguda/epidemiologia , Leucemia Mieloide Aguda/etiologia , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/epidemiologia , Estudos Retrospectivos , Fatores de Risco , Transplante Autólogo , Irradiação Corporal TotalRESUMO
PURPOSE: The role for high-dose therapy and autologous stem-cell transplantation in mantle-cell lymphoma (MCL) is unknown. We retrospectively analyzed patients with chemosensitive disease who underwent high-dose chemoradiotherapy and anti-B-cell monoclonal antibody-purged autologous bone marrow transplantation (ABMT) for MCL in first remission, as well as following relapse from conventional therapy. PATIENTS AND METHODS: Between August 1985 and April 1996, 28 patients underwent ABMT using a uniform ablative regimen with cyclophosphamide and total-body irradiation (TBI) and a bone marrow-purging regimen. Re-review of original tissue demonstrated that all patients had morphologic, phenotypic, and genotypic characteristics of MCL. MCL was the original diagnosis in 21 patients, whereas seven patients had a prior diagnosis of diffuse small cleaved-cell lymphoma. RESULTS: Twenty patients received multiple regimens before ABMT, while eight underwent ABMT in first complete remission (CR)/partial remission (PR) following CHOP induction. At bone marrow harvest, only 18% of patients were in CR and overt BM infiltration was present in 57%. Following cyclophosphamide/TBI, no treatment-related deaths were seen. Nineteen of 28 patients have relapsed at a median time of 21 months (range, 3 to 70). Of eight patients transplanted in first CR/PR, five have relapsed. Nine patients are in continuous CR with a median follow-up time of 24 months (range, 10 to 135). Disease-free survival (DFS) and overall survival (OS) are estimated to be 31% and 62% at 4 years, respectively. CONCLUSION: ABMT using cyclophosphamide/TBI conditioning may at best be effective in only a small fraction of patients with relapsed MCL. The lack of plateau with a median follow-up time of 24 months suggests cure may not be achievable. The role of this therapy in patients in first remission requires more study using better induction therapy to enhance the CR rate before ABMT.
Assuntos
Anticorpos Monoclonais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfócitos B/imunologia , Purging da Medula Óssea/métodos , Transplante de Medula Óssea/métodos , Linfoma não Hodgkin/terapia , Adulto , Idoso , Terapia Combinada , Ciclofosfamida/administração & dosagem , Doxorrubicina/administração & dosagem , Feminino , Humanos , Linfoma não Hodgkin/patologia , Masculino , Pessoa de Meia-Idade , Prednisona/administração & dosagem , Prognóstico , Estudos Retrospectivos , Transplante Autólogo , Resultado do Tratamento , Vincristina/administração & dosagemRESUMO
The cellular transcriptional repressor RBP-Jkappa associates with the Epstein-Barr virus nuclear antigens (EBNAs) determined to be essential for transformation of human primary B lymphocytes. It was demonstrated through genetic analysis that interaction between the viral transactivator EBNA2 and RBP-Jkappa is essential for EBV immortalization of primary B lymphocytes. We have shown that the association of RBP-Jkappa with intracellular NOTCH1 differs significantly in B and T cells. Immunoprecipitation analyses with antibodies to both the intracellular forms of NOTCH1 and to RBP-Jkappa demonstrated that little or no RBP-Jkappa is associated with NOTCH1 in B cell lines compared to the RBP-Jkappa associated with NOTCH1 in T cell lines and was further demonstrated in human primary lymphocytes. Additionally, EBNA2 can compete with intracellular NOTCH1 for binding to GST-RBP-Jkappa in vitro. Northern blot for the cellular gene hairy enhancer of split (HES1) demonstrated that HES1 is upregulated in the EBV transformed lymphoblastoid cells expressing high levels of EBNA2 and in a T cell line SupT1 overexpressing intracellular activated NOTCH1. Hence, EBNA2 may be able to compete for the available pool of RBP-Jkappa more effectively in human B cells than in T cells and provides a possible explanation for the ability of EBV to potently and efficiently infect and immortalize human B cells. Leukemia (2000) 14, 84-92.
Assuntos
Linfócitos B/metabolismo , Proteínas de Ligação a DNA/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr , Proteínas de Membrana/metabolismo , Proteínas Nucleares , Receptores de Superfície Celular , Linfócitos T/metabolismo , Fatores de Transcrição , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Ligação Competitiva , Transformação Celular Neoplásica , Proteínas de Homeodomínio/biossíntese , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina , Ligação Proteica , Receptor Notch1 , Fatores de Transcrição HES-1 , Células Tumorais Cultivadas , Proteínas Virais/metabolismoRESUMO
Five missense mutations in the FCH/LCCL domain of the COCH gene, encoding the protein cochlin, are pathogenic for the autosomal dominant hearing loss and vestibular dysfunction disorder, DFNA9. To date, the function of cochlin and the mechanism of pathogenesis of the mutations are unknown. We have used the biological system of transient transfections of the entire protein coding region of COCH into several mammalian cell lines, to investigate various functional properties of cochlin. By western blot analysis of lysates prepared from transfected cells, we show that cochlin is a secreted protein. Immunocytochemistry shows concentrated localisation of cochlin in perinuclear structures consistent with the Golgi apparatus and endoplasmic reticulum, showing intracellular passage through these secretory compartments. We detected that cochlin is proteolytically cleaved between the FCH/LCCL domain and the downstream vWFA domains, resulting in a smaller cochlin isoform of approximately 50 kDa. Interestingly, this isoform lacks the entire mutation bearing FCH/LCCL domain. We have also shown that cochlin is N-glycosylated in its mature secreted form. Previous studies of the FCH/LCCL domain alone, expressed in bacteria, have demonstrated that three of four DFNA9 mutations cause misfolding of this domain. Characteristic eosinophilic deposits in DFNA9 affected inner ear structures could be the result of aberrant folding, secretion, or solubility of mutated cochlins, as in certain other pathological states in which misfolded proteins accumulate and aggregate causing toxicity. To examine the biological consequences of cochlin misfolding, we made separate constructs with three of the DFNA9 mutations and performed parallel studies of the mutated and wild type cochlins. We detected that mutated cochlins are not retained intracellularly, and are able to be secreted adequately by the cells, through the Golgi/ER secretory pathway, and also undergo proteolytic cleavage and glycosylation. These results suggest that DFNA9 mutations may manifest deleterious effects beyond the point of secretion, in the unique environment of the extracellular matrix of the inner ear by disrupting cochlin function or interfering with protein-protein interactions involving the FCH/LCCL domain. It is also possible that the mutations may result in aggregation of cochlin in vivo over a longer time course, as supported by the late onset and progressive nature of this disorder.