RESUMEN
EWS/FLI1, a fusion gene found in Ewing's sarcoma, encodes a transcriptional regulator and promotes cellular transformation by modulating the transcription of specific target genes. We have found that EWS/FLI1 and structurally related fusion proteins upregulate manic fringe (MFNG), a recently described member of the Fringe gene family instrumental in somatic development. MFNG is also expressed in human tumour-derived cell lines expressing EWS/FLI1. Overexpression of MFNG in NIH 3T3 cells renders them tumorigenic in mice with severe combined immunodeficiency disease (SCID). These data demonstrate that part of the oncogenic effect of EWS/FLI1 is to transcriptionally deregulate a member of a family of morphogenic genes.
Asunto(s)
Transformación Celular Neoplásica/genética , Proteínas de Fusión Oncogénica/fisiología , Proteínas/genética , Sarcoma de Ewing/genética , Factores de Transcripción/fisiología , Células 3T3 , Animales , Regulación Neoplásica de la Expresión Génica , Glucosiltransferasas , Ratones , Ratones SCID , Trasplante de Neoplasias , Proteínas de Fusión Oncogénica/genética , Biosíntesis de Proteínas , Proteína Proto-Oncogénica c-fli-1 , Proteína EWS de Unión a ARN , Sarcoma de Ewing/etiología , Factores de Transcripción/genética , Células Tumorales CultivadasRESUMEN
More than 85% of Ewing's family tumors carry a specific chromosomal translocation that fuses the NH(2) terminus of the EWS gene to the COOH terminus of the FLI1 transcription factor. It has been shown previously that both the transactivation domain encoded by EWS and the DNA binding domain of FLI1 were necessary for transforming cells to anchorage independence. We now report that a COOH-terminal domain in addition to the FLI1 DNA binding domain is necessary to promote cellular transformation. NIH 3T3 cells expressing a COOH-terminal deletion mutant (EWS/FLI1 DeltaC) have a greatly reduced capability to form colonies in soft agar and tumors in severe combined immunodeficient mice. The rate of tumor formation for NIH 3T3 that express EWS/FLI1 DeltaC is 50 days, whereas EWS/FLI1 forms tumors within 22 days. In addition, cells expressing the EWS/FLI1 DeltaC mutant failed to completely demonstrate the round-cell histology that is seen in both Ewing's tumor cell lines and NIH 3T3 cells expressing full-length EWS/FLI1. Northern and microarray analyses were performed to assess the effect of loss of the FLI1 COOH terminus on transcriptional modulation of EWS/FLI1 target genes. We found that although EWS/FLI1 DeltaC up-regulates smaller numbers of genes (21 genes) compared with EWS/FLI1 (34 genes), 41% of the EWS/FLI1 targets were also up-regulated by EWS/FLI1 DeltaC. On the other hand, EWS/FLI1 DeltaC is unable to down-regulate genes (3 genes) as efficiently as EWS/FLI1 (39 genes) with only one target gene repressed by both fusion constructs. Our study indicates that the EWS/FLI1 transcription factor has strong transcriptional activating as well as repressing properties and suggests that transcriptional activation and repression of target genes may occur through biochemically different mechanisms.
Asunto(s)
Transformación Celular Neoplásica , Proteínas de Unión al ADN/fisiología , Proteínas de Fusión Oncogénica/fisiología , Proteínas Proto-Oncogénicas , Transactivadores/fisiología , Factores de Transcripción/fisiología , Células 3T3 , Secuencia de Aminoácidos , Animales , Adhesión Celular/fisiología , División Celular/fisiología , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas de Fusión Oncogénica/genética , Estructura Terciaria de Proteína , Proteína Proto-Oncogénica c-fli-1 , Proteína EWS de Unión a ARN , Alineación de Secuencia , Relación Estructura-Actividad , Transactivadores/genética , Factores de Transcripción/genética , Activación Transcripcional/fisiologíaRESUMEN
Tumor-associated chromosomal translocations lead to the formation of chimeric fusions between the EWS gene and one of five different ETS transcription factors in Ewing's family tumors (EFTs). The resultant EWS/ETS proteins promote oncogenesis in a dominant fashion in model systems and are necessary for continued growth of EFT cell lines. EWS belongs to a family of genes that encode proteins that may serve as adapters between the RNA polymerase II complex and RNA splicing factors. EWS/ETS fusions have biochemical characteristics of aberrant transcription factors and appear to promote abnormal cellular growth by transcriptionally modulating a network of target genes. Early evidence suggests that EWS/ETS proteins may also impact gene expression through alteration in RNA processing. Elucidation of EWS/ETS target gene networks in the context of other signaling pathways will hopefully lead to biology based therapeutic strategies for EFT.
Asunto(s)
Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Ribonucleoproteínas/química , Sarcoma de Ewing/genética , División Celular , Ribonucleoproteínas Nucleares Heterogéneas , Cariotipificación , Modelos Biológicos , Modelos Genéticos , Familia de Multigenes , Neoplasias/etiología , Neoplasias/genética , Unión Proteica , Estructura Terciaria de Proteína , ARN/metabolismo , Proteína EWS de Unión a ARN , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Translocación GenéticaRESUMEN
Ewing's sarcomas express chimeric transcription factors resulting from a fusion of the amino terminus of the EWS gene to the carboxyl terminus of one of five ETS proteins. While the majority of tumors express EWS/FLI1 fusions, some Ewing's tumors contain variant chimeras such as EWS/ETV1 that have divergent ETS DNA-binding domains. In spite of their structural differences, both EWS/ETS fusions up regulate EAT-2, a previously described EWS/FLI1 target gene. In contrast to EWS/FLI1, NIH3T3 cells expressing EWS/ETV1 cannot form colonies in soft agar though coexpression of a dominant negative truncated ETV1 construct attenuates EWS/FLI1 mediated anchorage independent growth. When EWS/ETV1 or EWS/FLI1 expressing NIH3T3 cells are injected into SCID mice, tumors form more often and faster than with NIH-3T3 cells with empty vector controls. The tumorigenic potency of each EWS/ETS fusion is linked to its C-terminal structure, with the FLI1 C-terminus confering a greater tumorigenic potential than the corresponding ETV1 domain. The resulting EWS/ETV1 and EWS/FLI1 tumors closely resemble each other at both a macroscopic and a microscopic level. These tumors differ greatly from tumors formed by NIH3T3 cells expressing activated RAS. These data indicate that in spite of their structural differences, EWS/ETV1 and EWS/FLI1 promote oncogenesis via similar biologic pathways.
Asunto(s)
Neoplasias Óseas/genética , Transformación Celular Neoplásica/genética , Proteínas de Fusión Oncogénica/fisiología , Sarcoma de Ewing/genética , Factores de Transcripción/fisiología , Células 3T3/patología , Células 3T3/trasplante , Proteínas Adaptadoras Transductoras de Señales , Animales , Regulación Neoplásica de la Expresión Génica , Genes ras , Humanos , Ratones , Ratones SCID , Trasplante de Neoplasias , Proteínas de Fusión Oncogénica/genética , Fenotipo , Proteína Proto-Oncogénica c-fli-1 , Proteína EWS de Unión a ARN , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
The EWS/FLI1 fusion gene found in Ewing's sarcoma and primitive neuroectodermal tumor, is able to transform certain cell lines by acting as an aberrant transcription factor. The ability of EWS/FLI1 to modulate gene expression in cells transformed and resistant to transformation by EWS/FLI1, was assessed by Representational Difference Analysis (RDA). We found that the cyclin selective ubiquitin conjugase murine E2-C, was up regulated in NIH3T3 cells transformed by EWS/FLI1 but not in a nontransformed NIH3T3 clone expressing EWS/FLI1. We also found that mE2-C is upregulated in NIH3T3 cells transformed by other genes including activated cdc42, v-ABL and c-myc. We demonstrated that expression of mE2-C in both the EWS/FLI1 transformed and parent NIH3T3 lines varies with the cell cycle. Finally, dominant-negative mE2-C, created by changing a catalytic cysteine to serine, inhibits the in vitro ubiquitination and degradation of cyclin B in human HeLa cell extracts. These data suggest that part of the biologic effect of EWS/FLI1 could be to transcriptionally modulate genes involved in cell cycle regulation.
Asunto(s)
Proteínas Portadoras/biosíntesis , Ciclina B/metabolismo , Ligasas , Proteínas de Fusión Oncogénica/metabolismo , Factores de Transcripción/metabolismo , Enzimas Ubiquitina-Conjugadoras , Regulación hacia Arriba , Células 3T3 , Secuencia de Aminoácidos , Animales , Ciclo Celular , Línea Celular Transformada , Transformación Celular Neoplásica , Sistema Libre de Células , Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas de Fusión Oncogénica/genética , Proteína Proto-Oncogénica c-fli-1 , Proteína EWS de Unión a ARN , Factores de Transcripción/genéticaRESUMEN
The EWS/FLI1 fusion protein is created by the translocation between chromosomes 11 and 22 that appears in most Ewing's sarcomas. This chimeric protein has been demonstrated to be an aberrant transcription factor. Genes up regulated by EWS/FLI1 but not by full-length FLI1 were identified by representational difference analysis (RDA). We have characterized a novel gene, EWS/FLI1 activated transcript 2 (EAT-2) that was cloned from a murine cDNA library using a differentially expressed RDA fragment. EAT-2 expression is seen within 4-8 h of EWS/FLI1 induction. Its expression correlates with transformation of NIH3T3 cells by chimeric proteins related to EWS/FLI1 but not by unrelated genes. EAT-2 is expressed in normal murine tissues and contains a unique but biochemically functional SH2 domain. An homologous sequence in the human genome has been identified and mapped to chromosome 1q22. Human EAT-2 transcripts were identified by reverse transcriptase-polymerase chain reaction (RT-PCR) in Ewing's sarcoma cell tumour cell lines. EAT-2's unique structure and correlation with transformation make it a candidate for playing a role in the transformation of NIH3T3 cells and the oncogenesis of Ewing's sarcoma.
Asunto(s)
Cromosomas Humanos Par 1/genética , Proteínas de Unión al ADN/genética , Proteínas Proto-Oncogénicas , Proteínas Recombinantes de Fusión/genética , Proteínas Oncogénicas de Retroviridae/genética , Ribonucleoproteínas/genética , Sarcoma de Ewing/genética , Transactivadores/genética , Factores de Transcripción/genética , Transcripción Genética , Dominios Homologos src/genética , Células 3T3 , Proteínas Adaptadoras Transductoras de Señales , Animales , Secuencia de Bases , Mapeo Cromosómico , Proteínas de Unión al ADN/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas , Humanos , Ratones , Datos de Secuencia Molecular , Fosfotirosina/metabolismo , Reacción en Cadena de la Polimerasa , Proteína Proto-Oncogénica c-fli-1 , Proteína EWS de Unión a ARN , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Oncogénicas de Retroviridae/metabolismo , Ribonucleoproteínas/metabolismo , Sarcoma de Ewing/metabolismo , Transducción de Señal/genética , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
Specific chromosomal translocations involving the ews gene and one of five members of the ets family of transcription factors create ews/ets fusion genes that are found in approximately 85% of Ewing's family of tumors. ews/ets fusion genes consistently maintain an intact and functional ets DNA binding domain (DBD) in all of these cases. We demonstrate here, however, that EWS/FLI1, the most prevalent EWS/ETS fusion, activates oncogenic pathways independent of its DBD. In in vivo tumor assays, EWS/FLI1 molecules with either point mutations or a large deletion in the ets DBD retain the ability to accelerate tumors in NIH 3T3 cells, whereas they lose the ability to bind DNA in vitro. Additionally, whereas inhibition of DBD functions of EWS/FLI1 with a dominant negative form of FLI1 is sufficient to inhibit anchorage-independent growth in NIH 3T3 cells, it is ineffective in inhibiting tumor growth in SCID mice. Usage of this dominant negative construct in a Ewing's tumor cell line, however, does reduce the rate of tumor formation, supporting the need for a functional DBD in this context. Together, these results suggest that EWS/FLI1 induces both DBD-dependent and DBD-independent oncogenic pathways.
Asunto(s)
ADN/metabolismo , Neoplasias Experimentales/etiología , Proteínas de Fusión Oncogénica/fisiología , Factores de Transcripción/fisiología , Células 3T3 , Animales , Sitios de Unión , Transformación Celular Neoplásica , Femenino , Ratones , Ratones SCID , Proteínas de Fusión Oncogénica/química , Proteína Proto-Oncogénica c-fli-1 , Proteína EWS de Unión a ARN , Sarcoma de Ewing/etiología , Factores de Transcripción/químicaRESUMEN
Today, rotary pumps are routinely used for extracorporeal circulation in different clinical settings and applications. A review of these applications and specific limitations in extracorporeal perfusion was performed and served as a basis for the development of the DeltaStream. The DeltaStreams is a miniaturized rotary blood pump of a new and unique design with an integrated drive unit. Despite its small design, the pump maintains a sufficient hydraulic capacity, which makes the DeltaStream very flexible for intra- and perioperative applications. It also opens the field for short-term ventricular assist devices (VAD) applications or use as a component in extracorporeal life support systems (ECLS). The DeltaStream and, specifically, its impeller design have been optimized with respect to haemolysis and nonthrombogenicity. Also, the pump facilitates an effective pulse generation in VAD applications and simulates heart action in a more physiological way than other rotary pumps or roller pumps. Hydraulic and haematological properties have been tested in vitro and in vivo. In a series of seven animal experiments in two different setups, the pump demonstrated its biocompatibility and applicability. Basic aspects of the DeltaStream pump concept as well as important console features are presented. A summary of the final investigation of this pump is given with focus on hydraulic capabilities and results from animal studies.
Asunto(s)
Circulación Extracorporea/instrumentación , Corazón Auxiliar , Animales , Diseño de Equipo , Hemoglobinas/análisis , Humanos , Flujo Pulsátil , OvinosRESUMEN
The DeltaStream blood pump has been developed for extracorporeal circulation with one focus on potential integration into simplified bypass systems (SBS). Its small size and an embedded electric motor are the basic pump properties. A variation of the impeller design has been performed to optimize hydraulic and hematologic characteristics. A simple impeller design was developed which allows flow and pressure generation for cardiopulmonary bypass applications. The option of a pulsatile flow mode for ventricular assist device applications also was demonstrated in vitro. Impeller washout holes were implemented to improve nonthrombogenicity. The pump was investigated for potential thermal hazards for blood caused by the integrated electric motor. It could be demonstrated that there is no thermal risk associated with this design. Durability tests were performed to assess the lifetime of the pump especially with regard to the incorporated polymeric seal. Seal lifetimes of up to 28 days were achieved using different blood substitutes. In animal tests using either the pump as a single device or in an SBS setup, biocompatibility, low hemolysis, and nonthrombogenicity were demonstrated. In summary, the DeltaStream pump shows great potential for different extracorporeal perfusion applications. Besides heart-lung machine and SBS applications, ventricular assist and extracorporeal membrane oxygenation up to several days also appear promising as potential applications.