RESUMEN
Cellular trafficking of growth factor receptors, including cross-talk among receptors at the cell surface, may be important for signal transduction in normal hematopoietic cells. To test this idea, the signaling domain of Mpl (the thrombopoietin receptor) was targeted to the plasma membrane, or to the cytoplasm of murine marrow cells, and the ability of the cells to proliferate and differentiate in response to Mpl dimerized at the plasma membrane or free in the cytoplasm was assessed. Constructs encoding the signaling domain of Mpl linked to an FK506 binding protein domain (to permit dimerization by the membrane-permeable ligand AP20187) with or without a myristylation sequence (to target the receptor to the plasma membrane) and a hemagglutinin epitope tag were generated and introduced into murine marrow cells using a murine stem cell virus (MSCV)-based retroviral vector. Both populations of transduced marrow cells proliferated in Iscoves modified Dulbecco medium-10% FCS-100 nM AP20187 without exogenous growth factors for more than 100 days and achieved greater than a 10(7)-fold expansion of cells by day 50 (n = 4 transductions). Growth was dimerizer dependent, and myeloid, erythroid, and megakaryocytic progenitors were generated. Activation of Mpl either at the plasma membrane or in the cytoplasm allowed for the terminal maturation of transduced progenitor cells. Introduction of membrane-targeted or cytoplasmic Mpl into fetal liver cells from homozygous JAK2 knock-out mice or wild-type littermates demonstrated that both forms of Mpl require JAK2 for signaling. These data show that the activation of Mpl independent of its normal plasma membrane location can support production of the full range of normal hematopoietic progenitor cells in vitro.
Asunto(s)
Membrana Celular/metabolismo , Células Madre Hematopoyéticas/efectos de los fármacos , Proteínas de la Leche , Proteínas de Neoplasias , Proteínas Proto-Oncogénicas/fisiología , Receptores de Citocinas , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular , Membrana Celular/química , Citoplasma/química , Proteínas de Unión al ADN/farmacología , Proteínas de Unión al ADN/fisiología , Dimerización , Células Madre Hematopoyéticas/citología , Janus Quinasa 2 , Ratones , Microscopía Fluorescente , Transporte de Proteínas , Proteínas Tirosina Quinasas/farmacología , Proteínas Tirosina Quinasas/fisiología , Proteínas Proto-Oncogénicas/farmacología , Receptores de Trombopoyetina , Factor de Transcripción STAT5 , Transducción de Señal , Transactivadores/farmacología , Transactivadores/fisiologíaRESUMEN
STAT5 is activated in a broad spectrum of human hematologic malignancies. We addressed whether STAT5 activation is necessary for the myelo- and lymphoproliferative disease induced by TEL/JAK2 using a genetic approach. Whereas mice transplanted with bone marrow transduced with retrovirus expressing TEL/JAK2 develop a rapidly fatal myelo- and lymphoproliferative syndrome, reconstitution with bone marrow derived from Stat5ab-deficient mice expressing TEL/JAK2 did not induce disease. Disease induction in the Stat5a/b-deficient background was rescued with a bicistronic retrovirus encoding TEL/JAK2 and Stat5a. Furthermore, myeloproliferative disease was induced by reconstitution with bone marrow cells expressing a constitutively active mutant, Stat5a, or a single Stat5a target, murine oncostatin M (mOSM). These data define a critical role for Stat5a/b and mOSM in the pathogenesis of TEL/JAK2 disease.
Asunto(s)
Proteínas de Unión al ADN/genética , Trastornos Linfoproliferativos/fisiopatología , Proteínas de la Leche , Trastornos Mieloproliferativos/fisiopatología , Proteínas de Fusión Oncogénica/genética , Transactivadores/genética , Animales , Southern Blotting , Células de la Médula Ósea/patología , Trasplante de Médula Ósea , Ensayo de Unidades Formadoras de Colonias , ADN de Neoplasias/análisis , Fibrosis , Citometría de Flujo , Técnicas de Transferencia de Gen , Trastornos Linfoproliferativos/genética , Ratones , Ratones Mutantes , Mutagénesis/fisiología , Trastornos Mieloproliferativos/genética , Trasplante de Neoplasias , Oncostatina M , Péptidos/genética , Fenotipo , Retroviridae/genética , Factor de Transcripción STAT5RESUMEN
Many receptors activate phospholipase Cgamma1 or -gamma2. To assess the role of PLCgamma2, we derived enzyme-deficient mice. The mice are viable but have decreased mature B cells, a block in pro-B cell differentiation, and B1 B cell deficiency. IgM receptor-induced Ca2+ flux and proliferation to B cell mitogens are absent. IgM, IgG2a, and IgG3 levels are reduced, and T cell-independent antibody production is absent. The similarity to Btk- or Blnk-deficient mice demonstrates that PLCgamma2 is downstream in Btk/Blnk signaling. FcRgamma signaling is also defective, resulting in a loss of collagen-induced platelet aggregation, mast cell FcepsilonR function, and NK cell FcgammaRIII and 2B4 function. The results define a signal transduction pathway broadly utilized by immunoglobulin superfamily receptors.
Asunto(s)
Linfocitos B/fisiología , Isoenzimas/fisiología , Receptores de IgG/fisiología , Fosfolipasas de Tipo C/fisiología , Proteínas Adaptadoras Transductoras de Señales , Agammaglobulinemia Tirosina Quinasa , Animales , Linfocitos B/metabolismo , Plaquetas/fisiología , Proteínas Portadoras/metabolismo , Hematopoyesis/fisiología , Isoenzimas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfolipasa C gamma , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de IgG/metabolismo , Fosfolipasas de Tipo C/genéticaRESUMEN
SOCS1 is an SH2-containing protein that is primarily expressed in thymocytes in a cytokine- and T cell receptor-independent manner. SOCS1 deletion causes perinatal lethality with death by 2-3 weeks. During this period thymic changes include a loss of cellularity and a switch from predominantly CD4+ CD8+ to single positive cells. Peripheral T cells express activation antigens and proliferate to IL-2 in the absence of anti-CD3. In addition, IFNgamma is present in the serum. Reconstitution of the lymphoid lineage of JAK3-deficient mice with SOCS1-deficient stem cells recapitulates the lethality and T cell alterations. Introducing a RAG2 or IFNgamma deficiency eliminates lethality. The results demonstrate that lymphocytes are critical to SOCS1-associated perinatal lethality and implicate SOCS1 in lymphocyte differentiation or regulation.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Regulación del Desarrollo de la Expresión Génica , Linfocitos/fisiología , Proteínas Represoras , Factores de Edad , Animales , Animales Recién Nacidos , Proteínas de Unión al ADN , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Interferón gamma/farmacología , Janus Quinasa 3 , Ratones , Ratones Mutantes , Proteínas Tirosina Quinasas/fisiología , Proteína 1 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas , Linfocitos T/metabolismo , Timo/embriología , Timo/metabolismo , Distribución TisularRESUMEN
SOCS3 (CIS3/JAB2) is an SH2-containing protein that binds to the activation loop of Janus kinases, inhibiting kinase activity, and thereby suppressing cytokine signaling. During embryonic development, SOCS3 is highly expressed in erythroid lineage cells and is Epo independent. Transgene-mediated expression blocks fetal erythropoiesis, resulting in embryonic lethality. SOCS3 deletion results in an embryonic lethality at 12-16 days associated with marked erythrocytosis. Moreover, the in vitro proliferative capacity of progenitors is greatly increased. SOCS3-deficient fetal liver stem cells can reconstitute hematopoiesis in lethally irradiated adults, indicating that its absence does not disturb bone marrow erythropoiesis. Reconstitution of lymphoid lineages in JAK3-deficient mice also occurs normally. The results demonstrate that SOCS3 is critical in negatively regulating fetal liver hematopoiesis.
Asunto(s)
Eritropoyesis/fisiología , Regulación del Desarrollo de la Expresión Génica , Hígado/embriología , Proteínas/genética , Proteínas/fisiología , Proteínas Represoras , Factores de Transcripción , Animales , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Hematopoyesis/fisiología , Hibridación in Situ , Interleucina-2/farmacología , Interleucina-4/farmacología , Hígado/fisiología , Ratones , Ratones Mutantes , Modelos Genéticos , Mutagénesis , Fenotipo , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas , Factores de Tiempo , TransfecciónAsunto(s)
Citocinas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Leche , Proteínas Represoras , Linfocitos T/citología , Linfocitos T/inmunología , Animales , Proteínas Portadoras/metabolismo , Ciclo Celular , Diferenciación Celular , Proteínas de Unión al ADN/metabolismo , Humanos , Interleucina-7/metabolismo , Factor de Transcripción STAT5 , Transducción de Señal , Proteína 1 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas , Transactivadores/metabolismoRESUMEN
Bcl-2 family proteins are key regulators of apoptosis and function as cell death antagonists (e.g., Bcl-2, Bcl-XL, and Mcl-1) or agonists (e.g., Bax, Bad, and Bak). Here we report that among the Bcl-2 family of proteins tested (Bcl-2, Bcl-XL, Mcl-1, Bax, Bad, and Bak), Bcl-XL was unique in that its protein levels were tightly regulated by hemopoietins in both immortal and primary myeloid progenitors. Investigating signaling pathways utilized by cytokine receptors established that the regulation of Bcl-XL protein levels is mediated by the Jak kinase pathway and is independent of other signaling effectors including STATs, PI-3' kinase, and Ras. Moreover, we provide the first direct evidence that Bcl-X is altered in cancer, because bcl-X expression was activated selectively by retroviral insertions in murine myeloid and T-cell hemopoietic malignancies. Tumors harboring bcl-X insertions had altered bcl-X RNAs, expressed elevated levels of Bcl-XL protein, and lacked the requirements for cytokines normally essential for cell survival. Finally, overexpression of Bcl-XL effectively protected IL-3-dependent myeloid cells from apoptosis following removal of trophic factors. Therefore, Bcl-XL functions as a key cytokine regulated anti-apoptotic protein in myelopoiesis and contributes to leukemia cell survival.
Asunto(s)
Factores de Crecimiento de Célula Hematopoyética/fisiología , Leucemia Mieloide/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas , Animales , Apoptosis , Secuencia de Bases , Transformación Celular Neoplásica , Regulación Neoplásica de la Expresión Génica , Janus Quinasa 2 , Leucemia Mieloide/genética , Leucemia de Células T/genética , Leucemia de Células T/metabolismo , Ratones , Datos de Secuencia Molecular , Mutagénesis Insercional , Proteínas Proto-Oncogénicas c-bcl-2/genética , Retroviridae/genética , Alineación de Secuencia , Células Tumorales Cultivadas , Proteína bcl-XRESUMEN
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.
Asunto(s)
Citocinas/farmacología , Proteínas Tirosina Quinasas/fisiología , Proteínas Proto-Oncogénicas , Receptores de Citocinas/fisiología , Transducción de Señal/fisiología , Animales , Linfocitos B/citología , Diferenciación Celular , Factores Estimulantes de Colonias/farmacología , Proteínas de Unión al ADN/genética , Eritropoyesis/genética , Fibroblastos/citología , Células Madre Hematopoyéticas/citología , Factor 1 Regulador del Interferón , Janus Quinasa 2 , Hígado/citología , Hígado/embriología , Ratones , Ratones Noqueados , Fenotipo , Fosfoproteínas/genética , Fosforilación , Proteínas Tirosina Quinasas/genética , ARN Mensajero/análisis , Linfocitos T/citología , Trombopoyetina/farmacología , Timo/inmunología , Tirosina/metabolismoRESUMEN
A variety of cytokines that regulate functions of multiple lineages share the utilization of receptors that are structurally and functionally related and are referred to as the cytokine receptor superfamily. These receptors associate with one or more of the four mammalian Janus kinases (Jaks) and ligand-induced receptor aggregation results in their activation. Critical roles for Jak3 and Jak2 are demonstrated by the phenotypes of mice that lack each gene. Among the substrates of the Jaks are one or more of the seven members of the signal transducers and activators of transcription (Stats). Each Stat family member plays a critical role in the biological functions of specific cytokines as demonstrated by the phenotype of mice lacking one or more of these genes.
Asunto(s)
Citocinas/fisiología , Proteínas Proto-Oncogénicas , Receptores de Citocinas/fisiología , Transducción de Señal/fisiología , Animales , Proteínas de Unión al ADN/metabolismo , Humanos , Janus Quinasa 2 , Janus Quinasa 3 , Mamíferos , Ratones , Proteínas Tirosina Quinasas/metabolismo , Factor de Transcripción STAT1 , Factor de Transcripción STAT2 , Transactivadores/metabolismoAsunto(s)
Agranulocitosis/genética , Mutación , Neutropenia/genética , Receptores de Factor Estimulante de Colonias de Granulocito/genética , Secuencia de Bases , Cartilla de ADN , Exones , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , SíndromeRESUMEN
Structural alterations occur in the long arm of chromosome 3 in approximately 2% of patients with acute myelogenous leukemia (AML) or myelodysplastic syndrome (MDS). The major alterations are inv(3)(q21q26) and t(3:3)(q21;q26) and are often classified as the 3q21q26 syndrome. We previously reported that the EVI1 gene is transcriptionally activated in AMLs with t(3;3)(q21;q26) and inv(3)(q21q26) and that the chromosomal breakpoints at 3q26 in the translocations were 5' of the EVI1 gene, whereas the breakpoints in the inversion cases were 3' of the gene. In these studies, four additional cases of AML with inv(3)(q21q26) are shown to express the EVI1 gene and to have breakpoints 3' of the gene. To characterize the 3q21 breakpoint region, cosmid and phage clones were isolated that cover approximately 100 kb. At 3q21, the breakpoints for both AMLs with t(3;3)(q21;q26) and inv(3)(q21q26) were found to cluster over a region of approximately 50 kb downstream of the Ribophorin I gene. The results indicate a common mechanism for the translocations and inversions and support the hypothesis that the transcriptional activation of the EVI1 gene is mediated by enhancer elements associated with the Ribophorin I gene.
Asunto(s)
Aberraciones Cromosómicas , Cromosomas Humanos Par 3 , Proteínas de Unión al ADN/genética , Leucemia Mieloide Aguda/genética , Proteínas de la Membrana/genética , Proto-Oncogenes , Transcripción Genética , Adulto , Secuencia de Bases , Southern Blotting , Preescolar , Femenino , Humanos , Proteína del Locus del Complejo MDS1 y EV11 , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Mapeo Restrictivo , Factores de Transcripción/genéticaRESUMEN
The EVI1 gene encodes a zinc-finger, DNA-binding protein originally described as the transforming gene associated with a common ecotropic viral insertion site in myeloid leukemias. Previous studies demonstrated EVI1 expression in human leukemias in cases with 3q26 translocations, but not in normal blood or bone marrow. These studies also suggested an association between EVI1 expression and chromosome 7 deletion (del). Because of this association, we examined expression of EVI1 using RNA polymerase chain reaction (PCR) in patients with myelodysplastic syndromes (MDS) and acute leukemia with and without 3q26 translocations. EVI1 RNA was expressed in 29% of 34 (95% confidence interval, 20% to 50%) patients with the MDS subtypes refractory anemia (RA), refractory anemia with excess blasts (RAEB), or refractory anemia with excess blasts in transformation (RAEB-T). The vast majority of these cases occurred in patients with RAEB and RAEB-T. EVI1 expression was not detected in patients with chronic myelomonocytic leukemia (CMML), normal bone marrow or cord blood, or a variety of other hematologic malignancies. EVI1 RNA was detected in three of 18 patients with acute myelogenous leukemia (AML) and in two of four patients with acute promyelocytic leukemia (APL). Karyotypes showed that only one AML patient had karyotype 3q26 abnormalities, indicating that EVI1 expression is associated with cases that do not have structural abnormalities involving chromosome 3q26. These studies document for the first time the abnormal expression of EVI1 RNA by patients with MDS, and suggest an important role for EVI1 in the pathogenesis or progression of some myeloid malignancies.
Asunto(s)
Cromosomas Humanos Par 3 , Proteínas de Unión al ADN/biosíntesis , Expresión Génica , Leucemia/metabolismo , Síndromes Mielodisplásicos/metabolismo , Proto-Oncogenes , Factores de Transcripción , Translocación Genética , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Médula Ósea/metabolismo , Médula Ósea/patología , Línea Celular , Deleción Cromosómica , Mapeo Cromosómico , Cromosomas Humanos Par 7 , Cartilla de ADN , Proteínas de Unión al ADN/genética , Femenino , Humanos , Cariotipificación , Leucemia/genética , Proteína del Locus del Complejo MDS1 y EV11 , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Síndromes Mielodisplásicos/genética , Reacción en Cadena de la Polimerasa , Células Tumorales Cultivadas , Dedos de ZincRESUMEN
Two genes have been implicated in leukemias of patients with abnormalities of chromosome 3, band q26: EVI1, which can be activated over long distances by chromosomal rearrangements involving 3q26, and EAP, a ribosomal gene that fuses with AML1 in a therapy-related myelodysplasia patient with a t(3;21)(q26.2;q22). AML1 was identified by its involvement in the t(8;21)(q22;q22) of acute myeloid leukemia. Here we report the consistent identification of fusion transcripts between AML1 and EAP or between AML1 and previously unidentified sequences that we named MDS1 (MDS-associated sequences) in the leukemic cells of four patients with therapy-related myelodysplasia/acute myeloid leukemia and in one patient with chronic myelogenous leukemia in blast crisis, all of whom had a t(3;21). In addition, we have identified a third chimeric transcript, AML1/EVI1, in one of the therapy-related acute myeloid leukemia patients. Pulsed-field gel electrophoresis established the order of the genes as EAP, the most telomeric, and EVI1, the most centromeric, gene. The results indicate that translocations could involve multiple genes and affect gene expression over long distances.
Asunto(s)
Leucemia/genética , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogénicas , Proto-Oncogenes , Proteínas Ribosómicas , Factores de Transcripción , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos Par 21 , Cromosomas Humanos Par 3 , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Cartilla de ADN/química , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Genes , Humanos , Proteína del Locus del Complejo MDS1 y EV11 , Datos de Secuencia Molecular , Síndromes Mielodisplásicos/genética , Empalme del ARN , ARN Mensajero/genética , ARN Neoplásico/genética , Proteínas de Unión al ARN/genética , Proteínas Recombinantes de Fusión/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Translocación GenéticaRESUMEN
Specific rearrangements involving 3q21 and 3q26 are well documented in acute myeloid leukemia (AML). Aberrant expression of the Ecotropic virus integration-1 (EVI1) gene, located at 3q26, has been reported in individuals with AML and translocations or inversions of chromosome 3 long arm. We have studied six individuals with AML and inv(3)(q21q26) for disruptions to the EVI1 locus by in situ hybridization and long-range mapping. EVI1 transcripts have been detected in the blast cells of the two individuals available for expression studies. We derived a YAC containing the EVI1 gene and showed that it crossed the 3q26 inversion breakpoints in three of four cases examined. Pulsed field analysis detected aberrant fragments 3' of the EVI1 gene in all six patients. The orientation of the gene was established and the locations of the breakpoints were refined by in situ hybridization using phage clones from this region.
Asunto(s)
Cromosomas Humanos Par 3 , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Leucemia Mieloide Aguda/genética , Proto-Oncogenes , Factores de Transcripción , Secuencia de Bases , Aberraciones Cromosómicas/genética , Trastornos de los Cromosomas , Inversión Cromosómica , Cromosomas Artificiales de Levadura , Clonación Molecular , Cartilla de ADN/química , Reordenamiento Génico , Humanos , Hibridación Fluorescente in Situ , Proteína del Locus del Complejo MDS1 y EV11 , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Neoplásico/genética , Mapeo Restrictivo , SíndromeRESUMEN
Retroviral activation of Evi-1 gene expression is one of the most common transforming events in murine myeloid leukemias. To evaluate the role of the EVI1 gene in human acute myelogenous leukemia (AML), leukemic blasts or cell lines from 116 patients were examined. In eight patients the EVI1 gene was expressed and all but one had cytogenetically detectable translocations of chromosome 3q26 where the EVI1 gene has been localized. To identify breakpoints, a restriction map that spans 1700 kilobases (kb) of the EVI1 locus was developed by pulsed-field gel electrophoresis. In one case, t(3;3)(q21;q26), a rearrangement was localized to 170-330 kb 5' of the gene. In a second case, t(3;3)(q21;q26), there was a rearrangement 13 kb 5' of the gene. This rearrangement was cloned and shown to be due to the fusion of sequences from 3q21-22 with the EVI1 locus. In the third case, ins(3)-(q21q25q27), there was a rearrangement that mapped 150 kb downstream from the 5' end of the gene.
Asunto(s)
Cromosomas Humanos Par 3 , Proteínas de Unión al ADN/genética , Leucemia Mieloide Aguda/genética , Proto-Oncogenes , Factores de Transcripción , Secuencia de Bases , Aberraciones Cromosómicas/genética , Trastornos de los Cromosomas , ADN de Neoplasias/genética , Expresión Génica , Reordenamiento Génico , Humanos , Proteína del Locus del Complejo MDS1 y EV11 , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , ARN Mensajero/genética , Mapeo Restrictivo , Translocación Genética , Dedos de ZincRESUMEN
Expression of the Evi-1 gene is frequently activated in murine myeloid leukemias by retroviral insertions immediately 5' or 90 kb 5' of the gene. The Evi-1 gene product is a nuclear, DNA-binding zinc finger protein of 145 kDa. On the basis of the properties of the myeloid cell lines in which the Evi-1 gene is activated, it has been hypothesized that its expression blocks normal differentiation. To explore this proposed role, we have constructed a retrovirus vector containing the gene and examined its effects on an interleukin-3-dependent myeloid cell line that differentiates in response to granulocyte colony-stimulating factor (G-CSF). Expression of the Evi-1 gene in these cells did not alter the normal growth factor requirements of the cells. However, expression of the Evi-1 gene blocked the ability of the cells to express myeloperoxidase and to terminally differentiate to granulocytes in response to G-CSF. This effect was not due to altered expression of the G-CSF receptor or to changes in the initial responses of the cells to G-CSF. These results support the hypothesis that the inappropriate expression of the Evi-1 gene in myeloid cells interferes with the ability of the cells to terminally differentiate.
Asunto(s)
Diferenciación Celular/genética , Proteínas de Unión al ADN/genética , Factor Estimulante de Colonias de Granulocitos/fisiología , Granulocitos/citología , Proto-Oncogenes , Factores de Transcripción , Dedos de Zinc/genética , Animales , División Celular , Transformación Celular Neoplásica/genética , Expresión Génica , Leucemia Mieloide , Proteína del Locus del Complejo MDS1 y EV11 , Ratones , Células Tumorales CultivadasRESUMEN
Activation of the Evi-1 zinc finger gene is commonly associated with the transformation of murine leukemias and is involved in some cases of human AML involving rearrangements at chromosome 3q25. To determine the normal function of the gene, we have looked for expression in a variety of cell lines and tissues. The predominant sites of expression of the gene are in the kidney and ovary. In the kidney, expression is localized to the renal tubules in the corticomedullary junction. In the ovary, high levels of the Evi-1 protein are found in the cytoplasm of developing oocytes. The latter result suggests a potential role for the Evi-1 gene product in early oocyte development.
Asunto(s)
Proteínas de Unión al ADN/análisis , Expresión Génica , Riñón , Oocitos , Proto-Oncogenes , Factores de Transcripción , Dedos de Zinc/genética , Animales , Línea Celular , Proteínas de Unión al ADN/genética , Femenino , Humanos , Riñón/química , Túbulos Renales/química , Proteína del Locus del Complejo MDS1 y EV11 , Ratones , Oocitos/química , Oocitos/crecimiento & desarrollo , ARN/genéticaRESUMEN
Retroviral insertional activation of the expression of the Evi-1 is one of the most common events associated with transformation in murine myeloid leukemia. The murine Evi-1 gene encodes a 145 kDa nuclear, DNA binding protein that contains two domains containing seven and three sets of repeats of the zinc finger motif. During studies to determine the role of the Evi-1 gene in the transformation of human cells, we have found that the Evi-1 gene is uniquely expressed at low levels in HEC-1-A cells and at high levels in HEC-1-B cells, two related human endometrial carcinoma cell lines. cDNA clones were isolated and sequenced from the HEC-1-B cell line. The human gene is highly homologous to the murine gene and shows 91% and 94% homology in nucleotide or amino acid sequence respectively. In addition an alternatively spliced form of the gene was identified that encodes a protein with an internal deletion 315 amino acids including two of the zinc finger repeats. The possible basis for the unique expression of the Evi-1 gene by HEC-1 cells could not be determined by karyotype or Southern blot analysis.
Asunto(s)
Carcinoma/genética , Proteínas de Unión al ADN/genética , Genes Relacionados con las Neoplasias , Metaloproteínas/genética , Proto-Oncogenes , Factores de Transcripción/genética , Neoplasias Uterinas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , ADN/genética , ADN de Neoplasias/genética , Femenino , Expresión Génica , Humanos , Cariotipificación , Proteína del Locus del Complejo MDS1 y EV11 , Datos de Secuencia Molecular , Empalme del ARN , ARN Mensajero/genética , ARN Neoplásico/genética , Mapeo Restrictivo , Transcripción Genética , Células Tumorales CultivadasRESUMEN
The murine Evi-1 gene encodes a protein that has multiple 28-amino acid repeats containing the consensus sequence found in the zinc finger domains of many transcriptional regulatory proteins. Activation of the expression of the Evi-1 gene is frequently found in murine myeloid leukemias and leukemia cell lines and is due to retroviral insertions in the 5' region of the gene in either the Evi-1 or the CB-1/FIM3 common sites of viral integrations. To examine the role of the Evi-1 gene in human leukemias we have cloned regions of the human locus corresponding to the coding region of the gene and regions corresponding to the Evi-1 and CB-1/FIM3 common sites of integrations. Using these probes we demonstrate that the human Evi-1 gene maps to chromosome 3q24-q28 in a region that is translocated in acute nonlymphocytic leukemias with a t(3;5)(q25;q34). By in situ hybridization with metaphase chromosomes from one patient with a 3;5 translocation, the Evi-1 gene was found to be translocated to the derivative 5 chromosome. However, no rearrangements were detected by Southern blot analysis with DNAs from three patients with a t(3;5) using probes from the Evi-1 or CB-1/FIM3 loci. No Evi-1 transcripts were detected with RNA from leukemic blasts of one patient with a t(3;5).