RESUMEN
Hox genes have been identified in chromosomal translocations involving the nucleoporin gene NUP98. Though the resulting chimeric proteins directly participate in the development of leukemia, the long latency and monoclonal nature of the disease support the requirement for secondary mutation(s), such as those leading to overexpression of Meis1. Models to identify such events and to study leukemic progression are rare and labor intensive. Herein, we took advantage of the strong transforming potential of NUP98-HOXD13 or NUP98-HOXA10 to establish preleukemic myeloid lines from bone marrow cells that faithfully replicate the first step of Hox-induced leukemogenesis. These lines contain early granulomonocytic progenitors with extensive in vitro self-renewal capacity, short-term myeloid repopulating activity and low propensity for spontaneous leukemic conversion. We exploit such lines to show that Meis1 efficiently induces their leukemic progression and demonstrate a high frequency of preleukemic cells in the cultures. Furthermore, we document that the leukemogenic potential of Meis1 is independent of its direct binding to DNA and likely reflects its ability to increase the repopulating capacity of the preleukemic cells by increasing their self-renewal/proliferative capacity. The availability of lines with repopulating potential and capacity for leukemic conversion should open new avenues for understanding progression of Hox-mediated acute myeloid leukemia.
Asunto(s)
Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Leucemia Mieloide/fisiopatología , Células Progenitoras Mieloides/trasplante , Proteínas de Neoplasias/metabolismo , Proteínas de Fusión Oncogénica/genética , Enfermedad Aguda , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Trasplante de Médula Ósea/métodos , Diferenciación Celular , División Celular , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Leucemia Mieloide/genética , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide , Células Progenitoras Mieloides/citología , Células Progenitoras Mieloides/fisiología , Proteínas de Neoplasias/genética , Retroviridae/genética , Factores de TranscripciónRESUMEN
PBX1 is a homeodomain protein that functions in complexes with other homeodomain-containing proteins to regulate gene expression during developmental and/or differentiation processes. A yeast two-hybrid screen of a fetal liver-hematopoietic cDNA library using PBX1a as bait led to the discovery of a novel non-homeodomain-containing protein that interacts with PBX1 as well as PBX2 and PBX3. RNA analysis revealed it to be expressed in CD34(+) hematopoietic cell populations enriched in primitive progenitors, as is PBX1; search of the expressed sequence tag data base indicated that it is also expressed in other early embryonic as well as adult tissues. The full-length cDNA encodes a 731-amino acid protein that has no significant homology to known proteins. This protein that we have termed hematopoietic PBX-interacting protein (HPIP) is mainly localized in the cytosol and in small amounts in the nucleus. The region of PBX that interacts with HPIP includes both the homeodomain and immediate N-terminal flanking sequences. Strikingly, electrophoretic mobility shift assays revealed that HPIP inhibits the ability of PBX-HOX heterodimers to bind to target sequences. Moreover, HPIP strongly inhibits the transactivation activity of E2A-PBX. Together these findings suggest that HPIP is a new regulator of PBX function.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Transformación Celular Neoplásica , Clonación Molecular , Proteínas Co-Represoras , Hematopoyesis , Ratones , Datos de Secuencia Molecular , Factor de Transcripción 1 de la Leucemia de Células Pre-B , Factores de Transcripción/química , Activación Transcripcional/efectos de los fármacos , TransfecciónRESUMEN
In this report, we demonstrate that the Src homology 2 domain-containing inositol-5-phosphatase (SHIP) plays a critical role in regulating both B cell development and responsiveness to antigen stimulation. SHIP(-/-) mice exhibit a transplantable alteration in B lymphoid development that results in reduced numbers of precursor B (fraction C) and immature B cells in the bone marrow. In vitro, purified SHIP(-/)- B cells exhibit enhanced proliferation in response to B cell receptor stimulation in both the presence and absence of Fcgamma receptor IIB coligation. This enhancement is associated with increased phosphorylation of both mitogen-activated protein kinase and Akt, as well as with increased survival and cell cycling. SHIP(-/)- mice manifest elevated serum immunoglobulin (Ig) levels and an exaggerated IgG response to the T cell-independent type 2 antigen trinitrophenyl Ficoll. However, only altered B cell development was apparent upon transplantation into nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice. The in vitro hyperresponsiveness, together with the in vivo findings, suggests that SHIP regulates B lymphoid development and antigen responsiveness by both intrinsic and extrinsic mechanisms.
Asunto(s)
Linfocitos B/inmunología , Monoéster Fosfórico Hidrolasas/inmunología , Proteínas Serina-Treonina Quinasas , Dominios Homologos src/inmunología , Animales , Apoptosis , Células de la Médula Ósea/inmunología , Trasplante de Médula Ósea , Ciclo Celular , Ficoll/análogos & derivados , Ficoll/inmunología , Citometría de Flujo , Inmunidad Celular , Inmunoglobulina M , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos NOD , Ratones Mutantes , Ratones SCID , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Trastornos Mieloproliferativos , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Fosforilación , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Receptores de Antígenos de Linfocitos B , Transducción de Señal , Bazo/citología , Bazo/inmunología , Trinitrobencenos/inmunologíaRESUMEN
Huntington's disease (HD) is a neurodegenerative disease associated with polyglutamine expansion in huntingtin, a widely expressed protein. The function of huntingtin is unknown although huntingtin plays a fundamental role in development since gene targeted HD (-) (/-)mouse embryos die shortly after gastrulation. Expression of huntingtin is detected in spleen and thymus but its role in hematopoiesis has not been examined. To determine the function of huntingtin and to provide insight into potential pathologic mechanisms in HD, we analyzed the role of huntingtin in hematopoietic development. Expression of huntingtin was analyzed in a variety of hematopoietic cell types, and in vitro hematopoiesis was assessed using an HD ( +/-)and several HD( -) (/-)embryonic stem (ES) cell lines. Although wild-type, HD ( +/-)and HD( -) (/-)ES cell lines formed primary embryoid bodies (EBs) with similar efficiency, the numbers of hematopoietic progenitors detected at various stages of the in vitro differentiation were reduced in HD ( +/-)and HD( -/-)() ()ES cell lines examined. Expression analyses of hematopoietic markers within the EBs revealed that primitive and definitive hematopoiesis occurs in the absence of huntingtin. However, further analysis using a suspension culture in the presence of hematopoietic cytokines demonstrated a highly significant gene dosage-dependent decrease in proliferation and/or survival of HD ( +/-)and HD( -) (/-)cells. Enrichment for the CD34(+)cells within the EB confirmed that the impairment is intrinsic to the hematopoietic cells. These obser- vations suggest that huntingtin expression is required for the generation and expansion of hematopoietic cells and provides an alternative system in which to assess the function of huntingtin.