RESUMEN
The blood system serves as a key model for cell differentiation and cancer. It is orchestrated by precise spatiotemporal expression of crucial transcription factors. One of the key master regulators in the hematopoietic systems is PU.1. Reduced levels of PU.1 are characteristic for human acute myeloid leukemia (AML) and are known to induce AML in mouse models. Here, we show that transcriptional downregulation of PU.1 is an active process involving an alternative promoter in intron 3 that is induced by RUNX transcription factors driving noncoding antisense transcription. Core-binding factor (CBF) fusions RUNX1-ETO and CBFß-MYH11 in t(8;21) and inv(16) AML, respectively, activate the PU.1 antisense promoter that results in a shift from sense toward antisense transcription and myeloid differentiation blockade. In patients with CBF-AML, we found that an elevated antisense/sense transcript and promoter accessibility ratio represents a hallmark compared with normal karyotype AML or healthy CD34+ cells. Competitive interaction of an enhancer with the proximal or the antisense promoter forms a binary on/off switch for either myeloid or T-cell development. Leukemic CBF fusions thus use a physiological mechanism used by T cells to decrease sense transcription. Our study is the first example of a sense/antisense promoter competition as a crucial functional switch for gene expression perturbation by oncogenes. Hence, this disease mechanism reveals a previously unknown Achilles heel for future precise therapeutic targeting of oncogene-induced chromatin remodeling.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad beta del Factor de Unión al Sitio Principal/genética , Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/genética , Proteínas Proto-Oncogénicas/genética , Transactivadores/genética , Elementos sin Sentido (Genética)/genética , Línea Celular Tumoral , Fusión Génica , Humanos , Proteínas de Fusión Oncogénica/genética , Regiones Promotoras Genéticas , Proteína 1 Compañera de Translocación de RUNX1/genética , Células Tumorales CultivadasRESUMEN
Mixed lineage leukemia (MLL) fusion proteins directly activate the expression of key downstream genes such as MEIS1, HOXA9 to drive an aggressive form of human leukemia. However, it is still poorly understood what additional transcriptional regulators, independent of the MLL fusion pathway, contribute to the development of MLL leukemia. Here we show that the transcription factor PU.1 is essential for MLL leukemia and is required for the growth of MLL leukemic cells via the promotion of cell-cycle progression and inhibition of apoptosis. Importantly, PU.1 expression is not under the control of MLL fusion proteins. We further identified a PU.1-governed 15-gene signature, which contains key regulators in the MEIS-HOX program (MEIS1, PBX3, FLT3, and c-KIT). PU.1 directly binds to the genomic loci of its target genes in vivo, and is required to maintain active expression of those genes in both normal hematopoietic stem and progenitor cells and in MLL leukemia. Finally, the clinical significance of the identified PU.1 signature was indicated by its ability to predict survival in acute myelogenous leukemia patients. Together, our findings demonstrate that PU.1 contributes to the development of MLL leukemia, partially via crosstalk with the MEIS/HOX pathway.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Leucemia Bifenotípica Aguda/genética , Leucemia Bifenotípica Aguda/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Análisis por Conglomerados , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Humanos , Leucemia Bifenotípica Aguda/mortalidad , Ratones , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , PronósticoRESUMEN
Mixed lineage leukemia (MLL)-fusion proteins can induce acute myeloid leukemias (AMLs) from either hematopoietic stem cells (HSCs) or granulocyte-macrophage progenitors (GMPs), but it remains unclear whether the cell of origin influences the biology of the resultant leukemia. MLL-AF9-transduced single HSCs or GMPs could be continuously replated, but HSC-derived clones were more likely than GMP-derived clones to initiate AML in mice. Leukemia stem cells derived from either HSCs or GMPs had a similar immunophenotype consistent with a maturing myeloid cell (LGMP). Gene expression analyses demonstrated that LGMP inherited gene expression programs from the cell of origin including high-level Evi-1 expression in HSC-derived LGMP. The gene expression signature of LGMP derived from HSCs was enriched in poor prognosis human MLL-rearranged AML in three independent data sets. Moreover, global 5'-mC levels were elevated in HSC-derived leukemias as compared with GMP-derived leukemias. This mirrored a difference seen in 5'-mC between MLL-rearranged human leukemias that are either EVI1 positive or EVI1 negative. Finally, HSC-derived leukemias were more resistant to chemotherapy than GMP-derived leukemias. These data demonstrate that the cell of origin influences the gene expression profile, the epigenetic state and the drug response in AML, and that these differences can account for clinical heterogeneity within a molecularly defined group of leukemias.
Asunto(s)
Leucemia Mieloide Aguda/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Adulto , Animales , Antineoplásicos/farmacología , Citarabina/farmacología , Perfilación de la Expresión Génica , N-Metiltransferasa de Histona-Lisina , Humanos , Ratones , Ratones Endogámicos C57BLRESUMEN
RAF kinase inhibitor protein (RKIP) is a negative regulator of the RAS-mitogen-activated protein kinase/extracellular signal-regulated kinase signaling cascade. We investigated its role in acute myeloid leukemia (AML), an aggressive malignancy arising from hematopoietic stem and progenitor cells (HSPCs). Western blot analysis revealed loss of RKIP expression in 19/103 (18%) primary AML samples and 4/17 (24%) AML cell lines but not in 10 CD34+ HSPC specimens. In in-vitro experiments with myeloid cell lines, RKIP overexpression inhibited cellular proliferation and colony formation in soft agar. Analysis of two cohorts with 103 and 285 AML patients, respectively, established a correlation of decreased RKIP expression with monocytic phenotypes. RKIP loss was associated with RAS mutations and in transformation assays, RKIP decreased the oncogenic potential of mutant RAS. Loss of RKIP further related to a significantly longer relapse-free survival and overall survival in uni- and multivariate analyses. Our data show that RKIP is frequently lost in AML and correlates with monocytic phenotypes and mutations in RAS. RKIP inhibits proliferation and transformation of myeloid cells and decreases transformation induced by mutant RAS. Finally, loss of RKIP seems to be a favorable prognostic parameter in patients with AML.
Asunto(s)
Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/genética , Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Diferenciación Celular/genética , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/genética , Genes ras , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidad , Monocitos/citología , Monocitos/metabolismo , Mutación , Células Mieloides/metabolismo , Proteínas de Unión a Fosfatidiletanolamina/deficiencia , Proteínas de Unión a Fosfatidiletanolamina/genética , PronósticoRESUMEN
Overexpression of the ecotropic virus integration-1 (EVI1) gene (EVI1+), localized at chromosome 3q26, is associated with adverse outcome in adult acute myeloid leukemia (AML). In pediatric AML, 3q26 abnormalities are rare, and the role of EVI1 is unknown. We studied 228 pediatric AML samples for EVI1+ using gene expression profiling and RQ-PCR. EVI1+ was found in 20/213 (9%) of children with de novo AML, and in 4/8 with secondary AML. It was predominantly found in MLL-rearranged AML (13/47), monosomy 7 (2/3), or FAB M6/7 (6/10), and mutually exclusive with core-binding factor AML, t(15;17), and NPM1 mutations. Fluorescent in situ hybridization (FISH) was performed to detect cryptic 3q26 abnormalities. However, none of the EVI1+ patients harbored structural 3q26 alterations. Although significant differences in 4 years pEFS for EVI1+ and EVI1- pediatric AML were observed (28%+/-11 vs 44%+/-4, P=0.04), multivariate analysis did not identify EVI1+ as an independent prognostic factor. We conclude that EVI1+ can be found in approximately 10% of pediatric AML. Although EVI1+ was not an independent prognostic factor, it was predominantly found in subtypes of pediatric AML that are related with an intermediate to unfavorable prognosis. Further research should explain the role of EVI1+ in disease biology in these cases. Remarkably, no 3q26 abnormalities were identified in EVI1+ pediatric AML.
Asunto(s)
Biomarcadores de Tumor/genética , Cromosomas Humanos Par 3/genética , Proteínas de Unión al ADN/genética , Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/genética , Proto-Oncogenes/genética , Factores de Transcripción/genética , Niño , Preescolar , Aberraciones Cromosómicas , Femenino , Perfilación de la Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Leucemia Mieloide Aguda/metabolismo , Proteína del Locus del Complejo MDS1 y EV11 , Masculino , Nucleofosmina , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de SupervivenciaRESUMEN
FHL2 is a multifunctional LIM domain protein that acts as a transcriptional modulator mediating proliferation and apoptosis in a tissue-specific manner. Upregulation of FHL2 has been detected in a variety of cancers. We demonstrate that upregulation of FHL2 is associated with a subset of acute myeloid leukemia with a characteristic gene-expression signature, and abnormalities of chromosome 5. In mice, expression of endogenous Fhl2 is downregulated coordinately during the differentiation of hematopoietic cells. Upregulation of FHL2 enhances proliferation of myeloid progenitor cells, and serial-replating efficiency of hematopoietic cells in vitro. Chimeric mice with enforced expression of FHL2 in bone marrow cells, are characterized by an expanded pool of myeloid progenitor cells, enhanced granulopoiesis and megakaryocytopoiesis. In addition, enhanced expression of FHL2 promotes cell-cycle entry of myeloid progenitor cells and increases the frequency of apoptosis of bone marrow cells in vivo. These results raise the possibility that deregulation of FHL2 contributes to the development of human myeloid disorders.
Asunto(s)
Proteínas de Homeodominio/fisiología , Leucemia Mieloide Aguda/etiología , Proteínas Musculares/fisiología , Mielopoyesis , Factores de Transcripción/fisiología , Animales , Proliferación Celular , Hematopoyesis , Proteínas de Homeodominio/genética , Proteínas con Homeodominio LIM , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas Musculares/genética , Células Progenitoras Mieloides , Trombopoyesis , Factores de Transcripción/genéticaRESUMEN
Id1 is frequently overexpressed in many cancer cells, but the functional significance of these findings is not known. To determine if Id1 could contribute to the development of hematopoietic malignancy, we reconstituted mice with hematopoietic cells overexpressing Id1. We showed for the first time that deregulated expression of Id1 leads to a myeloproliferative disease in mice, and immortalizes myeloid progenitors in vitro. In human cells, we demonstrate that Id genes are expressed in human acute myelogenous leukemia cells, and that knock down of Id1 expression inhibits leukemic cell line growth, suggesting that Id1 is required for leukemic cell proliferation. These findings established a causal relationship between Id1 overexpression and hematologic malignancy. Thus, deregulated expression of Id1 may contribute to the initiation of myeloid malignancy, and Id1 may represent a potential therapeutic target for early stage intervention in the treatment of hematopoietic malignancy.
Asunto(s)
Células Madre Hematopoyéticas/citología , Proteína 1 Inhibidora de la Diferenciación/fisiología , Trastornos Mieloproliferativos/etiología , Animales , Células de la Médula Ósea/citología , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Factor Estimulante de Colonias de Granulocitos/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Ratones , Ratones Endogámicos C57BLAsunto(s)
Análisis Mutacional de ADN , Perfilación de la Expresión Génica , Leucemia Mieloide Aguda/genética , Mutación , Monoéster Fosfórico Hidrolasas/genética , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Exones , Humanos , Inositol Polifosfato 5-Fosfatasas , Intrones , Leucemia Mieloide Aguda/etnología , Modelos Biológicos , Modelos Genéticos , Datos de Secuencia Molecular , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Población BlancaAsunto(s)
Proteínas Potenciadoras de Unión a CCAAT/genética , Leucemia Mieloide Aguda/genética , Mutación , Disomía Uniparental , Alelos , Análisis Mutacional de ADN , Eliminación de Gen , Genoma , Homocigoto , Humanos , Cariotipificación , Pérdida de Heterocigocidad , Modelos Genéticos , Estructura Terciaria de ProteínaRESUMEN
The mechanism underlying p210(BCR/ABL) oncoprotein-mediated transformation in chronic myelogenous leukemia (CML) is not fully understood. We hypothesized that p210(BCR/ABL) suppresses expression of genes which may explain at least some of the pathogenetic features of CML. A subtractive cDNA library was created between BCR/ABL-enhanced-green-fluorescent-protein (GFP)-transduced umbilical cord blood (UCB) CD34+ cells and GFP-transduced UCB CD34+ cells to identify genes whose expression is downregulated by p210(BCR/ABL). At least 100 genes were identified. We have confirmed for eight of these genes that expression was suppressed by quantitative real-time-RT-PCR (Q-RT-PCR) of additional p210(BCR/ABL)-transduced CD34+ UCB cells as well as primary early chronic phase (CP) bone marrow (BM) CML CD34+ cells. Imatinib mesylate reversed downregulation of some genes, to approximately normal levels. Several of the genes are implicated in cell adhesion and motility, including L-selectin, intercellular adhesion molecule-1 (ICAM-1), and the chemokine receptor, CCR7, consistent with the known defect in adhesion and migration of CML cells. Compared with GFP UCB or normal (NL) BM CD34+ cells, p210 UCB and CML CD34+ cells migrated poorly towards the CCR7 ligands, CCL19 and CCL21, suggesting a possible role for CCR7 in the abnormal migratory behavior of CML CD34+ cells.
Asunto(s)
Movimiento Celular/fisiología , Quimiocinas CC/fisiología , Proteínas de Fusión bcr-abl/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Receptores de Quimiocina/fisiología , Adhesión Celular/genética , Adhesión Celular/fisiología , Línea Celular , Movimiento Celular/genética , Quimiocina CCL19 , Quimiocina CCL21 , Quimiocinas CC/genética , Regulación hacia Abajo , Proteínas de Fusión bcr-abl/genética , Humanos , Molécula 1 de Adhesión Intercelular/genética , Células K562 , Selectina L/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ligandos , ARN Mensajero/genética , Receptores CCR7 , Receptores de Quimiocina/genéticaRESUMEN
Aberrant expression of the Evi1 (ecotropic virus integration site 1) proto-oncogene has been associated with hematopoietic malignancies in both mice and man. To determine the effect of enforced expression of Evi1 in vivo, we developed a transgenic mouse model utilizing the murine Sca-1 (Ly-6E.1) promoter. Here, we describe the generation and analysis of three independent lines of Evi1 transgenic mice. Transgenic animals of two founder lines developed normally. These mice did not show any obvious hematological abnormalities but showed a significant reduction in the number of bone marrow colony-forming unit erythroid (CFU-E)-derived colonies. This implies a defect of normal erythroid hematopoiesis affecting relatively late erythroid progenitor cells. We also show that when newborn Evi1 transgenic mice of these two lines were infected with Cas-Br-M MuLV, tumor incidence was greatly enhanced in comparison with nontransgenic littermates, indicating an increased susceptibility for leukemia development. Interestingly, analysis of a third founder line revealed that all male progeny consistently displayed severely impaired erythropoiesis with major defects in the bone marrow, spleen and peripheral blood. Taken together, our results present the first evidence of Evi1 disturbing normal erythropoiesis in vivo and provides evidence for cooperative potential of Evi1 in tumor progression.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Leucemia Experimental/etiología , Factores de Transcripción/fisiología , Dedos de Zinc/fisiología , Animales , Antígenos Ly/genética , Células Sanguíneas/patología , Médula Ósea/patología , Proteínas de Unión al ADN/genética , Células Precursoras Eritroides/patología , Eritropoyesis/genética , Predisposición Genética a la Enfermedad , Virus de la Leucemia Murina/patogenicidad , Leucemia Experimental/genética , Leucemia Experimental/virología , Proteína del Locus del Complejo MDS1 y EV11 , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos , Modelos Animales , Regiones Promotoras Genéticas , Proto-Oncogenes , Proteínas Recombinantes de Fusión/genética , Infecciones por Retroviridae/genética , Bazo/patología , Factores de Transcripción/genética , Infecciones Tumorales por Virus/genética , Dedos de Zinc/genéticaRESUMEN
Cas-Br-M murine leukemia virus (MuLV) is a slow-transforming retrovirus that potently induces leukemias in mice and therefore is well suited for retroviral insertional mutagenesis. We used Cas-Br-M MuLV in NIH/Swiss mice to establish a new panel of mainly myeloid leukemias. All tumors found in leukemic animals were classified by gross pathology, morphology, and immunophenotype, as well as the incidence of known common virus integration sites (VISs) in MuLV-induced myeloid malignancies (i.e., Evi1, Evi11/Cb2, Evi12, Fli1, and c-Myb). Interestingly, male mice were more susceptible than females to the induction of leukemia by Cas-Br-M MuLV. Seventy-four of the Cas-Br-M MuLV-inoculated mice developed a severe splenomegaly, sometimes in association with a thymoma. Although most of the immunophenotyped Cas-Br-M MuLV tumors were of myeloid origin (58%), numerous T-cell leukemias (21%) and mixed myeloid/T-cell leukemias (21%) were found. The myeloid leukemias and myeloid compartment of the mixed leukemias were further characterized by immunophenotyping with stem cell-, myeloid-, and erythroid-specific antibodies. The known Cas-Br-M MuLV common VISs (Evi1, Evi11/Cb2, and Evi12) were demonstrated in 19%, 12%, and 20% of the cases, respectively, whereas no Fli1 and c-Myb rearrangements were found. Integrations into Evi1 were restricted to myeloid leukemias, whereas those in Evi11/Cb2 and Evi12 were identified in myeloid as well as T-lymphoid leukemias. This panel of well characterized Cas-Br-M MuLV-induced hematopoietic tumors may be useful for the isolation and characterization of new proto-oncogenes involved in myeloid or T-cell leukemias.
Asunto(s)
Transformación Celular Viral , Proteínas de Unión al ADN/genética , Virus de la Leucemia Murina/genética , Leucemia Experimental/virología , Proto-Oncogenes , Infecciones por Retroviridae/virología , Factores de Transcripción , Infecciones Tumorales por Virus/virología , Integración Viral/genética , Células 3T3 , Animales , Femenino , Leucemia Experimental/genética , Leucemia Experimental/patología , Proteína del Locus del Complejo MDS1 y EV11 , Masculino , Ratones , Fenotipo , Proteínas Proto-Oncogénicas/aislamiento & purificación , Proteínas Proto-Oncogénicas/fisiología , Infecciones por Retroviridae/genética , Infecciones por Retroviridae/patología , Infecciones Tumorales por Virus/genética , Infecciones Tumorales por Virus/patologíaRESUMEN
INTRODUCTION: Anandamide (ANA) is an endogenous ligand for the cannabinoid receptors Cb1 and Cb2 that is able to synergistically stimulate the proliferation of hematopoietic growth factor-dependent blood cells in serum-free culture. To elucidate the mechanisms by which ANA enhances the proliferative responses of hematopoietic cells, we investigated the ANA-mediated effects on proliferation, cell cycling, apoptosis and intracellular signaling of erythropoietin-stimulated 32D/EPO cells. MATERIALS AND METHODS: 32D/EPO cells were cultured serum free to determine the effects of EPO and anandamide on these cells. Proliferation was analyzed by tritiated thymidine incorporation. Apoptosis as well as cell cycle analysis was carried out by flow cytometry. MAPKinase activation was determined by Western blotting, using phospho-specific MAPK antibodies. RESULTS: Simultaneous addition of erythropoietin (EPO) and ANA enhanced DNA synthesis and increased 32D/EPO cell numbers in serum-free culture. Interestingly, ANA did not alter the G1/S transition but it accelerated each of the successive cell cycle phases of EPO-stimulated 32D/EPO cells. Percentages of apoptotic 32D/EPO cells were equally low in cultures supplemented with EPO alone or a combination of EPO and ANA. Both cultures showed enhanced activation of two mitogen-activated protein kinases, namely, extracellular factor responsive kinases 1 and 2 (ERK1/2), as well as the MAPK-target gene protein c-Fos. This fully correlated with the synergistic stimulation of proliferation of 32D/EPO cells by EPO and ANA. ANA had no effect on EPO-induced STAT-5 activation of 32D/EPO cells. Experiments with the Cb2 receptor-specific antagonist SR144528 demonstrated that the synergistic stimulation of proliferation by ANA was partially Cb2 receptor-mediated. CONCLUSION: These data suggest that the positive effects of ANA on the erythropoietin-induced proliferation of 32D/EPO cells are mediated by receptor-dependent as well as receptor-independent mechanisms, both of which involve activation of the mitogen-activated protein kinases, ERK1/2.
Asunto(s)
Ácidos Araquidónicos/farmacología , Eritropoyetina/farmacología , Proteínas de la Leche , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Animales , Apoptosis/efectos de los fármacos , Canfanos/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular/efectos de los fármacos , Medio de Cultivo Libre de Suero , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Sinergismo Farmacológico , Endocannabinoides , Activación Enzimática , Genes fos , Ratones , Proteína Quinasa 3 Activada por Mitógenos , Alcamidas Poliinsaturadas , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Pirazoles/farmacología , ARN Mensajero/biosíntesis , Receptores de Cannabinoides , Receptores de Droga/antagonistas & inhibidores , Receptores de Droga/biosíntesis , Receptores de Droga/efectos de los fármacos , Receptores de Droga/genética , Factor de Transcripción STAT5 , Transducción de Señal/efectos de los fármacos , Transactivadores/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The common virus integration site (VIS) Evi11 was recently identified within the gene encoding the hematopoietic G-protein-coupled peripheral cannabinoid receptor Cnr2 (also referred to as Cb2). Here we show that Cnr2 is a frequent target (12%) for insertion of Cas-Br-M murine leukemia virus (MuLV) in primary tumors in NIH/Swiss mice. Multiple provirus insertions in Evi11 were cloned and shown to be located within the 3' untranslated region of the candidate proto-oncogene Cnr2. These results suggest that proviral insertion in the Cnr2 gene is an important step in Cas-Br-M MuLV-induced leukemogenesis in NIH/Swiss mice. To isolate Evi11/Cnr2 collaborating proto-oncogenes, we searched for novel common VISs in the Cas-Br-M MuLV-induced primary tumors and identified a novel frequent common VIS, Evi12 (14%). Interestingly, 54% of the Evi11/Cnr2-rearranged primary tumors contained insertions in Evi12 as well, which suggests cooperative action of the target genes in these two common VISs in leukemogenesis. By interspecific backcross analysis it was shown that Evi12 resides on mouse chromosome 10 in a region that shares homology with human chromosomes 12q and 19p. Sequence analysis demonstrated that Evi12 is located upstream of the gene encoding the molecular chaperone Tra1/Grp94, which was previously mapped to mouse chromosome 10 and human chromosome 12q22-24. Thus, Tra1/Grp94 is a candidate target gene for retroviral activation or inactivation in Evi12. However, Northern and Western blot analyses did not provide evidence that proviral insertion had altered the expression of Tra1/Grp94. Additional studies are required to determine whether Tra1/Grp94 or another candidate proto-oncogene in Evi12 is involved in leukemogenesis.
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Cannabinoides , Proteínas Portadoras , Proteínas HSP70 de Choque Térmico/genética , Proteínas de la Membrana/genética , Virus Inductores de Focos en Células del Visón/genética , Proteínas de Transferencia de Fosfolípidos , Proteínas/genética , Receptores de Droga/genética , Integración Viral , Células 3T3 , Animales , Secuencia de Bases , Sitios de Unión , Femenino , Genes Virales , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Proto-Oncogenes Mas , Receptores de Cannabinoides , Células Tumorales CultivadasRESUMEN
Following retroviral insertional mutagenesis we recently identified the gene encoding the peripheral cannabinoid receptor (Cb2) near a common virus integration site (VIS), Evi11. In 13 out of 105 Cas-Br-M murine leukemia virus (MuLV) induced leukemias retroviral integrations occured either in the 5' or 3' part of the Cb2 gene. The Cb2 receptor protein is 44% homologous to the central cannabinoid receptor Cb1, which belongs to the superfamily of seven transmembrane (7TM) receptors. Cb1 is mainly expressed in brain, whereas Cb2 encodes the hematopoietic form. Besides the natural cannabinoids, delta9-tetrahydrocannabinol (delta9-THC) and cannabinol, and the many synthetic agonists that have been generated, e.g CP55,940 or WIN55,212-2, several endogenous ligands have recently been identified. These include the arachidonic acid derivatives anandamide and 2-arachidonylglycerol as well as the fatty acid palmitoylethanolamide. Although in the past many studies described growth inhibitory effects of cannabinoid agonists on the in vitro proliferation of hematopoietic cells, recent studies demonstrated that activation of Cb2 may have growth stimulatory effects on blood precursor cells. We demonstrated that many murine hematopoietic growth factor (HGF) dependent cell lines also require the presence of anandamide for optimal growth in serum free culture. Thus, the Cb2 receptor may be an important regulator of normal hematopoietic growth and development. These results strengthen our finding that Cb2 is a proto-oncogene and may implicate a growth advantage for leukemia cells that aberrantly express Cb2. Here we briefly review the mechanisms and application of retroviral insertional mutagenesis in leukemic transformation in mice and discuss the role of the peripheral cannabinoid receptor in leukemia development and normal hematopoiesis.
Asunto(s)
Hematopoyesis/fisiología , Leucemia Experimental/metabolismo , Receptores de Droga/metabolismo , Animales , Cannabinoides/farmacología , División Celular/efectos de los fármacos , Transformación Celular Viral , Mapeo Cromosómico , Expresión Génica , Hematopoyesis/efectos de los fármacos , Hematopoyesis/genética , Humanos , Mutagénesis Insercional , Proto-Oncogenes Mas , Receptores de Cannabinoides , Receptores de Droga/agonistas , Receptores de Droga/genética , Retroviridae , Transducción de Señal/fisiología , Integración Viral/genéticaRESUMEN
Proto-oncogenes in retrovirally induced myeloid mouse leukemias are frequently activated following retroviral insertion. The identification of common virus integration sites (VISs) and isolation of the transforming oncogene is laborious and time consuming. We established a rapid and simple PCR based procedure which facilitates the identification of VISs and novel proto-oncogenes. Complementary DNA fragments adjacent to retrovirus integration sites were selectively isolated by applying a reverse transcriptase (RT) reaction using an oligo(dT)-adaptor primer, followed by PCR using the adaptor sequence and a retrovirus long terminal repeat (LTR) specific primer. Multiple chimeric cDNA fragments suitable for Southern and northern blot analysis were isolated.
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ADN Complementario/análisis , Reacción en Cadena de la Polimerasa/métodos , Retroviridae/genética , Integración Viral/genética , Animales , Cartilla de ADN , ADN Complementario/genética , Leucemia Mieloide , Ratones , Proto-Oncogenes/genética , Células Tumorales CultivadasRESUMEN
A new common region of virus integration, Evi11, has been identified in two retrovirally induced murine myeloid leukemia cell lines, NFS107 and NFS78. By interspecific backcross analysis, it was shown that Evi11 is located at the distal end of mouse chromosome 4, in a region that shows homology with human 1p36. The genes encoding the peripheral cannabinoid receptor (Cnr2) and alpha-L-fucosidase (Fuca1) were identified near the integration site by using a novel exon trapping system. Cnr2 is suggested to be the target gene for viral interference in Evi11, since proviruses are integrated in the first intron of Cnr2 and retroviral integrations alter mRNA expression of Cnr2 in NFS107 and NFS78. In addition, proviral integrations were demonstrated within the 3' untranslated region of Cnr2 in five independent newly derived CasBrM-MuLV (mouse murine leukemia virus) tumors, CSL13, CSL14, CSL16, CSL27, and CSL97. The Cnr2 gene encodes a seven-transmembrane G-protein-coupled receptor which is normally expressed in hematopoietic tissues. Our data suggest that the peripheral cannabinoid receptor gene might be involved in leukemogenesis as a result of aberrant expression of Cnr2 due to retroviral integration in Evi11.
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Mapeo Cromosómico , Receptores de Droga/genética , Integración Viral , alfa-L-Fucosidasa/genética , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN , Exones , Femenino , Humanos , Virus de la Leucemia Murina/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Receptores de Cannabinoides , Homología de Secuencia de Aminoácido , Transcripción Genética , Células Tumorales CultivadasRESUMEN
The imprinted mouse insulin-like growth factor type 2 receptor (Igf2r) maps to the middle of a gene-rich region in band A2 of mouse chromosome 17. The t(Lub2) chromosome 17 variant contains a small deletion that removes at least seven genes including Igf2r. We have constructed a YAC contig spanning the entire t(Lub2) deletion and created a restriction map that covers 700 kb. The position, transcription orientation, and imprinted status of the genes immediately flanking Igf2r have been assessed. We show here that the Mas gene, which lies 65 kb upstream to Igf2r, contains a novel 5' exon and is not imprinted in adult tissues. We further show that the recently identified Lx1 gene lies immediately downstream and is also expressed from both parental alleles in adult tissues. The remaining genes in this region have previously been shown to be biallelically expressed.
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Alelos , Cromosomas Artificiales de Levadura/genética , Cromosomas/genética , Expresión Génica/fisiología , Genes/fisiología , Impresión Genómica , Receptor IGF Tipo 2/genética , Animales , Secuencia de Bases , Northern Blotting , Mapeo Cromosómico , Clonación Molecular , Cósmidos/genética , Cósmidos/aislamiento & purificación , Sondas de ADN/genética , Sondas de ADN/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II , Exones/genética , Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica , Genes/genética , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/genética , Receptores Acoplados a Proteínas G , Mapeo Restrictivo , Análisis de Secuencia de ADNRESUMEN
We recently demonstrated that the gene encoding the peripheral cannabinoid receptor (Cb2) may be a proto-oncogene involved in murine myeloid leukemias. We show here that Cb2 may have a role in hematopoietic development. RNAse protection analysis showed that Cb2 is normally expressed in spleen and thymus. Cb2 mRNA is also expressed in 45 of 51 cell lines of distinct hematopoietic lineages, ie, myeloid, macrophage, mast, B-lymphoid, T-lymphoid, and erythroid cells. The effect of the fatty acid anandamide, an endogenous ligand for cannabinoid receptors, on primary murine marrow cells and hematopoietic growth factor (HGF)-dependent cell lines was then investigated. In vitro colony cultures of normal mouse bone marrow cells showed anandamide to potentiate interleukin-3 (IL-3)-induced colony growth markedly. Whereas HGFs alone stimulate proliferation of the various cell lines in serum-free culture only weakly, anandamide enhances the proliferative response of the cell lines to HGFs profoundly. This was apparent for responses induced by IL-3, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and erythropoietin. Anandamide was already effective at concentrations as low as 0.1 to 0.3 micromol/L and plateau effects were reached at 0.3 to 3 micromol/L. The addition of anandamide as single growth factor had no effect. The costimulatory effect of anandamide was not evident when cells were cultured with fetal calf serum (FCS), suggesting that FCS contains anandamide or another ligand capable of activating the peripheral cannabinoid receptor. Other cannabinoid ligands did not enhance the proliferative responsiveness of hematopoietic cells to HGFs. Transfection experiments of Cb2 in myeloid 32D cells showed that anandamide specifically activates proliferation through activation of the peripheral cannabinoid receptor. Anandamide appears to be a novel and synergistic growth stimulator for hematopoietic cells.
Asunto(s)
Ácidos Araquidónicos/fisiología , Cannabinoides/metabolismo , Sustancias de Crecimiento/fisiología , Células Madre Hematopoyéticas/citología , Receptores de Droga/metabolismo , Animales , Ácidos Araquidónicos/metabolismo , Células de la Médula Ósea , División Celular/efectos de los fármacos , Medio de Cultivo Libre de Suero , Sinergismo Farmacológico , Endocannabinoides , Eritropoyetina/farmacología , Factor Estimulante de Colonias de Granulocitos/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Sustancias de Crecimiento/metabolismo , Células Madre Hematopoyéticas/metabolismo , Interleucina-3/farmacología , Ligandos , Ratones , Alcamidas Poliinsaturadas , ARN Mensajero/metabolismo , Receptores de Cannabinoides , Receptores de Droga/genéticaRESUMEN
The EVI1 gene is activated by chromosomal translocations and inversions in approximately 5% of human acute myeloid leukemia (AML) and by retroviral insertion in approximately 20% of murine myeloid leukemias. EVI1 encodes a nuclear DNA-binding protein having 10 zinc finger motifs in two noncontiguous domains consisting of an amino-terminal domain of seven fingers and a carboxyl domain containing three fingers. To evaluate the sequence specificity of Evi-1 binding and potentially identify genomic targets, whole-genome PCR was utilized to isolate multiple Sau3A fragments which specifically bind to the amino-terminal zinc finger domain. The majority of these clones represented single copy sequences and virtually all contained variable numbers of repeats of the GATA motif, the target sequence for the erythroid-specific transcription factor GATA-1. GST/Evi-1 fusion proteins containing the amino-terminal domain of zinc fingers bound the GATA motif in these clones as well as to those present in the human gamma-globin promoter, similar to the binding of purified GATA-1 protein. By obtaining corresponding large genomic clones for eight of these fragments, transcription units were found associated with two. One corresponded to the glyceraldehyde-3-phosphate dehydrogenase gene and its expression was not affected by Evi-1. The second is a novel gene whose expression is repressed in murine myeloid cell lines that express Evi-1.