RESUMO
NRAS is frequently mutated in hematologic malignancies. We generated Mx1-Cre, Lox-STOP-Lox (LSL)-Nras(G12D) mice to comprehensively analyze the phenotypic, cellular, and biochemical consequences of endogenous oncogenic Nras expression in hematopoietic cells. Here we show that Mx1-Cre, LSL-Nras(G12D) mice develop an indolent myeloproliferative disorder but ultimately die of a diverse spectrum of hematologic cancers. Expressing mutant Nras in hematopoietic tissues alters the distribution of hematopoietic stem and progenitor cell populations, and Nras mutant progenitors show distinct responses to cytokine growth factors. Injecting Mx1-Cre, LSL-Nras(G12D) mice with the MOL4070LTR retrovirus causes acute myeloid leukemia that faithfully recapitulates many aspects of human NRAS-associated leukemias, including cooperation with deregulated Evi1 expression. The disease phenotype in Mx1-Cre, LSL-Nras(G12D) mice is attenuated compared with Mx1-Cre, LSL-Kras(G12D) mice, which die of aggressive myeloproliferative disorder by 4 months of age. We found that endogenous Kras(G12D) expression results in markedly elevated Ras protein expression and Ras-GTP levels in Mac1(+) cells, whereas Mx1-Cre, LSL-Nras(G12D) mice show much lower Ras protein and Ras-GTP levels. Together, these studies establish a robust and tractable system for interrogating the differential properties of oncogenic Ras proteins in primary cells, for identifying candidate cooperating genes, and for testing novel therapeutic strategies.
Assuntos
Genes ras , Hematopoese/genética , Leucemia Mieloide Aguda/genética , Mutação , Substituição de Aminoácidos , Animais , Citocinas/biossíntese , Eritropoese/genética , Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Linfopoese/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Mielopoese/genética , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismo , Transtornos Mieloproliferativos/patologia , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Gain of chromosome 8 is the most common chromosomal gain in human acute myeloid leukemia (AML). It has been hypothesized that gain of the MYC protooncogene is of central importance in trisomy 8, but the experimental data to support this are limited and controversial. In a mouse model of promyelocytic leukemia in which the MRP8 promoter drives expression of the PML-RARA fusion gene in myeloid cells, a Myc allele is gained in approximately two-thirds of cases as a result of trisomy for mouse chromosome 15. We used this model to test the idea that MYC underlies acquisition of trisomy in AML. We used a retroviral vector to drive expression of wild-type, hypermorphic, or hypomorphic MYC in bone marrow that expressed the PML-RARA transgene. MYC retroviruses cooperated in myeloid leukemogenesis and suppressed gain of chromosome 15. When the PML-RARA transgene was expressed in a Myc haploinsufficient background, we observed selection for increased copies of the wild-type Myc allele concomitant with leukemic transformation. In addition, we found that human myeloid leukemias with trisomy 8 have increased MYC. These data show that gain of MYC can contribute to the pathogenic effect of the most common trisomy of human AML.
Assuntos
Cromossomos Humanos Par 8 , Genes myc , Leucemia Promielocítica Aguda/genética , Trissomia , Animais , Células Cultivadas , Modelos Animais de Doenças , Humanos , Leucemia Promielocítica Aguda/etiologia , Camundongos , Proteínas de Fusão Oncogênica/genética , RecidivaRESUMO
Monosomy 7 and del(7q) are associated with adverse features in myeloid malignancies. A 2.5-Mb commonly deleted segment (CDS) of chromosome band 7q22 is implicated as harboring a myeloid tumor suppressor gene (TSG); however, molecular analysis of candidate TSGs has not uncovered loss of function. To determine whether haploinsufficiency for the 7q22 CDS contributes to myeloid leukemogenesis, we performed sequential gene targeting to flank a region of orthologous synteny on mouse chromosome band 5A3 with loxP sites. We then generated Mx1-Cre, 5A3(fl) mutant mice and deleted the targeted interval in vivo. Although excision was inefficient, we confirmed somatic deletion of the 5A3 CDS in the hematopoietic stem cell compartment. Mx1-Cre, 5A3(fl) mice show normal hematologic parameters and do not spontaneously develop myeloid malignancies. The 5A3(fl) deletion does not cooperate with oncogenic Kras(G12D) expression, Nf1 inactivation, or retroviral mutagenesis to accelerate leukemia development and did not modulate responsiveness to antileukemia drugs. These studies demonstrate that it is feasible to somatically delete a large chromosomal segment implicated in tumor suppression in hematopoietic cell populations in vivo; however, our data do not support the hypothesis that the 7q22/5A3 CDS interval contains a myeloid TSG.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 7/genética , Leucemia Experimental/genética , Leucemia Mieloide/genética , Animais , Antineoplásicos/uso terapêutico , Sequência de Bases , Bandeamento Cromossômico , Mapeamento Cromossômico , Primers do DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Marcação de Genes , Genes da Neurofibromatose 1 , Genes Supressores de Tumor , Engenharia Genética/métodos , Humanos , Leucemia Experimental/tratamento farmacológico , Leucemia Mieloide/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Modelos Genéticos , Mutagênese Insercional , Proteínas Proto-Oncogênicas p21(ras)/genética , Recombinação Genética , Especificidade da EspécieRESUMO
Loss of heterozygosity (LOH) involving chromosome 16q23.4 occurs frequently in breast tumors, which suggests that this region may contain a tumor suppressor gene. Since ZFP276 is located in this region, we have therefore cloned and performed mutation analysis of its coding region in 70 breast tumors. One silent polymorphism and two alterations predicted to result in amino acid changes were detected. Absence of the wild-type allele in tumors carrying the E530D variant suggests a possible role for this change in tumorigenesis.
Assuntos
Neoplasias da Mama/genética , Genes Supressores de Tumor , Polimorfismo Genético , Proteínas Supressoras de Tumor/genética , Alelos , Sequência de Aminoácidos , Northern Blotting , Neoplasias da Mama/patologia , Cromossomos Humanos Par 16 , Clonagem Molecular , Análise Mutacional de DNA , DNA Complementar/metabolismo , Feminino , Humanos , Modelos Genéticos , Dados de Sequência Molecular , Proteínas Nucleares , Polimorfismo Conformacional de Fita Simples , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Dedos de ZincoRESUMO
Fanconi Anemia (FA) is an autosomal recessive disorder characterized by cellular hypersensitivity to DNA cross-linking agents. Recent studies suggest that FA proteins share a common pathway with BRCA proteins. To study the in vivo role of the FA group A gene (Fanca), gene-targeting techniques were used to generate Fanca(tm1Hsc) mice in which Fanca exons 1-6 were replaced by a beta-galactosidase reporter construct. Fanca(tm1.1Hsc) mice were generated by Cre-mediated removal of the neomycin cassette in Fanca(tm1Hsc) mice. Fanca(tm1.1Hsc) homozygotes display FA-like phenotypes including growth retardation, microphthalmia and craniofacial malformations that are not found in other Fanca mouse models, and the genetic background affects manifestation of certain phenotypes. Both male and female mice homozygous for Fanca mutation exhibit hypogonadism, and homozygous females demonstrate premature reproductive senescence and an increased incidence of ovarian cysts. We showed that fertility defects in Fanca(tm1.1Hsc) homozygotes might be related to a diminished population of primordial germ cells (PGCs) during migration into the gonadal ridges. We also found a high level of Fanca expression in pachytene spermatocytes. Fanca(tm1Hsc) homozygous males exhibited an elevated frequency of mispaired meiotic chromosomes and increased apoptosis in germ cells, implicating a role for Fanca in meiotic recombination. However, the localization of Rad51, Brca1, Fancd2 and Mlh1 appeared normal on Fanca(tm1Hsc) homozygous meiotic chromosomes. Taken together, our results suggest that the FA pathway plays a role in the maintenance of reproductive germ cells and in meiotic recombination.
Assuntos
Proteínas de Ligação a DNA , Anemia de Fanconi/genética , Células Germinativas/patologia , Meiose/genética , Proteínas/genética , Proteínas/metabolismo , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Éxons , Proteína do Grupo de Complementação A da Anemia de Fanconi , Feminino , Retardo do Crescimento Fetal , Marcação de Genes , Infertilidade Feminina/genética , Infertilidade Masculina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microftalmia/genética , FenótipoRESUMO
Fanconi anemia (FA) is a chromosomal instability syndrome characterized by the presence of pancytopenia, congenital malformations and cancer predisposition. Six genes associated with this disorder have been cloned, and mice with targeted disruptions of several of the FA genes have been generated. These mouse models display the characteristic FA feature of cellular hypersensitivity to DNA cross-linking agents. Although they do not develop hematological or developmental abnormalities spontaneously, they mimic FA patients in their reduced fertility. Studies using these animal models provide valuable insights into the involvement of apoptotic pathways in FA, and help characterize the defects in FA hematopoietic cells. In addition, mouse models are also useful for testing treatments for FA.
