RESUMEN
Homeostasis of hematopoietic stem and progenitor cells is a tightly regulated process. The disturbance of the balance in the hematopoietic progenitor pool can result in favorable conditions for development of diseases such as myelodysplastic syndromes and leukemia. It has been shown recently that mice lacking p15Ink4b have skewed differentiation of common myeloid progenitors toward the myeloid lineage at the expense of erythroid progenitors. The lack of p15INK4B expression in human leukemic blasts has been linked to poor prognosis and increased risk of myelodysplastic syndromes transformation to acute myeloid leukemia. However, the role of p15Ink4b in disease development is just beginning to be elucidated. This study examines the collaboration of the loss of p15Ink4b with Nup98-HoxD13 translocation in the development of hematological malignancies in a mouse model. Here, we report that loss of p15Ink4b collaborates with Nup98-HoxD13 transgene in the development of predominantly myeloid neoplasms, namely acute myeloid leukemia, myeloproliferative disease, and myelodysplastic syndromes. This mouse model could be a very valuable tool for studying p15Ink4b function in tumorigenesis as well as preclinical drug testing.
Asunto(s)
Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Proteínas de Homeodominio/genética , Leucemia Mieloide/genética , Síndromes Mielodisplásicos/genética , Proteínas de Complejo Poro Nuclear/genética , Factores de Transcripción/genética , Enfermedad Aguda , Animales , Médula Ósea/metabolismo , Médula Ósea/patología , Complejo CD3/metabolismo , Proliferación Celular , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/deficiencia , Progresión de la Enfermedad , Inmunohistoquímica , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Síndromes Mielodisplásicos/metabolismo , Síndromes Mielodisplásicos/patología , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Fusión Oncogénica/genética , Bazo/metabolismo , Bazo/patología , Análisis de SupervivenciaRESUMEN
The tumor suppressor p15Ink4b is frequently inactivated by methylation in acute myeloid leukemia and premalignant myeloid disorders. Dendritic cells (DCs) as potent APCs play critical regulatory roles in antileukemic immune responses. In the present study, we investigated whether p15Ink4b can function as modulator of DC development. The expression of p15Ink4b is induced strongly during differentiation and activation of DCs, and its loss resulted in significant quantitative and qualitative impairments of conventional DC (cDC) development. Accordingly, ex vivo-generated BM-derived DCs from p15Ink4b-knockout mice express significantly decreased levels of the antigen-presenting (MHC II) and costimulatory (CD80 and CD86) molecules and have impaired immunostimulatory functions, such as antigen uptake and T-cell stimulation. Reexpression of p15Ink4b in progenitors restored these defects, and confirmed a positive role for p15Ink4b during cDC differentiation and maturation. Furthermore, we have shown herein that p15Ink4b expression increases phosphorylation of Erk1/Erk2 kinases, which leads to an elevated activity of the PU.1 transcription factor. In conclusion, our results establish p15Ink4b as an important modulator of cDC development and implicate a novel function for this tumor suppressor in the regulation of adaptive immune responses.
Asunto(s)
Diferenciación Celular/genética , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/fisiología , Células Dendríticas/fisiología , Inmunidad Adaptativa/genética , Animales , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/fisiología , Células Cultivadas , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/metabolismo , Metilación de ADN/fisiología , Células Dendríticas/metabolismo , Eliminación de Gen , Regulación de la Expresión Génica/inmunología , Regulación de la Expresión Génica/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Regiones Promotoras Genéticas/fisiología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
Inactivation of p15INK4b, an inhibitor of cyclin-dependent kinases, through DNA methylation is one of the most common epigenetic abnormalities in myeloid leukemia. Although this suggests a key role for this protein in myeloid disease suppression, experimental evidence to support this has not been reported. To address whether this event is critical for premalignant myeloid disorders and leukemia development, mice were generated that have loss of p15Ink4b specifically in myeloid cells. The p15Ink4b(fl/fl)-LysMcre mice develop nonreactive monocytosis in the peripheral blood accompanied by increased numbers of myeloid and monocytic cells in the bone marrow resembling the myeloproliferative form of chronic myelomonocytic leukemia. Spontaneous progression from chronic disease to acute leukemia was not observed. Nevertheless, MOL4070LTR retrovirus integrations provided cooperative genetic mutations resulting in a high frequency of myeloid leukemia in knockout mice. Two common retrovirus insertion sites near c-myb and Sox4 genes were identified, and their transcript up-regulated in leukemia, suggesting a collaborative role of their protein products with p15Ink4b-deficiency in promoting malignant disease. This new animal model demonstrates experimentally that p15Ink4b is a tumor suppressor for myeloid leukemia, and its loss may play an active role in the establishment of preleukemic conditions.
Asunto(s)
Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Leucemia Mieloide/genética , Leucemia Mieloide/patología , Lesiones Precancerosas/genética , Lesiones Precancerosas/patología , Animales , Línea Celular Tumoral , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/metabolismo , Modelos Animales de Enfermedad , Genes Supresores de Tumor/fisiología , Genes myb/fisiología , Predisposición Genética a la Enfermedad , Ratones , Ratones Noqueados , Células Precursoras de Monocitos y Macrófagos/patología , Células Precursoras de Monocitos y Macrófagos/fisiología , Monocitos/patología , Monocitos/fisiología , Retroviridae/genética , Factores de Transcripción SOXC/genética , Bazo/patologíaRESUMEN
The c-Myb transcription factor is a major regulator that controls differentiation and proliferation of hematopoietic progenitor cells, which is frequently deregulated in hematological diseases, such as lymphoma and leukemia. Understanding of the mechanisms regulating the transcription of c-myb gene is challenging as it lacks a typical promoter and multiple factors are involved. Our previous studies identified some distal regulatory elements in the upstream regions of c-myb gene in murine myeloid progenitor M1 cells, but the detailed mechanisms still remain unclear. In the present study, we found that a cell differentiation-related DNase1 hypersensitive site is located at a -28k region upstream of c-myb gene and that transcription factors Hoxa9, Meis1 and PU.1 bind to the -28k region. Circular chromosome conformation capture (4C) assay confirmed the interaction between the -28k region and the c-myb promoter, which is supported by the enrichment of CTCF and Cohesin. Our analysis also points to a critical role for Hoxa9 and PU.1 in distal regulation of c-myb expression in murine myeloid cells and cell differentiation. Overexpression of Hoxa9 disrupted the IL-6-induced differentiation of M1 cells and upregulated c-myb expression through binding of the -28k region. Taken together, our results provide an evidence for critical role of the -28k region in distal regulatory mechanism for c-myb gene expression during differentiation of myeloid progenitor M1 cells.
Asunto(s)
Regulación Leucémica de la Expresión Génica , Proteínas de Homeodominio/genética , Interleucina-6/farmacología , Células Progenitoras Mieloides/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myb/genética , Proteínas Proto-Oncogénicas/genética , Transactivadores/genética , Animales , Sitios de Unión , Factor de Unión a CCCTC , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/inmunología , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/inmunología , Desoxirribonucleasa I/genética , Desoxirribonucleasa I/inmunología , Proteínas de Homeodominio/inmunología , Ratones , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide , Células Progenitoras Mieloides/inmunología , Células Progenitoras Mieloides/patología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas/inmunología , Proteínas Proto-Oncogénicas c-myb/inmunología , Proteínas Represoras/genética , Proteínas Represoras/inmunología , Transducción de Señal , Transactivadores/inmunología , CohesinasRESUMEN
The Ink4b gene (Cdkn2b) encodes p15(Ink4b), a cyclin-dependent kinase inhibitor. It has been implicated in playing a role in the development of acute myeloid leukemia (AML) in man, since it is hypermethylated with high frequency. We provide evidence that the gene is a tumor suppressor for myeloid leukemia in mice. The evidence is twofold: (1) retrovirus-induced myeloid leukemias of the myelomonocytic phenotype were found to have hypermethylation of the 5' CpG island of the Ink4b gene, and this could be correlated with reduced mRNA expression, as demonstrated by TaqMan real-time PCR. p15(Ink4b) mRNA expression in a leukemia cell line, with hypermethylation at the locus, was induced following treatment with 5-aza-2'-deoxycytidine. (2) Targeted deletion of one allele in mice by removal of exon 2 increases their susceptibility to retrovirus-induced myeloid leukemia. Mice deficient in both alleles were not more susceptible to myeloid disease than those deficient in one allele, raising the possibility that there are opposing forces related to the development of myeloid leukemia in Ink4b null mice.
Asunto(s)
Azacitidina/análogos & derivados , Proteínas de Ciclo Celular/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Metilación de ADN , Leucemia Mieloide Aguda/genética , Proteínas Supresoras de Tumor , Animales , Antimetabolitos Antineoplásicos/farmacología , Azacitidina/farmacología , Southern Blotting , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Islas de CpG , Inhibidor p15 de las Quinasas Dependientes de la Ciclina , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Decitabina , Exones , Eliminación de Gen , Genotipo , Intrones , Ratones , Ratones Transgénicos , Fenotipo , ARN Mensajero/metabolismo , Retroviridae/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
CIZ1 is a nuclear protein involved in DNA replication and is also implicated in human diseases including cancers. To gain an insight into its function in vivo, we generated mice lacking Ciz1. Ciz1-deficient (Ciz1(-/-)) mice grew without any obvious abnormalities, and Ciz1(-/-) mouse embryonic fibroblasts (MEFs) did not show any defects in cell cycle status, cell growth, and DNA damage response. However, Ciz1(-/-) MEFs were sensitive to hydroxyurea-mediated replication stress and susceptible to oncogene-induced cellular transformation. In addition, Ciz1(-/-) mice developed various types of leukemias by retroviral insertional mutagenesis. These results indicate that CIZ1 functions as a tumor suppressor in vivo.
Asunto(s)
Proteínas Nucleares/fisiología , Proteínas Supresoras de Tumor , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Fibroblastos/fisiología , Ratones , Ratones Noqueados , Modelos Biológicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
Retroviruses can be used to accelerate hematopoietic cancers predisposed to neoplastic disease by prior genetic manipulations such as in transgenic or knockout mice. The virus imparts a second neoplastic "hit," providing evidence that the initial hit is transforming. In the present study, a unique retrovirus was developed that can induce a high incidence of myeloid disease and has a broad host range. This agent is a Moloney murine leukemia virus (Mo-MuLV)-based virus that has most of the U3 region of the long terminal repeat (LTR) replaced with that of retrovirus 4070A. Like Mo-MuLV, this virus, called MOL4070LTR, is NB-tropic and not restricted by Fv1 allelles. MOL4070LTR causes myeloid leukemias in ca. 50% of mice, a finding in contrast to Mo-MuLV, which induces almost exclusively lymphoid disease. The data suggest that the LTR of the 4070A virus expands the tissue tropism of the disease to the myeloid lineage. Interesting, MCF recombinant envelope was expressed in the lymphoid but not the myeloid neoplasms of BALB/c mice. This retrovirus has the potential for accelerating myeloid disease in genetically engineered mice.
Asunto(s)
Leucemia Experimental/virología , Virus de la Leucemia Murina de Moloney/patogenicidad , Recombinación Genética , Retroviridae/patogenicidad , Secuencias Repetidas Terminales/genética , Animales , Ingeniería Genética/métodos , Leucemia Mielomonocítica Aguda/virología , Ratones , Ratones Endogámicos BALB C , Virus de la Leucemia Murina de Moloney/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/virología , Retroviridae/genética , Infecciones por Retroviridae/virología , Infecciones Tumorales por Virus/virologíaRESUMEN
Alternative splicing in Mo-MuLV recruits a splice donor site, SD', within the gag that is required for optimal replication in vitro. Remarkably, this SD' site was also found to be utilized for production of oncogenic gag-myb fusion RNA in 100% of murine-induced myeloid leukemia (MML) in pristane-treated BALB/c mice. Therefore, we investigated the influence of silent mutations of SD' in this model. Although there was no decrease in the overall incidence of disease, there was a decrease in the incidence of myeloid leukemia with a concomitant increase in lymphoid leukemia. Importantly, there was a complete lack of myeloid tumors associated with 5' insertional mutagenic activation of c-myb, suggesting the specific requirement of the SD' site in this mechanism.
Asunto(s)
Expresión Génica , Genes myb , Virus de la Leucemia Murina de Moloney/fisiología , Regiones Promotoras Genéticas , Empalme del ARN , Integración Viral , Animales , Northern Blotting , Southern Blotting , Modelos Animales de Enfermedad , Leucemia Linfoide/patología , Leucemia Linfoide/virología , Leucemia Mieloide/patología , Leucemia Mieloide/virología , Ratones , Ratones Endogámicos BALB C , Virus de la Leucemia Murina de Moloney/genética , Virus de la Leucemia Murina de Moloney/patogenicidad , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Viral/análisis , ARN Viral/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/patologíaRESUMEN
Cancer is a multistep process resulting from an accumulation of several genetic changes. The determination of cooperating events in experimental models can help scientists decipher specific neoplastic pathways and place genes with similar functions in complementation groups. In leukemia models, retrovirus tagging is a powerful approach to determine genes that cooperate with oncogenic transgenes or tumor suppressors that have undergone targeted deletion. Experimental models for B and T cell leukemias involving transgenic c-myc were the first to show the utility of retroviral tagging. Here we review these experiments and present examples of new models of myeloid leukemia where retroviruses have collaborated with a transgene [Cbfbeta-MYH111 from Inv(16)] and with loss of a tumor suppressor (Ink4b) mice to induce disease.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Leucemia/etiología , Retroviridae/fisiología , Proteínas Supresoras de Tumor/fisiología , Animales , Proteínas de Ciclo Celular/genética , Inhibidor p15 de las Quinasas Dependientes de la Ciclina , Leucemia/genética , Leucemia/virología , Leucemia de Células B/etiología , Leucemia de Células B/genética , Leucemia de Células B/virología , Leucemia Mieloide/etiología , Leucemia Mieloide/genética , Leucemia Mieloide/virología , Leucemia de Células T/etiología , Leucemia de Células T/genética , Leucemia de Células T/virología , Ratones , Ratones Transgénicos , Células Mieloides/metabolismo , Transgenes , Proteínas Supresoras de Tumor/genéticaRESUMEN
Mml loci have been identified as provirus integration sites among a subset of monocytic tumours induced by murine leukaemia virus (MuLV) infection of BALB/c and DBA/2 mice. These myeloid leukaemias contain a retrovirus integrated on chromosome 10 in proximity to the c-myb locus; however, c-myb expression was not altered. Detailed physical mapping enabled placement of the retroviral integration sites approximately 25 kb (Mml1), approximately 51 kb (Mml2), and approximately 70 kb (Mml3) upstream of the c-myb locus. Furthermore, the Fti1 (fit-1) locus, a common integration site in feline leukaemia virus-induced T cell lymphomas, was mapped upstream of Mml3. Sequence analysis of Mml1, Mml2 and Mml3 loci (39.6, 16.4 and 5.9 kb, respectively) in conjunction with the BLAST (basic local alignment search tool) homology searches against the expressed sequence tag (EST) database and the use of gene/exon prediction programs revealed potential coding sequences that were not confirmed by Northern analysis or RT-PCR. The sequences between c-myb and Fti1, which were shown to include two potential scaffold/matrix attachment regions (S/MARs), are most likely regulatory in nature. An extended search for transcribed sequences far upstream of Mml3 revealed five genes, four of which were expressed in multiple tissues in mice. These genes could not be linked to tumour formation by the virus but their homologous sequences were found on human chromosome 6, thus allowing extension of the syntenic region on mouse chromosome 10 to approximately 250 kb.