RESUMEN
There are still gaps in our understanding of the complex processes by which p53 suppresses tumorigenesis. Here we describe a novel role for p53 in suppressing the mevalonate pathway, which is responsible for biosynthesis of cholesterol and nonsterol isoprenoids. p53 blocks activation of SREBP-2, the master transcriptional regulator of this pathway, by transcriptionally inducing the ABCA1 cholesterol transporter gene. A mouse model of liver cancer reveals that downregulation of mevalonate pathway gene expression by p53 occurs in premalignant hepatocytes, when p53 is needed to actively suppress tumorigenesis. Furthermore, pharmacological or RNAi inhibition of the mevalonate pathway restricts the development of murine hepatocellular carcinomas driven by p53 loss. Like p53 loss, ablation of ABCA1 promotes murine liver tumorigenesis and is associated with increased SREBP-2 maturation. Our findings demonstrate that repression of the mevalonate pathway is a crucial component of p53-mediated liver tumor suppression and outline the mechanism by which this occurs.
Asunto(s)
Ácido Mevalónico/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Transportador 1 de Casete de Unión a ATP/metabolismo , Animales , Línea Celular , Colesterol/metabolismo , Femenino , Genes Supresores de Tumor , Células HCT116 , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Neoplasias/genética , Regiones Promotoras Genéticas , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Terpenos/metabolismoRESUMEN
Radiation-induced gastrointestinal syndrome (RIGS) is a limiting factor for therapeutic abdominopelvic radiation and is predicted to be a major source of morbidity in the event of a nuclear accident or radiological terrorism. In this study, we developed an in vivo mouse-modeling platform that enables spatial and temporal manipulation of potential RIGS targets in mice following whole-abdomen irradiation without the confounding effects of concomitant hematopoietic syndrome that occur following whole-body irradiation. We then tested the utility of this platform to explore the effects of transient Wnt pathway activation on intestinal regeneration and animal recovery following induction of RIGS. Our results demonstrate that intestinal epithelial suppression of adenomatous polyposis coli (Apc) mitigates RIGS lethality in vivo after lethal ionizing radiation injury-induced intestinal epithelial damage. These results highlight the potential of short-term Wnt agonism as a therapeutic target and establish a platform to evaluate other strategies to stimulate intestinal regeneration after ionizing radiation damage.
Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/antagonistas & inhibidores , Enfermedades Gastrointestinales/prevención & control , Intestinos/citología , Traumatismos Experimentales por Radiación/prevención & control , Regeneración , Irradiación Corporal Total/efectos adversos , Proteínas Wnt/metabolismo , Animales , Enfermedades Gastrointestinales/etiología , Enfermedades Gastrointestinales/metabolismo , Intestinos/efectos de la radiación , Ratones , ARN Interferente Pequeño , Traumatismos Experimentales por Radiación/etiología , Traumatismos Experimentales por Radiación/metabolismo , SíndromeRESUMEN
DNA methyltransferase 3A (DNMT3A) is mutated in hematologic malignancies affecting myeloid, mixed, and lymphoid lineages, and these mutations are associated with poor prognosis. Past studies in mice revealed Dnmt3a-knockout (KO)hematopoietic stem cells (HSCs) had increased self-renewal, but no leukemia was observed. Here, all lethally irradiated mice transplanted with Dnmt3a-deleted HSCs died within 1 year. Animals were diagnosed with a spectrum of malignancies similar to those seen in patients with DNMT3A mutations, including myelodysplastic syndrome, acute myeloid leukemia, primary myelofibrosis, and T- and B-cell acute lymphocytic leukemia. In some cases, acquired malignancies exhibited secondary mutations similar to those identified in patients. Loss of Dnmt3a led to disturbed methylation patterns that were distinct in lymphoid and myeloid disease, suggesting lineage-specific methylation aberrations promoted by Dnmt3a loss. Global hypomethylation was observed in all of the malignancies, but lymphoid malignancies also exhibited hypermethylation, particularly at promoter regions. This mouse model underscores the important role of Dnmt3a in normal hematopoietic development and demonstrates that Dnmt3a loss of function confers a preleukemic phenotype on murine HSCs. This model may serve as a tool to study DNMT3A mutation associated malignancies and for developing targeted strategies for eliminating preleukemic cells for prevention and treatment of hematologic malignancies in the future.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , ADN (Citosina-5-)-Metiltransferasas , Metilación de ADN , ADN de Neoplasias/metabolismo , Neoplasias Hematológicas/enzimología , Células Madre Hematopoyéticas/enzimología , Regiones Promotoras Genéticas , Animales , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , ADN Metiltransferasa 3A , ADN de Neoplasias/genética , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patología , Ratones , Ratones NoqueadosRESUMEN
Chelex-based DNA extractions are well suited for student DNA barcoding research because they are simple, safe, and inexpensive and can be performed without specialized laboratory equipment, allowing them to be performed in classrooms or at home. Extracted DNA is stable in Chelex solution for at least a week at ambient temperature, allowing collection of DNA samples from remote students. These extractions provide quality DNA for many taxa and are optimal for barcoding invertebrates, especially in combination with novel cytochrome c oxidase I (COI) primer cocktails and PCR cycling conditions.
Asunto(s)
Código de Barras del ADN Taxonómico , Complejo IV de Transporte de Electrones , Reacción en Cadena de la Polimerasa , Código de Barras del ADN Taxonómico/métodos , Animales , Complejo IV de Transporte de Electrones/genética , Reacción en Cadena de la Polimerasa/métodos , Invertebrados/genética , Invertebrados/clasificación , ADN/genética , ADN/aislamiento & purificaciónRESUMEN
Hematopoietic stem cells (HSCs) remain the most well-characterized adult stem cell population both in terms of markers for purification and assays to assess functional potential. However, despite over 40 years of research, working with HSCs in the mouse remains challenging because of the relative abundance (or lack thereof) of these cells in the bone marrow. The frequency of HSCs in murine bone marrow is about 0.01% of total nucleated cells and â¼5,000 can be isolated from an individual mouse depending on the age, sex, and strain of mice as well as purification scheme utilized. Adding to the challenge is the continual reporting of new markers for HSC purification, which makes it difficult for the uninitiated in the field to know which purification strategies yield the highest proportion of long-term, multilineage HSCs. In this updated version of our review from 2009, we review different strategies for hematopoietic stem and progenitor cell identification and purification. We will also discuss methods for rapid flow cytometric analysis of peripheral blood cell types, and novel strategies for working with rare cell populations such as HSCs in the analysis of cell cycle status by BrdU, Ki-67, and Pyronin Y staining. The purpose of updating this review is to provide insight into some of the recent experimental and technical advances in mouse hematopoietic stem cell biology.
Asunto(s)
Células de la Médula Ósea/citología , Citometría de Flujo/métodos , Células Madre Hematopoyéticas/citología , Animales , Ciclo Celular , Proliferación Celular , Colorantes , Humanos , RatonesRESUMEN
Cancer cells rely on altered metabolism to support abnormal proliferation. We performed a CRISPR/Cas9 functional genomic screen targeting metabolic enzymes and identified PDXK-an enzyme that produces pyridoxal phosphate (PLP) from vitamin B6-as an acute myeloid leukemia (AML)-selective dependency. PDXK kinase activity is required for PLP production and AML cell proliferation, and pharmacological blockade of the vitamin B6 pathway at both PDXK and PLP levels recapitulated PDXK disruption effects. PDXK disruption reduced intracellular concentrations of key metabolites needed for cell division. Furthermore, disruption of PLP-dependent enzymes ODC1 or GOT2 selectively inhibited AML cell proliferation and their downstream products partially rescued PDXK disruption induced proliferation blockage. Our work identifies the vitamin B6 pathway as a pharmacologically actionable dependency in AML.
Asunto(s)
Leucemia Mieloide Aguda/enzimología , Fosfotransferasas/metabolismo , Fosfato de Piridoxal/metabolismo , Vitamina B 6/metabolismo , Animales , Sistemas CRISPR-Cas , Línea Celular Tumoral , Proliferación Celular , GTP Fosfohidrolasas/metabolismo , Regulación Leucémica de la Expresión Génica , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Proteínas de Unión al GTP Monoméricas/metabolismo , Fosfotransferasas/genética , Fosfotransferasas (Aceptor de Grupo Alcohol) , Poliaminas/metabolismo , ARN Interferente Pequeño/metabolismoRESUMEN
Mutations in the TP53 tumor suppressor gene are common in many cancer types, including the acute myeloid leukemia (AML) subtype known as complex karyotype AML (CK-AML). Here, we identify a gain-of-function (GOF) Trp53 mutation that accelerates CK-AML initiation beyond p53 loss and, surprisingly, is required for disease maintenance. The Trp53R172H mutation (TP53R175H in humans) exhibits a neomorphic function by promoting aberrant self-renewal in leukemic cells, a phenotype that is present in hematopoietic stem and progenitor cells (HSPC) even prior to their transformation. We identify FOXH1 as a critical mediator of mutant p53 function that binds to and regulates stem cell-associated genes and transcriptional programs. Our results identify a context where mutant p53 acts as a bona fide oncogene that contributes to the pathogenesis of CK-AML and suggests a common biological theme for TP53 GOF in cancer. SIGNIFICANCE: Our study demonstrates how a GOF p53 mutant can hijack an embryonic transcription factor to promote aberrant self-renewal. In this context, mutant Trp53 functions as an oncogene to both initiate and sustain myeloid leukemia and suggests a potential convergent activity of mutant Trp53 across cancer types.This article is highlighted in the In This Issue feature, p. 813.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Mutación con Ganancia de Función , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/patología , Proteína p53 Supresora de Tumor/genética , Animales , Línea Celular Tumoral , Plasticidad de la Célula/genética , Factores de Transcripción Forkhead/genética , Perfilación de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , Células Madre Neoplásicas/metabolismo , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/patología , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoAsunto(s)
Proteínas del Citoesqueleto/genética , Atresia Esofágica/genética , Dosificación de Gen , Cardiopatías Congénitas/genética , Proteínas con Dominio LIM/genética , Deformidades Congénitas de las Extremidades/genética , Polimorfismo de Nucleótido Simple , Fístula Traqueoesofágica/genética , Alelos , Canal Anal/anomalías , Atresia Esofágica/diagnóstico , Esófago/anomalías , Estudios de Asociación Genética , Cardiopatías Congénitas/diagnóstico , Humanos , Riñón/anomalías , Deformidades Congénitas de las Extremidades/diagnóstico , Columna Vertebral/anomalías , Tráquea/anomalías , Fístula Traqueoesofágica/diagnósticoRESUMEN
Our understanding of the mechanisms that regulate hematopoietic stem/progenitor cells (HSPCs) has been advanced by the ability to genetically manipulate mice; however, germline modification is time consuming and expensive. Here, we describe fast, efficient, and cost-effective methods to directly modify the genomes of mouse and human HSPCs using the CRISPR/Cas9 system. Using plasmid and virus-free delivery of guide RNAs alone into Cas9-expressing HSPCs or Cas9-guide RNA ribonucleoprotein (RNP) complexes into wild-type cells, we have achieved extremely efficient gene disruption in primary HSPCs from mouse (>60%) and human (â¼75%). These techniques enabled rapid evaluation of the functional effects of gene loss of Eed, Suz12, and DNMT3A. We also achieved homology-directed repair in primary human HSPCs (>20%). These methods will significantly expand applications for CRISPR/Cas9 technologies for studying normal and malignant hematopoiesis.
Asunto(s)
Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Células Madre Hematopoyéticas/metabolismo , Animales , Reparación del ADN , Eliminación de Gen , Técnicas de Inactivación de Genes , Humanos , Antígenos Comunes de Leucocito/metabolismo , RatonesRESUMEN
DNMT3A, the gene encoding the de novo DNA methyltransferase 3A, is among the most frequently mutated genes in hematologic malignancies. However, the mechanisms through which DNMT3A normally suppresses malignancy development are unknown. Here, we show that DNMT3A loss synergizes with the FLT3 internal tandem duplication in a dose-influenced fashion to generate rapid lethal lymphoid or myeloid leukemias similar to their human counterparts. Loss of DNMT3A leads to reduced DNA methylation, predominantly at hematopoietic enhancer regions in both mouse and human samples. Myeloid and lymphoid diseases arise from transformed murine hematopoietic stem cells. Broadly, our findings support a role for DNMT3A as a guardian of the epigenetic state at enhancer regions, critical for inhibition of leukemic transformation.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN , Leucemia/genética , Tirosina Quinasa 3 Similar a fms/genética , Animales , ADN Metiltransferasa 3A , Elementos de Facilitación Genéticos , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Ratones , Mutación , Neoplasias ExperimentalesRESUMEN
Hematopoietic development and homeostasis are based on hematopoietic stem cells (HSCs), a pool of ancestor cells characterized by the unique combination of self-renewal and multilineage potential. These two opposing forces are finely orchestrated by several regulatory mechanisms, comprising both extrinsic and intrinsic factors. Over the past decades, several studies have contributed to dissect the key role of niche factors, signaling transduction pathways, and transcription factors in HSC development and maintenance. Accumulating evidence, however, suggests that a higher level of intrinsic regulation exists; epigenetic marks, by controlling chromatin accessibility, directly shape HSC developmental cascades, including their emergence during embryonic development, maintenance of self-renewal, lineage commitment, and aging. In addition, aberrant epigenetic marks have been found in several hematological malignancies, consistent with clinical findings that mutations targeting epigenetic regulators promote leukemogenesis. In this review, we will focus on both normal and malignant hematopoiesis, covering recent findings that illuminate the epigenetic life of HSCs.
Asunto(s)
Linaje de la Célula/fisiología , Cromatina/metabolismo , Epigénesis Genética/fisiología , Neoplasias Hematológicas/metabolismo , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/fisiología , Modelos Biológicos , Metilación de ADN/fisiología , Histonas/metabolismo , Humanos , Proteínas del Grupo Polycomb/metabolismoRESUMEN
Epigenetic regulation of hematopoietic stem cells (HSCs) ensures lifelong production of blood and bone marrow. Recently, we reported that loss of de novo DNA methyltransferase Dnmt3a results in HSC expansion and impaired differentiation. Here, we report conditional inactivation of Dnmt3b in HSCs either alone or combined with Dnmt3a deletion. Combined loss of Dnmt3a and Dnmt3b was synergistic, resulting in enhanced HSC self-renewal and a more severe block in differentiation than in Dnmt3a-null cells, whereas loss of Dnmt3b resulted in a mild phenotype. Although the predominant Dnmt3b isoform in adult HSCs is catalytically inactive, its residual activity in Dnmt3a-null HSCs can drive some differentiation and generates paradoxical hypermethylation of CpG islands. Dnmt3a/Dnmt3b-null HSCs displayed activated ß-catenin signaling, partly accounting for the differentiation block. These data demonstrate distinct roles for Dnmt3b in HSC differentiation and provide insights into complementary de novo methylation patterns governing regulation of HSC fate decisions.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/enzimología , Animales , Apoptosis , Diferenciación Celular/genética , Proliferación Celular , Islas de CpG/genética , Metilación de ADN/genética , ADN Metiltransferasa 3A , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Isoenzimas/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias/metabolismo , Neoplasias/patología , beta Catenina/metabolismo , ADN Metiltransferasa 3BRESUMEN
Hematopoietic stem cells (HSCs) represent one of the first recognized somatic stem cell types. As such, nearly 200 genes have been examined for roles in HSC function in knockout mice. In this review, we compile the majority of these reports to provide a broad overview of the functional modules revealed by these genetic analyses and highlight some key regulatory pathways involved, including cell cycle control, Tgf-ß signaling, Pten/Akt signaling, Wnt signaling, and cytokine signaling. Finally, we propose recommendations for characterization of HSC function in knockout mice to facilitate cross-study comparisons that would generate a more cohesive picture of HSC biology.