RESUMEN
Mice deficient in the transcription factor Runx3 develop a multitude of immune system defects, including early onset colitis. This paper demonstrates that Runx3 is expressed in colonic mononuclear phagocytes (MNP), including resident macrophages (RM) and dendritic cell subsets (cDC2). Runx3 deletion in MNP causes early onset colitis due to their impaired maturation. Mechanistically, the resulting MNP subset imbalance leads to up-regulation of pro-inflammatory genes as occurs in IL10R-deficient RM. In addition, RM and cDC2 display a marked decrease in expression of anti-inflammatory/TGF ß-regulated genes and ß-catenin signaling associated genes, respectively. MNP transcriptome and ChIP-seq data analysis suggest that a significant fraction of genes affected by Runx3 loss are direct Runx3 targets. Collectively, Runx3 imposes intestinal immune tolerance by regulating maturation of colonic anti-inflammatory MNP, befitting the identification of RUNX3 as a genome-wide associated risk gene for various immune-related diseases in humans, including gastrointestinal tract diseases such as Crohn's disease and celiac.
Asunto(s)
Colitis/inmunología , Colon/inmunología , Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Sistema Mononuclear Fagocítico/inmunología , Animales , Diferenciación Celular , Colitis/genética , Modelos Animales de Enfermedad , Humanos , Ratones , Receptores de Interleucina-10/genética , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia Arriba , beta Catenina/metabolismoRESUMEN
Leo Sachs spent almost his entire scientific career in Israel, at the Weizmann Institute of Science, and became a worldwide renowned scientist for his pioneering studies in normal hematopoiesis, its breakdown in leukemia and the suppression of malignancy by inducing differentiation, thereby bypassing genetic defects that give rise to malignancy. The cell culture system he established in the early 1960s for the clonal development of normal hematopoietic cells, made it possible to discover the proteins that regulate the viability, proliferation and differentiation of different blood cell lineages, the molecular basis of normal hematopoiesis and the changes that drive leukemia. His studies established significant general concepts including: a) the value of a multi-gene cytokine network in regulating the viability, number and development of different cell types; b) the existence of alternative pathways that give flexibility to development in both normal and cancer cells; c) the response of some cancer cells to normal regulators of development; d) suppression of myeloid leukemia by inducing differentiation, bypassing malignancy-driving genetic defects; e) identification of chromosomes that control tumor suppression; f) discovering apoptosis as a major mechanism by which WT-p53 suppresses malignancy and g) the ability of hematopoietic cytokines to suppress apoptosis in both normal and leukemic cells. It is gratifying that Leo had the good fortune to witness his pioneering discoveries and ideas move from the basic science stage to effective clinical applications, augmenting normal hematopoiesis in patients with various hematopoietic deficiencies, in patients requiring hematopoietic stem cell transplantation and in the suppression of malignancy by inducing differentiation and apoptosis.
Asunto(s)
Biología Evolutiva/historia , Hematopoyesis , Animales , Apoptosis , Células Sanguíneas/citología , Técnicas de Cultivo de Célula , Diferenciación Celular , División Celular , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Factores Estimulantes de Colonias , Citocinas/metabolismo , Células Madre Hematopoyéticas/citología , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Israel , Leucemia/metabolismoRESUMEN
In this chapter we summarize the pros and cons of the notion that Runx3 is a major tumor suppressor gene (TSG). Inactivation of TSGs in normal cells provides a viability/growth advantage that contributes cell-autonomously to cancer. More than a decade ago it was suggested that RUNX3 is involved in gastric cancer development, a postulate extended later to other epithelial cancers portraying RUNX3 as a major TSG. However, evidence that Runx3 is not expressed in normal gastric and other epithelia has challenged the RUNX3-TSG paradigm. In contrast, RUNX3 is overexpressed in a significant fraction of tumor cells in various human epithelial cancers and its overexpression in pancreatic cancer cells promotes their migration, anchorage-independent growth and metastatic potential. Moreover, recent high-throughput quantitative genome-wide studies on thousands of human samples of various tumors and new investigations of the role of Runx3 in mouse cancer models have unequivocally demonstrated that RUNX3 is not a bona fide cell-autonomous TSG. Importantly, accumulating data demonstrated that RUNX3 functions in control of immunity and inflammation, thereby indirectly influencing epithelial tumor development.
Asunto(s)
Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Inmunidad/genética , Inflamación/genética , Neoplasias/genética , Neoplasias/patología , Animales , Humanos , Inflamación/patologíaRESUMEN
Inactivation of tumor suppressor genes (TSG) in normal cells provides a viability/growth advantage that contributes cell-autonomously to cancer. More than a decade ago claims arose that the RUNX3 member of the RUNX transcription factor family is a major TSG inactivated in gastric cancer, a postulate extended later to other cancers. However, evidence that Runx3 is not expressed in normal gastric and other epithelia has challenged the RUNX3-TSG paradigm. Here we critically re-appraise this paradigm in light of recent high-throughput, quantitative genome-wide studies on thousands of human samples of various tumors and new investigations of the role of Runx3 in mouse cancer models. Collectively, these studies unequivocally demonstrate that RUNX3 is not a bona fide cell-autonomous TSG. Accordingly, RUNX3 is not recognized as a TSG and is not included among the 2000 cancer genes listed in the "Cancer Gene Census" or "Network for Cancer Genes" repositories. In contrast, RUNX3 does play important functions in immunity and inflammation and may thereby indirectly influence epithelial tumor development.
Asunto(s)
Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Inmunidad Innata/genética , Inflamación/genética , Neoplasias/genética , Subunidad alfa 3 del Factor de Unión al Sitio Principal/inmunología , Genes Supresores de Tumor , Humanos , Inflamación/inmunología , Inflamación/patología , Neoplasias/inmunología , Neoplasias/patología , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Glandulares y Epiteliales/inmunología , Neoplasias Glandulares y Epiteliales/patologíaRESUMEN
Carcinogen-induced skin tumorigenesis depends heavily on proinflammatory tumor-promoting processes. Here, we show that leukocytic Runx3 expression is central to the two-stage DMBA/TPA-induced skin tumorigenesis. Runx3-null mice were highly resistant to this process and concomitant ablation of Runx3 in dendritic and T cells fully recapitulated this resistance. Mechanistically, this resistance was associated with a shift in the skin cytokine milieu toward a tumor nonpermissive microenvironment. Specifically, leukocytic Runx3 loss substantially increased the antitumorigenic cytokine thymic stromal lymphopoietin (TSLP) and profoundly decreased two protumorigenic cytokines, interleukin-17a and osteopontin. Therefore, inflammation-mediated tumor promotion requires leukocytic Runx3 expression, as its loss creates a unique cytokine composition that polarizes the tumor microenvironment to a potent antitumorigenic state.
Asunto(s)
Carcinógenos/toxicidad , Subunidad alfa 3 del Factor de Unión al Sitio Principal/biosíntesis , Leucocitos/metabolismo , Neoplasias Cutáneas/metabolismo , 9,10-Dimetil-1,2-benzantraceno/toxicidad , Animales , Citocinas/biosíntesis , Modelos Animales de Enfermedad , Citometría de Flujo , Immunoblotting , Inmunohistoquímica , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa , Piridinas/toxicidad , Neoplasias Cutáneas/inducido químicamenteRESUMEN
Natural killer cells belong to the family of innate lymphoid cells comprising the frontline defense against infected and transformed cells. Development and activation of natural killer cells is highly dependent on interleukin-15 signaling. However, very little is known about the transcription program driving this process. The transcription factor Runx3 is highly expressed in natural killer cells, but its function in these cells is largely unknown. We show that loss of Runx3 impaired interleukin-15-dependent accumulation of mature natural killer cells in vivo and under culture conditions and pregnant Runx3(-/-) mice completely lack the unique population of interleukin-15-dependent uterine natural killer cells. Combined chromatin immunoprecipitation sequencing and differential gene expression analysis of wild-type versus Runx3-deficient in vivo activated splenic natural killer cells revealed that Runx3 cooperates with ETS and T-box transcription factors to drive the interleukin-15-mediated transcription program during activation of these cells. Runx3 functions as a nuclear regulator during interleukin-15-dependent activation of natural killer cells by regulating the expression of genes involved in proliferation, maturation, and migration. Similar studies with additional transcription factors will allow the construction of a more detailed transcriptional network that controls natural killer cell development and function.
Asunto(s)
Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Interleucina-15/genética , Células Asesinas Naturales/metabolismo , Activación de Linfocitos/genética , Factores de Transcripción/genética , Animales , Antígenos de Protozoos/genética , Antígenos de Protozoos/metabolismo , Movimiento Celular/genética , Proliferación Celular , Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Femenino , Interleucina-15/metabolismo , Interleucina-2/genética , Interleucina-2/metabolismo , Ratones , Regiones Promotoras Genéticas/genética , Factores de Transcripción/metabolismo , Transcripción Genética/genéticaRESUMEN
The transcription factor Runx3 is highly expressed in CD8(+) T and NK cytotoxic lymphocytes and is required for their effective activation and proliferation but molecular insights into the transcription program regulated by Runx3 in these cells are still missing. Using Runx3-ChIP-seq and transcriptome analysis of wild type vs. Runx3(-/-) primary cells we have now identified Runx3-regulated genes in the two cell types at both resting and IL-2-activated states. Runx3-bound genomic regions in both cell types were distantly located relative to gene transcription start sites and were enriched for RUNX and ETS motifs. Bound genomic regions significantly overlapped T-bet and p300-bound enhancer regions in Runx3-expressing Th1 helper cells. Compared to resting cells, IL-2-activated CD8(+) T and NK cells contain three times more Runx3-regulated genes that are common to both cell types. Functional annotation of shared CD8(+) T and NK Runx3-regulated genes revealed enrichment for immune-associated terms including lymphocyte activation, proliferation, cytotoxicity, migration and cytokine production, highlighting the role of Runx3 in CD8(+) T and NK activated cells.
Asunto(s)
Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Regulación de la Expresión Génica , Linfocitos T Citotóxicos/metabolismo , Transcripción Genética , Animales , Elementos de Facilitación Genéticos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Histonas/metabolismo , Interleucina-2/metabolismo , Interleucina-2/farmacología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Ratones Noqueados , Motivos de Nucleótidos , Posición Específica de Matrices de Puntuación , Unión Proteica , Fase de Descanso del Ciclo Celular/genética , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Factor de Transcripción AP-1/metabolismo , Sitio de Iniciación de la TranscripciónRESUMEN
The t(8;21) and inv(16) chromosomal aberrations generate the oncoproteins AML1-ETO (A-E) and CBFß-SMMHC (C-S). The role of these oncoproteins in acute myeloid leukemia (AML) etiology has been well studied. Conversely, the function of native RUNX1 in promoting A-E- and C-S-mediated leukemias has remained elusive. We show that wild-type RUNX1 is required for the survival of t(8;21)-Kasumi-1 and inv(16)-ME-1 leukemic cells. RUNX1 knockdown in Kasumi-1 cells (Kasumi-1(RX1-KD)) attenuates the cell-cycle mitotic checkpoint, leading to apoptosis, whereas knockdown of A-E in Kasumi-1(RX1-KD) rescues these cells. Mechanistically, a delicate RUNX1/A-E balance involving competition for common genomic sites that regulate RUNX1/A-E targets sustains the malignant cell phenotype. The broad medical significance of this leukemic cell addiction to native RUNX1 is underscored by clinical data showing that an active RUNX1 allele is usually preserved in both t(8;21) or inv(16) AML patients, whereas RUNX1 is frequently inactivated in other forms of leukemia. Thus, RUNX1 and its mitotic control targets are potential candidates for new therapeutic approaches.
Asunto(s)
Inversión Cromosómica , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Leucemia Mieloide Aguda/genética , Translocación Genética , Apoptosis/genética , Línea Celular Tumoral , Cromosomas Humanos Par 16 , Cromosomas Humanos Par 18 , Cromosomas Humanos Par 21 , Perfilación de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/patología , TransfecciónRESUMEN
RUNX1 transcription factor (TF) is a key regulator of megakaryocytic development and when mutated is associated with familial platelet disorder and predisposition to acute myeloid leukemia (FPD-AML). We used mice lacking Runx1 specifically in megakaryocytes (MK) to characterized Runx1-mediated transcriptional program during advanced stages of MK differentiation. Gene expression and chromatin-immunoprecipitation-sequencing (ChIP-seq) of Runx1 and p300 identified functional Runx1 bound MK enhancers. Runx1/p300 co-bound regions showed significant enrichment in genes important for MK and platelet homeostasis. Runx1 occupied genomic regions were highly enriched in RUNX and ETS motifs and to a lesser extent in GATA motif. Megakaryocytic specificity of Runx1/P300 bound enhancers was validated by transfection mutagenesis and Runx1/P300 co-bound regions of two key megakaryocytic genes Nfe2 and Selp were tested by in vivo transgenesis. The data provides the first example of genome wide Runx1/p300 occupancy in maturating primary FL-MK, unravel the Runx1-regulated program controlling MK maturation in vivo and identify a subset of its bona fide regulated genes. It advances our understanding of the molecular events that upon RUNX1mutations in human lead to the predisposition to familial platelet disorders and FPD-AML.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/fisiología , Regulación de la Expresión Génica , Megacariocitos/fisiología , Transcripción Genética , Animales , Secuencia de Bases , Sitios de Unión , Diferenciación Celular , Células Cultivadas , Inmunoprecipitación de Cromatina , Ratones , Ratones Noqueados , Cultivo Primario de Células , Regiones Promotoras Genéticas , Unión Proteica , Análisis de Secuencia de ADN , Factores de Transcripción p300-CBP/metabolismoRESUMEN
The Runx3 transcription factor regulates cell fate decisions during embryonic development and in adults. It was previously reported that Runx3 is strongly expressed in embryonic and adult gastrointestinal tract (GIT) epithelium (Ep) and that its loss causes gastric cancer. More than 280 publications have based their research on these findings and concluded that Runx3 is indeed a tumour suppressor (TS). In stark contrast, using various measures, we found that Runx3 expression is undetectable in GIT Ep. Employing a variety of biochemical and genetic techniques, including analysis of Runx3-GFP and R26LacZ/Runx3(Cre) or R26tdTomato/Runx3(Cre) reporter strains, we readily detected Runx3 in GIT-embedded leukocytes, dorsal root ganglia, skeletal elements and hair follicles. However, none of these approaches revealed detectable Runx3 levels in GIT Ep. Moreover, our analysis of the original Runx3(LacZ/LacZ) mice used in the previously reported study failed to reproduce the GIT expression of Runx3. The lack of evidence for Runx3 expression in normal GIT Ep creates a serious challenge to the published data and undermines the notion that Runx3 is a TS involved in cancer pathogenesis.
Asunto(s)
Subunidad alfa 3 del Factor de Unión al Sitio Principal/deficiencia , Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Mucosa Gástrica/metabolismo , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/metabolismo , Animales , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Células Epiteliales/citología , Células Epiteliales/metabolismo , Mucosa Gástrica/citología , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Noqueados , Especificidad de Órganos , Proteínas/metabolismo , ARN no Traducido , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Supresoras de Tumor/genética , beta-Galactosidasa/metabolismoRESUMEN
Activation of naive CD8(+) T cells with antigen induces their differentiation into effector cytolytic T lymphocytes (CTLs). CTLs lyse infected or aberrant target cells by exocytosis of lytic granules containing the pore-forming protein perforin and a family of proteases termed granzymes. We show that effector CTL differentiation occurs in two sequential phases in vitro, characterized by early induction of T-bet and late induction of Eomesodermin (Eomes), T-box transcription factors that regulate the early and late phases of interferon (IFN) gamma expression, respectively. In addition, we demonstrate a critical role for the transcription factor Runx3 in CTL differentiation. Runx3 regulates Eomes expression as well as expression of three cardinal markers of the effector CTL program: IFN-gamma, perforin, and granzyme B. Our data point to the existence of an elaborate transcriptional network in which Runx3 initially induces and then cooperates with T-box transcription factors to regulate gene transcription in differentiating CTLs.
Asunto(s)
Subunidad alfa 3 del Factor de Unión al Sitio Principal/fisiología , Regulación de la Expresión Génica , Proteínas de Dominio T Box/fisiología , Linfocitos T Citotóxicos/metabolismo , Animales , Northern Blotting , Western Blotting , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Citotoxicidad Inmunológica/inmunología , Granzimas/genética , Granzimas/metabolismo , Interferón gamma/metabolismo , Interleucina-2/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Modelos Biológicos , Perforina/genética , Perforina/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The transcription factor Runx1 is a key regulator of definitive hematopoiesis in the embryo and the adult. Lineage-specific expression of Runx1 involves transcription and post-transcription control through usage of alternative promoters and diverse 3'UTR isoforms, respectively. We identified and mapped microRNA (miR) binding sites on Runx1 3'UTR and show that miR-27a, miR-9, miR-18a, miR-30c, and miR-199a* bind and post-transcriptionally attenuate expression of Runx1. miR-27a impacts on both the shortest (0.15 kb) and longest (3.8 kb) 3'UTRs and, along with additional miRs, might contribute to translation attenuation of Runx1 mRNA in the myeloid cell line 416B. Whereas levels of Runx1 mRNA in 416B and the B cell line 70Z were similar, the protein levels were not. Large amounts of Runx1 protein were found in 70Z cells, whereas only minute amounts of Runx1 protein were made in 416B cells and overexpression of Runx1 in 416B induced terminal differentiation associated with megakaryocytic markers. Induction of megakaryocytic differentiation in K562 cells by 12-o-tetradecanoylphorbol-13-acetate markedly increased miR-27a expression, concomitantly with binding of Runx1 to miR-27a regulatory region. The data indicate that miR-27a plays a regulatory role in megakaryocytic differentiation by attenuating Runx1 expression, and that, during megakaryopoiesis, Runx1 and miR-27a are engaged in a feedback loop involving positive regulation of miR-27a expression by Runx1.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/fisiología , Regulación de la Expresión Génica/fisiología , Megacariocitos/citología , MicroARNs/fisiología , Trombopoyesis/genética , Diferenciación Celular , Línea Celular , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Retroalimentación Fisiológica , Humanos , MicroARNs/genética , Unión Proteica , ARN Mensajero/análisisRESUMEN
We have analyzed gene expression in different normal human tissues and different types of solid cancers derived from these tissues. The cancers analyzed include brain (astrocytoma and glioblastoma), breast, colon, endometrium, kidney, liver, lung, ovary, prostate, skin, and thyroid cancers. Comparing gene expression in each normal tissue to 12 other normal tissues, we identified 4,917 tissue-selective genes that were selectively expressed in different normal tissues. We also identified 2,929 genes that are overexpressed at least 4-fold in the cancers compared with the normal tissue from which these cancers were derived. The overlap between these two gene groups identified 1,340 tissue-selective genes that are overexpressed in cancers. Different types of cancers, including different brain cancers arising from the same lineage, showed differences in the tissue-selective genes they overexpressed. Melanomas overexpressed the highest number of brain-selective genes and this may contribute to melanoma metastasis to the brain. Of all of the genes with tissue-selective expression, those selectively expressed in testis showed the highest frequency of genes that are overexpressed in at least two types of cancer. However, colon and prostate cancers did not overexpress any testis-selective gene. Nearly all of the genes with tissue-selective expression that are overexpressed in cancers showed selective expression in tissues different from the cancers' tissue of origin. Cancers aberrantly expressing such genes may acquire phenotypic alterations that contribute to cancer cell viability, growth, and metastasis.
Asunto(s)
Perfilación de la Expresión Génica , Neoplasias/genética , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Especificidad de ÓrganosRESUMEN
BACKGROUND: Alternative promoters usage is an important paradigm in transcriptional control of mammalian gene expression. However, despite the growing interest in alternative promoters and their role in genome diversification, very little is known about how and on what occasions those promoters are differentially regulated. Runx1 transcription factor is a key regulator of early hematopoiesis and a frequent target of chromosomal translocations in acute leukemias. Mice deficient in Runx1 lack definitive hematopoiesis and die in mid-gestation. Expression of Runx1 is regulated by two functionally distinct promoters designated P1 and P2. Differential usage of these two promoters creates diversity in distribution and protein-coding potential of the mRNA transcripts. While the alternative usage of P1 and P2 likely plays an important role in Runx1 biology, very little is known about the function of the P1/P2 switch in mediating tissue and stage specific expression of Runx1 during development. RESULTS: We employed mice bearing a hypomorphic Runx1 allele, with a largely diminished P2 activity, to investigate the biological role of alternative P1/P2 usage. Mice homozygous for the hypomorphic allele developed to term, but died within a few days after birth. During embryogenesis the P1/P2 activity is spatially and temporally modulated. P2 activity is required in early hematopoiesis and when attenuated, development of liver hematopoietic progenitor cells (HPC) was impaired. Early thymus development and thymopoiesis were also abrogated as reflected by thymic hypocellularity and loss of corticomedullary demarcation. Differentiation of CD4/CD8 thymocytes was impaired and their apoptosis was enhanced due to altered expression of T-cell receptors. CONCLUSION: The data delineate the activity of P1 and P2 in embryogenesis and describe previously unknown functions of Runx1. The findings show unequivocally that the role of P1/P2 during development is non redundant and underscore the significance of alternative promoter usage in Runx1 biology.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Regulación del Desarrollo de la Expresión Génica , Hematopoyesis Extramedular/genética , Regiones Promotoras Genéticas , Timo/embriología , Alelos , Animales , Apoptosis , Diferenciación Celular , Ensayo de Unidades Formadoras de Colonias , Cartilla de ADN , Embrión de Mamíferos , Células Madre Embrionarias/citología , Citometría de Flujo , Genes Letales , Células Madre Hematopoyéticas/citología , Hibridación in Situ , Ratones , Ratones Noqueados , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timo/citologíaRESUMEN
We have analyzed gene expression data from three different kinds of samples: normal human tissues, human cancer cell lines, and leukemic cells from lymphoid and myeloid leukemia pediatric patients. We have searched for genes that are overexpressed in human cancer and also show specific patterns of tissue-dependent expression in normal tissues. Using the expression data of the normal tissues, we identified 4,346 genes with a high variability of expression and clustered these genes according to their relative expression level. Of 91 stable clusters obtained, 24 clusters included genes preferentially expressed either only in hematopoietic tissues or in hematopoietic and one to two other tissues; 28 clusters included genes preferentially expressed in various nonhematopoietic tissues such as neuronal, testis, liver, kidney, muscle, lung, pancreas, and placenta. Analysis of the expression levels of these two groups of genes in the human cancer cell lines and leukemias identified genes that were highly expressed in cancer cells but not in their normal counterparts and, thus, were overexpressed in the cancers. The different cancer cell lines and leukemias varied in the number and identity of these overexpressed genes. The results indicate that many genes that are overexpressed in human cancer cells are specific to a variety of normal tissues, including normal tissues other than those from which the cancer originated. It is suggested that this general property of cancer cells plays a major role in determining the behavior of the cancers, including their metastatic potential.
Asunto(s)
Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/genética , Neoplasias/genética , Regulación hacia Arriba/genética , Adenocarcinoma/genética , Línea Celular , Línea Celular Tumoral , Salud , Humanos , Leucemia/clasificación , Leucemia/genética , Familia de Multigenes/genética , Neoplasias/clasificaciónRESUMEN
NAD(P)H:quinone oxidoreductase 1 (NQO1) regulates the stability of the tumor suppressor WT p53. NQO1 binds and stabilizes WT p53, whereas NQO1 inhibitors including dicoumarol and various other coumarins and flavones induce ubiquitin-independent proteasomal p53 degradation and thus inhibit p53-induced apoptosis. Here, we show that curcumin, a natural phenolic compound found in the spice turmeric, induced ubiquitin-independent degradation of WT p53 and inhibited p53-induced apoptosis in normal thymocytes and myeloid leukemic cells. Like dicoumarol, curcumin inhibited the activity of recombinant NQO1 in vitro, inhibited the activity of endogenous cellular NQO1 in vivo, and dissociated NQO1-WT p53 complexes. Neither dicoumarol nor curcumin dissociated the complexes of NQO1 and the human cancer hot-spot p53 R273H mutant and therefore did not induce degradation of this mutant. NQO1 knockdown by small-interfering RNA induced degradation of both WT p53 and the p53 R273H mutant. The results indicate that curcumin induces p53 degradation and inhibits p53-induced apoptosis by an NQO1-dependent pathway.
Asunto(s)
Curcumina/farmacología , NAD(P)H Deshidrogenasa (Quinona)/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Células Cultivadas , Humanos , Ratones , Mutación Missense/genética , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Neoplasias/genética , Timo/citología , Timo/efectos de los fármacos , Timo/metabolismo , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Ubiquitina/metabolismoRESUMEN
Using DNA microarray and cluster analysis of expressed genes in a cloned line (M1-t-p53) of myeloid leukemic cells, we have analyzed the expression of genes that are preferentially expressed in different normal tissues. Clustering of 547 highly expressed genes in these leukemic cells showed 38 genes preferentially expressed in normal hematopoietic tissues and 122 other genes preferentially expressed in different normal nonhematopoietic tissues, including neuronal tissues, muscle, liver, and testis. We have also analyzed the genes whose expression in the leukemic cells changed after activation of WT p53 and treatment with the cytokine IL-6 or the calcium mobilizer thapsigargin. Of 620 such genes in the leukemic cells that were differentially expressed in normal tissues, clustering showed 80 genes that were preferentially expressed in hematopoietic tissues and 132 genes in different normal nonhematopoietic tissues that also included neuronal tissues, muscle, liver, and testis. Activation of p53 and treatment with IL-6 or thapsigargin induced different changes in the genes preferentially expressed in these normal tissues. These myeloid leukemic cells thus express genes that are expressed in normal nonhematopoietic tissues, and various treatments can reprogram these cells to induce other such nonhematopoietic genes. The results indicate that these leukemic cells share with normal hematopoietic stem cells the plasticity of differentiation to different cell types. It is suggested that this reprogramming to induce in malignant cells genes that are expressed in different normal tissues may be of clinical value in therapy.
Asunto(s)
Expresión Génica , Leucemia Mieloide/genética , Animales , Línea Celular Tumoral , Análisis por Conglomerados , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Genes p53 , Interleucina-6/farmacología , Leucemia Mieloide/patología , Ratones , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Tapsigargina/farmacología , Distribución TisularAsunto(s)
Apoptosis/fisiología , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Dicumarol/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Transcripción Genética , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Runx3 transcription factor regulates cell lineage decisions in thymopoiesis and neurogenesis. Here we report that Runx3 knockout (KO) mice develop spontaneous eosinophilic lung inflammation associated with airway remodeling and mucus hypersecretion. Runx3 is specifically expressed in mature dendritic cells (DC) and mediates their response to TGF-beta. In the absence of Runx3, DC become insensitive to TGF-beta-induced maturation inhibition, and TGF-beta-dependent Langerhans cell development is impaired. Maturation of Runx3 KO DC is accelerated and accompanied by increased efficacy to stimulate T cells and aberrant expression of beta2-integrins. Lung alveoli of Runx3 KO mice accumulate DC characteristic of allergic airway inflammation. Taken together, abnormalities in DC function and subset distribution may constitute the primary immune system defect, which leads to the eosinophilic lung inflammation in Runx3 KO mice. These data may help elucidate the molecular mechanisms underlying the pathogenesis of allergic airway inflammation in humans.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Células Dendríticas/metabolismo , Neumonía/inmunología , Factores de Transcripción/fisiología , Factor de Crecimiento Transformador beta/fisiología , Animales , Líquido del Lavado Bronquioalveolar/citología , Antígenos CD18/biosíntesis , Células Cultivadas , Subunidad alfa 3 del Factor de Unión al Sitio Principal , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Células Dendríticas/patología , Eosinófilos/patología , Ratones , Ratones Noqueados , Moco/metabolismo , Neumonía/patología , Alveolos Pulmonares/patología , Transducción de Señal , Linfocitos T/metabolismo , Linfocitos T/patología , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
Proteasomal degradation of p53 is mediated by two alternative pathways that are either dependent or independent of both Mdm2 and ubiquitin. The ubiquitin-independent pathway is regulated by NAD(P)H: quinone oxidoreductase 1 (NQO1) that stabilizes p53. The NQO1 inhibitor dicoumarol induces ubiquitin-independent p53 degradation. We now show that, like dicoumarol, several other coumarin and flavone inhibitors of NQO1 activity, which compete with NAD(P)H for binding to NQO1, induced ubiquitin-independent p53 degradation and inhibited wild-type p53-mediated apoptosis. Although wild-type p53 and several p53 mutants were sensitive to dicoumarol-induced degradation, the most frequent "hot-spot" p53 mutants in human cancer, R175H, R248H, and R273H, were resistant to dicoumarol-induced degradation, but remained sensitive to Mdm2-ubiquitin-mediated degradation. The two alternative pathways for p53 degradation thus have different p53 structural requirements. Further mutational analysis showed that arginines at positions 175 and 248 were essential for dicoumarol-induced p53 degradation. NQO1 bound to wild-type p53 and dicoumarol, which induced a conformational change in NQO1, inhibited this binding. Compared with wild-type p53, the hot-spot p53 mutants showed increased binding to NQO1, which can explain their resistance to dicoumarol-induced degradation. NQO1 thus has an important role in stabilizing hot-spot p53 mutant proteins in human cancer.