RESUMEN
Grasp55 is a ubiquitous Golgi stacking protein involved in autophagy, protein trafficking, and glucose deprivation sensing. The function of Grasp55 in protein trafficking has been attributed to its PDZ-mediated interaction with the C-terminal PDZ-binding motifs of protein cargos. We have recently shown that such an interaction occurs between Grasp55 and the adhesion molecule Jam-C, which plays a central role in stemness maintenance of hematopoietic and spermatogenic cells. Accordingly, we have found that Grasp55-deficient mice suffer from spermatogenesis defects similar to Jam-C knockout mice. However, whether Grasp55 is involved in the maintenance of immunohematopoietic homeostasis through regulation of protein transport and Jam-C expression remains unknown. In this study, we show that Grasp55 deficiency does not affect hematopoietic stem cell differentiation, engraftment, or mobilization, which are known to depend on expression of Grasp55-dependent protein cargos. In contrast, using an Myc-dependent leukemic model addicted to autophagy, we show that knockdown of Grasp55 in leukemic cells reduces spleen and bone marrow tumor burden upon i.v. leukemic engraftment. This is not due to reduced homing of Grasp55-deficient cells to these organs but to increased spontaneous apoptosis of Grasp55-deficient leukemic cells correlated with increased sensitivity of the cells to glucose deprivation. These results show that Grasp55 plays a role in Myc-transformed hematopoietic cells but not in normal hematopoietic cells in vivo.
Asunto(s)
Aparato de Golgi/patología , Proteínas de la Matriz de Golgi/metabolismo , Leucemia/metabolismo , Animales , Apoptosis/genética , Autofagia , Carcinogénesis , Supervivencia Celular , Proteínas de la Matriz de Golgi/genética , Hematopoyesis/genética , Leucemia/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-myc/metabolismo , Carga TumoralRESUMEN
Spermatogenesis is a dynamic process that is regulated by adhesive interactions between germ and Sertoli cells. Germ cells express the Junctional Adhesion Molecule-C (JAM-C, encoded by Jam3), which localizes to germ/Sertoli cell contacts. JAM-C is involved in germ cell polarity and acrosome formation. Using a proteomic approach, we demonstrated that JAM-C interacted with the Golgi reassembly stacking protein of 55 kDa (GRASP55, encoded by Gorasp2) in developing germ cells. Generation and study of Gorasp2-/- mice revealed that knock-out mice suffered from spermatogenesis defects. Acrosome formation and polarized localization of JAM-C in spermatids were altered in Gorasp2-/- mice. In addition, Golgi morphology of spermatocytes was disturbed in Gorasp2-/- mice. Crystal structures of GRASP55 in complex with JAM-C or JAM-B revealed that GRASP55 interacted via PDZ-mediated interactions with JAMs and induced a conformational change in GRASP55 with respect of its free conformation. An in silico pharmacophore approach identified a chemical compound called Graspin that inhibited PDZ-mediated interactions of GRASP55 with JAMs. Treatment of mice with Graspin hampered the polarized localization of JAM-C in spermatids, induced the premature release of spermatids and affected the Golgi morphology of meiotic spermatocytes.
Asunto(s)
Proteínas Portadoras/metabolismo , Moléculas de Adhesión Celular/metabolismo , Aparato de Golgi/metabolismo , Inmunoglobulinas/metabolismo , Proteínas de la Membrana/metabolismo , Espermatogénesis , Espermatogonias/metabolismo , Animales , Sitios de Unión , Proteínas Portadoras/química , Proteínas Portadoras/genética , Células Cultivadas , Aparato de Golgi/ultraestructura , Péptidos y Proteínas de Señalización Intracelular , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Transporte de Proteínas , Espermatogonias/citologíaRESUMEN
Most cancer deaths result from metastasis, which is the dissemination of cells from a primary tumor to distant organs. Metastasis involves changes to molecules that are essential for tumor cell adhesion to the extracellular matrix and to endothelial cells. Junctional Adhesion Molecule C (JAM-C) localizes at intercellular junctions as homodimers or more affine heterodimers with JAM-B. We previously showed that the homodimerization site (E66) in JAM-C is also involved in JAM-B binding. Here we show that neoexpression of JAM-C in a JAM-C-negative carcinoma cell line induced loss of adhesive property and pro-metastatic capacities. We also identify two critical structural sites (E66 and K68) for JAM-C/JAM-B interaction by directed mutagenesis of JAM-C and studied their implication on tumor cell behavior. JAM-C mutants did not bind to JAM-B or localize correctly to junctions. Moreover, mutated JAM-C proteins increased adhesion and reduced proliferation and migration of lung carcinoma cell lines. Carcinoma cells expressing mutant JAM-C grew slower than with JAM-C WT and were not able to establish metastatic lung nodules in mice. Overall these data demonstrate that the dimerization sites E66-K68 of JAM-C affected cell adhesion, polarization and migration and are essential for tumor cell metastasis.
Asunto(s)
Movimiento Celular , Molécula C de Adhesión de Unión/metabolismo , Multimerización de Proteína , Secuencia de Aminoácidos , Animales , Adhesión Celular , Línea Celular Tumoral , Polaridad Celular , Proliferación Celular , Células Epiteliales/patología , Molécula B de Adhesión de Unión/metabolismo , Molécula C de Adhesión de Unión/química , Molécula C de Adhesión de Unión/genética , Pulmón/patología , Ratones Endogámicos NOD , Ratones SCID , Proteínas Mutantes/metabolismo , Mutación/genética , Metástasis de la Neoplasia , Fenotipo , Unión ProteicaRESUMEN
Hematopoietic stem cells (HSCs) located in adult bone marrow or fetal liver in mammals produce all cells from the blood system. At the top of the hierarchy are long-term HSCs endowed with lifelong self-renewal and differentiation properties. These features are controlled through key microenvironmental cues and regulatory pathways, such as Wnt signaling. We showed previously that PTK7, a tyrosine kinase receptor involved in planar cell polarity, plays a role in epithelial Wnt signaling; however, its function in hematopoiesis has remained unexplored. In this article, we show that PTK7 is expressed by hematopoietic stem and progenitor cells, with the highest level of protein expression found on HSCs. Taking advantage of a Ptk7-deficient mouse strain, we demonstrate that loss of Ptk7 leads to a diminished pool of HSCs but does not affect in vitro or in vivo hematopoietic cell differentiation. This is correlated with increased quiescence and reduced homing abilities of Ptk7-deficient hematopoietic stem and progenitor cells, unraveling novel and unexpected functions for planar cell polarity pathways in HSC fate.
Asunto(s)
Movimiento Celular , Proliferación Celular , Hematopoyesis , Células Madre Hematopoyéticas/citología , Proteínas Tirosina Quinasas Receptoras/genética , Animales , Adhesión Celular , Línea Celular , Polaridad Celular , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de SeñalRESUMEN
The junctional adhesion molecules Jam-b and Jam-c interact together at interendothelial junctions and have been involved in the regulation of immune response, inflammation, and leukocyte migration. More recently, Jam-c has been found to be expressed by hematopoietic stem and progenitor cells (HSPC) in mouse. Conversely, we have reported that Jam-b is present on bone marrow stromal cells and that Jam-b-deficient mice have defects in the regulation of hematopoietic stem cell pool. In this study, we have addressed whether interaction between Jam-b and Jam-c participates to HSPC mobilization or hematopoietic reconstitution after irradiation. We show that a blocking monoclonal antibody directed against Jam-c inhibits hematopoietic reconstitution, progenitor homing to the bone marrow, and induces HSPC mobilization in a Jam-b dependent manner. In the latter setting, antibody treatment over a period of 3 days does not alter hematopoietic differentiation nor induce leukocytosis. Results are translated to human hematopoietic system in which a functional adhesive interaction between JAM-B and JAM-C is found between human HSPC and mesenchymal stem cells. Such an interaction does not occur between HSPC and human endothelial cells or osteoblasts. It is further shown that anti-JAM-C blocking antibody interferes with CD34(+) hematopoietic progenitor homing in mouse bone marrow suggesting that monoclonal antibodies inhibiting JAM-B/JAM-C interaction may represent valuable therapeutic tools to improve stem cell mobilization protocols.
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Anticuerpos Monoclonales/farmacología , Moléculas de Adhesión Celular/metabolismo , Movilización de Célula Madre Hematopoyética/métodos , Células Madre Hematopoyéticas/metabolismo , Inmunoglobulinas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Animales , Moléculas de Adhesión Celular/antagonistas & inhibidores , Comunicación Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Endoteliales/citología , Células Endoteliales/metabolismo , Femenino , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Humanos , Masculino , Células Madre Mesenquimatosas/citología , Ratones , Ratones Noqueados , Osteoblastos/citología , Osteoblastos/metabolismoRESUMEN
ABSTRACT: The leukemic stem cell (LSC) score LSC-17 based on a stemness-related gene expression signature is an indicator of poor disease outcome in acute myeloid leukemia (AML). However, it is not known whether "niche anchoring" of LSC affects disease evolution. To address this issue, we conditionally inactivated the adhesion molecule JAM-C (Junctional Adhesion Molecule-C) expressed by hematopoietic stem cells (HSCs) and LSCs in an inducible mixed-lineage leukemia (iMLL)-AF9-driven AML mouse model. Deletion of Jam3 (encoding JAM-C) before induction of the leukemia-initiating iMLL-AF9 fusion resulted in a shift from long-term to short-term HSC expansion, without affecting disease initiation and progression. In vitro experiments showed that JAM-C controlled leukemic cell nesting irrespective of the bone marrow stromal cells used. RNA sequencing performed on leukemic HSCs isolated from diseased mice revealed that genes upregulated in Jam3-deficient animals belonged to activation protein-1 (AP-1) and tumor necrosis factor α (TNF-α)/NF-κB pathways. Human orthologs of dysregulated genes allowed to identify a score that was distinct from, and complementary to, the LSC-17 score. Substratification of patients with AML using LSC-17 and AP-1/TNF-α genes signature defined 4 groups with median survival ranging from <1 year to a median of "not reached" after 8 years. Finally, coculture experiments showed that AP-1 activation in leukemic cells was dependent on the nature of stromal cells. Altogether, our results identify the AP-1/TNF-α gene signature as a proxy of LSC anchoring in bone marrow niches, which improves the prognostic value of the LSC-17 score. This trial was registered at www.ClinicalTrials.gov as #NCT02320656.
Asunto(s)
Moléculas de Adhesión Celular , Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda , Células Madre Neoplásicas , Animales , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Humanos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Moléculas de Adhesión Celular/metabolismo , Moléculas de Adhesión Celular/genética , Eliminación de Gen , Modelos Animales de Enfermedad , Células Madre Hematopoyéticas/metabolismo , InmunoglobulinasRESUMEN
In adult mammals, hematopoietic stem cells (HSCs) reside in the bone marrow (BM) and are maintained in a quiescent and undifferentiated state through adhesive interactions with specialized microenvironmental niches. Although junctional adhesion molecule-C (JAM-C) is expressed by HSCs, its function in adult hematopoiesis remains elusive. Here, we show that HSCs adhere to JAM-B expressed by BM stromal cells in a JAM-C dependent manner. The interaction regulates the interplay between HSCs and BM stromal cells as illustrated by the decreased pool of quiescent HSCs observed in jam-b deficient mice. We further show that this is probably because of alterations of BM stromal compartments and changes in SDF-1α BM content in jam-b(-/-) mice, suggesting that JAM-B is an active player in the maintenance of the BM stromal microenvironment.
Asunto(s)
Células de la Médula Ósea/fisiología , Moléculas de Adhesión Celular/fisiología , Células Madre Hematopoyéticas/fisiología , Inmunoglobulinas/fisiología , Animales , Médula Ósea/metabolismo , Médula Ósea/fisiología , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Adhesión Celular/genética , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Proliferación Celular , Hematopoyesis/genética , Hematopoyesis/fisiología , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/metabolismo , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Masculino , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
The development and maintenance of secondary lymphoid organs, such as lymph nodes, occur in a highly coordinated manner involving lymphoid chemokine production by stromal cells. Although developmental pathways inducing lymphoid chemokine production during organogenesis are known, signals maintaining cytokine production in adults are still elusive. In this study, we show that thrombomodulin and platelet-derived growth factor receptor α identify a population of fibroblastic reticular cells in which chemokine secretion is controlled by JAM-C. We demonstrate that Jam-C-deficient mice and mice treated with Ab against JAM-C present significant decreases in stromal cell-derived factor 1α (CXCL12), CCL21, and CCL19 intranodal content. This effect is correlated with reduced naive T cell egress from lymph nodes of anti-JAM-C-treated mice.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Quimiocinas/metabolismo , Fibroblastos/metabolismo , Homeostasis/inmunología , Inmunoglobulinas/fisiología , Ganglios Linfáticos/metabolismo , Trombomodulina/biosíntesis , Animales , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/deficiencia , Línea Celular Transformada , Movimiento Celular/genética , Movimiento Celular/inmunología , Fibroblastos/inmunología , Regulación de la Expresión Génica/inmunología , Células HEK293 , Homeostasis/genética , Humanos , Inmunoglobulinas/biosíntesis , Inmunoglobulinas/deficiencia , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratas , Ratas Endogámicas F344 , Células del Estroma/inmunología , Células del Estroma/metabolismoRESUMEN
B-cell acute lymphoblastic leukemia (B-ALL) reflects the malignant counterpart of developing B cells in the bone marrow (BM). Despite tremendous progress in B-ALL treatment, the overall survival of adults at diagnosis and patients at all ages after relapse remains poor. Galectin-1 (GAL1) expressed by BM supportive niches delivers proliferation signals to normal pre-B cells through interaction with the pre-B cell receptor (pre-BCR). Here, we asked whether GAL1 gives non-cell autonomous signals to pre-BCR+ pre-B ALL, in addition to cell-autonomous signals linked to genetic alterations. In syngeneic and patient-derived xenograft (PDX) murine models, murine and human pre-B ALL development is influenced by GAL1 produced by BM niches through pre-BCR-dependent signals, similarly to normal pre-B cells. Furthermore, targeting pre-BCR signaling together with cell-autonomous oncogenic pathways in pre-B ALL PDX improved treatment response. Our results show that non-cell autonomous signals transmitted by BM niches represent promising targets to improve B-ALL patient survival.
RESUMEN
BACKGROUND AIMS: X-linked agammaglobulinemia (XLA) is a rare primary immunodeficiency associated with mutations of the BTK (Bruton agammaglobulinemia tyrosine kinase) gene. Non-functional BTK leads to a severe decline in peripheral B cells and profound pan-hypogammaglobulinemia. Substitutive immunoglobulin replacement therapy improves long-term survival but remains a symptomatic rather than curative treatment that does not provide an optimal quality of life. Hematopoietic stem cell gene therapy represents a potentially curative treatment. Thorough pre-clinical testing of innovative therapies requires that adequate disease models are available. Invalidation of the murine btk gene produces a phenotype that is less severe than the human disease; alternatively, xenotransplantation of human hematopoietic progenitors obtained from XLA patients may provide a model for testing new treatment procedures. METHODS: The standard of care for XLA patients rarely offers an opportunity to collect peripheral blood or bone marrow (BM) hematopoietic progenitors; however, we had access to two BM samples obtained from such individuals. We analyzed hematopoietic engraftment of immunoselected CD34(+) cells from these samples in NOD/SCID/ gammac(null) (NOG) mice. RESULTS: In both cases, human hematopoietic cells were readily detected in BM and thymus, and at low levels in spleen and peripheral blood. Unexpectedly, the early defect in B-lymphoid differentiation associated with XLA was not accurately reproduced in NOG mice, as large amounts of pre-B cells were found in BM. CONCLUSIONS: These results support the existence of differences in environmental regulation of B-cell ontogeny between mice and humans. This questions the relevance of the NOG xenograft model for pre-clinical study of XLA gene therapy.
Asunto(s)
Agammaglobulinemia/terapia , Linfocitos B/patología , Diferenciación Celular , Enfermedades Genéticas Ligadas al Cromosoma X/terapia , Células Madre Hematopoyéticas/citología , Agammaglobulinemia Tirosina Quinasa , Agammaglobulinemia/sangre , Agammaglobulinemia/inmunología , Animales , Antígenos CD34/biosíntesis , Linfocitos B/metabolismo , Médula Ósea , Proteínas Portadoras , Separación Celular , Modelos Animales de Enfermedad , Citometría de Flujo , Enfermedades Genéticas Ligadas al Cromosoma X/sangre , Enfermedades Genéticas Ligadas al Cromosoma X/inmunología , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/fisiología , Humanos , Sistema Inmunológico/citología , Sistema Inmunológico/crecimiento & desarrollo , Ratones , Ratones Noqueados , Ratones SCID , Proteínas Tirosina Quinasas/deficiencia , Trasplante HeterólogoRESUMEN
In the bone marrow, CXCL12 and IL-7 are essential for B cell differentiation, whereas hematopoietic stem cell (HSC) maintenance requires SCF and CXCL12. Peri-sinusoidal stromal (PSS) cells are the main source of IL-7, but their characterization as a pro-B cell niche remains limited. Here, we characterize pro-B cell supporting stromal cells and decipher the interaction network allowing pro-B cell retention. Preferential contacts are found between pro-B cells and PSS cells, which homogeneously express HSC and B cell niche genes. Furthermore, pro-B cells are frequently located in the vicinity of HSCs in the same niche. Using an interactome bioinformatics pipeline, we identify Nidogen-1 as essential for pro-B cell retention in the peri-sinusoidal niche as confirmed in Nidogen-1-/- mice. Finally, human pro-B cells and hematopoietic progenitors are observed close to similar IL-7+ stromal cells. Thus, a multispecific niche exists in mouse and human supporting both early progenitors and committed hematopoietic lineages.
Asunto(s)
Células Madre Hematopoyéticas/inmunología , Glicoproteínas de Membrana/inmunología , Células Precursoras de Linfocitos B/inmunología , Nicho de Células Madre/inmunología , Animales , Células Madre Hematopoyéticas/citología , Interleucina-7/genética , Interleucina-7/inmunología , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Células Precursoras de Linfocitos B/citología , Células del Estroma/citología , Células del Estroma/inmunologíaRESUMEN
Acute myeloid leukemia (AML) originates from hematopoietic stem and progenitor cells that acquire somatic mutations, leading to disease and clonogenic evolution. AML is characterized by accumulation of immature myeloid cells in the bone marrow and phenotypic cellular heterogeneity reflective of normal hematopoietic differentiation. Here, we show that JAM-C expression defines a subset of leukemic cells endowed with leukemia-initiating cell activity (LIC). Stratification of de novo AML patients at diagnosis based on JAM-C-expressing cells frequencies in the blood served as an independent prognostic marker for disease outcome. Using publicly available leukemic stem cell (LSC) gene expression profiles and gene expression data generated from JAM-C-expressing leukemic cells, we defined a single cell core gene expression signature correlated to JAM-C expression that reveals LSC heterogeneity. Finally, we demonstrated that JAM-C controls Src family kinase (SFK) activation in LSC and that LIC with exacerbated SFK activation was uniquely found within the JAM-C-expressing LSC compartment. Cancer Res; 77(23); 6627-40. ©2017 AACR.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Moléculas de Adhesión Celular/metabolismo , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/patología , Familia-src Quinasas/metabolismo , ADP-Ribosil Ciclasa 1/metabolismo , Animales , Antígenos CD34/metabolismo , Biomarcadores de Tumor/genética , Moléculas de Adhesión Celular/genética , Línea Celular Tumoral , Activación Enzimática , Femenino , Perfilación de la Expresión Génica , Humanos , Subunidad alfa del Receptor de Interleucina-3/metabolismo , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Trasplante de Neoplasias , Células Madre Neoplásicas/citología , Trasplante HeterólogoRESUMEN
OBJECTIVE: The aim of this report is to demonstrate the feasibility and safety of genetically modifying autologous human blood CD34(+) cells in vitro, with a retroviral vector that encodes a marker gene. The fate of genetically modified cells and their progeny was followed in vivo, after reinfusion in patients treated with high-dose chemotherapy for poor-prognosis breast or ovarian carcinomas. PATIENTS AND METHODS: Six patients received genetically modified autologous peripheral blood progenitors, together with unmanipulated aphereses, following high-dose chemotherapy. CD34(+) cells were immunoselected from aphereses, and retrovirally transduced by coculture with the retroviral vector producing cell line, to express a nuclear localized version of E. coli beta-galactosidase, encoded by a defective Moloney-murine leukemia virus-derived retroviral vector. Cells were reinfused to the patients after myeloablation, without prior ex vivo selection. RESULTS: Five out of six patients showed the transient presence of low numbers of beta-galactosidase(+) cells, as detected with an immunocytochemical assay, in the peripheral blood, during the first month following infusion. One patient had beta-galactosidase(+) clonogenic progenitors in her marrow at two months after transplantation, including HPP-CFC; intriguingly, this patient had the lowest percentage of X-gal(+) cells in her graft. Patients experienced side effects that are often observed after high-dose chemotherapy. CONCLUSIONS: Feasibility and safety of genetic modification of human hematopoietic stem and progenitor cells are demonstrated by this study. Ex vivo or in vivo selection is not mandatory, even in clinical situations where transduced cells have no survival advantage over wild-type cells; however, significant improvements in gene transfer technology are needed to achieve potentially useful levels of expression in such clinical situations.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/terapia , Vectores Genéticos , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/fisiología , Virus de la Leucemia Murina de Moloney/genética , Neoplasias Ováricas/terapia , Transducción Genética , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/patología , Terapia Combinada , Femenino , Expresión Génica , Humanos , Persona de Mediana Edad , Neoplasias Ováricas/patología , Trasplante Autólogo , beta-Galactosidasa/genéticaRESUMEN
Biomarkers and novel therapeutic targets are urgently needed in colorectal cancer (CRC). The pseudo tyrosine kinase receptor 7 (PTK7) is involved in planar cell polarity and it is deregulated in various malignancies, including CRC. Yet, little is known about its protein expression in human CRC, or about a possible correlation of its expression with clinical endpoints. Using a clinically annotated Tissue MicroArray (TMA) produced from from 192 consecutive CRC patients treated by initial surgery, we examined PTK7 expression by immunohistochemistry in tumoral tissue and matched normal mucosae, and correlated its expression with clinico-pathological features and patient outcome. PTK7 depletion by specific shRNA in HCT116 and HCT15 CRC cell lines was found to affect cell proliferation, resistance to drugs and cell migration. Tumor growth and metastatic phenotype were investigated in vivo using a xenograft mouse model of CRC cells with modulated expression of PTK7 levels. PTK7 was significantly up-regulated in CRC tissue as compared to matched healthy mucosae, and significant overexpression was found in 34% of patients. PTK7 overexpression was significantly associated with a reduced metastasis-free survival in non-metastatic patients. In HCT116 and HCT15 cells, shRNA PTK7 reduced migration but did not affect cell proliferation and resistance to drugs. In a xenograft mouse of HCT15 cells, downregulation of PTK7 led to reduced tumor growth, whereas its overexpression in PTK7-negative cancer cells led to increased metastatic events. PTK7 expression thus represents a potential prognostic biomarker and a novel therapeutic target in CRC.
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Moléculas de Adhesión Celular/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/mortalidad , Expresión Génica , Proteínas Tirosina Quinasas Receptoras/genética , Animales , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Neoplasias Colorrectales/patología , Modelos Animales de Enfermedad , Regulación hacia Abajo , Resistencia a Antineoplásicos/genética , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Masculino , Melanoma Experimental , Ratones , Metástasis de la Neoplasia , Estadificación de Neoplasias , Pronóstico , ARN Interferente Pequeño/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Carga Tumoral/genética , Regulación hacia ArribaRESUMEN
Metastasis is a major clinical issue and results in poor prognosis for most cancers. The Junctional Adhesion Molecule-C (JAM-C) expressed by B16 melanoma and endothelial cells has been involved in metastasis of tumor cells through homophilic JAM-C/JAM-C trans-interactions. Here, we show that JAM-B expressed by endothelial cells contributes to murine B16 melanoma cells metastasis through its interaction with JAM-C on tumor cells. We further show that this adhesion molecular pair mediates melanoma cell adhesion to primary Lung Microvascular Endothelial Cells and that it is functional in vivo as demonstrated by the reduced metastasis of B16 cells in Jam-b deficient mice.
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Moléculas de Adhesión Celular/metabolismo , Células Endoteliales/metabolismo , Inmunoglobulinas/metabolismo , Melanoma Experimental/metabolismo , Animales , Adhesión Celular , Moléculas de Adhesión Celular/genética , Línea Celular Tumoral , Movimiento Celular , Células Cultivadas , Técnicas de Cocultivo , Células Endoteliales/citología , Femenino , Citometría de Flujo , Inmunoglobulinas/genética , Inmunohistoquímica , Pulmón/irrigación sanguínea , Pulmón/metabolismo , Masculino , Melanoma Experimental/genética , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Metástasis de la Neoplasia , Unión Proteica , Interferencia de ARNRESUMEN
Adult stem cells are critical for maintaining cellular homeostasis throughout life, yet the effects of age on their regenerative capacity are poorly understood. All lymphoid and myeloid blood cell lineages are continuously generated from hematopoietic stem cells present in human bone marrow. With age, significant changes in the function and composition of mature blood cells are observed. In this study, we report that age-related changes also occur in the human hematopoietic stem cell compartment. We find that the proportion of multipotent CD34(+) CD38(-) cells increases in the bone marrow of elderly (>70 years) individuals. CD34(+) CD38(+) CD90(-) CD45RA(+/-) CD10(-) and CD34(+) CD33(+) myeloid progenitors persist at the same level in the bone marrow, while the frequency of early CD34(+) CD38(+) CD90(-) CD45RA(+) CD10(+) and committed CD34(+) CD19(+) B-lymphoid progenitors decreases with age. In contrast to mice models of aging, transplantation experiments with immunodeficient NOD/SCID/IL-2Rγ null (NSG) mice showed that the frequency of NSG repopulating cells does not change significantly with age, and there is a decrease in myeloid lineage reconstitution. An age-related decrease in the capacity of CD34(+) cells to generate myeloid cells was also seen in colony-forming assays in vitro. Thus, with increasing age, human hematopoietic stem/progenitor cells undergo quantitative changes as well as functional modifications.
Asunto(s)
Envejecimiento , Antígenos CD/análisis , Células de la Médula Ósea/citología , Médula Ósea/fisiología , Células Madre Hematopoyéticas/citología , Células Mieloides/citología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antígenos CD/inmunología , Linfocitos B/citología , Linfocitos B/inmunología , Células de la Médula Ósea/inmunología , Recuento de Células , Diferenciación Celular , Linaje de la Célula , Ensayo de Unidades Formadoras de Colonias , Femenino , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/inmunología , Humanos , Masculino , Ratones , Ratones SCID , Células Mieloides/inmunologíaRESUMEN
Ikaros is a transcription factor that acts both as an activator and as an inhibitor of gene expression in several hematopoietic lineages. Ikaros functions in hematopoiesis have mostly been studied in mice, and are notably crucial for lymphopoiesis. Deregulation of Ikaros expression was evidenced in several leukemia subtypes, including pre-B-ALL. Here, we studied the role of Ikaros in human B lymphoid differentiation through xeno-transplantation of genetically modified cord blood (CB) human hematopoietic progenitor cells (HPC) in NOD/SCID mice. We used lentiviral vectors to force expression of Ikaros 6 (Ik6), a known dominant negative (DN) protein that interferes with normal Ikaros and structurally related proteins in HPC and their progeny. Two types of vectors were used: a vector containing the EF1alpha promoter which produces strong gene expression in all hematopoietic lineages, and a recently validated B-specific vector containing an enhanced CD19 derived promoter that strongly favors expression in the B-cell lineage. Ik6 transduction of CB CD34(+) cells with these vectors produced distinct consequences in the B-cell differentiation profiles of xenografted human cells. While the ubiquitous vector favored a specific block at the early pro-B/pre-B stage of differentiation, with an increase in Lambda Like transcript expression in the bone marrow (BM), B-specific Ik6 expression provoked a global decrease in the CD19(+) cell population in both BM and spleen, associated with a decrease in IgM+ immature B-cells in the spleen. We conclude that Ikaros proteins are active throughout human B-cell differentiation, before and after CD19 appearance.
Asunto(s)
Linfocitos B/fisiología , Diferenciación Celular/genética , Factor de Transcripción Ikaros/fisiología , Animales , Antígenos CD34/metabolismo , Linfocitos B/metabolismo , Línea Celular , Femenino , Genes Dominantes/fisiología , Humanos , Factor de Transcripción Ikaros/genética , Factor de Transcripción Ikaros/metabolismo , Células Jurkat , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Embarazo , Factores de Tiempo , Transducción Genética , Trasplante Heterólogo , Regulación hacia Arriba/genéticaRESUMEN
Ikaros--a factor that positively or negatively controls gene transcription--is active in murine adult erythroid cells, and involved in fetal to adult globin switching. Mice with Ikaros mutations have defects in erythropoiesis and anemia. In this paper, we have studied the role of Ikaros in human erythroid development for the first time. Using a gene-transfer strategy, we expressed Ikaros 6 (Ik6)--a known dominant--negative protein that interferes with normal Ikaros activity-in cord blood or apheresis CD34(+) cells that were induced to differentiate along the erythroid pathway. Lentivirally induced Ik6-forced expression resulted in increased cell death, decreased cell proliferation, and decreased expression of erythroid-specific genes, including GATA1 and fetal and adult globins. In contrast, we observed the maintenance of a residual myeloid population that can be detected in this culture system, with a relative increase of myeloid gene expression, including PU1. In secondary cultures, expression of Ik6 favored reversion of sorted and phenotypically defined erythroid cells into myeloid cells, and prevented reversion of myeloid cells into erythroid cells. We conclude that Ikaros is involved in human adult or fetal erythroid differentiation as well as in the commitment between erythroid and myeloid cells.
Asunto(s)
Células Eritroides/citología , Células Eritroides/metabolismo , Eritropoyesis , Factor de Transcripción Ikaros/metabolismo , Muerte Celular , Células Cultivadas , Citocinas/farmacología , Células Eritroides/efectos de los fármacos , Eritropoyesis/efectos de los fármacos , Sangre Fetal/citología , Sangre Fetal/metabolismo , Expresión Génica , Regulación de la Expresión Génica , Globinas/genética , Globinas/metabolismo , Humanos , Factor de Transcripción Ikaros/genética , Mielopoyesis , FenotipoRESUMEN
Hematopoietic progenitor cell trafficking is an important phenomenon throughout life. It is thought to occur in sequential steps, similar to what has been described for mature leukocytes. Molecular actors have been identified for each step of leukocyte migration; recently, CD99 was shown to play a part during transendothelial migration. We explored the expression and role of CD99 on human hematopoietic progenitors. We demonstrate that (1) CD34+ cells express CD99, albeit with various intensities; (2) subsets of CD34+ cells with high or low levels of CD99 expression produce different numbers of erythroid, natural killer (NK), or dendritic cells in the in vitro differentiation assays; (3) the level of CD99 expression is related to the ability to differentiate toward B cells; (4) CD34+ cells that migrate through an endothelial monolayer in response to SDF-1alpha and SCF display the highest level of CD99 expression; (5) binding of a neutralizing antibody to CD99 partially inhibits transendothelial migration of CD34+ progenitors in an in vitro assay; and (6) binding of a neutralizing antibody to CD99 reduces homing of CD34+ progenitors xenotransplanted in NOD-SCID mice. We conclude that expression of CD99 on human CD34+ progenitors has functional significance and that CD99 may be involved in transendothelial migration of progenitors.
Asunto(s)
Antígenos CD/metabolismo , Moléculas de Adhesión Celular/metabolismo , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/fisiología , Antígeno 12E7 , Animales , Anticuerpos Monoclonales/administración & dosificación , Antígenos CD34/sangre , Línea Celular , Movimiento Celular/inmunología , Ensayo de Unidades Formadoras de Colonias , Endotelio Vascular/citología , Endotelio Vascular/inmunología , Sangre Fetal/citología , Sangre Fetal/inmunología , Hematopoyesis/inmunología , Movilización de Célula Madre Hematopoyética , Humanos , Técnicas In Vitro , Recién Nacido , Ratones , Ratones Endogámicos NOD , Ratones SCID , Pruebas de Neutralización , Trasplante de Células Madre de Sangre Periférica , Trasplante HeterólogoRESUMEN
Development of gene transfer strategies will necessitate improved efficiency and control of transduction and transgene expression. We here provide evidence that targeting expression of the GFP reporter gene to the B-lymphoid progeny of genetically modified human hematopoietic progenitor cells can be achieved through the insertion of regulatory sequences from the human CD19 gene promoter into a lentiviral vector. Based on a bioinformatics approach, three human CD19-derived sequences were designed and inserted into a self-inactivated lentiviral vector backbone upstream of the GFP gene: S.CD19 (230 bp), M.CD19 (464 bp), and L.CD19 (1274 bp). These new lentiviral vectors efficiently transduced cord blood CD34(+) cells. The M.CD19 and especially L.CD19 sequences preferentially targeted GFP expression to in vitro and in vivo differentiated CD19(+) progeny; moreover, transgene expression was detected from the CD34(+) pro/pre-B cell to the mature peripheral IgM(+) B cell stage. In contrast, GFP expression was weak or absent in primary T-lymphoid and uncommitted progenitor cells or in erythroid, natural killer, or myeloid differentiated cells. Such B-lineage-specific lentiviral vectors may be useful for correcting inherited disorders that affect B-lymphoid cells or for deciphering the transcriptional program that controls B cell commitment and differentiation.