RESUMEN
The development and emergence of the hematopoietic stem cell involves a series of tightly regulated molecular events that are not well characterized. The hematopoietically expressed homeobox (Hhex) gene, a member of the homeobox gene family, is an essential regulator of embryogenesis and hematopoietic progenitor development. To investigate the role of Hhex in hematopoiesis we adapted a murine embryonic stem (ES) cell coculture system, in which ES cells can differentiate into CD41(+) and CD45(+) hematopoietic progenitors in vitro. Our results show that in addition to delayed hemangioblast development, Hhex(-/-) ES-derived progeny accumulate as CD41(+) and CD41(+)c-kit(+) cells, or the earliest definitive hematopoietic progenitors. In addition, Hhex(-/-) ES-derived progeny display a significantly reduced ability to develop into mature CD45(+) hematopoietic cells. The observed reduction in hematopoietic maturation was accompanied by reduced proliferation, because Hhex(-/-) CD41(+)CD45(-)c-kit(+) hematopoietic progenitors accumulated in the G(2) phase of the cell cycle. Thus, Hhex is a critical regulator of hematopoietic development and is necessary for the maturation and proliferation of the earliest definitive hematopoietic progenitors.
Asunto(s)
Embrión de Mamíferos/citología , Células Madre Embrionarias/metabolismo , Regulación de la Expresión Génica/fisiología , Hematopoyesis , Proteínas de Homeodominio/genética , Factores de Transcripción/genética , Animales , Apoptosis , Western Blotting , Ciclo Celular , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Embrión de Mamíferos/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Ratones , Ratones Noqueados , Glicoproteína IIb de Membrana Plaquetaria/metabolismo , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/metabolismoRESUMEN
The vitelline artery is a temporary structure that undergoes extensive remodeling during midgestation to eventually become the superior mesenteric artery (also called the cranial mesenteric artery, in the mouse). Here we show that, during this remodeling process, large clusters of hematopoietic progenitors emerge via extravascular budding and form structures that resemble previously described mesenteric blood islands. We demonstrate through fate mapping of vascular endothelium that these mesenteric blood islands are derived from the endothelium of the vitelline artery. We further show that the vitelline arterial endothelium and subsequent blood island structures originate from a lateral plate mesodermal population. Lineage tracing of the lateral plate mesoderm demonstrates contribution to all hemogenic vascular beds in the embryo, and eventually, all hematopoietic cells in the adult. The intraembryonic hematopoietic cell clusters contain viable, proliferative cells that exhibit hematopoietic stem cell markers and are able to further differentiate into myeloid and erythroid lineages. Vitelline artery-derived hematopoietic progenitor clusters appear between embryonic day 10 and embryonic day 10.75 in the caudal half of the midgut mesentery, but by embryonic day 11.0 are sporadically found on the cranial side of the midgut, thus suggesting possible extravascular migration aided by midgut rotation.
Asunto(s)
Arterias/embriología , Hematopoyesis , Sistema Hematopoyético/citología , Sistema Hematopoyético/embriología , Conducto Vitelino/irrigación sanguínea , Animales , Endotelio Vascular/embriología , Mesodermo/citología , Mesodermo/ultraestructura , RatonesRESUMEN
OBJECTIVE: Perostin (PN) has been found to be overexpressed in a variety of human malignancies including ovarian cancer. In the present study, we investigated PN expression status in a large cohort of ovarian tumors with the focus on biological influence of PN related on ovarian tumor angiogenesis and metastasis. METHODS: PN expression was determined by cDNA microarray, PN northern blot and PN IHC tissue array analyses. Exogenous PN expression in ovarian cancer cells OVCAR-3 and OV2008 were achieved through retroviral transfection and confirmed by PN western blot and ELISA. The effects of exogenous PN expression on tumor angiogenesis and metastatic growth were accessed in orthotopic mouse models. The in vitro cell adhesion, migration and invasion assays were performed to investigate the potential mechanisms involved in PN's in vivo effects. RESULTS: PN was frequently overexpressed in ovarian tumors. Higher PN levels significantly correlated with clinical late stages (III/IV) and cancer recurrence. PN was produced by engineered PN-overexpressing cells at levels comparable to that of A2780 cells, an ovarian carcinoma cell line with endogenous PN expression. PN overexpression did not change cell growth rates in vitro; however it significantly promoted intraperitoneal tumor metastatic growth in immunodeficient mice, which was associated with increased tumor angiogenesis and decreased tumor cell apoptosis. In vitro purified PN promoted cell adhesion, migration, and invasion of both human umbilical endothelial cells (HUVECs) and/or ovarian cancer cells. CONCLUSIONS: Our data indicate PN plays a critical role in both ovarian tumor angiogenesis and metastasis. Thus PN may represent a clinically effective new target for therapy of ovarian cancer.
Asunto(s)
Moléculas de Adhesión Celular/biosíntesis , Neoplasias Ováricas/metabolismo , Animales , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Femenino , Humanos , Inmunohistoquímica , Ratones , Ratones Desnudos , Ratones SCID , Metástasis de la Neoplasia , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Neoplasias Ováricas/irrigación sanguínea , Neoplasias Ováricas/genética , Neoplasias Ováricas/patologíaRESUMEN
Tyrosine phosphorylation has emerged as a mechanism to control cellular events in the nucleus. The c-Fes protein-tyrosine kinase is an important regulator of cell growth and differentiation in several cell types, and is found in the nucleus of hematopoietic cells. In this study, we showed nuclear localization of c-Fes in both hematopoietic (K562, TF-1, HEL, U937, and HL-60) and nonhematopoietic cell lines (293T, CaOv3, TfxH, MG-63, HeLa, DU-145) by immunofluorescence and confocal microscopy. c-Fes showed striking changes in subcellular localization at specific stages of mitosis. In interphase cells, the intranuclear distribution of c-Fes was diffuse with occasional bright foci. Some c-Fes was present in the cytosol after breakdown of the nuclear membrane, in prometaphase. At prometaphase and metaphase c-Fes was also associated with the chromosomes, in a punctate pattern that partially overlapped with the centromere. Further comparison with proteins that are known components of the kinetochore suggested that some c-Fes protein was located at the centromeric alpha-satellite DNA, between the kinetochores. At anaphase and telophase, c-Fes was entirely cytoplasmic and no protein was found associated with the chromosomes. The timing of c-Fes' appearance at the centromere coincides with the period of kinetochore assembly. These data suggest that c-Fes is recruited to the kinetochore during mitosis.
Asunto(s)
Núcleo Celular/metabolismo , Expresión Génica , Cinetocoros/metabolismo , Mitosis/fisiología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Células Cultivadas , Centrómero/metabolismo , Citoplasma/metabolismo , Cartilla de ADN , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Immunoblotting , Inmunoprecipitación , Ratones , Microscopía Confocal , Proteínas Proto-Oncogénicas c-fesRESUMEN
The cellular fes gene encodes a 93-kilodalton protein-tyrosine kinase (p93) that is expressed in both normal and neoplastic myeloid cells. Increased c-Fes expression is associated with differentiation in normal myeloid cells and cell lines. Our hypothesis was that primary leukemia cells would show a similar pattern of increased expression in more differentiated cells. Therefore, we compared c-Fes expression in cells with an undifferentiated, blast phenotype (acute myelogenous leukemia--AML) to cells with a differentiated phenotype (chronic myelogenous leukemia--CML). Instead of differences in p93 expression levels, we found complex patterns of c-Fes immunoreactive proteins that corresponded with differentiation in normal and leukemic myeloid cells. The "blast" pattern consisted of c-Fes immunoreactive proteins p93, p74, and p70; the "differentiated" pattern showed two additional c-Fes immunoreactive proteins, p67 and p62. Using mRNA from mouse and human cell lines, we found deletion of one or more exons in the c-fes mRNA. Those deletions predicted truncation of conserved domains (CDC15/FCH and SH2) involved in protein-protein interactions. No deletions were found, however, within the kinase domain. We infer that alternative splicing generates a family of c-Fes proteins. This may be a mechanism to direct the c-Fes kinase domain to different subcellular locations and/or substrates at specific stages of myeloid cell differentiation.
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Diferenciación Celular , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielomonocítica Aguda/metabolismo , Isoformas de Proteínas/metabolismo , Proto-Oncogenes , Animales , Antígenos CD34/metabolismo , Línea Celular , Línea Celular Tumoral , Técnica del Anticuerpo Fluorescente Indirecta , Células HL-60 , Células Madre Hematopoyéticas/enzimología , Células Madre Hematopoyéticas/metabolismo , Humanos , Immunoblotting , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielomonocítica Aguda/genética , Ratones , Células 3T3 NIH , Neutrófilos/enzimología , Neutrófilos/metabolismo , Reacción en Cadena de la Polimerasa , Isoformas de Proteínas/genética , ARN Mensajero/genética , Células U937RESUMEN
The in vitro differentiation of ES cells towards a hematopoietic cell fate is useful when studying cell populations that are difficult to access in vivo and for characterizing the earliest genes involved in hematopoiesis, without having to deal with embryonic lethalities. The ES/OP9 co-culture system was originally designed to produce hematopoietic progeny, without the over production of macrophages, as the OP9 stromal cell line is derived from the calvaria of osteopetrosis mutant mice that lack functional M-CSF. The in vitro ES/OP9 co-culture system can be used in order to recapitulate early hematopoietic development. When cultured on OP9 stromal cells, ES cells differentiate into Flk-1+ hemangioblasts, hematopoietic progenitors, and finally mature, terminally differentiated lineages. The standard ES/OP9 co-culture protocol entails the placement of ES cells onto a confluent layer of OP9 cells; as well as, periodic replating steps in order to remove old, contaminating OP9 cells. Furthermore, current protocols involve evaluating only the hematopoietic cells found in suspension and are not optimized for evaluation of ES-derived progeny at each day of differentiation. However, with replating steps and the harvesting of only suspension cells one potentially misses a large portion of ES-derived progeny and developing hematopoietic cells. This issue becomes important to address when trying to characterize hematopoietic defects associated with knockout ES lines. Here we describe a modified ES/mStrawberry OP9 co-culture, which allows for the elimination of contaminating OP9 cells from downstream assays. This method allows for the complete evaluation of all ES-derived progeny at all days of co-culture, resulting in a hematopoietic differentiation pattern, which more directly corresponds to the hematopoietic differentiation pattern observed within the embryo.
Asunto(s)
Técnicas de Cocultivo/métodos , Células Madre Embrionarias/citología , Células Madre Hematopoyéticas/citología , Animales , Diferenciación Celular/fisiología , Hematopoyesis/fisiología , RatonesRESUMEN
Periostin, an extracellular matrix protein, is reported to be overexpressed in a variety of human cancers and its functions seem to be linked to tumor metastasis. Our previous results show that engineered periostin overexpression promotes ovarian tumor growth and dissemination in vivo. In this study, we developed a neutralizing monoclonal antibody to periostin, named MZ-1, and investigated its effects on human ovarian tumor growth and metastasis. Our in vivo studies showed significant growth inhibition by MZ-1 on both subcutaneous and intraperitoneal (i.p.) tumors derived from the periostin-expressing ovarian cancer cell line A2780. In addition, MZ-1 treatment led to a reduction of the metastatic potential of these A2780 i.p. tumors. The in vivo antitumor effects of MZ-1 were linked to its specific inhibition of anchorage-independent growth and survival of periostin-expressing cells, as well as its neutralizing effects on periostin-induced cancer cell migration and invasion. The data suggest that blocking periostin expression may be a novel approach for treating the subset of invasive ovarian tumors that overexpress periostin protein.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/uso terapéutico , Antineoplásicos/uso terapéutico , Moléculas de Adhesión Celular/antagonistas & inhibidores , Neoplasias Ováricas/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/inmunología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones SCID , Metástasis de la Neoplasia , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Hematopoietic stem cells (HSCs) originate within the aortic-gonado-mesonephros (AGM) region of the midgestation embryo, but the cell type responsible for their emergence is unknown since critical hematopoietic factors are expressed in both the AGM endothelium and its underlying mesenchyme. Here we employ a temporally restricted genetic tracing strategy to selectively label the endothelium, and separately its underlying mesenchyme, during AGM development. Lineage tracing endothelium, via an inducible VE-cadherin Cre line, reveals that the endothelium is capable of HSC emergence. The endothelial progeny migrate to the fetal liver, and later to the bone marrow, and are capable of expansion, self-renewal, and multilineage hematopoietic differentiation. HSC capacity is exclusively endothelial, as ex vivo analyses demonstrate lack of VE-cadherin Cre induction in circulating and fetal liver hematopoietic populations. Moreover, AGM mesenchyme, as selectively traced via a myocardin Cre line, is incapable of hematopoiesis. Our genetic tracing strategy therefore reveals an endothelial origin of HSCs.
Asunto(s)
Linaje de la Célula/genética , Desarrollo Embrionario/genética , Células Endoteliales/metabolismo , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Diferenciación Celular/genética , Movimiento Celular/genética , Proliferación Celular , Células Cultivadas , Células Endoteliales/citología , Regulación del Desarrollo de la Expresión Génica/genética , Estratos Germinativos/embriología , Integrasas/metabolismo , Mesodermo/fisiología , Ratones , Ratones Transgénicos , Biología Molecular/métodos , Coloración y Etiquetado/métodosRESUMEN
In normal hematopoiesis, proliferation is tightly linked to differentiation in ways that involve cell-cell interaction with stromal elements in the bone marrow stem cell niche. Numerous in vitro and in vivo studies strongly support a role for Notch signaling in the regulation of stem cell renewal and hematopoiesis. Not surprisingly, mutations in the Notch gene have been linked to a number of types of malignancies. To better define the function of Notch in both normal and neoplastic hematopoiesis, a tetracycline-inducible system regulating expression of a ligand-independent, constitutively active form of Notch1 was introduced into murine E14Tg2a embryonic stem cells. During coculture, OP9 stromal cells induce the embryonic stem cells to differentiate first to hemangioblasts and subsequently to hematopoietic stem cells. Our studies indicate that activation of Notch signaling in flk+ hemangioblasts dramatically reduces their survival and proliferative capacity and lowers the levels of hematopoietic stem cell markers CD34 and c-Kit and the myeloid marker CD11b. Global gene expression profiling of day 8 hematopoietic progenitors in the absence and presence of activated Notch yield candidate genes required for normal hematopoietic differentiation, as well as putative downstream targets of oncogenic forms of Notch including the noncanonical Wnts Wnt4 and 5A. Disclosure of potential conflicts of interest is found at the end of this article.