RESUMEN

Genomic prediction has emerged as a pivotal technology for the genetic evaluation of livestock, crops, and for predicting human disease risks. However, classical genomic prediction methods face challenges in incorporating biological prior information such as the genetic regulation mechanisms of traits. This study introduces a novel approach that integrates mRNA transcript information to predict complex trait phenotypes. To evaluate the accuracy of the new method, we utilized a Drosophila population that is widely employed in quantitative genetics researches globally. Results indicate that integrating mRNA transcript data can significantly enhance the genomic prediction accuracy for certain traits, though it does not improve phenotype prediction accuracy for all traits. Compared with GBLUP, the prediction accuracy for olfactory response to dCarvone in male Drosophila increased from 0.256 to 0.274. Similarly, the accuracy for cafe in male Drosophila rose from 0.355 to 0.401. The prediction accuracy for survival_paraquat in male Drosophila is improved from 0.101 to 0.138. In female Drosophila, the accuracy of olfactory response to 1hexanol increased from 0.147 to 0.210. In conclusion, integrating mRNA transcripts can substantially improve genomic prediction accuracy of certain traits by up to 43%, with range of 7% to 43%. Furthermore, for some traits, considering interaction effects along with mRNA transcript integration can lead to even higher prediction accuracy.

Asunto(s)

Drosophila , Genómica , ARN Mensajero , Animales , ARN Mensajero/genética , Masculino , Genómica/métodos , Femenino , Drosophila/genética , Fenotipo

RESUMEN

Non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease and is a leading cause of cirrhosis and hepatocellular carcinoma. Due to its complex pathophysiology, there is currently no approved therapy. Polysaccharide, a kind of natural product, possesses a wide range of pharmacological activities. Numerous preclinical studies have confirmed that polysaccharides could interfere with the occurrence and development of NAFLD at multiple interrelated levels, such as improvement of glucose and lipid metabolism, antioxidation, anti-inflammation, and regulation of gut-liver axis, thus showing great potential as novel anti-NAFLD drugs. In this paper, we reviewed the polysaccharides with anti-NAFLD effect in recent years, and also systematically analyzed their possible pharmacological mechanisms.

RESUMEN

Central-to-axial chirality conversion provides efficient access to axially chiral compounds, and several examples regarding the conversion of one, two or four stereocenters to one axis have been reported. Herein, we report the conversion of two stereocenters to one or two chiral axes for the first time. In this study, a new class of enantiomerically enriched 2,3-diarylbenzoindoles was efficiently synthesized using a chiral phosphoric acid-catalyzed [3 + 2] formal cycloaddition and a mild DDQ oxidation strategy. Moreover, a speculative model of the central-to-axial chirality conversion outcome was proposed based on preliminary mechanistic studies and DFT calculations. Potentially, using this strategy, useful chiral phosphine ligand can be synthesized smoothly (99% ee).

RESUMEN

An efficient dehydrogenative Diels-Alder reaction of prenyl derivatives with dienophiles has been developed. The reaction exhibits broad substrate scope and provides efficient access to cyclohexene derivatives with good to excellent yields. A reasonable mechanism involving a metal-free thermal reversible process is proposed.

RESUMEN

OBJECTIVES: To investigate the interventional effects of 16-week aerobic exercises on the elderly's arteriosclerosis and its mechanism. METHODS: Twenty-seven elderly people with the average age of 62. 70 ±3. 26 joined a 16-week square dance/taijiquan exercise program that conducted 60 minutes each time, six times per week. Arterial stiffness and its related indexes such as systolic pressure(SBP), diastolic pressure(DBP), left brachial-ankle pulse wave velocity (L-baPWV), right brachial-ankle pulse wave velocity(R-baPWV), left ankle brachial index (L-ABI), right ankle brachial index(R-ABI), serum triglyceride(TG), total cholesterol(TC), high density lipoprotein cholesterol(HDL-c), low density lipoprotein cholesterol(LDL-c), superoxide dismutase(SOD), malondialdehyde(MDA) and glutathione peroxidase (GSH-Px) were detected at 3 time points including before exercise program, by the end of exercise for 8 weeks and 16 weeks. RESULTS: ① Compared with pre-exercise, the R-baPWV and R-ABI of the elderly people were decreased at the end of the 8th week, and the L-baPWV, RbaPWV, R-ABI and L-ABI were decreased significantly at the end of the 16th week. ②Compared with pre-exercise, SBP and DBP were declined markedly (P<0.01, P<0.05) at the end of the 8th week, SBP, DBP and pulse pressure were decreased significantly (P<0.01, P<0.05) at the end of the 16th week. ③Compared with pre-exercise, TC and LDL-c were declined markedly (P<0.01) at the end of the 8th and the 16th week, and there was no difference of the level of TG and LDL-c between pre-exercise and post-exercise. ④There was no evident difference of serum level of SOD, GSH-Px, MDA between pre-exercise and post-exercise at the end of the 8th week. Compared with pre-exercise, the level of serum SOD, GSH-Px was increased evidently while the content of serum MDA was decreased significantly (P<0.01). CONCLUSIONS: Sixteen-week aerobic exercises could reduce baPWV and ABI levels, regulate blood pressure, blood lipids and lipid peroxides levels of the elderly evidently, thus improve the controlling quality of atherosclerosis.

Asunto(s)

Índice Tobillo Braquial , Presión Sanguínea , Ejercicio Físico , Análisis de la Onda del Pulso , Anciano , Tobillo , Arteriosclerosis/terapia , Colesterol/sangre , Glutatión Peroxidasa/sangre , Humanos , Malondialdehído/sangre , Persona de Mediana Edad , Superóxido Dismutasa/sangre , Triglicéridos/sangre

RESUMEN

A new linear bismuth(III) coordination polymer, catena-poly[[chloridobismuth(III)]-µ3-1,10-phenanthroline-2,9-dicarboxylato-κ(6)O(2):O(2),N(1),N(10),O(9):O(9)], [Bi(C14H6N2O4)Cl]n, has been obtained by an ionothermal method and characterized by elemental analysis, energy-dispersive X-ray spectroscopy, IR spectroscopy, thermal stability studies and single-crystal X-ray diffraction. The structure is constructed by Bi(C14H6N2O4)Cl fragments in which each Bi(III) centre is seven-coordinated by one Cl atom, four O atoms and two N atoms. The coordination geometry of the Bi(III) cation is distorted pentagonal-bipyramidal (BiO4N2Cl), with one bridging carboxylate O atom and one Cl atom located in the axial positions. The Bi(C14H6N2O4)Cl fragments are further extended into a one-dimensional linear polymeric structure via subsequent but different centres of symmetry (bridging carboxylate O atoms). Neighbouring linear chains are assembled via weak C-H···O and C-H···Cl hydrogen bonds, forming a three-dimensional supramolecular architecture. Intermolecular π-π stacking interactions are observed, with centroid-to-centroid distances of 3.678 (4) Å, which further stabilize the structure. In addition, the solid-state fluorescence properties of the title coordination polymer were investigated.

Asunto(s)

Bismuto/química , Complejos de Coordinación/síntesis química , Fenantrolinas/síntesis química , Polímeros/química , Complejos de Coordinación/química , Cristalografía por Rayos X , Fluorescencia , Enlace de Hidrógeno , Estructura Molecular

RESUMEN

PURPOSE: To identify mediators of glioblastoma antiangiogenic therapy resistance and target these mediators in xenografts. EXPERIMENTAL DESIGN: We conducted microarray analysis comparing bevacizumab-resistant glioblastomas (BRG) with pretreatment tumors from the same patients. We established novel xenograft models of antiangiogenic therapy resistance to target candidate resistance mediator(s). RESULTS: BRG microarray analysis revealed upregulation versus pretreatment of receptor tyrosine kinase c-Met, which underwent further investigation because of its prior biologic plausibility as a bevacizumab resistance mediator. BRGs exhibited increased hypoxia versus pretreatment in a manner correlating with their c-Met upregulation, increased c-Met phosphorylation, and increased phosphorylation of c-Met-activated focal adhesion kinase and STAT3. We developed 2 novel xenograft models of antiangiogenic therapy resistance. In the first model, serial bevacizumab treatment of an initially responsive xenograft generated a xenograft with acquired bevacizumab resistance, which exhibited upregulated c-Met expression versus pretreatment. In the second model, a BRG-derived xenograft maintained refractoriness to the MRI tumor vasculature alterations and survival-promoting effects of bevacizumab. Growth of this BRG-derived xenograft was inhibited by a c-Met inhibitor. Transducing these xenograft cells with c-Met short hairpin RNA inhibited their invasion and survival in hypoxia, disrupted their mesenchymal morphology, and converted them from bevacizumab-resistant to bevacizumab-responsive. Engineering bevacizumab-responsive cells to express constitutively active c-Met caused these cells to form bevacizumab-resistant xenografts. CONCLUSION: These findings support the role of c-Met in survival in hypoxia and invasion, features associated with antiangiogenic therapy resistance, and growth and therapeutic resistance of xenografts resistant to antiangiogenic therapy. Therapeutically targeting c-Met could prevent or overcome antiangiogenic therapy resistance.

Asunto(s)

Inhibidores de la Angiogénesis/farmacología , Resistencia a Antineoplásicos , Neovascularización Patológica/genética , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Transcriptoma , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Bevacizumab , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Análisis por Conglomerados , Resistencia a Antineoplásicos/genética , Activación Enzimática/genética , Regulación Neoplásica de la Expresión Génica , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/mortalidad , Humanos , Ratones , Invasividad Neoplásica/genética , Neovascularización Patológica/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Interferencia de ARN , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Autophagy is a lysosomal degradation pathway that can sequester cytosolic material, including organelles, nonspecifically in a process called nonselective macroautophagy, or target specific protein aggregates designated for destruction in a process called selective autophagy. Autophagy is one mechanism that enables tumor cells to survive stressors in the tumor microenvironment, as well as injuries caused by treatments such as chemotherapy and radiation therapy. The complexity of the role of autophagy in cancer is underscored by evidence that autophagy can allow premalignant cells to escape the genotoxic stress and inflammation that promote tumorigenesis, and that some tumor cells exhibit loss of autophagy capacity altogether through molecular mechanisms that have not yet been defined. Efforts to understand and modulate the autophagy pathway will be crucial to maximize the full therapeutic potential of cancer therapies that are currently hindered by tumor cell autophagy as a resistance mechanism.

Asunto(s)

Adaptación Biológica , Inhibidores de la Angiogénesis/uso terapéutico , Antineoplásicos/uso terapéutico , Autofagia , Resistencia a Antineoplásicos , Neoplasias/tratamiento farmacológico , Animales , Autofagia/efectos de los fármacos , Cloroquina/farmacología , Humanos , Neoplasias/metabolismo , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Microambiente Tumoral

RESUMEN

While anti-angiogenic therapy was initially greeted enthusiastically by the cancer community, initial successes with this therapeutic modality were tempered by the failure of angiogenesis inhibitors to produce sustained clinical responses in most patients, with resistance to the inhibitors frequently developing. We recently reported that hypoxia increases after the devascularization caused by anti-angiogenic therapy, consistent with the goals of these therapies, but that some tumor cells become resistant and survive the hypoxic insult elicited by anti-angiogenic therapy through autophagy by activating both AMPK and HIF1A pathways. These findings suggest that modulating the autophagy pathway may someday allow anti-angiogenic therapy to fulfill its therapeutic potential. However, further work will clearly be needed to develop more potent and specific autophagy inhibitors and to better understand the regulators of autophagy in malignant cells.

Asunto(s)

Inhibidores de la Angiogénesis/uso terapéutico , Autofagia , Resistencia a Antineoplásicos , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Inhibidores de la Angiogénesis/farmacología , Animales , Autofagia/efectos de los fármacos , Hipoxia de la Célula/efectos de los fármacos , Ensayos Clínicos como Asunto , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Modelos Biológicos , Neoplasias/irrigación sanguínea , Células Madre Neoplásicas/patología , Microambiente Tumoral/efectos de los fármacos

RESUMEN

PURPOSE: To identify mechanisms and mediators of resistance to antiangiogenic therapy in human glioblastoma. EXPERIMENTAL DESIGN: We carried out microarray gene expression analysis and immunohistochemistry comparing 21 recurrent glioblastomas progressing during antiangiogenic treatment with VEGF neutralizing antibody bevacizumab to paired pretreatment tumors from the same patients. RESULTS: Microarray analysis revealed that bevacizumab-resistant glioblastomas (BRG) had two clustering patterns defining subtypes that reflect radiographic growth patterns. Enhancing BRGs (EBRG) exhibited MRI enhancement, a long-established criterion for glioblastoma progression, and expressed mitogen-activated protein kinases, neural cell adhesion molecule-1 (NCAM-1), and aquaporin 4. Compared with their paired pretreatment tumors, EBRGs had unchanged vascularity and hypoxia, with increased proliferation. Nonenhancing BRGs (NBRG) exhibited minimal MRI enhancement but had FLAIR-bright expansion, a newer criterion for glioblastoma recurrence since the advent of antiangiogenic therapy, and expressed integrin α5, laminin, fibronectin1, and PDGFRß. NBRGs had less vascularity, more hypoxia, and unchanged proliferation than their paired pretreatment tumors. Primary NBRG cells exhibited more stellate morphology with a 3-fold increased shape factor and were nearly 4-fold more invasive in Matrigel chambers than primary cells from EBRGs or bevacizumab-naive glioblastomas (P < 0.05). CONCLUSION: Using microarray analysis, we found two resistance patterns during antiangiogenic therapy with distinct molecular profiles and radiographic growth patterns. These studies provide valuable biologic insight into the resistance that has limited antiangiogenic therapy to date.

Asunto(s)

Anticuerpos Monoclonales Humanizados/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Resistencia a Antineoplásicos/genética , Perfilación de la Expresión Génica , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Anticuerpos Monoclonales Humanizados/farmacología , Acuaporina 4/biosíntesis , Acuaporina 4/genética , Bevacizumab , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Antígeno CD56/biosíntesis , Antígeno CD56/genética , Hipoxia de la Célula , Proliferación Celular , Células Cultivadas , Progresión de la Enfermedad , Fibronectinas/biosíntesis , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Integrina alfa5/biosíntesis , Laminina/biosíntesis , Proteínas Quinasas Activadas por Mitógenos/biosíntesis , Proteínas Quinasas Activadas por Mitógenos/genética , Neovascularización Patológica , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Microambiente Tumoral , Factor A de Crecimiento Endotelial Vascular

RESUMEN

Antiangiogenic therapy leads to devascularization that limits tumor growth. However, the benefits of angiogenesis inhibitors are typically transient and resistance often develops. In this study, we explored the hypothesis that hypoxia caused by antiangiogenic therapy induces tumor cell autophagy as a cytoprotective adaptive response, thereby promoting treatment resistance. Hypoxia-induced autophagy was dependent on signaling through the hypoxia-inducible factor-1α (HIF-1α)/AMPK pathway, and treatment of hypoxic cells with autophagy inhibitors caused a shift from autophagic to apoptotic cell death in vitro. In glioblastomas, clinically resistant to the VEGF-neutralizing antibody bevacizumab, increased regions of hypoxia and higher levels of autophagy-mediating BNIP3 were found when compared with pretreatment specimens from the same patients. When treated with bevacizumab alone, human glioblastoma xenografts showed increased BNIP3 expression and hypoxia-associated growth, which could be prevented by addition of the autophagy inhibitor chloroquine. In vivo targeting of the essential autophagy gene ATG7 also disrupted tumor growth when combined with bevacizumab treatment. Together, our findings elucidate a novel mechanism of resistance to antiangiogenic therapy in which hypoxia-mediated autophagy promotes tumor cell survival. One strong implication of our findings is that autophagy inhibitors may help prevent resistance to antiangiogenic therapy used in the clinic.

Asunto(s)

Adaptación Fisiológica , Inhibidores de la Angiogénesis/uso terapéutico , Autofagia/fisiología , Neoplasias Encefálicas/tratamiento farmacológico , Hipoxia de la Célula , Glioblastoma/tratamiento farmacológico , Proteínas Quinasas Activadas por AMP/fisiología , Animales , Anticuerpos Monoclonales Humanizados/uso terapéutico , Bevacizumab , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Supervivencia Celular , Cloroquina/farmacología , Glioblastoma/patología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Proteínas de la Membrana/análisis , Ratones , Proteínas Proto-Oncogénicas/análisis , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

HOXA9-mediated up-regulation of miR-155 was noted during an array-based analysis of microRNA expression in Hoxa9(-/-)bone marrow (BM) cells. HOXA9 induction of miR-155 was confirmed in these samples, as well as in wild-type versus Hoxa9-deficient marrow, using northern analysis and qRT-PCR. Infection of wild-type BM with HOXA9 expressing or GFP(+) control virus further confirmed HOXA9-mediated regulation of miR-155. miR-155 expression paralleled Hoxa9 mRNA expression in fractionated BM progenitors, being highest in the stem cell enriched pools. HOXA9 capacity to induce myeloid colony formation was blunted in miR-155-deficient BM cells, indicating that miR-155 is a downstream mediator of HOXA9 function in blood cells. Pu.1, an important regulator of myelopoiesis, was identified as a putative down stream target for miR-155. Although miR-155 was shown to down-regulate the Pu.1 protein, HOXA9 did not appear to modulate Pu.1 expression in murine BM cells.

Asunto(s)

Regulación de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Proteínas de Homeodominio/metabolismo , MicroARNs/metabolismo , Animales , Células Cultivadas , Proteínas de Homeodominio/genética , Ratones , MicroARNs/biosíntesis , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Regulación hacia Arriba

RESUMEN

While investigating the mechanism of action of the HOXA9 protein, we serendipitously identified Meis1 as a HOXA9 regulatory target. Since HOXA9 and MEIS1 play key developmental roles, are cooperating DNA binding proteins and leukemic oncoproteins, and are important for normal hematopoiesis, the regulation of Meis1 by its partner protein is of interest. Loss of Hoxa9 caused downregulation of the Meis1 mRNA and protein, while forced HOXA9 expression upregulated Meis1. Hoxa9 and Meis1 expression was correlated in hematopoietic progenitors and acute leukemias. Meis1(+/-) Hoxa9(-/-) deficient mice, generated to test HOXA9 regulation of endogenous Meis1, were small and had reduced bone marrow Meis1 mRNA and significant defects in fluorescence-activated cell sorting-enumerated monocytes, mature and pre/pro-B cells, and functional B-cell progenitors. These data indicate that HOXA9 modulates Meis1 during normal murine hematopoiesis. Chromatin immunoprecipitation analysis did not reveal direct binding of HOXA9 to Meis1 promoter/enhancer regions. However, Creb1 and Pknox1, whose protein products have previously been reported to induce Meis1, were shown to be direct targets of HOXA9. Loss of Hoxa9 resulted in a decrease in Creb1 and Pknox1 mRNA, and forced expression of CREB1 in Hoxa9(-/-) bone marrow cells increased Meis1 mRNA almost as well as HOXA9, suggesting that CREB1 may mediate HOXA9 modulation of Meis1 expression.

Asunto(s)

Genes Relacionados con las Neoplasias , Hematopoyesis , Proteínas de Homeodominio/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , Peso Corporal , Cruzamientos Genéticos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Implantación del Embrión , Femenino , Regulación del Desarrollo de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Heterocigoto , Proteínas de Homeodominio/genética , Humanos , Leucemia Mieloide/genética , Masculino , Ratones , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide , Proteínas de Neoplasias/genética , Células Precursoras de Linfocitos B/metabolismo , Células Precursoras de Linfocitos B/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulación hacia Arriba/genética

RESUMEN

The PicTar program predicted that microRNA-126 (miR-126), miR-145, and let-7s target highly conserved sites within the Hoxa9 homeobox. There are increased nucleotide constraints in the three microRNA seed sites among Hoxa9 genes beyond that required to maintain protein identity, suggesting additional functional conservation. In preliminary experiments, forced expression of these microRNAs in Hoxa9-immortalized bone marrow cells downregulated the HOXA9 protein and caused loss of biological activity. The microRNAs were shown to target their predicted sites within the homeobox. miR-126 and Hoxa9 mRNA are coexpressed in hematopoietic stem cells and downregulated in parallel during progenitor cell differentiation; however, miR-145 is barely detectable in hematopoietic cells, and let-7s are highly expressed in bone marrow progenitors, suggesting that miR-126 may function in normal hematopoietic cells to modulate HOXA9 protein. In support of this hypothesis, expression of miR-126 alone in MLL-ENL-immortalized bone marrow cells decreased endogenous HOXA9 protein, while inhibition of endogenous miR-126 increased expression of HOXA9 in F9 cells.

Asunto(s)

Genes Homeobox , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , MicroARNs/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células de la Médula Ósea/metabolismo , Línea Celular Tumoral , Secuencia Conservada , Regulación hacia Abajo , Humanos , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Ratones , Datos de Secuencia Molecular , Alineación de Secuencia

RESUMEN

The HOXA9 homeoprotein exerts dramatic effects in hematopoiesis. Enforced expression of HOXA9 enhances proliferation of primitive blood cells, expands hematopoietic stem cells (HSCs), and leads to myeloid leukemia. Conversely, loss of HOXA9 inhibits proliferation and impairs HSC function. The pathways by which HOXA9 acts are largely unknown, and although HOXA9 is a transcription factor, few direct target genes have been identified. Our previous study suggested that HOXA9 positively regulates Pim1, an oncogenic kinase. The hematologic phenotypes of Hoxa9- and Pim1-deficient animals are strikingly similar. Here we show that HOXA9 protein binds to the Pim1 promoter and induces Pim1 mRNA and protein in hematopoietic cells. Pim1 protein is diminished in Hoxa9(-/-) cells, and Hoxa9 and Pim1 mRNA levels track together in early hematopoietic compartments. Induction of Pim1 protein by HOXA9 increases the phosphorylation and inactivation of the proapoptotic BAD protein, a target of Pim1. Hoxa9(-/-) cells show increased apoptosis and decreased proliferation, defects that are ameliorated by reintroduction of Pim1. Thus Pim1 appears to be a direct transcriptional target of HOXA9 and a mediator of its antiapoptotic and proproliferative effects in early cells. Since HOXA9 is frequently up-regulated in acute myeloid leukemia, Pim1 may be a therapeutic target in human disease.

Asunto(s)

Regulación Enzimológica de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Apoptosis , Células de la Médula Ósea/metabolismo , Células Madre Hematopoyéticas/citología , Humanos , Células K562 , Leucemia Mieloide/metabolismo , Fosforilación , ARN Mensajero/metabolismo , Retroviridae/genética , Factores de Tiempo , Transgenes , Células U937

RESUMEN

The homeobox gene Hoxa-9 is normally expressed in primitive bone marrow cells, and overexpression of Hoxa-9 markedly expands hematopoietic stem cells, suggesting a function in early hematopoiesis. We present evidence for major functional defects in Hoxa-9-/- hematopoietic stem cells. Hoxa-9-/- marrow cells have normal numbers of immunophenotypic stem cells (Lin(-)c-kit(+)flk-2(-)Sca-1+ [KLFS] cells). However, sublethally irradiated Hoxa-9-/- mice develop persistent pancytopenia, indicating unusual sensitivity to ionizing irradiation. In competitive transplantation assays, Hoxa-9-/- cells showed an 8-fold reduction in multilineage long-term repopulating ability, a defect not seen in marrow cells deficient for the adjacent Hoxa-10 gene. Single-cell cultures of KLFS cells showed a 4-fold reduction in large high-proliferation potential colonies. In liquid cultures, Hoxa-9-deficient Lin(-)Sca-1(+) cells showed slowed proliferation (a 5-fold reduction in cell numbers at day 8) and delayed emergence of committed progenitors (a 5-fold decrease in colony-forming cells). Slowing of proliferation was accompanied by a delay in myeloid maturation, with a decrease in Gr-1hiMac-1hi cells at the end of the culture. Retroviral transduction with a Hoxa-9 expression vector dramatically enhanced the cytokine-driven proliferation and in vivo engraftment of Hoxa-9-/- marrow cells. Hoxa-9 appears to be specifically required for normal hematopoietic stem cell function both in vitro and in vivo.

Asunto(s)

Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Animales , Movimiento Celular/fisiología , Proliferación Celular , Citometría de Flujo , Expresión Génica , Hematopoyesis/genética , Ratones , Ratones Mutantes

RESUMEN

OBJECTIVE: Ovarian surface epithelium (OSE), the precursor of the epithelial ovarian carcinomas, has limited growth potential in culture. Epidermal growth factor+hydrocortisone (EGF+HC) enhances its growth but induces epitheliomesenchymal transition (EMT). This study was undertaken to define the effects of EGF+HC and their reversibility, to optimize growth-promoting media, and to relate OSE phenotypes in vitro to physiologic states in vivo. METHODS: OSE was cultured in media 199/MDCB105 or EBM (Clonetics) with 2% or 10% fetal bovine serum with or without 10 ng/mL EGF, 1.0 microg/mL HC, and 1.0 microg/mL bovine brain extract. Growth rates and growth potentials (population doublings [PD] to senescence) were defined, and growth patterns and expression of keratin and collagen types III and IV were compared with the ovarian cancer cell lines OVCAR3 and SKOV3. RESULTS: EGF+HC increased growth potentials from 12-14 PD to 40-42 PD and reduced PD time from 53 hours to 20 hours. Without EGF+HC, OSE cells remained uniformly epithelial. EGF+HC induced EMT (mesenchymal shapes, reduced keratin, and production of collagenous extracellular matrix), but the EMT response varied greatly among OSE from different women. EMT was reversed over 1-2 weeks by subculture into EGF+HC-free medium in passage 1, but inconsistently thereafter. EGF+HC had no effect on the differentiation of ovarian carcinoma lines. CONCLUSION: The phenotype of intact OSE in vivo is most closely reproduced in media without EGF+HC. EGF+HC enhances growth but initiates EMT, which likely mimics a repair response. Variations in EGF+HC-induced phenotypes point to the existence of OSE subpopulations with differing responsiveness to growth factors or steroids, which may relate to their susceptibility to malignant transformation.

Asunto(s)

Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Hidrocortisona/farmacología , Ovario/citología , Adulto , Células Cultivadas , Colágeno Tipo III/biosíntesis , Colágeno Tipo III/genética , Colágeno Tipo IV/biosíntesis , Colágeno Tipo IV/genética , Células Epiteliales/citología , Femenino , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Queratinas/biosíntesis , Queratinas/genética , Microscopía Fluorescente , Fenotipo , Células Tumorales Cultivadas

RESUMEN

BACKGROUND: Lysophosphatidic acid (LPA), at concentrations present in ascitic fluid, indirectly stimulates the growth of malignant ovarian tumors by increasing the expression of vascular endothelial growth factor (VEGF) in ovarian cancer cells. We investigated whether LPA could also directly promote ovarian tumor growth by increasing the level of cyclin D1, a key G1-phase checkpoint regulator, which thereby increases cell proliferation. METHODS: Expression of cyclin D1 and LPA receptors (EDG4 and EDG7) was determined in six ovarian cancer cell lines (including OVCAR-3 cells) and immortalized ovarian surface epithelial cells (IOSE-29). Cyclin D1 promoter activity was measured in LPA-treated OVCAR-3 cells cotransfected with cyclin D1 promoter-driven luciferase constructs and cDNA expression plasmids for IkappaBalphaM (a nuclear factor kappaB [NFkappaB] super-repressor). RESULTS: Four of six cancer cell lines, including OVCAR-3, overexpressed cyclin D1 protein relative to levels in IOSE-29 cells. LPA treatment increased cyclin D1 protein in a dose- and time-dependent manner in OVCAR-3 cells but not in IOSE-29 cells. LPA stimulated cyclin D1 promoter activity (3.0-fold, 95% confidence interval [CI] = 2.7-fold to 3.3-fold). Mutation of the NFkappaB-binding site in the cyclin D1 promoter to block NFkappaB binding and expression of IkappaBalphaM, which binds NFkappaB and inhibits its binding to the promoter, markedly diminished LPA stimulation of cyclin D1 promoter activity (activity stimulated only 1.4-fold, 95% CI = 1.1-fold to 1.7-fold, and 0.7-fold, 95% CI = 0.6-fold to 0.8-fold, respectively). EDG4 was overexpressed in all cancer cell lines studied relative to that in IOSE-29 cells, but EDG7 was overexpressed in only two lines. CONCLUSIONS: Dual mechanisms are probably involved in LPA stimulation of ovarian tumor growth in vivo. In addition to the previously characterized indirect mechanism that increases angiogenesis via VEGF, LPA may directly increase the level of cyclin D1 in ovarian cancer cells, increasing their proliferation.

Asunto(s)

Carcinoma/metabolismo , Ciclina D1/metabolismo , Lisofosfolípidos/metabolismo , Neoplasias Ováricas/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G , Northern Blotting , Western Blotting , División Celular/efectos de los fármacos , Ciclina D1/efectos de los fármacos , Ciclina D1/genética , Relación Dosis-Respuesta a Droga , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas I-kappa B/genética , Luciferasas/metabolismo , Lisofosfolípidos/farmacología , Mutación , FN-kappa B/genética , ARN Mensajero/metabolismo , ARN Neoplásico/metabolismo , Receptores del Ácido Lisofosfatídico , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Elemento de Respuesta al Suero/efectos de los fármacos , Factores de Tiempo , Transfección , Células Tumorales Cultivadas , Regulación hacia Arriba/efectos de los fármacos