RESUMEN
Ulcerative colitis (UC) is closely associated with disruption of intestinal epithelial tight junction proteins. A variety of studies have confirmed that resveratrol (RSV), a natural polyphenolic compound, has a potential anti-inflammatory effect and can regulate the expression of tight junction proteins. However, the mechanism by which RSV regulates the expression of tight junction proteins in the intestinal epithelium remains unclear. Therefore, we investigated the potential effect of RSV on tight junction proteins in an HT-29 cell model of inflammation induced by lipopolysaccharide (LPS) and explored its mechanism of action. First, the downregulated expression of the tight junction proteins occludin, ZO-1, and claudin-1 in the HT-29 cell model of inflammation induced by LPS was reversed by incubation with RSV, accompanied by a decrease in the expression of tumor necrosis factor α-converting enzyme (TACE). Additionally, the Notch1 pathway was attenuated and the expression of the inflammatory factors IL-6 and TNF-α was decreased by treatment with RSV. Second, after Jagged-1 was used in combination with RSV to reactivate the Notch1 pathway, the protective effects of RSV against the LPS-induced reductions in the expression of the tight junction proteins occludin, ZO-1, and claudin-1 and the decreases in the levels of the inflammatory factors IL-6 and TNF-α were abolished. These results suggest that RSV might regulate the expression of tight junction proteins by attenuating the Notch1 pathway.
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Inflamación , Receptor Notch1 , Resveratrol , Proteínas de Uniones Estrechas , Humanos , Claudina-1/metabolismo , Células HT29 , Inflamación/inducido químicamente , Inflamación/metabolismo , Interleucina-6/metabolismo , Mucosa Intestinal/metabolismo , Lipopolisacáridos , Ocludina/metabolismo , Receptor Notch1/metabolismo , Resveratrol/farmacología , Proteínas de Uniones Estrechas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
It has been extensively reported that long noncoding RNAs (lncRNAs) were closely associated with multiple malignancies. The aim of our study was to investigate the effects and mechanism of lncRNA POU6F2-AS1 in lung adenocarcinoma (LADC).The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets provided us the information of LADC clinical samples. High-regulation of POU6F2-AS1 was presented in LADC tissues compared with adjacent normal tissues, which was correlated with poor outcome of LADC patients. Functional experiments in Calu-3 and NCI-H460 cells showed that POU6F2-AS1 significantly promoted LADC cell proliferation, colony formation, invasion and migration. Moreover, through online prediction, luciferase reporter assay and Pearson's correlation analysis, we found that POU6F2-AS1 may act as a competing endogenous RNA (ceRNA) of miR-34c-5p and facilitated the expression of potassium voltage-gated channel subfamily J member 4 (KCNJ4). The promoting effect of cell aggressiveness induced by POU6F2-AS1 was enhanced by KCNJ4, whilst was abrogated due to the overexpression of miR-34c-5p. Collectively, POU6F2-AS1 might function as a ceRNA through sponging miR-34c-5p to high-regulate KCNJ4 in LADC, which indicates that POU6F2-AS1 might be a promising therapeutic target with significant prognostic value for LADC treatment.
RESUMEN
KCNJ4 (potassium voltage-gated channel subfamily J member 4) belongs to the inward rectifier potassium channel family, which is inhibited by novel anticancer agents. However, the biologic significance of KCNJ4 in lung adenocarcinoma (LADC) is largely unknown. Therefore, in this study, we evaluated the expression, clinical correlation, and prognostic value of KCNJ4 in LADC and normal lung tissues according to data from The Cancer Genome Atlas datasets. A small interfering RNA (siRNA)-mediated technology was used to inhibit the expression level of KCNJ4. Cell counting kit-8 and plate colony formation assays were used to measure cell proliferation. Wound-healing and transwell assays were applied to detect cell mobility and metastasis. Quantitative real-time polymerase chain reaction and western blot analysis were used to examine messenger RNA and protein expressions, respectively. It was found that KCNJ4 was significantly upregulated in LADC tissues and cells. The high level of KCNJ4 predicted shorter overall survival and was identified as an independent prognostic factor in patients with LADC. siRNA-mediated KCNJ4 silencing impeded LADC cell proliferation, migration, and invasion. Knockdown of KCNJ4 suppressed the expression of phosphorylated mitogen-activated protein kinase/extracellular signal regulated kinase (p-MEK) and phosphorylated extracellular signal-regulated kinase (p-ERK). Collectively, these results shed some light on the contribution of KCNJ4 functioning as a significant player in LADC, implying that KCNJ4 might be a valuable prognostic biomarker and a potential therapeutic target for LADC treatment.
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Neoplasias Pulmonares/patología , Canales de Potasio de Rectificación Interna/genética , Línea Celular Tumoral , Proliferación Celular , Progresión de la Enfermedad , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Metástasis de la Neoplasia , Pronóstico , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
After the discovery of activating mutations in EGFR, EGFR tyrosine kinase inhibitors (TKIs) have been introduced into the first-line treatment of non-small-cell lung cancer (NSCLC). A series of studies have shown that EGFR TKI monotherapy as first-line treatment can benefit NSCLC patients harbouring EGFR mutations. Besides, combination strategies based on EGFR TKIs in the first line treatment have also been proved to delay the occurrence of resistance. In this review, we summarize the scientific literature and evidence of EGFR TKIs as first-line therapy from the first-generation EGFR TKIs to conceptually proposed fourth-generation EGFR TKI, and also recommend the application of monotherapy and combination therapies of the EGFR-based targeted therapy with other agents such as chemotherapy, anti-angiogenic drugs and immunecheckpoint inhibitors.
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Acute graft-versus-host disease (aGVHD) continues to be a frequent and devastating complication of allogeneic hematopoietic stem cell transplantation (HSCT), posing as a significant barrier against the widespread use of HSCTs as a curative modality. Recent studies suggested serum/plasma microRNAs (miRs) may predict aGVHD onset. However, little is known about the functional role of circulating miRs in aGVHD. In this article, we show in two independent cohorts that miR-29a expression is significantly upregulated in the serum of allogeneic HSCT patients at aGVHD onset compared with non-aGVHD patients. Serum miR-29a is also elevated as early as 2 wk before time of diagnosis of aGVHD compared with time-matched control subjects. We demonstrate novel functional significance of serum miR-29a by showing that miR-29a binds and activates dendritic cells via TLR7 and TLR8, resulting in the activation of the NF-κB pathway and secretion of proinflammatory cytokines TNF-α and IL-6. Treatment with locked nucleic acid anti-miR-29a significantly improved survival in a mouse model of aGVHD while retaining graft-versus-leukemia effects, unveiling a novel therapeutic target in aGVHD treatment or prevention.
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Células Dendríticas/fisiología , Enfermedad Injerto contra Huésped/diagnóstico , Efecto Injerto vs Leucemia/genética , Trasplante de Células Madre Hematopoyéticas , MicroARNs/biosíntesis , Enfermedad Aguda , Estudios de Cohortes , Enfermedad Injerto contra Huésped/genética , Humanos , Inflamación/genética , Interleucina-6/metabolismo , MicroARNs/sangre , Persona de Mediana Edad , FN-kappa B/metabolismo , Pronóstico , ARN Interferente Pequeño/genética , Transducción de Señal , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 8/metabolismo , Trasplante Homólogo , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia ArribaRESUMEN
PURPOSE: Selinexor, a selective inhibitor of XPO1, is currently being tested as single agent in clinical trials in acute myeloid leukemia (AML). However, considering the molecular complexity of AML, it is unlikely that AML can be cured with monotherapy. Therefore, we asked whether adding already established effective drugs such as topoisomerase (Topo) II inhibitors to selinexor will enhance its anti-leukemic effects in AML. EXPERIMENTAL DESIGN: The efficacy of combinatorial drug treatment using Topo II inhibitors (idarubicin, daunorubicin, mitoxantrone, etoposide) and selinexor was evaluated in established cellular and animal models of AML. RESULTS: Concomitant treatment with selinexor and Topo II inhibitors resulted in therapeutic synergy in AML cell lines and patient samples. Using a xenograft MV4-11 AML mouse model, we show that treatment with selinexor and idarubicin significantly prolongs survival of leukemic mice compared with each single therapy. CONCLUSIONS: Aberrant nuclear export and cytoplasmic localization of Topo IIα has been identified as one of the mechanisms leading to drug resistance in cancer. Here, we show that in a subset of patients with AML that express cytoplasmic Topo IIα, selinexor treatment results in nuclear retention of Topo IIα protein, resulting in increased sensitivity to idarubicin. Selinexor treatment of AML cells resulted in a c-MYC-dependent reduction of DNA damage repair genes (Rad51 and Chk1) mRNA and protein expression and subsequent inhibition of homologous recombination repair and increased sensitivity to Topo II inhibitors. The preclinical data reported here support further clinical studies using selinexor and Topo II inhibitors in combination to treat AML. Clin Cancer Res; 22(24); 6142-52. ©2016 AACR.
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Antineoplásicos/farmacología , Reparación del ADN/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/metabolismo , Hidrazinas/farmacología , Carioferinas/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Triazoles/farmacología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Humanos , Leucemia Mieloide Aguda/metabolismo , Ratones , Ratones SCID , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Mensajero/metabolismo , Inhibidores de Topoisomerasa II/farmacología , Proteína Exportina 1RESUMEN
Statins possess potent immunomodulatory effects that may play a role in preventing acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic cell transplantation (allo-HCT). We performed a phase II study of atorvastatin for aGVHD prophylaxis when given to allo-HCT recipients and their HLA-matched sibling donors. Atorvastatin (40 mg/day) was administered to sibling donors, beginning 14 days before the anticipated start of stem cell collection. Allo-HCT recipients (n = 40) received atorvastatin (40 mg/day) in addition to standard aGVHD prophylaxis. The primary endpoint was cumulative incidence of grades II to IV aGVHD at day 100. Atorvastatin was well tolerated, with no attributable grades III to IV toxicities in donors or their recipients. Day 100 and 180 cumulative incidences of grades II to IV aGVHD were 30% (95% confidence interval [CI], 17% to 45%) and 40% (95% CI, 25% to 55%), respectively. One-year cumulative incidence of chronic GVHD was 43% (95% CI, 32% to 69%). One-year nonrelapse mortality and relapse incidences were 5.5% (95% CI, .9% to 16.5%) and 38% (95% CI, 18% to 47%), respectively. One-year progression-free and overall survival rates were 54% (95% CI, 38% to 71%) and 82% (95% CI, 69% to 94%). One-year GVHD-free, relapse-free survival was 27% (95% CI, 16% to 47%). These results did not differ from our historical control subjects (n = 96). Although safe and tolerable, the addition of atorvastatin did not appear to provide any benefit to standard GVHD prophylaxis alone.
Asunto(s)
Atorvastatina/administración & dosificación , Enfermedad Injerto contra Huésped/mortalidad , Enfermedad Injerto contra Huésped/prevención & control , Trasplante de Células Madre Hematopoyéticas , Prueba de Histocompatibilidad , Donantes de Tejidos , Enfermedad Aguda , Adulto , Anciano , Aloinjertos , Supervivencia sin Enfermedad , Femenino , Enfermedad Injerto contra Huésped/etiología , Humanos , Masculino , Persona de Mediana Edad , Tasa de SupervivenciaRESUMEN
The prognosis of acute myeloid leukemia (AML) is poor, highlighting the need for novel treatments. Hypomethylating agents, including decitabine are used to treat elderly AML patients with relative success. Targeting nuclear export receptor (exportin 1 [XPO1]) is a novel approach to restore tumor suppressor (TS) function in AML. Here, we show that sequential treatment of AML blasts with decitabine followed by selinexor (XPO1 inhibitor) enhances the antileukemic effects of selinexor. These effects could be mediated by the re-expression of a subset of TSs (CDKN1A and FOXO3A) that are epigenetically silenced via DNA methylation, and cytoplasmic-nuclear trafficking is regulated by XPO1. We observed a significant upregulation of CDKN1A and FOXO3A in decitabine- versus control-treated cells. Sequential treatment of decitabine followed by selinexor in an MV4-11 xenograft model significantly improved survival compared with selinexor alone. On the basis of these preclinical results, a phase 1 clinical trial of decitabine followed by selinexor in elderly patients with AML has been initiated.
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Antineoplásicos/uso terapéutico , Azacitidina/análogos & derivados , Hidrazinas/uso terapéutico , Carioferinas/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Triazoles/uso terapéutico , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Azacitidina/uso terapéutico , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Metilación de ADN/efectos de los fármacos , Metilasas de Modificación del ADN/antagonistas & inhibidores , Decitabina , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Humanos , Carioferinas/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Receptores Citoplasmáticos y Nucleares/metabolismo , Células Tumorales Cultivadas , Regulación hacia Arriba/efectos de los fármacos , Proteína Exportina 1RESUMEN
Nucleophosmin-mutated acute myeloid leukemia (NPM1mut-AML) patients have a high rate of complete remission (CR) to induction chemotherapy. However, the mechanisms responsible for such effects are unknown. Because miR-10 family members are expressed at high levels in NPM1mut-AML, we evaluated whether these microRNAs could predict chemotherapy response in AML. We found that high baseline miR-10 family expression in 54 untreated cytogenetically heterogeneous AML patients was associated with achieving CR. However, when we included NPM1 mutation status in the multivariable model, there was a significant interaction effect between miR-10a-5p expression and NPM1 mutation status. Similar results were observed when using a second cohort of 183 cytogenetically normal older (age ≥ 60 years) AML patients. Loss- and gain-of-function experiments using miR-10a-5p in cell lines and primary blasts did not demonstrate any effect in apoptosis or cell proliferation at baseline or after chemotherapy. These data support a bystander role for the miR-10 family in NPM1mut-AML.
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Resistencia a Antineoplásicos/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Glicoproteínas de Membrana/genética , Mutación , Proteínas Nucleares/genética , Receptores Inmunológicos/genética , Antineoplásicos/uso terapéutico , Humanos , Nucleofosmina , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Chromosome maintenance protein 1 (CRM1) is a nuclear export receptor involved in the active transport of tumor suppressors (e.g., p53 and nucleophosmin) whose function is altered in cancer because of increased expression and overactive transport. Blocking CRM1-mediated nuclear export of such proteins is a novel therapeutic strategy to restore tumor suppressor function. Orally bioavailable selective inhibitors of nuclear export (SINE) that irreversibly bind to CRM1 and block the function of this protein have been recently developed. Here we investigated the antileukemic activity of KPT-SINE (KPT-185 and KPT-276) in vitro and in vivo in acute myeloid leukemia (AML). KPT-185 displayed potent antiproliferative properties at submicromolar concentrations (IC50 values; 100-500 nM), induced apoptosis (average 5-fold increase), cell-cycle arrest, and myeloid differentiation in AML cell lines and patient blasts. A strong down-regulation of the oncogene FLT3 after KPT treatment in both FLT3-ITD and wild-type cell lines was observed. Finally, using the FLT3-ITD-positive MV4-11 xenograft murine model, we show that treatment of mice with oral KPT-276 (analog of KPT-185 for in vivo studies) significantly prolongs survival of leukemic mice (P < .01). In summary, KPT-SINE are highly potent in vitro and in vivo in AML. The preclinical results reported here support clinical trials of KPT-SINE in AML.
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Antineoplásicos/farmacología , Carioferinas/antagonistas & inhibidores , Leucemia Mieloide/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto , Acrilamidas/farmacología , Acrilatos/farmacología , Enfermedad Aguda , Adulto , Anciano , Anciano de 80 o más Años , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Estimación de Kaplan-Meier , Carioferinas/genética , Carioferinas/metabolismo , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Tiazoles/farmacología , Triazoles/farmacología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Adulto Joven , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/metabolismo , Proteína Exportina 1RESUMEN
BACKGROUND: CCAAT/enhancer binding protein-delta (C/EBP-delta) is a member of the highly conserved C/EBP family of basic region leucine zipper transcription factors. C/EBP family members regulate cell growth and differentiation and "loss of function" alterations in C/EBPs have been reported in a variety of human cancers. C/EBP-delta gene expression is upregulated by G0 growth arrest, IL-6 family cytokines and endotoxin treatments. C/EBP-delta exhibits properties of a tumor suppressor gene, including reduced expression and promoter methylation-induced silencing in transformed cell lines and primary tumors. In addition, C/EBP-delta gene expression is repressed by c-Myc, an oncogene that is over-expressed in a wide range of human cancers. "ChIP-chip" studies demonstrated that C/EBP-delta functions as a transcriptional activator of target genes that function in intracellular signal transduction, transcription, DNA binding/repair, cell cycle control, cell adhesion, and apoptosis. Despite progress in determining the biochemical functions of C/EBP-delta, the specific cellular defects that are induced by C/EBP-delta "loss of function" alterations are poorly understood. This study investigated the impact of C/EBP-delta "loss of function" alterations on growth arrest, migration/invasion and differentiation in nontransformed mouse mammary epithelial cells (MECs) and primary mouse embryo fibroblasts (MEFs). RESULTS: C/EBP-delta siRNA transfected MECs exhibited ~90% reduction in C/EBP-delta mRNA and protein levels. C/EBP-delta siRNA treatment resulted in defective growth arrest as demonstrated by persistently elevated BrdU labeling, 3H-thymidine incorporation and cyclin D1 levels in response to growth arrest treatments. C/EBP-delta siRNA treatment also resulted in increased migration/invasion and defective differentiation. C/EBP-delta knockout MEFs exhibited defective growth arrest and increased proliferation/migration. Re-introduction of C/EBP-delta expression restored the growth arrest response of C/EBP-delta knockout MEFs. Finally, deletion of the C/EBP-delta DNA binding domain or the C/EBP-delta bZIP domain resulted in the loss of C/EBP-delta growth inhibition in clonogenic assays. CONCLUSIONS: This study demonstrates that C/EBP-delta functions in the regulation of critical cell fate determining programs such as growth arrest, migration, and differentiation. These results support the tumor suppressor function of C/EBP-delta and identify potential mechanisms in which "loss of function" alterations in C/EBP-delta could promote cell transformation and tumorigenesis.
RESUMEN
The skin is the outer layer of protection against the environment. The development and formation of the skin is regulated by several genetic cascades including the bone morphogenetic protein (BMP) signaling pathway, which has been suggested to play an important role during embryonic organ development. Several skin defects and diseases are caused by genetic mutations or disorders. Ichthyosis is a common genetic skin disorder characterized by dry scaly skin. Loss-of-function mutations in the filaggrin (FLG) gene have been identified as the cause of the ichthyosis vulgaris (IV) phenotype; however, the direct regulation of filaggrin expression in vivo is unknown. We present evidence that BMP signaling regulates filaggrin expression in the epidermis. Mice expressing a constitutively active form of BMP-receptor-IB in the developing epidermis exhibit a phenotype resembling IV in humans, including dry flaky skin, compact hyperkeratosis, and an attenuated granular layer associated with a significantly downregulated expression of filaggrin. Regulation of filaggrin expression by BMP signaling has been further confirmed by the application of exogenous BMP2 in skin explants and by a transgenic model overexpressing Noggin in the epidermis. Our results demonstrate that aberrant BMP signaling in the epidermis causes overproliferation and hyperkeratinization, leading to an IV-like skin disease.
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Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Epidermis/metabolismo , Ictiosis Vulgar , Animales , Western Blotting , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Proteínas Portadoras/genética , Diferenciación Celular , Proliferación Celular , Epidermis/embriología , Epidermis/patología , Proteínas Filagrina , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Genotipo , Ictiosis Vulgar/genética , Ictiosis Vulgar/metabolismo , Ictiosis Vulgar/patología , Proteínas de Filamentos Intermediarios/genética , Queratinocitos/citología , Queratinocitos/metabolismo , Ratones , Ratones Transgénicos , Fenotipo , Reacción en Cadena de la Polimerasa , Transducción de SeñalRESUMEN
BMP signaling plays many important roles during organ development, including palatogenesis. Loss of BMP signaling leads to cleft palate formation. During development, BMP activities are finely tuned by a number of modulators at the extracellular and intracellular levels. Among the extracellular BMP antagonists is Noggin, which preferentialy binds to BMP2, BMP4 and BMP7, all of which are expressed in the developing palatal shelves. Here we use targeted Noggin mutant mice as a model for gain of BMP signaling function to investigate the role of BMP signaling in palate development. We find prominent Noggin expression in the palatal epithelium along the anterior-posterior axis during early palate development. Loss of Noggin function leads to overactive BMP signaling, particularly in the palatal epithelium. This results in disregulation of cell proliferation, excessive cell death, and changes in gene expression, leading to formation of complete palatal cleft. The excessive cell death in the epithelium disrupts the palatal epithelium integrity, which in turn leads to an abnormal palate-mandible fusion and prevents palatal shelf elevation. This phenotype is recapitulated by ectopic expression of a constitutively active form of BMPR-IA but not BMPR-IB in the epithelium of the developing palate; this suggests a role for BMPR-IA in mediating overactive BMP signaling in the absence of Noggin. Together with the evidence that overexpression of Noggin in the palatal epithelium does not cause a cleft palate defect, we conclude from our results that Noggin mediated modulation of BMP signaling is essential for palatal epithelium integrity and for normal palate development.
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Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Portadoras/metabolismo , Epitelio/embriología , Organogénesis , Hueso Paladar/embriología , Hueso Paladar/metabolismo , Transducción de Señal , Animales , Apoptosis , Proteínas Morfogenéticas Óseas/genética , Proliferación Celular , Fisura del Paladar/embriología , Fisura del Paladar/metabolismo , Fisura del Paladar/patología , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Epitelio/metabolismo , Epitelio/patología , Regulación del Desarrollo de la Expresión Génica , Mandíbula/anomalías , Mandíbula/patología , Ratones , Mutación/genética , Hueso Paladar/anomalías , Hueso Paladar/patología , Fenotipo , Proteínas Smad/metabolismoRESUMEN
BACKGROUND: "Loss of function" alterations in CCAAT/Enhancer Binding Proteindelta (C/EBPdelta) have been reported in a number of human cancers including breast, prostate and cervical cancer, hepatocellular carcinoma and acute myeloid leukemia. C/EBPdelta gene transcription is induced during cellular quiescence and repressed during active cell cycle progression. C/EBPdelta exhibits tumor suppressor gene properties including reduced expression in cancer cell lines and tumors and promoter methylation silencing. We previously reported that C/EBPdelta expression is inversely correlated with c-Myc (Myc) expression. Aberrant Myc expression is common in cancer and transcriptional repression is a major mechanism of Myc oncogenesis. A number of tumor suppressor genes are targets of Myc transcriptional repression including C/EBPalpha, p15INK4, p21CIP1, p27KIP1 and p57KIP2. This study investigated the mechanisms underlying Myc repression of C/EBPdelta expression. RESULTS: Myc represses C/EBPdelta promoter activity in nontransformed mammary epithelial cells in a dose-dependent manner that requires Myc Box II, Basic Region and HLH/LZ domains. Chromatin Immunoprecipitation (ChIP) assays demonstrate that Myc, Miz1 and Max are associated with the C/EBPdelta promoter in proliferating cells, when C/EBPdelta expression is repressed. EMSAs demonstrate that Miz1 binds to a 30 bp region (-100 to -70) of the C/EBPdelta promoter which contains a putative transcription initiator (Inr) element. Miz1 functions exclusively as a repressor of C/EBPdelta promoter activity. Miz1 siRNA expression or expression of a Miz1 binding deficient Myc (MycV394D) construct reduces Myc repression of C/EBPdelta promoter activity. Max siRNA expression, or expression of a Myc construct lacking the HLH/LZ (Max interacting) region, also reduces Myc repression of C/EBPdelta promoter activity. Miz1 and Max siRNA treatments attenuate Myc repression of endogenous C/EBPdelta expression. Myc Box II interacting proteins RuvBl1 (Pontin, TIP49) and RuvBl2 (Reptin, TIP48) enhances Myc repression of C/EBPdelta promoter activity. CONCLUSION: Myc represses C/EBPdelta expression by associating with the C/EBPdelta proximal promoter as a transient component of a repressive complex that includes Max and Miz1. RuvBl1 and RuvBl2 enhance Myc repression of C/EBPdelta promoter activity. These results identify protein interactions that mediate Myc repression of C/EBPdelta, and possibly other tumor suppressor genes, and suggest new therapeutic targets to block Myc transcriptional repression and oncogenic function.
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Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Proteína delta de Unión al Potenciador CCAAT/biosíntesis , Regulación de la Expresión Génica/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Glándulas Mamarias Animales/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Represoras/metabolismo , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Western Blotting , Proteína delta de Unión al Potenciador CCAAT/genética , Inmunoprecipitación de Cromatina , Ensayo de Cambio de Movilidad Electroforética , Células Epiteliales , Femenino , Expresión Génica , Inmunoprecipitación , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myc/genética , ARN Interferente Pequeño , Proteínas Represoras/genética , TransfecciónRESUMEN
The basic helix-loop-helix (bHLH) transcription factor Hand2 has been implicated in the development of multiple organs, including craniofacial organs. Mice carrying Hand2 hypomorphic alleles (Hand2(LoxP/-)) display a cleft palate phenotype. A specific deletion of the Hand2 branchial arch-specific enhancer also leads to a hypoplastic mandible and cleft palate formation in mice. However, the underlying mechanism of Hand2 regulation of palate development remains unknown. Here we show that Hand2 is expressed in both the epithelium and mesenchyme of the developing palate. While mesenchymal specific inactivation of Hand2 has no impact on palate development, epithelial specific deletion of Hand2 creates a cleft palate phenotype. Hand2 appears to exert distinct roles in the anterior and posterior palate. In the anterior palate of Hand2(LoxP/-) mice, premature death of periderm cells and a down-regulation of Shh are observed in the medial edge epithelium (MEE), accompanied by a decreased level of cell proliferation in the palatal mesenchyme. In the posterior palate, a lower dose of Hand2 causes aberrant periderm cell death on the surface of the epithelium, triggering abnormal fusion between the palatal shelf and mandible and preventing palatal shelf elevation. We further demonstrate that BMP activities are essential for the expression of Hand2 in the palate. We conclude that Hand2 is an intrinsic regulator in the epithelium and is required for palate development.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Epitelio/metabolismo , Hueso Paladar/embriología , Animales , Apoptosis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proliferación Celular , Epitelio/embriología , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Mesodermo/citología , Mesodermo/embriología , Mesodermo/metabolismo , Ratones , Hueso Paladar/citología , Hueso Paladar/metabolismo , Transducción de SeñalRESUMEN
Lung cancer is one of the most lethal cancers in China because of high incidence and high mortality. Cyclooxygenase-2 (COX-2) and vessel endothelial growth factor C (VEGF-C) were found to play an important role in lymphangiogenesis of malignant tumors. In this study, we investigated whether lymphatic microvessel density (LMVD) is related to the prognosis in non-small cell lung cancer (NSCLC) patients, and the expressions of COX-2 and VEGF-C so as to determine the possible role of COX-2 and VEGF-C in NSCLC lymphangiogenesis. Sixty-five formalin-fixed paraffin embedded tissue samples of NSCLC were evaluated for COX-2 and VEGF-C by immunohistochemical staining. To assess tumor lymphangiogenesis, LMVD was determined by immunohistochemical staining of VEGFR-3 polyclonal antibody. The relationship among COX-2 and VEGF-C expression, LMVD, and clinicopathologic parameters was analyzed. Among the 65 samples, high LMVD was significantly associated with lymph node metastasis and poor survival. Multivariate survival analysis showed that LMVD value and lymph node metastasis were independent prognostic factors. The expression level of COX-2 and VEGF-C was significantly higher than those of the adjacent tissues. COX-2 and VEGF-C expressions in NSCLC significantly correlated with lymph node metastasis, but not with patient gender, age, tumor size, or tumor, nodes, metastasis classification stage. The mean LMVD value of COX-2- or VEGF-C-positive tumors was higher than that of COX-2- or VEGF-C-negative tumors. A significant correlation was found between the expressions of COX-2 and VEGF-C. This study suggests that LMVD may be one of the important prognostic factors for NSCLC patients. VEGF-C might play an important role in the COX-2 lymphangiogenic pathway. COX-2 and VEGF-C may play an important role in tumor progression by stimulating lymphangiogenesis. The inhibition of lymphangiogenesis, COX-2, or VEGF-C activity may have an important therapeutic benefit in the control of NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Ciclooxigenasa 2/metabolismo , Vasos Linfáticos/metabolismo , Microvasos/metabolismo , Factor C de Crecimiento Endotelial Vascular/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/cirugía , Femenino , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Linfangiogénesis , Metástasis Linfática , Vasos Linfáticos/patología , Masculino , Persona de Mediana Edad , Pronóstico , Análisis de Supervivencia , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Left-sided expression of Nodal in the lateral plate mesoderm (LPM) during early embryogenesis is a crucial step in establishing the left-right (L-R) axis in vertebrates. In the chick, it was suggested that chick Cerberus (cCer), a Cerberus/Dan family member, induces Nodal expression by antagonizing bone morphogenetic protein (BMP) activity in the left LPM. In contrast, it has also been shown that BMPs positively regulate Nodal expression in the left LPM in the chick embryo. Thus, it is still unclear how the bilaterally expressed BMPs induce Nodal expression only in the left LPM. In this study, we demonstrate that BMP signaling is necessary and sufficient for the induction of Nodal expression in the chick LPM where the type I BMP receptor-IB (BMPR-IB) likely mediates this induction. Tissue grafting experiments indicate the existence of a Nodal inductive factor in the left LPM rather than the presence of a Nodal inhibitory factor in the right LPM. We demonstrate that cCer functions as a BMP agonist instead of antagonist, being able to enhance BMP signaling in cell culture. This conclusion is further supported by the immunoprecipitation assays that provide convincing biochemical evidence for a direct interaction between cCer and BMP receptor. Because cCer is expressed restrictedly in the left LPM, BMPs and cCer appear to act synergistically to activate Nodal expression in the left LPM in the chick.
Asunto(s)
Proteínas Aviares/metabolismo , Tipificación del Cuerpo , Proteínas Morfogenéticas Óseas/agonistas , Proteínas Morfogenéticas Óseas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína Nodal/metabolismo , Animales , Proteínas Aviares/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Diferenciación Celular , Línea Celular , Embrión de Pollo , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Mesodermo/embriología , Mesodermo/metabolismo , Ratones , Unión ProteicaRESUMEN
Tissue and molecular heterogeneities are present in the developing secondary palate along the anteroposterior (AP) axis in mice. Here, we show that Wnt5a and its receptor Ror2 are expressed in a graded manner along the AP axis of the palate. Wnt5a deficiency leads to a complete cleft of the secondary palate, which exhibits distinct phenotypic alterations at histological, cellular and molecular levels in the anterior and posterior regions of the palate. We demonstrate that there is directional cell migration within the developing palate. In the absence of Wnt5a, this directional cell migration does not occur. Genetic studies and in vitro organ culture assays further demonstrate a role for Ror2 in mediating Wnt5a signaling in the regulation of cell proliferation and migration during palate development. Our results reveal distinct regulatory roles for Wnt5a in gene expression and cell proliferation along the AP axis of the developing palate, and an essential role for Wnt5a in the regulation of directional cell migration.
Asunto(s)
Movimiento Celular , Mamíferos/embriología , Hueso Paladar/citología , Hueso Paladar/embriología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Wnt/metabolismo , Animales , Tipificación del Cuerpo , Bromodesoxiuridina/metabolismo , Proliferación Celular , Quimiotaxis , Fisura del Paladar/enzimología , Fisura del Paladar/patología , Epistasis Genética , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Mesodermo/enzimología , Mesodermo/patología , Ratones , Ratones Mutantes , Hueso Paladar/enzimología , Hueso Paladar/metabolismo , Fenotipo , Proteínas Tirosina Quinasas Receptoras/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa , Proteínas Wnt/deficiencia , Proteínas Wnt/genética , Proteína Wnt-5aRESUMEN
Many genes are known to function in a region-specific manner in the developing secondary palate. We have previously shown that Shox2-deficient embryos die at mid-gestation stage and develop an anterior clefting phenotype. Here, we show that mice carrying a conditional inactivation of Shox2 in the palatal mesenchyme survive the embryonic and neonatal lethality, but develop a wasting syndrome. Phenotypic analyses indicate a delayed closure of the secondary palate at the anterior end, leading to a failed fusion of the primary and secondary palates. Consistent with a role proposed for Shox2 in skeletogenesis, Shox2 inactivation causes a significantly reduced bone formation in the hard palate, probably due to a down-regulation of Runx2 and Osterix. We conclude that the secondary palatal shelves are capable of fusion with each other, but fail to fuse with the primary palate in a developmentally delayed manner. Mice carrying an anterior cleft can survive neonatal lethality.
Asunto(s)
Fisura del Paladar , Proteínas de Homeodominio/metabolismo , Hueso Paladar/anatomía & histología , Hueso Paladar/embriología , Animales , Fisura del Paladar/genética , Fisura del Paladar/mortalidad , Factor 10 de Crecimiento de Fibroblastos/genética , Factor 10 de Crecimiento de Fibroblastos/metabolismo , Proteínas del Grupo de Alta Movilidad/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas de Homeodominio/genética , Humanos , Hibridación in Situ , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Osteogénesis/fisiología , Hueso Paladar/anomalías , Fenotipo , Factor de Transcripción SOX9 , Tasa de Supervivencia , Técnicas de Cultivo de Tejidos , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Polycomb group (PcG) proteins are required for maintaining the repressed state of developmentally important genes such as homeotic genes. Polycomblike (Pcl), a member of PcG genes with two characteristic PHD finger motifs, was shown to strongly enhance the effects of PcG genes in Drosophila. Three Pcl genes exist in the mouse genome, with their function largely unknown. Our previous studies demonstrate that the chick Pcl2 is essential for the left-right asymmetry by silencing Shh expression in the right side of the node (Wang et al., [2004b] Development 131:4381-4391). To elucidate the in vivo role of mouse Pcl2, we generated Pcl2 mutant mice. Phenotypic analyses indicate the normal development of left-right asymmetry in the Pcl2 mutant mice. However, Pcl2 mutant mice exhibit posterior transformation of axial skeletons and other phenotypic defects, with a relatively low penetrance. These results demonstrate that Pcl2 is dispensable for the normal left-right axis development in mice.