RESUMEN
The fallopian tube, connecting the uterus with the ovary, is a dynamic organ that undergoes cyclical changes and is the site of several diseases, including serous cancer. Here, we use single-cell technologies to construct a comprehensive cell map of healthy pre-menopausal fallopian tubes, capturing the impact of the menstrual cycle and menopause on different fallopian tube cells at the molecular level. The comparative analysis between pre- and post-menopausal fallopian tubes reveals substantial shifts in cellular abundance and gene expression patterns, highlighting the physiological changes associated with menopause. Further investigations into menstrual cycle phases illuminate distinct molecular states in secretory epithelial cells caused by hormonal fluctuations. The markers we identified characterizing secretory epithelial cells provide a valuable tool for classifying ovarian cancer subtypes. Graphical summary: Graphical summary of results. During the proliferative phase (estrogen high ) of the menstrual cycle, SE2 cells (OVGP1 + ) dominate the fallopian tube (FT) epithelium, while SE1 cells (OVGP1 - ) dominate the epithelium during the secretory phase. Though estrogen levels decrease during menopause, SE post-cells (OVGP1 + , CXCL2 + ) make up most of the FT epithelium.
RESUMEN
We carried out specific-heat and ac-susceptibility experiments under hydrostatic pressure to investigate the interplay of spin-density-wave antiferromagnetism (A) and superconductivity (S) in single-crystalline AS-type CeCu(2)Si(2). We find evidence for a line of magnetic-field- and pressure-tuned quantum critical points in the normal state in the zero-temperature magnetic field-pressure plane. Our analysis suggests an extension of this line into the superconducting state and corroborates the close connection of the underlying mechanisms leading to the formation of the antiferromagnetic and the superconducting states in AS-type CeCu(2)Si(2).
RESUMEN
BACKGROUND: Reversible posterior leukoencephalopathy syndrome (RPLS) is a clinical and radiologic syndrome of heterogeneous etiology. Although it has been described in association with intravenous cytotoxic chemotherapy including cisplatin and molecularly targeted therapies such as bevacizumab and sorafenib, it has not been described in the setting of intraperitoneal chemotherapy. CASE: A 64-year-old woman with stage IIIC fallopian tube cancer developed acute mental status changes and radiologic findings consistent with RPLS in conjunction with hypertension after one cycle of intravenous paclitaxel, followed by intraperitoneal (IP) cisplatin. Symptoms resolved over the course of 4 days with no obvious residual effects. CONCLUSION: The use of an intravenous paclitaxel and intraperitoneal cisplatin regimen can be associated with RPLS. Hypertension should be controlled in patients prior to receiving this chemotherapy regimen due to potential toxicity.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Neoplasias de las Trompas Uterinas/tratamiento farmacológico , Síndrome de Leucoencefalopatía Posterior/inducido químicamente , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Cisplatino/administración & dosificación , Cisplatino/efectos adversos , Femenino , Humanos , Infusiones Intravenosas , Infusiones Parenterales , Persona de Mediana Edad , Paclitaxel/administración & dosificación , Paclitaxel/efectos adversosRESUMEN
Migration of cells requires interactions with the extracellular matrix mediated, in part, by integrins, proteases, and their receptors. Previous studies have shown that beta(3)-integrin interacts with the urokinase-type plasminogen activator receptor (u-PAR) at the cell surface. Since integrins mediate signaling into the cell, the current study was undertaken to determine if in addition beta(3)-integrin regulates u-PAR expression. Overexpression of beta(3)-integrin in CHO cells, which are avid expressers of the receptor, downregulated u-PAR protein and mRNA expression. The u-PAR promoter (-1,469 bp) that is normally constitutively active in CHO cells was downregulated by induced beta(3)-integrin expression. A region between -398 and -197 bp of the u-PAR promoter was critical for beta(3)-integrin-induced downregulation of u-PAR promoter activity. Deletion of the PEA3/ets motif at -248 bp substantially impaired the ability of beta(3)-integrin to downregulate the u-PAR promoter, suggesting that the PEA3/ets site acts as a silencing element. An expression vector encoding the transcription factor PEA3 caused inhibition of the wild-type but not the PEA3/ets-deleted u-PAR promoter. The PEA3/ets site bound nuclear factors from CHO cells specifically, but binding was enhanced when beta(3)-integrin was overexpressed. A PEA3 antibody inhibited DNA-protein complex formation, indicating the presence of PEA3. Downregulation of the u-PAR promoter was achieved by the beta(3)A-integrin isoform but not by other beta(3)-integrin isoforms and required the cytoplasmic membrane NITY(759) motif. Moreover, overexpression of the short but not the long isoform of the beta(3)-integrin adapter protein beta(3)-endonexin blocked u-PAR promoter activity through the PEA3/ets binding site. Thus, besides the physical interaction of beta(3)-integrin and u-PAR at the cell surface, beta(3) signaling is implicated in the regulation of u-PAR gene transcription, suggesting a mutual regulation of adhesion and proteolysis receptors.
Asunto(s)
Antígenos CD/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Receptores de Superficie Celular/genética , Secuencias Reguladoras de Ácidos Nucleicos , Transcripción Genética , Secuencia de Aminoácidos , Animales , Antígenos CD/genética , Secuencia de Bases , Sitios de Unión , Células CHO , Cricetinae , Citoplasma/metabolismo , Regulación hacia Abajo , Regulación de la Expresión Génica , Integrina beta3 , Datos de Secuencia Molecular , Proteínas Nucleares , Glicoproteínas de Membrana Plaquetaria/genética , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Proteínas/genética , Proteínas/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
A previous investigation (Matsumoto et al., J. Oral Pathol. Med., 18: 498-501, 1989) has shown that the in vitro invasion of a collagen gel by squamous cell carcinoma can be substantially augmented in the presence of fibroblasts. Therefore, we undertook a study to determine if the production of collagenase(s) by a squamous cell carcinoma cell line, UM-SCC-1, was up-regulated by fibroblasts. Cocultivation of UM-SCC-1 cells with MDA-TU-138 fibroblasts, both established from the oral cavity, resulted in a dose-dependent increase in the activity of a M(r) 92,000 gelatinase as shown by zymography. Augmented M(r) 92,000 gelatinase activity was a consequence of the stimulation of the UM-SCC-1 cells by a soluble, fibroblast-derived factor since this effect could be reproduced with fibroblast-conditioned medium but not with glutaraldehyde-fixed fibroblasts. The increased M(r) 92,000 gelatinolytic activity could be accounted for by an increase in M(r) 92,000 type IV collagenase (MMP-9) protein, as demonstrated by Western blotting for this metalloproteinase. Trypsin treatment of the fibroblast-conditioned medium abolished its ability to increase MMP-9 secretion by UM-SCC-1 cells. Furthermore, fractionation of the fibroblast-conditioned medium revealed a M(r) 3,000-10,000 soluble factor(s) which was responsible for the augmented production of MMP-9 by UM-SCC-1 cells. To determine if the increased production of MMP-9, in response to the fibroblasts, was a consequence of increased promoter activity, UM-SCC-1 cells were transiently transfected with a chloramphenicol acetyltransferase reporter driven by the MMP-9 promoter and plated on plastic or on a monolayer of MDA-TU-138 fibroblasts. A 4-5-fold stimulation of MMP-9 promoter activity was observed with UM-SCC-1 cells plated with the MDA-TU-138 fibroblasts, when compared with similarly transfected cells recultured on plastic. In conclusion, we have demonstrated that MMP-9 expression in a squamous cell carcinoma cell line is augmented by a fibroblast-derived protein(s). This finding indicates a role for stromal cells in the regulation of MMP-9 expression in squamous cell carcinoma. The ability of fibroblasts to regulate MMP-9 expression in tumor cells in vitro may explain the observation that the amount of M(r) 92,000 type IV collagenase mRNA in tumor cells is highest at the tumor:stromal interface of resected squamous cell carcinoma.
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Carcinoma de Células Escamosas/enzimología , Colagenasas/biosíntesis , Fibroblastos/fisiología , Colagenasas/metabolismo , Inducción Enzimática , Humanos , Metaloproteinasa 9 de la Matriz , Peso Molecular , Sensibilidad y Especificidad , Solubilidad , Tripsina/farmacología , Células Tumorales CultivadasRESUMEN
A cell line representative of human high-grade serous ovarian cancer (HGSOC) should not only resemble its tumour of origin at the molecular level, but also demonstrate functional utility in pre-clinical investigations. Here, we report the integrated proteomic analysis of 26 ovarian cancer cell lines, HGSOC tumours, immortalized ovarian surface epithelial cells and fallopian tube epithelial cells via a single-run mass spectrometric workflow. The in-depth quantification of >10,000 proteins results in three distinct cell line categories: epithelial (group I), clear cell (group II) and mesenchymal (group III). We identify a 67-protein cell line signature, which separates our entire proteomic data set, as well as a confirmatory publicly available CPTAC/TCGA tumour proteome data set, into a predominantly epithelial and mesenchymal HGSOC tumour cluster. This proteomics-based epithelial/mesenchymal stratification of cell lines and human tumours indicates a possible origin of HGSOC either from the fallopian tube or from the ovarian surface epithelium.
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Células Epiteliales/patología , Perfilación de la Expresión Génica , Neoplasias Ováricas/patología , Proteómica/métodos , Línea Celular Tumoral , Conjuntos de Datos como Asunto , Trompas Uterinas/citología , Trompas Uterinas/patología , Femenino , Humanos , Espectrometría de Masas/métodos , Clasificación del Tumor , Neoplasias Ováricas/genética , Ovario/citología , Ovario/patología , Cultivo Primario de Células , TranscriptomaRESUMEN
The 92 kDa type IV collagenase (MMP-9), which degrades type IV collagen, has been implicated in tissue remodeling. The purpose of the current study was to determine the role of Jun amino-terminal kinase (JNK)- and extracellular signal-regulated kinase- (ERK)-dependent signaling cascades in the regulation of MMP-9 expression. Towards this end, we first determined the transcriptional requirements for MMP-9 promoter activity in a cell line (UM-SCC-1) which is an avid secretor of this collagenase. Transfection of these cells with a CAT reporter driven by progressive 5' deleted fragments of the MMP-9 promoter indicated the requirement of a region spanning -144 to -73 for optimal promoter activity. DNase I footprinting revealed a protected region of the promoter spanning nucleotides -91 to -68 and containing a consensus AP-1 motif at -79. Mutation of this AP-1 motif practically abolished the activity of the MMP-9 promoter-driven CAT reporter. Mobility shift assays indicated c-Fos and Jun-D bound to this motif and transfection of the cells with a mutated c-Jun, which quenches the function of endogenous Jun and Fos proteins, decreased MMP-9 promoter activity by 80%. UM-SCC-1 cells contained a constitutively activated JNK and the expression of a kinase-deficient JNK1 reduced the activity of a CAT reporter driven either by the MMP-9 promoter or by three tandem AP-1 repeats upstream of a thymidine kinase minimal promoter. Conditioned medium collected from UM-SCC-1 cells transfected with the dominant negative JNK1 expression vector diminished 92 kDa gelatinolysis. Similarly, interfering with MEKK, which lies upstream of JNK1, using a dominant negative expression vector reduced MMP-9 promoter activity over the same concentration range which repressed the AP-1-thymidine kinase CAT reporter construct. UM-SCC-1 cells also contained a constitutively activated ERK1. MMP-9 expression, as determined by CAT assays and by zymography, was reduced by the co-expression of a kinase-deficient ERK1. Interfering with MEK1, which is an upstream activator of ERK1, either with PD 098059, which prevents the activation of MEK1, or with a dominant negative expression construct, reduced 92 kDa gelatinolysis and MMP-9 promoter activity respectively. c-Raf-1 is an upstream activator of MEK1 and a kinase-deficient c-Raf-1 expression construct decreased the activity of a promoter driven by either the MMP-9 promoter or three tandem AP-1 repeats. Conversely, treatment of UM-SCC-1 cells with PMA, which activates c-Raf-1, increased 92 kDa gelatinolysis. These data suggest that MMP-9 expression in UM-SCC-1 cells, is regulated by JNK- and ERK-dependent signaling pathways.
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Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Colagenasas/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos , Proteínas Proto-Oncogénicas c-jun/fisiología , Sitios de Unión , Huella de ADN , Proteínas de Unión al ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Regulación Enzimológica de la Expresión Génica , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , MAP Quinasa Quinasa 1 , Metaloproteinasa 9 de la Matriz , Proteína Quinasa 3 Activada por Mitógenos , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-raf , Transducción de Señal , Activación Transcripcional , Células Tumorales CultivadasRESUMEN
The urokinase-type plasminogen activator receptor (u-PAR) facilitates extracellular matrix degradation in part by accelerating plasmin formation at the cell surface. We previously reported that u-PAR expression is elevated in colon cancer cell lines characterized by their in vitro invasive capacity. Since, u-PAR expression is increased by a variety of growth factors, which signal through the extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2), we determined if these mitogen-activated protein kinases (MAPKs) regulate u-PAR expression in two cultured colon cancer cell lines. An in-gel kinase assay showed that ERK1 activity was considerably higher in RKO cells, which display > or = 10(5) receptors/cell, than the GEO cells which have approximately 10(4) urokinase receptors per cell. The expression of either an ERK-inactivating phosphatase (CL100), or a kinase-defective ERK1, decreased the activity of a u-PAR promoter-driven CAT reporter in RKO cells. Immune complex kinase assays indicated that the constitutive ERK1 activity in RKO cells was largely a result of an activated MEK1. Further, treatment of RKO cells with a specific inhibitor (PD 098059) of MEK1 activation, which diminished ERK1 activity, reduced the amount of urokinase specifically bound to the cell surface and this was associated with reduced laminin degradation. The expression of a dominant negative c-Raf-1 also reduced u-PAR promoter activity suggesting that MEK1 activation involved an activator at, or upstream, of this serine-threonine kinase. Transfection of the u-PAR-deficient GEO cells with a constitutively activated MEK1 expression construct up-regulated u-PAR promoter activity. Similarly treatment of GEO cells with a phosphatase inhibitor (sodium vanadate) caused a dose-dependent increase in ERK1 activity which paralleled increased cell surface binding of urokinase. Taken together, these data suggest that elevated u-PAR expression, in at least a sub-population of colon cancer, is partly a consequence of a constitutively activated ERK-1-dependent signaling cascade.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas de Ciclo Celular , Neoplasias del Colon/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos , Fosfoproteínas Fosfatasas , Activadores Plasminogénicos/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal/fisiología , Fosfatasa 1 de Especificidad Dual , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Genes ras/genética , Proteínas Inmediatas-Precoces , Laminina/efectos de los fármacos , Laminina/metabolismo , MAP Quinasa Quinasa 1 , Proteína Quinasa 3 Activada por Mitógenos , Proteína Fosfatasa 1 , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Fosfatasas , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-raf , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Factores de Tiempo , Células Tumorales Cultivadas , Vanadatos/farmacologíaRESUMEN
Rac1 is a member of the Ras superfamily of small guanosine triphosphatases (GTPases) that act as molecular switches to control cytoskeletal rearrangements and cell growth. Analogous to Ras, constitutively activating point mutations of Rac1 cause tumorigenic transformation of cell lines. However, there is no information about whether Rac1 is also mutated in vivo. After RT - PCR of Rac1, several clones of seven benign and 10 malignant breast cancer tissues as well as eight breast cancer cell lines were sequenced. Only single-nucleotide polymorphisms of Rac1 could be detected, and none of these corresponded to constitutively activating point mutations that have been used in cell lines for transformation. While sequencing Rac1 in breast tissues, a new Rac1 isoform with an insertion of 19 codons within the reading frame of Rac1 close to switch region II was identified and named Rac1b. The Rac1b protein acts like a fast cycling GTPase in GTP binding and hydrolysis assays. In Northern and Western blot experiments both Rac1 RNA and Rac1 protein had a significantly higher expression in breast cancer tissues compared to normal breast tissue samples. Immunohistochemical staining of Rac1 showed weak Rac1 expression in benign breast disease but high expression level in ductal carcinoma-in-situ, primary breast cancer, and lymph node metastases. In addition, breast tumor cells from patients with recurrent disease had Rac1 expression at the plasma membrane, suggesting activation of Rac1, in patients with aggressive breast cancer. Oncogene (2000).
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Neoplasias de la Mama/metabolismo , Isoformas de Proteínas/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Secuencia de Bases , Enfermedades de la Mama/genética , Enfermedades de la Mama/metabolismo , Enfermedades de la Mama/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , División Celular , Cartilla de ADN , Femenino , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Isoformas de Proteínas/genética , ARN Mensajero/genética , Proteína de Unión al GTP rac1/genéticaRESUMEN
The urokinase-type plasminogen activator plays a central role in tissue remodeling by controlling the synthesis of the extracellular matrix-degrading plasmin. Urokinase expression is transcriptionally regulated by a variety of cytokines including TNF-alpha. The present study was undertaken to identify key transcription factor binding sites in the urokinase promoter necessary for the TNF-alpha-dependent induction of urokinase expression. TNF-alpha treatment of a squamous cell carcinoma cell line, UM-SCC-1, which produces no detectable TNF-alpha, led to a dose-dependent increase in urokinase secretion, thus reflecting a more abundant mRNA. Transient transfections of UM-SCC-1 cells with a CAT reporter driven by 5' deletion fragments of the urokinase promoter indicated that a sequence spanning -2109 to -1870, which contained binding sites for AP-1 and PEA3 was required for the stimulation by TNF-alpha. Mutation of an AP-1 binding site at -1967 and a PEA3 motif at -1973 completely abrogated the inductive effect of TNF-alpha on urokinase promoter activity. Mobility shift assays indicated the presence of a jun-containing factor(s) which bound specifically to the AP-1 sequence present in the urokinase promoter. The amount and/or activity of this factor(s) was greatly enhanced by TNF-alpha treatment. UM-SCC-1 cells transiently transfected with a CAT reporter driven by 3 tandem AP-1 binding sites demonstrated increased CAT activity following TNF-alpha treatment. Thus, the induction of urokinase expression by TNF-alpha is likely to involve the altered expression and/or activity of transcription factors which bind to the AP-1 and PEA3 target sequences in the urokinase promoter.
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Regiones Promotoras Genéticas/genética , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Activador de Plasminógeno de Tipo Uroquinasa/biosíntesis , Secuencia de Bases , Sitios de Unión , Cloranfenicol O-Acetiltransferasa/biosíntesis , Cloranfenicol O-Acetiltransferasa/genética , ADN/metabolismo , Inducción Enzimática/efectos de los fármacos , Genes Reporteros/genética , Humanos , Datos de Secuencia Molecular , Mutación/fisiología , ARN Mensajero/biosíntesis , Células Tumorales Cultivadas , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
AIM: To report the role of liver angiography in the staging of medullary thyroid cancer (MTC) patients. MATERIAL AND METHODS: Sixty MTC patients with persistent or recurrent hypercalcitonemia (n=49), a characteristic general symptom (diarrhea, n=4) or a normal basal calcitonin level without general symptoms (n=7) were investigated by dynamic liver CT, MRI and angiography between 06/1998 and 06/2002. RESULTS: Dual-phase CT and MRI investigations identified hepatic metastases with relatively low frequency (8/58 on MRI, and 7/60 on CT). Angiography indicated liver involvement in 54/60 cases. The hepatic metastases were typically multiple, hypervascular, small foci (only 13 foci measured >/=10 mm). With one exception significant disease progression was not observed over 5 years of follow-up. CONCLUSIONS: Liver angiography is a powerful tool to reveal hepatic metastases in MTC patients. Frequent, inoperable liver metastases in hypercalcitoninemic MTC patients demonstrate that secondary lymph node dissection is an inefficient technique for restoration of a normal calcitonin level.
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Neoplasias del Tronco Encefálico/diagnóstico por imagen , Neoplasias del Tronco Encefálico/cirugía , Neoplasias Hepáticas/diagnóstico por imagen , Neoplasias Hepáticas/cirugía , Hígado/patología , Hígado/cirugía , Escisión del Ganglio Linfático , Neoplasias de la Tiroides/diagnóstico por imagen , Neoplasias de la Tiroides/cirugía , Angiografía , Biomarcadores de Tumor/sangre , Neoplasias del Tronco Encefálico/sangre , Calcitonina/sangre , Cuello del Útero/metabolismo , Cuello del Útero/patología , Cuello del Útero/cirugía , Femenino , Estudios de Seguimiento , Humanos , Hígado/metabolismo , Neoplasias Hepáticas/sangre , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/cirugía , Metástasis Linfática , Imagen por Resonancia Magnética , Masculino , Mediastino/patología , Mediastino/cirugía , Neoplasias de Tejido Óseo/sangre , Neoplasias de Tejido Óseo/secundario , Neoplasias de Tejido Óseo/cirugía , Neoplasias de la Tiroides/sangre , Tomografía Computarizada por Rayos X , Resultado del TratamientoRESUMEN
This study was performed to determine if keratin 18 (K18) has prognostic significance in breast cancer cell lines and patients with breast carcinoma. Paraffin sections of primary breast carcinoma tumors and human breast carcinoma cell lines were examined for K18 expression by immunohistochemical staining with the monoclonal antibody CK2. K18 protein expression was low in highly metastatic cell lines and, conversely, high in weakly metastatic cell lines, suggesting that it may function as a prognostic indicator. K18 expression was consequently examined in 134 patients with breast cancer. The staining intensity was compared with clinicopathological variables and follow-up data spanning 8 years. A definitive positive staining was observed in 22 (16.4%) women. The mortality rate was 4. 5% in the K18-positive group and 44.6% in the K18-negative group. Multivariate analysis found K18 expression to be an independent and significant predictor for overall survival.
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Neoplasias de la Mama/diagnóstico , Queratinas/biosíntesis , Proteínas de Neoplasias/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Femenino , Humanos , Inmunohistoquímica , Queratinas/análisis , Persona de Mediana Edad , Invasividad Neoplásica , Metástasis de la Neoplasia , Proteínas de Neoplasias/análisis , Evaluación de Resultado en la Atención de Salud , Pronóstico , Análisis de Supervivencia , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
Proteases are linked to the malignant phenotype of different solid tumors. Therefore, the expression of the matrix metalloproteinase (MMP)-2 and MMP-9 and of the serine protease urokinase-type plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor type 1 (PAI-1) in the progression of ovarian cancer was investigated. Gelatinolytic activity and protein expression of MMP-2 and MMP-9 were analyzed in tissue extracts of 19 cystadenomas and 18 low malignant potential (LMP) tumors, as well as 41 primary tumors of advanced ovarian cancer stage International Federation of Gynecology and Obstetrics IIIc/IV and their corresponding omentum metastases by quantitative gelatin zymography and Western blot. In the same tissue extracts, antigen levels of uPA and its inhibitor PAI-1 were determined by ELISA. Protein expression of pro-MMP-2 (72 kDa) and pro-MMP-9 (92 kDa as well as antigen levels of uPA and PAI-1 were low in benign ovarian tumors but increased significantly from LMP tumors to advanced ovarian cancers. The highest values of all of the proteolytic factors were detected in omentum metastases. Active MMP-2 enzyme (62 kDa) was detected only in ovarian cancer (66%) and corresponding metastases (93%) but never in benign or LMP tumors. The activation rate of MMP-2 to its active isoform was higher in the metastases. Comparing both proteolytic systems, higher PAI-1 concentrations were consistently found in cancers with high pro-MMP-9 expression. These data indicate that members of the plasminogen activator system, as well as the metalloproteinases MMP-2/9, increase with growing malignant potential of ovarian tumors. These findings are of particular relevance to the development of protease inhibitors as new therapeutic approaches in ovarian cancer.
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Endopeptidasas/biosíntesis , Neoplasias Ováricas/patología , Adulto , Anciano , Anciano de 80 o más Años , Western Blotting , Progresión de la Enfermedad , Femenino , Humanos , Inmunohistoquímica , Metaloproteinasa 2 de la Matriz/biosíntesis , Metaloproteinasa 9 de la Matriz/biosíntesis , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Ováricas/enzimología , Ovario/enzimología , Ovario/patología , Inhibidor 1 de Activador Plasminogénico/análisis , Estadística como Asunto , Activador de Plasminógeno de Tipo Uroquinasa/biosíntesisRESUMEN
OBJECTIVE: Activated platelets induce alterations of chemotactic and adhesive properties of endothelial cells, a critical initial step in atherogenesis. We investigated the effect of transient interaction of activated platelets with cultured human umbilical vein endothelial cells (HUVECs) on secretion of monocyte chemoattractant protein-1 (MCP-1), a key molecule in monocyte chemotaxis and transmigration. METHODS AND RESULTS: Transient interaction of alpha-thrombin-activated platelets with endothelial cells for 10-120 min substantially induced endothelial secretion of MCP-1, monocyte chemotaxis and adhesion to HUVECs. Platelet-induced secretion of MCP-1 and monocyte-endothelium adhesion was reduced by the MAP kinase p38-specific inhibitor SB203580, but not by other kinase inhibitors including PD98059, wortmannin, or rapamycin. In addition, activated platelets induced transcription of a luciferase reporter construct containing a MCP-1 promotor, an effect that could be inhibited by SB203580. Overexpression of dominant-negative mutants of MAP kinase p38, CSBP2-(D168A) and CSBP2-(T180E,Y182E) reduced platelet-induced expression of MCP-1. CONCLUSIONS: Activation of the p38 MAP kinase and consecutive endothelial secretion of MCP-1 induced through transient interaction of activated platelets might play an important role in atherogenesis.
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Arteriosclerosis/etiología , Plaquetas/fisiología , Quimiocina CCL2/metabolismo , Endotelio Vascular/metabolismo , Proteínas Quinasas Activadas por Mitógenos/fisiología , Antiinflamatorios/farmacología , Adhesión Celular/fisiología , Técnicas de Cultivo de Célula , Quimiotaxis de Leucocito/fisiología , Endotelio Vascular/citología , Inhibidores Enzimáticos/farmacología , Humanos , Imidazoles/farmacología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/genética , Monocitos/fisiología , Activación Plaquetaria/fisiología , Piridinas/farmacología , Translocación Genética , Venas Umbilicales/citología , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Cancer cells grow in an environment comprised of multiple components that support tumor growth and contribute to therapy resistance. Major cell types in the tumor microenvironment are fibroblasts, endothelial cells and infiltrating immune cells all of which communicate with cancer cells. One way that these cell types promote cancer progression is by altering the expression of microRNAs (miRNAs), small noncoding RNAs that negatively regulate protein expression, either in the cancer cells or in the associated normal cells. Changes in miRNA expression can be brought about by direct interaction between the stromal cells and cancer cells, by paracrine factors secreted by any of the cell types or even through direct communication between cells through secreted miRNAs. Understanding the role of miRNAs in the complex interactions between the tumor and cells in its microenvironment is necessary if we are to understand tumor progression and devise new treatments.
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Comunicación Celular/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Neoplasias/genética , Neoplasias/patología , Microambiente Tumoral/genética , Animales , HumanosRESUMEN
The cross-talk between ovarian cancer (OvCa) cells and the metastatic microenvironment is an essential determinant of successful colonization. MicroRNAs (miRNAs) have several critical roles during metastasis; however, the role of microenvironmental cues in the regulation of miRNAs in metastasizing cancer cells has not been studied. Using a three-dimensional culture model that mimics the human omentum, one of the principal sites of OvCa metastasis, we identified and characterized the microenvironment-induced downregulation of a tumor suppressor miRNA, miR-193b, in metastasizing OvCa cells. The direct interaction of the OvCa cells with mesothelial cells, which cover the surface of the omentum, caused a DNA methyltransferase 1-mediated decrease in the expression of miR-193b in the cancer cells. The reduction in miR-193b enabled the metastasizing cancer cells to invade and proliferate into human omental pieces ex vivo and into the omentum of a mouse xenograft model of OvCa metastasis. The functional effects of miR-193b were mediated, in large part, by the concomitant increased expression of its target, urokinase-type plasminogen activator, a known tumor-associated protease. These findings link paracrine signals from the microenvironment to the regulation of a key miRNA in cancer cells. Targeting miR-193b, which is essential for metastatic colonization of cancer cells could prove effective in the treatment of OvCa metastasis.
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Movimiento Celular , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Epiplón/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Microambiente Tumoral , Animales , Apoptosis , Western Blotting , Adhesión Celular , Proliferación Celular , Femenino , Humanos , Técnicas para Inmunoenzimas , Ratones , Ratones Desnudos , Epiplón/metabolismo , Neoplasias Ováricas/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Activador de Plasminógeno de Tipo Uroquinasa/genética , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We previously characterized the link between WNT7A and the progression of ovarian cancer. Other groups have identified FGF1 as a relevant risk factor in ovarian cancer. Here, we show a linkage between these two signaling pathways that may be exploited to improve treatment and prognosis of patients with ovarian cancer. High expression of WNT7A and FGF1 are correlated in ovarian carcinomas and poor overall patient survival. A chromatin immunoprecipitation assay demonstrated that WNT7A/ß-catenin signaling directly regulates FGF1 expression via TCF binding elements in the FGF1-1C promoter locus. In vitro gene manipulation studies revealed that FGF1 is sufficient to drive the tumor-promoting effects of WNT7A. In vivo xenograft studies confirmed that the stable overexpression of WNT7A or FGF1 induced a significant increase in tumor incidence, whereas FGF1 knockdown in WNT7A overexpressing cells caused a significant reduction in tumor size. Niclosamide most efficiently abrogated WNT7A/ß-catenin signaling in our model, inhibited ß-catenin transcriptional activity and cell viability, and increased cell death. Furthermore, niclosamide decreased cell migration following an increase in E-cadherin subsequent to decreased levels of SLUG. The effects of niclosamide on cell functions were more potent in WNT7A-overexpressing cells. Oral niclosamide inhibited tumor growth and progression in an intraperitoneal xenograft mouse model representative of human ovarian cancer. Collectively, these results indicate that FGF1 is a direct downstream target of WNT7A/ß-catenin signaling and this pathway has potential as a therapeutic target in ovarian cancer. Moreover, niclosamide is a promising inhibitor of this pathway and may have clinical relevance.
Asunto(s)
Resistencia a Antineoplásicos , Factor 1 de Crecimiento de Fibroblastos/genética , Niclosamida/farmacología , Neoplasias Ováricas , Proteínas Wnt/fisiología , beta Catenina/fisiología , Animales , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Células Cultivadas , Progresión de la Enfermedad , Resistencia a Antineoplásicos/genética , Femenino , Factor 1 de Crecimiento de Fibroblastos/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Vía de Señalización Wnt/fisiologíaRESUMEN
Keratinocytes synthesize and secrete urokinase-type plasminogen activator, which binds to its specific receptor on keratinocytes. When bound to urokinase-type plasminogen activator receptor, urokinase-type plasminogen activator proteolytically converts surface bound plasminogen to plasmin, which in turn cleaves many extracellular components leading to pericellular proteolysis. The activation of the urokinase system has been observed during re-epithelialization of skin wounds and in lesions of the autoimmune blistering skin disease pemphigus. As pemphigus is photoinducible, we investigated the effect of ultraviolet B on urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor expression in the epidermal keratinocyte cell line A431. Ultraviolet B increased cellular and secreted urokinase-type plasminogen activator protein (enzyme-linked immunosorbent assay) and urokinase-type plasminogen activator receptor cell surface expression (flow cytometry) 24 h postirradiation. Northern blot analysis indicated that ultraviolet B increased urokinase-type plasminogen activator receptor mRNA. Compared with a more rapid mRNA induction by epidermal growth factor (maximal after 4 h) the ultraviolet B response was maximal after 24 h and prolonged up to 36 h. The mRNA induction was not dependent on protein synthesis as judged by cycloheximide incubation. Ultraviolet B did not influence urokinase-type plasminogen activator receptor mRNA stability (actinomycin D incubation). A transiently transfected chloramphenicol acetyltransferase-reporter construct containing a -398/+51 urokinase-type plasminogen activator receptor promoter fragment was activated when cells were exposed to ultraviolet B. This induction was almost completely abolished by mutating a -182/-176 AP-1 binding sequence. Ultraviolet B increased the binding capacity at this AP-1 motif in electrophoretic mobility shift assays. These data identify a distinct transcriptional mechanism by which ultraviolet B induces urokinase-type plasminogen activator receptor. The epidermal induction of components of the proteolytic urokinase system by ultraviolet B may help explain the photoinducibility of pemphigus lesions.
Asunto(s)
Receptores de Superficie Celular/genética , Rayos Ultravioleta , Activador de Plasminógeno de Tipo Uroquinasa/genética , Sitios de Unión/genética , Expresión Génica/efectos de la radiación , Regulación de la Expresión Génica , Humanos , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de la radiación , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Mensajero/efectos de la radiación , Receptores de Superficie Celular/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/fisiología , Transcripción Genética/efectos de la radiación , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/efectos de la radiaciónRESUMEN
Gastric enterochromaffin-like cells produce histamine in response to the antral hormone gastrin and accumulate the biogenic amine in secretory organelles via vesicular monoamine transporter subtype 2. The putative effects of gastrin on vesicular monoamine transporter subtype 2 expression and promoter activity are poorly understood. In the present study we used highly enriched rat enterochromaffin-like cells (purity, >90%) and rat pheochromocytoma cells stably transfected with a gastrin/cholecystokinin B receptor to investigate the expression and transcriptional regulation of vesicular monoamine transporter subtype 2. Stimulation of vesicular monoamine transporter subtype 2 mRNA and protein expression was observed in isolated enterochromaffin-like cells after 3- to 7-h incubation with gastrin (10(-7) M), forskolin (10(-5) M), or ionomycin (10(-5) M). Deletion analysis of the rat vesicular monoamine transporter subtype 2 promoter defined the minimal promoter sequence necessary for full basal activity as a -121 bp segment upstream of exon 1 containing two Sp1 sites (-97 to -88 bp and -68 to -59 bp) and a cAMP-responsive element (-44 to -35 bp). Gastrin (10(-7) M) stimulated extracellular signal related kinase1/2 phosphorylation, activated Sp1 and cAMP-responsive element-binding protein, and further induced activity of the complete rat vesicular monoamine transporter subtype 2 promoter (-800 bp) in gastrin/cholecystokinin B receptor cells. The -121-bp fragment was able to confer full gastrin responsiveness, and site-directed mutagenesis of the Sp1 and cAMP-responsive element motifs demonstrated their crucial importance for basal and inducible activities. Comparison of promoter activity of histidine decarboxylase, chromogranin A, or vesicular monoamine transporter subtype 2 in transfected cell lines revealed significant differences in basal and gastrin-stimulated activities. Our current study provides the first evidence that gastrin directly stimulates the expression and promoter activity of vesicular monoamine transporter subtype 2. Sp1 and cAMP-responsive element-binding protein recognition motifs located within 121 bp upstream of exon 1 appear to be indispensable for full basal and inducible promoter activities. Diverging effects of gastrin on histidine decarboxylase, chromogranin A, and vesicular monoamine transporter subtype 2 promoter may account for the coordinated synthesis and storage of histamine in this neuroendocrine cell type.
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Gastrinas/farmacología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana , Neuropéptidos , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/fisiología , Animales , Cromogranina A , Cromograninas/genética , Células Enterocromafines/efectos de los fármacos , Células Enterocromafines/fisiología , Histidina Descarboxilasa/genética , Inmunohistoquímica , Glicoproteínas de Membrana/efectos de los fármacos , Células PC12 , ARN Mensajero/metabolismo , Ratas , Valores de Referencia , Estimulación Química , Transcripción Genética/fisiología , Proteínas de Transporte Vesicular de Aminas Biógenas , Proteínas de Transporte Vesicular de MonoaminasRESUMEN
The incidence of circulating immune complexes, anti-low density lipoprotein (LDL) autoantibodies and the anti-LDL activity of immune complexes was studied in healthy young and aged controls and in patients with vascular diseases. Circulating immune complexes (CIC) frequently occurred both in the young or old patient groups and in the aged healthy control groups, whereas they could not be found in the young controls. Marked differences were found in the incidence of anti-LDL antibodies between the groups tested. In both young and aged control groups such antibodies were very rarely observed (4-5%). In contrast anti-LDL antibodies were present in 35-45% in the aged, or young patients. Similarly, no anti-LDL activity was found in CIC of the controls, whereas in the patients with vascular diseases a significant CIC-associated anti-LDL activity was detected. These results suggest that the presence of anti-LDL antibodies are associated with the arteriosclerotic manifestations, while that of circulating immune complexes is connected by the ageing process itself.