RESUMEN
Embryonic neural tumors are responsible for a disproportionate number of cancer deaths in children. Although dramatic improvements in survival for pediatric malignancy has been achieved in previous years advancements seem to be slowing down. For the development of new enhanced therapy and an increased understanding of the disease, pre-clinical models better capturing the neoplastic niche are essential. Tumors of early childhood present in this respect a particular challenge. Here, we explore how components of the embryonic process in stemcell induced mature teratoma can function as an experimental in vivo microenvironment instigating the growth of injected childhood neuroblastoma (NB) cell lines. Three human NB cell lines, IMR-32, Kelly and SK-N-BE(2), were injected into mature pluripotent stem cellinduced teratoma (PSCT) and compared to xenografts of the same cell lines. Proliferative NB cells from all lines were readily detected in both models with a typical histology of a poorly differentiated NB tumor with a variable amount of fibrovascular stroma. Uniquely in the PSCT microenvironment, NB cells were found integrated in a nonrandom fashion. Neuroblastoma cells were never observed in areas with well-differentiated somatic tissue i.e. bone, muscle, gut or areas of other easily identifiable tissue types. Instead, the three cell lines all showed initial growth exclusively occurring in the embryonic loose mesenchymal stroma, resulting in a histology recapitulating NB native presentation in vivo. Whether this reflects the 'open' nature of loose mesenchyme more easily giving space to new cells compared to other more dense tissues, the rigidity of matrix providing physical cues modulating NB characteristics, or if embryonic loose mesenchyme may supply developmental cues that attracted or promoted the integration of NB, remains to be tested. We tentatively hypothesize that mature PSCT provide an embryonic niche well suited for in vivo studies on NB.
Asunto(s)
Neuroblastoma/terapia , Células Madre Pluripotentes/citología , Teratoma/patología , Microambiente Tumoral , Animales , Línea Celular Tumoral , Humanos , Mesodermo/citología , Ratones , Neuroblastoma/embriología , Neuroblastoma/patología , Células Madre/patología , Trasplante Heterólogo , Tropismo/genéticaRESUMEN
BACKGROUND: CRIPTO-1 (CR-1) is involved in the pathogenesis and progression of human carcinoma of different histological origin. In this study we addressed the expression and the functional role of CR-1 in cutaneous melanoma. METHODS: Expression of CR-1 protein in melanomas and melanoma cell lines was assessed by immunohistochemistry, western blotting and/or flow cytometry. Levels of mRNA were evaluated by real-time PCR. Invasion assays were performed in Matrigel-coated modified Boyden chambers. RESULTS: Expression of CR-1 protein and/or mRNA was found in 16 out of 37 primary human cutaneous melanomas and in 12 out of 21 melanoma cell lines. Recombinant CR-1 protein activated in melanoma cells c-Src and, at lesser extent, Smad signalling. In addition, CR-1 significantly increased the invasive ability of melanoma cells that was prevented by treatment with either the ALK4 inhibitor SB-431542 or the c-Src inhibitor saracatinib (AZD0530). Anti-CR-1 siRNAs produced a significant inhibition of the growth and the invasive ability of melanoma cells. Finally, a close correlation was found in melanoma cells between the levels of expression of CR-1 and the effects of saracatinib on cell growth. CONCLUSION: These data indicate that a significant fraction of cutaneous melanoma expresses CR-1 and that this growth factor is involved in the invasion and proliferation of melanoma cells.
Asunto(s)
Proteínas Ligadas a GPI/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Melanoma/metabolismo , Melanoma/patología , Proteínas de Neoplasias/metabolismo , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Receptores de Activinas Tipo I/antagonistas & inhibidores , Receptores de Activinas Tipo I/metabolismo , Benzamidas/farmacología , Benzodioxoles/farmacología , Western Blotting , Proteína Tirosina Quinasa CSK , Adhesión Celular , Movimiento Celular , Proliferación Celular/efectos de los fármacos , Dioxoles/farmacología , Citometría de Flujo , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/genética , Humanos , Técnicas para Inmunoenzimas , Péptidos y Proteínas de Señalización Intercelular/genética , Melanoma/genética , Invasividad Neoplásica , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Quinazolinas/farmacología , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Cutáneas/genética , Proteínas Smad/metabolismo , Células Tumorales Cultivadas , Familia-src QuinasasRESUMEN
Pigment epithelium-derived factor (PEDF) is a multifunctional secreted glycoprotein that displays broad anti-tumor activity based on dual targeting of the tumor microenvironment (anti-angiogenic action) and the tumor cells (direct anti-tumor action). Here, we show that PEDF expression is high in melanocytes, but it is lost during malignant progression of human melanoma. Using a high-throughput analysis of the data from microarray studies of molecular profiling of human melanoma, we found that PEDF expression is lost in highly invasive melanomas. In paired cell lines established from the same lesion but representing the high and low extremes of malignant potential, abundant PEDF expression was restricted to the poorly aggressive counterparts. We used RNA interference to directly address the functional consequences of PEDF silencing. PEDF knockdown in poorly aggressive melanoma cell lines augmented migration, invasion and vasculogenic mimicry, which translated into an increased in vivo metastatic potential. PEDF interference also significantly enhanced the migratory and invasive capability of normal melanocytes and moderately increased their proliferative potential. Our results show that loss of PEDF enables melanoma cells to acquire an invasive phenotype and, therefore, modulation of this multifunctional factor could be critical for the malignant progression of human melanoma.
Asunto(s)
Movimiento Celular , Proteínas del Ojo/metabolismo , Regulación Neoplásica de la Expresión Génica , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Serpinas/metabolismo , Línea Celular Tumoral , Proteínas del Ojo/genética , Perfilación de la Expresión Génica , Humanos , Melanoma/genética , Melanoma/patología , Invasividad Neoplásica , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Factores de Crecimiento Nervioso/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Serpinas/genéticaRESUMEN
Microvascular endothelial cells involved in angiogenesis are exposed to an acidic environment that is not conducive for growth and survival. These cells must exhibit a dynamic intracellular (cytosolic) pH (pHcyt) regulatory mechanism to cope with acidosis, in addition to the ubiquitous Na+/H+ exchanger and HCO3--based H+-transporting systems. We hypothesize that the presence of plasmalemmal vacuolar-type proton ATPases (pmV-ATPases) allows microvascular endothelial cells to better cope with this acidic environment and that pmV-ATPases are required for cell migration. This study indicates that microvascular endothelial cells, which are more migratory than macrovascular endothelial cells, express pmV-ATPases. Spectral imaging microscopy indicates a more alkaline pHcyt at the leading than at the lagging edge of microvascular endothelial cells. Treatment of microvascular endothelial cells with V-ATPase inhibitors decreases the proton fluxes via pmV-ATPases and cell migration. These data suggest that pmV-ATPases are essential for pHcyt regulation and cell migration in microvascular endothelial cells.
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Membrana Celular/enzimología , Movimiento Celular/fisiología , Endotelio Vascular/fisiología , ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Membrana Celular/fisiología , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/enzimología , Concentración de Iones de Hidrógeno , Inmunohistoquímica , Microcirculación/fisiología , Ratas , Ratas Endogámicas BB , Intercambiadores de Sodio-Hidrógeno/fisiologíaRESUMEN
Identification of factors that play a role in regulating the highly invasive ability of human placental cells throughout gestation will contribute to a better understanding of this unique developmental process. The aims of this study were to determine whether the tumour suppressor gene maspin is present in the human placenta and plays a putative role in the regulation of cytotrophoblast invasion during placental development. The data showed that the expression of maspin mRNA was maximum in term placentae compared to the first and second trimester tissues, and absent in the HTR-SVneo (immortalized extravillous cytotrophoblast), JEG-3 and JAR (choriocarcinoma) cell lines. Maspin protein, detected by Western blot analysis, was twofold higher in the second trimester and 4.4-fold higher in the third trimester compared to the first trimester. Maspin immunohistochemical staining was localized in cytotrophoblasts with increased and more diffuse staining in the second and third trimesters. Corresponding to the period of maximum maspin expression, cytotrophoblasts isolated from term placentae had significantly lower invasive ability as compared to first and second trimester cytotrophoblasts (P< 0.03). Further, addition of recombinant maspin significantly decreased cytotrophoblast invasion in vitro by 40-50 per cent in all three trimesters of gestation. This study provides the first evidence of the temporal expression of maspin during human gestation and suggests a putative role for maspin in regulating the invasive activity of cytotrophoblasts at term. The down-regulation of maspin expression may be critical at the time of implantation and early placental development, whereas upregulation of maspin may serve as a signal for the end of cytotrophoblast invasion and gestation.
Asunto(s)
Genes Supresores de Tumor , Placenta/metabolismo , Placentación , Proteínas/genética , Proteínas/metabolismo , Serpinas/genética , Serpinas/metabolismo , Trofoblastos/metabolismo , Línea Celular , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Inmunohistoquímica , Técnicas In Vitro , Embarazo , Tercer Trimestre del Embarazo , Proteínas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/farmacología , Serpinas/farmacología , Transducción de Señal , Trofoblastos/efectos de los fármacosRESUMEN
In vitro morphogenesis of epithelial cells to form tube-like structures is regulated by hepatocyte growth factor-scatter factor (HGF/SF). The placenta is a rich source of HGF/SF, and its absence in mice has been shown to lead to impaired placental growth and embryonic death. There is no information in the literature regarding in vitro morphogenesis of human cytotrophoblasts or the effect of HGF/SF on this process. In this study, cytotrophoblasts were isolated from human placentae obtained from all three trimesters of gestation and cultured on the recombinant basement membrane matrix (Matrigel). Under these conditions, cytotrophoblasts participated in morphogenetic events including formation of spheroid-like structures, radial linear processes with branching, and invaded Matrigel and formed large, tube-like structures. The presence of a developing lumen was documented in the linear projections arising from spheroids and in the tube-like structures by both confocal and transmission electron microscopy. Immunohistochemistry was used to characterize the phenotype of the cells, and staining with anti-cytokeratin and anti-E-cadherin antibodies confirmed the presence of cytotrophoblasts in both the spheroids and tube-like structures. Recombinant HGF (rHGF) significantly increased the invasive activity of cytotrophoblasts isolated from the first and second (P < 0.001) and third trimesters (P < 0.01). In addition, rHGF significantly increased the percentage of spheroids with branching processes in the first and second trimesters (P < 0.05). Anti-HGF antibody inhibited both these effects in a dose-dependent manner, indicating the specificity of the above findings. This study provides new evidence indicating that HGF/SF regulates invasion and branching morphogenesis of cytotrophoblasts throughout gestation, with maximum effects in the first and second trimester. These findings may help to elucidate the importance of the reduced expression of HGF/SF identified in placentae from women with preeclampsia or intrauterine growth restriction and suggest that HGF/SF may serve as an important candidate in therapeutic intervention strategies.
Asunto(s)
Factor de Crecimiento de Hepatocito/farmacología , Morfogénesis , Trofoblastos/fisiología , Cadherinas/análisis , Colágeno , Técnicas de Cultivo , Combinación de Medicamentos , Femenino , Edad Gestacional , Humanos , Inmunohistoquímica , Queratinas/análisis , Laminina , Microscopía Confocal , Microscopía Electrónica , Embarazo , Proteoglicanos , Proteínas Recombinantes/farmacología , Trofoblastos/citologíaRESUMEN
If we are to maintain public appreciation and support for our scientific enterprise, we need to pay more attention to translating the benefits and grandeur of science into the language of broader society. Both educators and journalists have a role to play in communicating the achievements of science, but scientists must recognize that we have a responsibility to increase the availability and salience of science to the public.
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Periodismo Médico , Medios de Comunicación de Masas , Ciencia del Laboratorio Clínico/educación , Relaciones Públicas , Ciencia/educación , Investigación/educaciónRESUMEN
Vasculogenic mimicry describes a process where aggressive tumor cells in three-dimensional matrices mimic embryonic vasculogenesis by forming extracellular matrix (ECM)-rich, patterned tubular networks. Microarray gene chip analyses revealed significant increases in the expression of laminin 5 (Ln-5, gamma2 chain) and matrix metalloproteinases (MMP)-1, -2, -9, and MT1-MMP (MMP-14) in aggressive compared with poorly aggressive melanoma cells. These components colocalized with developing patterned networks and antisense oligonucleotides to the Ln-5 gamma2 chain (but not sense oligonucleotides), and antibodies to MMP-2 or MT1-MMP (but not MMP-9) inhibited the formation of these networks. Cultures which did not receive antibodies to either MMPs-2 or -14 contained the Ln-5 gamma2 chain promigratory cleavage fragments. Poorly aggressive melanoma cells seeded on collagen I matrices preconditioned by the aggressive cells formed tubular networks along the Ln-5 gamma2 chain-enriched tracks deposited by the aggressive cells. These results suggest that increased expression of MMP-2 and MT1-MMP, along with matrix deposition of the Ln-5 gamma2 chain and/or its cleavage fragments, are required for vasculogenic mimicry by aggressive melanoma cells. Furthermore, the apparent recapitulation of laminin-rich, patterned networks observed in aggressive melanoma patients' tissue sections by aggressive melanoma tumor cells in three-dimensional culture may also serve as a model to help identify specific molecular targets which could function as templates for the coordinated migration of aggressive tumor cells and their proteolytic remodeling of the ECM and may have profound implications for the development of novel therapies directed at the ECM to alter tumor progression.
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Moléculas de Adhesión Celular/fisiología , Metaloproteinasa 2 de la Matriz/fisiología , Melanoma/irrigación sanguínea , Melanoma/patología , Metaloendopeptidasas/fisiología , Neovascularización Patológica/metabolismo , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/genética , Humanos , Metaloproteinasa 2 de la Matriz/biosíntesis , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasas de la Matriz Asociadas a la Membrana , Melanoma/genética , Melanoma/metabolismo , Metaloendopeptidasas/biosíntesis , Metaloendopeptidasas/genética , Imitación Molecular , Neovascularización Patológica/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacología , Neoplasias Cutáneas/irrigación sanguínea , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Células Tumorales Cultivadas , Neoplasias de la Úvea/irrigación sanguínea , Neoplasias de la Úvea/genética , Neoplasias de la Úvea/metabolismo , Neoplasias de la Úvea/patología , KalininaRESUMEN
Recently, the National Cancer Institute (NCI) established criteria for determination of microsatellite instability (MSI) in colorectal tumors. Although the best panel of markers for ovarian tumors is not known, we evaluated epithelial ovarian cancers for MSI based on the NCI recommendations. One hundred and nine ovarian tumors were analyzed for MSI by gel analysis of paired germ-line and tumor DNA. PCR amplification was performed using the panel of five microsatellite markers recommended by the NCI (BAT25, BAT26, D5S346, D2S123, and D17S250) and nine additional markers picked based on their genomic location (NME1, D10S197, D11S904, D13S175, DXS981, DXS6800, DXS6807, AR, and D3S1611). Tumors were characterized on the basis of: high-frequency MSI (MSI-H) if two or more of the five NCI markers showed instability or there was instability at 30% or more of all markers tested; or low-frequency MSI (MSI-L) if only one of the five NCI markers showed instability or <30% of all of the markers. All of the other tumors were considered microsatellite stable. On the basis of the NCI markers, 12 (11%) tumors demonstrated MSI-H, and 8 (7%) additional tumors had MSI-L. When all of the 14 markers were considered together, 13 (12%) tumors demonstrated MSI-H (based on 30% or more unstable loci), and 26 (24%) tumors had MSI-L. A single tumor identified to have MSI-H based upon all of the markers tested would have been classified as MSI-L based upon the NCI markers alone. Inclusion of an additional dinucleotide marker (NME1) to the NCI panel allowed detection of all of the tumors with MSI-H using only six markers. MSI-H occurs in approximately 12% of invasive ovarian tumors. For optimal detection of microsatellite instability in ovarian cancer, an additional marker (NME1) may be required, along with the five recommended by the NCI.
Asunto(s)
Repeticiones de Microsatélite/genética , Neoplasias Ováricas/genética , ADN de Neoplasias/sangre , ADN de Neoplasias/genética , Femenino , Marcadores Genéticos/genética , Humanos , Reacción en Cadena de la Polimerasa/normas , Reproducibilidad de los ResultadosRESUMEN
We recently have introduced the term vasculogenic mimicry to describe the unique ability of aggressive melanoma tumor cells to form tubular structures and patterned networks in three-dimensional culture, which "mimics" embryonic vasculogenic networks formed by differentiating endothelial cells. In the current study, we address the biological significance of several endothelial-associated molecules (revealed by microarray analysis) with respect to expression and function in highly aggressive and poorly aggressive human cutaneous melanoma cell lines (established from the same patient). In a comparative analysis, CD31 was not expressed by any of the melanoma cell lines, whereas TIE-1 (tyrosine kinase with Ig and epidermal growth factor homology domains-1) was strongly expressed in the highly aggressive tumor cells with a low level of expression in one of the poorly aggressive cell lines. Vascular endothelial (VE)-cadherin was exclusively expressed by highly aggressive melanoma cells and was undetectable in the poorly aggressive tumor cells, suggesting the possibility of a vasculogenic switch. Down-regulation of VE-cadherin expression in the aggressive melanoma cells abrogated their ability to form vasculogenic networks and directly tested the hypothesis that VE-cadherin is critical in melanoma vasculogenic mimicry. These results highlight the plasticity of aggressive melanoma cells and call into question their possible genetic reversion to an embryonic phenotype. This finding could pose a significant clinical challenge in targeting tumor cells that may masquerade as circulating endothelial cells or other embryonic-like stem cells.
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Cadherinas/biosíntesis , Melanoma/metabolismo , Antígenos CD , Cadherinas/genética , Diagnóstico Diferencial , Endotelio Vascular/patología , Endotelio Vascular/fisiología , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/genética , Melanoma/patología , Neovascularización Patológica/genética , Células Tumorales CultivadasRESUMEN
During development, the formation and remodeling of primary vascular networks occurs by vasculogenesis and angiogenesis. Recently, the term "vasculogenic mimicry" has been used by our laboratory and collaborators to reflect the embryonic-like ability of aggressive, but not nonaggressive, melanoma tumor cells to form a pattern of matrix-rich networks (containing channels) surrounding spheroids of tumor cells in three-dimensional culture, concomitant with their expression of vascular cell markers. Ovarian cancer is usually diagnosed as advanced stage disease in most patients when widespread metastases have already been established within the peritoneal cavity. In this study, we explored whether invasive ovarian carcinoma cells could engage in molecular vasculogenic mimicry reflected by their plasticity, compared with their normal cell counterparts. The data revealed that the invasive ovarian cancer cells, but not normal ovarian surface epithelial cells, formed patterned networks containing solid and hollow matrix channels when grown in three-dimensional cultures containing Matrigel or type I collagen, in the absence of endothelial cells or fibroblasts. Immunohistochemical analysis showed that matrix metalloproteinases (MMP)-1, -2, and -9, and MT1-MMP were discretely localized to these networks, and the formation of the networks was inhibited by treatment with MMP inhibitors. Furthermore, the RNase protection assay revealed the expression of multiple vascular cell-associated markers by the invasive ovarian cancer cells. In patient tumor sections from high-stage, high-grade ovarian cancers, 7 to 10% of channels containing red blood cells were lined by tumor cells. By comparison, all vascular areas in benign tumors and low-stage cancers were endothelial lined. These results may offer new insights and molecular markers for consideration in ovarian cancer diagnosis and treatment strategies based on molecular vascular mimicry by aggressive tumor cells.
Asunto(s)
Imitación Molecular , Neovascularización Patológica/fisiopatología , Neoplasias Ováricas/irrigación sanguínea , Neoplasias Ováricas/fisiopatología , Femenino , Humanos , Laminina/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Microscopía Electrónica de Rastreo , Invasividad Neoplásica , Neovascularización Patológica/patología , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patologíaRESUMEN
During embryogenesis, blood vessels are formed initially by the process of vasculogenesis, the in situ differentiation of mesenchymal cells into endothelial cells, which form a primitive, patterned vasculogenic network. This is followed by angiogenesis, the sprouting of new vessels from preexisting vasculature, to yield a more refined microcirculation. However, we and our collaborators have recently described a process termed "vasculogenic mimicry," which consists of the formation of patterned, tubular networks by aggressive melanoma tumor cells (in three-dimensional cultures in vitro), that mimics endothelial-formed vasculogenic networks and correlates with poor clinical prognosis in patients. Previous microarray analysis from our laboratory comparing the highly aggressive versus the poorly aggressive melanoma cells revealed a significant increased expression of tyrosine kinases associated with the aggressive melanoma phenotype. Because of the important role of protein tyrosine kinases in phosphorylating various signal transduction proteins that are critical for many cellular processes (e.g., cell adhesion, migration, and invasion), we examined whether protein tyrosine kinases are involved in melanoma vasculogenic mimicry. Immunofluorescence analysis of aggressive melanoma cells forming tubular networks in vitro showed that tyrosine phosphorylation activity colocalized specifically within areas of tubular network formation. A phosphotyrosine profile of the aggressive melanoma cells capable of forming tubular networks indicated differences in tyrosine phosphorylated proteins compared with the poorly aggressive melanoma cells (incapable of forming tubular networks). Most notably, we identified epithelial cell kinase (EphA2) as being one receptor tyrosine kinase expressed and phosphorylated exclusively in the aggressive metastatic melanoma cells. Furthermore, general inhibitors of protein tyrosine kinases hindered tube formation, and transient knockout of EphA2 abrogated the ability of tumor cells to form tubular structures. These results suggest that protein tyrosine kinases, particularly EphA2, are involved in the formation of tubular networks by aggressive melanoma tumor cells in vitro, which may represent a novel therapeutic target for further clinical investigation.
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Melanoma/enzimología , Melanoma/patología , Neovascularización Patológica/enzimología , Proteínas Tirosina Quinasas Receptoras/fisiología , Neoplasias de la Úvea/enzimología , Neoplasias de la Úvea/patología , Humanos , Melanoma/irrigación sanguínea , Neovascularización Patológica/patología , Oligonucleótidos Antisentido/farmacología , Fosforilación , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor EphA2 , Células Tumorales Cultivadas , Tirosina/metabolismo , Neoplasias de la Úvea/irrigación sanguíneaRESUMEN
To test the hypotheses that cyclic stretch of 1) cardiac myocytes produces factors that trigger angiogenic events in coronary microvascular endothelial cells (CMEC) and 2) CMEC enhances the expression of growth factors, cardiac myocytes and CMEC were subjected to cyclic stretch in a Flexercell Strain Unit. Vascular endothelial growth factor (VEGF) but not basic fibroblast growth factor mRNA and protein levels increased approximately twofold in myocytes after 1 h of stretch. CMEC DNA synthesis increased approximately twofold when conditioned medium from stretched myocytes or VEGF protein was added, and addition of VEGF neutralizing antibody blocked the increase. CMEC migration and tube formation increased with the addition of conditioned media but were markedly attenuated by VEGF neutralizing antibody. Myocyte transforming growth factor-beta [corrected] (TGF-beta) increased 2.5-fold after 1 h of stretch, and the addition of TGF-beta neutralizing antibodies inhibited the stretch-induced upregulation of VEGF. Stretch of CMEC increased VEGF mRNA in these cells (determined by Northern blot and RT-PCR) and increased the levels of VEGF protein (determined by ELISA analysis) in the conditioned media. Therefore, cyclic stretch of cardiac myocytes and CMEC appears to be an important primary stimulus for coronary angiogenesis through both paracrine and autocrine VEGF pathways. These data indicate that 1) CMEC DNA synthesis, migration, and tube formation are increased in response to VEGF secreted from stretched cardiac myocytes; 2) VEGF in CMEC subjected to stretch is upregulated and secreted; and 3) TGF-beta signaling may regulate VEGF expression in cardiac myocytes.
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Vasos Coronarios/fisiología , Factores de Crecimiento Endotelial/metabolismo , Endotelio Vascular/citología , Linfocinas/metabolismo , Neovascularización Fisiológica/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Anticuerpos/farmacología , Comunicación Autocrina/efectos de los fármacos , Comunicación Autocrina/fisiología , Northern Blotting , División Celular/efectos de los fármacos , División Celular/fisiología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Células Cultivadas , Vasos Coronarios/citología , Medios de Cultivo Condicionados/farmacología , Factores de Crecimiento Endotelial/genética , Factores de Crecimiento Endotelial/inmunología , Endotelio Vascular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Expresión Génica/fisiología , Linfocinas/genética , Linfocinas/inmunología , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/metabolismo , Miocardio/citología , Miocardio/metabolismo , Pruebas de Neutralización , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Estrés Mecánico , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/inmunología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Several studies have correlated escape from TGF-beta-mediated cell cycle arrest with the tumorigenic phenotype. Most often, this escape from growth control has been linked to dysfunctional TGF-beta receptors or defects in the TGF-beta-mediated SMAD signaling pathway. In this report, we found that highly metastatic 4T1 mammary carcinoma cells express functional TGF-beta receptors capable of initiating SMAD-mediated transcription, yet are not growth inhibited by TGF-beta1. We further observed that TGF-beta directly contributes to the metastatic behavior of this cell line. Exposure to TGF-beta caused 4T1 cells to undergo morphological changes associated with the metastatic phenotype and invade more readily through collagen coated matrices. Furthermore, expression of a dominant negative truncated type II receptor diminished TGF-beta signaling and significantly restricted the ability of 4T1 cells to establish distant metastases. Our results suggest that regardless of 4T1 resistance to TGF-beta-mediated growth inhibition, TGF-beta signaling is required for tumor invasion and metastases formation.
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Neoplasias Mamarias Animales/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Animales , Northern Blotting , Western Blotting , Ciclo Celular/efectos de los fármacos , División Celular , Movimiento Celular/efectos de los fármacos , Colágeno/metabolismo , Reactivos de Enlaces Cruzados/farmacología , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Citometría de Flujo , Genes Dominantes , Ratones , Ratones SCID , Microscopía Confocal , Invasividad Neoplásica , Fenotipo , Plásmidos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta1 , Células Tumorales CultivadasRESUMEN
Metastasis is the major cause of morbidity and death for cancer patients. A critical challenge to clinical and basic scientists is the development of improved prognostic methods to predict the metastatic aggressiveness of a patient's individual tumor and especially to control local invasion-a key step in the metastatic cascade. Before effective therapeutic regimens can be planned, we must invest more effort in understanding the pathogenesis of tumor cell dissemination in order to identify better targets for clinical intervention.
RESUMEN
OBJECTIVE: Despite increasing evidence regarding the significance of sex hormones in rheumatoid arthritis (RA), their etiopathological role and potential longterm effect on joint destruction remain unclear. We hypothesized that estrogen receptors (ER-alpha) are present in fibroblast-like synoviocytes, and 17beta-estradiol can modulate the production and activity of matrix degrading enzymes produced by these cells. Thus, depending on the endocrine balance, fibroblast-like synoviocyte activity can be suppressed or enhanced, leading to amelioration or exacerbation of the disease process, respectively. METHODS: By utilizing an in vitro cartilage invasion model, in combination with the molecular analyses of hormone receptors, matrix metalloproteinases (MMP) and their respective inhibitors, we investigated the effect of hormones (i.e., estrogen and progesterone) on fibroblast-like synoviocyte phenotypic changes, with particular emphasis on their functional interactions with cartilage. RESULTS: Our studies reveal the presence of functional ER-alpha in fibroblast-like synoviocytes. The findings indicate that estrogen exerts a stimulatory effect, while progesterone has an inhibitory effect on the expression of MMP, their tissue inhibitors (TIMP), and enzymatic activity of MMP produced by these cells. Furthermore, transfection of fibroblast-like synoviocytes with the ER-alpha gene resulted in the increased degradation and invasion of cartilage. CONCLUSION: We identified the presence of functional ER-alpha in fibroblast-like synoviocytes. This renders fibroblast-like synoviocytes as target cells for hormonal regulation. The regulatory effect of estrogen is partly targeted to the MMP and their respective inhibitors associated with fibroblast-like synoviocytes. Such studies provide a link between hormonal status and disease activity in RA and open new venues for future therapeutic intervention to combat this debilitating disease.
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Artritis Reumatoide/metabolismo , Artritis Reumatoide/fisiopatología , Estrógenos/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Progesterona/metabolismo , Membrana Sinovial/metabolismo , Membrana Sinovial/fisiopatología , Adolescente , Adulto , Anciano , Artritis Reumatoide/inducido químicamente , Cartílago/efectos de los fármacos , Cartílago/patología , Cartílago/fisiopatología , Células Cultivadas , Receptor alfa de Estrógeno , Estrógenos/efectos adversos , Femenino , Fibroblastos/citología , Humanos , Masculino , Metaloproteinasas de la Matriz/genética , Persona de Mediana Edad , Progesterona/efectos adversos , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Receptores de Estrógenos/efectos de los fármacos , Receptores de Estrógenos/metabolismo , Membrana Sinovial/efectos de los fármacos , Inhibidores Tisulares de Metaloproteinasas/genéticaRESUMEN
We previously identified a down-regulation in heterochromatin-associated protein 1 (HP1)Hsalpha expression in MDA-MB-231 breast carcinoma cells (highly invasive/metastatic) compared with MCF-7 cells (poorly invasive/nonmetastatic). In this study, we demonstrate that HP1Hsalpha, but not HP1Hsbeta or HP1Hsgamma, is down-regulated at the mRNA and protein levels in highly invasive/metastatic breast cancer cell lines. In agreement, little to no nuclear HP1Hsalpha staining was observed in these cell lines. In contrast, poorly invasive/nonmetastatic cell lines showed HP1Hsalpha localization to the nucleus and nuclear membrane. Transfection of MDA-MB-231 cells with a green fluorescent protein-HP1Hsalpha expression vector decreased their ability to invade a collagen IV/laminin/gelatin matrix compared with green fluorescent protein-transfected controls. Consistent with the cell culture studies, immunohistochemical analysis of HP1Hsalpha protein localization in distant metastatic tissues from breast cancer patients revealed a decrease in the staining intensity and percentage of cells expressing HP1Hsalpha in seven of nine distant metastatic lesions compared with normal mammary and primary tumors. These results demonstrate a role for HP1Hsalpha in breast cancer invasion and metastasis. Given the role of HP1 in transcriptional silencing in Drosophila, we propose a model in which HP1Hsalpha normally silences genes involved in breast cancer invasion and metastasis.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proteínas Cromosómicas no Histona/genética , Regulación Neoplásica de la Expresión Génica , Mama/citología , Mama/metabolismo , Núcleo Celular/patología , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/análisis , Femenino , Proteínas Fluorescentes Verdes , Humanos , Lactancia , Proteínas Luminiscentes/análisis , Invasividad Neoplásica , Metástasis de la Neoplasia/genética , Fenotipo , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/biosíntesis , Transcripción Genética , Transfección , Células Tumorales CultivadasRESUMEN
Maspin is a tumor suppressor whose expression is lost in many advanced breast cancers. Maspin has been shown to inhibit cell motility, invasion and metastasis; however, its precise role in normal mammary epithelium remains to be elucidated. Although expression of maspin mRNA is low or absent in most human breast cancer cells, the maspin gene is rarely re-arranged or deleted. We hypothesized that aberrant cytosine methylation and chromatin condensation of the maspin promoter participates in the silencing of maspin expression during neoplastic progression. To test this hypothesis, we compared cultured normal human mammary epithelial cells (HMECs) to 9 cultured human breast cancer cell lines. HMECs expressed maspin mRNA and displayed a completely non-methylated maspin gene promoter with an open chromatin structure. In contrast, 7 of 9 breast cancer cell lines had no detectable maspin expression and 6 of these 7 maspin-negative breast cancer cell lines also displayed an aberrant pattern of cytosine methylation of the maspin promoter. Interestingly, the maspin promoter was completely methylated in maspin-negative normal peripheral blood lymphocytes. This indicates that the maspin promoter is not a functional CpG island and that cytosine methylation of this region may contribute to normal tissue-restricted gene expression. Chromatin accessibility studies with MCF-7 cells, which lack maspin expression and have a methylated maspin promoter, showed a closed chromatin structure compared with HMECs. Moreover, maspin gene expression could be re-activated in MCF-7 cells by treatment with 5-aza-2;-deoxycytidine, a DNA demethylating agent. Thus, aberrant cytosine methylation and heterochromatinization of the maspin promoter may silence maspin gene expression, thereby contributing to the progression of human mammary cancer.