RESUMEN
PURPOSE: Prostate specific antigen has limited performance in detecting prostate cancer. The transcription factor GATA2 is expressed in aggressive prostate cancer. We analyzed the predictive value of urine extracellular vesicle GATA2 mRNA alone and in combination with a multigene panel to improve detection of prostate cancer and high risk disease. MATERIALS AND METHODS: GATA2 mRNA was analyzed in matched extracellular vesicles isolated from urines before and after prostatectomy (16) and paired urine and tissue prostatectomy samples (19). Extracellular vesicle GATA2 mRNA performance to distinguish prostate cancer and high grade disease was tested in training (52) and validation (165) cohorts. The predictive value of a multigene score including GATA2, PCA3 and TMPRSS2-ERG (GAPT-E) was tested in both cohorts. RESULTS: Confirming its prostate origin, urine extracellular vesicle GATA2 mRNA levels decreased significantly after prostatectomy and correlated with prostate cancer tissue GATA2 mRNA levels. In the training and validation cohort GATA2 discriminated prostate cancer (AUC 0.74 and 0.66) and high grade disease (AUC 0.78 and 0.65), respectively. Notably, the GAPT-E score improved discrimination of prostate cancer (AUC 0.84 and 0.72) and high grade cancer (AUC 0.85 and 0.71) in both cohorts when compared with each biomarker alone and PT-E (PCA3 and TMPRSS2-ERG). A GAPT-E score for high grade prostate cancer would avoid 92.1% of unnecessary prostate biopsies, compared to 61.9% when a PT-E score is used. CONCLUSIONS: Urine extracellular vesicle GATA2 mRNA analysis improves the detection of high risk prostate cancer and may reduce the number of unnecessary biopsies.
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Vesículas Extracelulares/química , Factor de Transcripción GATA2/genética , Próstata/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , ARN Mensajero/análisis , Anciano , Anciano de 80 o más Años , Biopsia , Humanos , Masculino , Persona de Mediana Edad , Estudios ProspectivosRESUMEN
BACKGROUND: The molecular chaperone heat shock protein-90 (Hsp90) is a promising cancer drug target, but current Hsp90-based therapy has so far shown limited activity in the clinic. METHODS: We tested the efficacy of a novel mitochondrial-targeted, small-molecule Hsp90 inhibitor, Gamitrinib (GA mitochondrial matrix inhibitor), in the Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model. The TRAMP mice receiving 3-week or 5-week systemic treatment with Gamitrinib were evaluated for localised or metastatic prostate cancer, prostatic intraepithelial neoplasia (PIN) or localised inflammation using magnetic resonance imaging, histology and immunohistochemistry. Treatment safety was assessed histologically in organs collected at the end of treatment. The effect of Gamitrinib on mitochondrial dysfunction was studied in RM1 cells isolated from TRAMP tumours. RESULTS: Systemic administration of Gamitrinib to TRAMP mice inhibited the formation of localised prostate tumours of neuroendocrine or adenocarcinoma origin, as well as metastatic prostate cancer to abdominal lymph nodes and liver. The Gamitrinib treatment had no effect on PIN or prostatic inflammation, and caused no significant animal weight loss or organ toxicity. Mechanistically, Gamitrinib triggered acute mitochondrial dysfunction in RM1 cells, with loss of organelle inner membrane potential and release of cytochrome-c in the cytosol. CONCLUSIONS: The Gamitrinib has pre-clinical activity and favourable tolerability in a genetic model of localised and metastatic prostate cancer in immunocompetent mice. Selective targeting of mitochondrial Hsp90 could provide novel molecular therapy for patients with advanced prostate cancer.
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Adenocarcinoma/prevención & control , Antineoplásicos/uso terapéutico , Proliferación Celular/efectos de los fármacos , Guanidinas/uso terapéutico , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Lactamas Macrocíclicas/uso terapéutico , Neoplasias de la Próstata/prevención & control , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Antineoplásicos/farmacología , Células Cultivadas , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Evaluación Preclínica de Medicamentos , Femenino , Predisposición Genética a la Enfermedad , Guanidinas/farmacología , Proteínas HSP90 de Choque Térmico/metabolismo , Lactamas Macrocíclicas/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Terapia Molecular Dirigida/métodos , Metástasis de la Neoplasia , Neoplasia Intraepitelial Prostática/genética , Neoplasia Intraepitelial Prostática/patología , Neoplasia Intraepitelial Prostática/prevención & control , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patologíaRESUMEN
Human umbilical vein endothelial cells attach and spread on laminin-coated substrates. Affinity chromatography was used to identify the attachment receptor. Fractionation of extracts from surface-iodinated endothelial cells on human laminin-Sepharose yielded a heterodimeric complex, the subunits of which migrated with molecular sizes corresponding to 160/120 kD and 160/140 kD under nonreducing and reducing conditions, respectively. The purified receptor bound to laminin and slightly less to fibronectin and type IV collagen in a radioreceptor assay. This endothelial cell laminin receptor was classified as an alpha 2 beta 1 integrin because monoclonal and polyclonal antibodies directed against the alpha 2 and bet 1 subunits immunoprecipitated the receptor. Cytofluorometric analysis and immunoprecipitation showed that the alpha 2 subunit is an abundant integrin alpha subunit in the endothelial cells and that the alpha subunits associated with laminin binding in other types of cells are expressed in these cells only at low levels. The alpha 2 beta 1 integrin appears to be a major receptor for laminin in the endothelial cells, because an anti-alpha 2 monoclonal antibody inhibited the attachment of the endothelial cells to human laminin. These results define a new role for the alpha 2 subunit in laminin binding and suggest that the ligand specificity of the alpha 2 beta 1 integrin, which is known as a collagen receptor in other types of cells, can be modulated by cell type-specific factors to include laminin binding.
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Endotelio Vascular/citología , Integrinas/inmunología , Laminina/fisiología , Receptores Inmunológicos/fisiología , Animales , Anticuerpos Monoclonales , Adhesión Celular , Cromatografía de Afinidad , Colágeno , Endotelio Vascular/inmunología , Fibronectinas , Citometría de Flujo , Humanos , Integrinas/aislamiento & purificación , Cinética , Ratones , Receptores de LamininaRESUMEN
Human umbilical vein endothelial cells (ECs) have been shown to attach to a substratum of fibrinogen (fg). Later, ECs undergo spreading, organization of thick microfilament bundles of the stress fiber type, and formation of focal contacts (adhesion plaques) that correspond to accumulation of vinculin at the cytoplasmic aspect of the ventral membrane. The rate of attachment to fg and the type of spreading is virtually identical to that obtained on substrata coated with fibronectin (FN). Antibodies to fg, but not to FN, prevent EC adhesion to fg; conversely, antibodies to FN, but not to fg, prevent adhesion of ECs to a FN-coated substratum. The removal of residual FN contamination from fg preparations by means of DEAE-cellulose chromatography does not result in any difference in EC adhesion on fg. Moreover, pretreatment of cells with inhibitors of synthesis and release of proteins does not impair their adhesion capacity on an fg-coated substratum. In contrast, human arterial smooth muscle cells do not adhere and spread on fg substrata but do so on FN. The synthetic peptides (Gly-Arg-Gly-Asp[GRGD] and Gly-Arg-Gly-Asp-Ser-Pro[GRGDSP]) containing the tripeptide Arg-Gly-Asp (RGD), originally found to be responsible for the cell binding activity of FN, have been found to inhibit EC spreading and the redistribution of their cytoskeleton, including the formation of stress fibers and the localization of vinculin either on fg or on FN. Conversely, the synthetic peptide Arg-Gly-Gly (RGG) was completely uneffective in inhibiting the adhesion and the sequence of events leading to spreading and cytoskeletal organization. These results indicate that ECs, but not smooth muscle cells, specifically adhere and spread on an fg substratum and this occurs by recognition mechanisms similar to those reported for FN.
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Citoesqueleto de Actina/ultraestructura , Adhesión Celular , Citoesqueleto/ultraestructura , Endotelio/citología , Fibrinógeno/fisiología , Aprotinina/farmacología , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Endotelio/efectos de los fármacos , Endotelio/ultraestructura , Femenino , Hirudinas/farmacología , Humanos , Biosíntesis de Proteínas , Venas Umbilicales/citología , Venas Umbilicales/ultraestructuraRESUMEN
Integrins regulate cell growth, differentiation, and behavior in many systems. Integrin beta 1C (beta 1S) is an alternatively spliced variant of integrin beta 1 with a specific cytoplasmic domain and is expressed in several human tissues. Human beta 1c transiently expressed in mouse 10T1/2 fibroblasts showed a diffuse pattern of cell surface staining, whereas beta1 localized to focal adhesions. Moderate concentrations of beta 1C had no effect on actin stress fibers or focal adhesions, but markedly inhibited DNA synthesis. Inhibition by beta 1C mapped to the late G1 phase of the cell cycle, near the G1-S boundary. Thus, alternative splicing of beta1 results in transmission of distinct signals that may regulate growth in vivo.
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Empalme Alternativo , Fase G1 , Integrinas/fisiología , Secuencia de Aminoácidos , Animales , Adhesión Celular , División Celular , Línea Celular , Tamaño de la Célula , ADN/biosíntesis , Humanos , Integrina beta1 , Integrinas/química , Integrinas/genética , Ligandos , Ratones , Datos de Secuencia Molecular , Eliminación de Secuencia , Transducción de Señal , TransfecciónRESUMEN
Survivin is a dual regulator of cell proliferation and cell viability overexpressed in most human tumors. Although strategies to lower survivin levels have been pursued for rational cancer therapy, the molecular circuitries controlling survivin expression in tumors have not been completely elucidated. Here, we show that stimulation with insulin-like growth factor-1 (IGF-1) results in increased survivin expression in prostate cancer cells. This response is independent of de novo gene transcription, changes in mRNA expression or modifications of survivin protein stability. Instead, IGF-1 induced persistence and translation of a pool of survivin mRNA, in a reaction abolished by the mTOR (mammalian target of rapamycin) inhibitor, rapamycin. Forced expression of the mTOR target p70S6K1 reproduced the increase in survivin expression in prostate cancer cells, whereas acute ablation of endogenous p70S6K1 by small interfering RNA downregulated survivin levels. Rapamycin, alone or in combination with suboptimal concentrations of taxol reduced survivin protein levels, and decreased viability of prostate cancer cells. Therefore, IGF-1/mTOR signaling elevates survivin in prostate cancer cells via rapid changes in mRNA translation. Antagonists of this pathway may be beneficial to lower an antiapoptotic threshold maintained by survivin in prostate cancer.
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Regulación Neoplásica de la Expresión Génica , Factor I del Crecimiento Similar a la Insulina/farmacología , Proteínas Asociadas a Microtúbulos/genética , Proteínas de Neoplasias/genética , Neoplasias de la Próstata/metabolismo , Proteínas Quinasas/metabolismo , Transducción de Señal , Animales , Apoptosis , Ciclo Celular , Proliferación Celular , Regulación hacia Abajo , Células HeLa , Humanos , Proteínas Inhibidoras de la Apoptosis , Masculino , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Regiones Promotoras Genéticas , Neoplasias de la Próstata/genética , Proteínas Quinasas/genética , Estabilidad del ARN , ARN Interferente Pequeño/farmacología , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/fisiología , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Sirolimus , Survivin , Serina-Treonina Quinasas TOR , Transcripción Genética , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Glomerular accumulation of extracellular matrix is a prominent feature of progressive glomerulonephritis. Previously, we have shown that transforming growth factor-beta (TGF-beta) is unique among growth factors in regulating the production of the proteoglycans biglycan and decorin by glomerular mesangial cells in vitro. We now provide evidence of an elevated expression of TGF-beta, proteoglycans, and fibronectin in glomerulonephritis induced in rats by injection of anti-thymocyte serum (ATS). Glomeruli were cultured from rat kidneys at 1, 4, 7, 14, and 28 d after ATS administration. Increased proteoglycan synthesis was detected beginning on day 4, which peaked at a 4,900% increase compared with control on day 7, and returned toward control levels by day 28. The increased proteoglycan synthesis by cultured nephritic glomeruli, as well as that of fibronectin, were greatly reduced by addition of antiserum raised against a synthetic peptide from TGF-beta. Conditioned media from ATS glomerular cultures, when added to normal cultured mesangial cells, induced elevated proteoglycan synthesis that also peaked on day 7 and that mimicked the response to added exogenous TGF-beta. The stimulatory activity of the conditioned media was blocked by addition of TGF-beta antiserum. Prior addition of the immunizing peptide to the antiserum abolished the blocking effect. The main induced proteoglycans were identified as biglycan and decorin by immunoprecipitation with antiserum made against synthetic peptides from the proteoglycan core proteins. Glomerular histology showed mesangial matrix expansion in a time course that roughly paralleled both the elevated proteoglycan synthesis by the ATS glomeruli and the ability of the conditioned media from these glomeruli to induce proteoglycan synthesis. At the same time there was an increased expression of TGF-beta mRNA and TGF-beta protein in the glomeruli. These results suggest a central role for TGF-beta in the accumulation of pathological extracellular matrix in glomerulonephritis.
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Glomerulonefritis/metabolismo , Proteoglicanos/biosíntesis , Factores de Crecimiento Transformadores/metabolismo , Animales , Suero Antilinfocítico/farmacología , Northern Blotting , Técnicas de Cultivo , Matriz Extracelular/metabolismo , Fibronectinas/biosíntesis , Técnica del Anticuerpo Fluorescente , Mesangio Glomerular/metabolismo , Glomerulonefritis/patología , Microscopía Electrónica , ARN Mensajero/genética , Ratas , Factores de Crecimiento Transformadores/genéticaRESUMEN
The role of thrombospondin, a multifunctional matrix glycoprotein, in platelet adhesion is controversial: both adhesive and antiadhesive properties have been attributed to this molecule. Because shear flow has a significant influence on platelet adhesion, we have assessed thrombospondin-platelet interactions both under static and flow conditions. The capacity of thrombospondin to support platelet adhesion depended upon its conformation. In a Ca(2+)-depleted conformation, such as in citrated plasma, thrombospondin was nonadhesive or antiadhesive as it inhibited platelet adhesion to fibrinogen, fibronectin, laminin, and von Willebrand factor by 30-70%. In a Ca(2+)-replete conformation, however, thrombospondin effectively supported platelet adhesion. Shear rate influenced this adhesion; percent surface coverage on thrombospondin increased from 5.4 +/- 0.3 at 0 s-1 to 41.5 +/- 6.7 at 1,600 s-1. In contrast to the extensive platelet spreading observed on fibronectin at all shear rates, platelet spreading on thrombospondin occurred only sporadically and at high shear rates. GPIa-IIa, GPIIb-IIIa, GPIV, and the vitronectin receptor, which are all proposed platelet receptors for thrombospondin, were not solely responsible for platelet adhesion to thrombospondin. These results suggest that thrombospondin may play a dual role in adhesive processes in vivo: (a) it may function in conjunction with other adhesive proteins to maintain optimal platelet adhesion at various shear rates; and (b) it may serve as a modulator of cellular adhesive functions under specific microenvironmental conditions.
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Plaquetas/metabolismo , Adhesividad Plaquetaria , Glicoproteínas de Membrana Plaquetaria/metabolismo , Antígenos CD36 , Cationes Bivalentes , Adhesión Celular , Endotelio Vascular/citología , Proteínas de la Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Humanos , Técnicas In Vitro , Glicoproteínas de Membrana Plaquetaria/ultraestructura , Conformación Proteica , Receptores de Citoadhesina/metabolismo , Reología , TrombospondinasRESUMEN
Integrins are a large family of transmembrane receptors that, in addition to mediating cell adhesion, modulate cell proliferation. The beta1C integrin is an alternatively spliced variant of the beta1 subfamily that contains a unique 48-amino acid sequence in its cytoplasmic domain. We have shown previously that in vitro beta1C inhibits cell proliferation and that in vivo beta1C is expressed in nonproliferative, differentiated epithelium and is selectively downregulated in prostatic adenocarcinoma. Here we show, by immunohistochemistry and immunoblotting analysis, that beta1C is coexpressed in human prostate epithelial cells with the cell-cycle inhibitor p27(kip1), the loss of which correlates with poor prognosis in prostate cancer. In the 37 specimens analyzed, beta1C and p27(kip1) are concurrently expressed in 93% of benign and 84%-91% of tumor prostate cells. Forced expression of beta1C in vitro is accompanied by an increase in p27(kip1) levels, by inhibition of cyclin A-dependent kinase activity, and by increased association of p27(kip1) with cyclin A. beta1C inhibitory effect on cell proliferation is completely prevented by p27(kip1) antisense, but not mismatch oligonucleotides. beta1C expression does not affect either cyclin A or E levels, or cyclin E-associated kinase activity, nor the mitogen-activated protein (MAP) kinase pathway. These findings show a unique mechanism of cell growth inhibition by integrins and point to beta1C as an upstream regulator of p27(kip1) expression and, therefore, a potential target for tumor suppression in prostate cancer.
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Adenocarcinoma/metabolismo , Proteínas de Ciclo Celular , Integrina beta1/biosíntesis , Proteínas Asociadas a Microtúbulos/biosíntesis , Neoplasias de la Próstata/metabolismo , Proteínas Supresoras de Tumor , Adenocarcinoma/genética , Adenocarcinoma/patología , División Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Regulación hacia Abajo , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Humanos , Integrina beta1/genética , Masculino , Proteínas Asociadas a Microtúbulos/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Células Tumorales CultivadasRESUMEN
It has been suggested that fibrinogen (fg) or its physiological derivatives influence the motility and growth of endothelial cells (ECs), but direct support for this concept is still lacking. In the present study, the capacity of fg to interact with ECs and induce the migration of ECs was examined. The capacity of fg to induce EC migration was studied by means of a modification of the Boyden chamber technique. fg in the lower compartment of the chamber caused a time- and concentration-dependent migration of ECs across filters. fg present in equal concentrations above and below the filter increased EC migration, but the maximal effect invariably occurred in the presence of a gradient between the lower and the upper compartments. Trypsin or plasmin digestion of fg and preincubation of fg with Fab fragments from specific antibody completely abolished fg-induced EC migration. Dialysis of fg to eliminate small peptides that might contaminate the preparation did not modify fg-induced migration. Plasma obtained from healthy donors induced EC migration, but plasma from an afibrinogenemic patient was completely ineffective. The addition of purified fg to afibrinogenemic plasma restored plasma-induced EC migration. Plasmin degradation fragments D and E, of 100,000 and 50,000 mol wt, respectively, did not induce EC migration. However, fragment E caused dose-related inhibition of fg-induced EC migration Direct interaction of highly purified radioiodinated human fg with cultured human and bovine Ecs was observed. The binding was time dependent and plateaued at 10 min. Nonlabeled fg in a large molar excess inhibited the interaction, but unrelated proteins, including fibronectin, ovalbumin, and myoglobin, did not. Monospecific Fab fragments directed to fg inhibited binding by 38% at a 50 to 1 molar ratio whereas nonimmune Fab caused only 2% inhibition at a similar concentration. The binding of 125I-fg with ECs was saturable, and an apparent dissociation constant of 0.23 x 10(-6) M was estimated from binding isotherms. After 30 min of incubation the interaction between 125I-fg and the cells was completely reversible and displaceable by a large molar excess of unlabeled fg. Autoradiography of the display of EC-bound 125I on polyacrylamide gel showed the constitutive B beta- and gamma-chains of the fg molecule, with a partial loss of the A alpha-chain. Purified fragment E and E were tested for their capacity to inhibit fg binding. At a 1 to 400 125I-fg-to-fragment molar ratio, fragment E, which also inhibited migration, competed for binding by 44%, but fragment D was completely ineffective. These data show that fg may specifically associate with ECs and induce migration of these cells; it also appears that the structural requirement of this activity is located in the N-terminal part of the molecule.
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Endotelio/citología , Fibrinógeno/fisiología , Movimiento Celular/efectos de los fármacos , Quimiotaxis , Electroforesis en Gel de Poliacrilamida , Endotelio/fisiología , Humanos , Cinética , Unión Proteica , Conformación ProteicaRESUMEN
The integrin cytoplasmic domain modulates cell proliferation, adhesion, migration, and intracellular signaling. The beta(1) integrin subunits, beta(1C) and beta(1A), that contain variant cytoplasmic domains differentially affect cell proliferation; beta(1C) inhibits proliferation, whereas beta(1A) promotes it. We investigated the ability of beta(1C) and beta(1A) to modulate integrin-mediated signaling events that affect cell proliferation and survival in Chinese hamster ovary stable cell lines expressing either human beta(1C) or human beta(1A). The different cytodomains of either beta(1C) or beta(1A) did not affect either association with the endogenous alpha(2), alpha(V), and alpha(5) subunits or cell adhesion to fibronectin or TS2/16, a mAb to human beta(1). Upon engagement of endogenous and exogenous integrins by fibronectin, cells expressing beta(1C) showed significantly inhibited extracellular signal-regulated kinase (ERK) 2 activation compared with beta(1A) stable cell lines. In contrast, focal adhesion kinase phosphorylation and Protein Kinase B/AKT activity were not affected. Selective engagement of the exogenously expressed beta(1C) by TS2/16 led to stimulation of Protein Kinase B/AKT phosphorylation but not of ERK2 activation; in contrast, beta(1A) engagement induced activation of both proteins. We show that Ras activation was strongly reduced in beta(1C) stable cell lines in response to fibronectin adhesion and that expression of constitutively active Ras, Ras 61 (L), rescued beta(1C)-mediated down-regulation of ERK2 activation. Inhibition of cell proliferation in beta(1C) stable cell lines was attributable to an inhibitory effect of beta(1C) on the Ras/MAP kinase pathway because expression of activated MAPK kinase rescued beta(1C) antiproliferative effect. These findings show that the beta(1C) variant, by means of a unique signaling mechanism, selectively inhibits the MAP kinase pathway by preventing Ras activation without affecting either survival signals stimulated by integrins or cellular interactions with the extracellular matrix. These findings highlight a role for beta(1)-specific cytodomain sequences in maintaining an intracellular balance of proliferation and survival signals.
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Integrina beta1/fisiología , Integrinas/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Proteínas ras/metabolismo , Animales , Células CHO , Adhesión Celular , Cricetinae , Citoplasma/metabolismo , Activación Enzimática , Fibronectinas/metabolismo , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Expresión Génica , Humanos , Integrina beta1/genética , Integrina beta1/metabolismo , Integrinas/genética , Integrinas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt , ConejosRESUMEN
The highly invasive human prostate cancer PC3 cell line was found to express the alpha(v)beta3 integrin; in contrast, the noninvasive LNCaP prostate cancer cell line did not express alpha(v)beta3. PC3 cells adhered to and migrated on vitronectin (VN), an alpha(v)beta3 ligand expressed in mature bone where prostate cancer cells preferentially metastasize. In contrast, LNCaP cells did not adhere to or migrate on VN. Analysis of primary human prostate cancer cells isolated from 16 surgical specimens, showed that these cells expressed alpha(v)beta3, whereas normal prostate epithelial cells did not. In addition, only primary prostate cancer cells adhered to and migrated on VN. The role of alpha(v)beta3 in mediating prostate epithelial cell migration was confirmed using LNCaP cell transfectants expressing beta3 (beta3-LNCaP). Exogenous expression of alpha(v)beta3 induced LNCaP cells to adhere to and migrate on VN. In response to alpha(v)beta3 engagement, increased tyrosine phosphorylation of focal adhesion kinase (FAK), a signaling molecule activated by integrins and able to modulate cell migration, was detected. Transfection of FAK-related nonkinase, known to compete with FAK for its correct localization and phosphorylation, caused inhibition of beta3-LNCaP cell migration, specifically on VN. These data indicate that de novo expression of alpha(v)beta3 integrin in prostate cancer cells generates a migratory phenotype that is modulated by a FAK signaling pathway. This study points to alpha(v)beta3 as potential target in prostate cancer cell invasion and metastasis.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Neoplasias de la Próstata/patología , Proteínas Tirosina Quinasas/fisiología , Receptores de Vitronectina/fisiología , Movimiento Celular , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Humanos , Masculino , Fosforilación , Células Tumorales Cultivadas , Vitronectina/fisiologíaRESUMEN
A large number of studies have underscored a major role for the integrin alpha beta cytoplasmic domains in the modulation of cell functions. Cytoplasmic domain variants of the beta 1, beta 3, beta 4, alpha 3, alpha 6 and alpha 7 subunits have been described. These molecules are generated by alternative splicing events and are expressed in a cell- or tissue-type specific manner. Some of these variants (beta 1C, beta 1D, alpha 6A and alpha 7A) are predominantly expressed upon differentiation and have been shown to be regulated during development. The studies on the structure-function relationship of the integrin variant subunits, published between 1989 and now, will be reviewed here for the first time. The results demonstrate that differences in the cytoplasmic domain do not affect either the alpha beta heterodimer formation or the ligand specificity. Instead, alternatively spliced integrin cytoplasmic domains appear to be essential modulators of receptor localization, cell proliferation and migration, as well as phosphorylation of signaling molecules. These observations lead to the current hypothesis that cell-type specific regulation of alternatively spliced integrin cytoplasmic domains may provide a highly specialized mechanism to control cell growth and intracellular signaling pathways.
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Empalme Alternativo , Integrinas/genética , Integrinas/fisiología , Transducción de Señal , Relación Estructura-Actividad , Secuencia de Aminoácidos , Animales , Citoplasma/fisiología , Humanos , Integrinas/metabolismo , Ligandos , Datos de Secuencia Molecular , Especificidad por SustratoRESUMEN
The adherence of human 3H-adenine-labeled platelets to rat subendothelium was quantitated using a rotating probe device. Platelet adhesion increased in relation to the rotation time, reaching a plateau value in about 4-6 min without any further increase. A non-linear fitting analysis of experimental data allowed calculations of initial rate and plateau value of platelet adhesion. Increasing the shear rates (from 35 to 150 sec-1) or the hematocrit (from 10% to 40%), both the adhesion rate and the plateau value were increased. When different platelet concentrations were used the adhesion rate and the plateau calculated increased with platelet concentration. Different plateau values were obtained in the experimental conditions considered. This suggests that the plateau was not reached for the complete occupation of the subendothelial surface by the adherent platelets. Experiments using two different vessels rotated in the same platelet suspension or, viceversa, the same vessel rotated successively in two fresh platelet suspensions, showed that the plateau was not determined by reduced platelet reactivity. Rotating the same vessel first in radiolabeled platelets, until the plateau was reached, and secondly in non labeled platelets, or viceversa, showed that the plateau was indeed a dynamic condition where the number of platelets adhering and detaching reached equilibrium. These observations suggest that the platelet adhesion to subendothelium is the final equilibrium of two platelet fluxes, one adhering to the surface and another detaching from the surface.
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Adhesividad Plaquetaria , Animales , Aorta Torácica/citología , Aorta Torácica/fisiología , Fenómenos Biomecánicos , Viscosidad Sanguínea , Endotelio/citología , Endotelio/fisiología , Hematócrito , Humanos , Técnicas In Vitro , Recuento de Plaquetas , Ratas , Flujo Sanguíneo RegionalRESUMEN
The differential expression of laminin receptors has been shown to modulate the invasive capability of malignant cells. We have investigated the reactivity of human pulmonary squamous carcinomas (SSC, n = 20) and adenocarcinomas (ADC, n = 20) with monoclonal antibodies to the cytoplasmic and extracellular domains of the integrin subunits alpha3 and alpha6. Integrins containing these subunits are laminin receptors. Monoclonal antibodies to beta1 and beta4 subunits, the beta1C splice variant of beta1, as well as to Ki-67, were also used. Reverse transcription polymerase chain reaction (PCR) single-strand conformational polymorphism analysis was done to detect possible mutations in the cytodomains. All carcinomas expressed alpha3 extensively; alpha3 expression predominated (40 of 40) over alpha6 (25 of 40). In all alpha6-positive carcinomas, alpha6A was expressed, whereas alpha6B was weakly expressed only in some of them. No mutations of the intracytoplasmic domain A of alpha3 and of the A or B intracytoplasmic domains of alpha6 were shown. Notably, in normal bronchial epithelium, alpha6 colocalized with beta4, whereas in the tumors, alpha6A frequently overlapped with beta1 in a circumferential pattern; alpha6beta1 coexpression was also shown by coprecipitation experiments. Strong and extensive beta4 reactions were invariably polarized at the cell/stroma interface in SCC and ADC. An inverse correlation was found between the expression of beta1C and Ki-67. The prevalence of alpha6A in pulmonary SCC and ADC is in contrast with previous results in colonic ADC in which alpha6B prevails, and alpha6 predominates over alpha3. The absence of mutations of the cytodomains suggests that the integrin subunits of these carcinomas are potentially active. Predominance of alpha3 over alpha6 and of alpha6A over alpha6B may contribute to explain the aggressive and metastatic behavior of lung carcinomas.
Asunto(s)
Adenocarcinoma/metabolismo , Antígenos CD/metabolismo , Carcinoma de Células Escamosas/metabolismo , Integrinas/metabolismo , Neoplasias Pulmonares/metabolismo , Receptores de Laminina/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patología , Antígenos CD/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Humanos , Inmunohistoquímica , Integrina alfa3 , Integrina alfa6 , Integrina beta1/metabolismo , Integrina beta4 , Integrinas/genética , Antígeno Ki-67/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Mutación , Polimorfismo Conformacional Retorcido-Simple , Receptores de Laminina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The action of AD6 as an anti-thrombotic agent was studied in a model of coronary artery thrombosis and on platelet aggregation in the dog. AD6 (10-100 microM) in vitro inhibited aggregation induced by ADP, epinephrine, collagen and PAF (platelet aggregating factor) used at their threshold concentration for maximal aggregation. Arterial thrombosis was induced in a coronary vessel by critically reducing (about 70%) the vessel lumen. Thrombus formation was estimated by measuring coronary flow in the stenosed vessel. Using this procedure on the left descending coronary artery (LAD), we obtained reproducible blood flow changes in 18 dogs. AD6 was given i.v. at three different doses. At 0.25 mg/kg two out of four dogs showed decreased thrombus formation at the stenosis site. Seven out of eleven dogs treated with 0.5 mg/kg and two out of three treated with 1.5 mg/kg showed decreased thrombus formation. Major decreases in coronary resistance, evaluated by measuring blood flow in the unstenosed left circumflex artery (LCX), were evident only after the highest dose. We conclude that AD6 has an inhibitory action on dog platelet aggregation and reduces thrombus formation in a stenosed coronary vessel.
Asunto(s)
Cromonar/farmacología , Enfermedad Coronaria/tratamiento farmacológico , Cumarinas/farmacología , Agregación Plaquetaria/efectos de los fármacos , 6-Cetoprostaglandina F1 alfa/sangre , Adenosina Difosfato/farmacología , Animales , Cromonar/análogos & derivados , Cromonar/uso terapéutico , Colágeno/farmacología , Modelos Animales de Enfermedad , Perros , Epinefrina/farmacología , Masculino , Factor de Activación Plaquetaria/farmacología , Flujo Sanguíneo Regional/efectos de los fármacos , Tromboxano B2/sangreRESUMEN
A rotating probe device was used to quantitate platelet adhesion to rat aorta subendothelial surface. Platelet adhesion increased in relation to the time of exposure to the subendothelial surface, the shear rate, the percentage of hematocrit and the number of platelets in the suspending media. Human umbilical arteries or collagen-coated glass can be used as adhesion surfaces with results similar to those obtained with rat aorta subendothelium. Platelet-rich plasma or washed platelets can be used in this system with comparable results. The test is sensitive to a series of antiaggregating agents and could be used in clinical studies of platelet adhesion defect.
Asunto(s)
Vasos Sanguíneos/citología , Adhesividad Plaquetaria , Animales , Endotelio , Hematócrito , Humanos , Indometacina/farmacología , Agregación Plaquetaria/efectos de los fármacos , RatasRESUMEN
Runx2, a bone-specific transcriptional regulator, is abnormally expressed in highly metastatic prostate cancer cells. Here, we identified the functional activities of Runx2 in facilitating tumor growth and osteolysis. Our studies show that negligible Runx2 is found in normal prostate epithelial and non-metastatic LNCaP prostate cancer cells. In the intra-tibial metastasis model, high Runx2 levels are associated with development of large tumors, increased expression of metastasis-related genes (MMP9, MMP13, VEGF, Osteopontin) and secreted bone-resorbing factors (PTHrP, IL8) promoting osteolytic disease. Runx2 siRNA treatment of PC3 cells decreased cell migration and invasion through Matrigel in vitro, and in vivo shRunx2 expression in PC3 cells blocked their ability to survive in the bone microenvironment. Mechanisms of Runx2 function were identified in co-culture studies showing that PC3 cells promote osteoclastogenesis and inhibit osteoblast activity. The clinical significance of these findings is supported by human tissue microarray studies of prostate tumors at stages of cancer progression, in which Runx2 is expressed in both adenocarcinomas and metastatic tumors. Together these findings indicate that Runx2 is a key regulator of events associated with prostate cancer metastatic bone disease.