RESUMEN
Smad4/DPC4 (deleted in pancreatic carcinoma, locus 4) is a tumor suppressor gene lost at high frequency in cancers of the pancreas and other gastrointestinal organs. Smad4 encodes a key intracellular messenger in the transforming growth factor beta (TGF-beta) signaling cascade. TGF-beta is a potent inhibitor of the growth of epithelial cells; thus, it has been assumed that loss of Smad4 during tumor progression relieves this inhibition. Herein, we show that restoration of Smad4 to human pancreatic carcinoma cells suppressed tumor formation in vivo, yet it did not restore sensitivity to TGF-beta. Rather, Smad4 restoration influenced angiogenesis, decreasing expression of vascular endothelial growth factor and increasing expression of thrombospondin-1. In contrast to the parental cell line and to control transfectants that produced rapidly growing tumors in vivo, Smad4 revertants induced small nonprogressive tumors with reduced vascular density. These data define the control of an angiogenic switch as an alternative, previously unknown mechanism of tumor suppression for Smad4 and identify the angiogenic mediators vascular endothelial growth factor and thrombospondin-1 as key target genes.
Asunto(s)
Antineoplásicos/metabolismo , Proteínas de Unión al ADN/metabolismo , Genes Supresores de Tumor , Neovascularización Patológica , Neoplasias Pancreáticas/irrigación sanguínea , Transactivadores/metabolismo , Animales , División Celular/efectos de los fármacos , Movimiento Celular , Proteínas de Unión al ADN/genética , Resistencia a Antineoplásicos , Factores de Crecimiento Endotelial/genética , Factores de Crecimiento Endotelial/metabolismo , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Genes Supresores de Tumor/genética , Humanos , Linfocinas/genética , Linfocinas/metabolismo , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína Smad4 , Trombospondina 1/genética , Trombospondina 1/metabolismo , Transactivadores/genética , Transfección , Factor de Crecimiento Transformador beta/farmacología , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
We recently identified DPC4/Smad4 as a candidate tumor suppressor gene mutated or lost in one half of pancreatic carcinomas and in a subset of colon and biliary tract carcinomas. DPC4 plays a key role in signal transduction of the TGF-beta superfamily of molecules and inactivation of TGF-beta mediated growth inhibition is supposed to be the driving force for DPC4 inactivation in human tumors. However, DPC4 mediated tumor suppression by reconstitution of defective cells has not yet been reported. Here we show suppression of tumorigenicity in nude mice by stable reexpression of DPC4 in SW480 colon carcinoma cells. In vitro growth of DPC4-transfected cells was not affected and resistance towards TGF-beta mediated growth inhibition was retained. Instead, cells exhibited morphological alterations and adhesion and spreading were accelerated. These phenotypic changes were associated with reduced expression levels of the endogenous urokinase-type plasminogen activator (uPA) and plasminogen-activator-inhibitor-1 (PAI-1) genes, the products of which are implicated in the control of cell adhesion and invasion. In patients, high expression levels of uPA and PAI-1 correlate with poor prognosis. Thus, reduced expression of uPA and PAI-1 is consistent with suppression of tumorigenicity in DPC4 reconstituted cells. These results demonstrate DPC4's tumor suppressive function and suggest a potential role for DPC4 as a modulator of cell adhesion and invasion.
Asunto(s)
Neoplasias del Colon/patología , Proteínas de Unión al ADN/metabolismo , Transactivadores/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Adhesión Celular , División Celular , Movimiento Celular , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , Proteínas de Unión al ADN/genética , Humanos , Ratones , Ratones Desnudos , Fenotipo , Inhibidor 1 de Activador Plasminogénico/genética , Transducción de Señal , Proteína Smad4 , Transactivadores/genética , Factor de Crecimiento Transformador beta/metabolismo , Células Tumorales Cultivadas , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
A method is described to separate and characterize neutral and acidic lactose-derived oligosaccharides without prior derivatization or reduction by high-pH anion-exchange chromatography and pulsed amperometric detection (HPAEC-PAD). This method has been applied to human milk oligosaccharides from donors with different blood group specificity (A, Le(a) and A, Le(b). Neutral and acidic components were separated from each other by anion-exchange chromatography. A distinct separation of individual components was obtained by size-exclusion chromatography on Fractogel TSK HW 50S (acidic oligosaccharides) or Fractogel TSK HW 40S (neutral oligosaccharides containing up to 6 monomers) and Bio-Gel P-4 size exclusion (neutral oligosaccharides containing more than 6 monomers). Furthermore the moral response factors after HPAEC-PAD have been determined for 28 components.
Asunto(s)
Cromatografía Liquida/métodos , Leche Humana/química , Oligosacáridos/análisis , Secuencia de Carbohidratos , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Cromatografía en Capa Delgada , Femenino , Humanos , Concentración de Iones de Hidrógeno , Antígenos del Grupo Sanguíneo de Lewis , Oligosacáridos/químicaRESUMEN
According to published data the group of urinary total 17-ketosteroid sulfates appears to represent an index of overall adrenal androgen production, at least before the onset of puberty. To quantify total 17-ketosteroid sulfates a modified colorimetric assay based on the Zimmermann reaction was validated. 17-ketosteroid sulfates were measured without previous hydrolysis (as conjugated Zimmermann chromogens against authentic dehydroepiandrosterone sulfate (DHEAS) as assay standard) after C18 reversed-phase extraction and LH-20 chromatography. Intra- and inter-assay coefficients of variation were 8.4% (15.0%) and 5.9% (17.6%), respectively, at urinary 17-ketosteroid sulfate concentrations of 10.8 (1.9) nmol/ml. Recoveries observed in spiking and parallelism experiments varied between 88 and 102%. In a group of 4-year-old children showing a renal DHEAS output of less than 0.1 mumol/d/1.73 m2 (measured by radioimmunoassay) a relatively high median 17-ketosteroid sulfate excretion of 1.29 mumol/d/1.73 m2 was found. Older children aged 8 years as well as a group aged 12-14 years demonstrated only moderately higher urinary 17-ketosteroid sulfates whereas excretion of DHEAS/d/1.73 m2 more than tripled from age group to age group. For children from 8 years onwards, adolescents, and adults, linear regression analysis indicated that urinary DHEAS elevations seem to contribute with a constant proportion of approximately 70% to the increments of total urinary 17-ketosteroid sulfates. These findings suggest that the attainment of such a constant relationship (between the total 17-ketosteroid sulfates and their major component) from about 8 years of age onwards could represent the hormonal correlate of the completion of the continuous zona reticularis in the adrenal gland (developing around this age from a focal reticularis zone).