RESUMEN
TGFbeta mediates cell cycle arrest in late G(1) phase of the cell cycle with a simultaneous peak in the levels of the cyclin-dependent kinase inhibitor, p27(kip1) (p27). In this report, we show that whereas p27 resides in the cytoplasm in the endometrial carcinoma (ECA) cell line HEC-1A, TGFbeta increases the total levels and translocation of p27 into the nucleus. Concomitantly, TGFbeta activates the transcription factors Smad2 and Smad3, inhibits proliferation, and blocks Cdk2 activity; all these events are blocked by an inhibitor of TbetaRI serine kinase activity (SD208). In addition, we show that inhibiting p27 transcription with a specific siRNA completely blocks TGFbeta-mediated growth inhibition in these cells. These data suggest that TGFbeta inhibits cellular proliferation by increasing p27 levels through Smad2/3 signaling in HEC-1A cells. We further show that TGFbeta decreases the levels of components of the SCF(Skp2) targeting complex for ubiquitin-mediated degradation of p27 in proteasomes, at the protein but not the mRNA level. Therefore, TGFbeta accumulates nuclear p27 by preventing its degradation to enable G(1) arrest in HEC-1A cells. Importantly, these data suggest a novel mechanism for TGFbeta/Smad mediated growth inhibition that might be inoperable in the numerous human cancers demonstrating early dysregulated TGFbeta signaling and loss of growth inhibition. The TGFbeta/p27 axis might provide novel therapeutic targets for cancer.
Asunto(s)
Ciclo Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Línea Celular Tumoral , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Fase G1 , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , ARN Interferente Pequeño/metabolismo , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína smad3/genética , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/genéticaRESUMEN
PURPOSE: Two major functions of thrombin observed in the cornea are activation of thrombin-sensitive, proteinase-activated receptors and cleavage of fibrinogen to fibrin. The purpose of this study was to determine whether the normal human cornea itself is competent to convert prothrombin to thrombin and synthesizes the mRNA for the proteins required. METHODS: Human corneas were processed for immunolocalization studies or separated into epithelial, stromal, and endothelial layers for proteins and RNA isolation. The protein extracts were used for Western blots, prothrombin time, and activated partial thromboplastin time assays and fibrinopeptide A generation tests. RNA was used for RT-PCR. Apoptosis of cultured human corneal cells was induced with sodium nitroprusside or camptothecin and activation of prothrombin tested. RESULTS: Prothrombin and its mRNA were present in all three layers of human donor cornea. It was found to be associated with the cells and the extracellular matrix at similar levels across the cornea. With corneal stromal extracts, activation of either the intrinsic or extrinsic coagulation pathways resulted in thrombin activation and fibrin formation with fibrinopeptide A release. Detection of key components of the coagulation cascades confirmed noninjured human corneas contain factors required for prothrombin activation. In addition, mRNAs for representative factors and inhibitors were detected by RT-PCR and confirmed by sequencing. Apoptotic corneal stromal cells provide a surface for prothrombin activation. CONCLUSIONS: These studies suggest that the normal avascular human cornea contains and synthesizes the components required for thrombin generation and that this process does not depend on a breech in the limbal vascular endothelium.
Asunto(s)
Factores de Coagulación Sanguínea/fisiología , Córnea/fisiología , Protrombina/metabolismo , Trombina/biosíntesis , Western Blotting , Camptotecina/farmacología , Permeabilidad Capilar , Células Cultivadas , Sustancia Propia/efectos de los fármacos , Sustancia Propia/metabolismo , Sustancia Propia/patología , Endotelio Vascular/metabolismo , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática , Epitelio Corneal/efectos de los fármacos , Epitelio Corneal/metabolismo , Epitelio Corneal/patología , Fibrinopéptido A/metabolismo , Humanos , Técnicas para Inmunoenzimas , Limbo de la Córnea/irrigación sanguínea , Nitroprusiato/farmacología , Tiempo de Tromboplastina Parcial , Protrombina/genética , Tiempo de Protrombina , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
PURPOSE: To determine whether the cornea contains and expresses, at the gene level, the major plasmin inhibitor alpha2-antiplasmin. METHODS: Corneal sections were immunostained for alpha2-antiplasmin. Extracts of human corneal stroma, epithelium, and endothelium were subjected to immunodot blot and Western blot analysis. Total RNA and alpha2-antiplasmin specific primers were used for RT-PCR. The cDNA was sequenced. RESULTS: Alpha2-antiplasmin was observed in all three corneal layers by immunolocalization and Western blots. The major alpha2-antiplasmin form observed in most extracts was the 70-kDa form. Total alpha2-antiplasmin was present at 0.119 +/- 0.014 microg/epithelium (n = 10) and 1.45 +/- 0.47 microg/stroma (n = 10). Alpha2-antiplasmin mRNA was detected in epithelial and stromal extracts and cultured human corneal stromal cells. The sequences of the PCR products were identical to that for human alpha2-antiplasmin. CONCLUSIONS: Alpha2-antiplasmin and its mRNA are present in the cornea and may serve to regulate corneal plasmin activity.
Asunto(s)
Antifibrinolíticos/metabolismo , Sustancia Propia/metabolismo , Endotelio Corneal/metabolismo , Epitelio Corneal/metabolismo , alfa 2-Antiplasmina/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Western Blotting , Humanos , Immunoblotting , Técnicas para Inmunoenzimas , Persona de Mediana Edad , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , alfa 2-Antiplasmina/genéticaRESUMEN
CD8+ tumor-infiltrating lymphocytes (TIL) are severely deficient in cytolysis, a defect that may permit tumor escape from immune-mediated destruction. Because lytic function is dependent upon TCR signaling, we have tested the hypothesis that primary TIL have defective signaling by analysis of the localization and activation status of TIL proteins important in TCR-mediated signaling. Upon conjugate formation with cognate target cells in vitro, TIL do not recruit granzyme B+ granules, the microtubule-organizing center, F-actin, Wiskott-Aldrich syndrome protein, nor proline rich tyrosine kinase-2 to the target cell contact site. In addition, TIL do not flux calcium nor demonstrate proximal tyrosine kinase activity, deficiencies likely to underlie failure to fully activate the lytic machinery. Confocal microscopy and fluorescence resonance energy transfer analyses demonstrate that TIL are triggered by conjugate formation in that the TCR, p56lck, CD3zeta, LFA-1, lipid rafts, ZAP70, and linker for activation of T cells localize at the TIL:tumor cell contact site, and CD43 and CD45 are excluded. However, proximal TCR signaling is blocked upon conjugate formation because the inhibitory motif of p56lck is rapidly phosphorylated (Y505) and COOH-terminal Src kinase is recruited to the contact site, while Src homology 2 domain-containing protein phosphatase 2 is cytoplasmic. Our data support a novel mechanism explaining how tumor-induced inactivation of proximal TCR signaling regulates lytic function of antitumor T cells.