RESUMEN
The anaplastic lymphoma kinase (ALK), tyrosine kinase oncogene is implicated in a wide variety of cancers. In this study we used conditional onco-ALK (NPM-ALK and TPM3-ALK) mouse MEF cell lines (ALK+ fibroblasts) and transgenic models (ALK+ B-lymphoma) to investigate the involvement and regulation of angiogenesis in ALK tumor development. First, we observed that ALK expression leads to downregulation of miR-16 and increased Vascular Endothelial Growth Factor (VEGF) levels. Second, we found that modification of miR-16 levels in TPM3-ALK MEF cells greatly affected VEGF levels. Third, we demonstrated that miR-16 directly interacts with VEGF mRNA at the 3'-untranslated region and that the regulation of VEGF by miR-16 occurs at the translational level. Fourth, we showed that expression of both the ALK oncogene and hypoxia-induced factor 1α (HIF1α) is a prerequisite for miR-16 downregulation. Fifth, in vivo, miR-16 gain resulted in reduced angiogenesis and tumor growth. Finally, we highlighted an inverse correlation between the levels of miR-16 and VEGF in human NPM-ALK+ Anaplastic Large Cell Lymphomas (ALCL). Altogether, our results demonstrate, for the first time, the involvement of angiogenesis in ALK+ ALCL and strongly suggest an important role for hypoxia-miR-16 in regulating VEGF translation.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Hipoxia/metabolismo , Linfoma Anaplásico de Células Grandes/genética , Linfoma Anaplásico de Células Grandes/metabolismo , MicroARNs/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Quinasa de Linfoma Anaplásico , Animales , Northern Blotting , Western Blotting , Estudios de Casos y Controles , Adhesión Celular , Movimiento Celular , Células Cultivadas , Metilación de ADN , Regulación hacia Abajo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Hipoxia/genética , Hipoxia/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Técnicas para Inmunoenzimas , Linfoma Anaplásico de Células Grandes/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , MicroARNs/metabolismo , Neovascularización Patológica , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Tirosina Quinasas Receptoras/genética , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
HIV-1 integrase (IN) catalyzes the integration of the proviral DNA into the cellular genome. The catalytic triad D64, D116 and E152 of HIV-1 IN is involved in the reaction mechanism and the DNA binding. Since the integration and substrate binding processes are not yet exactly known, we studied the role of amino acids localized in the catalytic site. We focused our interest on the V151E152S153 region. We generated random mutations inside this domain and selected mutated active INs by using the IN-induced yeast lethality assay. In vitro analysis of the selected enzymes showed that the IN nuclease activities (specific 3'-processing and non-sequence-specific endonuclease), the integration and disintegration reactions and the binding of the various DNA substrates were affected differently. Our results support the hypothesis that the three reactions may involve different DNA binding sites, enzyme conformations or mechanisms. We also show that the V151E152S153 region involvement in the integration reaction is more important than for the 3'-processing activity and can be involved in the recognition of DNA. The IN mutants may lead to the development of new tools for studying the integration reaction, and could serve as the basis for the discovery of integration-specific inhibitors.
Asunto(s)
Integrasa de VIH/química , Integrasa de VIH/metabolismo , Sitios de Unión , Dominio Catalítico , ADN/metabolismo , Análisis Mutacional de ADN , Endonucleasas/metabolismo , Ácido Glutámico/genética , Integrasa de VIH/genética , Mutagénesis , Fenotipo , Levaduras/citologíaRESUMEN
Current treatments of human immunodeficiency virus type 1 (HIV-1) infection consist in the combination of drugs targeting reverse transcriptase (RT) and protease (PR). Despite the multiple clinical benefits of this combination therapy, the emergence of resistance highlights the need for new anti-HIV agents. Agents able to interfere with additional steps of viral replication, such as integration of viral DNA in the host genome, would improve the antiviral potency of the treatment. In this regard, we have focused our interest on peptide-based compounds that have been shown to exhibit potential inhibition of RT and integrase (IN) activities in vitro and in vivo. Recently, the expansion of powerful technologies which allow the selection of peptides exhibiting high affinity for a target protein have provided a new approach to selecting potential anti-HIV drugs. Furthermore, efforts to characterize the protein-protein interactions involved in efficient reverse transcription and integration, as well as the determination of the enzyme structure, have generated a very useful source of data for the development of peptide inhibitors. Finally, while this class of compounds has long been considered as poor drug candidates, current knowledge on improving the stability and bioavailability of these agents would lead to the effective use of peptides in therapy.
Asunto(s)
Fármacos Anti-VIH/farmacología , Inhibidores de Integrasa VIH/farmacología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/efectos de los fármacos , Péptidos/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Fármacos Anti-VIH/química , Fármacos Anti-VIH/uso terapéutico , Diseño de Fármacos , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , Inhibidores de Integrasa VIH/química , Inhibidores de Integrasa VIH/uso terapéutico , VIH-1/enzimología , Humanos , Modelos Biológicos , Biblioteca de Péptidos , Péptidos/química , Péptidos/uso terapéutico , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/uso terapéuticoRESUMEN
The retroviruses are a large, diverse family of enveloped RNA viruses defined by their structure, composition and replicative properties. The hallmark of the family is its replicative strategy, essential steps of which include reverse transcription of the viral RNA and the subsequent integration of this DNA into the genome of the cell. These steps are performed by two viral-encoded enzymes, reverse transcriptase (RT), which possesses DNA polymerase and ribonuclease H (RNase H) activities, and integrase (IN). These enzymes are excellent targets for retroviral therapy since they are essential for viral replication. Numerous substances capable of inhibiting the DNA polymerase activity of HIV-1 RT are available, while few specific inhibitors of RNase H activity have been described. IN is absolutely necessary for stable and productive infection of cells. Some IN inhibitors have been recently reported and are available demonstrating the potential of IN as an antiviral target. This paper is an overview of the inhibitors of RNase H and IN and describes the most promising inhibitors.