RESUMEN
Amitozyn (Am) is a semi-synthetic drug produced by the alkylation of major celandine (Chelidonium majus L.) alkaloids with the organophosphorous compound N,N'N'-triethylenethiophosphoramide (ThioTEPA). We show here that the treatment of living cells with Am reversibly perturbs the microtubule cytoskeleton, provoking a dose-dependent cell arrest in the M phase. Am changed the dynamics of tubulin polymerization in vitro, promoted the appearance of aberrant mitotic phenotypes in HeLa cells and induced apoptosis by the activation of caspase-9, caspase-3 and PARP, without inducing DNA breaks. Am treatment of HeLa cells induced changes in the phosphorylation of the growth suppressor pRb that coincided with maximum mitotic index. The dose-dependent and reversible anti-proliferative effect of Am was observed in several transformed cell lines. Importantly, the drug was also efficient against multidrug-resistant, paclitaxel-resistant or p53-deficient cells. Our results thus open the way to further pre-clinical evaluation of Am.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Benzofenantridinas/farmacología , Segregación Cromosómica/efectos de los fármacos , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Compuestos Organotiofosforados/farmacología , Benzofenantridinas/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de los fármacos , Células HeLa , Humanos , Compuestos Organotiofosforados/química , Fenotipo , Multimerización de Proteína/efectos de los fármacos , Proteína de Retinoblastoma/metabolismo , Transducción de Señal/efectos de los fármacos , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismoRESUMEN
Specifically expressed at intercellular adherens junctions of endothelial cells, VE-cadherin is a receptor that exhibits particular self-association properties. Indeed, in vitro studies demonstrated that the extracellular part of VE-cadherin elaborates Ca(++)-dependent hexameric structures. We hypothesized that this assembly could be at the basis of a new cadherin-mediated cell-cell adhesion mechanism. To verify this assumption, we first demonstrated that VE-cadherin can elaborate hexamers at the cell surface of confluent endothelial cells. Second, mutations were introduced within the extracellular part of VE-cadherin to destabilize the hexamer. Following an in vitro screening, three mutants were selected, among which, one is able to elaborate only dimers. The selected mutations were expressed as C-terminal green fluorescent protein fusions in CHO cells. Despite their capacity to elaborate nascent cell-cell contacts, the mutants seem to be rapidly degraded and/or internalized. Altogether, our results suggest that the formation of VE-cadherin hexamers protects this receptor and might allow the elaboration of mature endothelial cell-cell junctions.
Asunto(s)
Uniones Adherentes/metabolismo , Antígenos CD/química , Antígenos CD/metabolismo , Cadherinas/química , Cadherinas/metabolismo , Adhesión Celular/fisiología , Uniones Intercelulares/metabolismo , Animales , Antígenos CD/genética , Células CHO , Cadherinas/genética , Calcio/metabolismo , Comunicación Celular , Cricetinae , Cricetulus , Reactivos de Enlaces Cruzados/farmacología , Citoplasma/metabolismo , Dimerización , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Técnica del Anticuerpo Fluorescente , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inmunoprecipitación , Microscopía Fluorescente , Mutación/genética , Venas Umbilicales/citología , Venas Umbilicales/metabolismoRESUMEN
BACKGROUND: Angiogenesis assays are important tools for the identification of regulatory molecules and the potential development of therapeutic strategies to modulate neovascularization. Although numerous in vitro angiogenesis models have been developed in the past, they exhibit limitations since they do not recapitulate the entire angiogenic process or correspond to multi-step procedures that are not easy to use. Convenient, reliable, easily quantifiable and physiologically relevant assays are still needed for pharmacological screenings of angiogenesis. RESULTS: Here, we have optimized an angiogenesis model based on ES cell differentiation for screening experiments. We have established conditions leading to angiogenic sprouting of embryoid bodies during ES cell differentiation in type I three-dimensional collagen gels. Immunostaining experiments carried out during these cultures showed the formation of numerous buds comprising CD31 positive cells, after 11 days of culture of ES cells. Moreover, this one-step model has been validated in response to activators and inhibitors of angiogenesis. Sprouting was specifically stimulated in the presence of VEGF and FGF2. Alternatively, endothelial sprouting induced by angiogenic activators was inhibited by angiogenesis inhibitors such as angiostatin, TGFbeta and PF4. Sprouting angiogenesis can be easily quantified by image analysis after immunostaining of endothelial cells with CD31 pan-endothelial marker. CONCLUSION: Taken together, these data clearly validate that this one-step ES differentiation model constitutes a simple and versatile angiogenesis system that should facilitate, in future investigations, the screening of both activators and inhibitors of angiogenesis.
Asunto(s)
Inductores de la Angiogénesis/administración & dosificación , Inhibidores de la Angiogénesis/administración & dosificación , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Células Madre/citología , Células Madre/efectos de los fármacos , Animales , Bioensayo/métodos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Células Endoteliales/fisiología , Ratones , Neovascularización Fisiológica/fisiología , Células Madre/fisiologíaRESUMEN
Transmigration of neutrophils across the endothelium occurs at the cell-cell junctions where the vascular endothelium cadherin (VE cadherin) is expressed. This adhesive receptor was previously demonstrated to be involved in the maintenance of endothelium integrity. We propose that neutrophil transmigration across the vascular endothelium goes in parallel with cleavage of VE cadherin by elastase and cathepsin G present on the surface of neutrophils. This hypothesis is supported by the following lines of evidence. 1) Proteolytic fragments of VE cadherin are released into the culture medium upon adhesion of neutrophils to endothelial cell monolayers; 2) conditioned culture medium, obtained after neutrophil adhesion to endothelial monolayers, cleaves the recombinantly expressed VE cadherin extracellular domain; 3) these cleavages are inhibited by inhibitors of elastase; 4) VE cadherin fragments produced by conditioned culture medium or by exogenously added elastase are identical as shown by N-terminal sequencing and mass spectrometry analysis; 5) both elastase- and cathepsin G-specific VE cadherin cleavage patterns are produced upon incubation with tumor necrosis factor alpha-stimulated and fixed neutrophils; 6) transendothelial permeability increases in vitro upon addition of either elastase or cathepsin G; and 7) neutrophil transmigration is reduced in vitro in the presence of elastase and cathepsin G inhibitors. Our results suggest that cleavage of VE cadherin by neutrophil surface-bound proteases induces formation of gaps through which neutrophils transmigrate.