RESUMEN
Novel ADCC effector cells expressing the V-variant or F-variant of FcγRIIIa (CD16a) and firefly luciferase under the control of a chimeric promoter incorporating recognition sequences for the principal transcription factors involved in FcγRIIIa signal transduction, together with novel target cells overexpressing a constant high level of the specific antigen recognized by rituximab, trastuzumab, cetuximab, infliximab, adalimumab, or etanercept, confer improved sensitivity, specificity, and dynamic range in an ADCC assay relative to effector cells expressing a NFAT-regulated reporter gene and wild-type target cells. The effector cells also contain a normalization gene rendering ADCC assays independent of cell number or serum matrix effects. The novel effector and target cells in a frozen thaw-and-use format exhibit low vial-to-vial and lot-to-lot variation in their performance characteristics reflected by CVs of 10% or less. Homologous control target cells in which the specific target gene has been invalidated by genome editing providing an ideal control and a means of correcting for nonspecific effects were observed with certain samples of human serum. The novel effector cells and target cells expressing noncleavable membrane-bound TNFα have been used to quantify ADCC activity in serum from patients with Crohn's disease treated with infliximab and to relate ADCC activity to drug levels.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos CD20/genética , Enfermedad de Crohn/inmunología , Receptores ErbB/genética , Técnicas Inmunológicas/métodos , Factores de Transcripción NFATC/genética , Receptor ErbB-2/genética , Receptores de IgG/genética , Linfocitos T/fisiología , Factor de Necrosis Tumoral alfa/genética , Antígenos CD20/inmunología , Cetuximab/metabolismo , Receptores ErbB/inmunología , Etanercept/metabolismo , Genes Reporteros/genética , Células HEK293 , Humanos , Infliximab/metabolismo , Células Jurkat , Receptor ErbB-2/inmunología , Receptores de IgG/inmunología , Rituximab/metabolismo , Transducción de Señal , Transgenes/genética , Trastuzumab/metabolismo , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Cell-cell contacts inhibit cell growth and proliferation in part by activating the Hippo pathway that drives the phosphorylation and nuclear exclusion of the transcriptional coactivators YAP and TAZ. Cell density and Hippo signaling have also been reported to block transforming growth factor ß (TGF-ß) responses, based on the ability of phospho-YAP/TAZ to sequester TGF-ß-activated SMAD complexes in the cytoplasm. Herein, we provide evidence that epithelial cell polarization interferes with TGF-ß signaling well upstream and independent of cytoplasmic YAP/TAZ. Rather, polarized basolateral presentation of TGF-ß receptors I and II deprives apically delivered TGF-ß of access to its receptors. Basolateral ligand delivery nonetheless remains entirely effective to induce TGF-ß responses. These data demonstrate that cell-type-specific inhibition of TGF-ß signaling by cell density is restricted to polarized epithelial cells and reflects the polarized distribution of TGF-ß receptors, which thus affects SMAD activation irrespective of Hippo pathway activation.
Asunto(s)
Citoplasma/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Aciltransferasas , Western Blotting , Recuento de Células , Proteínas de Ciclo Celular , Proliferación Celular , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Vía de Señalización Hippo , Humanos , Técnicas para Inmunoenzimas , Inmunoprecipitación , Proteínas Nucleares/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/genéticaRESUMEN
YAP and its paralog protein TAZ are downstream effectors of the Hippo pathway. Both are amplified in many human cancers and promote cell proliferation and epithelial-mesenchymal transition. Little is known about the status of the Hippo pathway in cutaneous melanoma. We profiled Hippo pathway component expression in a panel of human melanoma cell lines and melanocytic lesions, and characterized the capacity of YAP and TAZ to control melanoma cell behavior. YAP and TAZ immuno-staining in human samples revealed mixed cytoplasmic and nuclear staining for both proteins in benign nevi and superficial spreading melanoma. TAZ was expressed at higher levels than YAP1/2 in all cell lines and in those with high invasive potential. Stable YAP or TAZ knockdown dramatically reduced the expression of the classical Hippo target CCN2/connective-tissue growth factor (CTGF), as well as anchorage-independent growth, capacity to invade Matrigel, and ability form lung metastases in mice following tail-vein injection. YAP knockdown also reduced invasion in a model of skin reconstruct. Inversely, YAP overexpression increased melanoma cell invasiveness, associated with increased TEA domain-dependent transcription and CCN2/CTGF expression. Together, these results demonstrate that both YAP and TAZ contribute to the invasive and metastatic capacity of melanoma cells and may represent worthy targets for therapeutic intervention.