RESUMEN
Immortalized keratinocytes can offer a low-cost experimental platform for human skin research, with increased cell yield compared to primary cultures. However, the usefulness of these surrogate cell models is highly dependent on their ability to retain the phenotypic attributes of the parent cells. Keratins K14 and K5 are the hallmarks of undifferentiated, mitotically active basal keratinocytes. We observed occasional progressive loss of K14 expression in growing keratinocyte cell lines, with persistent retention of K5 and an epithelial phenotype, and investigated possible reasons for this. We show that K14 repression occurs by DNA promoter methylation of KRT14 gene and is compounded by histone deacetylation and by the presence of EGF. In vivo, keratinocytes shut down K14 synthesis as they commit to terminal differentiation and move from the basal to spinous layer, but by laser-capture microdissection of human epidermis we could detect no evidence of increased selective KRT14 methylation in this normal process. Loss of K14 expression suggests that epidermal identity of cultured keratinocytes can be compromised in certain tissue culture situations, possibly due to the immortalization method and persistent EGF supplementation.
Asunto(s)
Metilación de ADN , Queratina-14 , Queratinocitos , Regiones Promotoras Genéticas , Queratinocitos/metabolismo , Humanos , Queratina-14/genética , Queratina-14/metabolismo , Diferenciación Celular , Queratina-5/genética , Queratina-5/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Epidermis/metabolismo , Histonas/metabolismoAsunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/metabolismo , Queratinocitos/fisiología , Oxígeno/metabolismo , Fenómenos Fisiológicos de la Piel , Proliferación Celular , Células Cultivadas , Análisis por Conglomerados , Proteínas Filagrina , Regulación de la Expresión Génica , Humanos , Hipoxia/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Interleucina-1/genética , Proteínas de Filamentos Intermediarios/genética , Queratina-10/genética , Cultivo Primario de Células , Precursores de Proteínas/genéticaRESUMEN
Single wavelength fluorescence cross-correlation spectroscopy (SW-FCCS), introduced to study biomolecular interactions, has recently been reported to monitor enzyme activity by using a newly developed fluorescent protein variant together with cyan fluorescent protein. Here, for the first time to our knowledge, SW-FCCS is applied to detect interactions between membrane receptors in vivo by using the widely used enhanced green fluorescent protein and monomeric red fluorescent protein. The biological system studied here is the epidermal growth factor/ErbB receptor family, which plays pivotal roles in the development of organisms ranging from worms to humans. It is widely thought that a ligand binds to the monomeric form of the receptor and induces its dimeric form for activation. By using SW-FCCS and Förster resonance energy transfer, we show that the epidermal growth factor receptor and ErbB2 have preformed homo- and heterodimeric structures on the cell surface and quantitation of dimer fractions is performed by SW-FCCS. These receptors are major targets of anti-cancer drug development, and the receptors' homo- and heterodimeric structures are relevant for such developments.