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A. Vugler, J.M. Lawrence, P.J. Coffey, J. Greenwood; Expression of neuronal–associated proteins in Retinal Pigment Epithelial cell lines . Invest. Ophthalmol. Vis. Sci. 2004;45(13):642.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Recent work has indicated the potential for fenretinide to induce neuronal differentiation in ARPE19 cells. To further assess this phenomenon, we investigate the effect of this drug on the expression of neuronal markers in ARPE19 cells and compare patterns of expression to those found in untreated primary human RPE and an SV40 immortalised RPE cell line (h1RPE7). Methods:ARPE19 cells were cultured with or without the addition of 3µM fenretinide. After 7 days, cells were fixed and processed for immunocytochemistry using antibodies against neurofilament 200 (nf–200), calretinin, synaptophysin and the voltage gated (vg) sodium channel. Primary human RPE were passaged twice before fixation, while additional untreated ARPE19 and h1RPE7 cells were fixed at various densities approaching confluence. Results: Fenretinide–treated ARPE19 cells exhibited dramatic morphological changes characterised by the extension of neuron–like processes. This was in contrast to control dishes in which cells had grown to confluence and appeared normal. The expression of nf–200 and calretinin was enhanced by fenretinide treatment but there were distinct regions of positive staining in control dishes, with up to 50% of control cells expressing nf–200 to some degree. Synaptophysin and voltage gated sodium channels were expressed at high levels in control dishes of ARPE19 cells. Synaptophysin immunoreactivity (IR) appeared diffusely distributed throughout the cell, whereas vg sodium channel–IR was largely restricted to junctional regions between cells. Treatment with fenretinide did not appear to upregulate the expression of either of these two proteins. Synaptohysin and vg sodium channel–IR was present along processes of fenretinide treated ARPE19 cells, although the intensity of the latter was much reduced compared to controls. In untreated primary human and sub–confluent ARPE19 / h1RPE7 cells, the expression of synaptophysin was ubiquitous, with numerous cells co–expressing nf–200. The expression of nf–200 was often associated with dividing cells, this being most evident with an antibody against the phosphorylated form of this protein. Conclusions: The addition of fenretinide to ARPE19 cells would appear to enhance the expression of some proteins normally found in RPE cells during various stages of their cell cycle in vitro. However, Synaptophysin and vg sodium channels, two important components of retinal ganglion cells would appear to be constitutively expressed by cultured RPE cells and are not induced by addition of a synthetic retinoid.
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