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L. Pignataro, C. Condello, V. Sarthy; Glutamate Transporter Expression in Differentiated Retinoblastoma Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):5154.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The retinoblastoma cell line, Y79, is known to express both glial and neuronal markers, indicating that despite its cone-photoreceptor specific origin, the cell line is at an intermediate stage between neuronal and glial differentiation. However, Y79 cells can be induced to differentiate into "neuronal’-like cells by treatment with dibutyryl cAMP, sodium butyrate or pigment epithelium-derived growth factor (PEDF). Because Y79 cells express both glial (EAAT1) as well as neuronal (EAAT2, 3, 5) glutamate transporters, the present study was designed to examine whether morphological differentiation of Y79 is accompanied by changes in the pattern of glutamate transporter expression. Methods: Y79 cells were cultured in suspension in defined medium with PEDF, dbcAMP or sodium butyrate for five days. Next, they were seeded on poly-L-ornithine coated dishes to promote attachment and kept for ten days in the defined medium containing the same agents. The expression of different marker proteins, EAAT1, EAAT5, GFAP (glial marker), and neuronal enolase (NSE, neuronal marker) was studied by fluorescence immunocytochemistry using specific antibodies. Results: Y79 cells were immunostained by both EAAT1 and EAAT5 antibodies with EAAT5 Ab showing a more robust staining. In cultures exposed to 200ng/ml of PEDF, many cells showed extensive processes with a profusion of filopodia. In these cells, there was an increase in NSE staining. There was also an increase in EAAT5 labeling and a significant decrease in EAAT1 immunostaining. Dibutyryl cyclic AMP induced cell differentiation to a lesser extent than PEDF, but did not change the immunostaining pattern for GFAP, NSE, EAAT1 and EAAT5. Finally, cells treated with sodium butyrate behaved similarly to the dbcAMP-treated cells except that there was an increase in EAAT5 immunolabeling. Conclusions: The neuron-like morphological differentiation induced in Y79 cells by PEDF, is accompanied by continued expression of the neuronal glutamate transporter, EATT5, while the glial glutamate transporter appears to be down regulated under these conditions. Results with dbcAMP and sodium butyrate suggest that these agents are less effective in promoting morphological and biochemical differentiation of Y79 cells. Supported by NIH grant EY-13125.
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