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L.J. Frassetto, C.R. Schlieve, N. Agarwal, L.A. Levin; Kinase–Dependent Differentiation of a Retinal Ganglion Cell Line . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4011.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Primary cultures of retinal ganglion cells (RGC) are used to study the pathophysiology of optic nerve and retinal diseases. However, these cells are difficult to obtain in large quantities, and purification requires exposure to antibodies against a cell surface antigen. RGC–5 cells, a transformed retinal precursor cell line, have some attributes of RGCs, but are inherently mitotic, a property not shared by RGCs. We studied the ability of kinase inhibition to differentiate RGC–5 cells into a nonproliferating state and with the morphological appearance of primary RGCs. Methods: RGC–5 cells were plated on 12 mm round cover glass and treated with varying concentrations of the broad–spectrum protein kinase inhibitor staurosporine (SS) or other, more specific protein kinase inhibitors. Following varying lengths of treatment cells were fixed and visualized. Morphological changes were quantified by counting neurites equal to or greater in length than the cell soma. Proliferation was assessed based on the incorporation of BrdU. Changes in phosphorylation state of protein targets of several kinases were assessed by western blotting. Results:SS treatment induced concentration–dependent differentiation of RGC–5 cells into cells morphologically similar to RGCs. The number of neurites per cell after treatment for 24 hours with SS was 0.16 ± 0.05 (0 nM), 0.27 ± 0.06 (100 nM), 1.43 ± 0.10 (316 nM), 2.94 ± 0.11 (1 µM) and 3.88 ± 0.13 (1.78 µM). Even 60 seconds of exposure to SS followed by 24 hours of incubation was enough to differentiate RGC–5 cells. Some of the differentiating effects of SS were also seen with inhibitors of Rho–kinase (H–1152) and protein kinase A (H–89), but were not as complete. Differentiated cells did not proliferate and did not undergo apoptosis. Western blotting demonstrated a 90% increase in the S722 target of focal adhesion kinase. Other kinases with phosphorylation targets having notable differences from control were c–Kit (+43%), ERK1 (not present in control), ERK2 (–59%), CDK1 (+64%), MAP1 (–100%) and PKR (–100%). Conclusions:Morphological differentiation of an RGC–like retinal precursor cell line appears to depend on changes in activity of one or more protein kinases. Not only does this result provide a more realistic RGC–like cell for biological studies, but may also suggest a general mechanism for differentiation of neuronal precursor cells, potentially including stem cells.
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