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Maximilian Schultheiss, Sven Schnichels, Kapka Miteva, Katrin Warstat, Peter Szurman, Martin S. Spitzer, Sophie van Linthout; Staurosporine Induced "Differentiation" of the RGC-5 Cell Line Leads to Apoptosis and Cell Death at the Lowest Cell-Morphology-Changing Concentration. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4608.
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© ARVO (1962-2015); The Authors (2016-present)
Supplementation of staurosporine is thé method of choice to differentiate the solely existing retinal ganglion cell (RGC)-5 cell line. This "differentiation" is claimed to be in the absence of apoptosis. In respect to the inconsistencies of RGC-5 and the known apoptosis-inducing effect of staurosporine, we investigated whether RGC-5 cells could be differentiated by staurosporine without the induction of apoptosis.
50-, 200-, 300- and 600 nM of staurosporine were supplemented on RGC-5 cells for 24 hours. Cell morphology and cell death, via propidium iodide staining, were investigated with phase contrast and fluorescence microscopy, respectively. Cell amount, cell proliferation and cell viability were analyzed by crystal violet staining, CFSE flow cytometry and MTS assay, respectively. Apoptosis was determined by analyzing caspase 3/7 activity, Annexin-V+/ 7AAD cells and the mRNA expression of the Bax/ Bcl-2 quotient via caspase 3/7 activity assay, flow cytometry and real-time PCR, respectively.
RGC-5 cells started to "differentiate" at 50 nM of staurosporine resulting in neurite formation. This was associated with apoptosis and cell death. Apoptosis was indicated by a 2.1-fold (p<0.0005) increase in caspase 3/7 activity, a 1.2-fold (p<0.05) induction of Annexin-V+/ 7AAD- cells, whereas cell death was indicated by a 11.7-fold (p<0.0005) increase in propidium iodide positive cells. Furthermore staurosporine led to a dose-dependent increase in apoptosis and reduction in cell viability, cell density and cell proliferation.
The lowest staurosporine concentration changing RGC-5 cell`s morphology is accompanied with apoptosis and cell death. Therefore, its use as differentiation-agent is at least very doubtful.
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